Hem f 0tk ^mt <50ltep of ^^timltuu Hibrary Date Due mmmmmamtmaaama Cornell University Library QR 115.T3 C.2 Bacteriology and niycojogy of f oo 3 1924 003 214 552 BACTERIOLOGY AND MYCOLOGY OF FOODS FRED WILBUR TANNER, M.S., Ph.D. Associate in Bacteriology, University of Illinois FIJUST F.DITXOM NEW YORK JOHN WILEY & SONS, Inc. London: CHAPMAN c Fig. 5. — ^Types of Holders for Inoculating Needles and Loops* At Special Aluminum and Wooden Handle; S, Aluminum Rod; C, Glass Rod with Fused Wire. was from 0.017 to 0.023 gm. The size of the drop canied by this loop is about 0.020 c.c. Baskets. These should be made of wire with a mesh small enough to retain most of the test tubes used in a laboratory. Any special shape or size may be made at the average tin shop. General Technique Isolation of Microorganisms. This necessitates the plating of bacteria in decimal dilutions on solid media. Agar or gelatin may be used but the latter gives more data. If the organism which is sought in pure culture is known to be mixed with other forms, greater care must be exercised. The plating of decimal dilutions is advisable, especially where little is known with regard to the number of cells which are present. All apparatus must be sterile. Where careful work is 8 BACTERIOLOGICAL APPARATUS . j id K^jkimM'm'^'>^w7 Fig. 6.— Various Types of Wire Baskets for tlie Bacteriological Laboratory. COUNTING APPARATUS 9 being done, the sample of food should be thoroughly agitated on a shaking machine with glass beads or broken glass. Fig. 7. — ^Wolffhuegel Counting Apparatus. D E Fig. 8. — ^Types of Counting Devices. A, Petri Dish with the Bottom Divided into Squares; JS, Lafar's Counting Plate; C and I>, RoBzahegyi's Counting Flask; E, Jeffer's Counting Plate. Potirmg of Plates. A series of these should be poured with different concentrations of the sample. These may be obtained by thoroughly shaking a portion of the sample in sterile water and transferring a loop 10 BACTERIOLOGICAL APPARATUS of this suspension to a second tube of stenle water. This procedure is repeated until seveial concentrations of the sample ar6 secured. Portions of these are placed in sterile Petri dishes or under these condi- tions put into tubes of melted medmm. These are then put into the Petri dishes. Isolation of Pure Cultures. A pure culture may be defined in different ways but in this relation it may be regarded as the descend- ants of a single cell. Each single cell on the Petri dishes poured m the above paragraph will have developed into a colony. In most cases this colony will contain a pure culture if the plate does not contain too many colonies. When there are but a few colonies on the plates, each one that IS isolated from the others will be pure. If it is found to be a mixed culture the plating operation must be repeated. In fact in careful work it is essential to replate two or three times. This makes the probability of a pure culture more certain and also serves to '' tune- up " or strengthen the organism. Fig 9 — Bottcher's Counting Chamber. It consists of an ordinary glass slide with a glass ring 20 mm m diameter and 8 mm high The c over glass is ruled into 100 squares» 19" of which are numbered The isolated colonies are picked by means of a sterile platinum wire. A small portion of the colony is removed to either an agar slant or broth tube. Before any extensive investigations are carried out with the organism, it should be studied in hanging drop and stained smear to demonstrate its purity. Transferring of Cultures. This procedure should be carefully carried out in a room with little dust. Sterile platinum wires should be used which should be sufficiently cooled. If a wire of too great diameter is used, it will retain its heat longer and there is then greater danger that the culture will be killed or altered. Also, if much trans- ferring is done at the same time there will be a tendency for some of the material to cake on the wire. This should be removed. A piece of emery paper or No. 00 sand paper will be found to be a valua- ble aid. Dilutions. These are essential in the quantitative enumeration of bacteria. The kind of containers to be used has been described above. ENUMERATION OF MICROORaANLSMS 11 The dilution water in deiSmte aniountb may be put into them befoie sterilization but the quantity of water has been found to be altered dunng this procedure. To obviate this difficulty, the containers may be plugged and sterilized empty while the water may be sterihzed in a larger bottle. This may then be transferred to the dilution bottles with a sterile pipette. Fig. 10 — Stewart's Counting Apparatus The Petri clisb is illuminated by oblique rays of light commg from an incandescent light. Enumeration of Bacteria Several methods have been devised for counting the number of bacteria which are present in any substance. For convenience they may be classified as follows: A. Plate cultures. (a) Petri dish cultures. (b) Frost's microscopic plate method. B. Direct microscopic method. (a) Breed's method. (&) Haemocytometer methods. The plate method is probably the oldest and involves the use of the Petri dish. Any transparent sohd medium may be used in the Petri dish, but the dilution of sample introduced is important. Hill (1908) and Breed (1916) have both shown that over-crowded plates 12 BACTERIOLOGICAL APPARATUS will not give accurate results. Breed, in studying plates made from milk, found that those having more than thirty and less than four hundred colonies gave satisfactory results. Frost's microscopic plate method and Breed's microscopic method have been described in the Chapter on Milk. These methods were Fig. 11. — Hsemocytometer with Pipettes in Case. originally divised for milk but undoubtedly could be adapted to other food substances. Hsemocytometer. The hsemocytometer, which is used for counting leucocytes and erythrocytes, is now apphed to the enumeration of bacteria in very heavily populated materials. W B J^D Fig. 12. — Showing Hsemocytometer Chamber. The instrument consists of a glass sUde upon which is fastened pieces of glass in such a way as to form a chamber. The outer glass is square with a round hole in the center. In this is put a round piece of glass of smaller diameter so that there is a shelf, moat and disk formed. The upper surface of the disk is ruled into squares. These rulings are of various kinds as indicated in Fig. 11, the Thoma ruling being as com- BLOOD COUNTS 13 mon as any. This Thoma ruling, however, is more suitabte for counting red blood cells. The pipettes are two in number and are graduated to deliver differ- ent amounts. The pipette for erythrocytes delivers dilution of xio* or 2^0-; the pipette for leucocytes delivers a dilution of xV or ^V- The hsemocytometer was originally devised for counting the blood corpuscles. From this it has been adapted to the enumeration of bac- teria in substances containing large numbers. It is best for a person not famihar with the technique to apply it to the counting of blood corpuscles before attempting to count bacteria. Procedure, Red Corpuscles. Collect a drop of blood on a clean slide and draw part of it up to the mark on the red corpuscle pipette (101). The blood may be secured by cutting the tip of the finger (across the lines) or by a syringe from a vein in the forearm. By drawing the blood to the 0.5 mark a dilution of 200 is secured; when drawn to the 1.0 mark the dilution is 1 to 100. This first measurement of blood is very important since a slight variation from the mark introduces a larger error in the final results. Should the blood be drawn up too far in the pipette it may be shaken down carefully to the mark. After this the diluting fluid should be drawn into the pipette up to the 101 mark and the pipette thoroughly shaken. The blood is now suf- ficiently diluted for counting the red blood corpuscles. The contents of the end of the pipette below the bulb should be discarded since it is filled mostly with diluting fluid. A drop of the diluted blood is now placed on the disk of the counting chamber. This should be just suf- ficient to cover the disk after the cover glass has been put on. The cover glass should be carefully applied with a very slight sliding motion. This should be placed on the chamber as soon as possible in order to secure as even a distribution of corpuscles over the bottom of the chamber as possible. The chamber must be left on a level surface for about a half hour, after which the corpuscles will have settled to the bottom and will be resting on the ruled surface. The number of red corpuscles is now determined by putting the chamber imder a micro- scope fitted with a mechanical stage. The small squares cover an area -^hu sq. mm. since they are ^ mm. on a slide. The depth of the chamber from the bottom of the cover glass to the top of the disk is iV mm., hence the volume of Uquid above a square is ^VX^VXxV equals i-^Vo nim. 4000 X dilution (200) equals red corpuscles per cubic millimeter. A sufficient number of squares should be counted to give a fairly correct average per square. To accomplish this it is best to count about one hundred squares. By BACTERIOLOGICAL APPAEATUS ^^^^11 It I '^l^^l M— llllillllilll illl — B Biiiiii Thoma Ruling Neubauer Ruling Zappert Ruling Breuer RuKng Fuchs and Rosenthal Ruling Turk Ruling ' Biirker No. 1 Ruling Zappert-Ewing Ruling Zappert-Neubauer Ruling Bass Ruling (15x) Fig. IS.—Diagrams Showing the Commonest Hfcmocytometer EuHm BLOOD COUNTS 15 means of the mechanical stage the counting chamber should be moved carefully from square to square. It is useful to always count the cells when they he on the lines, which are above and to the left. By fol- lowing some definite system the chance of counting the same corpuscle twice is eliminated. White Corpuscles, The blood should be draw^n into the pipette up to the 0.5 mark after which the special diluting fluid for white corpuscles should be drawn up to the mark. This will give a dilution of -io. A special diluting fluid which will destro}^ the red and leave the white corpuscles, must be used. The preparation of the disk of the counting chamber is identical with that for counting red corpuscles. A systematic procedure should be followed in going over the shde in order not to count the same square twice. In a well-prepared mount, the number of cells per square should not vary very much from the average for 100 squares. Fig. 14. — Diluting Pipettes for Haemocytometer. A counting chamber of more recent construction has been made for the Arthur H. Thomas Co., Philadelphia, known as the Thoma- Levy hsemacytometer. This has certain advantages over the types of the older construction and is described as follows: " In the Levy construction a rectangular depression is cut into the slide itself extending across the entire width. In the middle of this depression is perma- nently fixed a rectangular strip of glass also extending entirely across the slide, and on this are the rulings. When the cover glass is placed in position on the slide itself the solution over the ruled areas is of the required depth. The Levy method of construction avoids the cemented cell and the attendant danger of its loosening by the drying out of the balsam cement, and the possibility of the loosening of the ruled counting surface is also greatly reduced by this construc- tion. The parallel form of cell greatly facilitates cleaning as compared with the circular type and the method of ruling used in the manufacture of these chambers provides a line with absolutely cleancut edges and of distinctly increased visi- bility when the chamber is filled with solution for the count. This increase in visibihty of the ruling greatly lessens the eye fatigue experienced in making repeated counts.' ' The manufacturers <31aim for this "counting chamber the following advantages: Increased visibility of the rulings when chamber is filled with solution. 16 BACTERIOLOGICAL APPARATUS The new method of construction entirely avoids the cemented cell and the attendant danger of its loosening by the drymg out of the balsam cement, and the possibility of the loosening of the ruled counting surface is also greatly reduced by this construction. The parallel form of cell first suggested by Burker and used m all Levy counting chambers provides a more uniform distribution of corpuscles over the ruled area and entirely removes the effect of atmospheric pressure upon the depth of the solution, a source of considerable error in the circular-form chambers. The parallel form of cell also greatly facilitates cleaning as compared with the circular type. Critical tests have shown the Levy rulings to be more accurate, both in the dimensions of the ruling and the depth of the cell, than any counting chambers of best European makes we have tested. All Levy counting chambers are Vertical longritudinal section of slide, with cover glass ijn ppsition, showing new methodi of construction ft 1 iB l ' ia I 1 n- ' l" """" .ii i c/.s ^ Arthte;'^Thoi;nas;C!o i RMI^NTAIJPIO (ioR- AA± w '"i" ' 'ttii ' '"" • mtmmmmmm Fig. 15. — ^Thoma-Levy Counting Chamber, guaranteed to be well within such Hmit of error as may be later established by the U. S. Bureau of Standards for instruments ojf this type. Application of Haemocytometer to the Enumeration of Bacteria. Since the samples which are used in the hsemocytometer are small, the bacteria must be present in large numbers. A slight error in making dilutions will cause a large error in the determination of the number of bacteria per cubic centimeter since the factor is large which converts the reading of the microscope into terms of cubic centimeters. It is also an easy matter to confuse organic matter with bacteria. This is especially true in the applications of this apparatus to the enumera- tion of bacteria in feces and tomato products. These are taken up in other places. Cleaning of Haemocytometer Apparatus. This is one of the most important essentials in the use. of the apparatus. Pipettes should be cleaned by attaching them to a suction pump and drawing dilute alkali through them. After this alcohol and distilled water may be used. If they become stopped up a small wire may be run through, ANAEROBIC METHODS 17 but this should be used with care since the })ottom of the pipette may chip off. The counting chamber should be washed with dilute alkali or soap. It should be thoroughly nnsed in distilled w^ater and wiped dry and should not be sterilized by heating because the cement which holds the disk will be melted. Anaerobic Methods For the cultivation of those microorganisms which grow under anaerobic conditions, some special apparatus and methods are neces- sary to remove free oxygen. Many procedures have been proposed, a few of which will be given here. Boiling. Air is very soluble in water at room temperature. Wink- ler has found that 1 Uter of water at 20° C. will dissolve about 9 mg. of oxygen. By boiling water for some time this dissolved oxygen may be driven off. Special methods are then necessary to prevent re- absorption of air. In liquid media where heating will cause no change, this may be done by a layer of sterile vaseline or ligroin. Displacement by an Inert Gas. For this purpose several gases, such as nitrogen, hydrogen, carbon dioxide, coal gas, etc., have been used. Hydrogen has had the widest application on account of its ease of preparation and lack of harmful effects on bacterial develop- ment. When these gases are used, they are bubbled through the medium and should be carefully washed by the proper solutions to render them pure. Gas wash bottles of the Bunsen or Friedrich types will allow satisfactory results. Absorption of Oxygen. For this purpose alkaUne pyrogallol which has great affinity for oxygen is usually used. It becomes reduced to compounds which are not definitely known. In applying this chemical to the growth of anaerobes many devices have been used. Any one is quite satisfactory which prevents the ingress of more oxygen after the initial oxygen has been absorbed. Buchner's Method. In this method the culture tube is placed in a larger one which contains the alkaline pyrogallol. It has the disadvantage that if some special support is not used to keep the cul- ture tube above the surface of the pyrogallol, it is difficult to follow growth during incubation. These tubes may be prepared as follows: Introduce a few grams of pyrogallol into the bottom of a large test tube and place therein a support for the culture tube. This may be made from wood, cotton, glass, etc. After the culture tube has been 18 BACTERIOLOGICAL APPARATUS placed on its support, introduce the NaOH by means of a pipette into the bottom of the tube and seal the tube immediately with a rubber stopper. Wright's Method, This is another absorption method and is different from the others only in method of procedure. It is especially adaptable to the isolation of anaerobic spore-forming bacteria. Decimal dilutions of the sample are introduced into sterile litmus milk. About 1 in. above the meniscus of the milk a tight cotton cylinder is forced upon which are put about 2 gms. of pyrogallol. This is treated with strong NaOH and the original plug replaced in the tube. This tube should then be heated for fifteen minutes at 80° to kill all vegetative bacteria. Later incubation will give the characteristic changes. Lentz's Method. This requires a glass plate 125 mm. square and cellulose absorbent rings. Before using the rings are soaked in pyrogal- lic acid and immediately before the culture is started in 1 per cent potassium hydroxide. The plate with the culture is inverted over the cellulose ring, being sealed to the glass with plasticine. Fig. 16.-— Lentz's Anaerobic Fig. 17.~-Kuster's Anaerobic Apparatus Culture Apparatus. Kuster*s Method. This depends upon the same principle as McLeod's and Lentz's methods. A glass absorption chamber is used having a small hole in the top through which is introduced the absorp- tion substance. The Petri dish containing the culture is inverted over this and sealed with plasticine. McLeod's Method. This method requires a porcelain dish con- sisting of two separate chambers for containing the pyrogallic acid and potassium hydroxide. The special Petri dish has its free edge turned inward and upward. Around the upper cover of the porcelain dish is a groove to contain plasticine which prevents the ingress of air. About 7 c.c. of a 10 to 20 per cent pyrogaUic acid is run into the chamber marked A in Fig. 18. Into part B are put 7 c.c. of a 10 per cent potas- sium hydroxide. The Petri dish is then pressed down into the plas- ANAEROBIC METHODS 19 ticine. After this is done air is excluded and the potassium hydroxide and pyrogallic acid are mixed by tilting the dish. Zinsser's Method. The main apparatus involved in the apphca- tion of this method is a set of circular glass dishes similar to a Petri dish except that they are much deeper and a larger space is left between the sides. The agar which has been inoculated with the sample is Fig. 18. — McLeod's Anaerobic Culture Apparatus. poured into the smaller of the dishes. This is allowed to harden and the excess of moisture is evaporated. Into the other dish, is placed a quantity of pyrogallol, over which the smaller dish is inverted. So- dium hydroxide is poured into the larger dish. While this is reacting with pyrogallol, an oil such as abolin is dropped into the same place. This successfully seals the chamber which is formed by the two dishes. Torrey's Method, This method was especially successful in the hands of Torrey for the isolation of B. Mfidus and B. acidophilus. Y////y///y//^^/////y//y/^^y^^^ m >^^^^^^^^>jy^'j'^^^'^^^jj'jjj^^^A'>^^^^j^>^^A/j^'^j^/>j'^yyy^jjwrA^. ■A C D -B Fig. 19. — Cross^section of Torrey's Anaerobic Chamber, A, Solid Medium Containing the Sample, B, Agar which has been Heavily Inoculated with B subtihs or B mesentencus , C and Z>, Petri Dishes which Form the Chamber It is similar to the method devised by Zmsser. A Petri dish about 10 cm. in diameter and about 2 cm. high is poured and inoculated with the sample under investigation. It is placed in an incubator to remove excess moisture and dry the sides of the dish. This is important since too much moisture will allow contamination with the culture which is used to absorb the oxygen. This dish after drying is inverted into another dish, containing agar heavily inoculated with BaalliLS cereuSj of at least 12 cm. in diameter. The agar in the lower plate forms a seal, and the strict aerobic organism Bacillus cereus will use up the free oxygen. This apparatus is placed in an incubator in a moist chamber to prevent evaporation of the agar in the larger dish. 20 BACTERIOLOGICAL APPARATUS Smillie's Method. This method depends upon the catalytic action of platinized asbestos upon oxygen and hydrogen when they are brought into contact. The author claims that the method is well adapted to the study of strict anaerobes such as B, botuhnus. The method adapt- able to cultures in test tubes is described as follows by the author: *' Platinized asbestos is first prepared in the usual way, or it may be purchased from any laboratory supply house. A small mass of the catalyzer is firmly fixed at the end of a platinum wire by coiling the wire about it. The other end of the wire is inserted in the end of a short glass rod; and the rod is inserted into a No. 1 one-hole rubber stopper. The apparatus is wrapped in a package and autoclaved. '^ The water of condensation is removed from a plain agar slant the tube inoculated, inverted, the cotton plug removed and the tube filled with hydrogen by means of a sterile capillary pipette. The hydrogen may be obtained from a Kipp generator, or more satisfactorily from a hydrogen tank. It should be passed-through a series of wash bottles con- taining silver nitrate, sulphuric acid potassium permanganate, and lead acetate to remove all impurities. ^' After allowing the hydrogen to fill the inverted inoculated test tube, the platinized asbestos is heated for a few minutes in a free flame, the rubber stopper is inserted firmly into the inverted tube and the end of the tube Fig. 20 —-Sketch of Anae- dipped into melted paraffin, robic Culture Apparatus n m^^ ± ^ i sr i for the Cultivations of ^^^ catalyzer glows for a second or Obligate Anaerobes m *wo as the hydrogen and oxygen are actively Test Tubes According united, and the water formed is deposited to Smillie's Method. on the surface of the tube. The process is now complete and the tube is ready for incubation.'' Smillie states that this method is satisfactory for the usual anaerobe but does not remove all traces of oxygen. Another method had to be devised. This is described as follows: '' Two lengths of nichrome wire No. 22, 6 cm. long, are separately fused into a glass tube so that they are insulated (see Fig. 20a) and the glass tube B closed at each end is passed through a one-hole rubber stopper C, To the lower end of the nichrome wire D thus completing SMILLIE'S ANAEROBIC METHOD the circuit. In the coil of the fine nichrome wire is placed a small mass of platinized asbestos E, The apparatus is placed in a package and autoclaved." Smillie has apphed the same method to the culture of large amounts of bacteria in Blake bottles. This method applied to the culture of bacteria in anaerobic jars is as follows : Smillie experienced some trouble in getting jars which would stand up under great vacuum. He finally adopted the ordmary specimen d < — Rubber Stopper, •Connecting rubber tube. Glass Bulb containing plaflnlzed asbestos. 2 mm, perforations. Fig. 21.* -Detail of Platinized Asbestos Bulb for Anaerobic Jar According to Snailhe's Method. jar about 30 cm. high and an inside diameter of 12.5 cm. In the cover of this were ground two holes into which were fitted No. 4 one-hole rubber stoppers, carrying a glass " angle " stop-cock. A rubber tube extending to the bottom of the jar was attached to one of these. To the other stop-cock, a glass bulb with perforations is attached by means BACTERIOLOGICAL APPARATUS of rubber tubing. This ^Uush bulb is filled with platinized asbestos. The cultures are placed in a glass tumbler which is put into the jar. Fig. 21 taken from Smillie's original pubhcation will give the details of the asbestos bulb. Exclusion of Oxygen Seattle's Method. Ordinary test tubes containing plain or car- bohydrate broths are used. Melted sterihzed vaseline is poured into the tube until a plug about | in. long is formed. The broth and vase- line are then boiled for twenty to thirty minutes in order to expel any dissolved air and allowed to cool. These tubes may be kept for an Fig. 22. — Typos of Novy Jars. indefinite time and are air tight. When they are to be inoculated they may be melted in a water bath at 55° C. The sample may be intro- duced by means of a sterile pipette. The vaseline may then be heated over a flame to reform the solid plug. The tube may then be placed in the incubator. In order to remove any portion of the culture, a sterile sealed pipette (capillary) is thrust through the plug. This may be broken by forcing the end against the bottom of the tube after which any portion of the culture may be removed. By melting the vaseline plug again the culture tube is sealed for further incubation. Novy's Method. The apparatus used in the application of this method consists of a large glass jar with a removable top (Fig. 22). The culture may be grown away from oxygen by using an inert gas vacuum or alkalin pyrogallol. The jar is constructed with a glass stop- cock which will allow the removal of air with a vacuum pump. This THE MICROSCOPE 23 may be combined with alkalin pyrogallol and give an atmosphere which is entirely free from oxygen. Petri Dish 10 cm diatn. ■Rubber Stopper^ liU Tl^ ') j:;^ t Air Outlet Annular flroove 3ram. Deep s 4 mm Wide. n 3n "un Fig. 23. — Jones Anaerobic Culture Apparatus. Cross-section of the Stone Base with Petri Dish in Position. —Eye Lens ' The Microscope A compound microscope is necessary for a study of bacteria. The success which a student has in the pursuit of microbiology is often dependent upon thorough knowledge of the microscope. It is desirable to study the microscope with much care in order to under- stand its limitations and the sig- nificance of each part. It is much better to regard it as a delicate piece of mechanism through whose intelligent use an entire world of living organisms will be made visi- (E Diaphragm ■Field Lens — DiawTuhe of the ^ Ocular Bulloch's Method. This is very similar to Novy's and needs no special attention here. The air in the jar should be replaced by an inert gas. Jones' Method. This method allows the observation of growth and requires very little gas. It is described by the author as follows: ^' The apparatus consists of one-half of a Petri dish sealed with paraffin on a square stone or metal base provided for an inlet of the inert gas and outlet of air. The figure gives the general construction of the apparatus. !1„ ■ Body Tube -Draw Tube Diaphgram with Society Screw Cover Glass. Society Screw • Mount ■Back Lens 'Middle Lens -Front Lens -Working- Distance . of the Objeetive Slide Object Fig. 24. — Showing Cross- section of a Microscope. (Courtesy of the Spencer Lens Company) 24 BACTERIOLOGICAL APPARATUS ble. The following pages have been prepared from a number of sources. Direct reference has been given in a few cases. Most of the data, however, have been taken with permission from microscope manuals which are furnished by the Bausch & Lomb Optical and Spencer Lens Companies. Don'ts Don't allow dust and dirt to settle on the microscope. Don't carry the microscope by the arm, unless the fine adjustment is protected. Don't use alcohol on the microscope. Don't expect toe great a range in the fine adjustment. Don't take the fine adjustment apart. Don't bring the objective into contact with the cover glass. Don't fail to focus up before turning the nosepiece unless you know the objectives are parfocal. Don't forget that high powers have short working distances. Don't focus down with the eye at the eyepiece. Don't fail to secure good even illumination. Don't drop the objectives and oculars. Don't try to take an objective apart. Don't try to clean the lens with a dirty cloth. Don't fail to clean oil from an immersion lens immediately after using. Don't try to work with* an immersion lens when there are air bubbles in the oil. Don't use high powers when low ones will do. Don't use higher oculars than necessary. Don't expect a lens to work at its best unless used on a cover thick- ness, and with a tube length, for which it is corrected. Don't shut one eye. Don't get discouraged if desired results do not come immediately. The instrument should always be kept in a case, either the one in which it was received from the factory or one which is specially constructed to hold a series of them in the laboratory. When carry- ing a microscope or removing it from its case, grasp it by the pillar and not by any other part. Finger marks and stains should be carefully rubbed with soft cloth or lens paper. If this is not sufficient, a little ether, chloroform or xylol may be used. Alcohol should never be used since this will remove the lacquer. PARTS OF THE MICROSCOPE 25 N Stage. This deserves little attention. It may be cleaned as above described. Jl Inclination Joint. By means of this joint the body of the micro- scope may be inchned at an angle. Should the joint become so loose that the microscope will not remain at the desired angle, it may be tightened by tightening a nut. ^OOf y "^OSg: '»s«:e. -**.Ct;v^j, ' "f-m '*'S fit. „ '^»<-J'vri, SJUiCK SCREW von fOGUSiNO CONDENSED? M»RHOS SAft MIRROR _ ^„_„ „ •/_ .^='*«^fr. ^Kistfi^ -., ST'AGE Clips '~>NA,rrr *OltiT - - "Oflse s Hoe 04 $s Fig. 25. — Showing the Different Parts of a Microscope. Course Adjustment. This is accomplished in one of two ways: The sliding tube and the rack and pinion. The former is found only on the cheaper microscopes, and is practically out of date. In it the body tube is made to slide inside of a sleeve which is fastened to the arm of the microscope. It is very hard to keep in order, because the exposed surfaces of the body tube and the inside of the sleeve 26 BACTEKIOLOGICAL APPARATUS become gummed and corroded from exposure to the atmosphere and reagents of the laboratory, and particularly from the perspiring or dirty hand of the operator in focusing. In this condition the tube works hard and unevenly, so that one is apt to force an objective into the slide. The tube and sleeve can generally be cleaned by rubbing with a coarse towel after moistening with xylol. Do not grease the tube or sleeve because it gums and corrodes the tube, and is very unsightly. Should the tube be too loose in the sleeve remove the tube and press in the top edge of the sleeve on either side of the one or more slots running down from the top edge. All of the better microscopes are provided with the rack and pinion coarse adjustment. The bearings must necessarily fit very closely. Any foreign matter on them interferes seriously. Do not strain the teeth of the rack and pinion by forcing the bearings back and forth over one another when they are not clean. A little xylol or chloro- form rubbed on the surfaces will clean them. Do not use emery in any form. When the bearings are perfectly clean, oil them slightly with a good acid-free lubricant (paraffin oil or watch oil). If the bear- ings become so loose that the tube will not stay in place tighten the little screws at the back of the pinion box. All makers have a provision here for taking up lost motion and wear. Do not fill the teeth of the rack with paper paraffin or any other foreign substance. If anything should accumulate in these teeth clean it out. Fine Adjustment. The fine adjustment is necessarily of limited range and delicate in its mechanism. If, when looking into the eye- piece, no change of focus is noticed by turning the micrometer head or, if the micrometer head ceases to turn, the adjustment has reached its limit. Turn the micrometer back to bring the fine adjustment mid- way within its range. When the fine adjustment head stops do not force it. All of the better microscopes are made so that the head stops at both ends of the range and so that the micrometer threads cannot be removed from their bearings. Other microscopes do not have this safeguard, and great care should be exercised not to remove the thread. If by chance it should be removed, exercise great care in replacing it to see that the threads are started properly so that they do not ^' run.^' Do not force them if they run at all hard. If they are started to '^ run- ning " they must go to a machinist. On some microscopes these threads are " left-handed, '^ which should be noted in trying to start the threads. In some microscopes a Uttle steel pin is fitted loosely into the hollow end of the micrometer thread. Be careful to see that this little pin PAKTS OF THE MICR08COPE 27 is in its place before starting the micrometer thread- In some cases this pin even drops out of place before the micrometer thread is entirely out of its bearings and the defect is not noticed until the fine adjust- ment fails to respond. In such a case the top of the fine adjustment must be opened to secure the pin and put it in place. This ought to be done by the maker or experienced mechanic. In some cases, especially in microscopes (Continental type) where the prism is used in the fine adjustment, the lubricant in the prism becomes gummed so that the adjustment fails to respond promptly and then jumps. On the best microscopes provision is now made against this. The bearings should be thoroughly cleaned and oiled with paraffin oil or watch oil. This ought to be done by the manu- facturers, because the mechanism is so deHcate that even though safely taken apart it would be put together and adjusted with great difficulty. All the modern microscopes have their fine adjustments so arranged that the fine adjustment ceases to work when the objective rests on the cover glass. This feature should be insisted upon Draw Tube. This should work easily and smoothly and should be kept clean and dry. In sliding this draw tube, do so with a revolving motion. The draw tube should always be drawn out to give the proper focal length for which the microscope was standardized. All micro- scopes give best results if used with cover glasses of definite thick- nesses. Substage. This should be kept dry and clean to prevent cutting and rusting of the delicate bearings. Should the leaves of the iris diaphragm become rusted, they may be cleaned with xylol and oiled. When they become bent, an expert is necessary. Lenses. Absolute cleanliness is necessary with the lenses. Japa- nese lens paper of highest quality should be used for cleaning. It is best to use no other material on the lenses. Great care should be used in cleaning them since they are very easily marred and scratched. Objectives. If these become soiled rub with lens paper using a little chloroform if necessary. Hard rubbing must be avoided. The immersion objective should always be cleaned by wiping with lens paper. If the oil is old and tends to be hard, xylol may have to be used. All dry objectives are corrected for a definite thickness of the cover glass. (Bausch & Lomb, 0.18 mm.; Spencer Lens Co., 0.18 mm.; Zeiss, 15 mm.: Leitz, 0.17 mm.) The objectives are corrected to a certain tube length, which is 160 mm. by most makers and 170 mm. by Leitz, Such tube lengths should always be used for which the objectives have 28 BACTERIOLOGICAL APPARATUS been standardized. Variations in the length of this draw tube should be made when cover glasses of a different thickness are used. Numerical Aperture. N.A. = ?i sin u. n-the lowest refractive index that appears between the object and the front lens of the objective; 2^=half the angular aperture. The numerical aperture is important since it determines the resolv- ing power definition and illumination. It is especially important with reference to the resolving power. The resolving power is directly pro- portional to the numerical aperture. The higher the numerical aper- ture the greater the resolving power and the finer the detail. Bausch has pointed out the significance of the N.A. (numerical aperture) as follows: '' If a very narrow central pencil is used for illumination, the finest detail that can be shown by a microscope, with high ^enough magni- fication, is equal to — — , where X is the wave length of the light used for illumination. The wider the pencil used for illumination, the greater the resolving power, until a maximum is reached, when the width of the pencil is suflScient to fill the whole aperture of the objective. In this ease the resolving power is twice as great, the finest detail that the objective can show being now equal to 2 N.A. This same hmit is reached when a narrow pencil of greatest possible obliquity is used. For example, the wave length of the brightest part of the spectrum may be assumed to equal 0.00053 mm. Consequently an objective of N.A. equal to 1.00 will resolve two lines separated by a distance of 0.00053 \ ^^ equal to 0.00053 with a narrow central illumination cone, and 1.00 ^~—- equal 0.000265, with a cone filling the whole aperture, or with a narrow obUque cone. " A 4 mm. 0.85 N.A. objective will resolve fines separated by dis- tance ranging between 0.00062 and 0.00031, dependent upon the aperture employed. For a 4 mm., 0.65 N.A. objective the limiting values are 0.00081 and 0.000405. » " The N.A. can also be expressed by the equation. ISr A — J^= effective aperture of back lens 2/ 2 X equivalent focus " Two objectives of the same equivalent focal length (E.F.) and the same N.A. should show the same illuminated area in the back lens, USE OF THE MICROSCOPE 29 when viewed without an eyepiece and illuminated with the widest cone of light they can take in. ^' The foregoing explanation shows the importance of the N.A. to the efficiency of an objective. ^' It, is also evident that an objective cannot show its full efficiency if it is not used with a condenser of a N.A. large enough to fill the back of the objective with light.'' The depth of sharpness of an objective is in inverse ratio to the N.A. great penetration goes along with a low N.A. and low penetration but great resolving power with a high N.A. Illuminating Power. The brilliancy of the objective increases with the square of the numerical aperture of the objective. An objec- tive of 0.40 N.A. will give an image four times as brilliant as one of 0.20 N.A., provided the magnification is the same and the full cone of the illumination is used in both cases. Magnifying Power. The magnifying power of an objective is in inverse ratio to its focal distance. An objective of 2 mm. focal distance will give, with the same ocular, a magnification eight times greater than one of 16 mm. focal distance. Numerical aperture and magnify- ing power are of little advantage if the definition is not good. Chromatic Aberration. This is due to the fact that a ray of white light passing from one medium to another of different refractive index at any angle other than 90° to the surface between them is refracted and dispersed into its component colors. Spherical Aberration. This is due to the fact that a spherical surface cannot bring a beam of Hght which passes through its vertex to the same focus as that of a beam of light passing through any other zone. Both aberrations are corrected by the use of different kinds of glass (crown and flint) combined as double and triple lenses in the objective. Neither can be corrected absolutely for all colors in an achromatic objective. Apochromatic objectives approach the ideally corrected objective almost to perfection. An objective can be tested for chromatic correction by using a narrow cone of obHque light and a coarse grating. Abbe's test plate is best. Diatoms are good. No stained object should be used. If the spherical correction is perfect (see next paragraph) and one side of a line passing through the center of the field shows a clear, narrow greenish yellow border, while the other side is fringed with a violet red (secondary colors) the objective is chromatically corrected. The colors shown in the higher power objectives are of a more primary 30 BACTERIOLOGICAL APPARATUS character, i.e., nearer the yellow and blue. Apochromatic objectives show no color borders in this test. The spherical correction of an objective is perfected for a certain thickness of cover glass and a certain tube length, and is influenced greatly by any variation in either. This is especially true with the high-power dry objectives. The homogeneous immersion objectives arc not sensitive to the variation in the cover thickness, because the immersion oil between the cover glass and the lens is of the same refrac- tive index as the glass. They must be used, however, with the proper tube length. In testing an objective for its spherical correction it is therefore very important to supply the proper thickness of cover and tube length. It is manifestly unfair to judge an objective on this point without complying with these conditions. The test for spherical correction can be made on the same object as used for the chromatic test. If the edges of the lines in the center of the field appear equally sharp and clear when illuminated by either a narrow central cone of light or a narrow obhque cone without having to change the fine adjust- ment the objective is spherically corrected. The color remnants men- tioned above will be clear and transparent, while if the lens is poorly corrected spherically, these borders will appear muddy and turbid. Defects in spherical corrections can often be corrected by using cover glasses suitable to them, also by changing the tube length. The fact that the periphery of the field is not in focus at the same time as the center does not bespeak a lack of spherical correction, but a lack of flatness of field with which it is often confused. Flatness of field depends not only upon the objective itself, but upon the ocular and the cone of light used, whereas the spherical aberration is inherent in the objective itself. No field is absolutely flat. It is a desirable quality in a lens, but spherical and chromatic corrections should never be sacrificed for it. Some lenses appear to be " flatter " than they really are, because their corrections are so poor that little contrast is noticed between objects in the center of the field and at the edge. Narrow cones of light give a flatter field than wide ones. Thin objects are more critical tests for flatness of field than thick ones. Working distance is the free distance between the cover glass and the objective when the latter is focused. It decreases generally with increasing power and numerical aperture of the objective. Of two lenses with the same focal distance the one with the higher N.A. will have the shorter working distance. The working distance also depends on the mounting of the front lens. If the lens has a prominent mount- USE OF THE MICROSCOPE ing projecting beyond its surface the working distance is lessened thereby. Oculars. These have a simpler structure than the objectives. They serve as collective lenses, making all of the light constituting the image from the objective enter the eye of the observer. Illumination. Practically all microscopic studies are carried out by transmitted light, and some special attachment is necessary to direct this Hght along the optical axis of the microscopOo For this purpose substage or Abbe condensers are used. This condenser allows the utiUzation of all light which enters the object space. Light is sent through the object at an angle great enough to fill the objective. The condenser is constructed to bring the rays of light to a focus above the upper surface of the uppermost lens. When the concave Fig. 26. — Showing Incidence of Light Rays in Condenser. (After Bausch.) mirror is used the rays of light are brought to a focus within the con- denser. The concave mirror acts as a lens and together with the con- verging effect of the lens in the condenser causes the light to converge more rapidly than that for which the microscope was standardized. Bausch has depicted this in Fig. 26. Bausch makes the following statement with regard to the use of the condenser: "In the use of the condenser with oil immersion objectives the custom prevails of using the condenser dry. It is well to point out, however, that both the condenser and the objective lose in their eflSciency when the former is used dry, and for critical work the condenser should be in immersion contact with the slide. To make immersion contact between condenser and slide place a drop of oil on the top of condenser^ drop the slide upon the stage, first turning the slips to one side. With immersion objectives the proper focusing of the condenser becomes a matter of nice distinction to obtain best results and can 32 BACTERIOLOGICAL APPARATUS only be reliably accomplished by considerable practice and experience. To obtain best position: Use a 16-mm. (f in.) objective; focus upon the object; adjust con- denser until image of window-Bash or flame is in the same plane with object. Practically all substages are provided with means for focusing the condenser: The rays of light should be focused sharply on the object. A little experience will indicate when the proper aperture is Fig. 27. — Showing Action of Different Parts of the Condenser. (After Bausch.) being used. The iris-diaphragm when used with dry lenses should not be more than half open. With the oil inamersion objective, how- ever, the full aperture should be used. Final Hints ''Sometimes the worker may have faithfully carried out all the directions heretofore given and been assured that his lenses possess the above-named quahties as they ought, yet be unable to obtain the desired results. He may be working with a water mount and dry objective become ' immersed ' in some water which has worked to the top of the cover glass. His objective may be dirty from a previous ' immer- sion ^ or it may have some other dirt upon the front lens. The field may be covered with specks which revolve when the ocular is turned. The field may be dim or hazy, due to dirt on the back of the objec- tive or a film on the inner surfaces of the lenses of the ocular, or because of moisture settling on the lenses because they have just been brought from a cold into a warm room. He may see great streaks on his field, which are due to his own eyelashes, or he may see small slowly moving bodies floating across the field. With the exception of this last, the ailment has only to be mentioned to suggest the remedy. The musc» volintantes, as these last-named bodies are called, are little specks of MAGNIFICATION TABLES shreds in the vitreous humor of the eye which cannot be removed, but which can easily be disregarded. '' In water mounts and fresh balsam mounts one is apt to find air bubbles. To be sure that the object is an air bubble, focus up with central light. The bright spot in the center will become clearer while the edge will become darker. With oblique light the bright spot will be thrown to one side. In studying water, blood or any fluid, always cover the drop with a cover glass. The objectives are cor- rected for rays passing through media with parallel surfaces. If such a mount is not kept horizontal, currents will be set up, due to gravita- tion, and they will be seen with a magnified velocity seemingly running up hill. '^ The fact that the microscope reverses every movement and magni- fies it may be mentioned again. '' Beside any movement due to currents there is sometimes a pecu- liar indefinite to and fro movement of particles from one position to another. This is called Brownian movement. " In studying sections a true idea of the structure of the tissue can only be obtained by moving the shde about to bring different parts into the optical axis and by focusing with the fine adjustment to bring different levels, or optical planes, successively into view Where serial sections are used each section must be studied in relation to its neighbors. X AnXt£j X MAGNIFICATION TABLE ^ Bausch ^i) feiSwHa iiiMi^ Slftl AREOLATE GRUMO^e MQRUUOIO CLOUDED GYROSE IVURMORATED RETJCULATE TYPES OF INTERNAL STRUCTURE OF COLONIES Fig. 33.-— Showing'the Various Types of Elevation, Margins, and Internal Structure of Colonies. FORMS OF GROWTH 103 r''^<::> Kiy viy ^ -/ vU CRATERIFORM NAPIFORM INFUNDIBULl SACCATE STRATIFORM TTPE5 OF LIQUEFACTION IN GELATIN STAB CULTURES /'"^^^^T""**^ /'**''*\*''***\- /'****" v_y \^ A_/ x^ vy A^^ v^ riLIFORM ECHINULATE BEADED CFfVSt PLUM05E AR60RE3CeNT RHlZOID FORMS OF GROWTH ON 6TREAK CULTURES iWa Fig. 34; 104 CLASSIFICATION AND DESCRIPTION OF BACTERIA discussion in the previous paragraph. From this it will be seen that those who have really used the Chart regard it as an aid in the study of bacteria. Barring certain recognized limitations it is a convenient Structure of colonies: 1, conglomerate colony; 2, toruloid colony; 3, alveolate structure; 4, grumose m center; 5, moruloid; 6, clouded; 7, reticulate; 8, marmorated; 9, gyrose. (From Moore's Laboratory Directions for Beginners in Bacteriology, Ginn s. Reduction, removing oxygen from a chemical compound. Refers to the conversion of nitrate to nitrite, ammonia, or free nitrogen, and to the de- colorization of Htmus. Rhizoid, growth of an irregular branched or root-like character, as in B. nnjcoides. Ring, growth at the upper margin of a liquid culture, adhering to the glass. Repand, wrinkled. Rapid, developing in twenty-four to forty-eight. Rugose, wrinkled. Saccate, liquefaction in form of an elongated sac, tubular, cylindrical. Scum, floating islands of bacteria, an interrupted pellicle or bacterial membrane. Slow, requiring five or six days for development. Short, applied to time, a few days, a week. Spindled, larger at the middle than at the ends. Applied to sporangia, refers to the forms frequently called clostridea. Sporangia, cells containing endospores. Spreading, growth extending much beyond the line of inoculation, i.e., several millimeters or more. Stratiform, liquefying to the walls of the tube at the top and then proceeding downwards horizontally. Thermal Death-point, the degree of heat required to kill young fluid cultures of an organism exposed for ten minutes (in thin-walled test tubes of a diam- eter not exceeding 20 mm.) in the thermal water-bath. The water must be kept agitated so that the temperature shall be uniform during the e;xposure. Transient, lasting a few days. Truncate, ends abrupt, square. Turbid, cloudy with flocculent particles, i.e., cloudy plus flocculence. Umbonate, having a button-like, raised center. Undulate, border wavy, with shallow sinuses. Verrucose, growth wart-like, with wart-like prominences. Vermiform-contoured, growth like a mass df worms, or intestinal coils. Villous, growth beset with hair-Hke extensions. Viscid, growth follows the needle when touched and withdrawn; sediment on shaking rises as a coherent swirl. Zoogloese, firm gelatinous masses of bacteria, one of the most typical examples of which is the streptococcus mesenterioides of sugar vats (leuconostoc mesenterioides), the bacterial chains being surrounded by an enormously thickened firm covering inside of which there may be one or many groups of the bacteria. Suggested Procedure for Studying Bacteria According to the Descriptive Chart of the Society of American Bacteriologists. The following procedures are suggested for use with the Descriptiye Chart. 108 CLASSIFICATION AND DESCRIPTION OF BACTERIA One of the factors which has iiihi])ile(l a more extondocl use of the Chart has been the laek of standard methods to use with it. Several of the tests nec^d more study. The Committee on the Chart for the Identi- fication of Bacterial Species of the Society of American Bacteriologists his made a prehminary report on methods to be used with the Chart (Conn, H. J., 1918). Since their report represents the attempt on the part of an organized society to standardize technique, parts of their report will be reproduced here. Media. The importance of using carefully prepared media does not need emphasis. Greatest care should be used in adjusting the reaction to true neutrality by means of brom thymol blue. All media should be prepared after the Standard Methods of the American Public Health Association. Invigoration of Cultures. The Committee* has made the following recommendation with regard to this important factor: Provided a medium can be found upon which the organism to be studied grows vigorously, it should be invigorated before study, even though freshly isolated from its natural habitat. The procedure to be employed is as follows: Prepare duplicate subcultures in standard gluclo&e broth, and on standard agar slopes, placing cultures of each at 37° and 25°. On the basis of resulting growth the organism falls into one of the following series: Senes I. Organisms which produce good growth (surface growth, dis- tinct turbidity, or heavy precipitate) in twenty-four hours at 37° in glucose broth. Senes II. Organisms which do not produce good growth in twenty-four hours as above, but do in forty-eight hours at 25° in glucose broth. Senes III. Organisms which do not grow well in glucose broth but do produce good growth on the surface of agar in twenty-four hours at 37°. Senes IV. Organisms excluded from the above groups but which produce good growth on the surface of agar in forty-eight hours at 25° C. Record the series number on the chart at the proper place and proceed with the mvigoration by inoculating into another tubfe of glucose broth for organisms of Series III and IV. Incubate this tube at the temperature, and for the time, called for in the series which it belongs; then transfer from this tube to a third tube and incubate as before. From this third culture make a gelatin or agar plate and incubate at the temperature previously used until colonies of sufficient size for isolation are obtained. Transfer from a typical colony to one or more agar slants and incubate one day at 37° or for two days at 25° according to the temperature relation of the organism studied. * The term '' Committee " when used in this chapter will refer to the Committee on the Chart for Identification of Bacterial Species of the Society of American Bac- teriologists. PURE CULTURE METHODS 109 In case the organism does not produce growth on either of these media at either temperature, it should be invigorated with any medium and at any tem- perature known to be adapted to its growth. Under such circumstances, invigorate by the procedure just outhned but using the medium and tempera- ture found most favorable for the organibm in question, recordmg on the chart the method of invigoration adapted. If no conditions are known under which the organism in question produces vigorous growth, it should be studied without preliminary cultivation as soon as possible after isolation from its natural habitat. Such an organism is not likely to give good growth on any ordinary media, and the results of the study called for by the chart will have little significance. Motility. Some care is necessary in interpreting the results of motility determinations. Positive results are satisfactory but negative results are convincing only after a number of attempts. The safest method to follow is to make flagella stains for if the organism possesses flagella, it is reasonable to assume that it is motile. The Committee states that even negative results do not absolutely prove that the organism is immotile. Vegetative CeUs. Some bacteria go into the spore stage so quickly that in order to secure vegetative cells, as young a culture as possible should be used. For making the various determinations it is necessary to have a young and vigorous culture. This may be secured by passing it through a process of rejuvenation which should consist of continued transferring to a medium which is best adapted for growth. It may be necessary to determine the optimum temperature and incubate all cultures at this temperature. Endospores. These may usually be observed on the smears which have been stained with the ordinary alcoholic-aqueous stains. On account of a different type of protoplasm, possibly, they resist the staining process and appear on the smear as colorless bodies usually round or oval in shape. In sonie celh they will be observed in the ends or middle. In this case the cell has not decomposed to set them free. Special stains may be Uvsed for indicating the presence of spores, the procedure for which has been given in the Chapter on Staining. Very often these special methods are not attended with much success. Another method which is often rehable is to kill the vegetative cells by heating in broth culture for thirty minutes at 80° C. If after this procedure growth is secured by subculturing into other media, it is good indication that the culture was a spore former. Capsules. The presence of capsules may be indicated either by special staining procedures or the growth in the common media. The 110 CLASSIFICATION AND DESCRIPTION OF BACTERIA special staining procedures have been described in the Chapter on Staining. Capsulated bacteria usually grow with a sticky, slimy growth which when touched with a needle, will pull out into a thread. In hquid media the sediment in the bottom of the culture tube will often show a slimy appearance. On the ordinary smears stained with aque- ous alcohohc solutions of the anihne dyes, the capsulated bacteria often show a halo about each cell. This seems to resist the staining procedure and may often be made more visible by slightly moving the fine adjustment of the microscope. Staining Reactions. The theory of staining and the preparation of smears has been fully described elsewhere. To make a complete study of the stainilig properties of an organism, all of the stains men- tioned on the Descriptive Chart should be used. The common special stains, such as the acid-fast, should also be tried. Oxygen Relations. The relation to oxygen may be determined from the closed arm of the fermentation tubes. Before using, however, it is advisable to reduce the dissolved oxygen to a minimum. This may be done either by a vacuum pump or boiling. The incubation of streak plates by means of Torrey's method will give good results. Also agar streaks of the organism may be incubated in a Novy jar. Nutrient Broth. After this medium has been inoculated from a young culture of the organism, it should be incubated at the optimum temperature for the organism. The characteristics of growth should be recorded on the Chart and the culture saved for other tests as here- after described. Before these observations are made the tube should be shaken as Uttle as possible in order not to destroy any ring or pellicle formation. Agar Stroke. Streak the agar slant in a straight line from the bottom of the slant to the top. Care should be exercised not to cut through the surface of the slant. . If the inoculum is deposited within the agar when it is intended for the surface anaerobic conditions may be established. With freshly prepared agar streaks, in the bottom of which is water ^of condensation, care must be used not to lay them flat on the desk since a spreading growth will usually result. Agar Colonies. Pour agar plates with a dilution which will allow few colonies on the plate. Ten to twenty are sufficient when cultural characteristics are to be determined. Spreading colonies should be avoided either by inverting the plates during incubation or the use of HilFs porous covers. PURE CULTURE METHODS 111 Gelatin Colonies, See the directions given for agar colonies. If the culture is a liquefier, observations should be made at frequent intervals before the colonies have spoiled the plate. Gelatin Stab. A straight needle should be used and the tube stabbed to the bottom. It should be incubated at 20® C. and frequent observations made to determine the amount of liquefaction. The amount of liquefaction may be determined by means of a millimeter rule. The Descriptive Chart adopted at the 1907 meeting of the Society of American Bacteriologists recommended that gelatin tubes should be held for six weeks to determine liquefaction. This is a test upon which some work must be carried out in order to secure better methods for determining gelatin liquefaction in the shortest time. There is little trouble or error involved in reporting positive results but the time is an important element with negative results. Some bacteria will liquefy gelatin only after several months. This indicates that another method, by which to more sharply distinguish between the liquefiers and the non-liquefiers, is needed. The Committee has proposed to make the method of Rothberg (1917) provisional until it may be tried out. Rothberg gives the organism a preliminary cultivation in a 1 per cent gelatin solution at 25° or 37°; then inoculate the surface of gelatin in a test tube and incubate for 15 days at 20° C. Potato. The inoculation of potato slants should be made in the same way as for agar slants. After the observations called for on the Descriptive Chart have been made, the potato may be tested for diastasic action according to the directions given under " Potato Starch Jelly.'' Potato Starch Jelly. Iodine should be used to determine the presence of diastasic action. Pour the contents of the culture tube into a beaker and dilute with distilled water. If the dilution is sufficient and if the organism possesses a diastase, the various colors of the starch decom- position products with iodine will be obtained. Some of these arc as follows: Starch blue color with iodine Soluble starch blue color with iodine Erythordextrin red color with iodine Achroodextrin no color with iodine Maltose no color with iodine Dextrose no color with iodine 112 CLASSIFICATION AND DESCRIPTION (/F B\CTERIA To secure success with this method both the iodine and starch sohitions must ho veiy (hhite The same method has been apphed to potato slants. In this piocedure the slants aie thoroughly mashed and after dilution with water aic tieated as above. Allen (1918) has described another convenient method which has had rather extended use. This involves the use of a 0.2 per cent thymol starch agar which is poured into a sterile Petri dish and allowed to harden. Stieaks are made on this and after incubation the dish may be flooded with iodine solution. A clear halo about the growth indicates diastasic action on starch. In order to satisfy the requirements of the Descriptive Chart Allen has stated that a clear zone of more than 2 mm. width should be regarded as strong While a feeble action is denoted by a width of less than 2 mm. This differentiation called for on Descrip- tive Chart is rather ambiguous since to distinguish between feeble and strong diastasic action is probably unnecessary. Edson and Carpenter (1912) to determine the presence or absence of diastasic action added a 2 per cent thymol starch paste to a 10-day- old broth culture. After incubation for about eight houis, the culture was tested for reducing sugais by means of Fehhng's solution. Temperature Relations. The determination of this characteristic has been usually limited to 37° C. and 20° C. Other temperatures may be used as desired. The organism should be inoculated into a medium in which it will grow well. This culture should then be incubated at different temperatures. The possibility of other factors inhibiting growth should be carefully guarded against. It may be necessary to study this characteristic before starting the other culture work in order to determine the optimum temperature. Cohn's and XJschinsky*s Media. After inoculation they should be incubated at the optimum temperature. The observations which should be made are mentioned on the Chart. These media may also be used as a base to which other compounds, such as fats, proteins, etc., may be added. Loeffler's Blood Sertun. Treat in the same way as described for ^^ Agar Stroke." Make the necessary records on the Chart. Milk. Freshly skimmed milk should be sterilized either in test tubes or Erlenmeyer flasks. The various observations should be recorded on the Chart. The proteolysis of casein may be determined either by the appearance of the tube or by means of the Hastings (1904) milk agar plate. For preparing this milk agar plate, sterile skimmed milk is added to melted plain agar which has been cooled to 50° C. and poured into a sterile Petri dish to harden. The strain which is STARCH AGAR PLATES 113 Fig. 36. — Showing Strong, Feeble and Weak Diastasic Action by Bacteria. (After Allen, 1918). 114 CLASSIFICATION AND DESCRIPTION OF BACTERIA being studied should be streaked across the surface of the agar. After incubation a clear zone will be visible, which, if due to true proteolysis, will not be rendered opaque if treated with weak acid. If a clear zone is present after incubation, it may be due to either proteolysis or the acid formed in the metabolism of the organism. To prove that proteo- lytic enzymes produced it, flood the plate with dilute acetic acid and if the clear zone remains, the presence of a protease is indicated. The Descriptive Chart calls for the determination of the reaction on certain days. In the past, this has been determined by titrating 5 c.c. of the culture with N/20 NaOH or HCl. That this method is not accurate has been shown by Clark and Lubs. In order to take advantage of their work the Committee has advised the following procedure: Acid production in milk can be detected by adding brom crcsol purple to the culture and comparing with the color obtained by adding the same propor- tionate quantity of the indicator to sterile milk. (Brom thymol blue does not give satisfactory results in milk.) Four degrees of acidity that can be recog- nized in milk are listed in Table 11. They correspond closely to those listed in Table I, differing only on that brom cresol purple is used instead of brom thymol blue to show " neutrahty '' and that the curdling point (Pi/= 4.7) is used to separate between ^' moderate ^' and '' strong ^' acidity instead of the less definite point of maximum red to methyl red. The same method of expression used in recording acidity in clear media should be used in recording that of milk. Table VI DEGREES OF ACIDITY EASILY RECOGNIZED IN MILK (After Conn et aL, 1918) Acidity. lEdicator Reaction, etc. Approximate P^ Value. Neutral Same color with brom cresol purple * as sterile milk; i.e., blue to gray green. 6.2-6.8 Weak Color with brom cresol purple lighter than in sterile milk; i.e., gray green to greenish yellow. 5.2-6.0 Moderate Yellow with brom cresol purple. Not curdled. .... 4,7-5.0 Strong Curdled. Blue or green to brom phenol blue 3.2-4.6 Very strong. . . . Yellow to brom phenol blue. Under 3.0 * Use a 0,04 per cent solution. PURE CULTURE METHODS 115 Table VII SHOWING DEGREES OF ACIDITY EASILY RECOGNIZED IN CLEAR MEDLl (After Conn ct al., 1918) Acidity. Indicator Reactions. Neutral .... Weak M(xleralc. . . Strong Very strong. Blue or green to hrom thymol blue. Yellow to brom thymol blue. . . Purple to t)rom cresol purple.* Yellow to brom eresol purple Orange to methyl red.f Maximum red to methyl red. Blue or green to brom phenol blue.'* Yellow to bromphenol blue , A">;>roximate Fji Value. Over 6.2 5.2-6.0 4.6-5.0 3.2-4.4 Under 3.0 ^ Use a 01 per cent alcoholic holution. t UbG a 0.02 per cent alcoholic boiution. Litmus Milk. Growth in this medium is not much different from that in plain milk. If the tube turns to a white with a pink layer at the surface which is in contact with air, the presence of a reductase for htmus is indicated. Clark and Lubs (1917) have stated that brom eresol purple may be used as a substitute for Utmus in milk. The committee regards this as not always to be recommended since this dye docs not show the reduction phenomena. Indol Production. Indol is formed from tryptophane and is pro- duced in media which contains proteins .or their split products con- taining this amino acid. Under routine conditions the production of indol may be determined in Dunham's medium or in the plain broth culture. Zipfcl (1912) has shown that trytophane in an inorganic medium will show indol formation in twenty-four hours. Since tryp- tophane is so expensive Cannon (1916) has prepared a hydrolyzed casein medium with which it is claimed that good indol formation is secured. This is prepared according, to directions which are given in the Chapter on Media. The following tests for indol may be used: Nitroso-Indol Nitrate Test: Add 0.5 c.c of concentrated sulphuric acid and 0.5 c.c. of dilute sodium nitrite to the culture. If a red color is obtained the presence of indol is indicated. Ehriich's Test: Add 0.5 c.c. of paradimethylamidobenzaldehyde to the culture and if indol is present, a deep crimson color will be formed. 116 CLASSIFICATION AND DESCRIPTION OF BACTERIA Hydrogen Sulphide. Hydrogen may be formed from many of the sulphur-containing compounds. When formed from proteins it probably comes from cystine or some other sulphur linkage. It may be detected by suspending a strip of bibulous paper saturated with lead acetate to which a Httle glycerol has been added. This method has been found to yield satisfactory results by the author. Ammonia Production. Dilute the culture with ammonia-free water and add 1 c.c. of Nessler's reagent. The presence of a yellowish-green color indicates the formation of ammonia. Nitrate in Nitrate Broth. Ammonia, see above. Nitrate, The amount of nitrate decomposed by an organism may be determined quantitatively by the aluminum-reduction method which has been outhned in the Chapter on Water Analysis. For qualitative results the presence of nitrites or ammonia in a medium which was free from these substances before inoculation may be taken as sufficient evidence of nitrate reduction. Test for Nitrites. The same method may be used for the quahta- tive estimation of the presence of nitrites that is used in the quantitative determination. The Griess method has had much application and depends on the formation of azobenzolnaphthylamin whenever naph- thylamin and sulphanilic acid are present in an acid solution of nitrites. Mason (1912) gives the reaction as follows: N=N NH2-~-C6H4-~HS03 + HNO2 = CoPL g Q + 2H2O CbH4 N— N -S— JdL \y\/ NH2 H Jtl N==N N TT HSO3 NH2 fi Jn. XT xl » This is a very delicate test and is regarded by a few bacteriologists as too dehcate for bacteriological work. If a control tube is made accurate results should be secured by its use. To determine nitrites, dilute 2 or 3 c.c. of the culture with ammonia- free water and add J c.c. each of sulphanihc acid and naphthylamin hydrochloride. The presence of a red color when viewed the long way of the Nessler tube indicates the presence of nitrites. PURE CULTURE METHODS 117 Smith (1905) regards the Iodine starch test as most satisfactory for microbiologists. He gives the procedure.as follows: " Twenty-five c.c. of distilled water are added to | gm. (more or less) of pure potato starch and the fluid boiled. One cubic centimeter of this starch water and 1 c.c. of freshly prepared potassium iodide water (1:250) are now put into the culture fluid, to which is then added a few drops of strong sulphuric acid water (2:1). If any appreciable quantity of nitrite is present the culture immediately becomes blue black from the libera- tion of free iodine which acts upon the starch. Old potassium iodide should never be used v/ithout first testing carefully as it usually con- tains some free iodine.'^ Blank determination should be made. Silicate Jelly. Make the required observations and record on the Chart. Fermentation Reactions. The various compounds upon which it is desired to study the action of microorganisms, are added to plain broth. Great care should be used in the sterihzation of these media since Mudge (1917) and Hasseltine (1917) have shown that polysaccharides are easily hydrolyzed to monosaccharides. The latter of these investi- gators found that serious errors were introduced in the study of B. proteuSj for instance, on common carbohydrates. For investigations requiring accurate data other methods of sterilization than moist heat should be used. The broth may be sterilized by filtration after the carbohydrates have been added, or the carbohydrate solutions may be sterilized by filtration and added to the broth by means of a sterile pipette I'ust before the tubes are inoculated. In determinating the reaction of the fermentation tubes, it has been the custom to titrate 5 c.c. of the medium with N/20 NaOH or HCI with phenolphthalein as the indicator. The work of Clark and Lubs has shown that this method does not allow accurate results. They have advised the deter- mination of H-ion concentration, but such a procedure is not adapted to routine work on account of the time consumed. In place of this, these workers have prepared a series of indicators with a sufiicient range to meet the requirements of bacteriological media. These should be used where it is possible. They are given in the appendix. Pathogenicity to Animals. The suspected organism should be fed to different laboratory animals and also injected into different tissues. The animal should be kept under observation for a short period before the experiment begins. After injection, it should be constantly watched and weighed. The Committee recommends the use of these indicators and makes the following statement: 118 CLASSIFICATION AND DESCRIPTION OF BACTERIA In careful research work the exact shade of the indicator should be compared with that obtained in standard ^' buffer " solutions, and the results recorded in terms of Ph- In laboratories where these solutions cannot be obtained, it is better to record the results as simply + or — according to the reaction of the culture to litmus, than to use the titration method. Under such conditions it is possible, however, to obtain a rough idea of the hydrogen-ion concentration by the use of Clark and Lubs' series of indicators without making accurate determinations of Pb, Four different degrees of acidity can be easily distin- guished by this simple method in sugar broth with initial reaction of neutrahty. The indicator reactions for these different degrees of acidity are listed in table II together with the approximate range of Pb to which each corresponds. In the absence of accurate determinations, these degrees of acidity may be recorded by the indefinite terms, ^' weak,'' '' moderate," '^ strong," and " very strong," or by the symbols +, ++, ++4.^ and ++++. If this procedure is used in order to secure data, it is possible that a more satisfactory method may be developed whereby the actual acidities of the culture tubes may be defined. Pathogenicity to Plants, Apply a broth culture of the organism to that part of the plant for which it is supposed to be specific. Loss of Virulence on Culttire Media. The loss of any characteristic may be determined by comparing it before and after prolonged culti- vation on artificial media. Attempts should also be made to bring it back after it has been demonstrated to have been lost. Ferments. The presence of enzymes may be determined by tho decompositions which the organisms accomplish. In searching for the presence of any special enzyme, a substrate containing the special compound should be used. Frankel's medium or any other such medium may be used as a base. Toleration to Acids or Alkalis. This characteristic may be deter- mined by adding different amounts of normal acid to alkali to plain broth before inoculating with the organism. Quite often a large amount of acid or alkali may precipitate the proteins which are in the media. This precipitation must not be confused with growth. Thermal Death-point. The Committee on Identification of Species of the Society of American Bacteriologists has recommended the expo- sure of the organism in nutrient broth for ten minutes. The medium should be prepared according to standard methods and the time very carefully limited to ten minutes. BIBLIOGRAPHY 119 BIBLIOGRAPHY Allen, P. W. 1918. A Simple Method for the Classification of Bacteria as to Diastase Production. Jour. Bact., 3, 15-17. Cannon, P. R. 1916. A Rapid and Smiple Indol Tebt. Journal of Bacteriology, 1, 535-536. Chester. 1901. Manual of Determinative Bacteiiology. New York. 1901. Claek, W. M. and Lubs, H. A. 1917. A Substitute for Litmus for Use in Milk Cultures. Jour. Agr. Research, 10, 105-111. CoHN, F. 1872 Untersuchungen uber Bakterien. Cohn's Beitrage zur BioL der Pflanzen. Bd. I. Hefte, 2, 127-224. Conn, H. W. 1906. Classification of Dairy Bacteria. Ann. Rept. Storrs Agr. Exp. Sta., 107. Conn, H. J. 1915. A Study of B. Subtilis by Means of the Classification Card. Science N. S., 41, 618* Conn, H. J. 1917. Soil Flora Studies. Part L Jour. Bact., 2, 35-45. DeBary. 1888. Comparative Morphology Biology of the Fungi, Mycetozoa, and Bacteria. Edson, H. a. and Carpenter, C. W. 1912. Microorganisms in Maple Sap. Vermont Agr. Exp. Sta , Bulletin 167. Ebson, H. a. and Carpenter, C. W. 1912. The Green Fluorescent Bacteria Occurring in Maple Sap. Vermont Agr. Exp. Sta., Bulletin 167, 521-599. Eisenberg. 1891. Bakteriologische Diagnostilc III. Ellis, H. 1909. Outlines of Bacteriology. Longmans, Green & Co., New " York. Fisher. 1900. The Structure and Functions of Bacteria. Oxford. Flugge. 1886. Die Mikroorganismen. II Auf., 1886. Gage, S. M., and Phelps, E. B. 1903. On the Classification and Identifica- tion of Bacteria with a Description of the Card System in Use at the Law- rence Experiment Station with Record of Species. Proceedings, American PubHc Health Association, 28, 494-505. Harding, H. A. 1910. The Constancy of Certain Physiological Characters in the Classification of Bacteria, Tech. Bull. 13., New York Agr. Exp. Sta., Geneva. Harding, H. A., Morse, W. J. and Jones, L. R. 1909. The Bacterial Soft Rot of Certain Vegetables. Tech. Bull., N. Y. Agr. Exp. Sta., Geneva. Harding, H. A. and Prucha, M. J. 1908. The Bacterial Flora of Cheddar Cheese. Tech. BulL, N. Y. Agr. Exp. Sta., Geneva. Hasseltine, H. E. 1917. The Bacteriological Examination of Water. Reprint No. 43 from PubHc Health Reports, Nov. 9, 1917. Hastings, E. G. 1904. The Action of Various Classes of Bacteria on Caseia as Shown by Milk Agar Plates. Cent. Bakt. Abt. II, 12, 590-592. Hort, E. C. 1917. Morphological Studies in the Life-Histories of Bacteria. Proc. Royal Soc. B., Vol 89, 468-480. 120 CLASSIFICATION AND DESCRIPTION OF BACTERIA Jensen, 0. 1909. Die Hauptlinien des naturlicheix Bakterien systems. Cent. Bakt. II, Abt., 22, 305. Jensen, 0. The Main Lines of the Natural Bacteria System and the Bacteri- ological Nomenclature. VII Intern Cong. Applied Chem., Part IV, 176-18L Johnston, W. 1894. On the Grouping of Water Bacteria. Amer. Pub. Health Assn. Proc, 20, 445-449. Jordan, E. 0. 1912. General Bacteriology. 107-109. Kendall, A. I. 1903. Reported by Gage and Phelps. On the Classification and Identification of Bacteria with a Description of the Card System in Use at the Lawrence Experiment Station for Records of Species. Amer. Pub Health Assn. Proc, 28, 494-505. Lehmann and Hetjmann. 1901. Atlas and Principles of Bacteriology Philadelphia. Lister, J. 1872. On the Germ Theory of Fermentation and Other Fermenta- tive Changes. Nature, July 10, and 17, 1872. Lister, J. 1873. A Further Contribution to the Natural History of Bacteria and the Germ Theory of Fermentation Changes. Quarterly Jour. Micro- scop. Soc, 380. LoHNis, F. 1916. Life Cycles of Bacteria. Jour. Agr. Research, 6, 675-702. Mason, W. P. 1912. Examination of Water. New York, John Wiley & Sons. MiGULA, Die Bakterien. MuDGE, C. S. 1917. The Effect of Sterilization upon Sugars in Cultmes Media. Jour. Bact. 2, 403-415. Rahn, 0. 191L Marshall's Microbiology. P. Blakiston's Son & Co, Philadelphia. Rahn, O and Harding, H. A. 1916. Die Bemuhumgen zur einheitlichen Beschreibung der Bakterien im Amerika. Cent. Bakt. Abt. II, 42, 385-393. RosENOW, E. C. 1914. Transmutation within the Streptococcus-pneumo- coccus Group. Jour. Inf. Diseases, 14, 1. Rosenow, E. C. and Towne, E B. 1917. Bacteriological Observatioiib m Experimental Poliomyehtis. Jour. Med. Research, 36. RoTHBERG, W. 1917. Observations on Some Methods for the Study of Gelatin Liquefaction. Paper read before the Society of American Bacteri- ologists, December, 1917. Smith, I. F. 1905. Bacteria in Relation to Plant Diseases. Tanner, F. W. 1918. A Study of the Green Fluorescent Bacteria from Water. Jour. Bact , 3, 63-101 Winslow, C. E. a. 1917. The Families and Genera of Bacteria. Jour, Bact., 2, 505-566. ZiPFEL. Cent. Bakt. Grig. Abt., I, 64, 65. ZoPF. 1884, Die Bakterien I. Aufl., 1885. CHAPTER V STERILIZATION AND DISINFECTION Bacteria do not exist in nature as pure cultures. Since there are so many varieties, the bacteriologist must use media and apparatus which are free from not only bacteria but all forms of life. This is accom- phshed by steriUzation, which is to be distinguished from disinfection— the removal of pathogenic bacteria only. The methods for steriUzatioi* may be classified as follows: I. Dry heat: a. Flaming and incineration. .b. Hot air oven. II. Moist heat: a. Inspissator. h. Streaming steam in the Arnold. c. High pressure steam in the autoclave. d. Boiling. III. Filtration: a. Through liquids. 1. Sodium hydroxide, sulphuric acid, etc. 6. Through solids. 1. Sand, cotton, glass wool, porcelain, etc. IV. Light: a. Sunlight. fe. Ultraviolet. Dry Heat Death by drying may be the result of two processes. The cell protoplasm may decompose or oxidation may take place. This has been studied by Paul (1909) and his co-workers who found, in general, that the rate of death in dry heat is proportional to the oxygen concentration. This would seem to indicate that death in dry heat was an oxidation process, although other reactions may enter. With regard to moist 121 122 STERILIZATION AND DISINFECTION bacteria, the oxidation pi'occsses would be limited by the solubility of the oxygen in water. According to Winkler (1889) this is 10.14 parts per million at 15° C. and 760 mm. Incineration. As a means of sterilization, this method needs no discussion. It is plainly a process of oxidation and very ejficient. Small incinerators may be purchased which will handle practically all ordinary material. Flaming. This simple method is a serviceable one for the bac- teriologist. Platinum wires may be sterilized by this method. Watch glasses, slides, etc., may be completely sterilized if a little care is used during the flaming process. The Bunsen burner or alcohol lamp may furnish the heat. Hot-air Oven. The apparatus for this method is constructed much like the ordinary baking oven. Quite often an attempt is made to insulate it either by means of a double wall or some special material. The tempei^aturc is usually maintained by means of a gas flame or electricity. Some hot-air sterilizers are so well insulated that after the temperature has been raised to 180° C, this temperature is main- tained for a long time even though the source of heat is removed. The hot-air oven is used for sterihzing all dry glassware. This should be put into the sterilizer when it is cool and gradually raised to the sterilization temperature. A temperature of 180° C. for from one hour to one hour and a half will suffice for all ordinary apparatus. This method has definite limitations and should not be applied to the sterili- zation of media, thick glassware, etc, Moist Heat The theory of sterilization by moist heat has been studied by Chick (1910). Here three essentially different processes take place: 1. Direct effect of heat on bacterial protein. 2. Effect of water possibly hydrolytic on these proteins at high temperature. 3. Desiccation of bacteria. It was pointed out by Miss Chick that an analogy exists between the disinfection of bacteria by hot water and the " heat coagulation '^ of proteins. This may help to account for the difficulty with which spores are disinfected by hot water when compared with the vegetative cells. The protein in the spores may be more resistant to hydrolysis than that in vegetative cells. Both changes follow the monomolecular law and both are greatly increased by the presence of minute amounts STERILIZATION BY MOIST HEAT 123 X of acid. Chick (1910) states, " The striking similarity between the effects of temperature (dry) on the one hand and hot water on the other indicate that disinfection by the latter is due to the action of water (coagulation and alteration) upon some one protsin which is essential for life of the bacterium and that the reaction is conditioned by the chemical action of water upon its constituent proteins.'' The follow- FiG. 37. — Hot-air Sterilizer, Lautenschlager Type. ing facts with regard to disinfection by moist heat were established by Chick: 1. Disinfection proceeds according to the logarithmic law the rate of disinfection being proportional, at any moment, to the concentration of surviving bacteria. 2. The presence of minute quantities of acid or alkaH too small to produce any direct disinfectant action increases the reaction greatly. The acid, however, gave the greatest increase. This presents close analogy to the '' heat coagulation " of proteins. 124 STERILIZATION AND DISINFECTION Rubner (1913) hab reviowed the application of steam to sterili- zation. He points out that vegetative cells contain much more water than spores and thus succumb more quickly to the action of heat. Spores contain hygroscopic water which soon evaporates leaving dry bacterial protein. This will resist heating for some time. Steam at 100° C. is an important chemical agent since hydrogen sulphide, ammonia and carbon dioxide are liberated from keratin, casein, and dried bacteria when they are subjected to its influence. Rubner found the saturation of the steam to be about as important as the temperature. Steam at 100*" C with a saturation of 80 per cent requires five times as long to kill bacteria as does saturated steam and steam with a saturation of 70 per cent requires twenty-two times as long as saturated steam. If steam at 100° C. is superheated, it is altered in two ways. The temperature is raised which makes it a more powerful disinfectant and the saturation is lowered which makes it a weaker disinfectant. In superheated steam at 110" C. made from steam at 100° C. spores Hved twice as long and at 127° C. ten times as long. This suggests the correlation between the temperature and saturation which are ^y important factors in sterilization. According to the laws of disin- fection, time is the other factor and it cannot be separated from the first two. High-presstire Steam. This method has extensive use in bacteri- ology. The apparatus is called an autoclave or dressing sterilizer. Several types are used, the most convenient of which are connected to a steam main from a power plant. Where this is impossible, it may be necessary to generate the steam under the steriKzer. This usually requires more time since it takes some time to get up the pressure. Often it is necessary to keep water in the bottom of the sterilizer in order to prevent the superheating of the steam. Under practical conditions this is usually not necessary since the steam will be saturated. The following table will show the relation between the usual pressures and temperatures required for sterilization in the autoclave: Gage Pressure. Temperature, Centigrade, Gage Pressure. Temperature, Centigrade. 5 10 100 109 115 5 15 20 40 121 5 126 141 In the presence of water. Chick has shown that the temperature is an important factor in sterilization. The temperature coefficient HIGH PRESSURE STEAM 125 was determined for B. typhosus and was reported to be 1.635 per I'' C. or 136 per 10° C. Here again the killing of bacteria in the presence of water was found to be analogous to the '' heat coagulation '^ of their constituent proteins. /_ Fig. 38. — Autoclave or Dressing Sterilizer, Kny-Schereer Type. The autoclave may be used for sterilizing many pieces of apparatus and many media. It has the advantage over other methods of taking less time. It has been recently shown that less hydrolysis of poly- saccharides is secured in the autoclave than in the Arnold. This is contrary to what was believed, for it is stated in many places that 123 STERILIZATION AND DISINFECTION N the xArnold steam sterilizer too should be used on such substances as igars to prevent hydrolysis. - Boiling. Practically no special apparatus is required by this method and any that is demanded may be quickly secured. Probably fifteen minutes is sufficient, for vegetative cells by spores are too resistant to be quickly killed. Surgical instruments may be sterilized by boiling. The water should be boiled for ten minutes before they are put in in order to prevent the possibility of rusting. Streaming Steam. The apparatus which is used in this method is much like the ordinary steamer which is available in the kitchen. Fig. 39. — Arnold Steam Sterilizer. (Boston Board of Health Pattern.) In bacteriology the apparatus is known as the Arnold steam sterihzer. It was devised by Tyndall, and improved by Koch and Arnold. It consists of essentially a copper box with a false bottom through which the steam rises to escape at the top. ' There is no superheating in the Arnold as with the autoclave and it requires less attention. It may be used in two ways — continuous or intermittently: With the continuous method the material to be sterilized is heated for from thirty minutes to a hour and a half. This method has the disadvantage that such prolonged heating may cause changes in the materials. Di-, tri-, and poly-saccharides may be hydrolyzed proteins STERILIZATION BY FILTRITION 127 caogulatcd, etc. Such has been found to be the case as mentioned elsewhere. With the intermittent method the material is heated for twenty to thirty minutes on three successive days. On the first day the vegetative cells are killed. The second day's heating will destroy those vegetative cells which have come from spores which passed through the first day's heating. On the third day the process is simply checked up. Fig 40 ■ — Bath for Sterilization at Low Temperatures (Inspissator). Inspissator. Some medja which are used by .bacteriologists must be sterilized at low temperatures. For this purpose a serum coagulator or inspissator is used. The media is heated at from 57° to 60° C. for different lengths of time. Blood serum may be heated for periods of one hour or for a longer period at one time. Sterilization by Filtration Filtration is an efficient method of sterilization which has certain distinct advantages over other methods. It leaves the filtrate sterile 128 STERILIZATION AND DISINFECTION and unchanged. Body extracts and sera may be thus treated to render them sterile. Filtration through Liquids. The action is entiiely mechanical unless some strong hquid such as sodmm hydroxide or sulphuric acid are used. In these cases the bacterial protein is decomposed by the chemical. Rettger appUed this method to the bacterial examination of air. Filtration through Solids. The same principle is involved here that is involved in the use of the cotton plug in test tubes and culture flasks. Different substances have been used but most attention has Pig. 41. — ^Diatomaceous Filters for the Sterilization of Bacterial Extracts and Body Fluids, been given to cotton, glass, wool and sand. Sand is used in the standard method for the bacterial examination of air and also in the purification of water. Stone or porcelain filters are the best substances for labora- tory use. They may be secured in many sizes and are easily sterilized and cleaned. Dialysis. In this medium the semi-permeable membrane is used as the filtering medium. Collodion sacs have had quite a little use in bacterial work. Light Although light does exert an antiseptic action on bacteria and may even totally destroy them after a long period, it is scarcely used STERILIZATION BY LIGHT 129 by the bacteriologist to sterilize media or apparatus. The work on the germicidal action of light is extensive and those wishing bibliographies on the subject should consult the papers of Dieudonne (1894), Ward (1895) and Weinzirl (1914). Ward (1895) using anthrax baciUi and spores found that exposures of one-half to one hour were necessary for inhibition of growth and that one and a half hours were necessary for stenhzation. Dieudonne (1894) worked with pathogenic bacteria. With B, fluorescens puditus and B, prodigtosus he found that one and a half hours were required in March, July and August and nearly twice as long in November. Kruse found that an atmospliere of oxygen shortened the length of life by one-half over that of hydrogen when bacteria were exposed to sunlight. Weinzirl (1914) devised a new technique which involved exposure of the bacteria on paper directly to the sunlight. He stated that non- spore-formmg bacteria were killed in from two to ten minutes. Such conditions are rarely secured in nature where the organisms are often accompanied with organic matter. For spores from two to eight hours were necessary. The various colors of the spectrum exert a different action on bac- teria. The intensity of this action increases as one goes from the red toward the violet. Apparent destruction is noticed only after the yellow is passed. From the yellow through the violet and ultra- violet, the germicidal action is at its maximum. In fact it is now well established that the shorter the wave length the greater is the bacteri- cidal action. Newcomer (1917) has reported experiments which indi- cate that X-ray fluorescence is very bactericidal. X-rays alone exerted a partial bactericidal effect on suspensions of typhoid bacilli. Kempster (1917) exposed tubercle bacilU to X-rays and secured a reduction in their multiplication. Inoculations of tubercle bacilli, which have been exposed to X-rays, into guinea pigs caused no tubercular lesions to develop. He stated that the phagocytes were able to overcome the bacteria if their multiphcation was reduced. Ultra Violet Light. This light on account of its short wave length possesses decided bactericidal effects. Ward (1892) in studying the effect of arc spectra on bacteria found that the infected plates were sterihzed whenever exposed to the violet or ultra violet rays. He successively treated Thames River water by submitting it the effects of ultra violet rays. Houghton and Davis (1913) give the following conclusions to their work: 130 STERILIZATION AND DISINFECTION u CD o >4 ft o o ft 3 CO m O 0) a 13 ft O « (D ,^ u o 1 CD ■P TJ TO 03 m o '3 o a K to pi u +> CD ^ a (D HJ M O M o 00 g DO o o o o o CO o CO o > CO ♦ I— I r-* c3 ^ a 5 O f-H f-i Ci «^ tH S CD d CO o ^ o3 A. The ultra viokt rays produced by the Cooper Hewitt mercury arc have a strong bactericidal power. B. Certain varieties of bacteria in aqueous suspension, including spore-forming organ- isms, are killed by exposure to the rays. Molds, however, are only partially de- stroyed by the ultra violet Hght. C. The action seems to be photo-mechani- cal and is in all proba- bihty due to absorp- tion of ultra violet __ 3 rays by bacterial pro- .s (^ toplasm. bC O g g other bodies of high •S ^ molecular weight in- terfere with the action of the rays. Turbidity, ^ g, both organic and in- organic, has a similar action. Color within certain limits seems to have no action. Ultra violet Hght was first used for the sterilization of milk and since this time its use has been extended to other materials. Von Recklinghausen studied the action of ultra violet light on bacteria in water. Spores are said to be D. Proteins and r^ i^ \U -fj ^ -^ n ^2 ;3 s .S I t^ o 'S 'ft -5 ^ ^ ^ U H ^ CO a g |Lmm| o u ci m o o o O ^ ^ ^ . ai a J3 . iS "ss . p3 S S xjih ^\^^x. Initial Dilution In'tial Dilution +Watei (c c) 1 20 1 40 1 80 1 1 . 160 1 :320 1 :64 Final Dilution 4+4 1 :40 1 :80 1 : 160 1 : 320 :640 1 : 1280 4+5 1 :4o 1 :90 1 : 180 1 :360 :720 1 : 1440 4+6 1 :50 1 : 100 1 : 200 1 :400 :800 1 : 1600 4+7 1 :65 1 : 110 1 :220 1 : 440 :880 1 : 1760 4+8 1 :60 1 : 120 1 :240 1 :480 :960 1 : 1920 4+9 1 :65 1 : 130 1 :260 1 : 520 : 1040 1 : 2080 4+10 1 :70 1 : 140 1 :280 1 : 560 : 1120 1 : 2240 Temperature. The standard temperature at which the organism is exposed to the action of the disinfectant is 20° C, but a variation of not more than 0.5° on either side of this figure is allowable. The tem- perature should be maintained by the use of a water bath, the design of which may be left to the individual operator. It is important, how- ever, that the bath be so arranged that the water will rise to a height greater than that of the dilution of disinfectant contained in these seed- ing tubes. The cultures and dilutions of disinfectants should be brought to a temperature of 20° C. before starting the test. In very warm weather, it will be found that there is a material increase in the tempera- ture of the water bath during the twenty-minute period. If, however, the bath is brought at the start to a temperature of 19,5° C. it will not generally exceed 20.5° C. during the test. For the determination of the temperature coefficient a temperature of 30° C. is also employed. All subcultures are incubated at 37° C. for forty-eight hours. The temperature of the incubator should be maintained between 36° and 38°. Teclmique. In the test of a new preparation the coefficient of which is unknown it will be necessary to make a set of range-finding tests. For this purpose use the series 1 : 40, 1 : 80, etc., at the head of the col- umns in the table of final dilutions and select for the actual test with phenol a series of final dilutions ranging from the highest dilution which killed in five minutes to the lowest dilution which failed to kill in twenty minutes. If some approximate idea of the coefficient is available, or if the preliminary test indicates a too extensive range of dilutions for the 170 STERILIZATION AND DISINFECTION final test, a closer range may be obtained by restricting the test to fewer columns and using the middle row of dilutions together with the top row. The seeding tubes having been properly sterilized are brought to the bench and placed in the seeding racks to the number required. Five c.c. of each of the dilutions of phenol and disinfectant to be tested are placed in ordei in these seeding tubes, which are appropriately num- bered, a separate 5-c.c. delivery tube being used for each dilution. The tube containing the filtered culture is next placed in one of the holes of the seeding-tube holder which is then placed in the water bath and allowed to stand there for sufficient time to bring the contents to tem- perature. The inoculator is then filled with culture and, at the beginning of an even five-minute period the first seeding tube is inoculated with 0.1 c.c. of culture and the succeeding tubes are then inoculated at appropriate intervals. If the manipulator is sufficiently skilled to make inoculations and transfers at fifteen-second intervals the total number of dilutions including the phenol dilutions that can be carried through a test simultaneously is 20. Five of these are necessary for the phenol, 1 : 80 to 1 : 120, inclusive, leaving a maximum of 15 for the disinfectant. This will permit the employment of two of the columns of the table of final dilutions, if necessary. In practice, however, it will seldom be found necessary to extend the range over more than one complete col- umn or eight dilutions which with five phenol dilutions gives thirteen in all. This will permit the making of inoculations and transfers at twenty-second intervals, with one minute leeway at the end of each round, a safer and more comfortable schedule. For the beginner thirty- second intervals are recommended which permit the carrying of ten dilutions simultaneously. Whatever the interval decided upon it is quite desirable that a plan of the test showing the dilutions to be used, intervals between inoculations, and times of making subcultures be carefully laid out on paper so that there shall be no confusion in these matters'during the acLal test.^ When the tubes have been inoculated, the inoculator is pushed away diagonally and the loop holder is drawn up to the edge of the bench and just before the expiration of the time interval, subculture tube No. 1 is taken in the hand, the plug withdrawn, and a loop dipped perpen- dicularly into the first seeding tube. At the expiration of the time interval, the loop is withdrawn, and the transfer consummated. The loop is returned to its groove on the loop holder, the burner moved in place under it and the subculture tube given a rapid whirling movement to mix its contents and replaced. Another tube and another loop are then taken in hand for the next transfer. At the completion of the test COAGUIATION COEFFICIEXT 171 the subculture tubes are placed in the incubator and forty-eight hours later they are removed, read and recorded. Determining the Coefficient. The concentrations of the highest dilu- tions which gave negative results in five, ten, fifteen and twenty minutes, respectively, are divided by the corresponding concentrations of phenol to obtain a series of four coefficients. The arithmetical mean of the four is taken as the " Mean Phenol Coefficient against Typhoid 20"" C.,' five to twenty minutes, A. P, H. A." It is recommended that a final report be based upon the results of not less than three separate tests. The coefficient will be known for brevity as the ''A. P. H. A. Standard Phenol Coefl&cient,^' but for any other organism temperature, time ranges that may be employed the word '^ Standard " will be omitted and the full expression given. The determination of these special coefficient for special uses is recommended. Determination of Coagulation Coefficient. (After Schneider, 1912). Certain chemicals which are used as disinfectants precipitate proteins and may be used up in that way. Mercuric chloride, for instance, is such a disinfectant and does not do its maximum work under such con- ditions. Many of the disinfectants do not coagulate albumin in the dilutions which are used for disinfecting. There seems to be no relation between the " phenol " coefficient and the ^' coagulating coefficient " but it is often desirable to know just how active a certain substance is in the precipitation of proteins. For this purpose Schneider (1912) devised the following procedure; Albumen Test Solution Based upon the results of the preKminary experiments a 1 per cent aqueous (distilled water) solution of pure dried egg albumen is recom- mended as the substance upon which the different strength solutions of the various disinfectants is to act. The following methods for making the albumen solution are sub- mitted for consideration, hoping that other investigators may try them out comparatively: (a) Gravimetric Method A, Place 2 gms. of pure powdered egg albumen in 100 c.c. of boiled distilled water, shake and set aside for six to twelve hours, shaking frequently. Filter through a tared filter paper which has been dried (at 100° C.) to constant weight. Filtering is slow, requiring perhaps one -hour's time. When the last drop has filtered through, dry the filter paper with the unfiltered albumen residue upon it to constant weight and weigh. Deduct fronDi this weight the 172 STERILIZATION AND DISINFECTION weight of ihc albumin residue. From 1 gm. of egg albumen dried to constant weight detciniine the percentage of moisture. From the data thus obtained it is easy to determine the amount of boiled distilled water which must be added to the filtrate (100 cc.) to make 1 per cent dried albumen solution. We will suppose that the dried filter paper to be used m filtering the albumen solution weighs 1.570 gms. and this same paper with the undissolved albumen residue (also dried at 100° C. to constant weight) weighs 1.965 gms., then the weight of the undissolved dried albumen residue equals 0.395 gm. We will suppose that 1 gm. of albumen loses 0.126 gm. on drying, or 12.6 per cent moisture. 0.395 gm. raised to its normal air moisture (0.395 gm. + 12.6 per cent of 0.395 gm. = 0.444 gm.) and subtracted from 2.00 gms, leaves 1.556 gms., the amount of albumen that passed through the filter paper. 12.6 per cent of 1.556 gms. = 0.196 gm. and 1.446 gms. less 0.196 gm-== 1.360 gms. which represents the amount of albumen, dried to constant weight, that passed into solution. Therefore to make a 1 per cent solution it is necessary to add enough boiled distilled water to the filtrate to make 1/100, in this case add water up to the 136 cc. mark. We now have a 1 per cent solution suflSciently accurate for all practical purposes. This albumen test solution is now ready for use but it must be kept in mind that it is readily attacked by microbes. However, if carefully prepared with pure albumen, boiled distilled water, in sterile vessels, and put on ice or in a cool place, it will keep for perhaps four days. Any quantity of albumen solution may be made, it merely being advised not to prepare more than may be required for the tests contem- plated. (6) Gravimetric Method B. In a dried and tarred platinum dish place 5 cc. of the albumen filtrate (2 gms. in 100 cc of boiled distilled water), evaporate over water bath and dry to constant weight, and from this determine the percentage of albumen in the solution and the amount of water that must be added to the albumen filtrate to make 1 per cent. (c) Nitrogen Determination. By means of the Kjeldahl apparatus determine the percentage of nitrogen representing the albumen in solution. Having prepared the egg albumen solution the next step is to make the phenol control solution, the primary stock solutions of the disin- fectants to be tested for coagulating powers, and, from these, the secondary or sub-stock solutions, from which the final test dilutions are made as the experiment progresses. COAGULATION COEFFICIENT 173 The standard of comparison is the opacity produced in 5 c.c. of the 1 per cent egg albumen solution when 5 c.c. of 5 per cent phenol solu- tion is added (in a standard test tube of about 15 c.c. capacity). This phenol tube is placed against a black background. In making a test, varying dilutions of the disinfectant are added to the egg albumen solution until the opacity produced is the same as thai in the phenol tube. In each test 5 c.c. of the dilution is added to 5 c.c. of the egg albumen in a standard test tube and the two tubes compared, placed against the black background. The following tentative procedure for making dilutions is suggested: DiLTTTioNS OF Disinfectants to be Used The phenol control solution (5 per cent) is made as for the Anderson- McClintic method of standardizing disinfectants, using only pure phenol crystals. Of the disinfectants to be tested 10 per cent and 1 per cent primary stock solutions are made; 10 per cent solutions of liquid disinfectants as alcohol, formalin, and acids, and 1 per cent solutions of the salts of heavy metals and of soluble substances generally. From these primary stock solutions the following secondary dilutions or «ub-stock solutions axe made, always in those amounts which will serve the purpose, that is, m amounts for perhaps ten sub-dilutions for each and every disinfectant to be tested: 1/10 (of liquids only) 1/100 . 1/1000 1/10,000 1/100,000. Method of Testing (a) Phenol Standard. Pour 5 c.c. of the egg albumen solution in a standard test tube, using a 5-c.c. pipette having a free outflow. Add to this 5 c.c. of the phenol stock solution (5 per cent). Set the tube in the standard test rack (with black background made of cardboard covered with black tissue paper). The degree of opacity developed is to serve as the standard of comparison. (b) Preliminary Testing, The albumen coagulating P9wer of the disinfectant being unknown, much time and labor can be saved by testing with the four or five sub-stock solutions, adding 5 c.c. to 5 c.c. of 174 STERILIZATION AND DISINFECTION the egg albumen test solution, in order to find that dilution of the disin- fectant which fails to show any opacity. We will suppose that the 1/1000 sub-stock solution shows very marked opacity or precipitation, then the 1/10,000 solution might be tried, which may also show quite marked opacity, then the 1/100,000 may be tried. If this gives nega- tive results then we know that the phenol standard lies between 1/10,000 and 1/100,000, with the probabilities that it is nearer 1/10,000. ± A!BXj£i xxJV.x PLAN FOR TEN DILUTIONS AT THIRTY-SECOND INTERVALS i Seeding Dilution. Inoculate 5-MiN Test lO-MiN Test 15-MlN TEtoT 20-MiN Test Tube Mm Sec Mm Sec Mm See Mm Sec Mm Sec 1 1 :80 00 00 5 00 10 00 15 00 20 00 2 1 :90 00 30 5 30 10 30 15 30 20 30 3 1 : 100 1 00 6 00 11 00 16 00 21 00 4 1 :110 1 30 6 30 11 30 16 30 21 30 5 1 :120 2 00 7 00 12 00 17 00 22 00 6 1 :360 2 30 7 30 12 30 17 30 22 30 7 1 :400 3 00 8 00 13 00 18 00 23 00 8 1 :440 3 30 8 30 13 30 18 30 23 30 9 1 :480 4 00 9 00 14 00 19 00 24 00 10 1 :520 4 30 9 30 14 30 19 30 24 30 (c) Concluding Testing, Going back to the 1/10,000 dilution make ten sub-dilutions, increasing the dilutions by a difference of 1000 by simply adding the required parts of distilled water, using small quan- tities, thus: 10 parts of 1/10,000+1 part water= 1/11,000. 10 parts of 1/10,000+2 parts water =1/12,000, 10 parts of 1/10,000+3 parts water = 1/13,000, etc. Any other quantity proportions may be used, however, as 10, 15 20, etc., parts of the sub-stock solution with the required parts of dis- tilled water. If the 1/1000 sub-stock solution is to be used then the dilutions should be increased by 100 as follows: 10+1 = 1/1100 10+2 = 1/1200 10+3 = 1/1300 10+4 = 1/1400; HALE TOXICITY COEFFICIENT 175 or any other equal proportion of stock solution and distilled water may be used, as 5+0.5, 100+10, or 1000+100, etc. If the highest stock dilution (1/100,000) is to be used, then the increase should be by 10,000, thus: 10+1 = 1/110,000 10+2=1/120,000 10+3 = 1/130,000, etc. Determination of the Phenol Coefficient Having determined that dilution which gives the same coagulation opacity as the 5 per cent carbohc acid, it is a very simple matter to determine the phenol albumen coagulating coejB&cient by simply dividing the strength of the dilution of the disinfectant tested by the phenol dilution (1/20). We conclude by giving the coagulating coefficients of a few disin- fectants: Name of Disinfectant. Phenol Coefficient Phenol Copper sulphate. Mercuric chloride. Silver nitrate Alcohol 1.00 750.00 500.00 475.00 0.15 The Hale Toxicity Coefficient of Disinfectants. Hale has attempted in this procedure to establish the toxicity of different substances and to give them a definite numerical relation to other toxic agents. He further proposed that this toxicity coefficient be established for groups of substances having, in general, a similar pharmacological action. The following procedure was proposed for determining the toxicity coefficient of phenol-like compounds: The animal upon which the substance in question is to be tested shall be the white mouse of not less than 15 nor more than 30 gms. weight. The dose is to be calculated per gram of body weight and should, when diluted, equal between 0.03 and 0.04 c.c. per gram weight; that is, 0.06 to 0.08 c.c. for a 20-gm. mouse. The diluent is to be dis- 176 STERILIZATION AND DISINFECTION tilled water and primary dilutions are to be made of such strength that the dose is easily measured with a 1-c.c. pipette graduated in hun- dredths. This is most easily accomplished by the use of the substance in greater concentration than that required to kill in the above volume dose. After the required dose of the diluted disinfectant has been estimated it is measured into a suitable dish and is then diluted further to the required volume by adding water in sufficient quantity. A series of mice are then to be injected with varying amounts of the substance until the least fatal dose is determined, the mice being kept under observation for a period of twenty-four hours unless death results in a shorter time. Mice of the same lot are similarly injected with pure phenol prop- erly diluted to make the measurement of the dose easy and then further diluted in a small dish to equal a volume dose of 0.03 to 0.04 c.c. per gram of body weight and the least fatal dose determined as above. The dose thus obtained is considered unity and the least fatal dose of the sub- stance in question is estimated in per cent of this, as is illustrated in the table following: Mouse Weight. Dose per Gram of Body Weight. Results. Time. Disinfectant A 21.13 20.64 18.32 19.05 18.46 20.10 19.23 18.90 0.0012 0.0016 0.0018 0.0020 0.00035 0.00040 0.00045 0.00050 Survived Survived Lethal Lethal Survived Survived Lethal Lethal Hrs. Min. Phenol. .. • • 10 30 2 15 1 15 - 25 The least fatal dose of disinfectant A was estimated to be 0.0018; that of phenol 0.00045. The phenol toxicity coefficient of disinfectant A therefore is, according to the proportion 4.5 : 18:: X : 100, 25 per cent. The following are some toxicity coefficients secured by Hale in the application of his procedure to some commercial disinfectants. For the sake of comparison, the '' phenol coefficients ^' as determined by McClintic have been included: BIBLIOGRAPHY ON DISINFECTION 177 Toxicity Coefficient. Phenol Coefficient. Without Or- ganic Matter. With Or- ganic Matter. Bacteriol Benetal Creola Creolin-Pearson Cresoleum Germol Hycol Kreotas Kreso Lysol Phenol sodique . Trikresol 45 33 12.8 18 11 16 32 5.6 45 4.5 90 58 23 3.25 2.90 12.30 1 10 3.92 2.62 1.34 0.92 2.52 1.75 1 79 9.37 0.30 2.32 1.57 2.50 BIBLIOGRAPHY Anderson, J. F. and McClintic, T. B. 1912. I. Method of Standardizing Disinfectants with or without Organic Matter. BulL 82, Hygienic Labora« tory U. S. Pub. Health Service, Wash. Bazzoni, C. B. 1914. The Destruction of Bacteria through Light. Amer. Jour. Pub, Health, 4, 975-992. Beyer, A. 1912. Alcohol as a Disinfectant. Zeit. Hyg., 70, 225. BiASSiOTi, A. 1910. The Action of Colloidal Metals on Pathogenic Bacteria. Cent. Bakt. Abt. L, Ref. 45, 680. Bitter, L. 1912. The Destruction of Bacteria by the Heavy Metals and Building Materials. Zeit. Hyg., 69, 483-512. Browning, C. H. and Russ, S. 1917. Germicidal Action of the Ultra-violet Radiation and its Correlation with Selective Absorption. Proc. Roy. Soc, London, (B) 90, 33-38. BuRGE, W. E. 1917. The Action of XJltra-\iolet Light in Ealling Living Cells such as Bacteria. American Journal Physiology, 43, 429-432. BxjRGE, W. E. and Neill, A. 1915. The Comparative Rate at which Fluores- cent and Non-fluorescent Bacteria are Killed on Exposure to Ultra-violet Light. Amer. Jour. Physiology, 38, 399-403. Chick, H. 1908. An Investigation of the Laws of Disinfection. Jour. Hygiene, 8, 92. Chick, H. 1910. The Process of Disinfection by Chemical Agencies and Hot Water. Journal of Hygiene, 10, 237. Chick, H. 1912. The Factors Influencing the Velocity of Disinfection. Eighth Intern. Cong. Appl Chem., 26, 167-197. 178 STERILIZATION AND DISINFECTION Chick, H, and Martin, C. F. 1908. The Principles Involved in the Stand- ardization of Disinfectants and the Influence of Organic Matter on the Germicidal Value. Journal Hygiene, 8, 654. Chick, H. and Martin, C. J. 1910. On the Heat Coagulation of Proteins. Journal of Physiology, 40, 404. Chick, H and Martin, C. F. 1908. A Comparison of the Power of a Germi- cide Emulsified or Dissolved with an Interpretation of their Superiority of the Emulsified Form. Journal of Hygiene, 8, 698. Churchman, J, W. 1912. The Selective Bactericidal Action of Gentian Violet. Journal Experimental Medicine, 16, 221--247, Cooper, E. A. 1912. Bactericidal Action of Cresols and Allied Substances and the Best Means of Employing Them. Brit. Med. Journal, 1912, I. 1234-1240. Cooper, E. A. 1912. Relations of Phenols and their Derivatives to Proteins. A Contribution to Our Knowledge of the Mechanism of Disinfection. Biochem Jour., 6, 362-387; 7 (1913), 175-96. Dakin, H. D., Cohen, J. B., Daxjfresne, M., and Kenyon, J. 1916. Pro- ceedings Royal Society, London, Series B, 89, 232-251. Dakin, H. D., Cohen, J. B., and Kenyon, J. 1916. Chloramin, Its Prepara- tion, Properties, and Use. Brit. Med. Journal No. 2874, 160-162. Dakin, H. D., Lee, W. E., Sweet, J. E., and Hendrix, B. M. 1917. A Report of the Use of Dichloramin-T (Toluene-parasulphondichloramin) in the Treatment of Infected Wounds. Jour Amer. Med. Assn., 69, 27-31 . DeWitt, L. M. and Sherman, H. 1916. The Bactericidal and Fungicidal Action of Copper. Journal Infectious Diseases, 18, 368-382. Dieudonnb, a. 1894. Beitrage zur Beurtheilung der Einwirkung des Lichtes auf Bakterien. Art. a. d. kais. Gesundheitsamte, 9, 405. Duyser, C. a. and Lewis, W. K. 1914. A New Method for Determining the Value of Disinfectants. Chem. Eng., 19, 113; Jour. Ind. Eng. Chem., 6, 198-200. EiJKMAN, C. 1908. Die Ueberlebungskurve bei Abtodtung von Bakterien durch Hitze. Biochem. Jour., 11, 12. Embrey, G. 1912. The Use of Copper Sulfate in Purifying Water SuppHes. Canadian Engineer, 23, 245-6. Eugling, M. 1912. The Disinfective Action of Iodoform and Novoidin. Cent. Bakt. Abt., I, 60, 397-416. Findlay, L. and Martin, W. B. M. 1915. The Effect of Daylight and Dry- ing on the Human and Bovine Type of Tubercle Bacilli. Brit. Med. J., 110-111. Frby, E. 1912. Why is Seventy Per Cent Alcohol so Strongly Bactericidal. Deut. Med. Wochenschr., 38, 1633-5. Chem. Absts., 6: 1912, 3843-4. Goodrich, H. P. 1917. Glycerol and Antiseptics. British Med. Jour., 1917, 647-648. GossL and Hbrzog. Theory of Disinfection. IL Disinfectants that Dissolve Lipooid. Zeit. Physiol Chem., SS (1), 103-8. BIBLIOGRAPHY ON DISINFECTION 179 Hamilton, H. C. 1917. Facts and Fallacies in Disinfection. Am. J. Pub, Health, 7, 282-95. Hamilton, H. C. and Ohno, T. 1913. Standardization of Disinfectants. Am. J. of Pub. Health, 3, 583. Hamilton, H. C. and Ohno, T. 1914. Bacteriological Standardization of Disinfectants. Amer. Jour. Pub. Health, 4, 486. Harris, F. J. and Hoit, H. S. Possible Origin of the Toxicity of Ultra Violet Light. Science, 46, 318-20. Herzog and Betzbl. 1911. Zur Theorie der Desinfection. Zeit. PhysioL Chemie, 67 (1910), 303, 74, 221. Hewlett. 1909. Milroy Lectures on Disinfection and Disinfectants. Lancet, March 13, 20-27. Hewlett and Hall. 1911. The Influence of Culture Media on the Germina- tion of Anthrax Spores with Special Reference to Disinfection- Experiments. Jour. Hyg., 11, 473. Hooker, A. H. 1913. Chloride of Lime in Sanitation. John Wiley & Sons, New York. Houghton, E., Maud, Davis L. 1913. A Study of the Germicidal Action of the Ultra Violet Rays. Amer. Jour. Pub. Health, 4, 224-240. Kendall, A. I. and Edwards, M. R. 1911. A Method for Determining the Germicidal Value and Penetrating Power of Liquid Disinfectants. Jour. Infectious Diseases, 8, 250-57. Kempster, C. 1917. The Effect of X-Rays upon Diseases of Bacterial Origin. Lancet, 1917, II, 423-424. Kingzett, C. T. and Woodcock, R. C. The Bacterial Testing of Disinfectants: A Practical Criticism. Analyst, 38, 190-200. Kingzett, C. T. and Woodcock, R. C. 1910. Bacteriological Testing of Certain Disinfectants and the Results as Effected by Varying Conditions. Pharm. Jour., 85, 157-8. Kligler, I. J. 1918* A Study of the Antiseptic Properties of Certain Organic Compounds. Journal of Experimental Medicine, 27, 463-478. Koch. 1901. The Physiological Action of Formaldehyde. Amer. Jour. Physiology, 6, 325-329. Ke5nig and Paul. 1897. Die chemischen Grundlagen der Lehre von der Giftwirkung und Disinfection. Zeit. f. Hyg., 25, 1. Lageb, H. 1916. Disinfection of Water with Calcium Hypochlorite. Zeit. Hyg., 81, 296-310. Chem. Abts., 12 (1918), 394. Lusini. 1912. Disinfecting Action of Cations According to the Law of the Periodic System. R. Acad, fisicrit. March 20, 1910. Chem. Abts., 6, 1912. 240. MahoN; T. and White, J. S. 1915. Germicidal Value of Iodine. J. Sec. Chem. Ind, 34 (1915), 244. Chem. and Druggists, 1916, 144. McClintic, T. B. 1905. Chloride of Zinc as a Deodorant, Antiseptic and Germicide. BuU. No. 22, Hyg. Lab. U. S. Pub, Health Service, Wash- ington. 180 STERILIZATION AND DISINFECTION McLaughlin, A. J. 1903. Inefficiency of Ferrous Sulfate as an Antiseptic and Germicide. Bull 15, Hyg. Lab. U. S. Pub. Health Service, Wash. Moore, G. T. and Kellerman, K. F. 1905. Copper as a Disinfectant and Algicide in Water Supplies. U. S. Dept. Agr. Bureau of Plant Industry Bull, 76. Madsen and Nyhan. 1907. Zur Theorie der Disinfection. Zeit. Hyg., 57, 388. Newcomer, H. S. 1917. Bactericidal Fluorescence Excited by X-Rays. Jour. Exper. Med., 26, 657-668. Newcomer, H. S. 1917. The Abiotic Action of Ultra Violet Light. Jour. Exper. Med., 26, 841-848. NissEN, F. 1890. Ueber die Desinficirende Eigenschaft des Ghlorkalks. Zeit. Hyg., 8, 62-77. Norton, J. F. The Coefficients of Germicides. Jour. Ind. Eng. Chem., 8, 197. Norton, J. F. and Hsu, P. H. 1916. Physical Chemistry of Disinfection. Jour. Inf. Diseases, 18, 180-195. OcHSNER, E. H. 1916. The Biochemistry of Topical Applications with Spe- cial Reference to the Use of Boric Acid in Septic Infections. Chemical Ab- stracts, 12 (1918), 191. Oker-Blom, M. 1913. The Nature of the Action of Ultra Violet Rays on Bacteria. Zeit. Hyg., 74, 242-247. Paul Birstein and Reuss. 1910. Beitrage zur Kinetik der Giftwirkung von Geloeteten Stoifen. I. Einfluss der Konzentration. II. Einfluss der Neu- tralsalze. Einfluss und der Temperature. Biochem. Zeit., 29, 202 and 249. Paul Birstein and Reuss. 1910. Beitrag zur Kinetik des Absterben tro- kener Bakterien in Sauerstoff \erschiedener Konzentration und ver- schiedenen Temperaturen. Biochem. Zeit., 25, 367. Phelps, E. B. 1911. On the AppHcation of Certain Laws of Physical Chem- istry in the Standardization of Disinfectants. Jour. Inf. Dis., 8, 27. Phelps, E. B. 1913. The Limitations of the Phenol Coefficient in the Stand- ardization of Disinfectants. Amer. Jour. Pub. Health, 3, 53-57. Reichel, H. 1909. Zur Theorie der Disinfection. Biochem Zeit,, 22, 149; 177 and 201. Renob, L. (et al), 1914. Antiseptic Role of Insoluble Substances. Comp. Rend. Soc. Biol., 76, 64-66. Reudiger, E. H. 1914, Germicidal Power of Glycerol on Various Micro- organisms under Various Conditions. Philippine Journal of Science., 9, B, 564-477. Ribeal, S. 1912. Suggested International Tests for Disinfectants. Eighth Intern. Cong. Applied Chemistry, 26, 261. Rideal, S. and Walker, A. 1903. The Standardization of Disinfectants. Jour. Roy. San. Institute, 24, 424. Rideal, (etal). 1915. A Note on Testing Disinfectants. Amer. Jour. Pub. Health, 5, 1175-1176. MBLIOGRAPHY ON DISIMFECTION 181 Robertson, T. B. 1914. Disinfection. Jour. Hyg., 14, 143-148. Robertson, T. B. 1914. On the Conditions under which Discontinuous Events may be Employed ah a Measure of Continuous Processes with Special Reference to the Killing of Bacteria by Disinfectants. Journal of Hygiene, 14, 143-148. RosENAU, M. J. The Antiseptic and Germicidal Properties of Glycerine. Bull., 16, Hyg. Lab. U. S. Pub. Health Service, Washington. RuBNER, M. 1913. Modern Steam Sterilization. Jour. Am. Med. Assoc, 60, 1344. Schmidt, E. W. 1910. Bactericidal Value of Thymol. Zeit. Physiol. Chem., 67, 412-432. Schneider, F. 1912. Albumin Coagulation Coefficient for Disinfectants. Pacific Pharmacist, 5, 335-339. Simpson, W. J. and Hewlett, R. T.. 1914. Germicidal Action of Colloidal Silver. Lancet, Dec. 12, 1914, 1359. Chemical Absts., 8 (1915), 640, 641. SoMMERViLLE, D. 1913. Antiscptics and Germicides. Jour. Roy. Soc. Arts, 61, 927-937; 945-952. Steenhauer, a. J. 1916. The Disinfecting Power of o-, m-, and para-Cresol and Basic Mercuric Cyanide. Pharm. Weekbladt, 53, 680-685. Straus. 1917. Disinfecting Properties of Alcohol. Schweiz. apoth. Ztg., 55, 36. SupFLE, K. and Deugeler, A. 1916. Culture Media for Use after Disin- fection Experiments. Arch. Hyg., 85, 189-197. Von Recklinghausen, M. 1914. Purification of Water by the Ultra Violet Rays. Jour. Amer. Water Works Assn., 1, 565-588. Walker, A. 1916. The Stand^-rdization of Disinfectants. New York Med. Journal, 1916, 500-505. ' Walters, A* L. 1917. The Specificity of Disinfectants and its Bearing on their Standardization. Amer. Jour. Pub. Health, 7, 1030-1036. Ward, H. M. 1895. The Action of Light on Bacteria. Phil. Trans. Roy. Soc. 1895, 961. Watson, H. E. 1908. A Note on the Variation of the Rate of Disinfection with Change in Concentration of the Disinfectant. Jour. Hygiene, 8, 536. Weinzirl, J. 1914. The Germicidal Action of Sunlight upon Bacterial Spores, Amer. Jour. Pub. Health, 4, 969-974. Winkler, L. W. 1888. Die Bestimmung des im Wasser gelosten Sauerstoffs. Berichte, 21, 2, 2843-2854. Wright, J. H. 1917. The Importance of Uniform Culture Media on the Bacteriological Exandination of Disinfectants. Jour. Bact., 2, 315-346. CHAPTER VI PROTEINS AND CARBOHYDRATES Classification of Proteins. Committees for the American Society of Biological Chemists and the American Physiological Society have made the following recommendations concerning the proteins: First. The word protetd should be abandoned. Second. The word protein should designate that group of sub- stances which consists, so far as at present is known, essentially of com- binations of the a-amino acids and their derivatives, e.g., a-amino acetic acid or glycocoll; a-amino propionic acid or alanin; phenyl-a-amino propionic acid or phenylalanin; guanidin-amino valerianic acid or arginin, etc., and are, therefore, essentially polypeptids. Third. That the following terms be used to designate the various groups of proteins: I. Simple Proteins. Protein substances which yield only a-amino acids or their derivatives on hydrolysis. Although no means are at present available whereby the chemical individuality of any protein can be established, a number of simple proteins have been isolated from animal and vegetable tissues which have been so well characterized by Constance of ultimate composition and uniformity of physical properties that they may be treated as chemical individuals until further knowledge makes it possible to char- acterize them more definitely. The various groups of simple proteins may be designated as follows: (a) Albu7nins, Simple proteins soluble in pure water and coagulable by heat. (6) Globulins. Simple proteins insoluble in pure water but soluble in neutral solutions of salts of strong bases with strong acids. (c) Glutelins. Simple proteins insoluble in all neutral solvents but readily soluble in very dilute acids and alkalies. (d) Alcohol Soluble Proteins. Simple proteins soluble in relatively strong alcohol (70 to 80 per cent) but insoluble in water, absolute alcohol, and other neutral solvents. (e) Albuminoids. Simple proteins which possess essentially the 182 CLASSIFICATION OF PROTEINS 183 same chemical structm-e as the other proteins, but are characterized by a great insolubihty in all neutral solvents. (/) Histories. Soluble in water and insoluble in very dilute ammonia and, in the absence of ammonium salts, insoluble even in an excess of ammonia; yield precipitates with solutions of other proteins and a coagulum on heating, which is easily soluble in very dilute acids. On hydrolysis they yield a large number of amino acids, among which the basic ones predominate. (g) Protmnins. Simpler polypeptids than the proteins including the preceding groups. They are soluble in water uncoagulable by heat, have the property of precipitating aqueous solutions of other proteins, possess strong basic properties and form stable salts with strong mineral acids, among which the basic amino acids predominate. II Conjugated Proteins. Substances which contain the protein molecule with nucleic acid. (a) I^ucleoprotetns, Compounds of one or more protein molecules with nucleic acid. (6) Glycoproteins, Compounds of the protein molecule with a sub- stance or substances containing a carbohydrate group other than a nucleic acid. (c) Phosphoproteins. Compounds of the protein molecule with some, as yet undefined, phosphorus containing substance other than a nucleic acid or lecithin. (d) Hemoglobins. Compounds of the protein molecule with hema- tin or some similar substance. (e) Lecitho-proteins. Compounds of the protein molecule with lecithins (lecithans, phosphatids). III. Derived Proteins. 1. Primary Protdn Derivatives, Derivatives of the protein mole- cule apparently formed through hydrolytic changes which involve only sHght alterations of the protein molecule. (a) Proteins, Insoluble products which apparently result from the incipient action of water, very dilute acids or enzymes. {6) Metaproteins, Products of the further action of acids and alka- lies whereby the molecule is so far altered as to form products soluble in very weak acids and alkalies but insoluble in neutral fluids. This group will include the familiar '* acid proteins '' and " alkali proteins " not the salts of proteins with acids. (c) Coagulated Proteins, Insoluble products which result from (1) the action of heat on their solutions, (2) the action of alcohols on the protein. 184 PROTEINS AND CARBOHYDRATES 2. Secondary Protein Derivatives. Products of the further hydrolytic cleavage of the protein molecule. (a) Proteoses. Soluble in water, uncoagulable by heat and pre- cipitated by saturating their solutions with ammonium sulphate or zinc sulphate. (b) Peptones. Soluble in water, uncoagulable by heat, but not precipitated by saturating their solutions with ammonium sulphate. (c) Peptids. Definitely characterized combinations of two or more amino acids, the carboxyl group of one being united with the amino group of the other, with the elimination of a molecule of water. Structure of Proteins. Proteins are combinations of amino acids the unions being between the carboxyl group of one acid and the amino group of the other. The following will serve to illustrate the linkage. H H H IOH~H H— C— H H H-™-C— H H— C— C=0 >N— C— CC =H— C— C— N— C— C=0 I H^ I \H I II I i \ H H NH2O H H ^ Glycine Alanin Glycyl-alanin The free amino and carboxyl groups on the glycyl-alanin molecule are free to react in the ^ame way. This yields infinite possibilities with regard to the size of the protein molecule. Fischer has done much toward synthesizing proteins from these smaller units the amino acids. The amino acids which have been isolated from proteins may be classified as follows: 1. Monoamine Monobasic Acids. GlycocoU, a-amino acetic acid. H H— C— C^OH NH2 Alanin, a-amino propionic acid. HIT H-C— C— C^OH H NH2 STRUCTURE 0^ AMINO ACIDS 185 Phenylalanin, a-amino, /9-phenyl propionic acid. / \_c— C— C^OH H NH2 Serine, a-amino, jS-hydroxy propionic acid. H H H— C— C— C^OH OHNH2 Tyrosine, a-amino, jS-para hydroxy phenyl propionic acid. H H W t OH ■C— G— C^OH H NH2 Cystine, a-diamino, ;8-dithiolactylic acid. S S H— C— H H— C— H H — C — NH2 H — C — NH2 I I COOH COOH Leucine, isobutyl a-amino acetic acid. >UJdL — ^±12 — ^-^-tl — l^UUxl CH3/ I NH 2 Isoleucine ethyl methyl, a-amino propionic acid. r^TT r^xj r^xT r^r\r\xx \^j[l2' /Uxl — L/JlL — ^OUUxi r^xx r^xj y I NH2 Valine, a-amino, isovalerianic acid. rvrt "XTTT UXX3 IN XI 2 Jtl — \j \j — Kj — UJtl o OTJ "tr Vyxl3 JjL 186 PROTEINS AND CARBOHYDRATES II. Dicarboxylic acids. Aspartic acids, a-amino succinic acid. TT Jtl -0 NH2— C— C^OH i /O Glutamic acid, a-amino, n-glutaric acid. NH2 H— C— C^OH H— C— H >0 H— C— C^OH XT III. Diamine acids. Lysine, a-e-diamino caproic acids. NH2 H H H NH3 / H— C C-C— C-C C^OH H H H H H Arginine, d-guanidine, amino valerianic acid. H H H NH3 N— C— C— C— C— C^OH H H H H C=NH NH2 IV. Heterocyclic amino acids. Tryptophane, j3-indole, a-amino propionic acid. Jtl /\ .C_C_C— C2-0H / -a HXTTJ N ^ Jtl ELEMENTS IN" ORGANIC COJNrPOUNDS 187 Histidine, a-amino imidazol propionic acid. H NH2 >0 H— C=C— C— C— C^OH Hivr IVT XT XT IN-V y^lN xl xl c XT JdL Proline, a-pyrrolidine carboxylic acid. 2vy v_/Xl2 H2C-. yC^C^OH N TT That amino acids are utilized by bacteria has been pointed out in other places. The commercial peptones which are used in the prepara- tion of media are, among other things, mixtures of amino acids. The changes induced by bacteria on certain amino acids in the intestinal tract are discussed in that chapter. Raistrick (1917) has shown that the bacteria of the coli-typhi group are able to change histidine into urocanic acid (iminazolylacrylic acid). Most of the other amino acids may be changed by bacterial enzymes to smaller molecules. Bacterial changes induced in other amino acids have been discussed elsewhere in this book. Qualitative Analysis for the Elements in Organic Compounds, The inorganic elements may be detected by heating some of the sample on a piece of platinum foil or porcelain. During this ignition the organic elements are driven off. The organic elements are determined by ignition with sodium. It often happens that an element is present in both organic and inorganic forms. Inorganic Elements, The substance is ignited on a piece of platinum foil or porcelain until all of the organic portion is destroyed. If the sample chars during fchis procedure, the presence of an organic substance is shown. If an inorganic substance is shown in the charring it may further be determined by the procedures of qualitative analysis. Organic Elements. Place a small piece of sodium in an ignition tube about 4 ins. long. Incline the tube in a slanting position and 188 PROTEINS AND CARBOHYDRATES heat the closed end very gently at first and later heat the bottom to a dull red. As soon as the sodium has begun to volatilize and condense on the sides of the tube, add a few drops of the sample or a few pieces of a solid, being careful to keep off the sides of the tube. As soon as the tube has cooled any excess of sodium may be removed by adding a little alcohol or if the sodium excess is not large the tube may be plunged into warm water. This solution must be filtered free of free carbon. The filtrate may then be tested for the halogens, nitrogen and sulphur. Hydrogen and oxygen are assumed to be present. Test for Organic Sulphur. Treat about 1 c.c. of the solution secured from the sodium ignition with lead acetate solution. If sulphur is present a black precipitate will be formed according to the following equation. The solution must be strongly alkaline. Na2S+Pb(C2H302)2 = PbS+2NaC2H302. Sodium nitro-prusside may also be used to test for organic sulphur. Add a few drops to about 1 c.c. of the filtrate secured from the sodium ignition and if a violet color results, the presence of sulphur is indicated. Test for Organic Nitrogen. Add to about 3 c.c. of the filtrate four drops of NaOH solution, four drops of FeS04 solution and one drop of FeCk. Add a little concentrated HCl to dissolve the precipitated ferrous and ferric hydroxides and boil. If N is present in the sample a precipitate of Prussian blue will come down. It may be necessary to heat the tube gently and leave it for a time. The following reactions take place: 6NaCN+FeS04 = Na4Fe(CN) 6+Na2S04, FeCls+NaOH =Fe(OH)3+3NaCL 4Fe(0H)3+3Na4Fe(CN)6+12HCl-Fe4(Fe(CN)6)3+ 12HoO+ 12NaCL Test for Sulphur and Nitrogen when Present Together. Acidify about 1 c.c. of the solution with HCl and add three drops of ferric chloride solution. A dark red indicates the presence of sulphur and nitrogen. 3NaSCN+FeCl3 = Fe(SCN)3+3NaCl To be certain of results the above tests for sulphur and nitrogen should be made along with this test. Test for Organic Halogens: Group Test This is carried out by removing H2S, and cyanide. To do this the solution is acidified with dilute nitric acid and boiling. The addition of silver nitrate at this ELElMENTS IN ORGANIC COMPOUNDS 189 point will precipitate the halids of silver which have the characteristic appearance. Tests for Bromine and Iodine. Acidify a few cubic centimeters of the sample and boil to remove H2S and the cyanids. Add a few drops of carbon bisulphide and a little chlorine water. If iodine is present, it is taken up by the heavier bisulphide which settle to the bottom of the tube with a distinct violet color. NaH-H3S04 = HI+NaHS04. 2HI+Cl3 = H40+l2. Qualitative Tests for Elements in Proteins. Carbon. Heat the protein 01^ a piece of platinum foil and if charring takes place the pres- ence of carbon is indicated. Hydrogen. Test for water in the top of the test tube. Nitrogen, Heat with soda lime and test for ammonia by smell and moist litmus paper. Sulphur. Heat with concentrated potassium hydroxide which will set free potassium sulphide. Cool and add lead acetate solution, which will precipitate black lead sulphide. After adding the lead acetate solu- tion acidify, boil and suspend lead acetate paper above the liquid. Detection of Proteins. (1) The coagulation of proteins by heat especially in shghtly acid solutions may be used in their detection. This precipitate does not disappear on continued addition of acid. (2) Alcohol in excess will form a precipitate which is soluble in water at first; if the precipitate is left in contact with the alcohol, it is changed. The proteins come down unchanged but are probably dehydrated when left in contact with the alcohol. The fixing of tissue in histological work rests upon this fact. (3) Heller^s Ring Test rests upon the fact that strong mineral acids will coagulate proteins. A small amount of con- centrated nitric acid is put into a test tube and some of the test solution is floated upon it. If proteins are present a whitish layer will be formed at the point of contact between the two liquids. Action of Bacteria on Proteins. The hydrolysis of proteins by bacteria has been studied by different investigators with the production of data which is not in agreement. Bainbridge (1911) found that bac- teria could not decompose '* purified native proteins." This was later verified by Sperry and Rettger (1915) who worked with purified serum albumin, egg albumin and edestin and the common proteolytic bacteria. They reported that when all other forms of nitrogen were removed, 190 PROTEINS AND CARBOHYDRATES bacteria could not start the decomposition of the proteins. Rettger, Herman and Sturges (1916) have continued this work and reported that proteoses and peptones follow essentially the same law of resistance to bacterial action as do the native proteins. They found that if there was sufficient nitrogen in other combinations to allow growth of the bacteria in order that proteoses could be secreted, the proteins were hydrolyzed. When no growth took place due to the lack of available nitrogen, no hydrolysis occurred. The bacteria used in the above experiment might be regarded as '^ starved " and thus not in a normal condition. Robinson and Tartar (1917) when studying this question made no attempts to use purified proteins: blood fibrin, egg albumin, casein, gliadin and peptone were split by bacteria to ammonia. The chief source of ammonia seemed to be the monoamino and diamino nitrogen. The work of these investigators simulates more closely the conditions in our common media and in nature. Action of Bacteria on Polypeptids. Sasaki (1912) repoited the B. coli spUt glycyl-Z-tyrosin and glycylglycine into their component acids and from this they conclude that bacteria probably plays a r61e in digestion. The same author in two other papers reports investigations wherein liquefying and non-hquefying bacteria were used. With non- Kquefying types such as typhi, dysentery, etc., glycyl-Z-tyrosine and glycylglycine were decomposed to their component acids. Sasaki regards this change as due to an ereptase-like enzyme, excluding pep- tase because synthetic polypeptids were used. The various liquefying bacteria which he used were also able to split the polypeptids. In 1914 Sasaki killed B. coli with toluene and, after action on polypeptids, demonstrated that they were split into their amino acids. Somewhat similar results have been reported by Mito (1918). He tested the enzymes of B. coli communis and Staph, aureus on di-leucylyblycine. Asymetric cleavages were secured. The copper salt of Z4eucine was isolated. The mother Hquor exhibited an optical activity compatible with c?-leucylglycine. Sasaki (1917) grew B. coli communis and B. proteus vulgaris in tyrosine containing media of two kinds. One of these media contained lactose to allow acid formation while the other con- tained a mixture of phosphates to maintain neutrality. In the lactose media both bacteria formed p-hydroxyphenyl»ethylamin. In the lac- tose free media no amine could be isolated but rather large amounts of d-p-hydroxyphenyllactic acid were secured. Bacterial Action on Other Bodies Related to Proteins. There exist a class of compounds which stand between the carbohydrates and pro- teins— glucosamin, REACTIONS OF PROTEINS 191 OH OH OH OH H I I I ! I H C C C C C C=0 I I I i I i H H H H NH2 H This compound possesses an amino group in place of a hydroxyl group and this amino group, to a certain extent, connects it with the protems although it retains many of its carbohydrate properties. In light of the controversy concerning the action of bacteria on proteins their action on this compound is interesting. Meyer, in attempting to explain the action of bacteria on d-glucosa- min, studied the action on acetyl derivatives. ' He believes that the NH2 group is probably split off after which a further degradation takes place. Color Reactions of Proteins. By means of these much information may be secured concerning the amino-acids in a protein. Biuret Reaction. The protein solution is warmed and a httle strong sodium hydroxide and dilute copper sulphate is added. Care should be exercised to use dilute copper sulphate two or three drops in a test tube of water. A violet color is obtained. The color is due to the formation of biuret, C — N — H N— H f; I / G— N— H The test is given by those substances which have two CONH2 groups. Xantho-proteic Reaction. The protein is treated with concentrated nitric acid. The solution or protein will turn yellow. The presence of a CbHs group is necessary. This reacts with the HNO3 to yield nitro derivatives of benzine. Nitro-benzine is formed. Millon's Reaction. The protein or protein solution is heated with Millon's reagent. The protein is turned red or, if a solution is, used a reddish precipitate is formed. The reaction is given by those proteins which possess a hydroxy-phenyl group. Since tyrosine is the only amino acid having this group which has been isolated from proteins, the presence of this amino acid is indicated by a positive test with Millon^s reagent. Care must be exercised in the use of Millon's reagent since it may be decomposed by some of the inorganic salts which precip- itate mercury. 192 PROTEINS AND CARBOHYDRATES Liebermann's Reaction. Boil the protein solution with about 4 c.c. of concentrated hydrochloric acid. A violet-lavender color will result if tryptophane is present. Bromine Reaction for Tryptophane. Uncombined tryptophane when treated with bromine water will give a violet color. Test for Tyrosine. Add a few drops of formol solution to concen- trated sulphuric acid. On warming with tyrosine, a brown red color is obtained, which, on addition of acetic acid becomes green. Neither proteins nor peptones give the reaction. (Nassers modification of Denige^s test. Quoted from Smith, 1905.) Schmidt Test for Tyrosine. Dissolve by boiling in water and add a solution of mercuric nitrate. The red reaction is sharper if a little fuming nitric acid diluted in water is added. Try also the violet reaction with neutral iron chloride. (Quoted from Smith, 1905.) Precipitation Reactions. The precipitation reactions of proteins are important in removing them from solution and also for detecting them in solution. Most of the metals will precipitate them. Mercury will throw down a heavy white precipitate of mercury proteinate. Cop- per, when added to a protein solution as the sulphate, will throw down a bluish precipitate. Iron and lead also give heavy precipitates, the one with iron being dissolved by an excess of that compound. Picric acid, trichloracetic acid, phosphotungstic acid, tannic acid and bromine also form precipitates in protein solutions. Carbohydrates The carbohydrates may be classified in different ways. The fol- lowing is a convenient method: I. Monosaccharides A, Pentoses, C5H10O5 1. Xylose 2. Arabinose B, Hexoses, C6H12O6 1. Dextrose 2. Levulose 3. Galactose 11. Disaccharides 1. Sucrose 2. Maltose 3. Lactose CLASSIFICATION OF CARBOHYDRATES 193 III. Trisaccharides 1. Raffinose IV. Polysaccharides A. Starches 1. Starch 2. Glycogen 3. Inulin JS. Celluloses 1. Cellulose 2. Hemicellulose a. Pentosans Gum arable 6. Hexosans Galactans Agar-agar Action of Bacteria on Carbohydrates. This class of substances serves bacteria for both energy and building purposes. The action of microorganisms on carbohydrates is a subject too large for extensive treatment here.* The enzymes which will attack carbohydrates are widely distributed among microorganisms. Foods containing car- bohydrates are especially susceptible to attack by bacteria unless the sugar is too concentrated. If there is too little moisture present the organisms are unable to carry on their activities. The changes induced in sugar by bacteria may be discussed in dif- ferent ways. It is quite natural to discuss them on a physiological basis. Lactic Acid Fermentation. This is brought about by bacteria by first splitting the lactose into two molecules of a monosaccharide, each of which is further decomposed to lactic acid according to the following equation: Ci2H220ii+HOH==2C6Hi206, C6H12O6 == CsHeOs. Since the lactic acid which is thus formed possesses so much latent energy, it may be further decomposed to other compounds such as butyric acid. The kind of lactic acid formed has been the subject of many investiga- tions. Four lactic acids are known, three of which have the same formula, * Those wishing an exhaustive treatment of the subject are referred to Kruse's AUgemeine Mikrobiologie, or Lafar's Handbuch der technischen Mycologie. 194 PROTEINS AND CARBOHYDRATES CH3CH2OHCOOH. The other has the formula CH2OHCH2COOH. The last-mentioned lactic acid since it does not possess an asymetric carbon atom is not optically active. The ethylidene lactic acids possess asymetric carbon atoms and are therefore optically active. Thomas (1916) has shown that the lactic acid produced by Matzoon in synthetic culture media is inactive but that it may be resolved into two active components. According to Heinemann (1907) milk which sours naturally at room temperature contains chiefly d-acid; at 37° C. chiefly r-acid with Z-acid in excess if allowed to stand a few days. He also found that the kind of organism present determined the kind of lactic acid which was formed. The temperature was also believed to have an effect. AlcohLolic Fermentation. These are those changes in sugar and decomposition products of sugar which result in the formation of large amounts of alcohol. Historically it has been known for a very long time and was not thoroughly analyzed until Pasteur proved that it was a biological phenomenon. The- chemistry of alcoholic fermentation is now fairly well understood and there is much data available In the literature to review at this time. Kruse beUeves that dextrose breaks up in a series of reactions while Harden maintains that this takes place in one reaction. Kruse's reactions will add up to equal that of Harden's : 3C6Hi206=2C2H50H+2C02; 2C6Hi206 = 3CH3COOH; 6C6Hi206 = 2C3H603; C6H12O6+6H2O-6CO2+6H2O; I2C6H12O6+H2O =12C3H603+6C2H50H+6 CH3-COOH -f- 12CO2 "j" I2H2O. Ethyl Alcohol. Iodoform Reaction. Add about an equal amount of iodine solution to the sample and treat with sodium hydroxide until decolorized. Warm the mixture very carefully and try to detect the odor of iodoform. A yellow precipitate of iodoform may result. This test is not specific as it is given by acetone, aldehyde, acetic ester, and other similar substances. Acetaldehyde Reaction. Add a few drops of potassium dichromate and* dilute sulphuric acid to the sample and heat. An odor of aldehyde will be observed if ethyl alcohol is present. REACTIONS OF CARBOHYDRATES 195 Ethyl Acetate Reaction, Add a little sodium acetate and concen- trated sulphuric acid to the sample and heat. The fruity odor of ethyl acetate will be detected if alcohol was present in the sample. Quanti- tative methods for this may be obtained from chemical texts. Acetone. Iodoform Reaction. This is the same test as is used to detect ethyl alcohol except that it is carried out in the cold. Add to the same a few drops of sodium hydroxide and ver}^ slowly iodine- potassium-iodide solution until the solution is colored yellow. If acetone is present iodoform will settle out. Nitroprusside Test. Add a few drops of a freshly prepared solution of sodium nitroprusside and about 1 c.c. of sodium hydi oxide. A red color is produced which will fade to a yellow on standing. Moliscli Reaction. Add a few drops of Molisch reagent to the sugar solution in a test tube. Incline the tube and pour a few cubic centimeters of concentrated H2SO4 down the side of the test tube. A lavender color will be formed if sugars are present. If the H2SO4 is put into the test tube first, and the sugar-Molisch reagent mixture run in on top, a ring test may be observed. Seliwanoff's Reaction for Ketoses. Add a small amount of the sugar or unknown solution to a few cubic centimeters of Seliwanoff's reagent and heat to boihng. A reddish coloration indicates the presence of ketose sugars. Fehling's Test. This depends upon the action of reducing sugars. This action is generally attributed to the presence of an aldehyde or ketone group. Some regard other parts of the sugar molecule as in- volved in this reduction. Two solutions are used in the test. These are kept apart until used. If they are mixed and allowed to stand, the copper will be slowly reduced by the tartrate. The solutions have the following composition: Solution I. Copper sulphate 34 65 gms. Distilled water. 500.00 c.c. Solution II. Potassium hydroxide 125 00 gms. Sodium potassium tartrate 173 00 gms. Distilled water 500. 00 c.c. In testing for reducing sugars, mix equal parts of these two solutions and heat to boiling. This will determine whether the solutions are good. If any copper is precipitated by this heating, the solutions must be dis- 196 PROTEINS AND CARBOHYDRATES carded. If the solutions are satisfactory, the sugar solution should be added very slowly to the hot FehUng's solution with boiling after each addition. The presence of reducing sugars will be indicated by a yellow- brown precipitate. Benedict's Method for Reducing Sugars. This is a modification of Fehling's test for reducing sugars. Instead of using the strong NaOH, Benedict has substituted lSra2C03. Sodium citrate is used to keep the Cu(0H)2 in solution. The method for conducting the test is much like that for FehHng's test. The solution should be boiled before using and allowed to cool. If reducing sugar is present the solution will turn first a green and later a yellow color. Benedict's test is more delicate than the original FehUng's method. The solutions are prepared as follows: Solution I. Copper sulphate 34 . 65 gms. Distilled water 500.00 c.c. Solution II, Anhydrous sodium carbonate... . 100.00 gms. Rochelle salt 173 . 00 gms. Distilled water 500.00 c.c. Nylander's Test for Reducing Sugars. Heat a few cubic centimeters of the solution under examination with I c.c. of Nylander's reagent. A black color is formed if a reducing sugar is formed which is due to a precipitation of the bismuth in the reagent. Phenyl-Hydrazine Reaction. With phenyl hydrazine osazones are formed by action with certain sugars. These are yellow crystalline substances which are characterized by a definite form typical for each different sugar. The test is carried out as follows: Put about 5 c.c. of the sugar solution into a small beaker or test tube and add about 1.5 c.c. of phenyl-hydrazine acetate solution. Heat on a water bath for three- quarters of an hour. Cool and examine the crystals under the micro- scope. Quantitative Determination of Dextrose. (AUihn's Method.) Reagents, (a) Copper sulphate solution : Copper sulphate CuS04-5H20 34,639 gms. Distilled water 500.000 c.c. (&) Alkahn tartrate solution: Rochelle salts 173 gms. Potassium hydroxide 125 gms. Distilled water 500 c.c. DETERMINATION OF DEXTROSE 197 Procedure, Place 30 c.c. of the copper sulphaie solution, 30 ^.c. of the alkaline tartrate solution and 60 c.c. of water in a beaker and heat to boiling. Add 25 c.c. of the solution of the material to be examined, which must be so prepared as not to contain more than 0.250 gm. of dextrose, and boil for two minutes. Filter immediately through asbes- tos without diluting. Wash the precipitate with hot water. Dry, ignite and weigh as cupric oxide. To calculate the amount of copper and its equivalent in dextrose consult AUihn's table in any chemical hand-book. Benedict's Method for Determination of Dextrose. Twenty-five c.c. of Benedict's special reagent (see Appendix) are measured into a porcelain evaporating dish, 25-30 cm. in diameter, 10-12 gms. of crystalline sodium carbonate, and a small amount of pumice, are added. The solution is boiled vigorously over a free flame and the sugar solution is run in until a white precipitate is formed and the blue color is dimin- ished. After this the sugar solution is run in a few drops at a time until the blue color has entirely disappeared. Water may be added to replace that driven off by evaporation. Twenty-five c.c. of the reagent =0.05 gm. glucose or .053 gm. levulose. Fats The fats or lipins are esters of glycerol and certain organic acids which have come to be known as fatty acids. When three molecules of palmitic acid unite with glycerol, the fat plamitin is secured, as follows: H \ H — C — OH HO — C — C15H31 \ H — C — OH HO — C — C15H31 \ H — C — OH HO — C — C15H31 H * I ^ H — C — — C — C15H31 H— C— 0— C— C15H31 +3H2O H Glycerol Palmitic acid H Palmitin Water When fats are broken up either by bacterial enzyme or other action, they are resolved into glycerol and fatty acids. This process is known as saponification. Action of Bacteria on Fats. There is much evidence that fats are split t9 fatty acids and glycerol by bacteria, although many text books 198 PROTEINB \ND CARBOHYDRATES state that lipase is not widely distributed in the bactciial world or, at least, this subject is little or almost not at all treated. Rancidity in butter has been attributed by some to the formation of butyric acid (Duclaux, 1887) . Others maintain that the fat in butter is not attacked, Reinmann (1900) tried the effect of pure cultures on sterile butter and found that most bacteria did not attack fat. In cheese Laxa (1902) demonstrated that Ps. fluorescens decomposed fat quite extensively. Two other peptonizing bacteria and a yeast were also found which de- composed fat. Rahn (1905) stated that Penicillium glaucum and a few bacteria possessed strong fat-splitting properties. Kendall and Simonds (1914) found that sterile filtrates of plain and dextrose broth cultures of B. typhi liberated acid from ethyl butyrate. This was also demon- strated in connection with acid-fast bacteria. Determination of the Iodine Absorption Number (Hantis Method.) The unsaturated fatty acids will react with the halogens. The amount of iodine differs according to the fat, and consequently this iodine absorption number is of some value for identification of fats. It (Iodine number) may be defined as the number of grams of iodine absorbed by 100 gms. of fat. Reagents. Eanus Iodine Solution, Dissolve 13.2 gms. of iodine in 1000 c.c. of glacial acetic acid (99.5 per cent) showing no reduction with bichromate and sulphuric acid. Add 3 c.c. of bromine to the cold solution. Starch Paste. Boil a gram of starch in 200 c.c. of distilled water for ten minutes and cool. Sodium Thiosulphate, Standardize a N/10 solution. Procedure. Weigh about 0.5 gm. of fat into a 250-c.c. glass-stoppered bottle and dissolve by adding 10 c.c. of chloroform. After solution add 30 c.c. of the iodine monobromide solution. Place the bottle in the dark and allow to stand for thirty minutes. The time factor is impor- tant. The excess of iodine should be at least as much as is absorbed. One or two blanks should be made under identical conditions. If the first addition of iodine is used up, another addition should be made. Add 10 c.c. of potassium iodide solution and shake after which add 100 c.c. of distilled water washing any iodine on the stopper of the bottle and sides back into the bottle. Titrate the iodine with the N/10 sodium thiosulphate to a yellow color. Then add a few drops of the starch solution and continue the addition of sodium thiosulphate until the blue color has disappeared. Toward the end of the reaction stopper the bottle and shake violently so that any iodine remaining in the chloroform may be taken up by the potassium iodide solution. The DETERMINATIONS ON FATS 199 number of cubic centimeters of iodine used in the blank minus the num- ber used for the sample gives the thiosulphate value of the sample. Saponification Number or Koettstorfer Number. Since the amount of alkali which will react with a fat is dependent upon the glycerides in it, the saponification number is valuable for identifying fats and oils. The saponification is the number of milligrams of sodium hydroxide neces- sary to saponify 1 gm. fat. Reagents. Sodium Hydroxide, Use a N/10 solution each cubic centimeter of which contains 0.0040 gm. NaOH and neutralizes 0.0088 gm. of butyric acid. Alcoholic Potash Solution. Dissolve 40 gms. of pure potassium hydroxide in 1000 c.c. 95 per cent alcohol. Acid Solutio7i. N/2 HCl. Indicator, Phenolphthalein. Procedure. Conduct the saponification in wide-mouth Erleximcyer flasks of 250 c.c. capacity. Place about 5 gms. of the fat in a tarred flask and weigh. Add exactly 2 cubic centimeters of the alcoholic potash solution, connect with a reflux condenser and boil for thirty minutes or until the fat is completely saponified. Cool and titrate with the N/2 hydrochloric acid. The Koettstorfer number is determined as follows: Subtract the number of cubic centimeters of hydrochloric acid used to neutrahze the excess of alkali after saponification from the number of cubic centimeters necessary to neutralize the 50 c.c. of alkali added; multiply the result by 28.06 and divide by the number of grams of fat used. Conduct two blanks using the same pipettes and con- ditions. (Ofiicial and Provisional Methods, 1912. A. 0. A. C.) Determination of Volatile Fatty Acids (Reichert-Meissl Method). This determination must be carried out under standard conditions. Under these, the Reichert-Meissl number is the number of cubic cen- timeters of N/10 sodium hydroxide required by the soluble fatty acids distilled from 5 gms. of fat. Procedure. Into two clean Erlcnmeyer flasks weigh 5 gms. of the fat. Add 10 c.c. of 95 per cent alcohol and 2 c.c. of sodium hydrox- ide (100 gms. in 100 c.c. H2O). Attach to a reflux condenser and heat on the steam bath until saponification is complete. After the sapon- ification, in case alcohol was used, remove this by dipping the flasks in a steam bath up to their necks. When the alcohol is nearly gone fi-othing may occur. Dissolve the soap by adding 135 c.c. of boiled water and warming on the water bath until a clear solution results. After the solution has cooled to about 60° C, free the fatty acids by adding 8 c.c. of dilute sulphuric acid (200 c.c. in 1000 c.c. of water). Connect to a 200 PROTEINS AND CARBOHYDRATES condenser and heat slowly on a water bath until the fatty acids form a transparent layer on the surface. Cool to room temperature and add a few pieces of pumice stone and distill at a rate that will allow about 110 c.c. to be driven over in thirty minutes. The pumice should be prepared by heating small pieces to a white heat and dropping them into distilled water. They should be kept under distilled water to keep them free from air. Mix the distillate, filter and titrate 100 c.c. with standard N/10 sodium hydroxide solution using phenolphthalein. To obtain the Reichert-Meissl number, increase the cubic centimeters of N/10 sodium hydroxide used by 100 c.c. of filtrate, by one-tenth, divide by the weight of fat taken, and multiply by 5» BIBLIOGRAPHx BAiNBEmGE, F. A. 1911. The Action of Certain Bacteria on Proteins. Jour- nal of Hygiene, 11, 341. DucLAUX, E. 1887. Le Lait Etudes Chimique et Biologiques, Paris. Heinemann, p. G. Kinds of Lactic Acid Produced by Lactic Acid Bacteria. Jour. Biological Chemistry, 2, 612. Kendall, A. I. and Simonds, J. P. 1914. The Esterase Activity of Plain and Dextro&e Broth Cultures of the Typhoid Bacillus. Jour. Inf. Diseases, 15, 354r-356. Laxa, 0. Ueber die Spaltung des Butterfetts durch Mikroorganismen. Arch. Hyg., 41, 119. Meyer, K. 1914. Bacterial Decomposition of (^-Glucosamine. Bichem. Zeit., 58, 415-416. Meyer, K. 1914. The Behavior of Some Bacteria Towards (^-Glucosamine. Biochem. Zeit., 57, 297-299. MiTO, L L. 1918. The Asymetric Decomposition of Racemic Polypeptids by Dead Bacteria. Chem. Abstracts, 12, 816. Raistrick, H. 1917. A New Type of Chemical Change Produced by Bac- teria. The conversion of histidine into urocanic acid by the bacteria of the coh-typhosus group. Biochem. Jour., 11, 71-77. Reinmann, R. 1900. Investigations on the Causes of Rancidity in Butter. Cent. Bakt. Abt., II, 6, 31. Rettgbr, L. F., Berman, N. and Sturges, W. T. 1916. The Utihzation of Protein and Non-protein Nitrogen. Jour. Bact., 1, 15. Robinson, R. H. and Tartar, H. V. 1917. The Decomposition of Protein Substances through the Action of Bacteria. Jour. Biol. Chem., 30, 135-144. Sasaki, T. 1912. Cleavage, of Polypeptids by Bacteria I. Bacterium Coli Commune. Biochem. Zeit., 41, 174-179. Sasaki, T. 1913. The Decomposition of Certain Polypeptids by Bacteria. III. Investigations with Liquefying Bacteria. Biochem. Zeit., 47, 472-481. BIBLIOGRAPHY ON FATS 201 Sasaki, T. 1913. The Decomposition of Certain Polypepticls by Bacteria. 11. Investigations with Non-liquefying Bacteria. Biochem. Zeit., 47, 462-471. Sasaki, T. 1916. Bacterial Decomposition of Polypeptids. Physiol. Ab- stracts, 1917, 2, 15. Sasaki, T. 1917. The Influence of Conditions of Badterial Cleavage of Pro- teins on the Cleavage Products. Jour. Biol. Chem., 32, 527-532. Sasaki, T., -and Otsuka, I. 1917. The Stereochemistry of the Bacterial Decomposition of Albumin. Jour. Biol. Chem., 32, 533-8. Sperry, J. A., and Rettger, L. F. 1915. The Behavior of Certain Bacteria toward Animal an<5 Vegetable Proteins. Jour. Biol. Chem,, 20, 445. Taylor, J. W., and Hall, I. W. 1912. Action of Saliva, Tissue Fluids, Bacteria and Bacterial Extracts upon Polypeptids. Jour, Path, and Bact., 17, 121-123. Thomas, S. J. 1916. A Study of Stereoisomerism of Fermentation Lactic Acid. Jour. Ind. Eng. Chem., 8, 821-823. TsuDji, M. 1918. The Asymetric Decomposition of Racemic Tyrosine by Bacillus Proteus Vulgaris and B. Subtihs (also a Biological Preparation of c?-tyrosine). Chem. Abstracts, 12, 816. CHAPTER VII YEASTS AND MOLDS The molds may be responsible for many chatges in foods. Their action, however, should be considered from two standpoints since they bring about desirable and undesirable changes. A type of the former m the manufacture of Camembert and Roquefort cheese while the spoilage of strawberries and citrus fruits may be offered as an example of the latter. Tas Y,u, the Chinese drink, is also said to be fermented m part by molds. Fungi. These are plants which possess no chlorophyl and conse- quently are unable to utihze the energy in sunlight. All of it must come from analytic processes. They do not construct their own starch but use that which has been built up by other organisms. For convenience, fungi have been divided into the following main groups. To the food microbiologist, these may not be so important but may be helpful in the identification of unknown fungi. Ascomycetes. This class produces ascospores or spores in an ascus. The yeasts since they possess this characteristic are put into this group. The aspergillace^ are ascomycetes. Some of these form perithecia which contain asci. Basidiomycetes. The spore-bearing bodies are termed basidia and possess a typical morphology. The conidia are produced from this on the sterigmata which may be either branched or unbranched. Phycom.ycetes. In this group are placed the algae. None of the fungi belonging to this group are commonly met with in food micro- biology. Fungi Imperfecti. Into this group are placed many fungi which do not come under any of the above groups. The fungi imperfecti are characteri^ied by no definite fruiting bodies. Otdium lactis is the best example and in this fungus each mycelial thread breaks up to form oidia which may develop into a new plant. Structure of Molds. The body of the mold is made up of a cotton- like structure called the mycelium. Each individual thread is called a mycelial thread or hypha. The hyphse are of two kinds— fertile and 202 FRUCTIFICATION IN MOLDS 203 vegetative. Fertile IiyphiB are those wliieli bear the fruiting body and produce the spores. The vegetative hyph^e secure the nutriment and get rid of the excess moisture. The structure of the hyphse differs among the molds. Some have cross walls or septa. In other molds these have not been observed. The presence or absence of these septa is an important factor in the n ..jff^fS. ^pj-w Fig. 47 — Rhizopus nigricans Ehrenberg (After Brefeld ) (a) is the extremity of a stolon, which has developed into the appressonum {h) This latter is the starting point of the sporangiophores (t) four of which are shown with the sporangia (&) unbroken, whilst the columella («) is all that remains of the fifth Magn 30 identification of molds. The mucoraceae do not possess these cross walls ordinarily. Fructib&cation. This takes place on the fertile hyphse which bear the fruiting bodies. Enormous numbers of spores are formed each of which may develop into a typical jnold plant. The spores ^re of two kinds, sexual and asexual. The sexual spores are formed by the union of two cells. The result- ing cell then possesses the characteristics which are algebraic sixms of 204 YEASTS AND MOLDS the characteristics inherent in the parent cells. When a-sexual spore is formed on a non-septate hyph^, it is termed a zygospore; if on a sep- tate hyphse it is an ascospore. Zygospores are formed by the union of two terminal cells on adjacent hyphse. The ascospore is formed by the M i Fig. 48. — Mucor Mucedo. Formation of the Zygospores. (After Brefeld.) 1, two hyphse in, terminal contact; 2, articulation into gamete a and suspensor 6, 3, fusion of the gametes a; the membrane thickens; 4, ripe zygospore b supported by the suspensors aa; 5» germination of the zygospore to a sporangmm stem. Mag. about 60. union of adjacent cells. In this mass are formed the asci which hold the spores. Most of the spores formed by molds are asexual being formed mostly at the ends of the fertile hyphae from typical fruiting bodies. Classification. The molds are thread fungi and it is often difficult to draw the line between them and some other classes of fungi. Oidium RHIZOPUS— MUCOR-OIDIUM 205 lactis (oospora lactis) is often classified with yeasts and has then been given the name Mycoderma lactis. The molds are set apart from bac- teria and yeasts in that they are multicellular. The following varieties may be met with in a microbiological examination of foods. Rhizopus. The most common member of this group is Rhizopus nigricans. It is the common black bread mold and may be responsible for other food spoilage. Stevens (1917) has shown that this fungus causes the rot of strawberries during shipment. This work verifies Pig. 49. — Oidimn Lactis. (After Thorn.*) o, 5, dichotomous branching of growing liyphae; c, d, g, simple chains of oidia breaking through substratum at dotted hne s~y, dotted portions submerged; e, /, chains of oidia from a branching outgrowth of a submerged cell; A., branching chain of oidia, k, ly m, n, o, p, s, types of gernoination of oidia under varying conditions; t, diagram of a portion of a colony showing habit of Oidium lochs as seen m culture media. some done by other investigators on the same subject. Schneider- Orelli (1911) have shown that R, nigricans is important in bringing about the decay of over-ripe pears during storage. Mucor. The mucors have the same structure as Rhizopus molds. They are separated, however, by the fact that the sporangiophores spring from the stolons singly. Oidium. The sour milk mold Oidium lactis is a common variety of this genus. The most of this mold is below the surface of the medium. 206 YEASTS AND MOLDS That part which is not submerged resembles a layer of velvet on the surface of the medium. Penicillium. Penicillium molds are important in the manufacture of certain cheeses. Thorn (1906, 1905) has given much study to this question. Dox (1910) has reported an extended study of the intracellu- lar enzymes in Penicillium camemberti. The following enzymes were demonstrated to be present: erepsin, nuclease, amidase, lipase, emulsin. WW/ \«>\! .■'.';''•' \i.> !.•.'/'•.'.•/ 1/ "& K'i.''' -^l JJi^'i^' Fig. 50.— Roquefort Penicillium (P. roqueforti), (After Thorn, 1906.) a, part of conidiophore and of bas of fructification, highly magnified showing the production of basidia on the sides as well as at the apex of the basidiophore; fe, c, other types of branching; d, young conidiophore just branching; e, /, basidia and the formation of conidia, highly magnified; g^'h, i, diagrams of ty^es of fructification as seen under low power ( X80) ; k, Z, m, n, germination of coonidia and new conidia produced directly on the first hyphse. amylase, inulase, rafhnase, sucrase, maltase, and lactase. The presence of these enzymes seems to indicate an ability to utilize various food products. Penicillia are widely distributed in nature and many of the molds which contaminate foods belong to this genus. Thorn (1910) has reported cultural studies on the species of penicillium. He has worked out a key which is of much assistance in studying molds and their relation to food spoilage. KEY TO PENCILLIA 207 KEY TO PENICILLIUM MOLDS (After Thorn, 1910) 4 . Species fruiting typically by coremia (vertical and definite). a. Coremia long (3-15 mm.)- 1. Conidial masses strictly terminal, olive green, fragrant P. claviforme 2. Upper third of coremia fertile, conidia green.. P. duclauxii aa. Coremia small. 1. Coremia definite, densely crowded, colony orange below P. granulatum 2. Coremiform characters indicated incultures by clustering of conidiophores, definite coremia only in old cultures becoming large and def- inite on apples P. expansum AA. Species not (or rarely) producing coremia in culture. B. Species constantly producing sclerotia or ascigerous masses. h. Producing asciginous masses, yellow or red- dish P. luteum bb. Sclerotia appearing as white masses in old cultures *. P. italicurai bbb. Sclerotia reddish or pink, globose or ellip- tical, 500 microns or less in diameter. c. Conidial fru Fig. 52. — ^Types of Cells in Common Yeasts. A, Saccharomyces cerevzsice, B, Saccharomyces elhpsoideus; C, Mycoderma iim Gmlliermond's Les Levures ) (Adapted from True and Pseudo Yeasts. True yeasts are budding fungi which produce endospores. The false yeasts resemble the true yeasts very closely but differ in the fact that no endospores are formed. These have some of the properties of the molds. The torulse (Turpin), mycodermse (Persoona) and cryptococcus (Kutzing-Vuillemin) and representatives of the false yeasts. Classification of Yeasts. The classification of the yeasts is rather confused. This is in part due to the different points from which the subject may be viewed. The fermentologist is concerned with the physiological changes which are brought about by yeasts and does not give much attention to nomenclature. His cultures may be designated 214 YEASTS AND MOLDS by such " pseucloscientific " names as ^' beer yeast/' ^' wine yeast/' etc., or they may be even numbered. The taxonomist, however, must be concerned with keeping this group of fungi classified and so far as pos- sible properly named. Many investigators have classified the budding fungi. Anderson (1917) has reviewed the various investigations and a student will find a good summary with discussion of the various classi- fications in his paper. Hansen has done most work on the classification of the yeasts. His classification which appeared in 1904 is now accepted as the basis for classification of the true yeasts. He made two families which are divided into nine genera. Not all of these have been accepted by microbiologists. Guillierimond (1912) does not recognize the schizo- saccharomycetes which Hansen separated from the saccharomycetes by the fact that they reproduced by fission. He divides the saccharo- mycetes into five groups. Hansen's classification is as follows: KEY TO GENERA OF TRUE YEASTS (Hansen after Buchanan) Family I. Vegetative reproduction by budding Saccharomycetacese A. Cells do not form a surface membrane at once on sugar media, i.e., do not grow exclusively at the top of the medium. 1. Spores having a single membrane. a. Cells fusing in pairs before spore formation Zygosaccharomycete 5. Cells not fusing in pairs before spore formation. (1) Spores germinate by or- dinary budding Saccharomyses (2) Spores germinate by means of promycel- ium Saccharomycodes 2. Spores having two membranes .... Saccharomycopsis B. Cells forming a surface membrane at once by sugar media. 1. Spores spherical, hemispherical or irregular Pichia 2. Spores lemon shaped with pointed ends Willia Family n* Vegetative reproduction by fission Schizosaccharomycetes KEY TO BUDDING FUNGI 215 FALSE YEASTS (After Guilliermoncl) L Cells generally spherical, often forming a pellicle but only after fermentation; pellicle always viscous without inter- vention of air Torula 2. Cells generally elongated. Pellicle ap- pears at the beginning of develop- ment with intervention of air Mycoderma 3i Yeasts without ascospores, parasitic to animals Cryptococcus Anderson (1917), after a careful study of the field, has proposed the following key, in which the budding phase is predominant. He has retained many of the names proposed by Hansen: KEY TO GENERA OF BUDDING FUNGI (Anderson, 1917) I. Ascospores known. Vegetative cells single or attached in irregular col- onies, mycelium not developed, ascospores formed within isolated cells (Saccharomycetacese)* Spores globose or void: Spores on germination forming typical yeast cells. Ascus formation preceded by the conjugation of gametes 1. Zygosaccharomyces Ascus formation not preceded by the conju- gation of gametes: Spore membrane single 2. Saccharomyces Spore membrane double 3. Sacchromycopsis Spores on germination forming a poorly de- veloped promycelium 4. Saccharomycodes Spores pileioform or limoniform, costate 5. WiUia Spores hemispheric, angular or irregular in form on germination forming an ex- tended promycelium 6. Pichia Vegetative cells produced predominately by budding but forming a mycelium under some conditions asci terminal or inter- calary, dif erentiatedfrom the myceHum.7. Endomyces * The genus Schizosaccharomyces, which does not bud, and the relatively unim- portant genera, Monospora and Nematospora, are not included in this key. 216 YEASTS AND MOLDS II. Ascospores not known, i.e., Fungi Imperfecti. Heavy dry pellicle formed on liquid media 8. Mycoderma No distinct pellicle formed: Vegetative cells forming a septate mycelium under exceptional conditions but predom- inately budding 9. Parasaccharomyces Vegetative cells formed only by budding. Cells apiculate, limoniform 10. Pseudosaccliaromyces Cells frequently elongated into narrow non- septate hyphal threads 11. Pseudomonilia Cells typically yeast-like 12. Cryptococcus Compressed Yeast. The greater part of the procedure in the manufacture of compressed yeast is concerned with the preparation of the medium or ^* mash." This is commonly prepared from a mixture of grains, rye, corn and barley being commonly used. After thorough cleaning, the barley is malted in the usual manner. The malt is later put into a kiln to stop germination. The mash is then prepared by mixing the ground grains with the malt and maintaining at a constant temperature of 60° until all of the starch has been changed to soluble sugars. The mash is then soured for two reasons. It renders the nitro- genous matter available to the yeasts and also prevents the changes induced by the putrefactive bacteria. The souring process is continued until about 1.5 per cent of acid has been produced. The mash is then passed through filter presses which gives a clear filtrate called the wort. This supplies the foods for the yeasts. It is inoculated either by a pure culture or a portion of the previous day's run. An optimum tempera- tm'e is maintained during which there is a vigorous multiplication of the yeast cells. The cells are carefully filtered out and pressed into cakes with or without starch. The steps in the preparation of com- pressed yeast are graphically shown in Fig. 53. Yeast Foods. The use of these substances in bread making has been investigated by Kohman (1916) and his co-workers. The inves- tigation apparently had its origin in the effect of different natural waters on the yeast fermentation in bread as noticed by a large baking company. Kohman showed that by the use of small quantities of ammonium and calcium salts and potassium bromide brought about a saving of from 50 to 65 per cent in the usual amount of yeast. Other distinct advantages connected with the use of these nutriments are also noted. Hoffmnn (1^17) in discussing the same subject has stated: ''Since there is so much evidence in favor of all the ammonium chloride being changed to protein by the yeast, we can calculate how much protein we DIAGRAM OF YEAST MANUFACTURE Barley Malt ^t Cleaner V Mm Rye ^r Cleaner 'f Mill Corn Cleaner Cooker Mash Tub /:: Sourmg" Tank Filter Tub Cooler € (c A 1 Fermenter where yeast grows Wntcr Filter 5) 3) Yeast Seperators 3) Seperated ^ Yeast 5) Coolei: Sprouts Cleaner Malt Rye Watu y- Small Lactic Acid Mash Shipping Boxes Fig. 53.— Diagram of Yeast Manufacture, (Fleisclimaan Co.) 218 YEASTS AND MOLDS gain in the form of yeast protein, from adthng 0,5 lb. of ammonium chloride in a dough batch containing 1000 Ibb. of flour and how much protem we lose by deducting one-half the quantity of yeafet (5 lbs. would be an average amount) regularly used; 0.5 *lb, ammonium chloride contains 0.131 lb. nitrogen; this times 6.25 gives 0.82 lb. protein gained by converting the salt into albuminous matter. Compressed bakers' yeast contains 27 per cent dry matter; 5 lbs. of yeast then contains 1.35 lbs. of dry matter, 50 per cent, or 0.68 lb. of which is albuminous in character. We have not only as much protein produced by the yeast from the ammonium salt but actually more than that lost by deducting half the yeast in the dough batch. The economic value of a process which can utilize such simple substances as sugar and ammonium salts and build there- from complex but useful food proteins cannot be overestimated." Table XXII is taken from his v^ork and shows clearly the effect of ammonium chloride on gas formation in bread. Table XXII SHOWING THE INCREASE IN GAS PRODUCTION IN DOUGH USING AMMONIUM CHLORIDE (Hoffman) Fermentation Hours. Control Dough, c c Difference. Dough Contain- ing 2 Gram NHCl per 100 Grams Flour Difference. 110 110 c.c. 5 180 80 180 80 1.0 285 105 280 100 1.5 390 105 390 110 2 470 80 500 110 2.5 570 100 640 140 3.0 665 95 785 145 3.5 790 125 955 170 4.0 880 90 1105 150 4.5 980 100 1260 155 5.0 1080 100 1400 140 5.5 1160 80 1520 120 6.0 1230 70 1625 105 Total gas in six hours 1120 c.c. • «*••«*•*«••« 1515 3 Per cent increase due to ammoniui n chloride. 35 3 Methods for Study of Yeasts. The study of yeasts demands a pure culture. The securing of this pure culture is not often an easy per- formance. Two methods have been used and are available to micro- biologists at the present time. IDENTIFICATION OF l^EASTS 219 Physiological Method. In this method an environment is created which is favorable to the desired fungus and unfavorable to the others. In applying this method to the yeasts, an acid medium may be used. Bacteria, unless they are acidophiles, are unable to grow in a 2 per cent tartaric acid solution. Pasteur and Hansen grew yeasts in such a medium to free them from bacteria. This method was accompanied with some uncertainty and was replaced by the dilution methods. Dilution Methods. These have been described before. They con- sist of diluting or breaking up the yeast^s masses in a definite amount of sterile water or physiological salt solution. From these diluted mix- tures definite portions are taken for plating on a soUd medium. After incubation and growth, cultures should be picked from the colonies and transferred to dextrose agar slants. The yeast is then ready for a cul- tural and morphological examination. Moist Chamber Preparations. These are essentially the hanging drop mounts which have been described before. A small portion of the pure culture of yeast should be gently emulsified in a drop of dex- trose broth or wort. This should be transferred to a clean cover glass and suspended over a depression in a concave slide or ring slide. The edges of the cover glass should be carefully lined with vaseline in order to make a tight seal and allow no evaporation during incubation. Such a mount will permit observations on a few cells to allow determine bud- ding and cell shape. Scheme for Identifjdng a Yeast. Whether the fungus is really a yeast or borderline mold should be determined first of all. The following points may be of assistance: I. Spore or non-spore former 4. Spore former 1. Shape, morphology 2. Scum or membrane 3. Top or bottom yeast 4. Per cent of alcohol formed B, Non-spore former 1. Non-scum former a. Color 6. Shape, morphology 2. Scum former a. Color 6. Morphology 220 YEASTS AND MOLDS Microscopic Examination of Compressed Yeasts. Prepare a thm mixture of the yeast in distilled water. To this mixture or a small por- tion of it add a httle of a 0.1 per cent solution of methylene blue. Mount a drop of this under a cover glass on a slide and enumerate the dead and living bacteria. The dead cells will stain but the living cells will not. Test for Adulteration with Beer Yeast. Practically all of the beer yeasts ferment rafiinose while the bread yeasts generally do not. One gm. of the yeast is mixed with about 10 c.c. of a 1 per cent solution of raflS-nose broth and placed into a fermentation tube. Incubate this tube for about twenty-four hours at 30° C. A blank tube without rafiinose should be prepared in order to test for gas produced from stored glycogen. If the difference between the two tubes is decided (about 50 per cent) adulteration with beer yeast is probable. A greater difference renders the test more conclusive. Efficiency of Bread Yeasts. This may be determined in different ways. A representative sample of the yeast should be obtained and divided into three parts. Store one part at 20°, 30°, and 40° C. At regular intervals test each part for dead ceUs, gas formation, and dough-rising power. Test once or twice for adulteration with beer yeast and with myco- derma. Dough-rising Power of Bread Yeast. Mix 100 gms. of flour, 2 gms. of yeast and 60 c.c. of distilled water by first making a thin batter of the yeast, water and a little of the flour. Then add the rest of the flour and knead for five minutes. About 3 gms. of flour should be kept with which to clean the glass rod. The dough should be formed into a cylinder and dropped into a warmed graduate cyl- inder which has been greased or powdered with flour. Press the dough down into the cylinder and place at 30° C. The volume is read every thirty minutes until the maximum is reached. Compute the maximum volume in terms of the original. Plot a curve which will show the activity of the yeast. Determination of the Fermenting Power of Yeasts, Meissl Method. Prepare a saccharose phosphate mixture as follows: 400 gms. of sac- charose, 25 gms. ammonium phosphate, 25 gms. potassium phosphate. Mix this thoroughly and weigh 4.5 gms. into an Erlenmeyer flask which Fig. 54. — Culture Flask for Fermentation Tests. Fitted with Alwood (19 8) Ventilation Tube. EXAMINATION OF YEAfcJTb iii^iM X carries a fermentation valve. Add 50 c.c. of distilled water and 1 gm. of the yeast to be tested and incu- bate at 30° C. Different types of valves are available. Alwood (1908) used a convenient type for studjdng the fermenting capacity of pure strains of yeasts. The valves are so con- structed that the activities of the yeast may be observed and the gases from the fermentation may escape. The moisture is retained by some absorbent such as calcium chloride or sulphuric acid. The apparatus should be weighed before and after tte run. The Alwood valve is shown in Figs. 54 and 55. Determination of the Ferment- ing Power of Yeasts, Kusserow*s Method. Put 400 c.c. of 10 per cent U —2.9 cm.- saccharose solution and 10 gms. of pio. 55.-~Detail of Ventilation Tubt the yeast under examination into the for Culture Flask. (Alwood, 1908.) fermentation flask of the Kusserow apparatus. Warm the flask and solution to 30° C. before starting. After the apparatus has stood for an hour at 30° C. it is connected and the amount of gas formed in the next half hour is measured; it shoxild be 250 c.c. The amount of gas for the first and second half hours is usually about 50 and 150 c.c, respectively. Five gms. of yeast in 400 c.c. of 10 per cent saccharose solution in a flask fitted with a fermentation valve will cause a loss of about 6.5 gms. of carbon dioxide in twenty- four hours if distilled water is used; with tap water the loss should approximate 8 to 12 gms. Determination of Mycoderma in Bread Yeast. Press the yeast with a sterile spatula into the bot- whichinaybeUsed ^^^ ^f ^ g^^^^ji p^^^.j ^jj^j^ ^^j incubate at 37° C. with Erlenmeyer _ , , ,^ ,, ^ . . , Flasks for Testing ^^^' ^^^ ^^y^' ^^ ^^^^ jeBBi contains a mycoderma, the Fermenting pure white characteristic colonies will appear on Power of Fungi. the yellowish background of the bread yeast. V^ Fig. 56. — Types of Ventilation Valves 222 YEASTS AND MOLDS Bread Making The flour is one of the first things to which the baker pays special attention. Miner (1917), in describing the manufacture of flour, divides It into three general types: I. Low grade II. Clear flour III. Patent flour The baker demands a uniform flour and to secure it he may resort to blending. Most of them use a baker's '^ patent " flour which may be superior to the domestic '^ patent." This is carefully sifted and the other ingredients added such as salt, sugar, lard, malt extract, milk, water, yeasts, etc. In the straight dough method all of these are mixed and allowed to ferment for about five hours in a long trough. The temperature of this room is maintained at 27° C. with a relative humidity of 75 per cent. From the fermenting room the dough is taken to a divider where it is cut into any desired size. It may be worked a little, allowed to rise and molded for baking. In the larger bakeries this may be entirely a machine process. Yeast in Bread Making. Bread is one of the oldest foods in the preparation of which fermentative changes are involved. In the early days before commercial yeasts were available, the housewife had to reply on spontaneous fermentations. The organisms which induced these chance fermentations got into the sponge either from the air or along with one of the ingredients. Quite often, a portion of one batch was taken out and saved over to inoculate the next batch of bread. To-day the baker does not have to rely on liquid brewers' yeast nor the housewife on the uncertain fermentations of wild yeasts. The pro- duction of compressed and dried yeasts caused a distinct advance to be made in the production of leavened foods. A mixture of yeast and flour will ferment, but the addition of other substances such as potato water, malt water, will greatly accelerate the action of the yeasts. All of the constituents of the flour which are given above are concerned in bread making. The carbohydrates are precursors of the carbon dioxide and in the baked loaf \hey impart a rich brown color to the crust. Accord- ing to Wardall (1910) the flavor of the bread is not dependent on the strain of yeast which is used as a leaven. She used thirty-three pure strains of yeasts and could observe no difference in the flavor of bread leavened by them. She regarded the time of incubation as too short for the development of any special flavor. SELF-RISING BREAD 223 Self-rising Bread. This has been known for generations under tlie name of " salt-rising " bread. As suggested by the name, the fermenta- tion is a spontaneous one. Hunt and Wessling (1917) give the following instructions for making: 1 cup milk 1 tablespoon sugar 2 tablespoons cornmeal, white Butter (if used) 1 tablespoon 1 teaspoon salt Flour. ^^ Scald the milk. Allow it to oool until it is lukewarm; then add salt, sugar and cornmeal. Place in a fruit can or heavy crock or pitcher and surround by water at about 120° F. Allow the mixture to stand six or seven hours or until it shows signs of fermentation. If it has been fermented sufficiently, the gas can be heard as it escapes.'' As shown in the above paragraph, the fermentation is a chance one. It is, then, almost an uncontrolled process. The agent inducing this fermentation has received much study. Kohman (1912) has given a review of the literature and some of the data and references mentioned here have been taken from his interesting paper. There seems to be two sides to the question of the organism which is concerned. One side argues that the fermentation is induced by wild yeasts which get in from the air. In support of this are the investigations of Mitchell (1908), Bailey (1914), and others. That it is a bacterial change is sup- ported by the work of Heinemann and Hefferan (1909) and Kohman (1912). The middle ground that both yeasts and bacteria are con- cerned is taken by Lehman (1894), Peters (1889) and perhaps Harrison (1902). Lehman isolated an organism which he called Bacillus levans, Heinemann and Hefferan isolated a bacillus from corn meal which had many of the characteristics of Bacillus bulgaricus. They proved that the organism came from the corn meal and believed that, in self -rising bread, the high amount of acid formed by this bacillus (1.65 per cent) from the milk, united with the inorganic salts to form carbon dioxide. The extensive work of Kohman (1912) indicated that bacteria were the important agents in the fermentation. He believes that members of the colon group are concerned. The gases produced by the salt-rising bacteria consisted of two-thirds hydrogen and about one-third carbon dioxide. These bacteria aerate the bread by decomposing some of the constituents possibly the carbohydrates. The organisms are probably of the type B, levans (Lehman). Woodward (1911) believed that the fermentation in salt-rising bread was due to the presence of organisms which are introduced with the corn meal. The organism which was isolated was not a yeast but belonged to another group. It was advised 224 YEASTS AND MOLDS that the milk be sterilized befoie the batter was mixed .since this allowed more uniform results and decreased the posfoil^ilities of the bad flavors which are so often inherent in this type of bread. Examination of Bread. The bacterial examination of bread is important for two reasons: first, the possibility of bread spreading dis- ease bacteria; and secondly, the relation of bacteria to undesirable fermentations and changes which may take place. Bread is generally regarded as an absolutely safe article in the diet since it is heated before eaten. Roussel (1908) stated that the mterior of the loaf reached 101-103° C; the crust reached 125-140.5° C. Such temperatures were regarded as sufficient to kill the vegetative cells but not the spores except they be present in the crust. Tubercle bacilli in the dough were reported to have retained their vitaUty. From these data Roussel states that mechanical mixers should be used in order to prevent the spread of disease. Auche (1909, 1910) introduced turbercle baciUi into dough before it was made into loaves. The loaves weighed up to 2 kg. The bacilli were killed in the baking process but the author regards it as a possibility that bacteria may pass through the baking process. In the latter paper cited, this same author added such bac- teria as the following to bread dough before baking: B. typJn, B. para- -ypM, B. dysentencB (Shiga), B. dysenterim (Flexner), B. coh, Strept. pyogenes, Staph, aureus and a variety of proteus. Cultures made from the loaves after baking were sterile. Auche states that possibly more resistant bacteria would pass through the* baking process. He states that the greatest care must be exercised to prevent contamination after the loaves have left the oven. Fenyvessy and Denies (1911) found that if the temperature of bread reached 94-104° C. during the baking process, the pathogenic bacteria, if accidentally present, would be destroyed. Gaujoux (1911), discusses some studies of this question. He found the Koch's bacillus (Bacillus tuberculosis) did not survive the temperature of baking bread but assumed that it might pass through the temperatures employed for pastries. Howell (1912) examined bread collected from various districts in Chicago. In general it was found that bread made under clean conditions had a lower bacteria content and fewer strepto- cocci. Jacobs, LeCIerc and Mason (1914) studied the effect of wrapping. Wrapped bread had fewer bacteria on the surface. From the above discussion it would seem that bread may carry infection. The evidence is not altogether convincing, however, that it does. Hansen (1913) has reported a typhoid fever epidemic which may have been caused by bread. In this case the bread may have been contaminated after baking. Slimy or ropy bread has received quite a little attention from the EXAMINATION OF BREAD 225 bacteriologists. Russell (1903) examined a sample of slimy bread and isolated B. mesentericus vulgatus. Temperature studies indicated that the organisms could pass through the baking temperature. Watkins (1906) has studied the question and advised that the flour be subjected to examination when this infection appears. Kornauth (1912) also advised an examination of the flour for the organism. Williams (1912) found a capsulated organism in a case of slimy bread. Kayser^(1911) found that the bacteria causing this infection were introduced with the yeast. He stated that 1 to 2 liters of vinegar added to each 100 kg. of the flour would prevent the infection. Kayser and Delavel (1912) isolated bacteria which were related to the mesentericus group. When a bakery once becomes infected with members of this group, it is disin- fected with great dijS&culty. Kayser suggests the use of acidulated water for washing all of the apparatus and even states that some appa- ratus may have to be discarded. Wright (1916), when studying musty bread, found an Aspergillus and Rhtzopus nigricans. Further experimental work indicated that the R. nigricans produced a mustiness in bread while Aspergillus produced a sourness. It was beheved that the mustiness resulted from proteolytic enzymes from the R. 7dgricans, Watkins' Method for Detecting in the Flour the Organisms which Cause Ropy Bread. This author (Watkins, 1906) finds the following procedure a dehcate one for determining whether a flour contains those bacteria which cause ropy bread. The test is rapid and the author states that there is little possibility of a sound failing to pass the test because they do not yield appearances of ropmess in the time proposed as a limit. Ten large test tubes (6 in. by 1 in ) are thoroughly boiled in water for one hour, washed and drained. When drained sterilize in the oven at 232"* C. for three hours The tubes should be thoroughly sterihzed. Cool and then place into each tube a finger of bread 3 m. by 2 in , cut from the center of the same two-day old loaf. The average weight of each loaf should be 5 gms. Moisten with distilled water and plug the tubes. Sterili^ie by boiling m water on three successive days. To test the flour, 2 gms. are well mixed wuth water and placed m boiling water for thirty minutes. To the series of ten tubes add successively from 1 to 7 c c. of the flour mixture, l^javing three tubes to serve as checks. Number the tubes m rotation. Incubate at 28° C. and at the end of twenty-four hours examine for ropiness. Komauth's Method for Detecting Organisms in Flour which Cause Ropy Bread. This is a modification of Kuhl's method and is carried out as follows: 280 gms. of flour, 140 c.c. of salt water, and 0.6 gm. of yeast are made into a loaf. After baking, the loaf should be stored at 25* C. for forty-eight hours. If the organisms causing ropy bread were present in the flour, the loaf will have a char- acteristic odor. Several loaves should be made at the same time with sound flour as a check. 226 YEASTS AND MOLDS BIBLIOGRAPHY Alwood, W. B. 1908. The Fermenting Power of Some Pure Yeasts and Some ^ Associated Fungi. U. S. Dept. Agriculture, Bureau of Chem., Bull. 111. Anderson, H. Wo 1917. Yeast-like Fungi of the Human Intestinal Tract. Jour. Infect. Dis., 21, 341-386. AucHE, B. 1910. Bacteria in Bread. Semaine Med , 35, Brit. Med. Jour., 1910, No. 2569, 771-772. AucHE, B. 1909. The Destruction of Tubercle Bacilli in Bread by Cooking. Comp. Rend. Soc. Biol, 66, 800-802. Bailey, E. H. S. 1914. Sanitary and Applied Chemistry. The Macmillan Co., New York. Balland, M. 1894. Sur la temperature interiure du pain sortant du four. Comp. Rend. Acad. Sci., 117, 519-521. Cadwell, H. V. 1917. Laboratory Control in Yeast Making. Amer. Food Jour., 12, 151. Fenyvessy, B. von. Denies, L. 1911. Is Baked Bread Sterile? Zeit. Hyg., 69, 223-224. Gaujoux, E. 1911. Concerning Bread and Pastry. Rev. Hyg. et Pol. Sanit., 33, 1176-1180. Gould, R. G. 1917. The Manufacture of Commercial Yeast. American Food Journal, 12, 143-148. Gould, R. Cf. The Manufacture of Bread. Amer. Food Jour,, 12, 195-201. Hansen, P. 1913. A Study of Typhoid Fever in Rockford, Illinois, in the Late Summer and Fall of 1913. Illinois State Water Survey Bull, 11, 384-430. Haerison, F. C. 1902. Yeast and its Household Use. Ontario Ag. Bull, 118, 17 pp. Heinemann, p. G., and Hbfferan, M. 1909. Note on a Lactic Acid-forming Bacillus Closely Resembling B, hulgaricus Isolated from Corn Meal. Science, 29, 1011. Hoffman, C. H. 1917. The UtiHzation of Ammonium Chloride by Yeast. Jour. Ind. Eng. Chem., 9, 148-153. Howell, K. 1912. The Bacterial Examination of Bread. Amer. Jour. Pub. Health, 2, 321-324. Jacobs, B. R., LeClerc, J. A., and Mason, Maud L. 1914. A report on a Chemical and Bacteriological Study of Wrapped Bread. Amer. Jour. Pub. Health, 4, 721-732. Kaysbr, E. 1912. Bacterial Changes in Bread. Chem. Abstracts, 6, 1067. Kayser, E., and Delavel, H. 1912. A Study of Stringy Bread. Comp. Rend., 153, 576-578. Chem. Abstracts, 6, 515. Kohman, H. 1912. Salt Rising Bread and Some Comparisons with Bread Made from Yeasts. Jour. Eng. Ind. Chem., 4, 20-30; 100-106. KoHMAN, H. A., et at 1916. On the Use of Certain Yeast Nutriments in Bread Making. Jour. Ind. Eng. Chem., 8, 781-789. BIBLIOGRAPHY 227 KoHMAN, H. A. 1917. Feeding the Yeast in Bread Making. American Food Journal, 12, 35-38. KoRNAUTH, K. 1912. Ropy Bread. Chem. Abstracts, 7, 847. Lehman, K. B. 1894. Ueber die Sauerteiggarung und Beziehungen des Bacillus levans zum Bacillus coli communiB. Cent. Bakt., 15, 350-354 Marchland, H. 1909. The Cooking of Bread. Meun. Franc, 25, 208-10. Exp. Sta. Rec, 22, 64. Miner, A. 1917. The Manufacture of Flour. Amer. Food Jour., 12, 27-31. Mitchell, M. 1908. Course in Cereal Foods and their Preparation for Mov- able Schools of Agriculture. U. S. Dept. Agric. Office Exp. Stations. Bull., 200. Peters, W. L. 1889. Die Organismen des Sauerteigs und Ihrer Bedeutung fur Brotgahrung. Botan. Zeit., 47, 405, 420 and 435. RoussEL, J. 1907. The Survival of Pathogenic Bacteria in Bread after Baking. Rev. Intend. MU., 20, 122-123. Chem. Absts., 2, 1168. Russell, H. L. 1903. Sticky and Slimy Bread and its Causes. Ann. Rept. Wis. Ag. Exp. Sta., 565. Schnoeder-Orblli, O. 1911. Investigations on the Growth and Spread of Decay Fungi in Storage Fruit. Landw. Jahrb. Schweiz., 25, 225-226. Cent. Bakt. Abt. II., 32, 161-169. Stevens, N. 1917. Rhizopus Rot of Strawberries in Transit. U. S. Dept. Agriculture, Bull., 531. Thom, C. 1905. Some Investigations from the Study of Dairy Fungi. Jour- nal of Mycology, 2, 117-124. Thom, C. 1906. Fungi in Cheese Ripening. U. S. Dept. Agriculture, Bureau of Animal Industry Bull., 82. Thom, C. 1910. Cultural Studies of Species Penicillium. U. S. Dept. Agri- culture, Bureau of Animal Industry Bull., 118. Wardall, Ruth A. 1*910. The Relation of Yeast to Flavor in Bread. Jour. Home Economics, 1910, 75-91. Watkins, E. J. 1906. Ropiness in Flour and Bread and its Detection and Prevention. Jour. Soc. Chem. Ind., 25, 350-357. Williams, Anna W. 1912. A Study of Ropy Bread. Biochem. Bull., 1, 529-534. Woodward, W. 1911. The Leavening Agent in Salt Rising Bread. Jour. Home Economics, 3, 100-101. Wright, A. M. 1916. A Cause of Mustiness in Bread. Jour. Soc. Chem. Ind., 35, 1045-1046. CHAPTER VIII T'lMr'T*'t70'PTTVT AT "DA /^ ^I ^I?T>T A IJNTESTINAL BACTERIA The flora of the mouth is dependent upon many factors and is, therefore, continually changing. Excluding the various infections of the mouth which will not be considered here, the bacteria in the mouth are not especially significant. There are, however, several features which, compared with other body fluids, are worthy of mention. The mouth often contains a large number of bacteria and many kinds. Germicidal Action of Saliva. The work which is available on this subject is neither conclusive nor extensive. Huggenschmidt (1896) studied the various factors which contribute to the protection of the oral cavity and from such factors as bactericidal action of the sputum, mechanical action of the saliva, action of the mucous membrane and bacterial antagonism concluded that the mechanical action of the sputum was the most important factor. SanarelH (1891) filtered saliva through a Chamberland filter and tested the bactericidal action of the filtrate. He found that saliva did possess a germicidal action quite similar to that exhibited by other body fluids, such as blood, aqueous humor, etc. The speed of the reaction depended upon the initial num- ber of bacteria introduced into the saliva. If a small number of cells (three or four hundred) were introduced, all were killed in a short time but when a few thousand cells were introduced, there was usually a decrease followed by an indefinite increase. With Bacterium diphtherise and the pneumococcus no germicidal action was noticed. They were found alive after twenty-eight and forty days. The cause of the ger- micidal action. was not determined. Gordon (1916) carried out his experiments on this subject in culture media and found a germicidal action against the meningococcus, which he attributed to the antagon- istic effect of other bacteria in the saliva. These were supposed to be mixed streptococci. Effects of Tobacco Smoke on Bacteria in the Mouth. This is a subject of more popular interest and has been of much use to those who wish to justify or decry the habit of smoking. Koerner (1896) found a decided reduction of bacteria in the mouth due to tobacco smoke. GERMICIDAL ACTION OF GASTRIC JUICE 229 From the facts secured, this author states that strong smokers (con- suming more than one dozen cigars a day) seldom have dental caries. Dunnon (1902) found no bactericidal action on B. tetanus, B. typhi or Leptothnx bucchalis and staphylococcus. Bacillus tuberculosis was inhibited. The action was not due to the nicotine content but to products of combustion. Arnold (1907) compared the bactericidal activity of hay and tobacco smoke. Rideal (1903) reported a slight bactericidal action of tobacco smoke. EUis (1909) has stated that any beneficial effects resulting from the use of tobacco, is easily overbalanced by the habit of spitting so prevalent among smokers. The Stomach The bacteriological aspects of the stomach have been concerned mostly with its diseases. The bacteria of the stomach gain entrance with the food and drink. The germicidal action of the saliva may probably be neglected as a factor influencing the number of bacteria passing through the mouth. Germicidal Action of Gastric Juice. The gastric juice is the normal secretion of the stomach. It contains between 2 to 3 per cent solids and about 0.2 per cent of free hydrochloric acid, which is the most powerful of any acid combination in the stomach. A condition of hyper- acidity exists when a decidedly greater amount of hydrochloric acid is present than normally, and hyperacidity when the amount of acid is below normal. During conditions of hypoacidity, fermentation may be active with the formation of lactic and butyric acids. Some hold that gastric ulcer and gastric catarrh may be due to a reduced acidity. That the gastric juice is germicidal was noticed by Spallanzani in 1734. When he moistened meat with gastric juice, it did not putrefy. Meat which was not so treated but which was moistened with other substances, such as water, developed a putrid odor very quickly. He opened a snake eighteen days after it had swallowed a lizard and while the lizard was partly digested, it had not decomposed. Other experi- ments by this early scientist demonstrated that putrefaction could be stopped by gastric juice. Koch (1884) fed cholera vibrios to dogs and after a few hours found that they had been destroyed. He stated that cholera vibrios were destroyed in the stomach under normal conditions and that if they passed through, it was due to some abnormal condition. Falk (1883) secured no action on JB. tuberculosis. Kurloff and Wagner (1889) found a selective action of the gastric juice. Cholera vibrio, B, typhi 230 INTESTINAL BACTERIA and Ps. pyocyaneus were destroyed but B, tuberculosis , B. anthrax, and B. tetanus (spores) were not affected. Strauss and Wurtz (1889) showed that anthrax bacilli were killed in fifteen minutes and that the destructive factor in the gastric juice was the hydrochloric acid. The same con- clusion was reached by Kabrhel (1890). Hamburger (1890) first called attention to the fact that some of the acid was bound and that the free acid had a stronger germicidal effect. This was proven by testing the action of a solution of HCl with one of the same strength containing peptone. Kianowsky (1891) showed a direct relation between the number of bacteria in stomachs and the amount of acid. With patients possessing a faulty acid secretion many bacteria were found. Cadeac and Bournay (1893) found that Ps, pyocyaneus was destroyed after six hours and that B. anthrax and B. tuberculosis were not killed. Pet- tenkofer (1893) reported some startling work which was carried out in 1893. This was done soon after the great epidemic of cholera in Ham- burg and Altoona. He proposed the X, Y, Z theory for cholera where -X'= bacterium; F = host or soil; 2'= environment. He stated that X without Y and Z would not cause cholera. So thoroughly did these workers believe in this theory that Pettenkofer and Emmerich drank pure cultures of the cholera vibrio. Each mixed 1 c.c. of a broth culture with 100 c.c. of 1 per cent sodium bicarbonate solution and drank it. Pettenkofer did not change his usual manner of living and, after having a diarrhoea for four days, came back to normal. Emmerich's case ran a longer time. In this case the sodium bicarbonate neutralized the HCl of the gastric juice. The bacteria thus passed through the stomach and produced the usual results. Stern (1908) does not regard the gastric juice as such a barrier against bacterial invasion as some of the former workers. He regards persons with hyperacidity as fortunate from this standpoint, but in no way absolutely protected. Bile was found to decrease the bacteri- cidal action of the gastric juice. When proteins were present, cholera vibrios were found to live a long time. Pepsin was found to strengthen the action of the acid. Hansen (1912) infected food with B, coli and secured only a slight decrease of bacteria in the stomach.. Schultz- Schultzenstein (1908) found the gastric juice to be more germicidal than hydrochloric acid solution of the same strength. Gregersen (1916) reported some of the more recent work on this subject. He studied the action of the acid in the juice and whether any other factors were GERMICIDAL ACTION OF GASTRIC JUICE 231 important. Tables XXIII and XXIV are taken from his paper and are quite signij&cant. Table XXIII STOMACH CONTENT U HOUR AFTER EWALD'S TEST BREAKFAST. TEMPERATURE 37° C. STAPHYLOCOCCUS PYOGENES AUREUS (After Gregerseiij 1916) Diagnosis Cancer ventriculi . . Neurasthenia Alcoholisnius chron . Cancer ventriculi. Alcoholismus chron. Vicus ventriculi . . . Cancer ventriculi. Arthroctis chron Anaemia Neurasthenia Ulcus ventriculi Ulcus ventriculi Cirrhosis hepatis . . . . Neurasthenia Colitis Neurasthenia Ulcus ventriculi Colitis Abstipatic Colitis Ulcus ventriculi Colitis Ulcus ventriculi Ulcus ventriculi Ulcus ventriculi Ulcus ventriculi Ulcus ventriculi G K -30 -14 -24 -10 -20 -12 -18 - 6 -16 -10 -14 - 3 6 - 3 10 6 12 23 8 14 3 10 5 14 5 18 5 14 6 12 8 22 10 22 13 34 20 28 20 40 26 48 32 42 46 58 50 64 Al Ph Pepsin Time of Destruction. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 2 4 1 14 17 10 24 35 12 34 16 20 22 20 16 29 30 50 30 54 5S 50 66 70 6 10 1 4 5 28 20 12 32 47 20 24 30 24 22 34 38 60 34 60 66 56 78 4 3 5 3 4 5 4 5 6 5 5 4.5 5 6 4 6 6 6 4.5 5.5 5 5 6 6 6.5 No destruction No destruction No destruction No destruction No destruction No destruction 90 minutes 90 mmutes 90 minutes 38 minutes 38 minutes 45 minutes 45 minutes 9 minutes 6 minutes 6 minutes 8 minutes 5 minutes 6 minutes 5 minutes 3 minutes 1 . 5 minutes 2 minutes 1 . 5 minutes 1 minute . 5 minute . 5 minute The letters at the top of the columns refer to the reaction as deter- mined by the following indicators: G==Gunzberg's reagent. Indicates free hydrochloric acid. if = Congo red. The blue color indicates free hydrochloric acid. Other mineral acids may also be indicated by this reagent. AZ= Sodium alizarin sulphonate, which indicates all available except that which is combined. P/i = Phenolphthalein, which indicates total acidity. 232 INTESTINAL BACTERIA It will be noticed from the table that, without free acid, the stomach may not be depended upon to kill bacteria. As the amount of free hydrochloric acid increases, however, the time required for the destruc- tion of bacteria is reduced. The same facts were obtained experi- mentally by this author and are reported in Table No. XXIV. Table XXIV STOMACH CONTENTS WITH OR WITHOUT HCl OR NaOH. STAPH. PYOGENES AUREUS. TEMPERATURE 37° C. (After Gregersen, 1916)) G K Al 1 7 19 24 29 12 35 40 45 55 75 14 34 39 44 54 2 15 20 30 30 70 Ph 4 11 23 27 32 24 47 52 57 67 87 20 40 45 50 60 6 19 24 34 38 ** 6.5 Pep- sin. 5 5.5 4.5 6 Destruction Time. No. 4. Without addition -18 -12 5 10 -6 17 22 No destruction After addition of HCl After addition of HCl After addition of HCl After addition of HCl No destruction 45 minutes 6 minutes 3 minutes No. 11. After addition of NaOH. . . Without addition After addition of PICl After addition of HCl After addition of HCl After addition of HCl -23 5 10 20 40 23 28 33 43 63 No destruction 38 minutes 6 minutes 3 minutes 1.5 minutes 1 minute No. 23. After addition of NaOH . . . After addition of NaOH . . . After addition of NaOH . . . After addition of NaOH . . . Without addition -20 5 10 20 20 25 30 40 IS 18 28 No destruction 45 minutes 8 minutes 3 minutes 1.5 minutes No. 22. After addition of NaOH . . . After addition of NaOH . . . After addition of NaOH . . . Without addition -8 5 10 20 No destruction 9 minutes 4 minutes « 1 . 5 minutes No. 27. After addition of NaOH . . Without addition 10 50* 24 64 2 minutes .5 minute The bacterial content of the stomach as shown above is quite depend- ent upon the acidity of the stomach. In order to standardize these determinations, the acidity of the stomach is usually examined after a 'test meal. The Ewald test breakfast is much used, which consists of two pieces of toast and 400 c.c. of water. The stomach is pumped -out at the end of about one hour. ACIDITY IN STOMACH 233 The contents should be carefully filtered and the following deter- minations made. Total Acidity. Filter 10 c.c. of the gastric contents and add about four drops of phenolphthalein. Titrate to a faint pink color with N/10 NaOH. Calculate the number of cubic centimeters of N/10 NaOH required for 100 c.c. of stomach contents. This is often termed degrees. Some prefer to express the results in terms of per cent hydro- chloric acid. (1 c.c. of N/10 HC1 = . 00365 gm. HCL) Free Acidity. This gives both the organic and inorganic free acid. Titrate 10 c.c. of the stomach contents with N/10 sodium alizarine sulphonate as the indicator. Titrate to a violet color. Determine and report the number of cubic centimeters of N/10 NaOH required by 100 c.c. of the juice. Combined Acidity. The difference between the total acidity and the free acidity is the combined acidity. This is also reported for 100 c.c. of the stomach contents. Detection of Lactic Acid. Prepare XJffelmann^s reagent by adding ferric chloride to a 1 per cent solution of phenol until a blue color results. Add 3 c.c. of this solution to 3 c.c. of the stomach contents. The pres- ence of lactic acid will be indicated by the formation of a yellow color. Organic Acids and Acid Salts. Subtract the free hydrochloric acid from the combined acidity and express the results in terms of 100 c.c. of gastric contents. Microscopical Examination. This may yield much valuable infor- mation. The microorganisms to be expected are yeasts, mold spores, rods and sarcinse. Other bacteria may be present under certain con- ditions. Other particles, such as cellulose epithelial cells, starch grains, etc., may also be present. Bacteria in Intestines and Feces, The bacteria which gain entrance to the intestines and feces are those which have passed through the stomach in the chyme or which may have been contributed, in rare cases from the blood stream. Relation of Bacteria to Life. This has been a much-debated ques- tion. Pasteur (1885) believed that bacteria were essential for normal life. Nuttal and Thierf elder (1895, 1896 and 1897) reared guinea pigs under aseptic conditions which had been delivered by Caesarian section. These increased in weight, but not as regularly as normal pigs. From these data, the authors concluded that bacteria were not necessary. Schotellius (1899, 1902) hatched chickens in a sterile environment and from the data which he secured concluded that chicks could not live in absence of bacteria. He stated also that sterilizing the grain may have 234 INTESTINAL BACTERIA destroyed some essential substance. Levin (1899, 1904) found the in- testines of most Arctic aninoials completely sterile. He draws no con- clusions, but his work is of interest on account of normal growth without bacteria. Metchnikoff (1901) grew tadpoles in sterile bread and water and secured no transformation into frogs in either the sterile or unster- ilized tadpoles. The non-sterile grew larger than the sterile. Portier (1905) determined that larvse of certain lepidoptera lived aseptically. Moro (1905) confirmed the work of Schotellius which has been men- tioned above. He used turtles instead of chicks. Bogdanow (1908) concluded that bacteria were necessary for normal development because sterile larvae of piptera did not do as well as those kept in an unsterile environment. Wolhnan (1911) criticized the work of Bogdanow, stating that his sterilization was carried out at too high a temperature. He concluded from his work on flies that bacteria were not necessary for normal development. Cohendy (1912) thought that the digestive processes could proceed without bacteria, but that they aided digestion. Life was thought to be possible without bacteria, but bacteria are not indispensable. Kianizine (1915) produced data which he thought weakened the argument of Cohendy. With chickens, Kianizine beUeved that bacteria were of great aid in digestion. They carried on analytic and synthetic processes which were of much value to the host. In a later paper by the same author (1916) he continues his argument. He made guinea pigs breathe sterile air and eat sterile food and found that even after a few days they were greatly weakened. The bad results from the deprivation of bacteria was thought to be due to reduced oxidation and accumulation of leucomaines in the body. Loeb and Northrop (1916-1917) attempted to determine the ability of flies to synthesize their body proteins without the aid of microorganisms. Larvae grown on sterile media (banana) did not do well, while those placed in a sterile yeast culture developed normally. It was thought that yeasts were essential for this species of fly although the essential element could not be isolated. This rather extensive presentation of the literature gives the data from which our present conclusions must be drawn. Obviously much of the data which have been used cannot be applied to conditions in the human intestinal tract. Action of Bile on Bacteria. This juice is poured into the intestine just below the pylorus. It is a thick liquid with a decidedly bitter taste. It is made up of salts of glycochoHc and taurocholic acids, pigments (bilirubin, biliverdin, etc.) fats, phosphatids and inorganic salts. The bile like most of the other body fluids is probably sterile when DIET AND BACTERIAL FLORA 235 secreted. Toicla (1914) found that bile of man and dogs was normally sterile and that it was not a satisfactory medium for all bacteria. It was only slightly suited to Staphylococcus pyogenes aureus and Ps, pyocyaneus. Streptococcus pyogenes and Dip. pneumonm were better adapted to it. The bactericidal action was found to vary with the origin. Just below the pomt of entrance into the intestine, the bacterial flora is much simpler. B. coli is often the predominating form. The regulation of carbohydrate fermentation by bile has been studied by Roger (1912, 1913, 1915). The hydrolysis of starch was increased up to 30-40 per cent but when smaller amounts (10 per cent) were used there was a retardation. Heating the bile did not destroy its activity. Moderate amounts of bile (5-20 parts per 100) retarded the action of intestinal bacteria on glycogen. Larger amoimts were erratic, causing sometimes a retardation and sometimes an increase in the action. The same was found to be true with regard to glucose. Pure cultures of Bacillus colon were inhibited in their action on glucose, moderate amounts exerting a more consistent retarding effect than larger amounts. Bile also exerts a retarding effect on putrefaction. Roger (1913) found that the intensity of the biuret reaction of protein media inocu- lated with intestinal bacteria in the presence of different amounts of bile, showed that the attack on proteins was retarded. This retardation was evident up to concentration of 20 per cent after which the retardation decreases. Roger (1915) regards the bile as a factor tending to diminish the production of bacterial enzymes and neutralizing toxins. Boudielle (1913) reported that bile as well as bile salts had an inhibiting action on the fermenting power of B. coli on glucose. Moderate amounts had more pronounced effects than greater amounts. Lagane (1914) added bile to bouillon cultures and noticed no retardation in growth of intes- tinal bacteria. He claimed that it favored the development of B. coli at the expense of other species. Whether the bile is a germicide seems hardly to have been settled. However, most of the data which have been accumulated seems to show that bile exerts a selective action. Toida found that under normal conditions the bile to be sterile. In this connection, it is interesting to remember that gall stones may be started by bacteria and that very often typhoid carriers liberate the bacilli in the bile. In these instances the bile seems to exert no bactericidal action, but perhaps a selective action. Effect of Diet on the Intestinal Flora. It has long been considered that diet was one^of the most important factors determining the intes- 236 INTESTINAL BACTERIA tinal flora. Much work has been done by various investigators from which facts have been well established with regard to changes induced in the intestines by variations in the diet. The changes in the flora resulting from various diets have been measured in terms of certain large groups and rarely in terms of any one or few organisms. These groups which have thus been used may be enumerated as follows: I. Putrefactive Group Bacillus colon, B. Welchii II. Aciduric Group jB. bulgaricus, B. acidophilus, B. hifidus III. Amylolytic Group Glycobacter amylolyhcus Herter and Kendall (1908), working with monkeys, studied the effect on the intestinal flora of sudden changes in the diet. After the flora from a distinctly protein diet had been established the diet was changed to a milk and sugar diet. From this change they were able to detect a change in the intestinal flora and mental condition of the animal. Also in the urine, putrefactive products were quite evident. In the intestines the acidolphile group of organisms was changed to a proteo- lytic group. This was one of the first papers from which definite con- clusions could be drawn. The authors suggest that in a disease where either the acidophile or putrefactive group tends to become estabHshed, a rapid alteration in the diet would tend to prevent either group of bacteria from becoming established. The same conclusions were reached by Herter (1910) in a later paper. Blatherwick and Hawk (1914) have studied the effect of fasting and of low- and high-protein intake on the bacteria in the intestines. The concentration of bacteria was measured by determining the amount of bacterial nitrogen in the feces according to the method mentioned else- where in this chapter and the output of urinary indican. They found that there was no relation between the output of bacteria in the feces and amount of indican in the urine. By means of a seven-day fast (Blatherwick and Hawk, 1914), the bacterial nitrogen excreted by a 76 kg. man was reduced from 1.571 gms. to 0.101* gm. The actual weight of excreted bacterial substance was reduced from 14.336 to 0.920 gm. per day. The output of bacterial nitrogen and bacterial sub- stance was about the same during a fast and on a low proten intake. Assumption of a high protein diet caused an increase in the above figures. FACTORS INFLUENCING INTESTINAL BACTEMA 237 Unfortunately a qualitative study of the bacteria in the feces was not made during this study and to secure such information it is necessary to consult the work of others. Herter and his colleagues, the work of whom has been summarized by Herter (1907), have given information to show that a protein diet induces the proteolytic group. Rettger and his colleagues have studied the effect of carbohydrate feeding. The experiments were made with laboratory animals and man. Hull and Rettger (1915) studied the effect of milk and carbohydrate feeding. They found that lactose when taken by adults along with their usual food, caused a shift in the flora to the acidophilli group of bacteria. B. acidophilus and B. bifidus appeared with the former more abundant. The most practical diet, however, to produce these results was a com- bination of milk and lactose. B. hulgaricus was not found which is in accord with the work of others. Fasting has a decided effect on intestinal bacteria. As quoted above Blatherwick and Hawk (1914), during fasting, noticed a decided reduc- tion in the amount of bacterial nitrogen and bacterial substance. Sisson (1917) has found that, after periods of starvation, there are definite changes in the number of bacteria in contrast to the usually vigorous growth- No change in the kind of organism after starvation was noted. The greatest decrease in the number of bacteria was noticed in the duo- denum. Sisson regards the condition produced by starvation as one of relative amicrobism. In this connection the influence of copious water drinking on fecal bacteria should be mentioned. Fowler and Hawk (1910) noticed a decrease in the total nitrogen appearing as bacterial nitrogen. The following weights of lecal bacteria were excreted per day during the periods mentioned: Preliminary 5.327 gms. per day Water period 4.579 gms. per day Final period 3 .280 gms. per day Hattrem and Hawk (1911) by means of copious water drinking obtained a decrease in putrefaction and stated that it was probably due to a decrease in the activities of indol-producing bacteria following accelerated absorption of products of protein digestion. Copious water drinking (1000 c.c.) caused a decrease of indican in the urine which was accompanied by an increase of ethereal sulphates. The drinking of distilled and softened waters were found by Sherwin and Hawk (1914) to cause a decrease in intestinal putrefaction as measured by urinary indican. 238 INTESTINAL BACTERIA Number of Bacteria in Feces. The different investigators are not in agreement on this question. This may be in part due to different methods of analysis and to the variations in samples. The securing of concordant results is hardly to be expected. The data which are on hand vary from 5 to 50 per cent. Strasburger (1896) stated that one- third of the feces of an individual on a normal diet was bacteria. The quantity of daily bacterial wastes dried in adults was as follows: a. Eight gms. under normal conditions. h. In dyspeptic intestinal conditions on an average of 14 gms. and even as much as 20 gms. Co In chronic constipation 5.5 gms. and, at times, even as little as 2.6 gms. The total number of bacteria eUminated by an individual in one day was believed by Strasburger to be 128,000,000,000. Among the deter- minations which have been made by different investigators are the fol- lowing collected by Mattill and Hawk (1911) : Daily Excretion of Dry Bacteria. Percentage of Dry Bacteria. Strasburger Berger and Tsuchiya McNeal, Latzer and Kerr Mattill and Hawk Sato Schittenhelm and Tollens. Lissauer Tobaya 8.0 gms. 3.023 5.34 8.27 8.54 24.3 % 12.60 26.90 27.97 24.39 42.00 8.67 11.22 Osborne and Mendel (1914) found that about 70 per cent of the nitrogen in feces of animals was caused by bacteria. Classification of Intestinal Bacteria. Since the intestinal flora is quite extensive it is not a simple matter to make a classification. The viewpoint and interests of the investigator might influence the kind of classification made. Ford classified the bacteria in the duodenum of infants. The following classification by Distaso (1912) is as satisfactory as any; INTESTINAL BACTERIA 239 THE INTESTINAL FLORA. BY A. DISTASO Lancet, 182 (1912), 496-98 A. Gram Negative Bacilli. 1. B. coll group, 2. B, variabilis group. (Anaerobic bacteria with round extremity.) 3. B. thetaiotnomicron group. (Bacilli very polymorphic, elliptical form.) 4. B. ngidis group. 5. B. preacutus group. (Bacillus swollen in the middle and pointed ex- tremities.) B. Gram Negative Cocci. 1. Sarcina citrea group. (Very common in the mouth and feces, similar in appearance to the gonococcus.) 2. Diplococcus obliculus group. (Strict anaerobes,) 3. Parvulus group. (Strictly anaerobic, small cocci.) C. Gram-Positive Bacilli. 1. B. Acetogenes group. The greater number of the gram-positive bacilli belong to this group. The bacillus acetogenes B is its chief repre- sentative, since the B. bifidus and B. acetogenes A are very rare in the feces examined in London. 2. Streptobacillus group. This microbe always exists in the intestinal flora. 3. Diploc, acuminatus group. An anaerobic bacillus. It produces burytic acid. 4. B, perfrigens group. An anaerobic microbe. It produces enormous quantities of lactic acid. 5. B. cedematic 7naligni group. With the B. sporogenes (Metchnikoff). 6- B. Todella group. Anaerobic group with very long bacilli. 7. A lemon-shaped bacillus, which is stained by iodine and is described by the author as being butyrous acid-producing microbe. It has not yet been obtained pure. D. Gram-positive Cocci. 1. Enterococci group, in chains, less frequently diplococci. 2. Small cocci group. Are also to be seen corresponding in size to the coccus Bananiy to the Staphylococcus pyogenes, and to the Staph, asaccharolyticus. In addition three kinds of spores are to be found. . 1. Oval spores; rather large, which may belong to B. sporogenes and to other. 2. Round spores, which may belong to B, putrificus (Bienstock-Tissier) or to the bacillus of Rodella and to the B. Alkaligines anaerohcus, 3. Very small spores, strongly refractile, belonging to the B. perfrigenes. 240 INTESTINAL BACTERIA Fermentation in the Intestinal Tract. Putrefaction is used in this case to indicate the bacterial dccouiposition of proteins. There has been much discussion with regard to the definition of the terms putre- faction and fermentation. Kendall (1911) has given a good discussion of these terms in their relation to the digestive tract. He quotes therein the definitions which have been given to the processes of putrefaction and fermentation by various workers and accepts the definition for fermentation which was given by Alfred Fischer. Fischer defines fer- mentation as the biochemical decomposition of nitrogen-free, organic compounds, especially the carbohydrates induced by fermenting organ- isms. If this definition is accepted there may then be different types of the process, depending on the main product: lactic acid, butyric acid, alcohoUc fermentations, etc. With regard to putrefaction more confu- sion exists. The term has been used in so many different ways that it is difficult to correlate them. Kendall states that the phenomena grouped together as putrefaction ^^ represent a scries of symbioses, in which the initial supeificial breakdown is brought about by the anaerobes, while the process is brought to its lowest terms by the facultative anaerobes.'^ The Germans have made a distinction between ^' Faulniss" putrefaction in the popular sense and '^ Verwesung " eremecausis. This latter is supposed to be an aerobic process carried on by many bacteria. That fermentation should mean decomposition of carbohydrates and putre- faction, decomposition of proteins are the opinions of the biological chemists. Certain microbiologists, however, have not accepted this definition. They define fermentation as an intracellular process which furnishes energy to the cell and pay no attention to the products which are decom- posed. The decomposition of soluble nitrogenous compounds within the cell protoplasm is regarded as fermentation. This seems to be the best definition for the term as has been suggested by Fischer (1902). He gives the following points as characteristic of such a conception of the term: 1. Fermentation is an intracellular process. 2. The products of the fermentation are essentially different from those in the substract and are not mere parts of these original substances. 3. The products are useless to the cell and may be harmful. 4. Vital energy is produced. According to this definition, then, and as it is stated by Fischer, the hydrolysis of starch to soluble sugars is not a fermentation, because it does not take place within the cell to yield energy to the cell. The burning of the sugar within the cell protoplasm, however, does consti- AEROBIC RESPIRATION 241 tute fermentation and the energy therefrom is available for cell use. Fermentation may then be regarded as respiration if the latter term is considered to cover those processes which yield energy. There are two kinds of respiration, aerobic respiration and anaerobic respiration. Aerobic Respiration. This involves the securing of energy from food substances by means of and in the presence of free oxygen. In order to present the facts required in a discussion of this nature, the carbohydrates lend themselves very well. It must be remembered, however, that there are certain well-known objections to the expression of bacterial changes by chemical equations and these should be borne in mind in considering the equations which follow. As a typical example of aerobic respiration the decomposition of dextrose may be taken C6H12O6+6O2 = 6CO2+6H2O+674 cal. In this change molecular oxy- gen has been used to burn dextrose completely.* In the products carbon dioxide and water there is no energy which is available for bacteria. With nitrogenous substances the oxidation of glycocoU takes place as foUdws: I H— C— C^OH + 30 - 2CO2 + H2O + NH3 + 152 Cal. NH2 In this equation allowance must be made for the combustion heat of ammonia since this compound may be further oxidized. Leucin is oxidized as follows: C6Hi3N02+150 = 602+5H20+NH3+755 cal. In this equation the same correction must also be made for the com- bustion heat of ammonia. Hydrogen sulphide is oxidized by the sulphur bacteria according to the following equation: H2S+2O2=H2SO4+207 cal. or to neutral sulphur as follows: H2S+0 = H20+65cal. * In all considerations of respiration, it must be remembered that the energy secured by complex molecules is latent in them, having been put into them by the organism which formed them. Such synthetic processes are endothermic for the organism which builds up these complex substances from the simpler compounds. The bacteria secure this energy when they split these complex compounds to simpler ones. 242 INTESTINAL BACTERIA Anaerobic Respiration. The products of anaerobic respiration are usually left in an incompletely oxidized condition. If dextrose is taken again to show this, any of the following equations might serve. CH20H(CHOH)4COH = CH3CHOH ■- COOH+ 15 cal CH20H(CHOH)4COH = 3CH3COOH+34 cal. CH2OH (CH0H)4C0H = C2H5OH + 2CO2 + 22 cal. The main product in each of these equations may be further oxidized to yield more energy. The amount of energy secured by aerobic and anaerobic bacteria from the same amount of food is striking. Take, for instance, the following equation as a typical one for aerobes: CH20H(CHOH)4COH+602 = 12Cp2l2H20+674 cal, and the following as typical for anaerobic bacteria: CH20H(CH0H)4C0H-CHjCH0H COOH+15 cal. The proportion would then exist as follows: Aerobic : anaerobic = 674. 15 Aerobic : anaerobic =45.1 That is, from the same amount of food the aerobes by complete oxidation will get forty-five times as much energy. Looking at it from the standpoint of energy, the anaerobes have to have forty-five times as much food as the aerobes. This explains why the anaerobic bacteria are so destructive in soil, sewage, etc. Putrefaction in the Intestinal Tract. As stated above the biological chemists have reserved this term for the decomposition of nitrogenous substances. According to the definition of fermentation which has been given above, some decompositions of nitrogenous substances would be regarded as fermentations. The combustion in the protoplasm of those peptones which are diffusible would be regarded as fermentation while the hydrolysis of the proteins and proteoses which goes on outside of the cell wall would not be regarded as fermentation. The chemistry of the putrefactive changes which take place in the intestines is fairly well known. Straight chain acids are among the first ANAEROBIC RESPIRATION jtC^'xO compounds which are attacked and the changes may be indicated as follows: I. Deaminization. H H C C'^OH - H — > H— C— C^OH + NH3 NH2 Glycocoil H Acetic acid Ammonia II. Decarboxylation. H H C C^OH - H -> H C H + CO2 NH2 Glycocoil NH2 Methylamm Carbon dioxide III. Oxidation. H ±. n ^ nn^ -u TTnO XT According to these reactions tyrosin may be putrefied as follows: X OH ^0 rrrr r^rr r^^y r\rr -O1I2 — v^-tl U — Uxl" NH2 Tyxosm /\_ CH2— CH2— COOH + NH3 OH /\_CH2— C^OH - Hydroxy phenyl acetic acid Para hydroxy phenyl propionic acid /\ — UXI3 \/ OH Para cresol OH Phenol The oxidation stops with phenol. These substances have no well- marked location in the intestine although it occurs in the large intestine. Their formation is not limited to the intestines for they have been found in other parts of the body. For excretion phenols are detoxicated. 244 INTESTINAL BACTERIA They are united with sulphuric acid in the Uver to form ethereal sul- phate. OH H— Ov /P H— 0/ ^0 — H / /\o This is usually excreted as a sodium or potassium salt. Putrefaction of Tryptophan. Hopkins and Cole have determined the steps in the decomposition of tryptophan by cells. /^ C-CHa— CH— C^OH -^ ^N C-CHa-CHa^C-OH-^ C NH: N H C N H Tryptophan — UJtl 2 — UJtls Indol propionic acid /\. c / N C— CHs C /V C— H N C / N N H JUL Indol aceticS acid Methyl mdol (skatol) Indol The final product is methyl indol or skatol. For excretion this may be united with sulphuric acid as follows: >0 /\- C— CHs C / N TT c N H TT C-CH2— 0~7S/ II ^ ^0 n / Indol is prepared for excretion in the same way. Putrefaction of Arginin. Putrecin in the end product and was supposed to be important in certain types of food poisoning. It is formed from arginin as follows: PUTREFACTION OF AMINO ACIDS 245 ^0 Nr^TX r^Tx r^Tj rixj c^'y r\xj C=NH NH2 NH2 Argimn NH2— CH2— CH2— CH2— CHNH2— COOH Ormthm ATTT C^TX f^TX f^TX f^Jj ATTJ IN n.2 — ^^0.2 — v^Jl12 — Vy±l2 V-/XI2 — IN x±2 Putrecin tetramethylene diamm Putrefaction of Lysin. Cadaverin is a common product of putre- faction. It was first observed by Brieger (1885). It is formed from lysin in the following manner: CH2NH2 — CH.2 — CH.2 — CH.2 — CH.NH2 — C — OH CH2NH;a;— CH2--CH2— CH2~CH2NH2 Pentamethylene diamm cadavenn Putrefaction of Cystine. /-ITT /"HTT ^hX pJLiJL j^ \»y Jin mill j|*j pTTXTTT PTTISITT COOH COOH ! — O — pJo. I ! H Cysteine first formed. I / H— C— N \h COOH H H— O-SH _ - 1 . " I TJ •XI H— C— N "^H COOH H2S may form m tWs way. CH3SH methyl mercaptan may form in this way. 246 INTESTINAL BACTERIA According to Mathews cystine goes to cysteine, and then as: SH-CH2-CHNH2 -> SHCH2— CH2NH2 -^ SHCH2-CH3 Thioethyl amm Ethyl mercaptan The action of bacteria and yeast-hke fungi has been studied by Tanner (1917, 1918). These microorganisms were quite able to split hydrogen sulphid from cystine. Some of these decomposition products of the amino acids and pro- teins may be detected as follows: The solution to be tested may be either a filtered bacterial culture or a fecal suspension. Indol Determinations. A number of methods have been devised for the detection of indole. Most of these yield good results when applied to bacteriology. Nitroso-indol Nitrate Reaction. Acidify a portion of the substance under examination with concentrated nitric acid. The addition of a few drops of potassium nitrate will produce a red coloration if indol is present. The red compound is nitroso-indol nitrate. Nitro-prusside Reaction. Add a few drops of a freshly prepared solu- tion of sodium nitro-prusside to a filtered sample. Add sodium hydrox- ide and examine for the production of a violet color. This may be changed to a blue by the addition of a few cubic centimeters of strong acetic acid. Ehrlich's Para-dimethyl Amino Benzaldehyde Test. Add about one-third the volume of a 2 per cent solution of para-dimethyl amino benzaldehyde in aclohol to the sample under examination. Then slowly add dilute hydrochlorine acid until a red color is evident. This color may be deepened by the addition of a few drops of sodium nitrite solution. Bergeim's (1917) Method for Determination of Fecal Indol. Rub 30 to 50 gms. of the fresh feces in a mortar with H2O. Transfer to a 1 liter Kjeldahl flask and add H2O to 400 c.c. Add 6 c.c. 10 per cent KOH and 2 c.c. paraffine and distill with steam lentil 500 c.c. have been obtained, bringing the volume of the fecal suspension down to about 100 c.c. Re-distill with steam after acidifying slightly with H2SO4 or else remove the NH3 with permutite. Mix an aliquot of the resulting solution (100 c.c.) in a 150-c.c. separatory funnel with 1 c.c. of a fresh 2 per cent solution of NaB naphthoquinonesulphonate and 2 c.c. of 10 per cent KOH. After fifteen minutes extract with CHCI3 using 10 and 7 c.c. Dilute extract to 15 c.c. and mix. Compare color in the colorimeter with that of a standard similarly prepared, using 0.1 mg. indol. The error appears to be only— 1 per cent. BACTEPJA IN FECES 247 Skatol Determinations. Skatol Is a product of putrefaction which has much in common with indol. Herter's Test (1907). Add a few cubic centimeters of acid para- dimethyl amino benzaldehyde (5 gms. to 100 c.c. concentrated H2SO4) to about 6 c.c. of the filtered sample. After heatmg to boiling a bluish- lavender color is obtained. This may be deepened by adding a small amount of hydrochloric acid. Phenol Determinations. Ferric Chloride Test. By adding a few drops of ferric chloride solution to the solution under examination a blue color will be obtained if phenol is present. Nitric Acid Test. Add a few drops of nitric acid to the sample and heat. If phenol is present, a yellow color is formed due to the formation of picric acid. Bromine Water Test. When bromine water is added to a solution containing phenol, mono-, di-, and tribromophenols are formed. The first two possess a very sharp odor. Tribromophenol precipitates as yellow white needles. Methobs for Deteemining Bacteria m Feces The accurate, rapid determination of the bacteria in feces is not a simple matter. Plating on ordinary media is known to give low results. On the other hand there are certain errors in the various microscopic methods. In these, the presence of pieces of organic matter hinder the counting. MacNeaPs (et al.) Modification of the Winterberg Method. The first employment of the Thoma-Zeiss blood-counting chamber for bac- teriological technic was made in the one-cell dilution method of obtaining a pure culture. By this method the number of cells per unit volume could be ascertained and the dilution per unit volume of a suspension with one cell in two to five drops required to obtain a suspension with one cell in two to five drops calculated. In this way the first pure bacterial culture was obtained. Henrich Winterberg was the first to use this method of bacterial counting, and to test its accuracy both as regards suspensions of varying dilutions and in comparison with the microscopic plate-counting method. He considers the method more accurate than the plate-counting method, but as a quantitative pro- cedure he would consider it unimportant. He says his determinations were too low. Winterberg's counts were made with suspensions of living bacteria in bacteria-free distilled water. " The method, as used by MacNeal, Latzer and Kerr, is as follows; 248 INTESTINAL BACTERIA a portion of the 1 : 100 suspension of feces is diluted ten times, and a portion of this is drawn up to the mark 1 in the capillary of a dilution pipette, ordinarily used in estimating the white blood cells. This is diluted to the mark 11 with a dilute solution of methylene blue in physio- logical-salt solution. (The staming solution consists of methylene blue, 1 gm.; glycerine, 25 c.c; distilled water, 75 c.c. A few drops of this are mixed with 10 c.c. of 0.8 per cent salt solution until the mix- ture is well colored, but not too opaque. This mixture is used as the diluting fluid, A little practice will show the proper depth of color to be employed.) The suspension is thoroughly mixed in the bulb by shaking and roUing in the usual manner. Several drops are blown out and then a very small drop is placed in the center of the circular elevated portion of the slide, which has been previously thoroughly cleansed by washing in distilled water and alcohol. A clean, thin, ground cover-slip is made slightly moist by breathing upon it and is immediately placed upon the shde. Slight pressure upon it causes the Newton color rings to appear and these remain after the pressure is removed if the preparation has been properly made. The sHde is allowed to stand one to two hours to allow the bacteria to settle. Then the bacteria in fifty small squares upon the marked scale are counted microscopically, the No. 7 Leitz objective and No. 3 ocular being used. The calculation is simple as an example will show. " Example, B252, Subject H, Julv 15, 1908. Fifty squares contain 400 bacteria. Therefore the average per square is 8.0 bacteria. One small square =1/4000 c.mn. One c.mn. contains 8X4000 = 32,000 bacteria. One cubic centimeter contains 32,000X1000 = 32,000,000 bacteria. Pipette dilution is 1 : 10. 32,000,000 X 10 = 320,000,000 bac- teria per cubic centimeter of 1/1000 suspension. One cubic centimeter of 1/1000 suspension is equivalent to 1 mg. feces. Therefore, there are 320,000,000 bacteria per milligram feces. " Counting by this method is an exacting process and much practice is required to see all the bacteria present. Careful adjustment of the light is important and best results have been obtained by illumina- tion with Welsbach Hght; constant focusing through the different layers is necessary. Perhaps the greatest source of error is the difficulty of distinguishing accurately the bacteria. Skill in this is acquired only by practice. The method has the advantage of simplicity. The results of the authors' seem to conform to those of Winterberg in that high dilutions give a relatively higli count." MacNeal's (et al.) Modification of the Eberle-Klein Method for Betermining the Number of Bacteria in Feces. Eberle, working in BACTERIA IX FECES 249 Escherich's laboratory, was the first to determine the quantity of fecal bacteria by counting them in stained films. His experiments were carried out with normal infant's stools. Coverglass preparations were made from fecal suspensions of known dilution and after being com- pletely dried were stained with freshly prepared aniline water fuchsin, then washed in water, air-dried, mounted m Canada balsam, and counted. The method was very much improved by Alex, Klein and Hehewerth. Klein had noticed that vegetative bacteria were more sensitive to disinfectants in a moist than a dry state, and this led him to believe that bacteria would also be stained more readily in a moist con- dition than after drying. Therefore he allowed the dye to act while the bacteria were suspended in a hquid and the coverglass preparation was made, dried, and fixed,, only after the bacteria were stained. Later the same investigator modified the method still further, using gelatin to fix the stained" bacteria to the coverglass. The aqueous solution of gelatin and the stained bacterial emulsion were put upon the cover- glass separately, then fixed and spread. When dry, the preparation was immediately mounted in Canada balsam without flaming. *' The procedures as employed by MacNeal, Latzer and Kerr have been considerably modified. In the addition of the gelatin to the bac- terial suspension, in spreading the films, in selecting the fields to be counted, and in counting individuals, rather than groups, as units, the technic differs from Klein's method. Of the 1 : 100 suspension of the feces prepared as described above (p. 248), 2^ c.c. are transferred to a clean, dry bottle, | c.c. of melted nutrient gelatin and 2 c.c. of aniline water gentian violet added, and the whole thoroughly mixed and allowed to stand for three to five minutes. Then, by means of a platinum loop, the carrying capacity of which has been determined with great care, a loopful of the mixture, well shaken immediately before, is transferred to a clean, flamed 20 mm. square No. 1 coverglass and deposited near the center of the glass. Immediately another coverglass of the same size is accurately placed on top of the first so that the two glasses are in contact over about three-quarters of their surfaces and the sides evenly fitted together. As soon as the liquid has spread evenly between the two coverglasses, they are quickly slipped apart and allowed to dry. The technic here is the same as ordinarily used in the preparation of cover- glass blood films. If the preparation does not appear evenly spread the process is to be repeated with two more coverglasses until a satis- factory result is obtained. The films are next accurately measured by a millimeter rule and then, without further treatment, mounted in Can- ada balsam upon one slide so that one diagonal of each rectangular 250 INTESTINAL BAOTEPvl \ film is parallel with the long axis of the slide. By laying the slide over coordinate paper these diagonals arc readily brought into the same line parallel with the edge of the slide. '^ The covers must be so mounted that Ihece diagonals, now in a straight hne, are those which crossed each other as the two coverglasses were originally put together in preparing the films. When properly made each diagonal measures almost exactly 25 mm. '^ For counting the bacteria in the preparation, the Leitz 1/12 oil immersion objective and the No. 3 ocular fitted with an Ehrlich ocular square to restrict the field to a convenient size, and a mechanical stage graduated in miUimeters are employed. Beginning at the end of the diagonal of one coverglass the bacteria are counted in each of 25 fields 1 mm. apart along this diagonal. In a similar way 25 fields are counted on the diagonal of the second coverglass, making a total of 50 fields, the average of which may be considered as representative of both films. The size of the square field is accurately measured by a stage microm- eter. From the data then at hand the number of bacteria per milligram feces is calculated. '' Example, B252, Subject H, July 15, 1908. In the preparation made, as described, each film measures 16.5X17.5 mm., total film area therefore, 33X17.5 mm.; the field employed measured 0.0445 mm. square; the amount carried by the loop 2.01 mg.; the number of bac- teria counted in 50 fields was 559 and the original 1 : 100 suspension was used (diluted to 1 : 200 by dye and gelatin). From these data, 559X33X17 5X200 Bacteria per milligram feces^ ^^;^^^^^^^^^^^^^^^ ^ 324,000.000. In this fraction all the members except the size of the films and the number of bacteria counted may be kept constant and the calculation simplified by the use of logarithmic tables. ^* With apparatus and reagents ready and the 1 : 100 suspension prepared, this entire estimation can be completed in about forty min- utes. The results cannot be considered very accurate as the platinum loop does not carry an exactly constant quantity. There is also some- times great difficulty in distinguishing micrococci from other fine par- ticles in the preparations. The concentration of the bacterial suspen- sion also influences the final result. The estimation is in general rela- tively higher when dilute suspensions are counted as we have obseived in applying it to enumeration of bacteria in pure cultures." * Steele's Modification of the Strassbtirger Procedure for the Quanti- tative Enumeration of Fecal Bacteria. Steele describes the method as * From MacNeal, Latzer and Kerr. J. Inf, Dis. 6 (1909), p. 127. BACTEEIA IN FECES 251 follows: '^^ The possibility of separating the bacteria from the rest of the feces depends on the fact that the bacteria are so nearly of the same specific gravity as distilled water that they cannot be centrifugalized out of a watery suspension of the feces, but remain suspended in the supernatant fluid. Taking advantage of this, the bacteria can be removed by washing with the centrifuge. Then, if the specific gravity of the wash-water is lowered by the addition of large amounts of alcohol, the relation of the bacteria to the fluid is changed to such an extent that the microorganisms can be readily centrifugalized out, separated, and weighed. Unless the period of passage of the feces has been ascer- tained to be normal, it is better to mark the beginning and end of each period of examination by carmine. The use of the Schmidt diet is not necessary. The whole stool is saved. Unless the feces are liquid they are rubbed up with a known amount of distilled water until they are smooth and semi-liquid and as homogeneous as it is possible to make them. '' Two portions of 5 c.c. are measured off with a pipette of large caliber, using for this purpose an ordinary 5 c.c. pipette with the tapering end cut off, and with the necessary correction made at the upper mark. One of these portions of 5 c.c. is put into a porcelain dish and dried over a water bath and later in a drying oven, in order to determine the dried weight. The addition of a little alcohol and thorough mixing will hasten the process of drying and prevent caking of the feces. '' The second portion is washed free from bacteria. This is done as follows: The wash-water is 0.5 of 1 per cent HCl solution in distilled water. The acid increases the solubility of the salts and soaps of the feces. One hundred c.c. of this solution is employed at the beginning of the washing The feces are thoroughly mixed with the wash solu- tion and then centrifugalized. The use of the water motor or electric centrifuge is almost essential. Each tube is centrifugalized for about IJ minutes, then the cloudy supernatant liquid is poured through a layer of gauze. This fluid contains the bacteria in suspension. All of the mixture (the wash-water and the feces) is centrifugalized the same way, and then the residue in the tubes is shaken up with more of the wash-water and centrifugalized again. This is repeated until the super- natant liquid after the centrifugalizing is transparent, showing that approximately all the bacteria have been washed out. If smear is made of the residue at this point, it will be found that the bacteria are not entirely washed away, but are evidently very much reduced. They occur singly, while in the unwashed feces they are in great lumps and masses. The suspension of bacteria is then mixed with a liberal 252 INTESTINAL B VCTT RI V portion of alcohol, and evaporated down slowly at a tenaperattire of 40° to 50° C. until it amounts to not more than 50 c.c. in all. This takes approximately twenty-four hours. It is then mixed with at least twice its volume of alcohol, preferably absolute alcohol, although this is not absolutely necessary. This lowers the specific gravity of the fluid to such an extent that now the bacteria readily centrifugalize out. The mixture is then centrifugalized until the supernatant liquid is quite clear. This takes thirty minutes or more for each tube. The residue, which consists of the bacteria, is washed with pure alcohol and is shaken up with ether to remove the fat; then it is again washed with alcohol. All of this washing is done by means of the centrifuge. The bacteria are next washed out of the tube with a little alcohol and evap- orated to dryness and dried in the oven at moderate heat, dried in the desiccator, and weighed. Smears of the final preparation show that it consists of bacteria with a very few minute particles of other material. These particles are only visible with high power, and are very few in number, perhaps two at each field of the 1/12-inch objective. They stain with methylene blue; Strasburger suggests that they are cellulose, which they may well be. At any rate, the error arising from the inclu- sion of these small particles in the dried weight of the bacteria must be very small, and is probably balanced by the bacteria that it is not pos- sible to wash out of the residue in the first washing. During the prep- aration of the bacteria the first portion of 5 c.c. has been dried and weighed. We then know the dried weight of 5 c.c, the weight of the dried bacteria in 5 c.c, the original volume of the stool, and the volume after the addition of a known amount of water. It is then easy to cal- culate the data that we desire, namely, the volume of the stool, its dried weight, the weight of the dried bacteria, and the percentage of bacteria in the dried weight." Mattil and Hawk's Method for Quantitative Determination of Fecal Bacteria. '^ The method is a simphfication of MacNeal's adaptation of the Strasburger procedure. About 2 gms. of feces are accurately weighed and placed in a 50-cc centrifuge tube. To the feces in the tube a few drops of 0.2 per cent hydrochloric acid are added, and the material is mixed to a smooth paste by means of a glass rod. Further amounts of the acid are added with continued crushing and stirring until the material is thoroughly suspended. The tube is then whirled in the centrifuge at high speed for one-half to one minute. The sus- pension is found sedimented in not more or less definite layers, the upper- most of which is fairly free from larger particles. The upper and more liquid portion of the suspension is now drawn off by means of a pipette BACTERIA IN FECES 253 and transferred to a beaker. The sediment remaining in the tube is again rubbed up with a glass rod with the addition of further amounts of dilute acid, and again centrifugalized for one-half to one minute. The supernatant liquid is pipetted off and added to the first, the same pipette being used for the one determination throughout. A third por- tion of the dilute acid is then added to the sediment^ which is again mixed by stirring and again centrifugalized. All the washings are added to the first one, and, during the process, care is taken to wash the material from the wall and mouth of the centrifuge down into it. Finally, when the sediment is sufficiently free from bacteria, the various remain- ing particles are visibly clean, and the supernatant liquid, after cen- trifugalization, remains almost clear. This is removed to the beaker in which are now practically all of the bacteria present in the original portion of feces, together with some soUd matter not yet separated. In the centrifuge tubes there is a considerable amount of bacteria-free solid matter. '' The suspension is now transferred to the same centrifuge tube, centrifugalized for a minute, and the supernatant Hquid transferred to a clean beaker by means of the same pipette. The tube is then refilled from the first beaker and thus all the suspension centrifugalized a second time. The beaker is finally carefully washed with the aid of a, rubber- tipped glass rod, the second sediment in the centrifuge tube is washed free of bacteria by means of this wash-water and by successive portions of the dilute acid, and the supernatant liquid aft;er centrifugalization is added to the contents of the second beaker. The second clean sediment is added to the first. The bacterial suspension now in the second beaker is again centrifugalized in the same way and a third portion of bacteria- free sediment is separated. Frequently a fourth serial centrifugalization is performed — always if the third sediment is of appreciable quantity. At all stages of the separation, small portions of the dilute hydrochloric acid are used, so that the final suspension shall not be too voluminous. Ordinarily it amounts to 125 to 200 c.c. At the same time, the final amount of fluid should not be too small, as shown by Ehrenpfordt, because the viscosity accompanying increased concentration prevents proper and complete sedimentation. '^ To the final bacterial suspension an equal volume of alcohol is added and the beaker set aside to concentrate. A water bath at 50° to 60° is very satisfactory. After two or three days, when the liquid is concentrated to about 50 c.c, the beaker is removed and about 200 c.c. of alcohol are added. The beaker is covered and allowed to stand at room temperature for twenty-four hours. At the end of this time 254 INTESTINAL BACTERIA the bacterial substance is generally seitlcd, so that most of the clear supernatant liquid, of dark brown color, can be directly siphoned off without loss of solid matter. The remainder is then transferred to centrifuge tubes, centrifugalized, and the remaining clear liquid pipetted off. The sediment consists of the bodies of the bacteria, and is trans- ferred to a Kjeldahl flask for nitrogen determination. This is the bac- terial nitrogen. Where a detefmination of bacterial dry substance is desired, the sediment of bacteria is extracted by absolute alcohol and ether in succession, transferred to a weighed porcelain crucible, and dried at 102° C. constant weight. This dried sample is then used in the nitrogen determination. Our procedure differs from that of MacNeal in that the bacterial dry matter is not determined. A saving of about seven days' time and of considerable labor is accomplished by this omission. '' Inasmuch as it has been shown by various investigators that such bacteria as are present in the feces contain on the average about 11 per cent of nitrogen, the values for bacterial nitrogen as determined by our method may conveniently serve as a basis for the calculation of the actual output of bacterial substance." Examination of Feces for Bacillus Typhosus Lumsden and Stimson's Method. "Place about 5 gms. of the feces in a conical glass or other suitable vessel, add about 15 or 20 c.c. of sterile physiological salt solution or bouillon and agitate. Let stand one-half to one hour either at room temperature or, preferably, at incubator temperature (37 "" C.) in order to permit the heavy particles to settle. Deposit one or two drops of the prenatant fluid in the center of an Endo plate. With a right-angled glass rod distribute the drop over the entire surface of the plate and then rub the rod over the surface of a second, third, fourth and fifth Endo plate. By carrying the spreader over the surfaces of several plates in this way, one or two of the plates will furnish abundant but sufficiently isolated colonies to permit " After inoculation place the plates inverted in the incubator, leave there for twenty to twenty-four hours, and then examine. On the plates colonies of typhoid and paratyphoid bacilli will be transparent, colorless, dew-drop like and usually from 1 to 2 mm. in diameter; while colonies of colon bacilli will be deep red, showing sometimes on the sur- face a sheen from precipitation of the fuchsin, and measure usually from BACILLUS TYPHOSUS IN FECES 255 3 to 4 mm. in diameter. If typhoid4ike colonies appear on the plates fish five or six of them, and inoculate tubes of Russell's medium in the following manner: " Touch the colony with a sterilized platinum needle, make two streaks along the slanted surface of the medium in the tube and then stab the needle down through the center of the block of medium to the bottom of the tube. Incubate the inoculated tubes of RusselFs medium for twenty to twenty-four hours and then examine. Kendall and Day's Method. '' The feces are collected preferably in a small rectal tube. A small portion of the feces (about a loopful) is thoroughly emulsified in 10 c.c. of sugar-free broth and preferably incubated one hour at 37° C. prior to the inoculation of the plates. This preliminary incubation does two things: the clumps of bacteria are thrown down, leaving a more uniform suspension of bacteria in the supernatant solution for inoculation and the bacteria undergo a slight development in a medium particularly suited for their growth. It is beheved that better growth is secured if the feces is put through broth than if it is added directly to the solid media. It is essential that sugar- free broth be used. '^ The fecal suspension is then rubbed over the surface of the Endo agar plates in the usual manner by means of the sterile glass rods. These plates are then incubated at 37° C. for eighteen hours. At the end of this time small dewdrops are seen which may be removed entire to broth tubes. (1 c.c. broth which have been held at 37° C.) These are then incubated for two hours at the end of which time sufficient growth will have taken place, with which to make the agglutination tests." Holt-Harris-Teague (1916) Method for Isolating jB. Typhi from Stools. These authors have devised a medium which they claim will give better results than Endows medium. They claim that a greater percentage of colorless colonies on this medium turn out to be B. typhi than on Endows medium. It is prepared as follows: Nutrient agar is made in the usual way, containing 1.5 per cent agar, 1 per cent Witters peptone, 0.5 per cent sodium chloride, and 0.5 per cent Liebig^s meat extract, to the liter of distilled water. It is cleared with egg-white, placed in flasks, and sterilized in the Arnold sterilizer on three successive days. The reaction is brought to +0.8. The agar is melted and saccharose (.5 per cent) and lactose (.5 per cent) are added. The medium is then heated for ten minutes in the Arnold. To every 50 c.c. of the medium are added 1 c.c. of 2 per cent yellowish eosin and 1 c.c. of 0.5 per cent methylene blue. We always add the eosin first 256 INTESTINAL BACTERIA and then the methylene blue. The mixture is shaken and plates are poured. The surface of the medium is dried in the usual way before the plates are inoculated. We have also obtained excellent results by sub- stituting for Liebig's extract, meat infusion lendered free from sugar by incubation with B. coh. Stock solutions of 2 per cent eosin and 0.5 per cent methylene blue in distilled water are kept in the dark. We have not sterilized these solutions, as we found that they could be kept m the ice-box for weeks without causing contaminations of the medium. Ordinarily we do not heat the agar after the dyes are added, but we have demonstrated that the stained agar can be heated a half hour in the Arnold sterilizer without injury. Carnot and Halle's Sand Tube Method. This method has had much praise. The method has been described by Gautier (1915) and Levy (1916) somewhat as follows: A pipette tube 33 cm. long by about 5 to 6 mm. in diameter is softened in the middle and the ends brought up to form a U. One of the arms thus formed is filled to a height of 10 cm. with very fine sand (passed through a No. 40 sieve). The other end is filled with hot bouillon and tinted with neutral red, which works up through the sand until the other tube is filled to about the same level. The arm in which there is no sand is inoculated with the sample and incubated for eighteen hours. .The most motile bacteris will penetrate the sand and appear in the other arm. Gautier found that B. coli occa- sionally did this. Agglutination reactions should be made to confirm the results of the sand tube. Other adaptations of the sand-tube method have been reported by Borzone and Carbone (1918) and Piazza (1916). Isolation of B. typhi from Feces. (Teague and Clurman's Method.) This method is especially adapted to specimens which must be kept for a short time before examination. The specimens are rubbed into a solution of 0.6 per cent sodium chloride containing 30 per cent of glycerol. This environment seems to be very destructive to the colon bacilli and other bacilli in stools but not harmful to the B. typhi. After this, the stool is streaked on plates made from Teague and Clurman's special eosin-brilHant green agar the preparation of which has been mentioned in the chapter on the preparation of media. Beckler (1918) reports that this method has given very satisfactory results at the labora- tories of the Massachusetts State Board of Health. One specimen is reported which gave negative results on immediate examination but positive results after holding in the presence of glycerol as advised by Teague and Clurman. This work is also confirmed by Benians (1918). MICROSPIRA CHOLERiE IN FECES 257 He claims that positive results are more likely after preserving the stool in glycerol especially in hot climates or countries. Isolation of Typhoid Bacilli from Urine. (Morishama and Teague's Method.) Streak two or three loops of the urine over an Endo or eosin- briUiant green medium plate; add to the urine about one-half its volume of nutrient broth and incubate the mixture over night. If the plate which has been inoculated directly with the urine is negative prepare dilution of the incubated urine next morning and streak on the above plates again. This latter procedure will furnish a higher percentage of positive results. Examination of Feces for Microspira CHOLBRiB Gedding's Method. It would be neither possible nor desirable to fix a technique limited by strict rules for the various operations of bac- teriological examinations, but the following general indicatipns may be recommended as permitting in the great majority of cases a positive diagnosis within twenty-four to thirty-five hours: 1. When mucous flakes are available for examination of microscopical investigation of the same, in stained preparations and in the hanging drop. 2. The isolation of the vibrios, employing for the purpose, agar media, at a temperature of 37° C. (a) Plant plates of ordinary suitably alkalinized agar and of Dieu- donne's medium, using, for the latter, a risiform particle, or an equiva- lent quantity of feces. (6) Plant in 50 c.c. of peptone solution 1 c.c. of fecal matter. After a stay of six hours in the incubator (or twelve to eighteen hours, if need be), at 37° C. take several loopfuls from the surface and plant with them several plates of Dieudonne medium and ordinary agar. (c) Investigate the agglutination reaction, using drops for the pur- pose, from the isolated colonies, the properties belonging to cholera vibrios, and secure pure cultures. 3. Demonstrate the character of the vibrios obtained in pure culture, by the reaction of agglutination or that of Pfeiffer. The conditions are much more favorable to the discovery of vibrios if pathological materials (feces or intestinal contents) are collected as early in the attack as possible, or secured from the cadaver as early as possible after death. Examinations made of the small quantity of material collected by a sound introduced into the rectum, in the living body, or from the cadaver are unreliable. It is sometimes possible to 258 INTESTINAL BACTERIA recognise that a person even in good health has undergone an attack of cholera by determining whether his blood serum gives with a genuine cholera vibrio the immunity reactions, viz., agglutination or the reaction of Pfeiffer. Teague and Travis' Method. Two pounds of beef are soaked in 2 liters of water over night. In the morning filter through cloth, heat in the Arnold steam sterihzer and filter through paper. Adjust the reaction to neutrality to litmus by means of sodium hydroxide. Inoc- ulate vdth B. coh and incubate for a few days. Then prepare nutrient agar from it by adding 1 per cent of Witte's peptone, 0.5 per cent of sodium chloride and clear with egg white. Adjust the reaction to 0.5 per cent alkaline, filter and add 0.25 per cent of nutrose. To 50 per cent of this nutrose agar add 1 per cent of sucrose and 1 c.c. of a 3 per cent solution of bluish eosin and 2 c.c. of a 1 per cent solution of Bismarck Brown. Microspira cholerce colonies will show brown centers after twenty-four hours, B, coK will show pale pink or yellowish colonies. Examination of Feces for B. Tuberculosis Petrof s Method. In order to remove the food particles, dilute with water and filter through gauze. Saturate the filtrate with sodium chloride and, at the end of a half hour, all of the bacteria will be found in this film. Collect the film and add sodium hydroxide, shake well and incubate at 22*^ C. for three hours. Then neutraUze to sterile Htmus paper, centrifuge and inoculate. Reh Method. Stir the lump of stool in an Esbach glass with suffi- cient sterile water to make a soft paste but solid enough not to flow when the glass is tilted. Add a Httle ether and shake after closing with a rubber stopper. Pour the ether into a centrifuge glass and centrifugahze. The ether is then decanted and the sediment examined after staining with ZiehFs method. Engleson's Method. Scrape the rectal mucosa with an ordinary sound. Make a smear and stain. IS0LA.TI0N OF Yeasts from Feces Anderson's Method. Prepare plates of Sabouraud's agar. By means of a platinum wire which has been dipped into the feces emulsion, touch one of these plates in Unes across the plate at distances of about 4 mm. Make as many rows as possible. The yeast colonies which develop in this way should be subjected to further study. BIBLIOGRAPHY 259 BIBLIOGRAPHY Anderson, H. W. 1917. Yeast-like Fungi of the Human Intestinal Tract. Jour. Inf. Diseases, 21, 341-386. Bacon, R. F. 1916. The Germicidal Efficiency of Some Copper Cements used in Dental Work. Dental Cosmos, 58, 4(1-49. Barker, L. F. 1914. The Diet of Typhoid Fever. Jour. Amer. Med. Assn., 63, 929-931. Basten, J. 1914, Methods of Determining the Bacterial Flora in Feceb of Suckling Infants and our Knowledge of the Bacterial Types. Zeit. Hygiene, 77, 282. Beckler, E. a. 1918. Use of Glycerine in the Examination of Feces for Typhoid Bacilli. Jour. Amer. Med. Assn., 70, 768. Benians, T. H. C. 1918. Preservation of Typhoid Bacilli in Stools. Lancet 1, 255, 1918. Jour. A. M. A., 70, 961; Amer. Jour. Pub. Health, 8 (1918), 396. Bergeim, 0. 1917. The Determination of Fecal Indole. Jour. Biol Chem., 32, 17-22. Blatherwick and Hawk. 1914. 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The Influence of Milk and Carbo- hydrate Feeding on the Character of Intestinal Flora. Cent. Bakt., 75, 365-388. Jour. Bact., 2, 47-71. Kendall, A. I. 1911-12. The Rapid Isolation of Typhoid, Paratyphoid and Dysentery BaciUi. Jour. Med. Res., 25, 95-99. Kendall, A. I. 1911. Activity of Bacteria in the Intestinal Tract* Jour. Med. Res., 25, 117-187. Kianowsky. 1891. Zur Frage uber die antimikrobiellen Eigengeshaft des Magensaftes. Cent. Bakt., 10, 236-237. Klein, A. 1900. Eine neue Mikroskopische Zahlungsmethode der Bakterien. Cent. Bakt. Orig., 27, 834. Klein, A. 1906. Ueber den Bakteriengehalt menschlicher und tierischer Feces. Arch. Hyg , 59, 283. KnGLER, I. J. 1915. Chemical Studies of the Relations of Oral Micro- organisms to Dental Caries. IV. A Biochemical Study and Differentiation of Oral Bacteria with Special Reference to Dental Caries. Jour. Allied Dental Societies, 10, 445-448, 282-330. KuRLOFF and Wagner. 1889. Ueber die Einwirkung des menschlichen Magensaftes auf Krankheits erregende Keime. Cent. Bakt,, 7, 448-450. Lagane, L, 1914. Action of Bile in Vitro on the Development of Intestinal Bacteria. Comp. Rend. Soc. Biol., 73, 242-243. Levy, M. D. 1916. The Use of the Sand Tube in the Isolation of the Typhoid Bacillus. Jour. Med. Assn., 66, 1022-1023. LuMSDEN, L. L. and Stimson, A. M. 1912. Examination of Excreta for Typhoid Bacilli. Reprint No. 80, Public Health Reports. MacConkey, a. 1907. Lactose Fermenting Bacteria in the Feces. Jour. Hyg., 7, 331; 5, 333. MacNeil, Latzee and Keee. Fecal Bacteria of Healthy Men. Jour. Inf. Dis., 6, 123-169; 571-609. Maeshall. 1916. An Acidimetric Study of the Saliva and its Relation to Dental Caries. Dental Items of Interest, 38, 116-127. Mattill, H. a. and Hawk, P. B. 1911. A Method for the Quantitative Estimation of Fecal Bacteria. Jour. Exper. Med., 14, 433-443. Mattill and Hawk. 1911. Studies on Water Drinking. IX. Distribution 262 INTESTINAL BACTERIA of bacterial and other forms of nitrogen and utilization of injected protein under the nifluence of copiou'^^ and moderate water drinking with meals. Jour. Amer. Chem. Soc , 33, 1999-2032. Neucki, M., Sieber, N., and Schoumowa, E. 1898. Die Entgiftung der Toxine durch die Verdauungssafte. Cent. Bakt., 23, 840-847. NiEDERGESAFS 1915. Anatomical, Bacteriological and Chemical Investi- gations of the Production of Dental Caries. Arch. Hyg., 84, 220-260. Osborne, T. B. and Mendel, L. B. 1914. The Contribution of Bacteria to the Feces after Feeding Diets Free from Indigestible Components. Jour. Biol. Chem., 18, 177. OzAKi. 1912. Zur Kenntniss der Anaeroben Bakterien der Mundhole. Cent. Bakt., 62, 76-98. Petroff. 1915. A New and Rapid Method for the Isolation and Cultivation of Tubercle Bacilli Directly from the Sputum and Feces with the Aid of Sodium Hydrate and Gentian Violet Egg Meat Juice Media. Science, 41, 661. Piazza. 1916. Sand Tube Method for Isolating Typhoid Bacilli from Stools. Jour. Amer. Med. Assn., 70, 1281-1282. Rahe. 1914. An Investigation into the Fermentation Activities of Aciduric Bacteria. Jour. Inf. Diseases, 15, 141-150. Rettger, L. F. 1915. The Influence of Milk Feeding on MortaHty and Growth, and on the Character of the Intestinal Flora. Jour. Exper. Med., 21, 365-388. Rettger, L. F. and Harton, G. D. A Comparative Study of the Intestinal Flora of White Rats Kept on Experimental and Ordinary Mixed Diet. Cent. Bakt. Abt. I., 73, 362-372, RoDELLA. 1908. Magen Carcinoma. Cent. Bakt. Abt. L, 47, 445-447. Roger, R. 1912. Influence on Microbic Fermentations of Carbohydrates. Arch. Med. Exp., 24, 461-488. Roger, R. 1913. Influence of Bile on the Putrefaction of Nitrogenous Mate- rial. Comp. Rend. Soc. Biol., 73, 274-276. Roger, R. 1913. Influence of Bile on Bacterial Fermentation. II. Fer- mentation of glycogen. Comp. Rend. Soc. Biol., 72, 544-545. Roger, R. 1913. Influence of Bile on Fermentation. IIL Fermentation of Glucose. Comp. Rend. Soc. Biol, 72, 603-604. Roger, R. 1913. Influence of Bile Extracts and Bile Salts on Bacterial Fermentations. Comp. Rend. Soc. BioL, 72, 656-657. Roger, R. 1915, Antiputrefactive Role of the Bile, Ann. Past. Inst., 29, 545-550. Sandberg. 1904. Ein Beitrag zur Bakteriologie der Milchsauer Garung im Magen mit besonderer Berucksichtigung der langen Bacillen. Zeit. f- klin. Med., 51, 80-94. Complete bibliography. Schmidt and Strassburger. 1905. Die Faeces des Menchen, Berlin. ScHMiTZ, C. 1893. Zur Kenntniss der Darmfaulniss. Zeit. PhysioL Che., 17, 401-403. BIBLIOGRAPHY 263 ScHULz-ScHULTZBNSTEiN. 1908. Zur Kenntni&s cler Einwirkung des mensch- lichen Magensekrete auf Choleravibrionen. Cent. Bakt. Orig., 30, 785. Smirnow. 1915. Germicidal Action of Dental Cements. Dental Cosmos. 57, 1209-1228. Steele, J. D. 1907. The Method of Determming the Total Amount of Fecal Bacteria by Weight and its Clinical Significance. Jour. Amer. Ivied. Assn., August 24, 1907. Stern. 1908. Ueber das Verhalten der Cholera-vibrionen dem menschlichen Mageninhalt gegenuber. Cent. Bakt., 47, 561, Abt. I. Str\ssburger. 1902. Untersuchungen uber die Bakterienmengen in mensch- lichen Feces. Zeit. klin. Med., 46, 413. Strauss. 1909, Ueber die Abhangigkeit der Milch sauer Gahrung vom HCl Gehalt des Magensaftes. Zeit. klin. Med., 28, 567-578. Tanner, F. W. 1917. Studies on the Bacterial Metabolism of Sulphur. I. Formation of hydrogen sulphid from certain sulphur compounds under aerobic conditions. Jour. Bact., 3, 565-593. Tanner, F. W. 1918. Studies on the Bacterial Metabolism of Sulphur. II. Formation of hydrogen sulphid from certain sulphur compounds by yeast- like fungi. Jour. Amer. Chem. Soc, 40, 663-669. Teague, 0. and Ci^tjrman, A. W. 1916. A Method of Preserving Typhoid Stools for Delayed Examination and a Comparative Study of the Efficacy of Eosin Brilliant Green Agar and Endo Agar for the Isolation of Typhoid Bacilli from Stools. Jour. Inf. Diseases, 18, 653-671. Teague, 0. and Clurman, A. W. 1916. An Improved Brilliant Green Cul- ture Medium for the Isolation of Typhoid Bacilli from Stools. Jour. Inf. Diseases, 18, 674-652. Teague, 0. and Travis, W, C. 1916. A Differential Culture Medium for the Cholera Vibrio. Jour. Inf. Dis., 18, 601-605. Toi0A, R. 1914. Sterility of the Bile under Normal Conditions and its Bac- tericidal Action on Pathogenic Bacteria. Chem. Abstracts, 8, 2558-2559. TuRESSON, G. 1916. The Presence and Significance of Molds in the Intestinal Canal of Man and Animals. Exp. Sta. Rec, 35, 559-560. Washburn and Goadby. 1896. Some Points in Connection with the Bac- teria in the Mouth. Trans. Odontological Soc. Great Brit., 1896. Williams. 1899. A Contribution to the Bacteriology of the Human Mouth. Dental Cosmos. 1899. Wohman, E. 1912. The Amylolytic Bacteria of the Intestines. Am. Past. Inst., 26, 610-624. CHAPTER IX BACTERIAL EXAMINATION OF AIR The bacteriological examination of air has not been of much sanitary significance. Many investigations have been carried out and different apparatuses devised to remove the bacteria from a definite quantity of air. A review of these methods has been given by Besson and those wishing a rather complete historical survey should consult that treatise. Ruehle (1915) has devised the most satisfactory technique and probably his method represents the best which has been devised up to the present. Much of the matter presented in this chapter has been taken from his pubUcation. This method involves the use of the aeroscope, which is regarded as an apparatus for gathering bacteria from the air. The other accessories to the procedure are not included in this procedure. Rettger (1910) made one of the first important advances in the bacterial examination of air. He used 5 c.c. of physiological salt solu- tion as the filtering medium in a special aeroscope described as follows: The entire apparatus consists of a glass tube with a small round bulb at the end. The bulb has eight or ten smaU perforations which serve the purpose of allowing the air to pass through at a rapid rate and divide the gas to such an extent that every particle of it is brought into close contact with the filtering fluid. This glass tube or aeroscope is fitted into a small, thick-walled test tube by means of a rubber stopper, which also bears, besides the aeroscope, a short glass tube bent at right angles. The upper end of the aeroscope is at an angle of about 45°, in order to prevent bacteria and particles of dust from falling into the open end of the tube, and still permit of the tube being drawn through the stopper without difiSiculty. The standard aeroscope has been described by Ruehle as follows: A 10 mm. layer of sand which has been passed through a 100-mesh sieve and has been retained by a 200-mesh sieve is supported within a cylindrical glass tube 70 mm. in length and 15 mm. in diameter upon a layer of bolting cloth folded over the end of a rubber stopper. Through a perforation in the stopper, there passes a tube 6 mm. in diameter and 40 mm. in length. This tube is attached to the aspirator bottle. The upper end of the cylindrical tube is closed by a perforated rubber stopper through which is passed a glass tube 40 mm. in 264 AEROSCOPES 265 length and 6 mm. in diameter bent at an angle of 45® in order to prevent precip- itation of bacteria or dust particles into the aeroscope. In using this aeroscope a measured volume of air is filtered through the tube, the sand shaken out into 10 c.c. of water and ahquot portions of this suspension plated on nutrient agar. The " modified " form of the aeroscope differs very little. The lower rubber stopper and the bolting cloth is eliminated by fusing a small tube into the large one. A layer of cotton supports the sand. standard Aeroscope Modified Standard Aeroscope Rettger Aeroscope Fig. 57. — ^Types of Aeroscopes. (After Ruehle.) In order to render the apparatus capable of being sterilized by hot air, the upper stopper is made of cork. The Committee on Standard Methods for the Examination of Air of the American PubUc Health Association recommended the following procedure: The number of bacteria in the air does not appear to be a factor of any great significance. In special cases, however, as in the study of dairy conditions, it may be of interest. The sand filter method originally used by Petri has proved essentially sound 266 BACTERIAL EXAMINATION OF AIR in principle. It wab recommended in our 1909 report and again, after careful studies of altertive processes in 1912. One modification has been made in the apparatus since 1912 which is so clearly an improvement as to warrant immediate adoption. The layer of sand in the filters as previously used was supported on a perforated rubber stopper in a straight section of glass tubing, and the aspiration was apphed to a small glass tube passing into the rubber stopper. It was clearly essential that the stopper should fit tightly, which meant that the apparatus must be sterilized in steam. Ruehle (1915) points out that the steam sterilization causes caking of the filter sand and thus introduced appreciable errors in analysis. Ruehle, therefore, made the suggestion that the rubber stopper should be eliminated and the small aspirating tube fused into the large one. We, therefore, recommend that for the study of bacteria in air there should be used a glass tube of 15 mm. in diameter and 70 mm. long with a smaller tube 6 mm. in diameter and 40 mm. long fused into one end. On the shoulder of the joint between the tubes rests a plug of cotton supporting a layer of sand 10 mm. deep. The sand should be capable of passing a 100-mesh sieve but not a 200-mesh sieve. The opposite or inlet end of the larger tube is stoppered by a cork stopper (which need not be exactly tight) perforated by a glass tube 6 mm. in diameter and 40 mm. long bent at an angle of 45° to prevent direct precipi- tation of dust particles into the filter tube. Five cu. ft. of air should be drawn through the filter by the use of an aspira- tor of known volume, preferably one of the double or continuous type, or by the' use of some form of pump or meter. A convenient and ingenious samphng pump was made by Wallace and Tiernan of New York City for the study of Basker- ville and Winslow (1913) of the air of New York City schools. After filtra- tion, the sand should be shaken out into 10 c.c. of sterile water, and, after thor- ough shaking, aliquot portions of the water should be plated on ordinary nutrient agar. Browne (1917) has proposed a tube which he claims has special advantages over the aeroscope used in standard methods. This con- sists of the filter tube and dilution tube fused together at right angles. During filtration the sand is placed over the stoppered end. After fil- tration the sand is shaken into the other end and the filter plug with the bolting cloth is replaced by a sterile stopper. Ten c.c. of sterile water are added and the sand thoroughly shaken. From this decimal dilutions are plated on standard media. BIBLIOGRAPHY Baskerville, C. and Winslow, C. E. A. 1913. The Air of New York City Schools. Report of the Committee on School Enquiry Board of Esti- mate and Apportionment, City of New York. Ill, 611. BIBLIOGRAPHY 267 Beowne, W. W. 1917. Improved Technique in Bacterial Air Analysis. Amer. Jour. Pub. Health, 7, 52-53. Gordon, M. H. 1902-3. Report of a Bacterial Test for Estimating Air Pol- lution. Annual Report of the Local Government Board, containing the Report of the Medical OfHcer for 1902-3, 421. Rettger, L. F. 1910. A New and Improved Method for Enumerating Air Bacteria. Jour. Med. Res., 17, N. S., 461-468. RuEHLE, G. L. A. 1915. Methods of Bacterial Analysis of Air. Jour. Ag. Res., 4, 343-368. RuEHLE, G. L. A. and Kulp, W. L. 1915. Germ Content of Stable Air and its Effect on the Germ Content of Milk. Bull. 409, N. Y. Agric. Exp. Sta., Geneva. WiNSLOW, C. E. A. and Browne?, W. W. 1914. The Microbic Content of Indoor and Outdoor air. Monthly Weather Review, 42, 452. mr A T>TT?T? 'V L/Xli\Jr 1 ShSX A. WATER HYGIENE That water has a close connection with the spread of disease needs no special emphasis. Johnson (1916) has given ample proof of this by collecting statistics from the large registration cities of the United States. (Fig. 59.) TYPHOID FEVER AT PITTSBURGH PA. 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 1900 1901 1902 1903 1904 " 1905 1906 1907 1908 1909 mO Fig. 58.— Showing the Effect of a Pure Water Supply on the Death Rate from a Typhoid Fever in Pittsburg. (After McLaughlin, 1912.) The ** Mills-Reincke Phenomenon '* and the " Hazen Theorem." In 1893, soon after tlie establishment of the Lawrence, Mass., filtration plant, Mills, the city engineer, noticed a reduction in the general death rate along with a reduction in the typhoid fever death rate. This was also noticed for Hamburg, Germany, by Reincke. When Sedgwick and 268 o Pi 8 r-i U 3G 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2h - — y ^ \ - ^ / "" "••-\ \CJ^/y - Y^%^ ^ - - \ /•I \ - - -x\ . ~ - Y<^ - - GROWTH OF WATER FILTRATION AND DECREASE OF TYPHOID FEVER DEATH RATE IN THE REGISTRATION CITIES OF UNITED STATES ^ C'- - - \ % y^ „ - \ \ \ N \ - - Vv - " \ - - 1! — ^ x. - - ./' V - - ./" - - ^^ ^ - ^o-t ml ^ - - -;^i\tO U:i (a^i. - - ■ - - - o tH CM CO -^ lO <© o o O o O o o OS OS o 05 CJi o OS T-l TH T-i iri y-\ I-l >-< o CD o e3s cs o (M CO ■<* tH rH tH OJ C5 OS r-l rH rH 30 34 32 30 28 26 22 20 18 16 14 12 10 8 6 4 tH OS Fig. 59. — Relation of Water Filtration to Typhoid Fever. (After Johnson.) AVERAGE TYPHOID FEVER DEATH RATE FOR FIVE YEARS BEFORE AND FIVE YEARS AFTER FILTRATION ALL CITIES OF U S. OVER 100 000 POPULATION IN 1915 ■■■Bl Pittsburs: \' a.^'/^a\ Albany U.^^^./^^ coiumbiis v:^v/; A Philadelphia YfTT^^^^A , ,,.. Z&4,y., IiOUlSVillO Cincinnati Washington Atlanta Beading Indianapolis New Haven l,:,:^,i^;\/'/ ;\ New Orleans yyyiy.ii^W//^A Toledo WW/Mt/MM Patersoa ^2^ Scranton f^T^ Providence ^^^'^^/^ mm Explanation ■■i Before Filtration X^rf/^^ After Filtration Average of Above M^J^2Wm Average Reduction =61 ^ Total Population of Above Cities 5,450,000 Fig. 60.—Typhoid Fever Death Rate in American Cities. (After Johnson.) CHEMICAL VS BACTERIAL EXAMINATION 271 Sawyer (1915) quotes an instance of a water-borne epidemic which could have been prevented had it been possible to cany out a sanitary inspec- tion. In a certain water supply indications of pollution appeared two months before the epidemic broke out. Could a sanitary inspection have been made to investigate the conditions indicated m the laboratory resultS; seven deaths and a large number of cases of typhoid fever could have been prevented. In many cases it would not be necessary to make laboratory examinations because the envn^onmental factors would indicate a bad water. In such a case, the laboratory might be used to check up the remedial measures which had been carried out. The field investigations would also yield information with regard to the endemic disease which occurs in a community. Where possible, both field studies and laboratory studies should be made but where one is possible, the sanitary inspection will make it unnecessary, very often to carry out the laboratory examination. Pure Water, Chemically water is a monoxide of hydrogen and is made chemically pure only with great difficulty. Only the greatest care in technique will yield a water which is free from all traces of metals and gases. Bacteriologically speaking, a water is pure when it contains no bac- teria which may cause disease or sickness. This is a difficult term to define since a water which is pure for one individual may be impure for another. The technique which is available for isolating certain path- ogenic bacteria is too crude to give accurate knowledge with regard to purity. Negative results may mean little more than that the certain bacteria sought for, were not foune extreme opinions and may not repre- sent the opinions of the sanitarians in each country. Race (1914, 1916) argues that B. coli is a reliable indicator of pollution and states that those cities which have many B. coli in their water usually have a high typhoid fever death rate. He warns that B. coli should be regarded as merely an inferential indicator. The fact that the presence of B. coli in a sample of water may not be sufficient evidence of serious pollution, it is, how- ever, a danger signal which must not be overlooked. Houston (1915) representing the English practice regards 5. coli as the best index for the control of purij&cation processes. Winslow (1916), in tracing the history of this organism as an indicator has advised the accumulation of as much data as possible to more definitely determine whether J5. coli is purely a fecal form and B. aerogenes a saprophytic form. The testing of water for this organism has taken some recent advances. Thanks to the research of Rogers and his co-workers in the laboratories of the Bureau of Animal Industry, we have now fairly rehable methods for differentiating between fecal and non-fecal B, coli. The methods of analysis which are given in the following pages have been taken with permission from the 1917 report of the committee on water and sewage analysis of the American Public Health Association. American Public Health Association Method for Determining the Presence of Members of the B. coli Group. It is recommended that the jB. coli group be considered as including all non-spore-forming bacilli which ferment lactose with gas formation and grow aerobically on standard solid media. The formation of 10 per cent or more of gas in a standard lactose broth fermentation tube within twenty-four hours at 37^ C. is presump-* tive evidence of the presence of members of the B. coli group, since the majority of the bacteria which give such a reaction belong to this group. The appearance of aerobic lactose-splitting colonies on lactose-litmus- agar or Endows medium plates made from a lactose-broth fermentation tube in which the gas has formed confirms to a considerable extent the presumption that gas-formation in the fermentation tube was due to the presence of members of the B, coli group. To complete the demonstration of the presence of B. coli, as above defined, it is necessary to show that one or more of these aerobic plate colonies consists of non-spore-forming bacilli which, when inoculated into a lactose-broth fermentation tube, forms gas. It is recommended that the standard tests for the S. coli group be TESTS FOR B COLI 301 either (A) the Presumptive, (B) the Partially Confirmed, or (C) the Completed test, as hereafter defined, each test being applicable under the circumstances specified, A. Prestjmptive Test 1. Inoculate a series of fermentation tubes with appropriate gradu- ated quantities of the water to be tested. 2. Incubate these tubes at 37° C. for forty-eight hours. Examine tube at twenty-four and forty-eight hours, and record gas-formation. The records should be such as to distinguish between: (a) Absence of gas-formation. (b) Formation of gas occupying less than 10 per cent (10%) of the closed arm. More detailed records of the amount of gas formed, though desirable for purposes of study, are not necessary for carrying out the standard tests prescribed. 3. The formation within twenty-four hours of gas occupying more than 10 per cent (10%) of the closed arm of fermentation tube con- stitutes a positive presumptive test, 4. If no gas is formed in twenty-four hours, or if the gas formed is less than 10 per cent (10%), the incubation shall be continued to forty- eight hours. The presence of a gas in any amount in such a tube at forty-eight hours constitutes a doubtful test, which, in all cases, requires confirmation. 5. The absence of gas-formation after forty-eight hours' incubation constitutes a negative test. (An arbitrary limit of forty-eight hours' observation doubtless excludes from consideration occasional members of the B. coli group which forms gas very slowly, but for the purposes of a standard test the exclusion of these occasional slow gas-forming organ- isms is considered immaterial) B. Partially Confirmed Test 1. Make one or more Endo's medium or lactose-litmus-agar plates from the tube which, after forty-eight hours' incubation, shows gas- formation from the smallest amount of water tested. For example, if the water has been tested in amounts of 10 c.c, 1 c.c, and 0.1 c.c, and gas is formed in 10 c.c, and 1 c.c, and not in 0.1 c.c, the test need be confirmed only in the 1 cc amount.) 2. Incubate the plates at 37*" C, eighteen to twenty-four hours. 302 WATER HYGIENE 3. If typical colon-like red colonies have developed upon the plate within this period, the confirmed test may be considered positive. 4. If, however, no typical colonies have developed within twenty- four hours, the test cannot yet be considered definitely negative, since it not infrequently happens that members of the B. coh group fail to form typical colonies on Endo's medium or lactose-litmus-agar plates, or that the colonies develop slowly. In such cases, it is always neces- sary to complete the test as directed under C 2 and 3. C. Completed Test 1. From the Endo's medium or lactose4itmus-agar plate, made as prescribed under B, fish at least two typical colonies, transferring each to an agar slant and a lactose broth fermentation tube. 2. If no typical colonies appear upon the plate within twenty-four hours, the plate should be re-incubated another twenty-four hours, after which at least two of the colonies considered to be most likely B. coh, whether typical or not, shall be transferred to agar slants and lactose broth fermentation tubes. 3. The lactose broth fermentation tubes thus inoculated shall be incubated until gas formation is noted; the incubation not to exceed forty-eight hours. The agar slants shall be incubated at 37^ C. for forty-eight hours, when a microscopic examination shall be made of at least one culture, selecting one which corresponds to one of the lactose broth fermentation tubes which has shown gas-formation. The formation of gas in lactose broth and the demonstration of non-spore-forming bacilli in the agar culture shall be considered a sat- isfactory completed test, demonstrating the presence of a member of the B, coll group. The absence of gas-formation in lactose broth or failure to demon- strate non-spore-forming baciUi in a gas-forming culture constitutes a negative test. Application of Presumptive, Partially Confirmed, and Completed Tests. A. The Presumptive Test. 1. When definitely positive, that is showing more than 10 per cent (10%) of gas in twenty-four -hours, is sufficient, (a) As applied to all except the smallest gas-forming portion of each sample in all examinations. (6) As applied to the smallest gas-forming portion in the examination of sewage or of water showing relatively TEST FOR B COLI 303 high pollution, such that its fitness for use as drink- ing water does not come into consideration. This applies to the routine examinations of raw water in connection with the control of the operation of puri- fication plants. 2. When definitely negative, that is, showing no gas in forty-eight hours, is final and, therefore, sufficient in all cases. 3. When doubtful, that is, showing gas less than 10 per cent (10%) (or none) in twenty-four hours, with gas either more or less than 10 per cent in forty-eight hours, must always be confirmed. jB. The Partially Confirmed Test. 1. When definitely positive, that is, showing typical plate colonies within twenty-four hours, is sufficient. (a) When applied to confirm a doubtful presumptive test in cases where the latter, if definitely positive, would have been sufficient. (6) In the routine examination of water-supplies where a sufficient number of prior examinations have estab- lished a satisfactory index of the accuracy and sig- nificance of this test in terms of the completed test. 2. When doubtful, that is, showing colonies of doubtful or negative appearance in twenty-four hours, must always be com- pleted. C. The Completed Test. The completed test is required as applied to the smallest gas- forming portion of each sample in all cases other than those noted as exceptions under the ^* presumptive " and the '' par- tially confirmed '' tests. The completed test is required in all cases where the confirmed test has been doubtful In order that tests for 5. coU may have a quantitative significance, the following general principles and rules should be observed: Ordinarily not less than three portions of each sample should be tested, the portions being even decimal multiples of fractions of a cubic centimeter; for example, 10 c.c, 1 c.c, 0.1 e.c, .01 c.c, etc. It is essential that the dilutions should be such that the largest amount gives a positive test (unless the water is such as to give negative tests in 10 c.c), and the smallest dilution, a negative result. To insure this result, it is often necessary to plant four or five dilutions, especially in 304 WATER HYGIENE the examination of a sample of cntn*ely unknown quantity. The quan- titative value of a series of tests is lost, unless all or, at least, a large portion of the smallest dilutions tested have given negative results. In reporting a single test, it is preferable merely to record results as observed, indicating the amounts tested and the result in each, rather than to attempt expression of the result in numbers of JS. coh per cubic centimeter. In summarizing the results of a series of tests, however, it is desirable for the sake of simplicity to express the results in terms of the numbers of J5. coli per cubic centimeter or per 100 c.c. To con- vert the result of fermentation tests to this form, the result of each test is recorded as indicating a number of B. coli per cubic centimeter equal to the reciprocal of the smallest decimal or multiple fraction of a cubic centimeter giving a positive result. For example the result: 10 c.c. plus; 1 c.c. plus; 0.1 c.c. minus would be recorded as indicating one B, coh per 3ubic centimeter. An exception should be made in the case where a negative result is obtained in an amount larger than the smallest portion giving a positive result; for example, in a result such as 10 c.c. plus; 1 c.c. minus; 0.1 c.c. plus. In such a case, the result should be recorded as indicating a number of B, coh per cubic centimeter equal to the reciprocal of the dilution next larger than the smallest one giving a positive test, this being a more probable result. Where tests are made in amounts larger than 1 c.c. giving an average of results less than one B, coh per cubic centimeter, it is convenient to express results in terms of the number of B, coU per 100 c.c. The following table illustrates the method of recording and aver- aging results of B, coh tests Result of Tests m Amounts Designated Indicated No of B coh 10 c.c. 1 c.c. 1 cc 01 c.c. per c c. per 100 c.c. plus minus minus minus 1 10 plus plus minus minus 1 100 plus plus plus minus 10 1000. plus plus plus plus 100 10,000 plus plus minus plus 10 1,000. 121 1 12,110 24 2,422 The above method of expressing results is not mathematically alto- gether correct. The average number of B, coh per cubic centimeter, as thus estimated is not precisely the most probable number calculated by ROUTINE PROCEDURES 305 application of the theory of probability. To apply this theory to a correct mathematical solution of any considerable series of results involves, however, mathematical calculations so complex as to be im- practicable of apphcation in general practice. The simpler method given is, therefore, considered preferable, since it is easily applied and the results so expressed are readily comprehensible. In order that the results as reported may be checked and carefully valuated, it is necessary that the report should show not only the aver- age number of B. coh per cubic centimeter, but also the number of sam- ples examined; and for each dilution, the total number of tests made, and the number (or per cent) positive. Routine Procedures for Examination of Samples of Water First Day. 1. Prepare dilutions as required. 2. Make two (2) gelatin plates from each dilution, and incubate at 20° C. 3. Make two (2) agar plates from each dilution, and incubate at 37° C. 4. Inoculate lactose broth fermentation tubes with appropriate amounts for B. coli tests, inoculating two (2) tubes with each amount. Note. Where repeated tests are made of water from the same source as is customary in the control of public suppUes, it is not necessary to make duplicate plates or fermentation tubes in each dilution. It is sufficient, in such circumstances, to make duplicate plates only from thb dilution which will most probably give from 25 to 250 colonies per plate. Second Day. 1. Count the agar plates made on the first day. 2. Eecord the number of additional fermentation tubes which show 10 per cent (10%) or more of gas. Note. In cases only the presumptive test for B. coli is required fermentation tubes showing more than 10 per cent (10%) of gas at this time may be discarded. Third Day. 1. Count gelatin plates made on the first day. 2. Record the additional number of fermentation tubes which show 10 per cent or more of gas. 306 WATER HYGIENE 3. Make a lactose-htmus-agar of Endo's medium plate from the smallest portion of each sample showing gas. Incubate plate at 37° C. Note. In case the smallest portion in which gas has been formed shows less than 10 per cent (10%) of gas it is well to make a plate also from the next largest portion so that in case the smallest portion gives a negative end result, it may still be possible to demonstrate B, coh in the next larger dilution. Fourth Day. 1. Examine Endo's medium on lactose-htmus-agar plates If typical colonies have developed, select two and transfer each to a lactosc-broth-fermentation tube and an agai slant, both of which are to be incubated at 37° C. 2. If no typical J3. coh colonics are found, incubate the plates another twenty-four hours. Fifth Day. 1. Select at least two colonies, whether typical or not, from the Endo's medium or lactose-htmus-agar plates which have been incubated an additional twenty-four hours; transfer each to a lactosc-broth-fermentation tube and an agar slant, and com- plete the test as for typical colonies. 2. Examine-lactose-broth fermentation tubes inoculated from plates on the previous day. Tubes in which gas has been formed may be discarded after the result has been recorded. Those in which no gas has formed should be incubated an additional twenty-four hours. Sixth, Day. L Examine lactosc-broth-fermentation tubes reincubated the previ- ous day. 2. Examine microscopically agar slants corresponding to lactose-fer- mentation tubes inoculated from plate colonies and showing gas formation. Methyl Red Test. As sanitary knowledge increased and it became apparent that B. coli or B. coZ^-like bacteria were present on substances which had not received fecal pollution, studies were made to detect those B. coh which' came from fecal sources. To this end Rogers (1915) and his co-workers found that the gas ratio separated B. coli of fecal origin from B. coh of non-fecal origin. B. coU from bovine feces fer- mented dextrose with the formation of equal volumes of carbon dioxide VOGES-PROSKAUER REACTION 307 and hydrogen while those from grains formed two or three times as much carbon dioxide. The next step was to j&nd a simple procedure for determinmg this ratio or findmg some procedure which was correlated with it since the determination of the gas ratio is too exacting for routine analyses. Clark and Lubs (1915) working in the same laboratory dis- covered a constant correlation between the gas ratio and the hydrogen ion concentration. The B. coh from bovine feces were found to possess a higher hydrogen ion concentration than those from grains or non- fecal origins. A study of indicators yielded the information that methyl red was yellow with the high ratio group and red or acid with the low ratio group. Levine (1916) correlated this test with the Voges-Pros- kauer reaction. Voges-Proskauer Reaction. This has been studied by Levine who correlated it with the methyl red test. He found the bacteria which give the Voges-Proskauer reaction were rarely found in feces and that the Voges-Proskauer reaction, like the high gas ratio and the alkalinity to methyl red, is characteristic of non-fecal strains and, therefore, of much sanitary significance. Harden (1901), in his various publications, has reported his studies on the chemistry of this reaction which are well reviewed by Levine. The red color which results is due to a definite end product in the fermentation of glucose. Voges and Proskauer (1898), in their original publication, describe the test as follows: ^' On addition of caustic potash, we observed a new and interesting color reaction. If the tube be allowed to stand twenty-four hours and longer at room temperature, after the addition of the potash, a beautiful fluorescent color somewhat similar to that of a dilute alcoholic solution of eosin forms in the culture fluid particularly at the open end of the tube exposed to the air. We have investigated a few of the properties of this coloring substance which is not produced by the action of the alkali on the sugar and havQ found that it is fairly resistant to the action of the external air. , After a time, however, it becomes paler, and finally gives place to a dirty greenish brown.'' Harden and Walpole (1905-6) could not account for all of the car- bon in the dextrose by the ordinary products (organic acids, alcohol, etc.) along with these substances was secured a glycol which was made up of 2 : 3 butylene glycol. HTT TT TT Jtl JJL JjL Vj V> Vv Kj 11 H OH OH H 308 WATER HYGIENE This was oxidized to acetyl-methyl-carbinol. H H OH H— C— C C— C— H H OH H When this was mixed with potassium hydroxide, in the presence of peptone, an eosin-Uke color was obtained after a period. Walpole (1910) found that under aerobic conditions Bacillus aerogenes gave a greater amount of acetyl-methyl-carbinol. The coloration was not secured when butylene glycol or acetyl-methyl-carbinol were mixed with potassium hyrdoxide. Harden (1905) later explained this by finding that the acetyl-methyl-carbinol was oxidized to diacetyl CH3CO — CO — CHc. This, in some way, acted with the pepton to give a pink color. Harden and Norris (1911) later secured the same pink color between diacetyl and arginine, dicyanamide, creatin and gaunidine acetic. They state that the reaction depends upon the group NH=C — N=HR, R has not NH2 been determined. Levine demonstrated that the Voges-Proskauer reaction had much sanitary significance and that it would distinguish between fecal and non-fecal strains of Bacillus colon. He states that the natural habitat of those strains which form the acetyl-methyl-carbinol is probably the soil. Bacillus coli from non-fecal origins would then be methyl red and V-P plus. Standard Methods gives the following procedure for the differen- tiation of fecal from non-fecal members of the B. coli group. American Public Health Association Method for the Differentiation of Fecal and Non-fecal B. coli. (1) At least ten cultures should be used. If possible these should be sub-cultured from plates made direct from the water since all the cultures obtained by plating from fermenta- tion tubes may be descendants of a single cell in the water. If cul- tures from water plates are not available those obtained from the plates made as prescribed under B may be used. (2) Inoculate each culture into dextrose potassium phosphate broth,* * The preparation of this medium has been described in the chapter on media. Since devismg this medium, these investigators (Clark and Lubs, 1917) have de- scribed the preparation of a substitute. The new medium is a synthetic solution without Witte's peptone. Aspartic acid is used in its place, thus satisfying the nitrogen requirements and acting as a buffer. The preparation of this new medium is also given in the chapter on media. FECAL AND NON-FECAL B. COLI 309 adonite broth and gelatin. For additional confirmatory evidence inoculation may be made into tryptophane broth and saccharose broth. The dextrose broth must be incubated at 30°. Other sugar broths may be incubated at 30° or 37° as convenient. Gelatin should be incubated at 20°. (3) After forty-eight hours record gas formation in adonite and saccharose broths. Determine indol formation in tryptophane broth by adding, drop by drop, to avoid mixing with the medium, about 1 c.c. of a 2 per cent alcoholic solution of p-dimethyl amido-benzaldehyde, then a few drops of concentrated hydrochloric acid. The presence of indol is indicated by a violet color. (4) After five days apply methyl red test and Voges-Proskauer test to dextrose broth. Methyl Red Test Indicator Solution. Dissolve 0.1 gm. methyl red in 300 c.c. alcohol and dilute to 500 c.c. with distilled water. Procedure in Test. 1. To 5 c.c. of each culture add five drops of methyl red solution. 2. Record distinct red color as methyl red+, distinct yellow color as methyl red—, and intermediate colors as ?. Voges-Proskauer Test To the remaining 5 c.c. of medium add 5 c.c. of a 10 per cent solution of potassium hydroxide. Allow to stand over night. A positive test is indicated by an eosin pink color. (5) Gelatin tubes should not be pronounced negative until they have been incubated at least fifteen days. The following group reactions indicate the source of the culture with a high degree of probability. Methyl red+ Voges-Proskauer — Gelatin- i ^ ,. .. . . . . , ., I B, coh of fecal origm Adonite— Indol, usually- Saccharose, usually— 310 WATER HYGIENE Meuhyl red— Voges-Eroskauer+ Gelatin— Adonite+ Indol, usually + Saccharose + Methyl red— Voges~Proskauer+ Gelatin— Adonite— Indol, usually— Saccharose + Methyl red— Voges-Proskauer+ Gelatin+ Adonite + Indol, usually— Saccharose + 5. aerogenes of fecal origin B. aerogenesj probably not of fecal origin ^ B. cloacce, or may not be of fecal origin The Treasury Department Standard for the Examination of Water on Interstate Common Carriers. The following method for the exam- ination of water on Interstate common carriers has been formulated by a committee of prominent sanitarians. The permissible limits of bacteriological impurity are stated as follows: 1. The total number of bacteria developing on standard agar plates, in- cubated twenty-four hours at 37° C, shall not exceed 100 per cubic centimeter; provided, that the estimate shall be made from not less than two plates, showing such numbers and distribution of colonies as to indicate that the estimate is reliable and accurate. 2. Not more than one out; of five 10 c.c. portions of any sample examined shall show the presence of organisms of the Bacillus colt group when tested as follows: (a) Five 10 c.c. portions of each sample tested shall be planted, each in a fermentation tube containing not less than 30 c.c. of lactose peptone broth. These shall be incubated forty-eight hours at 37° C. and observed to note gas formation. (b) From each tube showing gas, occupying more than 5 per cent of the closed arm of fermentation tube, plates shall be made after forty-eight hours' incubation, upon lactose litmus agar or Endows medium. (c) When plate colonies resembling B. coU develop upon either of these plate media within twenty-four hours, a well-isolated characteristic colony shall be TREASURY DEPARTMENT STANDARD 311 fished and transplanted into a lactose-broth fermentation tube, which shall be incubated at 37° C. for forty-eight hours. For the purposes of enforcing any regulations which may be based upon these recommendations the following may be considered sufficient evidence of the presence of organisms of the Bacillus colt group. Formation of gas in fermentation tube containing original sample of water (a). Development of acid-forming colonies on lactose-litmus-agar plates or bright red colonies on Endo's medium plates, when plates are prepared as directed above under (6). The formation of gas, occupying 10 per cent or more of closed arm of fer- mentation tube, in lactose peptone broth fermentation tube, inoculated with colony fished from twenty-four-hour lactose litmus agar or Endows medmm plate. These steps are selected with reference to demonstrating the presence in the samples examined of aerobic lactose-fermenting organisms. 3. It is recommended, as a routine procedure, that, in addition to five 10 c.c. portions, one 1 c.c. portion, and one 0.1 c.c. portion of each sample examined be planted in a lactose peptone broth fermentation tube in order to demonstrate more fully the extent of pollution in grossly polluted samples. 4. It is recommended that in the above-designated tests the culture media and methods used shall be in accordance with the specifications of the'Committee on Standard Methods of Water Analysis of the American Public Health Asso- ciation, as set forth in Standard Methods of Water Analysis (A. P. H. A., 1912), This standard has been appUed to the examination of water from trains by Bartow (1916), Creel (1914), Hanford (1916), and to water from boats by Cobb, Williams and Letton (1916). Bartow confirmed the presence of B. coli in 83 per cent of the samples examined. Creel found that an anaerobic bacillus which formed gas was responsible for gas in the presumptive test in 91 out of 421 samples. Such data indicate the significance of the confirmatory test. More data is required before it may be determined whether this method is the best. Letton (1917) has later stated that the requirement of '' not more than 100 colonies per cubic centimeter on agar '* of the Treasury Department standard is very lenient. He regards the limit of permissible B. coli (not more than two per 100 c.c.) as not too low. Lactose Bile in Water Analysis. Lactose bile was advocated for some time for use in the presumptive test for B, coli. It was supposed to inhibit other bacteria and a few of the weaker types of 5. colL For that reason it was deemed superior for the presumptive test than the ordinary carbohydrate broths. Jordan (1915) found that the typical as well as the typical B. coli were inhibited. The work of Cummings (1917) for the United States Public Health Service is interesting in this connection. He found, in his stream pollution studies on the Potomac 312 WATER HYGIENE river that lactose broth was the better medium. In 1851 parallel sets of lactose broth and lactose bile tubes, lactose broth gave higher results, confirmed as well as presumptive, than lactose bile. It was granted, however, by Cummings that, in the lower reaches of the river, remote from pollution, the bile tubes were more reliable. The opinion of other bacteriologists has been that for highly polluted waters the lactose broth was the better medium, and for sUghtly polluted waters lactose bile gives the better results. This is attributed to the presence of spore- forming lactose fermenting anaerobic bacteria which are not manifest when the number of B. coU are large. Obst (1916) also found that the bile inhibited about one-half of the typical B. coU, The general use, then of lactose broth in place of lactose bile seems to be justified. Hauser (1917) has described the method used for isolating and iden- tifying jB. coli at the Cincinnati filtration plant. This procedure is interesting and valuable because it is representative of the practice in one of the largest water-treatment plants. The B. coli group may be defined as aerobic non-spore forming Gram negative bacilli, fermenting lactose with production oi gas, and not liquefying gelatin. At the Cincinnati filtration plant the attempt is made to carry out the examina- tions so that the above definition is fulfilled as completely as possible, consistent with the speed and facility necessitated by routine water analysis. The particular water samples under examination are planted into lactose broth. Lactose broth is used rather than lactose bile, which is the medium recommended in the 1912 edition of the Standard Methods of Water Analyses of the A, P.H. A., for the reason that, not only at this laboratory, but at the labora- tory of the Ohio River Investigation of the United States Public Health Service and other laboratories, it has given consistently a higher percentage of con- firmed end-results from original inoculations. In planting the water sample decimal dilutions are used to facilitate computations of the B. coli index. In routine examinations, at least three dilutions are used in order to obtain, if pos- sible, a negative and a positive test. The formation of gas in any amount after forty-eight hours' incubation at 37^ C, is recorded as positive and considered as presumptive positive. The smallest quantity of water giving gas is then confirmed. A loopful of culture from the tube furnishing the presumptive test is stroked upon Endo's medium previously poured in plates. The plates are incubated at 37^ C. for twenty-four hours. If no aerobic colonies (sterile plate) develop, the respective gas-formation is considered due to anaerobes,and B.coliis recorded as not present. If aerobic colonies develop, the most typical of B. coli is trans- planted into lactose broth and gelatin and a microscopical examination made directly. As there are several varieties of typical B, coli colonies, experience alone will determine the choice. If in the lactose broth transplant, no gas is formed after the forty-eight CINCINNATI PilOCEDURE 313 hours at 37° C, the result is considered nepjative, and B. coli is recorded as not present. If gas is formed the result is considered positive for B. coli, subject to the results of the gelatin transplant and a microscopical examination. If after forty-eight hours' mcubation at 37° C. the gelatin transplant will solidify on cooling, the culture is considered as non-liquefying. If the gelatin does not solidify on cooling, and gas is formed in the lactose broth transplant and the microscopical examination demonstrates a bacillus, the organism is called B, cloacce and included in the B. coli numerical estimation as it has the same sani- tary significance. The microscopical examinatio*n consists of a smear made according to the method of Gram from the Endo plate colony selected for transplanting. A Varying- Quantities (0.01-0.1-1 .0 c cetc ) Planted into Lactose Broth Gas Formation-Presumptive Positive Smallest Quantity giving gas [^ is Plated onEndo's Medium Sterile Plate Negative Typical or Atypical Colony Fished and Planted into Lacto&e Broth B No Gas Foimcd Negative Gas Formed r Gelatine Liquefaction X Negative Microscopical Examination Not A - Bacillus Gram-negative Non-spore former Negative Non- Liquefaetion A - Bacillus Gram-negative Non-spore former B. Ooli-Confirmed Method Followed at Cincinnati Plant for Identification of B. coli. Gram negative non-spore- forming bacillus obtained following the above pro- cedure fulfills in every particular the definition for the Bacillus coli. If other than a gram-negative non-spore-forming bacillus is obtained, it is not considered B. coll and is so recorded, but such "a result is unlikely and has not been en- c:)untered in experiences at Cincinnati. The liquefaction of gelatin and dis- cordant results in the microscopical examination accompanying gas production in the lactose broth transplant are likely to be due to mixed cultures, in which case 'separation should be made by replating from the lactose broth transplant. " Estimation of the B. coli index is made according to Phelps' method — that is, the reciprocal of the smallest volume of water which gave a positive test is taken as the approximate number of B, coli per cubic centimeter and is recorded as such. In case of an anomaly it is assumed that the positive result should have been negative and the negative result positive and is so considered 314 WATER HYGIENE in obtaining the B. coli index. If a negative confirmation test is obtained, B. coli is considered present in the next larger quantity without confirmation. If a negative result is obtained from the largest quantity planted, either by no gas fc/rmation in the original planting or by a negative confirmation test, B. coli is considered present in the next larger amount that would have been planted, except that a negative result in 100 c.c. is recorded at zero. The assumption IS value only in the case of waters such as are encountered in water purification plants where the approximate coli content of the water is known. Such assump- tions are rarely necessary as the quantities of water chosen for examination can orcjinarily be changed in sufficient time to always obtain a positive test. It is probable that this assumption causes less error than would be introduced by either omitting the figure from the averages or calling it zero. "The laboratory work above described with the numerical estimations of B. coli derived, are recorded for the month under headings of waters in the consecutive stages of purification. The form used is convenient and compact and lends itself readily to a grasp of conditions throughout the plant at any time. Five days from the collection of the water sample, B. coli may, following the above scheme, be satisfactorily and conclusively isolated and identified to conform to the definition. The following table will show the number of samples examined, according to this method, from January to October, 1916, and the percentage of samples confirmed as B, coli. River Water. Settled Water. Applied Watei FiiTERED Water. Effluent from Filters. Outlet Clear Water Reservoir. Samples tested . . . Percentage confirmed . 271 97 4 257 91.0 266 96 6 258 *94.9 194 *86.1 * Includes one sample that showed B. Cloaco O CO lO o o CO O N o w oa tN o tH CO C* c3 «-* , o 1-1 lO SO '^ o « ^ s Ol tH rt5" ft ^ CO 4J . '(li^ tH rt* CO o ,_^ -^ii Tt< ■^ 00 rM iH »H CO o o o o O o oo o 00 o cq ■«* o d CO o CO O T-H o * 00 CO N U3 00 OS 1> r-t OS c ) rH o O CM tH o !=<<< * • ^ H M t— <£> o CC > CO XO CO a » »o i> O -* a iH © r- t- tH CO «-« r- rH (M O ° c s O tH O r<^ o c o O c o o o o ir. lO CM Q 1 >. U -P 5^ ^rt h3 OC (M N CO CSI Xi o . § : to 03 • -5J • -3 .a 03 ■ o • M d O •a : §■ 6 o o CQ ake Rice, | mile e Galesburg s . - bO Q> O ■§ ft ft «; O ,£ 1^ d O OJ 3ol opposite Moli: Coj's plant, Moli] O M si 9. ike Rice, east of burg. o g 6 .a O • p-4 1 yA ^ 1— 1 Ph HH ^. u M « o ►H OS i-i tH lO M CO tH CO cq "<*< 4 6 ?D t* M 04 (Nt (M CO CO O t- r>. Cft C31 OS OS CD OS o o T-H r-i (N (N C' » w— 0/ f ,«''■ 1 1 1 1 t 1 j Pearl 4 t I ' ii 1 t k 1 1 Beardstov ^ 1 I DISSOLVED OXYGEN ILLINOIS RIVER / 1 J 1 1 i / Havana 4 1 J Copperas >. 1 L t f J 1 Peoria / 'Bolow fc Vbova ewt g < 1 ( \ Clxihcothe i 1 •:^ / l^ ~i ^. deniiepin k V ^ 1 \ Peru \ V 1 \ V Starvea E ocl ^ \ Bel )W I 'am * 1 Ab( ve X )am ^ Morris -A -\a 4 -iCIi Dresden \ / »* C ? \ C 2 Lockport N 1 ^ S H •4 to >^. 1*?. o Parts per ^ MlUioa ■a 0=1 M O o , to Mississippi-Riverj Moutli of lUiiois' R, Parts per Million ^ o oo Pearl Beardsto Havana Peona Chilicothc Henry Hennepxn Spring Vajile^'' La Salle Starved B!oc1c Ottawa Marseillef Morris Dresden Lotkport O 00 Parts per Million Fig. 65.— Showing Dissolved Oxygen Concentration in Different Parts of the Illinois River. (From Illinois State Water Survey Bulletin.) this case has been summarized by Leighton (1907). Both sides of the case presented some very interesting opinions and data. Jordan (1900) who did valuable work for the defendants reported some of his data in PATHOGENIC BACTERIA IN RIVERS 343 a separate publication. Table XXXIII is taken from that paper. It will be seen from this table that there is a steady decrease in the chlorine content of the water and also in the bacteria content. The rise in the count at Wesley City may be explained by the fact that at Peoria largo amounts of distillery slops and animal refuse were dumped into the river. These would have a greater effect on the bacteria count than on the chlorine. Dissolved oxygen studies have been carried out by the Illinois State Water Survey, some of the data of which are presented in Fig. 65. From these data, it will be seen that there is evident puri- fication in the IlUnois River. Pathogenic Bacteria in River Water. There are many data available in the literature but in looking it over one is impressed with the diversity of opinion. Much of this disagreement may probably be explained by the different conditions under which each of the investigators worked. It is evident that the factors in the environment influence, to a certain extent, the longevity of bacteria in nature. These factors will not be taken up here, but those who are interested will find discussions in texts on water bacteriology. From the evidence which was presented in the Illinois River case, we know that the typhoid bacilli die rather rapidly in nature. Prob- ably after three or four weeks the water which contained them would be safe. Jordan, Russell and Zeit (1904) studied this question in both pure and polluted waters and their data are taken as the bases upon which our present knowledge rests. They suspended the bacteria in collodion sacs in both pure and polluted waters. In pure Lake Michigan water, the bacilli did not die out until after a week, but three days seemed to be the limit for the polluted Chicago Drainage Canal water. In 1906 Russell and Puller confirmed these results using pure Lake Men- dota water and sewage as the substrates. In this experiment the bacilli lived for about ten days in Lake Mendota water and three days in sew- age. Houston has studied this question in England. He points out that 1 c.c. samples were used by the American workers in most of the cases, and from these small samples conclusions were drawn with regard to the longevity of B. typhi. When no organisms were found in 1 c.c. samples, it was stated that the organism had died out. Probably American bacteriologists have been too much accustomed to use small amounts of water in analytical work. Houston carried on experiments using Thames, Lee and New River waters, using 100 c.c. quantities of the water as the maximum amounts examined. For 100 c.c, quantities in eighteen experiments, it required nine weeks for the total disappear- ance of B. typhi. Ninety-nine per cent of them disappeared in one week under laboratory conditions of the experiment. The above data of 344 WATER HYGIENE both American and English bacteriologists point out the value of storage. The rate of death of B. typhi and B, coli in pure natural water has been reported by Eahn and Hinds (1914) to follow the mono- molecular law. Under these conditions the rate increased with the temperature. Oxygen was found to be harmful to B. coli but bene- ficial to B, typhi. The viability of Microspira cholerce has also been investigated by Houston using the same technique as for B. typhi. Ninety-nine per cent of the cholera organisms perished in three days under the labora- tory conditions of the experiment. This is a much shorter period than required for B. typhi, Gelarie (1916) studied the longevity of Micro- spira choleroe in water of New York Bay. He stated that the survival of these organisms in water depends upon the strain which is used, the number of bacteria seeded and other factors. The organisms are said to live in native bay water from 7 to 45 days and in sterilized bay water up to 285 days. In sterile tap water they lived for from 1 to 18 days and in native tap water from 1 to 3 days. In competition with the other water organisms the life of the cholera organisms was found to be short* The other phase of the stream pollution question has been con- cerned with nuisances resulting from overloading the stream with sewage or organic industrial wastes. It is now fairly well established that rivers do purify themselves but it is more often a difficult question to determine the length of time required. Practically every river is able to handle a certain amount of organic matter. Phelps (1914) has stated that three factors are involved-the oxidizing bacteria, organic matter and oxygen. Since the organic matter and bacteria are always present, " the questions of stream purification and prevention of nui- sanco reduce to one of oxygen supply." Solution of Atmospheric Oxygen in Water. The amount of oxygen in the water which is available for the oxidizing bacteria is the important question in stream purification. Especially so is the extent to which the oxygen content may be reduced before putrefaction sets in. As long as there is sufficient oxygen, the bacteria will carry to completion the oxidation of organic matter but when this oxygen is reduced to a certain point, putrefaction sets in and this is always accompanied by foul odors. As long as the available oxygen is sufiicient, the bacteria will carry it over to the organic matter always driving the reaction in that direction and not allowing an equilibrium to be established. The greater the speed of this reaction, the greater will be the amount of re- aeration which will take place and Phelps (1916) has stated that this is the important factor in stream purification and that dams and rapids DISSOLVED OXYGEN 345 increase this by a mixing action. The balance between the available oxygen and the required oxygen must probably be established for each stream. Determination of Dissolved Oxygen. Different methods have been in use for determining the oxygen content of a water. The Winkler method has been included in standard methods and has certain distinct advantages over the Levy method which has been advocated by some. It is given in standard methods as follows: Reagents. 1. Sulphuric acid, concentrated. (Sp. Gr. 1.83-1.84.) 2. Potassium permanganate. Dissolve 6.32 gms. of the salt in water and dilute the solution to a Hter. 3. Potassium oxalate. A 2 per cent solution. 4. Manganous sulphate. Dissolve 480 gms. of the salt in water and dilute the solution to 1 liter. 5. Alkaline potassium iodide. Dissolve 700 gms. of potassium hydroxide and 150 gms. of potassium iodide in water and dilute to a Uter. 6. Hydrochloric acid, concentrated. (Sp. Gr. 1.18-1.19.) 7. Sodium thiosulphate. A N/40 solution. Dissolve 6.2 gms. of chemically pure recrystalUzed sodium thiosulphate in water and dilute the solution to 1 liter with freshly boiled distilled water. Each cubic centimeter is equivalent to 0.2 mg. of oxygen or to 0.1395 c.c. of oxygen at 0° C. and 760 mm. pressure. Inasmuch as this solution is not per- manent it should be standardized occasionally against a N/40 solution of potassium bichromate. The keeping qualities of the thiosulphate solution are improved by adding to each liter 5 c.c. of chloroform and 1.5 gms. of ammonium carbonate before diluting to the prescribed volume. 8. Starch solution. Mix a small amount of clean starch with cold water until it becomes a thin paste and stir this mass into 150 to 200 times its weight of boiling water. Boil for a few minutes, then sterilize. It may be preserved by adding a few drops of chloroform. Collection of the Sample. Collect the sample in a narrow-necked glass-stoppered bottle of 250 to 275 c.c. capacity. The following pro- cedure should be followed in order to avoid entrainment or absorption of atmospheric oxygen. In collecting from a tap, fill the bottle through a glass or rubber tube extending well into the tap and to the bottom of the bottle. To avoid air bubbles allow the bottle to overflow for several minutes, and then carefully replace the glass stopper so that no air bubble is entrained. In collecting from the surface of a pond or tank connect the sample bottle to a bottle of 1 liter capacity. Provide each 346 WATEK HYGIENE bottle with a two-hole stopper having one glass tube extending to the bottom and another glass tube entering, but not projecting, into the bottle. Connect the short tube of the sample bottle with the long tube of the liter bottle. Immerse the sample bottle in the water and apply suction to the outlet of the liter bottle. To collect a sample at any depth arrange the two bottles so that the outlet tube of the liter bottle is at a higher elevation than the outlet of the sample bottle. Lower the two bottles in any convenient form of cage properly weighted, to the desired depth. Water entering during the descent will be flushed through into the liter bottle with a glass stopper in such a manner as to avoid entraining bubbles of air. Procedure. Remove the stopper from the bottle and add, first, 0.7 c.c. of the concentrated sulphuric acid, and then 1 c.c. of the potassium permanganate solution. These and all other reagents should be intro- duced by pipette under the surface of the liquid. Insert the stopper and mix by inverting the bottle several times. After twenty minutes have elapsed, destroy the excess of permanganate by adding 1 c.c. of the potassium oxalate solution, the bottle being at once restoppered and its contents mixed. If a noticeable excess of potassium perman- ganate is not present at the end of twenty minutes again add 1 c.c. of potassium permanganate solution. If this is still insufficient use a stronger potassium permanganate solution. After the liquid has been decolorized by the addition of potassium oxalate add 1 c.c. of man- ganous sulphate solution and 3 c.c. of the alkaline potassium iodide solution. Allow the precipitate to settle. Add 2 c.c. of the hydro- cliloric acid and mix by shaking. The procedure to this point must be carried out in the field, but after the acid has been added and the stopper replaced there is no further change, and the rest of the test may be performed within a few hours as convenient. Transfer 200 c.c. of the contents of the bottle to a flask and titrate with N/40 sodium thiosulphate, using a few cubic centimeters of the starch solution as an indicator toward the end of the titration. Do not add the starch solution until the color has become a faint yellow and titrate until the blue color disappears. The use of potassium permanganate is made necessary by high nitrite or organic matter. The procedure outlined must be followed on all work on sewage and partly purified effiuents or seriously polluted streams or samples whose nitrite nitrogen exceeds 0.1 part per million. In testing other samples the procedure may be shortened by beginning with the addition of the manganous sulphate solution and proceeding DISSOLVED OXYGEN 347 from that point as outlined, except that only 1 c.c. of alkaline potas- sium iodide need be added. Calculatton of Results. Oxygen shall be reported in parts per million by weight. It is sometimes convenient to know the number of cubic centimeters per liter of the gas at 0° C. temperature and 760 mm. pressure and also to know the percentage which the amount of gas present is of the maximum amount capable of being dissolved by dis- tilled water at the same temperature and pressure. If 200 c.c of the sample is taken the number of cubic centimeters of Ny/40 thiosulphate used is equal to parts per million of oxygen. Corrections for volume of reagents added amount to less than 3 per cent and are not justified except in work of unusual precision. To obtain the result in cubic centimeters per liter, multiply the number of cubic centimeters of thiosulphate used by 0.698. To obtain the result in percentage of saturation consult the table on page 68 of the 1917 edition of Standard Methods for the Examination of Water and Sewage, and divide the number of cubic centimeters of thiosulphate used by the figure in this table opposite the temperature of the water and under the proper chlorine figure. The last column of this table permits interpolation for intermediate chlorine values. At elevations dijffering considerably from mean sea level and for accurate work, attention must be given to barometric pressure, the normal pressure in the region being preferable to the specific pressure at the time of sampling. The term ^' saturation " refers to a condition of equilibrium between the solution and an oxygen pressure in the atmosphere corresponding to 158.8 mm., or approximately one-fifth atmosphere. The true saturation or equilib- rium between the solution and pure oxygen is nearly five times this value and consequently values in excess of 100 per cent saturation fre- quently occur in the presence of oxygen-forming plants. When the oxygen concentration becomes too low, the stream becomes putrescible. This same condition may be duplicated in a bottle by allowing it to stand full of sewage tightly stoppered. The following things will be noticed: formation of evident amounts of hydrogen Bulphid; heavy deposits of black sediment; disappearance of dissolved oxygen. These are conditions which are not desired in streams and they indicate that the available oxygen is insuflScient to care for the organic matter. Stability and Relative Stability. A stream is stable when its oxygen demand has been satisfied; the products resulting from the decomposi- tion of the organic matter should be completely oxidized. Phelps has stated that this condition obtains when the available oxygen exceeds the 348 WATER HYGIENE required oxygen. He stated that available oxygen is used up according to the following order: Dissolved oxygen, nitrates, nitrites, sulphates, phosphates, etc. Since all streams are not stable it is necessary to use other terms to fit their condition. For such conditions the term relative stability is used. Stable streams have a relative stability of 100 since they have all of the oxygen that is demanded by the organic matter. A relative stability of 60 means that a stream has 60 per cent of the oxygen necessary to make it stable. This is determined in the following manner : Reagent. Methylene blue solution. A 0.05 per cent aqueous solu- tion of methylene blue, preferably the double zinc salt or commercial variety. Collection of Sample, Collect the sample in a bottle holding approxi- mately 150 c.c. If the dissolved oxygen is low, observe precautions similar to those used in collecting samples for dissolved oxygen (p. 162). Use rubber stoppers or a cork stopper of good grade which has been boiled in water. Procedure, Add 0.4 c.c. of the methylene blue solution to the sample in the 150 c.c, bottle. As methylene blue has a slightly antiseptic prop- erty be careful to add exactly 0.4 c.c. Add the methylene blue solution preferably below the surface of the Hquid after filling the bottle with the sample. If the methylene blue is added first do not allow the liquid to orverfiow as coloring matter will thus be lost. Incubate the sample at 20° C. for ten days. Four days' incubation may be considered as sufficient for all practical purposes in routine plant-control work. If quick results are desired incubate the sample at 37° C. for five days using suitable stoppers to prevent the loss and reabsorption of dissolved oxygen. The bacterial flora at 37° C. is different from the flora at 20° C. The lower temperature is more nearly the average temperature of sur- face waters and, therefore, the higher temperature should be used only when quick approximate results are essential. Observe the sample at least twice a day during incubation. Give a sample in which the methylene blue becomes decolorized a relative stability corresponding to the time required for reduction (see Table XXXIV). For routine filter control ordinary room or cellar temperature will give fairly satisfactory results. For accurate studies, room temperature incubation is very undesirable, as the fluctuations in temperature which are ordinarily not noticed are responsible for appreciable deviations from the true values of relative stability. If the samples are incubated less than ten days at 20° C. and are not decolorized, place a plus sign after the stability value in order to indicate that the stability might have been higher if more time had been allowed. In applying this test to river DISSOLVED OXYGEN 349 waters it often happens that the blue coloring matter is precipitated either partly or completely through absorption by the clay which many rivers carry in suspension. True relative stabilities cannot be obtained for such waters except by determining the initial available oxygen at the start and the biochemical oxygen demand on incubation at 20° C. for ten days. Germicides, such as calcium hypochlorite, if present in suf- ficient quantity vitiate the results. If a sample contains free chlorine, therefore, store it about two hours, or until the chlorine is gone, and then add methylene blue. Table XXXIV gives the relation between the time of reduction in days at 20° C. and the relative stability number. Table XXXIV RELATIVE STABILITY NUMBERS tzo 5 120 s 5 11 8 ai 1 21 9 87 1 5 30 10 90 2 37 11 92 2 5 44 12 94 3 50 13 95 4 GO 14 96 5 68 16 97 6 75 18 98 7 80 20 99 ;Sf = Relative stability or ratio of available oxygen to oxygen required for equilib- rium. Expressed m per cent. />o=Time in days to decolorize methylene blue at 20° C. The theoretical relation is, /S- 100(1-0.794) tio. The relation between the time of reduction at 20° C. and that at 37° C. is approximately two to one. It is desirable that each observer work out his own comparative 37° C. table or factor, but results should be reported in terms of 20° C* stability numbers. A relative stability of 75 signifies that the sample examined con- tains a supply of available oxygen equal to 75 per cent of the amount of oxygen which it requires in order to become perfectly stable. The available oxygen is approximately equivalent to the dissolved oxygen plus the available oxygen of nitrates and nitrites. The nitrites in sewage are usually so low as to be negligible. 350 ^VATER HYGIENE The theory of the test is unique. Methylene blue is very sensitive to products of putrefaction. Such compounds as hydrogen sulphide mercaptans, etc., and other reducing substances quickly reduce it to its colorless Icuco base. Consequently when this is nuxed with a polluted water, it will change from blue to white as soon as any of these com- pounds are formed. They are present when putrefaction sets in and, therefore, cause the change in the color of the dye. Phelps has stated that this occurs when the nitrites are used up. This reducing time rep- resents the time " required for the exhaustion of available oxygen." Lederer (1914, 1915) who, as bacteriologist and chemist for the Sanitary District of Chicago has had ample opportunity to study this subject, has pointed out that great care must be used in the test to secure comparable results. He has devised some unique methods for the study of required oxygen. They are given in Standard Methods as follows: Biochemical Oxygen Demand of Sewage and Effluents (Ledererj 1914) • Relative Stability Method, The relative stabihty method may be employed to obtain a measure of the putrescible material in sewages and effluents in terms of oxygen demand. Procedure for Effluents, Divide the total available oxygen, including the oxygen of nitrites and nitrates, by the relative stabihty expressed as a decimal. Procedure for Sewages, Make one or two dilutions with fully aerated distilled water of known dissolved oxygen content. Tap water may be employed if it is free from nitrates. Vary the relative proportions of sewage and water to be employed to give a relative stability of from 50 to 75. Unless proper seals are employed bring the water as well as the sewage to the temperature at which the mixtures are to be incubated before preparing the dilutions. During the manipulation avoid aeration. Having made the proper dilutions, determine the relative stability of each. Calculate the oxygen demand in parts per million by the following formula: O(l-p) Oxygen demand is Rp In this formula is the initial dissolved oxygen of the diluting water; p is the proportion of sewage; and R is the relative stabihty of the mix- ture. Ordinarily the available oxygen in crude sewages, septic tank effluents, settling tank effluents, and tradewastes can be neglected. Sodium Nitrate Method. For the determination of the biochemical oxygen demand the sodium nitrate method may be used. The method BIOCHEMICVL OXYGEN DEMAND 351 is based on the biochemical consumption of oxygen from sodium nitrate by a sewage or polluted water during an incubation period of ten days at 20° C. A reasonable excess of sodium nitrate does not give a higher oxygen demand, as do higher dilutions with aerated water. The oxygen absorbed from the air in applying the method to sewages is negligible. Reagent. Sodium nitrate solution. Dissolve 26.56 gms. of pure sodium nitrate in 1 liter of distilled water. One c.c. of this solution in 250 c.c. of sewage represents fifty parts per million of available oxygen. The strength of the sodium nitrate solution may be varied to suit con- ditions. Procedure for Sewages^. Ordinarily disregard the initial available oxygen as it is very small compared with the total biochemical oxygen demand. Add measured amounts of the sodium nitrate solution to the sewage in bottles holding approximately 250 c.c' which have been com- pletely filled and stoppered. Incubate for ten days at 20° C. A seal is not required during incubation. The appearance of a black sediment and the development of a putrid odor during incubation indicates that too little sodium nitrate has been added. Methylene blue solution in proper proportion may be added at the start to serve as an indicator during the incubation. Domestic sewage usually varies in its oxygen demand from 100 to 300 parts per million, approximately 30 per cent of which is used up at 20° C. in the first twenty-four hours. At the end of the incubation period determine the residual nitrite and nitrate. Determine the nitrate by the aluminium reduction method, followed by direct Nesslerization. To convert the nitrogen into oxygen equiv- alents, multiply the nitrite nitrogen by 1.7 and the nitrate nitrogen by 2.9. The difference between the available oxygen added as sodium nitrate and that found as nitrite and nitrate at the end of the incuba- tion period is the biochemical oxygen demand. Procedure far Trade^astes. Employ the same procedure using larger quantities of the sodimn nitrate solution. Make the reaction alkaline to methyl orange and acid to phenolphthalein. Adjust an acid reaction with sodium bicarbonate, and a caustic alkaline reaction with weak hydrochloric acid. If the liquid is devoid of sewage bacteria seed it with sewage after adjusting the reaction. Procedure for Polluted River Waters. Determine the initial available oxygen. Unless the river water is badly polluted add ten p^^rts per million of sodium nitrate oxygen. Collect carefully to avoid aeration, three samples in 250 cc. bottles. To one sample add a definite quan- tity of sodixmi nitrate solution and incubate. Incubate the other two samples for the determination of the residual free oxygenj nitrite aad 352 WATER HYGIENE nitrate. If there is free oxygen left, the bottle containing the sodium nitrate solution may be discarded. If there is no free oxygen determine residual nitrite and nitrate as diiectcd under the procedure for sewage, and calculate the oxygen demand, (Standard Methods of Water Analysis.) Examination of Sewage. The chemical examination of sewage and sewage effluents is probably more valuable than the bacterial examina- tion. The problem involved in controlling a sewage treatment plant is quite different than that involved in the control of a water treatment plant. As Lederer and Bachmann (1911) have stated the effluent from a water treatment plant is subjected to different requirements than the efSuent from a sewage treatment plant. In the first case the bac- teriologist is concerned m the presence of certain types of bacteria and their concentration. Little attention is given to the daily variations in the chemical content of the efHuent. With the sewage effluent, however, the question is a different one. It is necessary to watch carefully the variations in the chemical content and keep the organic matter reduced to a minimum. The question of stream pollution is involved if the effluent is to be emptied into a stream. It must not be putrescible and thus cause the nuisances which result from too heavily polluting a stream. Lederer and Bachmann have stated some of the fallacies connected with the bacterial control of sewage plants. They have stated that '^ to try to obtain rehable information on the number of bacteria in effluents from various devices in a large plant would require a staff of trained men entirely out of proportion to the value of the results obtained." The difficulties in securing careful sampling and the necessity of immediate plating since bacteria in sewage die very rapidly when stored at icebox temperatures, are also mentioned. For these reasons and others more attention is given to the determination of the organic matter content and the biochemical oxygen demand according to the methods which have been given before. The deposits from rivers and sewage sludges are examined in the following manner according to Standard Methods for the Examination of Water and Sewage, 1917. Analysis of Sewage Slujdge and Mud Deposits Collection of the Sample. Collect a representative sample. In general, more than one sample should be taken from a spot and a large number of samples should be collected rather than a few large samples. If the surface layer is darker and a lower layer consists of pure clay ANALYSIS OF SLUDGE 353 sample only the surface layer. Samples may be analyzed either sep- arately or as composites of careful mixtures. After the sample has settled a few minutes roughly drain or siphon the excess water. Allow sewage sludge to stand for one hour before draining it free from excess water unless it is essential to determine the moisture content of the sample originally collected. If sludge cannot be analyzed within twenty-four hours it is better not to use air-tight bottles and to add small quantities of chloroform to retard decomposition. At the time of collection carefully examine mud from the bottom of surface water for evidence of sewage pollution and macroscopic and microscopic animal and plant organisms. Record the predominant species. Note the physical appearance of the material, particularly its color, odor, and consistency. Express all analytical results in percentage on a dry basis. Specific Gravity. Weigh to the nearest tenth of a gram a wide- mouthed flask of 100 to 300 c.c. capacity, according to the quantity of material available. Then completely fill the flask with distilled water to the brim, and weigh it again. Empty and fill the flask completely with fresh sewage sludge or mud. If the material is of such consistency that it flows readily, fill the flask to the brim and weigh. The specific gravity is equal to the weight of the sludge or mud divided by the weight of an equal volume of distilled water. If the material does not flow readily, fill the weighed flask as com- pletely as possible without exerting pressure during the procedure. Weigh and then fill the flask to the brim with distilled water. Let it stand for a few minutes, until trapped air has escaped, then add more water if necessary and weigh. The specific gravity is equal to the weight of the material divided by the weight of the distilled water less the weight of the water added. Record the specific gravity only to the second decim.al place. Moisture, Heat approximately 25 gms. of sludge or mud in a weighed nickel dish on the water bath until it is fairly dry. Dry the residue in an oven at 100° C, cool, and weigh. Repeat to approximate constant weight. The loss in weight is moisture. Volatile and Fixed Matter, Ignite, in a hood, the residue from the determination of moisture until all the carbon has disappeared. Cool the residue in a desiccator and weigh it. The residue is the fixed matter. The volatile matter is the difference in weight between the original dried sludge and the ignited sludge. Total Organic Nitrogen. Preparation of Sample. For the deter- mination of organic nitrogen and fat dry approximately 50 to 75 gms. of the sludge or mud in a porcelain dish first oix the water bath and 354 WATER HYGIENE finally in the hot-water oven until all the moisture has disappeared. Grind the dry material to a fine powder and keep it in a glass-stoppered bottle. Reagents, 1. Sulphuric acid. Concentrated, nitrogen free. 2. Copper sulphate solution. Ten per cent. 3. Potassium permanganate. Crystals. Procedure, Weigh accurately 0.5 gm. of dried sludge or 5.0 gms of dried mud and put it in a 500 c.c. Kjeldahl flask. Digest it with 20 c.c. of sulphuric acid, or more if necessary, and 1 c.c. of copper sulphate solution to assist the oxidation. Boil for several hours until the hquid becomes colorless or sUghtly yellow. Oxidize the residue with 0.5 gm. of potassium permanganate, and follow the '' Procedure for Sewage '' (p. 165). The following method is convenient for routine work at sewage disposal plants. Digest 0.5 gm. of dried sludge or 5.0 gms. of dried mud with 20 c.c. of strong sulphuric acid and 1 c.c. of the copper sulphate solution in a 300 c.c. Kjeldahl flask. After digestion for several hours, cool, transfer to a glass-stoppered 100 c.c. flask, dilute with distilled water to 100 c.c, and mix well. Transfer 50 c.c. with a pipette into another 100 c.c. volumetric flask, and make this portion alkaline with 50 per cent sodium hydroxide, testing a drop of the hquid on a porcelain plate with phenolphthalein to insure neutraUzation. The formation of a floe usually indicates that the neutraUzation is complete. Pour the solution into a small glass-stoppered bottle and permit it to stand until the next day. Nesslerize an aliquot portion of the clear, supernatant liquid, and calculate the percentage of nitrogen in the material. Fats. Fats are usually determined only on sewage sludge, but some mud deposits contain small quantities due to the presence of trade wastes. Procedure. Weigh, according to the quality of the sewage or mud, 0.5 to 25 gms. of dry material. Add water to the weighed portion in a porcelain dish and acidify the mixture with N/50 sulphuric acid in the presence of litmus tincture or azolitmin solution indicator. Avoid adding too much acid as an excess gives too high results on account of fatty acid residues. Evaporate the acidified mixtures to dryness on the water bath, and heat it in the hot-air oven at 100"" C. for two to three hours. Extract the dry residue with boiling ether, rubbing the sides and bottom of the dish to insure complete solution of the fat. Three extractions with ether are usually sufficient. Filter the ether solution through a 5 cm. filter paper into a small flask. Evaporate the ether slowly, dry the fatty extract for half an hour at 100° C, cool in a des- ANALYSIS OF SLUDGE 355 iccator a:nd weigh. If it is desirable, particularly with certain trade wastes, to determine the quantity of soap fat, determine the fats with and without the addition of acid. The difference between the amounts found by the two determinations is the amount of soap fat present. Ferrous Sulphide. The liberation of hydrogen sulphide on adding dilute hydrochloric acid to a sludge indicates the presence of ferrous sulphide. As ferrous sulphide quickly oxidizes on exposure to air, a quantitative determination of this constituent must be made imme- diately after collection of the sample. Procedure, Heat a definite portion of the sludge with hydrochloride acid in a flask. Pass the liberated gas through bromine water or hydro- gen peroxide. Determine gravimetrically the sulphate in the oxidizing solution, and calculate the equivalent of ferrous sulphide by multiplying the weight of barium sulphate by 0.376. Biochemical Oxygen Demand. The quantity of river mud most suitable for the determination of the biochemical oxygen demand ranges within certain limits, largely according to the amount of deoxygenating matter present. For examinations of river mud prepare a 1 per cent stock solution in distilled water or tap water saturated with oxygen and free from nitrate; use in the test a dilution of this stock solution equivalent to a concentration of 1 to 10 gms. per liter of mud. For examinations of fresh sewage sludge prepare a 1 per cent stock solution in a similar manner, but use in the test a dilution equivalent to only 0.1 to 1.0 gm. per liter of wet material. For examinations of dried sludges, which have undergone more or less minerahzation, higher concentrations may be required. Procedure. Place a measured portion of the sample, or the proper amount of the 1 per cent stock solution of the sample, in a 300 c.c. narrow-mouth, glass-stoppered bottle, and dilute it to the desired dilu- tion with water saturated with oxygen. Determine the oxygen content at 20"^ C. of the waters that are used for dilution. This determination must be made before the mud or sludge is added, because iron sulphide in the mud or sludge rapidly consumes part of the dissolved oxygen. Incubate at 20° C for five days. Shortly before the determination of the oxygen remaining in solution at the end of five days rotate the bottle once or twice to mix its contents and allow sedimentation for about thirty minutes. Siphon the greater part of the hquid through a narrow-bore siphon into a 150 c.c. bottle, which has been filled with carbon dioxide. Reject the first 25 c.c. of the siphoned liquid and allow a Uttle to overflow at the end of siphoning. 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P. and Scott, R. D. 1914. Advantage of the Use of Lime in Water Purification. Monthly Bull. Ohio State Board of Health, December, 1914. * Houston, A. C. First Report on Research. The Vitality of the Typhoid Bacillus in Artificially Infected Samples of raw Thames, Lee and New River Waters, with Special Reference to the Question of Storage. Metro- politan Water Board, London. Houston, A. C. Fourth Report on Research Work. The Vitality of the Chol- era Vibrio in Artificially Infected Samples of raw Thames, Lee and New River Waters, with Special Reference to the Question of Storage. Metro- politan Water Board. Hutchins and Wheeler. 1903. An Epidemic of Typhoid Fever Due to Impure Ice. Amer. Jour. Science, 126, 680. Johnson, G. A. 1904. Isolation of B. coli communis from the Alimentary Tract of the Fish and the Significance Thereof. Journal of Infectious XyLseases, x, otco ootc, Johnson, G. A. 1910. Hypochlorite Treatment of Water Supplies: Its Sig- nificance and Limitations. Eng. Record, 62, 321. Johnson, G. A. 1913. The Purification of Public Water Supplies. U. S. Geological Survey, Water Supply Paper No. 315. Johnson, G. A. 1916. The Typhoid Toll. Jour. Amer. Water Works Assn., 3, 249-326. Johnston, W. 1894. On the Grouping of Water Bacteria. Proc. Amer. Pub. Health Assn., 20, 445-449. Jordan, E. 0. 1915. The Bacterial Examination of Water. Trans. XV. Intern. Cong. Hyg. and Demog., 2, 47-50. Jordan, E.O, 1900. The Self-purification of Streams, Jour.Exper. Med., 5,302. Jordan., E. 0. 1903. The Kinds of Bacteria Found in River Water. Jour- nal Hygiene, 3, 1-27. Jordan, E. 0., Russell and Zeit. 1904. The Longevity of Typhoid Bacillus in Water. Jour. Inf. Diseases, 1, 641. Jordan, E. 0. and Victorson, Ruth. 1917. Differentiation of the Para- typhoid Enteritidis Group 11. Jour. Inf. Diseases, 21, 554-555* BIBLIOGRAPHY 359 Klein, E. 1895. Ueber einen pathogenen anaeroben Darmbacillus, BacilluvS enteritidis sporogenes. Cent. Bakt., 18, 737. Klein, E. 1897-8. Morphology and Biology of Bacillus Enteritidis Sporo- genes; on Association of this Microbe with Infantile Diarrha^a and with Cholera Nostras; on its Relation with Milk and with Manure. Local Government Board; Medical Officer's Report, p. 210. Klein, E. 1898-9. Further Report on Bacillus Enteritidis Sporogenes. Local Government Board, Medical Officer's Report, p. 312. Klein, E. 1899. Ein Beitrag zur Bakteriologie der Leichenverwesung. Cent. Bakt., 25, 278. Klein, E. 1901-2. Differentiation of the Several Anaerobic Microbes Com- monly Present in the Intestinal Contents of Man and Other Animals. Local Government Board; Medical Officer's Report, p. 404. Kligler, I. J. 1917. A Simple Medium for the Differentiation of Members of the Typhoid Paratyphoid Group. Amer. Jour. Pub. Health, 7, 1042-1044, Krumwiede, C. and Kohn, L. A. 1917. A Triple Sugar Modification of the Russell Double Sugar Medium. Jour. Med. Research, 37, 225-227. Kruse, W. 1894. Kritische und Experimentale Beitrage zur Hygienischen Beurtheilung des Wassers. Zeit. Hyg., 17, 1894. Lederer, a. and Bachmann, F. 1913. Some Interesting Observations on the Disinfection of Lake Water with Calcium Hypochlorite. Proceedings Illinois Water Supply Assn., 1913, 184-201. Lederer, A. 1914. A Serious Fallacy of the "Standard" Methylene Blue Putrescibility Test. Amer. Jour. Pub. Health, 4, 241-248. Lederer, A. 1914. A New Method for Determining the Relative Stability of Sewage Effluent or PoUuted River Water. Jour. Inf. Diseases, 14, 482-497. Lederer, A. 1915. Notes on the Practical Application of the "Saltpeter" Method for Determining Strengths of Sewage. Amer. Jour. Pub. Health, 5, 354-361. Lederer, A. 1915. Determination of the Biochemical Oxygen Demand by the Saltpeter Method in Stockyards, Tannery and Corn Products Wastes. Jour. Ind. Eng. Chem., 7, 514, 6. Lbighton. 1907. Pollution of the Illinois and Mississippi Rivers by Chicago Sewage. A digest of the testimony taken in the case of the State of Mis- souri vs. the State of lUinois and Sanitary District of Chicago. XJ. S. Geological Survey Water Supply Paper No. 194. Letton, H. p. 1917. The Treasury Department Standard for Drinking Water : Its Value and Enforcement. Jour. Amer. Water Works Assn., 4, 478-481. Levine, M. 1916. The Significance of the Voges-Proskauer Reaction. Jour. Jt>aCl}., JL, J.0O'-"Xut:. Levine, M. 1916. The Correlation of the Voges-Proskauer Reaction and Methyl Red Reactions in the Colon-serogenes Group of Bacteria. Jour. Inf. Diseases, 18, 358-367. Levine, M. 1918. Presumptive Test for Bacillus Coli. Eng, Contractor, 49, 34. Also see Jour. Inf. Dis., 23, 43-47. 360 WATER HYGIENE Lewis, W. L. 1912. Evanston's Experience with Hypochlorite. Proc. Illinois Water Supply Association, 1912, 124-139. McCallie, S. W. 1904. Experiment Relating to Problems of Well Contam- ination at Quitman, Ga. U. S. Geol. Survey, Water Supply Paper, 110, 45-54. McLaughlin, A. J. 1912. The Necessity for Safe Water Supplies in the Control of Typhoid Fever. Proceeding Illinois Water Supply Association, 1912, 64-84. McLaughlin, A. J, 1914. What is a Safe Drinking Water? Public Health Reports, 29, June 26, 1914. MacNeal, W. J., Latzer, I. and Kerr, J. 1909. The Fecal Bacteria of Healthy Men. Jour. Inf. Diseases, 6, 571. Marboutin, F. 1901. Contribution h TEtude des Eaux Souterraines. Comp. Rend., 132, 365-368. Maroni. 1910. Bakterium CoH Commune. Arch. Exper. Med., 22, 261. Martel, E. a. 1903- Sur Fapplication de la fluorescein a I'hydrologie souterraine. Gompt. Rend., 137, 225-227. Metcalf, H. 1905, Organisms on the Surface of Grains with Special Refer- ence to Bacillus coli. Science, 22, 434~44L Montgomery, S. D. and Phelps, E. B. 1917. Stream Pollution; a Digest of Judicial Decisions and a Compilation of Legislation Relating to the Subject. U. S. Pubhc Health Service, Public Health Bulletin, 87. Moore, V. A. and Wright, F. R. A. 1900. A Comparison of B. Coh Com- munis from Different Species of Animals. Jour. Bost. Med. Soc, 4, 175. Morishima, K. a Simple and Practical Medium for Difterentiating B. Typho- sus, B. Paratyphosus, A. B. Paratyphosus B and B, Enteriditis. Journal of Bacteriology, 3, 19-22. MuiR, R. and Ritchie, J. 1914. Manual of Bacteriology, New York, 5th Edit. Nankivell, A. T. 1911. The Sand Filtration and Purification of Chalk Waters. Journal of Hygiene, 11, 235-258. Nichols, A. H. 1876. Report of an Outbreak of Intestinal Disorder Attrib- utable to the Contamination of Drinking Water by Means of Impure Ice. 7th Annual Report State Board of Health, Massachusetts, 467. Obst, M. M. Bile Compared with Lactose Broth for Determining the Presence of B. Coli in Water. Jour. Bact,, 1, 73-79. Orchard, W. J. 1918. Water Supply Standards and their Improvement. Jour. Am. Water Works Assn., 5, 49-58. Park, W. H. 1907. The Importance of Ice in the Production of Typhoid Fever. Jour. Amer. Med. Assn., 49, 731-732. Prescott, S. C. 1906. The Occurrence of Organisms of Sanitary Signific- ance on Grains. Biol. Studies of Pupils of W. T. Sedgwick, 208-222, Boston. Prescott, S. C. and Winslow, C. E. A. 1915. Elements of Water Bacteri- ology. New York. 3d Edit., .217. BBILIOGRAPHY 361 Phelps, E. B. 1909. The Disinfection of Sewage and Sewage Filter Effluents, with a Chapter on the Putre&cibility and Stability of Sewage Effluents. U. S. Geol. Survey, Water Supply Paper 229. Phelps, E. B. 1914. Studies on the Self-purification of Streams. Public Health Reports, 29, August 14, 1914. Phelps, E. B. 1916. Re-aeration Held to be Important in the Self-purifi- cation of Streams. Eng. Record, 74, 617-618. Race, J. 1914. The Significance of B. Coli in Water Examination. Amer. Jour. Pub. Health, 26, 758-759. Race, J. 1914. Bacillus Coh in Water Supply. Municipal Eng., 47, 12-14. Race, J. 1916. Bacterial Counts in Water Examination. Amer. Jour. Pub. Health, 6, 488-496. Race, J. 1918. Chlorination and Chloramine. Jour. Amer. Water Wks. Assn., 5, 63-68. Reynolds. 1902. Report of Stream Examination, Chemic and Bacteriologic, of the Water Between Lake Michigan and the Mississippi River at St. Louis, Chicago, 1902. Rogers, L. A., Clark, W. M. and Evans, A. 1915. Science, 41, 624. Royal Institute op Public Health. Committee Report. Jour. State Med., 22, 559-567. Savage, W. G. 1906. The Bacteriological Examination of Water. Chap. 9, Philadelphia. Savage, W. G. and Read, W. J. 1916. Significance of Streptococci in Water Supplies. Jour. Hyg., 5, 334r-351. Savage, W. G. and Wood, D. R. 1917. The Vitality and Viability of Strep- tococci in Water. Jour. Hygiene, 16, 227, 239. Sedgwick, W. T. and Winslow, C.-E. A. 1902. Experiments on the Effects of Freezing and Other Low Temperatures upon the Vitality of the Bacillus of Typhoid Fever with Consideration Regarding Ice as a Vehicle of Infec- tious Disease. Mem. Amer. Acad. Arts and Sciences, 12. Sedgwick, W. T. and MaoNutTj J. S. 1910. Study on the Hazen Theorem Jour. Inf. Diseases, 7, 489. Smith, E. G. B. 1905. Notes on the Occurrence on Grain of Organisms Resembling Bacillus Coli Communis. Science, 21, 710-711. Tanner, F, W. 1916. Comparison of the Number of Water Bacteria Growing on Agar at 37° C. and on Gelatin at 20° C. University of Illinois, Bulletin No. 13. Water Survey Series, No. 12, 242-8. Thresh, J. C. 1913. Examination of Water and Water Supplies. Blakistons, Philadelphia. Traube, M. 1894. Einf aches Verfahren Wasser in grossen Mengen Keim- frei zu machen. Zeit. Hyg., 149-188. Theasury Department. Bacteriological Standards for Drinking Water. Pub. Health Reports, 29, 2959-2966. Jour. Amer. Water Works Assn., 4, 927-946. 362 WATER HYGIENE Trillat, a. 1899. Sur Femploi matiores poloiirantes pour la recherche de Forigine deb vsources et des caux d'infiltration. Comp. Rend., 128, 698- 700. Ttjller, C. a. and Armstrong, V. A. 1913. The Differentiation of Fecal Streptococci by their Fermentative Reactions in Carbohydrat*e Media. Jour. Inf. Diseases, 13, 442-462. Van Brunt, G. A. 1911. The Action of Bleaching Powder in Water Puri- fication. University of Illinois Bulletin, Water Survey Series No 8, 53. Ward, M. 1897. Fifth Report to the Royal Society Water Research Com- mittee. Proc. Royal Soc, London, 61, 415-423. Watt, J. 1913. Purification of Water Supplies by the Excess Lime Method. Jour. State Medicine, 21, 489-499. Weissenfeld, J. 1900. Der Befund des Bakterium Coh im Wasser und das Thiere Experiment sind keine brauchbaren Hulfmittels fur die hygienische Beurtheilung des Wassers. Zeit. Hyg., 35, 78-86. Whipple, G. C. 1913. The 37° Bacterial Count. American Journal of Pub- lic Health* 3, 36-43. WxNSLOW, C.-E. A. and Hunnewell, M. P. 1902. Streptococci Charac- teristics of Sewage and Sewage Polluted Waters. Science, 15, 827. Winslow, C.-E. a. and Hunnewell, M. P. 1902. A Study of the Distri- bution of the Colon Bacillus of Escherich and of the Sewage Streptococci of Houston in Polluted and Unpolluted Waters. Journal of Medical Research, 8, 502. WiNSLOw, C.-E. A.^ 1916. Tests for Bacillus Coli as an Indicator of Water Pollution. Jour. Amer. Water Works Assoc, 3, 927-946. WoLMAN, A. 1917. The Quality of Water and Confirmatory Tests for B. Coli. Jour. Amer. Works Association, 4, 200-205. CHAPTER XI MILK AND MILK PRODUCTS Milk is the special secretion of the mammary glands and is generally used for food by man. Fresh milk is amphoteric to litmus with a spe- cific gravity heavier than water — 1.030. It has a yellowish color and a characteristic odor. On account of its chemical composition, which is mentioned later, it is susceptible to many undesirable changes. Many of these are brought about by microorganisms and, therefore, may be controlled by holding the milk in an environment, the factors of which are detrimental to bacterial development. Chemical Constituents of Milk The constituents of milk have been classified by Van Slyke and Bosworth (1914) in the following manner: [ilk constituents in 2. Milk constituents part- 3. Milk constituents en- true solution in ly in solution and tirely in suspen- milk serum. partly in suspen- sion or colloidal sion or colloidal solution. solution. a. Sugar a. Albumin a. Fat 6. Citric acid h. Inorganic phos- h. Casein c. Potassium phate d. Sodium c. Calcium e. Chlorine d. Magnesium Bosworth and Van Slyke (1915) report the composition of cow's, goat's and human milk, shown in Table XXXVI. From this presenta- tion of the chemical composition of milk, it will be seen that it is an excellent medium for bacterial growth. Van Slyke and Bosworth (1914) studied the composition of milk serum after the other constituents had been removed by filtration through a Pasteur-Chamberland filter. The analysis of the serum is given in Table XXXVII. 363 364 MILK AND MILK PRODUCTS Table XXX-VI COMPOUNDS IN COW'S, GOAT'S, AND HUMAN MILK (After Bosworth and Van Slyke, 1915) Compounds. Fat Milk sugar Proteins combined with calcium. Salts Di calcium phosphate Tri calcium phosphate Mono magnesium phosphate. . Di magnesium phosphate. . . , . Tri magnesium phosphate . . . Mono potassium phosphate. . . Di potassium phosphate Potassium citrate Sodium citrate Potassium chloride Sodium chloride Calcium chloride Cow's Milk. Goat's Milk. Per cent 3.90 4.90 3.20 0.901 175 000 103 000 0.000 0.000 0.230 052 0.222 0.000 0.000 0.119 Per cent 3.80 4.50 3.10 0.951 092 0.062 0.000 068 0.024 0.073 0.000 0.250 0.000 0.160 0.095 0.115 Human Milk. Per cent 3.30 6.50 1.50 0.313 0.000 0.000 0.027 0.000 0.000 0.069 O.OOO 0.103 0.05.^ O.OOO O.OOO 0.059 Table XXXVII COMPOSITION OF MILK SERUM (After Van Slyke and Bosworth, 1914) Constituents Sample No. 1, Original Milk, 100 c.c . Grams Sugar '. Casein Albumin Nitrogen in other compounds Citric acid Phosphorus (organic and in- organic) Phosphorus (inorganic) Calcium Magnesium , Potassium \ Sodium / Chlorine xxsn* r * •••.». , 3.35 0.525 0.125 0.096 0.128 0.012 * 354 0.081 Milk Serum, 100 c.c. Grams 0.00 0.369 0.067 0.067 0.045 0.009 *0 352 0.082 P(»rcentage of Milk Constitu- ents in Serum. Per cent 0.00 70.29 53.60 70.00 35.16 75.00 99.44 I 100.00 Sample No. 2. Original MUk, 100 cc. Grams 5.75 3.07 0.506 0.049 0.237 0.087 0.144 0.013 0.120 055 0.076 0.725 Milk Serum, 100 c.c. Percentage of Milk Constitu- ents in Serum. Grams 5-75 0.00 0.188 0.049 0.237 0.056 0.048 0.007 0.124 0.057 0.081 0.400 Per rent 100,00 0.00 37.15 100.00 100.00 64.40 33.33 53.85 100.00 100.00 100.00 55.17 * As chlorides. CHANGES IN MILK 365 Milk Proteins. According to the table from Bosworth and Van Slyke cow's niilk has a larger content of protein than goat's or human milk. Data from other sources confirm this fact. The following proteins are found in milk: Casein Lact-albumin Lact-globulin Changes in Milk Milk is subject to many types of changes. One of the most common and one which occupied the attention of chemists for a long time is the coagulation or clotting of the casein. The natural state of this milk constituent may be changed either by acid or the presence of a specific enzyme. Other proteins may be coagulated in the same way by enzymes. This change in the state of the casein is the foundation of the whole cheese industry. Souring of Milk. This usually refers to the normal changes in milk which are brought about by the continued growth of lactic acid bacteria. The lactic acid which is thus formed removes the calcium from the calcium caseinate. The casein thus formed is insoluble in the milk serum and is precipitated. The chemical changes which take place in the soluble and insoluble constituents of milk have been istudied by Van Slyke and Bosworth (1916). The milk was inoculated with a culture containing Bacterium ladis acidi and J5. lactis aerogenes and allowed to stand at room tempera- ture for sixty hours. At the end of this time it was filtered through a porcelain filter and subjected to analysis. A blank was made by pre- serving milk with chloroform and separating the serum in the same way. The changes which took place in sixty hours are given in Table XXXVIII. Coagulation by Rennin. It was believed for a long time that the coagulation of milk was due only to acid. Fremy, in 1839, one of the earliest workers on the subject, supposed that a special enzyme was present in the lining of calves* stomachs which formed acid from the lactose. Hammarsten (1872) proved that the production of acid from the lactose was not the cause of coagulation by rennin but that it was brought about by a special enzyme, which acted directly on the casein. The phenomenon involved in the action of rennin is the change of casein in milk into soluble paracasein along with another compound. However, before actual clotting takes place an insoluble calcium para- caseinate must be formed from calcium salts in the milk. Van Slyke 366 MILK AND MILK PRODUCTS and Bosworth (1913) have shown that the molecular weight of casein is 8888 and that of paracasein 4444. The valence of the molecule in basic cascinates is 8 and in basic paracaseinates 4. Bosworth (1913), in another paper states that these facts indicate three things: Iable -a.-A.-X-V ill CHANGES IN CONDITION OF MILK CONSTITUENTS AS A RESULT OF SOURING (After Van Slyke and Bosworth) Constituents. Sugar Casein Albumin Nitrogen in other compounds Citric acid. Phosphorus (inorganic) Calcium Magnesium Potassium. . , Sodium Chlorine Ash Lactic acid Original IMilk, 100 c.c. Grams 5.75 3.07 0.506 0.049 0.237 0.087 0.144 0.0*13 0.120 0.055 0.076 0.725 0.000 Sdrum from Fresh Milk. Serum 100 c.c. Grams. 5.75 0.00 0.188 0.049 0.237 0.056 0.048 0.007 0.124 0.057 0.081 0.400 0.000 Percent- age of Milk Con- stituents in the Serum. Per Cent. 100.00 0.00 37.15 100.00 100.00 64.40 33.33 53.85 100.00 100.00 100.00 55.17 0.00 Sfrum prom Sour Milk. Serum, 100 c.c. Grams. 4.48 0.00 0.506 0.049 0.000 0.090 0.148 0.014 0.120 0.058 0.079 0.690 1.124 Percent- age of Milk Con- stituents in the Serum. Per Cent. 0.00 100.00 100.00 0.00 100.00 100.00 100.00 100.00 100.00 100.00 95.17- First, that rennin action consists of the hydrolytic splitting of the casein molecule into two similar molecules of paracasein; perhaps in somewhat the same manner that maltose is split into two molecules of dextrose. Second, that, as a consequence of this cleavage, it would seem to be doubtful if Hammarsten's whey-protein could be one of the products of rennin action. Third, that rennin is not, strictly speaking, a coagulating ferment, the coagulation of paracasein being due to the fact that calcium para- caseinates are less soluble than the calcium caseinates, especially in the presence of soluble salts of calcium barium or strontium. CHEMICAL EXAMINATION 367 The action of rennin is then proteolytic since it destroys casein. It might be regarded as a preliminary process in digestion, Bosworth (1913) states that probably the action attributed to rennin may be produced by any proteolytic enzyme. In the bacterial world this has been known for some time that an organism which will split casein will also probably split gelatin and other proteins. This is especially well borne out in the following table compiled by Frost and McCamp- bell and taken from their text. From this table one might infer that there was a specificity even for the different proteins. Table XXXIX ACTION OF CERTAIN BACTERIA ON CERTAIN PROTEINS (Frost and McCampbell) Milk. Gelatin. Serum. Egg Albumin. Fibrin. Name of Organism. Coag. Digest. Bact. anthracis + + + + + + + + + + + + + + + + + -1- 4. Microspira comma. . . M. pyogenes aureus Pseudomonas pyocyaneus. Pseudomonas violacea Bacillus mycoides Bacillus prodigiosus Aspergillus niger Asoergillus oryzae + + + Chemical Examination The functions of the chemical and bacteriological examinations may be entirely different although, in many cases, they must go along together. A sanitarian must have all the knowledge which it is pos- sible to secure and should not exclude either data if he would come to an accurate conclusion. In the chemical examinations that are made on milk, data may be secured with regard to the normal or abnormal occur- rence of any milk constituent. The presence of preservatives may be directly determined while, in the bacteriological examination, they may be indirectly inferred if the milk does not sour when it is placed in a favorable environment for this change. Sampling of Milk* To secure accurate results from the chemical or bacterial examination of milk requires good sampling. Too much 368 MILK AND MILK PRODUCTS attention cannot be paid to this mipoitant step. The larger portion from which the sample is taken should be thoroughly stirred. If the sample is collected in a bottle, the contents should be very thoroughly mixed. Specific Gravity. This may be determined by any of the usual methods. Hydrometers are convenient and, in this form, are known as lactometers. They are of different types. The New York Board of Health has an arbitrary scale. This is divided into 120 parts with zero equal to the specific gravity of water and 120 equal to the specific gravity of 1.029. The length is usually about 12 ins. The Quevenne lactometer is graduated in accordance with the specific gravity scale with the first two figures omitted for sake of convenience (1.029 sp. gr. = 29 Que- ; p iBN..aia...ia, |fiii^^fe^ict-'i"."^ A lfMmm^iiiimwPF==\hr^ B Fig. 66 — Types of Lactometers. A, New York Board of Health Type, J5, Same as A Except with Thermometer, C, Quevenne Type venne). They are now made with blue and yellow columns. The fractions in the blue column indicate the percentage of water in the skimmed milk, and in the yellow column, in whole milk. Determination of Ash in Milk. Weigh about 20 gms. of milk in a tared dish and, after adding 6 c.c. of nitric acid, evaporate to dryness, ignite just below redness. Determination of Fat in Milk. Babcock Test. This rests on the fact that concentrated sulphuric acid will dissolve the sohds not fat in milk. The fat is unchanged and may be separated in a centrifuge as follows: Special bottles are necessary and into each should be placed 18 gms. (17.6 c.c.) of the well-shaken sample of milk. By means of a special pipette add 17.5 c.c. of commercial sulphuric acid with a spe- cific gravity of about 1.83. During the addition of the sulphuric acid, the contents of the bottle should be constantly shaken. The bottle should now be placed in a centrifuge and whirled for five minutes at about 1000 r.p.m. After this, boiling water should be added to the Babcock bottle, in order to raise the fat column into the neck of the FAT DETEEMINATIONS 369 bottle. The bottle should be' whirled again for about a minute and then the percentage of fat read by means of a pair of calipers. This test is adapted to ice cream and cream with the exception that a larger bottle is used. Leffmann-Beam Test. The distinctive feature is the use of fusel oil, the effect of which is to produce a greater difference in surface ten- sion between the fat and the liquid in which it is suspended, and thus promote its readier separation. This effect has been found to be heightened by the presence of a small amount of hydrochloric acid. The test bottles have a capacity of about 30 c.c. and are provided with a graduated neck, each division of which represents 9.1 per cent by weight of butter fat. Fifteen centimeters of the milk are measured into the bottle, 3 c.c. of a mixture of equal parts of amyl alcohol and strong hydrochloric acid added and mixed. Then 9 c.c. of concentrated sulphuric acid is added in portions of about 1 c.c; after each addition the liquids are mixed by giving the bottle a gyratory motion. If the fluid has- not lost all of its milky color by this treatment, a little more concentrated acid must be added. The neck of the bottle is now inimediately filled at about the zero point with one part sulphuric acid and two parts water, well mixed just before using. Both the liquid in the bottle and the diluted acid must be hot. The bottle is then placed at once in the centrifugal ma- chine; after rotation from one to two minutes, the fat will collect in the neck of the bottle and the percentage may be read off. Gerber's Test. This test is applied as follows: The test bottles are put into the stand with the mouths uppermost ; then, with the pipette designed for the purpose, or with an automatic measurer, 10 c.e. of • sulphuric acid are filled into the test bottle, care being taken not to allow any to come in contact with the neck. The few drops remaining in the tip of the pipette should not be blown out. Then 11 c.c. of milk are measured with the proper pipette and allowed to flow slowly onto the acid, so that the two liquids mix as little as possible. Finally, the amyl alcohol is added. (It is important to use the reagents in the proper order, which is — sulphuric acid, milk, amyl alcohol. If the sul- phuric acid is followed by amyl alcohol and the milk last, then the result is sometimes incorrect.) A rubber stopper, which must not be dam- aged, is then fitted into the mouth of the test bottle, and the contents are well shaken, the thumb being kept on the stopper to prevent it coming out. As a considerable amount of heat is generated by the action of the sulphuric acid on the milk, the test bottle should be wrapped in a cloth. 370 MILK AND MILK PRODUCTS The shaking of the sample must be done thoroughly and quickly, and the test bottle inverted several times, so that the hquid in the neck becomes thoroughly mixed. By pressing in the rubber stopper the height of the Hquid can be brought to about the zero point on the scale. If only a few samples have to be analyzed and the room is warm, the test bottles can be put into the centrifuge without any preliminary heating, otherwise the test bottles must be warmed for a few minutes (not longer) in the water bath at a temperature of 60° to 65° C. When the temperature rises higher than this, say above 70° C, the rubber stop- per is Hable to be blown out of the test bottle. After the test bottles have been heated they are arranged symmetrically in the centrifuge and whirled for three to four minutes at a speed of about 1000 r.p.m. When the centrifuge has a heating arrangement attached to it, the preliminary warming is not, of course, necessary. When the test bottles are taken out of the centrifuge, they are again placed in the water bath at a tem- perature of 60° to 65° C, and left there for several minutes before being read; where the centrifuge is heated, the tubes can be read off as taken from the centrifuge. By carefully screwing in the rubber stopper, or even by pressing it, the lower limit of the fat column is brought onto one of the main divisions of the scale, and then, by holding the test bottle against the light the height of the column of fat can be accurately ascertained. The lowest point of the meniscus is taken as the level when reading the upper surface of the fat in a sample of whole milk, and the middle of the meniscus for separated milk. If the column of fat is not clear and sharply defined, the sample must be again whirled in the centrifuge. Each division on the scale is equivalent to 0.1 per cent, so it is very * easy to read to 0.05 per cent, or, with a lens, to 0.025 per cent. If the number which is read off is multiplied by 0.1, then the percentage quan- tity of fat in the milk is obtained; e.g., if the number on the scale was 36.5, then the percentage of fat is 3.65. (Milk and Dairy Products, Barthel; translated by Goodwin, p. 71.) Aria-Jensen (1916) advises the use of chemically pure amyl alcohol. Pboteins in Milk Procedure. Measure 5 c.c. of the milk into a 500 c.c. Kjeldahl flask delivering it to the bottom of the flask. Add 25 c.c. of concentrated sulphuric acid and 0.7 gm. mercuric oxide or the equivalent of pure mercury. Heat the flask directly over a free flame in a hood until the TESTS FOR PRESERVATIVES 371 contents are yellow or white. After the flask has cooled add about 200 c.c. of ammonia-free water, 20 c.c. of a potassium sulphide solution (40 gms. potassium sulphide per liter of water) and a small piece of zinc. Connect the flask to a condenser and, after adding carefully 50 c.c. of a 60 per cent of sodium hydroxide solution, distill, receiving the distillate in standard acid solution. Titrate the excess of acid with standard alkali, using methyl orange and compute the amount of nitrogen in the milk. This percentage of nitrogen multiplied by 6.25 will roughly give the percentage of protein. Test for Coloring Matter in Milk. Shake about 10 c.c. of the milk with 5 or 10 c.c. of ether. Fresh milk will give a colorless supernatant ether layer, while, if coloring matter has been added to the milk, this ether layer will be colored. Detection of Preservatives. The refusal of milk to sour when placed in a warm place is evidence that a preservative has been added. The following preservatives are often found in milk and their detection may be carried out as follows: Benzoic Acid. Add a few drops of lime water to 300 c.c. of the sam- pie and reduce the volume to about 75 c.c. Mix powdered calcium sulphate with this to a paste and evaporate to dryness. When dry, grind and moisten with dilute sulphuric acid; shake out in 50 per cent alcohol. Repeat this three times and reduce the acid alcohol solution to small volume on the water bath after adding barium hydroxide to neutrality. Acidulate with sulphuric acid and extract with ether. When the ether is evaporated, the benzoic acid is left behind. This precipitate may be subjected to further study. Boric Acid. Add sodium hydroxide to 75 or 100 c.c. of the milk and evaporate to dryness. Fuse and dissolve the ash in water. Add a httle hydrochloric acid and filter. Moisten a strip of turmeric paper with the filtrate and dry on a watch glass. A red color indicates boric acid or its salts. Salicylic Acid. Add a little sulphuric acid to 15 or 20 c.c. of the milk and shake out with ether. Evaporate to dryness and treat the residue with alcohol and ferric chloride. The presence of salicylic acid is indi- cated by a violet color. Formaldehyde. To 5 c.c. of the milk add 5 c.c. of distilled water and 10 c.c. of hydrochloric acid, containing a little ferric chloride. The presence of formaldehyde is indicated by a violet color. Detection of Heated Milk. Storch's Method. Five c.c. of milk are poured into a test tube; a drop of weak solution of hydrogen dioxide (about 0.2 per cent) which contains about 0,1 per cent of sulphuric acid 372 MILK AND MILK PRODUCTS is added, and two drops of a 2 per cent solution of paraphenylendiamin (solution should be renewed quite often), then the fluid is shaken. If the milk or cream becomes, at once, indigo blue or the whey violet or reddish brown, then this has not been heated, or, at all events, it has not been heated higher than 78° C; if the milk becomes a light bluish gray immediately or in the course of half a minute, then it has been heated to 79° to 80° C. If the color remains white, the milk has been heated at least to 80° C. In the examination of sour milk or sour buttermilk, lime water must be added as the color reaction is not shown in acid solution. (Report of the Commission on Milk Standards.) Arnold's Guaiac Method. A little milk is poured into a test tube and a little tincture of guaiac is added, drop by drop. If the milk has not been heated to 80° C. (176° F.) a blue zone is formed between the two fluids; heated milk gives no reaction, but remains white. The guaiac tincture should not be used perfectly fresh, but should have stood a few days and its potency have been determined. Thereafter it can be used indeiSnitely. These tests for heated milk are only active in the case of milks which have been heated to 176° F. or 80° C. (Jensen's Milk Hygiene, Pearson's translation, p. 192.) Microscopic Test for Heated (Pasteurized) Milk. Frost and Ravenel (1911). About 15 c.c. of milk are centrifuged for five minutes, or long enough to throw down the leucocytes. The cream layer is then completely removed with absorbent cotton and the milk drawn off with a pipette, or a fine-pointed tube attached to a Chapman air pump. Only about 2 mm. of milk are left aboVe the sediment which is in the bottom of the sedimentation tube. The stain, which is an aqueous solution of safranin 0, soluble in water, is then added very slowly from an opsonizing pipette. The important thing is to mix stain and milk so slowly that clotting does not take place. The stain is added until a deep opaque rose color is obtained. After standing three minutes, by means of the opsonizing pipette, which has been washed out in hot water, the stained sediment is then transferred to slides. A small drop is placed at the end of each of several shdes and spread by means of a glass spreader, as in Wright's method for opsonic index determinations. In an unheated milk the polymorphonuclear leucocytes have their protoplasm slightly tinged or are unstained. In heated milk the polymorphonuclear leucocytes have their nuclei stained. In milk heated to 63° C. or above, practically all of the leu- cocytes have their nuclei definitely stained. When milk is heated at a TESTS FOR HEATED MILK 373 lower temperature the nuclei are not all stained above 60° C. The majoiity, however, are stained. Frost (1915) has pointed out bonie of the difficulties of the above method and devised a new one. Procedure. Add one part of aqueous niethylei:e blue (7 gms. of Gruebler's dry dye to 100 c.c. distilled water) to jfivc parts of milk. This should be added slowly to prevent coagulation of the milk. Allow the stain to remain in contact with the milk for from fifteen to thirty minutes. Centrifuge and spread the sediment on a slide. This may be done by using the edge of another slide. Frost reports the following appearance in raw and pasteurized milk, /fair; The entire field is stained a Hght blue, the depth of the stain depending on the thickness of the film. In this blue background may be clear areas. These may be either leucocytes or fat globules. The leucocytes are practically always colorless. Heeded Milk: Smears made from heated milk are not as deeply stained as those made from raw milk. The leucocytes are always more deeply stained than the background and appear as dark blue areas in a blue field. The background immediately about the leucocytes frequently shades off into the color of the background, thus forming a halo about them. Under the oil immersion objective the leucocytes are less regular in outline than those in raw milk. The nuclei are distinctly stained. Balaz's Test for BoUed Milk. To 5 c.c. of the milk; add 2 c.c. of copper sulphate solution which contains 69.26 gms. of copper sulphate per liter. Shake and filter. Then, to five drops of the transparent filtrate add Adamkiewicz's reagent (one part of sulphuric acid and two parts of acetic acid). Warm carefully without boiling, shake, and allow to stand for a while. The serum of the boiled milk remains colorless, while the raw milk shows a violet red color. Its greatest intensity occurs within ten or fifteen minutes. The '^ Commission on Milk Standards Appointed by the New York Milk Committee '^ has made a careful study of the production of safe milk. It is composed of the foremost authorities on the relation of milk to the pubhc health. Realizing the lack of uniformity with regard to milk standards, the committee made the following recommendations. Chemical Standards. The following are proposed and represent those which are enforced in many cities and States. The Babcock test makes easily practicable the determination of fat and solids-not-fat in milk. Such examinations of milk can be readily adopted and executed by any health board laboratory at a very moderate expense. It is believed that such chemical standards as are suggested 374 MILK AND MILK PRODUCTS will help to raise the standards of dairying in this country, and that the provision regarding substandard milk is a liberal one. Cow^s Milk. Standard milk should contain not less than 8.5 per cent of sohds-not-fat and not less than 3.25 per cent of milk fat. Skim Milk. Standard skim milk should contain not less than 8.75 per cent of milk solids. Cream. Standard cream should contain not less than 18 per cent of milk fat, and should be free from all constituents foreign to normal milk. The percentage of milk fat in cream over or under that standard should be stated on the label. Adjusted Milks. On the question of milks and creams in which the ratio of the fat to the solids-not-fat has been changed by the addition to or subtraction of cream or milk fat the commission has hesitated to take a position. On the one hand they are in favor of every procedure which will increase the market for good milk and make the most profit- able use of every portion of it. On the other, they recognize the sensitiveness of milk, the ease with which it is contaminated, and the difficulty of controUing such processes as standardizing, skimming, homo- genizing, souring, adjusting, etc., so as to prevent contamination and the use of inferior materials. On this subject the commission passed a resolution presented by a special committee, as follows: The committee believes that it is probably necessary to admit standardized and adjusted milk. They believe that such manipulation should be controlled and that such milk should be distinctly labeled as to its modifications. Milk in which the ratio of the fat to the solids-not-fat has been changed by the addition to or subtraction of cream should be labeled ^'adjusted milk"; the label should show the minimum guaranteed percentage of fat and should comply with the same sanitary or chemical requirements as for milk not so standardized or modified. The committee very carefully considered the subject of the agita- tion which has taken place regarding percentage of sohds-not-fat due to the fact that in some large cities much of the milk contains less than 8.5 per cent sohds-not-fat. While the commission is disposed to admit that these conditions may exist, yet it beheves that these conditions can be remedied, if not immediately at least gradually. On the other hand, experience has shown that to lower the standard would, in a few years, result in the lowering of 5he general quaUty of the milk placed on the market, since commerce always tends to approach the minimima stand- ard. The commission, therefore, thinks it is unwise to reduce the standard for sohds-not-fat below the percentage of 8.5. In those com- munities where such a standard can not be rigidly enforced at the BACTERIAL EXAMINATION 375 present time the commission suggests that the standard be gradually applied. The Bacterial Examination of Milk The value of the bacterial examination of milk is somewhat depend- ent upon the application of the results and the opinions of those who are interpreting them. The Commission on Milk Standards regards the bacterial content as being due to dirt, temperature and age. The bac- terial examination, th^n is supposed to yield information bearing on these three factors. Initial Contamination. Milk is such an excellent medium for bac« teria that the initial infection determines the number which will be present after a time. The factors which influence this may be men- tioned as follows: I. Cow a. Udder, 6. Coat, etc. II. Barn Conditions III. Milker IV. Utensils a. Pails, cans, etc. 6. Special apparatus 1. Separator 2. Clarifier 3. Pasteurizer 4. Cooler 6. Bottler It is fairly well established that milk as it is excreted from the udder is not sterile. Harding and Wilson (1913) and other workers to whom they refer in their paper furnish much information to confirm this state*» ment. An examination of 1230 samples from the udders of 78 cows showed an average of 428 bacteria per cubic centimeter. Evans (1916) studied 192 samples of milk from 161 cows in dairies which were widely separated. Three types of bacteria were found commonly present in milk from the five dairies which were studied: streptococci, staphy- lococci and bacilU. Streptococcus lacticus (Kruse) was not found in any of the samples. This author states that '' there is a definite udder flora comprising bacteria which belong to parasitic types. It is not 376 MILK AND MILK PRODUCTS surprising that the majority of udder bacteria should be of the same type as those common on the skin and mucous membranes of man and animals. The majority of the staphylococci on the skin are of the non- virulent variety which fails to produce pigment and fails to ferment mannite. But pathogenic varieties are also found on the skin where they ordinarily cause no trouble. . . . Whatever the variety may be, conditions in the udder are favorable to multipUcation and frequently large numbers are eliminated in the milk." Rogers and Dahlberg (1914) in studying the origin of streptococci in milk decided that they were "contributed by the feces; infected udders and the animals' mouths were also mentioned. Licking of the flanks and udders allow the bac- teria to reach the milk. Sherman (1915) found 19 out of 142 cows from 6 different herds that gave a milk with over 10,000 bacteria per cubic centimeter. Two of them had a bacterial content of 100,000 bacteria per cubic centimeter in their fresh milk. Sherman states that all of the cows were in a normal healthy condition but he does not state what data were secured to arrive at this conclusion. In 48 cows, in which there were no indications of disease, there were streptococci in the milk of 15. He regards the streptococci of milk as of Httle significance, which is a very interesting statement in light of the work on septic sore throat. Colwell (1917) studied the influence of high count and gargety cows on the number of bacteria in milk. Seventy-two per cent of the cows examined produced a milk with less than 10,000 bacteria per cubic centimeters. The other 28 per cent were classed as high-count animals. An interesting fact was established that, where one or more of the quarters were infected with an organism, the same organism could be isolated from the other quarters. Such data have much significance in the question of the relation of milk to septic sore throat; it points out that all of the milk fiom an animal with an infected udder must be excluded from the milk supply. From the literature which has beeen presented above, it will be seen that some difference of opinion exists about the udder flora. The pre- dominating organisms seem to be luicrococci and, as Evans pointed out, may come from the cow's body. These enter the teat and reproduce since they are in a favorable environment. With regard to high-count cows too little information is available to definitely understand them. They are excluded from certain herds when certified milk is being pro- duced. No exacting study is usually made to determine whether they are really healthy. If nothing is outwardly the matter with them, they are usually considered healthy without any other examination. It is possible that the large numbers of bacteria in the milk of certain cows FACTORS INFLUENCING BACTERIAL CONTENT 377 could be explained by a more careful investigation with regard to the condition of the animal. Barn Conditions. Some difference of opinion still exists with regard to the effect of barn conditions on the bacteria in milk. Harding et ah (1913) found that many procedures about the barn which were sup- posed to limit the number of bacteria contributed to milk might, under some conditions, increase the count. Ruehle and Kulp (1915) have shown that dusty air in a stable might increase the bacteria in milk but the increase, as such, would be of little sanitary importance. If these added bacteria developed, the increased count, at a later time, would come within sanitary considerations. Savage (1909-10) found a general agreement between the cleanliness of cows' stables and the number of bacteria in milk. This also was borne out by the work of Brainerd (1911). Utensils. Recent work points to the conclusions that utensils may be one of the most important factors determining the number of bacteria in milk. Pease (1916) claims that, where high counts have been obtained in a dairy which has operated for a long time, they are due to inefficiently cleaned apparatus and by incubation of the bacteria on the moist surfaces of the cans, pails, etc. Prucha, Harding and Weeter (1915) reported some interesting data to substantiate this contention of Pease. With sterile utensils and sterile bottles, milk leaving the barn was found to contain 2588 bacteria per cubic centimeter and the bottled milk 3875. Where the utensils were simply washed and with only the bottles sterile, there were increases due to the pails of 57,077, up to the clarifier of 15,353, due to the clarifier of 172,763, due to cooler of 19,841 and due to the bottler of 247,611. More data on the same subject have been since reported by Prucha, Weeter and Chambers (1918). Ayers, Cook and Clemmer (1918) in a careful study of the factors involved in the production of a milk of low bacterial content, state as follows: '^ Three simple factors were necessary for the pro- duction of milk with a low bacterial content, namely, sterilized utensils, clean cows with clean udders and teats, and the small top pail.^' A fourth factor, holding the milk at a temperature near 10° C. or lower is necessary in order to keep the bacterial count low. Even cow manure was found to be of less importance in influencing the bacterial count than utensils. Age. The bacterial count is supposed among other things to yield information with regard to the age of the milk. Up to certain limits this is true, but soon a limit is reaished beyond which no further growth may take place unless something happens to disturb the conditions. 378 MILK AND MILK PRODUCTS The number of bacteria is probably not proportional to the age of the milk. Temperature. The effect of temperature on the bacteria in milk has received attention from several standpoints. No single extensive piece of work has been carried out on this subject and consequently our knowledge must come from the various separate pieces of work. Lux- wolda (1911) found that milk kept at low temperatures contained an enormous number of bacteria. These did not cause an acid reaction in the milk but the changes were regarded as more dangerous than if the acid bacteria had been present. At 20° C. lactic acid bacteria appeared and exerted a restraining action on the other forms. This restraining action was steadily diminished as the temperature was lowered. Reed and Reynolds (1916) inoculated sterile fiasks of milk with pure cultures and incubated them at four different temperatures for six weeks. The results are shown in Table XL. Table XL EFFECT OF AGE AND TEMPERATURE ON BACTERIAL GROWTH IN MILK (After Reed and Reynolds) Name of Organism. Bacterium lactis acidi Sarcina lutea Bacillus coli Bacillus cyanogenes Bacillus proteus vulgaris Bacillus aerogenes Bacillus fluorescens liquefaciens . BaciUus puditum Microspira tyrogena BaciUus subtilis Micrococcus citrieus Oidium lactis Bacillus prodigiosus Age in Days, of Milk kept at Various Tempera- tures, WHICH Gave Maximum Counts on Nutrient Agar. Incubator, SS" c. Room, 15-28° C. Water Tank, Cold Stor- age, ~1°C. 1 3 5 3 1 42 42 21 3 5 5 2 2 21 4 42 2 2 4 3 3 5 42 3 2 5 42 3 3 4 5 21 3 21 42 42 3 42 42 21 21 5 4 4 42 42 5 42 3 42 42 42 Reed, H S and Reynolds, R. R. Some effects of temperature upon the growth and activity of bacteria m milk, Virginia Ag. Exp. Sta. Bull. (Tech.), 10, 1916. Conn and his co-worker Esten (1902, 1903, 1904) gave much atten- tion to this subject. Their work was some of the earUest on this sub- ject. In general, they found that at 20° G. the ordinary lactic acid ^^ QUALITY" IN MILK 379 bacteria predominated. At the end of forty hours, when the milk had curdled, Bacillus acidi ladici constituted about 90 per cent of the total bacteria. At 37° C, B. lactis aerogenes predominated over B, acidi ladici as did 5. coli when it was present. At 10*^ C. all types of bacteria developed uniformly. Neutral bacteria grew more rapidly and Uquefiers became abundant. Conn and Esten found Httle differ- ence in the flora at 10° C. and V C. Growth, however, was more rapid at 10° C. Ravenel et al. (1910) have studied the changes in milk at —9° C. and 0° C. At —9° C. there was no increase in the number of bacteria as determined on agar and gelatin plates. At 0° C. there was a decided increase. This was accompanied by an increase in acidity and other changes incident to marked bacterial development. The lactic acid bacteria were inhibited vA this temperature but putrefactive bacteria were not. Pennington et al. (1908, 19 13) in their studies on cold storage reported that milk stored at 0° C. underwent marked proteolysis which was noticeable at the end of two weeks. In a later paper it was stated that proteolysis of casein was due primarily to bacteria while the lact- albtmiin was destroyed by the enzymes in the milk. The numbers of bacteria greatly increased and this increase was most striking in the raw, untreated milk. The freezing-point was gradually lowered with the decomposition. Market milk held below 0° C. increased from 15,956 to 376,000,000 at the end of five weeks. These investigations tend to indicate that milk and other foods may not be held indefinitely even at low temperatures. Changes may eventually take place to render the food unfit for consumption. " What is Meant by Quality in Milk?" Quality in any food sub- stance may be defined in different ways. The definition often depends entirely on the viewpoint of the definer. The farmer may have quite another conception of quality in milk than the sanitarian; the chemist may define milk quality from the standpoint of food value while the bacteriologist is concerned with disease. It is a far easier proposition to define milk quality than to devise methods for measuring it. One could scarcely find a field in hygiene which is more complicated than that of dairy and milk sanitation. The difficulty of finding practical methods for measi^ing quality in milk has caused much contention among health authorities. Probably little difficulty would be experi- enced in reaching a definition for safe milk. A milk to be safe must be clean. In fact cleanliness is the slogan of modern sanitation. A number of different methods have been used or proposed for measuring quality in milk. Each one has its own special advantages and 380 MILK AND MILK PRODUCTS objections. Some have been discussed before in this book and will not be given further attention. The following methods have been pro- posed at one time or another: 1. Sanitary inspection of the dairies; 2. Bacterial examination of the milk; 3. Dirt or sediment test. Harding et al. (1917) consider that the elements of quality in city milk to consist of food value, healthfulness, cleanliness and keeping quality. The discussion of these factors leads these authors to the following combination of them to form a basis for grading milk. Park Grade Special milk Element of Quality Degeee of Excellence Table milk Food value Healthfulness Cleanliness ^ Keeping quality Food value Healthfulness Cleanliness Keeping quality Cooking milk ^ r Food value Healthfulness Cleanliness . Keeping quality Fat content as stated on package Medical supervision of health of men and animals, or proper pasteurization Sediment, not more than a trace Excellent Fat content as stated on package Properly pasteurized Sediment, not more than a small amount Good Fat content as stated on package Boiled May not be sufficient for table grade May not be sufficient for table grade (1918) does not approve some of the claims in this report. He calls attention to the fact that Harding and his coUaboratois are inter- ested in the dairy instead of the infant. / He further states, " The experts . . . were considering the best methods of grading milk and not the question as to whether excessive numbers of bacteria in market milk were wholesome or not. It would be, to my mind, a step backward to accept the views expressed in the body of the editorial." (From a letter discussing this report.) Park and his colleagues at the Research Laboratories of the New York City Department of Health have given much study to the relation of milk bacteria to disease in children. The Dairy Score Card. No attempt will be made to present in this place a complete discussion of the dairy score card. The score card had DAIRY SCORE CARD 381 its origin in 1904 when Doctor W. C. Woodward prepared one for use in Washington A year later Pearson at Cornell University devised a similar card. The score card received such approbation by sanitarians that the National Association of Dairy Instructors appointed a com- mittee which had much to do with the present official score card. Much has been written m favor of and against the card. It is regarded by some milk hygienists as one of the most important factors in the production of clean milk. Others, even though they represent large cities do not regard the card as of any great help in milk control. It must be empha- sized that the score card was not devised to give information with regard to bacteria in milk but merely to rate the production plants. It has been estabhshed, generally speaking, that a high scoring dairy will have a low number of bacteria in its milk. This is to be expected. Some of the recent work in dairy hygiene has emphasized the fact that the present score card lays too much stress on factors which are not directly concerned with the production of milk with few bacteria. Brew (1915) made a comparative study of the bacteria in the milk and the scores of thirty-four commercial dairies. He found no correlation between these two factors. The apparent reason for this was that a large number oi items called for on the score card have little or no effect on the bacteria in the milk. Too much emphasis is placed on procedures which do not affect the milk. This study indicates that the score card needs revision if it is to be an index of the bacteria in milk. As indicated by recent studies, within certain limits, the environment of the cow does not seem to exert much influence on the milk. Prucha, Harding and Weeter (1915) also confirm the contention of Brew that the score card needs revision. They found that utensils were important in relation to the number of bacteria in milk. These are not given an important place on the card. Ayers, Cook and Clem,mer (1918) reach the same con- clusion. Brainerd and Mallory (1914) in studying the Richmond, Virginia, milk supply by means of the bacterial count and score card found the count to be cumbersome — a high count not always being char- acteristic of a dangerous milk. For some reason which was not deter- mined, they found that the highest scoring dairy showed a larger count than the lowest scoring dairy. North (1917) believes that the life of the present score card is threatened if it is not changed to give a reason- ably close indication of the character of the product of dairies. North has presented a revised score card on which he has tried to place emphasis on factors which are concerned in keeping the number of bacteria in milk low. This to a certain extent may change the purpose of the card. 382 MILK AND MILK PRODUCTS BOAED OF HEALTH Racine, Wis. Sanitary hispectian of Dairies Dairy Score Card Owner or lessee or farm P. 0. Address State '. Total Number of cows Number milking Galloxis of milk produced daily Product is retailed by producer in Sold at wholesale to ! For milk supply of Permit No Date of Inspection ,191 Remarks (Signed) , Inspector^ DAIRY SCORE CARD 383 DETAILED SCORE Score. Equipment. COWS Health Apparently in good health 6 If tested with tuberculin once a year and no tu- berculosis is found, or if tested once in six months and all reacting animals removed .... 5 (If tested only once a year and reacting animals found and removed, 2.) Comfort Bedding 1 Temperature of stable. 1 Food (clean and wholesome) Water Clean and fresh 1 Convenient and abundant 1 Perfect. Allowed. STABLiES Location of stable Well drained 1 Free from contaminating surroundings 1 Construction of stable Tight, sound floor and proper gutter 2 Smooth, tight walls and ceiling 1 Proper stall, tie, and man- ger 1 Means of light: 4 sq.ft. of glass per cow (Three sq.ft., 3; 2 sq.ft., 2; 1 sq.ft., 1. Deduct for uneven distribution.) Ventilation: Automatic sys- tem Adjustable windows 1 Cubic feet of space for cow: 500 to 1000 ft (Less than 500 ft., 2; less than 400 ft., 1 ; less than 300 ft., 0; over xOOO ft., 0.) UTENSILS Construction and condition of utensils Water for cleaning. (Clean, convenient, and abundant.) Small-top milking pail Facilities for hot water or steam (Should be in milk house not in kitchen.) Milk cooler Clean milking suits , MILK ROOM ' Location of milk room Free from contaminating surroundings 1 Convenient 1 Construction of milk room. Floor, walls, and ceiling, 1 Light, ventilation, screens 1 Total. 40 iMethods. Score. COWS Cleanliness of cows STABLES Cleanliness of stables Floor 2 Walls 1 Ceiling and ledges 1 Mangers and partitions 1 Windows. 1 Stable air at milking time. Freedom from dust. . . 3 Freedom from odors. . . 3 Barnjrard clean and well drained Removal of manure daily to field or proper pit (To 50 ft. from stable, 1.) MILK ROOM Cleanliness of milk room . . . UTENSILS AND MILKING Care and cleanliness of uten- sils Thoroughlj^ washed.. . . 2 Sterilized in live steam for thirty minutes. . - 3 (Placed over steam jet, or thoroughly scalded with boiling water, 2.) Inverted in pure air 3 Cleanliness of milking. ..... Clean, dry hands 3 Udders washed and dried 6 (Udders cleaned with moist cloth, 4; cleaned with dry cloth at least fifteen min- utes before milking, 1.) HANDLING THE MILK Cleanliness of attendants in milk room Milk removed immediately from stable Prompt cooling (cooled im- mediately after milking each cow) Efficient cooling: below 50" F (SI*' to 55°, 4; 56° to 60% 2.) Storage: below 50° F (51° to 55°, 2; 56° to G0°» 1) Transportation: iced in sum- j mci' , (,For jacket or wet blan- ket, allow 2; dry blanket or 'covered wagon, 1.) Total , Perfect. Allowed. 60 Equipment + Methods ~ Final score. Note 1. — If any filthy condition is found, particularly dirty utensils, the total score shall be limited to 49. ♦ Note. 2. — If the water is exposed to dangerous contamination, or there is evidence of the presence of a dangerous disease in animals or attendants, the score shall be 0. 384 MILK AND MILK PEODUCTS Dirt or Sediment in Milk. The estimation of dirt in milk has been regarded by many as a reliable index of milk quality. Most attention has been given to insoluble dirt or that which may be removed from the milk by some mechanical means. Schroeder (1914) has given a resume of the different methods of determining dirt in milk. He stated that the amount of dirt in fresh milk approximates the number of bacteria. This was borne out by the work of Reed and Reynolds whose data bearing on this question have been presented in Table XLI. Campbell (1916) carried out an interesting study on the relation of the number of bac- teria to the amount of dirt and stated that the amount of dirt which appeared on the disks was no criterion of the number or kind of bacteria in the milk. Ayers, Cook and Clemmer (1918) found that'the " sedi- ment test bore a somewhat close relation to the number of bacteria in fresh, unstrained milk handled in sterilized utensils." The kind of dirt is, of course, important in this connection. Cow dung would probably be an objectionable type. Ayers and his colleagues, however, state that cow dung (fresh) " though an important source of contamination in general, is not so great a factor as unsterilized utensils in causing high bacterial counts.'' Kinyoun (1914) regards cleanliness in production as the most important factor in milk hygiene. From an examination of 3000 samples of milk he states that in good milks, there is 1 colon bacillus to every 50,000 bacteria while in dirty milk the ratio is 1 to 555. Table XLI RELATION BETWEEN THE AMOUNT OF DIRT IN MILK AND THE BACTERIAL CONTENT (After Reed and Reynolds) Amount of Dirt m 100 c.c. of Milk. Number of Bacteria per Cubic Centimeter of Milk. 10 milligrams 20 milligrams 4,833 6,750 12,000 17,625 18,375 32,875 30 milligrams , 40 milligrams 50 milligrams 100 milligrams The available data seem to indicate that milk may be produced under decidedly filthy conditions and yet have a low bacteria content. The utensils seem to be the contributing factor. These data should not be taken to prove that barns may be kept dirty. Milk, which is produced in dirty plants, is more liable to contain dirt; this dirt may be SIGNIFICANCE OF BACTERIAL CONTENT 385 very objectionable. It is reasonable that a farmer who is careful about the condition of his cows will be just as careful about the other features which are known to affect milk quaHty. Schroeder (1914) has classified the methods of determining dirt in milk as follows: Group I. (a) Type in which the sediment is obtained by gravity fil- tration. Conn's filter paper method. The Lorenz or Wisconsin tester. The Stewart tester. The Gerber tester. The Schroeder tester, or multiple filter. (6) Type in which pressure or suction is used. The Lorenz improved. The Wizard. The Gooch crucible. Group 11. Type in which sediment is obtained by means of the cen- trifuge. The Babcock. The Gerber. The Stewart-Slack. Conn's centrifugal method. Significance of the Bacterial Count. There is no agreement on this important question. The Commission on Milk Standards makes the following statement on the subject: Bacterial Counts and Decency, On this subject the comniission passed the following resolutions: (a) Because high bacterial counts indicate milk is either warm, dirty, or stale, the bacterial count is an indicator of decency in milk character, entirely apart from its significance as an indicator of the safety of milk. (6) In determining the sanitary character of milk and the grade in which it belongs, decency must be considered as desirable for its own sake, entirely apart from the consideration of safety. Decency is important as a characteristic of foods and drinks, because it gives pleasure to the consumption of food, while the lack of decency means distaste, displeasure, and even disgust. (c) The bacterial count is a sufficiently accurate measure of decency to justify the health ofiicer in condemning milk with a high bacterial count because it is lacking in this characteristic. Bacteriological Laboratory Testing of Milk, On the subject of laboratory examinations of milk for bacteria the commission beheves that the interest of 386 MILK AND MILK PRODUCTS public health demand that the control of milk supphes, both as to production and distribution, should include regular laboratory examinations of milk by bacteriological methods. They stated by resolution that — Among present available routine laboratory methods for determining the sanitary quality of milk, the bacterial count occupies first place, and that bac- terial standards should be a factor in classifying milk of different degrees of excellence. The adoption and enforcement of bacterial standards will be more effective than any other one thing in improving the sanitary character of public milk supplies. The enforcement of these standards can be carried out only by the regular and frequent laboratory examinations of milk for the numbers of bacteria it may contain. It is of the utmost importance that standard methods should be adopted by all laboratories for comparing the bacterial character of milks, since by this means only is it possible to grade and classify milks and properly enforce bac- terial standards. Concerning the methods which should be used by milU laboratories for determining the numbers of bacteria the commission unanimously resolved: That there be adopted as standards for making the bacterial count the stand- ard methods of the American Pubhc Health Association Laboratory Section. One of the chief objections raised against pasteurization is the claim that it is frequently employed to cover filthy methods, the milk producer using less care in his methods if he knows that the milk is to be subsequently pasteurized. To meet this objection the commission believes there should be bacterial standards for raw milk as well as bacterial standards for pasteurized milk. In the case of pasteurized milk, standards should be required of the milk before pasteurization as well as after pasteurization. ReUability of Bacterial Tests, The commission has considered the numerous criticisms that have been raised as to the unreliability of bacteriological analyses, and has made extensive inquiry as to the force of these criticisms. An opinion concerning the reliabihty of laboratory tests for numbers of bacteria has been reached based on voluminous statistics secured for the most part by groups of observers working together, as well as by individuals. One of these researches alone carried out by members of the commission in cooperation with others included the testing of over 20,000 samples of milk. In other instances repeat- edly the same sample of milk was tested one hundred times. Some variations in the analysis of duplicate samples are inevitable, due to the fact that the bac- teria are not in solution, but are floating in the milk more or less clustered to- gether in clumps, each of which will count only as a single colony. Under such conditions only an approximate agreement can be expected. The results of extensive study justify the commission in the conclusion that the analysis of duplicate samples of milk made by routine methods in different laboratories may be expected to show an average variation of about 28 per cent, with occasional samples of wider variation. In some good laboratories the varia- tion may not be greater than 10 per cent* Variations in results diminish with RELIABILITY OF BACTERIAL COUNTS 387 the numbers of samples analyzed. If five samples of the same milk are tested the results may be relied upon as fairly accurate, and always sufficiently accurate to place any particular milk supply unhesitatingly in Grade A, B, or C. The object of bacterial tests of milk samples for the numbers of bacteria should be primarily to determine the sanitary character of the milk supply from which the sample is taken, rather than the character of a single sample of milk. It is strongly urged by this commission that no grading of milk should be made upon the analysis of single samples, and that no prosecutions or court cases should be brought upon the bacterial analysis of a single sample of mEk. Interpretation of Bacterial Tests. The commission has put its opinions on this subject in the form of resolutions, as follows: Whereas milk is one of the most peribhable foods, being extremely suscep- tible to contamination and decomposition; and Whereas the milk consumer is justified in demanding that milk should be clean, fresh, and cold, in addition to having the element of safety; and Whereas milk which is from healthy cows and is clean, fresh, and which has been kept cold, will always have a low bacterial count; and Whereas milk that is dirty, stale, or has been left warm, will have a high bacterial count; therefore it is resolved: First. That the health oflicer is justified in using the bacterial count as an indicator of the degree of care exercised by the producer and dealer in securing milk from healthy cows and in keeping the same clean, fresh, and cold; and Second. 'That the health officer is justified in condemning milk with a high bacterial count as being either unhealthy or decomposed, or containing dirt, filth, or the decomposed material as a result of the multiplication of bacteria due to age and temperature. Third, That the health officer is justified in ruling that large numbers of bacteria are a source of possible danger, and that milk containing large numbers of bacteria is to be classed as unwholesome, unless it can be shown that the bac- teria present are of a harmless type, as, for example, the lactic acid bacteria in buttermilk or other especially soured milks. Grading by the Bacterial Count. Concerning the number of tests which should be made in order to determine the grade of a milk supply, the commission recommends that the grade into which a milk falls shall be determined bac- teriologically by at least G.Ye consecutive bacterial counts, taken over a period of n,ot less than one week, nor more than one month, and that at least four out of five of these counts (80 per cent) must fall below the limit or standard set for the grade for which classification is desired. The grading of milk has necessarily been based on its sanitary character, primarily as determined by the bacterial test. The enforcement of grading, therefore, requires the application of the bacterial test in a manner sufficiently comprehensive to fairly determine the sanitary character of milk so that it may be assigned to the grade in which it belongs. Such an administrative syBtem greatly modifies the former conception of milk inspection by public health officials. The inspection ser\dce under the grading system becomes subordinate 388 MILK AND MILK PRODUCTS to the bacterial laboratory, or at least must look to the bacterial laboratory as a guide. If bacterial tests are recognized as an indication of the sanitary charac- ter of milk, then the bacterial laboratory tests should precede the dairy inspec- tion since they will point out to the dairy inspector the location of unsanitary milk. In the enforcement of the grading system, therefore, the milk inspection service should be reorganized in such a manner that the bacterial laboratory makes its tests first, in order to determine the sanitary character of the various milks offered for sale on the city market, and the inspection service then takes up the task of discovering the location and causes of the defects which the laboratory has discovered and of remedying them. The laboratory service and inspection service consequently must be centralized under one head and their work thoroughly coordinated in order to give the greatest economy and effi- ciency. It is quite apparent from the above rather lengthy quotation from the report of the Commission on Milk Standards, that they regard the bacterial count as of much importance. Contrary to this is the opinion of Harding (1917) and a few others who maintain that the number of bacteria in milk has no relation to the conditions under which the milk was produced. In order to back up this stand the following argument has been used. '' The most offensive, and, at the same time, a typical form of filth that may get into milk is cow dung. The bac- terial plate count resulting under standard methods of milk analysis gives rather less than 5,000,000 per gram as the germ count of fresh cow dung. A gram of such material added to a liter (approximately 1 qt.) of milk would, accordingly, increase the germ count but 5000 per cubic centimeter. Hence, otherwise l6w-content milk might carry approxi- mately 2 gms. of cow dung per quart and still not exceed the ordinary limit of 10,000 per cubic centimeter for certified milk. Likewise a quart of Grade A milk, which is understood to be clean milk, according to the present accepted standards might contain 12 gms. of cow dung per quart and still be legal Grade A milk. ... In establishing bacterial count standards as an index of cleanliness, we are lending official sanc- tion to conditions which would outrage public decency and are creating a false sense of security and of cleanliness.^' It is common knowledge that fresh cow dung has a very variable bacteria content. Usually it is much higher than 5,000,000 per gram and may even approach or exceed a billion. Ayers et al. (1918) found an average bacterial content of 50,000,000 in 57 samples of fresh cow dung. This is probably dependent upon what the cow eats, for with human beings the diet has much to do in determining the bacterial flora. Then, again, fresh cow dung does not ordinarily gain entrance to milk. It is the dry dung BACTERIAL COUNTS 389 which happens to fall into the milk from the cow^s body and this has a bacteria content which is far different from fresh cow dung. In this connection, Taylor (1918) has shown that 85 per cent of fresh cow dung will dissolve in milk. His studies were carried out with the fresh (wet) cow feces and it required a Uttle restriction to apply his results directly to dirt in milk. Fresh cow dung contains about 85 per cent of moisture and this water ought not to be considered as cow feces. Prucha, Weeter and Chambers (1918) carried out a series of investigations on this subject using dried feces. In discussing this question of bacterial counts in milk Pease (1913, 1916) claims that ^' none of the bacteria found on our counting plates are even recognized by bacteriologists as belonging to the groups of those microorganisms which produce the well- known specific diseases.'^ His general opinion seems to be that the dairy score card representing sanitary inspection of the dairy should come before the bacterial count and that any attempt to make it of secondary importance to the count is a decided step backwards. North (1913, 1916) has stated that confusion exists over the question of dirt and disease and that this has caused some misunderstanding. North (1917) has presented a revised score card and has tried to obviate some of the discrepancies inherent in the present one. In his earher papers North states that there has been an undercurrent of competition between dairy inspection and bacterial testing and suggests that a system of cooperation be worked out between these two factors. One cannot help but be impressed with the fair-minded stand of this inves- tigator. Rogers (1915) claims that, how much a high bacterial count is due to contamination and how much is due to multiplication is uncertain. He suggests that it may become necessary to distinguish between measures to reduce the bacterial count and measures in the interest of decency and cleanliness. Conn (1917) has well stated the question. ^^The bacteriological analysis of milk is not to be taken as indicating in itself either a condition of safety or a condition of danger, but only as a warning. Good, clean, fresh milk will have a low bacterial count, and a high bacterial count means dirt, age, disease or temperature. A high bacterial count is, therefore, a danger signal and justifies the health officer in putting a source with a persistently high bacterial count among the class of unwholesome milkJ^ Rosenau (1912) makes the following statement on this subject: The enumeration of bacteria ia milk is, therefore, one of the readiest and cheapest methods at the disposal of the health officer to determine the general sanitary quality of the market milk supply. The laboratory results serve not 390 MILK AND MILK PRODUCTS only as a guide to direct the efforts of the health officer, but to confirm the con- clusions arrived at from an inspection of the dairies and dairy farms. A review of the literature with regard to the bacterial analysis of milk indicates a confused condition. There seem to be two distinct views with regard to the significance of the number of bacteria in milk. That side supporting the view that the bacterial content she aid not be used is made up of those who are interested in the production of milk and possibly the ^' protection '' of the farmer. The other side is supported by well-known physicians and public health officials who have been engaged for some time in protecting the consuming public from dirty foods. Park and Williams (1910) have shown a close relationship between infant death rate and the number of bacteria in milk. These data were secured after careful experimentation and observation. Numerous data have been secured in this connection when comparing the value of raw and pasteurized milk in infant feeding. In essentially all of the cases, the pasteurized milk has been shown to be superior and this IS probably not all due to destruction of certain harmful bacteria. A milk or any other food which contains large numbers of bacteria, is more liable to contain undesirable forms than one with a low count. If the streptococci are of any sanitary significance and there are data on record which support the theory that they are, they are more liable to be present in milks with a large number of bacteria. Breed and Brew (1917), in studying the control of market milk by the microscopic examination, found that one-fifth of all the milk which contained bacteria in excess of 1,000,000 per cubic centimeter contained large numbers of streptococci. They state that, " So far as was deter- mined, all of the milk of this type was originally infected from the udders of some one or more cows in a herd." In this case, then, the number of the bacteiia in the milk was closely related to undesira- ble conditions at the source of production. The greater part of the argument against the bacterial count seems to rest upon data which have been produced at several experiment stations to show that many of the barn conditions which have, in the past, been supposed to be very important, are not related to the number of bacteria in the milk when it reaches the consumer. These investigators argue that the quality of city milk is too complex a problem to be solved by the con- sideration of any one factor. They, therefore, put down the four essential factors as food value, healthfulness, cleanliness and keeping quality. From a bacteriological viewpoint the element of cleanliness is the most important because the others may depend upon this one. This element is supposed to be measured by the sediment or visible dirt. GRADES OF MILK 391 No attention is given to the '' invisible dirt '' which may be present. The sediment test of Weld (1907) detects only one kind of dirt— the insoluble or visible. It does not show the amoimt of invisible dirt or insoluble foreign matter, such as urine; this must be regarded as filth in the same way as other matter. Special mention is made of the ^^ extreme sensitiveness of the pubhc in this matter." It is open to question whether the public is sensitive to dirt in milk or any other food product. If it was so the filthy establishments where food is on sale would not be open for business very long. The absence of dirt in milk may not indicate that the milk has been produced under favorable conditions. That no dirt is visible, does not indicate a safe milk. This question, doubtless, needs more consideration from all standpoints. Grades of Milk. The commission beUeves that all milk should be classified by dividing it into three grades, which shall be designated by the letters of the alphabet. It is the sense of the commission that the essential part is the lettering and that all other words on the label are explanatory. In addition to the letters of the alphabet used on caps or labels, the use of other terms may be permitted so long as such terms are not the cause of deception. Caps and labels shall state whether milk is raw or pasteurized. The letter designating the grade to which the milk belongs shall be conspicuously displayed on the caps of bottles or the labels of cans. The requirements for the three grades shall be as follows: Grade A. Raw Milk, Milk of this class shall come from cows free from disease as determined by tuberculin tests and physical examinations by a qual- ified veterinarian, and shall be produced and handled by employees free from disease as determined by medical inspection of a qualified physician, under sanitary conditions, such that the bacterial count shall not exceed 10,000 per cubic centimeter at the time of delivery to the consumer. It is recommended that dairies from which this supply is obtained shall score at least eighty on the United States Bureau of Animal Industry score card. Pasteurized MilL Milk of this class shall come from cows free from dis- ease as determined by physical examinations by a qualified veterinarian, and shall be produced and handled under sanitary conditions, such that the bac- teria count at no time exceeds 200,000 per cubic centimeter. All milk of this class shall be pasteurized under official supervision, and the bacteria count shall not exceed 10,000 per cubic centimeter at the time of delivery to the consumer. It is recommended that dairies from which this supply is obtained shall score at least 65 on the United States Bureau of Animal Industry score card. Grade B, Milk of this class shall come from cows free from disease as determined by physical examinations, of which one each year shall be by a qualified veterinarian, and shall be produced and handled under sanitary con- 392 MILK AND MILK PRODUCTS ditions, such that the bacteria count at no time exceeds 1,000,000 per cubic centimeter. All milk of this class shall be pasteurized under official supervision, and the bacterial count shall not exceed 50,000 per cubic centimeter when deliv- ered to the consumer. It is recommended that dairies producing grade B milk should be scored, and that the health departments or the controlling departments, whatever they may be, strive to bring these sources up as rapidly as possible. Grade C. Milk of this class shall come from cows free from disease, as determined by physical examinations, and shall include all milk that is pro- duced under conditions such that the bacteria count is in excess of 1,000,000 per cubic centimeter. All milk of this class shall be pasteurized, or heated to a higher temperature, and shall contain less than 50,000 bacteria per cubic centimeter when delivered to the consumer. Whenever any large city or community finds it necessary, on account of the length of haul or other peculiar conditions, to allow the sale of grade C milk, its sale shall be surrounded by safeguards such as to insure the restriction of its use to cooking and manufacturing purposes. Milk Standards If it is difficult and intricate to formulate standards for various foods, it is especially true with regard to milk. To a great extent, this is due to the various number of standpoints from which the milk ques- tion may be considered. The farmer is interested more in securing a reasonable financial return on the investment in his milk production plant. The sanitarian, however, often gives little attention to the farmer's side of the question and considers only the effect of the milk on the health of the consumer. Undoubtedly, a broader consideration by any one of these specialists would greatly clear up some of the mis- understanding with regard to certain phases of the milk question. Milk standards are essential but the question with regard to who shall formulate them is still an open one. If a study is made of the milk standards which are being enforced by many of our large cities, the great discrepancy is apparent. There is essentially no agreement whatever in many of these standards. The Commission on Milk Stand- ards in their third report has attempted to estabUsh a simple system for grading milk and to distinguish between milks which are different in their sanitary and other characteristics. The variation in bacterial sBiandards for milk analysis is emphasized very well in Reprint No. 192, May 15, 1914, from the Pubhc Health Reports 29. The standards MILK STANDARDS 393 for many cities in the United Hiatos having a population of 10,000 or over are tabulated. The procedure for the bacteriological examination of milk has been standardized in the same way as has the bacteriological examination of water and sewage. Standard Methods of Bacteriological Analysis of Milk prepared by the Laboratory Section of the American Public Health Association are now used by all bacteriologists making routine analyses of milk and wishing their results to be comparable to those secured in other laboratories. For that reason, much of the following is adapted or copied, with permission, from that report : Routine Milk Analysis. This type of analysis is designed for the control of the public milk supply and for the purpose of grading milk. For routine analysis the work must be capable of being done quickly and cheaply, and in such a way as to give the most speedy results possible and by methods having the smallest possible expenditure of time and money consistent with fair uniformity of result. The demand for rapid methods, with the smallest possible expenditure of time and money has been forced on different laboratories in the past few years, and has brought about various short cuts from the standard methods as previously formulated. The necessity for such rapid and inexpensive methods must be recognized. But it is also evident that they should be uniform in' dif- ferent laboratories. Research Methods, Collection of Samples. All collecting apparatus, glass- ware, pipettes, collecting tubes, bottles, etc., shall be sterilized at a temperature of at least 175° C. for one hour. Each sample shall consist of at least 10 c.c. of milk. Before taking the sample the milk shall be mixed as thoroughly as pos- sible. If the original container can be inverted the mixing of the milk should be done by inverting it several times. If this is impossible, the milk should be stirred with some sterile stirrer. Any stirrer already in the container may be used. If there is none in the container, the sampling pipette (or any other sterile article) may be used; but it should be used for one container only until it is again sterilized. A sample simply poured from a large can is not a fair sample unless the milk in the can be thoroughly stirred. The sample shall be taken from the cans by means of a tube with straight sides long enough to reach to the bottom of the original container, and inserted not too rapidly, with the bottom of the tube left open. This will result in the tube containing a cylindrical section of the milk from top to bottom of the can. The finger then placed on the top of the tube will make it possible to withdraw the tube full of milk and transfer it to the sampling bottle. The sample bottle shall be large enough to hold the entire contents of the tube, all of which must be reserved as the sample. Each tube shall be used for collecting a single sample only and must be washed and sterilized again before being used again. An aluminum tube of the diameter of J in. and 21 ins. long is very convenient. If the sample is to be taken from the bottle^ 894 MILK AND MILK PRODUCTS the bottle should bo first shaken to insure thorough mixing and then the milk may be poured into the sample bottle, although it is better here to use a sampling tube. If the temperature of the milk is desired, it should be taken from a separate sample that should then be discarded. All records shall be made immediately after takmg the sample. The milk sample shall be placed in a properly labeled bottle. The most convenient kind of sample bottles are glass stoppered, or those closing with a cork-lined screw cap. Cotton plugs are not a satisfactory method of closure. The sample bottles shall be placed at once in a carrying case contain- ing cracked ice, so that the milk is cooled at once to near the freezing-point. The samples shall be transferred to the laboratory as quickly as possible, and shall be plated at once. If the samples can be placed in melting ice and water, they may be kept for several hours (12) without an increase in bacteria. If the plates are not made within four hours of the time of collection, the number of hours that have elapsed should be stated on the report. If the milk is kept at 40° F. a slight increase may be found in twelve to twenty hours. Up to twenty hours this will not be more than 20 per cent. Media. This is essentially identical with that used for water analysis and has been fully treated in the chapter on that subject. Sherman (1916) has demonstrated that lactose agar gives a much higher count than plain agar plates for enumerating bacteria in milk. Lactose agar also of some value in differentiating the types of bacteria which are present. Sherman (1915) has recommended that the reaction of agar media used in the bacterial analysis of milk should be 0.5 per cent plus. Plating. For miscellaneous samples the character of which is not known, three dilutions shall be made 1-100, 1-1000 and 1-1 0000. Where the character of milk is known the number of dilutions may be reduced. If the milk is pas- teurized, certified or known to be fresh, and of high grade, the 10,000 and 100 dilutions may be omitted; if the milk is known to be old, the 100 dilution may be omitted. In no case shall less than two plates be made of each sample. Any convenient method of making dilutions may be used always using pipettes and sterile water blanks. The water for dilutions may be placed in dilution bottles (99 c.c. and 9 c.c. are convenient sizes) a&ad sterihzed for one hour in an auto- clave at 15 lbs. pressure. These standard methods do not give the maximum number of bac- teria. Some research workers in order to secure the maximum number of bacteria incubate their plates at 37° C. for five days and at 20° C. for two days. Such an incubation period of seven days' duration is obvi- ously too long for routine work. Slack (1917) has advised the incuba- tion of agar plates made from milk in routine analysis for forty-eight MICR0SC\)P1(^ METHOD OF :MILIv ANALYSIS 395 hours rather than twenty-four hours bccauFie on an average the count is doubled or trebled. In this connection he advises meat infusion agar in place of meat extract agar. Sherman (1916) recommended the addition of dextrose or lactose to agar used in the examination of milk. A much higher count was obtained and the colonies grew much larger. Incubation. Only one period of incubation and one temperature is regarded as standard, forty-eight hours at 37.5°^ C. In crowded incubators ventilation shall be provided. Counting. If among the different dilutions, there are plates contain- ing from 30 to 300 colonies these should be counted, and the number multiplied by the dilution, be reported as the final count. All colonies on such plates should be counted and the numbers averaged. If there are no plates within these limits the one that comes nearest to 300 is to be counted, unless it happens that there are no other plates with a larger number of colonies, or, unless the numbers in the plates check with other dilutions. ' If the number of colonies on the plate is over 300, a part of the plate may be counted and the whole plate averaged. Counting shall be done with a lens magnifying 2.5 diameters (or what opticians call a 2.5 X lens. Near-sighted persons shall use their glasses in counting while far-sighted persons shall remove them. In case it is doubtful whether certain objects are colonies or dirt specks they should be examined under a compound microscope. Reports. In making reports it must be borne in mind that with high numbers obtained by routine methods, only an approximation to accuracy can be obtained- Only the left-hand figures of the final numbers are significant. It is best, therefore, to report only the two left-hand figures of the results, in order to avoid an unwarranted im- pression of accuracy. For example, where the numbers are in the mil- lions no figures smaller than millions have any significance in the routine analysis of milk. In making the report raise the number to the next highest round number, but never lower it. In no case shall the count of a single plate be regarded as sufficient for the purpose of grading milk. If a single sample of milk only is to be tested, there should be at least three plates counted before a report is made. Microscopic Method of Analysis. This method has been brought to such a stage by Breed and his colleagues that it is now included in the Standard Methods of Bacteriological Milk Analysis of the Labora- tory section of the American Public Health Association, 1917. Brew has summed up the advantages and disadvantages of the plate and direct microscopic method as follows: 396 MILK A.ND MILK PRODUCTS'^ ADVANTAGES AND DISADVANTAGES OF THE DIRECT MICROSCOPIC METHOD AS COMPARED WITH THE PLATE METHOD Disadvantages Direct Microscopic Method 1. Difficult to measure accurately such a small quantity of milk. 2. The sample measured is too small to be representative. 3. Dead bacteria may be counted. 4. Error of count is great where bacteria are few or many. 5. Cannot be used for quantitative work when bacteria are few in number. 6. Many fields must be counted, because of the imeven distribution, if an accurate count is desired. Plate Method 1. All bacteria do not grow on the plates because of changes in food tempera- ture relations, or other conditions of environment. 2. The difficulty of breaking up the clumps in the milk affects the ac- curacy of the count. 3. Requires from two to five days' in- cubation period. 4. Different species require different in- cubation temperatures. 5. Gives no idea of the morphology of the bacteria present. 6. More apparatus required, therefore more expensive. Technique com- plicated and difficult for the trained bacteriologists to use in such a way to secure consistent results. 7. Large compact clumps cannot be counted. 8. Bacteria may be lost in the process of preparing slides. Advantages Direct Microscopic Method 1. Less apparatus required, therefore less expensive. Technique simple. 2. The results on a given sample may be reported in a few minutes. Plate Method 1. Is necessary for isolation of pure cul- tures. 2. Gelatin shows the liquefiers and if litmus is used, the acid producing bacteria. 3. Shows the cell content, the presence 3. Shows the character of growth. or absence of streptococci and other important things necessary in esti- mating the sanitary quality of milk. 4. Gives a better idea with regard to the 4. Shows living organisms only. actual number of germs present. Samples. Milk samples collected, as above described, may be pre- served by icing and handled as in the case of the plate method. All samples on which the cream has risen to the surface must be vigorously shaken before preparations are made from them. Apparatus, In addition to a microscope and ordinary microscopic slides, stains, etc., the only special apparatus required is a pipette PREPARATION OF SMEARS 397 V \/ 0X1 Cj!. which measures 1/100 c.c. The most convenient form of pipette is the straight capillary pipette, calibrated to deliver 1/100 c.c, the graduation mark being If to 2| ins. from the tip. Such pipettes are now for sale by manu- facturers, and can be easily obtained. The cali- bration should be tested by weighing with chemical balances the amount of milk discharged from the tube. Only a single pipette is needed in making a series of tests, provided this is kept clean while in use. In this kind of work cleanliness rather than sterilization is required. Clean towels may be used for wiping the exterior of these pipettes, while their bores may be kept clean by rinsing them in clean water between each sample. The small amount of water left in the tube may be rinsed out into the milk sample under examination. This method of procedure, while adding a small number of bacteria to each sample, introduces only a theoretical error, tests showing that such bacteria cannot subsequently be detected, and make no difference in the final result. Preparation of Smears. One one-hundredth c.c. of milk or cream is deposited upon a clean glass slide by means of a pipette described above. By the use of a clean, stiff needle this drop of milk is spread over an area of 1 sq. cm. This may be most conveniently done by placing the slide upon any glass or paper ruled into areat 1 cm. square. These marks show- ing through the glass serve as guides. After uniform spreading the preparation is dried in a warm place upon a level surface. In order to prevent noticeable growth this drying must be accomplished within five to ten minutes; but excessive heat must be avoided or the dry films may crack and peel from the slides in later handling. After drying, the slides are to be dipped in xylol (gasoline may be used) for one minute, then drained and the slides dried. They are then immersed in 90 per cent grain or denatured alcohol, for one minute or more, ani then trans- ferred to a fresh aqueous solution of methylene W w B A Fig. 67.— -Breed's Capillary Pipettes. Several types of pi- pettes have been de- vised. Type A gives as much satisfaction as any P ru cha at the Illinois Agricultural Station uses ordinary capillary tubing and standardizes the amount ot milk deliv- ered by weighing it on an analytical balance. blue. Old or 398 MILK AND KIILK PRODUCTS imfiltered stains arc to be avoided, as they may contain troublesome precipitates. The shdes remain in this solution for five seconds to one minute or longer, depending upon the effect desired, and are then rinsed in water to remove the surplus stain, and decolorized in alcohol. The decolorization takes several seconds to a minute, during which time the slide must be under observation in order that the decol- orization may not proceed too far before they are removed from the alcohol. When properly decolorized the general background of the film should show a faint blue tint. Poorly stained sUdes may be decol- orized and restrained as many times as necessary, without aay apparent injury. After drying the slides may be examined at once, or they may be filed away and preserved for further reference. Standardization of the Microscope. The microscope to be used must be adjusted in such a way that each field of the microscope covers a certain known fraction of the total square centimeter^s area. This procedure is simple, with the proper materials at hand. The micro- scope should have a 1.9 mm. (1/12 in.) oil immersion objective, and an ocular giving approximately the field desired, and should preferably be fitted with a fnechanical stage. To standardize the microscope, place upon the stage a stage micrometer, and by the selection of oculars or adjusting the draw tube, or both, bring the diameter of the whole microscopic field to .205 mm. When so adjusted, the microscope field will cover almost exactly 1/300,000 of a cubic centimeter of the milk (actually 1/302,840). This means that if the bacteria in one field only are counted, the number should be multiplied by 30,000 to give the total number for a cubic centimeter. If the bacteria in a hundred fields are to be counted, the total should, of course, be multiplied by 3000. Inasmuch as it is difiicult to count bacteria lying near the margin of the microscopic field, it is much better to have an eyepiece micrometer, with a circular ruHng, 8 mm. in diameter, and divided into quadrants. This will give, in the microscopic field, a smaller area within which the bacteria may be seen most sharply, and which may be more easily counted. Such eyepiece micrometers are now manufactured by labora- tory supply houses, and may be easily obtained. In the use of this eye- piece micrometer the inner circle, by the adjustment of the draw tube, should be made to cover a circle with a diameter of .146 mm. In this case this inner circle will cover 1/600,000 of a cubic centimeter of milk, meaning, of course, that the number of bacteria in a single field should be multiplied by 600,000, or, if a hundred fields are counted, by 6000, to obtain the number per cubic centimeter. ussue cell seen GOOD QUALITY MILK JJacteria Seen. • Xw, ount =800,000 por cnbie <'.entimeter. , \ iilLK S^ *" ""^nMALLY. / Fig. 3.— Milk . . Nearly Soii-^ - -^-- ji iiiv bacteria are lactic u- . . ...cteria. One tis.,., ipfPT-;;.! ic centimeter. Cell count =400,000 per cubic centimr -7 To fane jxtge 3U Plate lA:~prawmgs pf Milk Smears as Seen mider Hie Microsu - (After Brew, 1914.) \ A!! prepared in suoli a way that each bacterium or tissue r-ell peon is equival^l p< £• lubic centiiiiotor. MILK OF FAIK QrAl.ITY Fig. 2, — ^I'wo PmIvs of Fiactie Acid Bacteria and One Single Bacteriuii One tissue cell. Bacterial count =2,000,000 per cubic ccntinieter. r^ell count =400,000 p cubic centimeter. ^ . Pf ,( »H f; I ' \ !" fl' V \; TI.K '\ liG. i. — Milk which is both nearlv Sonr and Suspicious in Sanitary Quctn rfeven tissue cells. Bacterial count =100,000,000 per cubic centimeter Cell count =2, per cubic centimeter. To fare page S98. INTERPRETATION OF RESULTS 399 The number of microscopic fields to be comiied will depend .some- what upon the kind of data that are dcbircd. If liiis method is to be used simply for the purpose of dividing milk into gi-ades, it will in mo>st eases be unnecessary to do the actual counting, smce a Grade A milk will show field after field without any bacteria at all, while a Grade C milk will show the field crowded with bacteria. In all doubtful cases, however^ counting should be done, and there should never be less than thirty fields counted in order to have reliable results. Counting thirty fields is not so tedious a task as would seem to be, since, in ordinary milk, the num- ber of bacteria in each field is small, and the counting may be done very rapidly. Counting. Counting the bacteria in such a smear may be done in two ways: 1. The number of groups of one or more bacteria present. 2. The number of individuals. The second, of course, is really the correct count of the number of bacteria, but the former will give a count much closer to that obtained by the plate count, since the colonies upon the plate represent groups of bacteria rather than individuals, each group growing into a single colony only. Extensive tests have shown that there is a fair correspondence between the number of groups re- ported by experienced observers and the number of colonies that may grow in plates made from the same milk, although there are occasionally discrepancies of considerable extent. These discrepancies are caused by variations in judgment as to what constitutes a group, variations in the extent to which groups break up in the dilution waters when the smears are made, and the presence of dead bacteria or of bacteria which do not grow on the plates. Some experience is needed by the micros- copist in determining just what should be counted. In high-grade milks, an inexperienced person is apt to fail to recognize differences between bacteria and other minute objects. This results, as a rule, in an overcount by inexperienced men. In milk containing many readily recognizable bacteria in each field the inexperienced man is apt to over- look some of them, giving an undercount. These difficulties are over- come, however, by training and experience. Interpretation of Results. It must be recognized that the results obtained from the microscopic record give a closer approximation to the actual number of bacteria present in the milk than those obtained by the plate method, since the plate method will count as one, either a single bacterium or a group which may sometimes contain a hundred or even more individuals. Inasmuch, however, as the plate count has become a method of analysis that is well known and commonly applied, it becomes desirable to know as closely as possible what relations there 400 MIT K AND MILK PRODXirTS may be between the plate count and the microscopic comit. Experi- ence has shown that the count of individual bacteria is ordinarily 1.5 to 8 times as great as the plate count, the ratio between the two being largely dependent upon the size of the clumps of bacteria present. Where the bacteria are mostly isolated, the ratio of the two counts would be much closer than where there are present long chains of strep- tococci or masses of cocci. After one has had a little experience in counting clumps it is found that the number of groups shown by the microscope, agrees fairly well with the number of colonies shown by the plate count, though even here there are occasionally discrepancies, due among other things to the appearance in the microscope of kinds of bacteria which fail to grow in the culture media used in making plates. In all cases, however, the direct count of raw milk will give a much closer approximation to the actual numbers of bacteria than the plate count. In view of these facts it is difficult to interpret one count in terms of the other; but a few suggestions will give a fairly satisfactory idea as to how the two may be related. Grade A raw milk, which should have less than 100,000 bacteria per cubic centimeter, will not show more than three to four small clumps of bacteria for each 30 fields of the microscope where the diameter of the fields is .205 mm. Such milk also ought not to contain more than 500,000 individual bacteria per cubic centimeter when counted by the microscope. For Grade A pasteurized milk (which should have less than 200,000 per cubic centimeter by the plate count before pasteuri- zation) the microscope should not show more than six to eight clumps per 30 microscopic fields, and not more than 1,000,000 individual bac- teria when counted with the microscope. Grade B milk, which is supposed not to have more than 1,000,000 bacteria before pasteurization, when counted by the plating method, should not show more than 20 individual bacteria per field, where the diameter of the fields is .205 mm., and not more than three to four groups of bacteria per field. While the above relation between the plate count and the microscopic counts cannot be relied upon as having a very great amount of accuracy, it will serve to give a general idea of the ratio between the two- under ordinary conditions, and may serve as a guide in the use of direct microscopic method. The direct microscopic method is not as yet recommended by this committee as a method of estimating the numbers of bacteria that are present in samples of milk. For this purpose the plate method, which has long been in use and is fairly well understood, is still recommended as FROST'S MICROSCOPIC PLATE METHOD 401 the standard method to be employed. For the purpose of rapidly dividing raw milk into a series of grades, in such a way that the results can be obtained in the quickest possible time, the direct microscopic method seems to be extremely useful, and the results which are obtained will, in nearly all cases, agree with those obtained by the plate count, and probably in all cases will give a closer approximation to the fair grading than the plate count can do. For these reasons the use of the direct microscopic method is extremely valuable at the dairy end of the milk route, where the farmer wishes to know the kind of milk he is pro- ducing, or the purchaser at the shipping station wishes to know the kind that he is receiving from the farmer. It is of less value at the city end of the milk route, especially if pasteurization of the milk has been intro- duced anywhere along the route. It is especially useful in the data it gives concerning the kinds of bacteria present in milk, since it sometimes enables the farmer quickly to pick out from his herds such cows as are discharging large numbers of streptococci, thus giving a very efficient means of protecting the milk supply from this type of organisms that are to-day recognized as suspicious and decidedly undesirable. Goodrich (1914) finds a marked correlation between the microscopic and plate counts. He regards the factor of 20,000 which is used to reduce the microscopic count to terms of the plate count as satisfactory. Breed and Brew (1917) claim that their microscopic method is as satis- factory for controlling city milk suppHes as is the agar plate method. Breed and Stocking (1917) found that the plate counts are characterized by greater regularity in the hands of laboratory assistants carrying out routine analyses of milk, than the microscopic method of Breed and Brew. Inexperienced workers are said to secure great errors in the use of the microscopic method. About the same conclusion was reached by Conn (1915). He stated that considerable experience was needed by the analyst to distinguish between bacteria and dirt particles. The Breed method has been demonstrated by the work of different investigators to be fairly accurate with milks which have large numbers of bacteria. It may scarcely be conceded that where the bacteria are so few that many of the fields of a Breed smear contain no bacteria, the count is very accnrate. In this connection the Frost microscopic plate method or Allen's microscopic method may be found to be more satis- factory. Brew has mentioned this .last fact as a disadvantage of the microscopic method. To secure the greatest possible accuracy a large number of fields should be counted. Frost's Microscopic Plate Method. This differs from the Breed Method of securing a microscopic count in that little plates are made 402 IVIILIv AND IVIILK PEODUCTS instead of smears; otherwise the method is somewhat the same. In general, some of the same objections which are appHed to Breed's method may be appUed to the Frost method. The method is outUned by Frost in the following words: Preparation of Glass Slides. The httle plates are made on the ordinary microscopic glass slides (2.5 by 7.5 cm.). These arc carefully cleaned and are then ruled with a grease pencil so that a square surface, over the center of the slide, is surrounded by a grease line. The included area is 4 sq. cm. The ruling is best done by having a wooden or card- board frame for the slide and some arrangement, as brads, to hold the straight-edge in place. These ruled slides ere then steriUzed by passing them through a gas flame, and then they arc placed on a warm box (45° C, 113*^ F.) provided with an overhanging top to protect them from dust. Preparation of the Plates. A few tubes of ordinary nutrient agar are then melted and placed in a water bath at 45° C. to cool. Some sterile plugged test tubes are also put in this water bath to warm up (one for each sample of mill^ to be tested). The milk sample to be tested is then thoroughly shaken and 1 c.c. is transferred to one of the warm sterile test tubes. To this is added an equal amount (1 c.c.) of the melted agar. The milk and agar are thoroughly shaken, care being taken to prevent the agar from cooling below 40° C. It is best to warm it up frequently by putting it into the 45° C. bath. One-tenth (0.1 c.c.) of the milk and agar mixture is then transferred to a warm glass slide and spread as quickly and evenly as possible over the ruled surface. This may be readily done with the help of a pipette while the shde is on the warm plate. The agar is allowed to set firmly by placing it on a level surface under cover for a few minutes. TMs makes a little plate culture containing 0.05 c.c. of milk. When the milk being examined is supposed to contain more than a million bacteria per cubic centimeter, it should be diluted with sterile water or sterile milk before it is mixed with the agar. The dilution may be 1 : 10. This would make the final dilution as much as 1 : 200. Incubation. This is accomplished by placing the little plates in an incubator in a specially prepared cabinet, or moist chamber with a layer of water in the bottom 'to maintain a saturated atmosphere. A Petri dish may be used for a few shdes. The incubation temperature is 37° C. for a varying length of time. Five or six hours should be allowed. For samples which contain many bacteria such as market milk which has been kept in a warm place three or four hours will be FROSTVS IMICROBCOPIG PLATE METHOD 403 sufficient. For pasteurized milk from ten to twelve hours may be necessary. Drying the Plates. When the colonies are sufficiently large the plates are dried. This should be done carefully. Too rapid drying will crack the films. They will dry well in the incubator or over a steam radiator. Staining. Thib is accomplished in the following manner: 1. Fix in the flame. 2. Put in a 10 per cent solution of acetic acid in 95 per cent alcohol. 3. Stain with Loeffler's methylene blue (1:4) for three minutes 4. Decolorize in 95 per cent alcohol for a few seconds or until the background is a pale blue and the colonies stand out prominently. 5. Dry without washing. Counting of Colonies. For the purpose of counting the colonies, the shdes may be examined with the low-power dry lens. If the struc- ture of the colonies is to be studied it is always best to use a moxmting medium and the cover glass. Cedar oil serves quite well and in that case the cover glass may be removed for use with the oil immersion but Canada balsam may be used in the usual way. A microscopic examination of these plates reveals colonies of considerable size stained a dark blue in a Ught blue field. The staining process should be so regulated that the colonies will be stained a dark blue in a light blue field. At least twenty fields should be counted on each plate and the plate should be gone over carefully in the selection of these twenty fields. Calculation. The number of bacteria per cubic centimeter may be determined by multiplying the number of colonies in a microscopic field by the number of times the area of this field is contained in 400 sq. mm. or the area of the little plates times the dilution used. The area of the microscopic field for any definite combination of lenses and tube length is a constant and may be determined by the formula: Area=E. The radius may be determined by a stage micrometer. The same tube length must be used that was used in the standardization of the microscope. When the microscope factor is known this formula may be used: Number of colonies counted ^^ . i r vi x- r -n — ^^ . = Reciprocal of dilution of milk. Number of fields counted Microscope factor = number of bacteria per cubic centimeter in the milk. Example: 404 MILK AND MILK PRODUCTS 5 — X 20X200=-^—— — = 120j000 bacteria per cubic centimeter, where 2J 20 600= number of colonies counted; 20 = number of fields counted; 20 = dilution— 1-20; 200 = microscope factor. Frost (1915, 1917) has secured a satisfactory agreement between his method and the standard plate method. In his second communication, this author reports some results secured with his method during a prac- tical test on the Boston milk supply. The table which is presented in this book shows that Frost's microscopic method gives results which are different than those obtained on the standard agar plate. Allen's Microscopic Method for Counting Bacteria in Milk. Allen (1918) has described his method as based on the fact that a water sus- pension of aluminum hydroxide readily collects bacteria in milk. This may then be thrown down in one end of a centrifuge tube leaving the normal milk constituents suspended. Allen has described his tech- nique as follows: Preparation of Hydroxide Suspension. Mix equal parts of N/20 A1K(S04)2 — I2H2O and N/20 sodium hydroxide. After precipitation is complete, wash the precipitate thoroughly by decanta- tion using scrupulously distilled water, keeping in mind' that any dirt in the wash water will be taken up by the aluminum hydroxide and will appear in the film under the microscope. Make up to one-half the original volume and continue to dilute until a proper amount of alu- minum hydroxide is obtained after centrifuging. Procedure. 1. Add to the sample of milk to be analyzed enough of the washed alu- minum hydroxide precipitate suspension so that it becomes 20 per cent of the mixture. Shake thoroughly for several minutes. 2. Add 2.5 c.c. of the above mixture to a centrifuge tube holding this amount when stoppered. (Use plain centrifuge tube open at both ends for stoppers.) 3. Centrifuge for fifteen minutes at 5000 r.p.m. in a centrifuge of 10-in. diameter or give equivalent centrifugalization. 4. Pull stopper at cream end of the tube and remove the cream with a needle allowing the milk to run out. Then carefully pull the stopper at the other end of the tube and transfer the plug of aluminum hydroxide to a clean glass shde laying over glass or cardboard ruled off in square centimeters. By use of a needle or loop grind up the precipitate on the ALLEN'S MICROSCOPIC METHOD 405 slide and spread it evenly over an area of 2 sq. em. using a loopful of sterile water if necessary for nniforni spreading. 5. Place m the incubator until nearly dry, then iSnish di'}ing at room temperature. 6. Heat slightly above the Bunsen flame. 7. Place in a tray of toluene and agitate the tray several minutes to remove butter fat, then take the slides out of the tray and allow the toluene to evaporate from them. 8. Place the shdes in 95 per cent alcohol to remove all traces of toluene. 9. Stain in 25 per cent saturated aqueous solution of methylene blue until the film reaches a proper depth. 10. Dry in the warm air above a Bunsen flame and examine under the microscope, using an oil immersion lens of which the number of fields per square centimeter has been determined. IL Count 20 representative fields on the square centimeter and obtain the average number of bacteria per field. 12. The number of bacteria per cubic centimeter of milk is obtained by multiplying the average number of bacteria per field by the number of microscopic fields in a square centimeter. Usually there are a little over 3000 fields per square centimeter when the ordinary 1.8 oil immer- sion lens is used but using 3000 as an even number to multiply by is satisfactory in counting. Allen also gives the following precautions: Care must be exercised not to use a suspension of aluminum hydroxide too strong to give too thick a smear. The thickness of the plug of aluminum hydroxide after centrifugalization should be about 1 mm. No fat or toluene should appear in the field imder the microscope. Some samples of pasteurized milk show fine particles of curd due to heating. These may be removed by filtering through a small amount of cotton. The film should be uniformly stained. Rubber stoppers should be used with the centrifuge tubes as cork stoppers will not hold the liquid in the centrifuge. If toe deep a stained film is secured, it may be decolorized sufficiently by dip- ping in alcohol. Interpretation of Results. ^' The interpretation of the bacteriologi- cal analysis of market milk must depend upon the history of the milk. It is, Jerefore. difficult to give any general interpretation. The fol- lowing are a few significant conclusions: 1. '' Where the analysis can be made immediately after the milking the number of bacteria enables conclusions to be drawn as to the cleanli- ness and care in the dairy and the thoroughness in the cleaning and 406 3MILK AND MILK PRODUCTS sterilizing of the milk vessels, or soiuolimes the presence of cows with infected udders. With propeily cleaned ajid ^teriUzed milk vessels and proper care in the farm and dairy the numbers of bacteria should not exceed 10,000, and may easily be brought down to 5000. Numbers beyond these in milk analyzed immediately after the milking may be regarded as an indication of unclean dairy methods, dirty and unsterile milking vessels, or to infected udders. Apart from infected udders the factors in dairying that most noticeably increase the bacteria count are unsterile milk vessels, unsterile strainers, unclean udders, and failure to cool the milk promptly. 2. ''If the milk is properly cooled with ice the numbers should not materially increase in five to seven hours. Communities within five to seven hours of their dairies should, if perfect conditions prevail, be able to obtain milk with nearly as low a count as above indicated. Hence, in such communities bacterial counts above these numbers should not be found in properly guarded milk. A count of 50,000 in such a com- munity is an indication either of unsatisfactory dairy conditions or of failure to properly cool the milk during transpoitation. Night's milk, if properly cooled, can also easily be brought within these limits if analyzed the next morning. A count of over 50,000 for a community close to the dairies must be regarded as unsatisfactory, and the number should approach the 10,000 mark for high-grade milk. In hot summer weather the difficulties of keeping low counts are greater, but even then they need not surpass 50,000 if the milk is properly cooled. 3. '' Where milk must be a longer time in transportation from the dairy there will inevitably be an increase in bacteria, depending on the length of time and the temperature. Experience has shown, however, that even in these conditions the excessively high numbers that have frequently been found in city milk are in reality due to diseased udders, to dirty dairy conditions, to dirty and unsterile milk utensils, or culpable neglect of cooling. Moreover, such high bacterial counts at the ship- ping station are frequently traceable to a few dirty dairies whose milk with an abnormally high count contaminates the rest of the supply. Dirty shipping cans and warm temperatures in shipping are respon- sible for most of the high bacterial counts in city milk. Where the milk from healthy cows reaches the city within twenty-four hours, however, the number should not be over 100,000 in winter or 200,000 in summer, and numbers in excess of this may be regarded as due either to improper dairy conditions, dirty milk vessels, insufficient cooling or, perhaps, to diseased udders. In larger cities where much of the milk is forty-eight hours in reaching the city, higher numbers may naturally be expected; MICROSCOPIC VS. PLATE COUNT 407 but even under these conditions there is no good reason why the number of bacteria should reach 1,000,000; and it may mostly be brought down to below 200,000. In such cities, therefore, milk with more than 1,000,- 000 bacteria must be regarded as improperly guarded either at the dairy or on its transit. 4. " For a Grade A milk higher demands should be made than for the ordinary grade. The standard set by the Milk Commission for Grade A, viz.; of 200,000 for milk to be subsequently pasteurized or for 100,000 to be used raw, is stated by that Commission to be an extreme limit for the most unfavorable conditions. Cities situated near the supplying dairies should demand a much higher standard, which should not allow over 10,000 in bacterial content in Grade A milk in communities favorably situated. 5. " For communities situated where ice is not available it may be necessary to accept a milk with a higher bacterial content; but as rapidly as possible the standard should be made to approach the limits as given above." Relation of the Microscopic to the Plate Count. The relation of Breed's microscopic to the standard plate count has received some study. Brew (1914), after a rather extensive study of the question pointed out some interesting facts in his summary. He stated that " the relative differences between the two counts are greater where the bacteria are few in nmnber." In such a case, there is probably an error in the microscopic count since it is difficult to see how it is well adapted to milks with few bacteria. Brew finds a greater difference when the indi- vidual bacteria on the smear are counted than when only groups of bacteria are counted. This is said to be due to the fact that a colony on a standard plate has developed either from a single bacterium or a group of bacteria. Goodrich (1914) reported a marked correlation between the two counts. He thinks that, for accurate work, more than one slide should be prepared. Brew again studied this relation with the aid of Dotterer (1917) and found that the plate counts were higher than the microscopic counts on milks with a small number of bacteria. Brew has made an attempt to explain this by " unrecognized contamination " on the standard agar plate which would increase this count and by over- looking bacteria under the microscope. This seems a rather feeble attempt to bolster up the Breed microscopic count in one of its apparent deficiencies. More reasonable does it seem that '' bacteria where they are very few in number, even though well stained and conspicuous may not occur in the microscopic fields examined." This is probably one of the major objections to Breed's microscopic method. Unlike Allen's 408 MILK AND MILK PRODUCTS microscopic method, no concentration of the bacteria in the sample is made. Breed and Brew (1917) compared again, the relation of the two counts when studying the appUcation of the microscopic method to the control of bacteria in market milk. No general agreement was observed. Frost's method seems to have had Uttle application under commercial conditions. Frost (1916, 1917) has carried some studies himself and reported a smaller variation with his '^ little plate '' method than was observed with the standard plate. The Allen microscopic method may overcome some of the disad- vantages of the Breed method. Allen has devised a procedure which is said to be apphcable to all grades of milk. "With the Breed smear gross errors are involved on milks with low counts. In his original contribu- tion Allen (1916) has shown that on milk with a count of 1000 and slightly over, he was able to check the standard plate count very closely. Ayers' Milk Tube Method. This is a very satisfactory method for studying the types of bacteria in milk. The milk is plated on agar plates which are incubated at 37° C. for six days. After counting the plates, each colony should be picked from the plates and transferred to a sterile tube of Utmus milk. After fourteen days' incubation these tubes are examined and as a result of the reactions shown, the bacteria picked from the plates are divided into five groups: the acid-forming, acid-forming and coagulating, inert, alkali-forming, and peptonizing. Ayers has used this method in studying the flora of different milk products. Leucocytes in Milk These cells in milk were first given much attention by Stokes and Wegefarth (1897). It was noticed that they had much resemblance to the white corpuscles and from this it was concluded that they repre- sented '^ pus " from the cow's udder. Breed (1914) has given a good summary of the literature besides reporting the results of his own inves- tigations. He used the direct microscopical method for counting the cells first suggested by Prescott and Breed (1911) and later applied to the counting of bacteria m milk by Breed (1911). No relationship, if any exists, was established between the number of cells discharged and bacterial infections of the udders. Breed (1913) reports a milk having the enormous number of 54,300,000 cells per cubic centimeter. This milk when partaken of was found to have a normal taste and the imbiber suffered no evil after effects. Stokes' Method for Leucocytes in Milk. Centrifugal sediments from 10 c.c. of milk are stained and examined under the one-twelfth oil SAVAGE'S METHOD FOR LEUCOCYTES 409 immersion objective. The presence of cells in such a field was regarded by Stokes as justification for excluding an animal from a herd. Reed's Method for Leucocytes in Milk. 1. Fill 10 c.c. centrifuge tubes with milk and heat for ten minutes at 70-75° C. 2. Centrifuge the tubes at high speed for ten minutes. Remove the upper layers of cream and milk with a pipette and refill the tubes with distilled water. Centrifuge again for three or four minutes. 3. Draw off all except J c.c. of liquid in the point of the centrifuge tube. Wipe out the upper part of the tube with a bit of absorbent cotton fastened to the end of a glass rod. Mix thoroughly the remaining liquid and sediment. 4. Transfer a drop of this mixture to a clean Thoma-Zeiss blood- counting cell and place the cover glass over it. Count the cells under a one-sixth objective. If the number of leucocytes is low, the entire area of the cell should be counted, using a mechanical stage to move the slide- If their number is large, five or six small squares may be counted and averaged. The average number per small square multipUed by 200,000 will give the number of leucocytes per cubic centimeter in the original milk. Savage's Method for Leucocytes in Milk. (Savage, 1914.) The ordinary Thoma-Zeiss blood-counting chamber is employed. Direct counting of the cells is impossible owing to the opacity caused by the large amount of fat. One c.c. of the milk is accurate!}" transferred to a centrifugal tube (about 15 c.c. capacity) of the usual pattern, and freshly filtered Toisson's solution is poured in to almost fill the tube. The two fluids are weU mixed and then centrifugalized for ten minutes. The cream is well broken up by a clean glass rod, to disentangle leucocytes carried to the surface, and the mixture centrifu- galized for an additional five minutes. All the fluid is then removed down to the 1 c.c. mark, great care being taken not to disturb the deposit. This can be conveniently and readily done by means of a fine glass tube connected to an exhaust pump. Theoretically, all the cellular elements present in the original 1 ci of milk are now present in the 1 c.c. of fluid. The deposit is thoroughly well mixed (with a wire), and disturbed through the 1 c.c. A sufficient quantity is placed on the ruled squares of the Thoma-Zeiss apparatus, and the cover glass put on. The number of cells is counted in a number of different fields of vision, moving reg- ularly from one field of vision to another. The diameter of the field of vision is ascertained before counting by drawing out the microscope tube until an exact number of sides of the squares spans a diameter of the field of vision. 410 INIILK AND MILK PRODUCTS The number of cellular elements per cubic millimeter of milk= -^~-~2^, where i/ = the average number per field of vision, d = the number X JLCi' of squares which just spans the diameter, d is determined once for all by marking the microscope draw tube so that only 20 fields have to be counted, and the figures substituted in the formula. Doane-Buckley Quantitative Method for Estimating Leucocytes in Milk. With this method 10 c.c. of milk are centrifuged for four min- utes in graduated sedimentation tubes, at an approximate speed of 2000 r.p.m. The cream is lifted out with a cotton swab, care bemg taken to get as much as possible of the fat. It is then centrifuged one minute more and the cream again removed with a cotton swab. Any fat re- maining in the milk interferes seriously with the counting, as, if there are more than a few globules they form a layer on the top of the hqiuid m the counting chamber, and, as the leucocytes settle to the bottom of the chamber, it is difficult to see through the fat. It is only with cows giving milk difficult of separation where this trouble is experienced, and with such animals considerable care is necessary in removing all the cream gathered at the top of the sedimentation tube. The method of removing the fat with cotton is the best one that has occurred to us, and it IS the only part of the process that does not operate with entire satis- faction m every instance - Following the removal of the cream, after the second centrifuging, the bottom of the tube will contain a portion of the sediment which is easily seen. This sediment may, in extreme cases of cows suffering from garget amount to as much as 1 c.c. Ordinarily it will be considerably less than | c.c. The amount varies considerably with the number of leucocytes, but not absolutely. The milk above this sediment is removed with a small siphon, which can be easily arranged with bent glass tubes drawn to a fine point and supplied with a small rubber end pinch cock. In using the siphon it is better to keep the point near the surface of the milk in the tube in order not to agitate the precipitated leucocytes and draw a number of them off with the milk. The milk in the tube may be siphoned within an eighth of an inch of the sediment in the tube. This will usually be below the |-c.c. mark. Two drops of saturated alcoholic solution of methylene blue are then added, thor- oughly mixed with the sediment by shaking, and then set in boiling water two or three minutes to assist the leucocytes in taking the color. The contents of the tube can be boiled by holding it directly in the flame, but it has no advantage over the use of the water bath, and it is very likely to break the glass. After heating, some water is added to the tube to DOANE^BUCKLEY METHOD 411 render the color less dense. Ordinarily filling the tube to the 1 c.c. mark will be sufficient, and this quantity gives an easy factor for cal- culating the final results. In putting this liquid containing the leucocytes into the blood counter considerable care is necessaiy, owing to the tendency of the leucocytes to sink to the bottom. At this place a capillary tube is used, and the cover glass was held in one hand ready to cover the chamber as soon as the drop *was transferred to the counting counter. After placing the glass cover over the chamber, about a minute is allowed the leucocytes to settle to the bottom of the chamber. There are very few foreign bodies likely to be mistaken m counting for leucocytes. Ordinarily the polynuclear leucocytes predominate and the stained nuclei with the unstained surrounding cell show up very distinctly. A few small leucocytes with large nuclei may be found and these may be confounded with yeast cells until the worker becomes familiar with the distinction. As regards counting we have taken a standard with a cubic centi- meter as a basis quantity of milk^ though we are, of course, aware that the corpuscles in the blood are enumerated with a cubic millimeter basis. We adopted the centimeter largely for two reasons. In counting bac- teria in the milk the cubic centimeter is always the basis employed. Simply because the leucocytes were derived from the blood seemed to be no reason why the same basis for counting should be employed as was used with the blood, while to the ordinary bacteriological worker to whom this work will fall, if ever adopted to any extent, the cubic cen- timeter standard would be a little more easily comprehended because more frequently used. The blood counter holJs Ac mm. and 1/10,000 c.c. If 10 c.c. of milk are used and the 1 c.c. of fluid is in the tube after siphoning, and the coloring matter and the water used to dilute has been added, then the resulting number of leucocytes in the counting multi- phed by 1000 will be the total number of leucocytes per cubic centi- meter in the milk. If a total of 75 leucocytes was counted in the chamber there would be 75,000 leucocytes per cubic centimeter in the mixix* In the actual counting under the microscope a square millimeter of the counting chamber will be found to be ruled off into 400 smaller equal squares. This facilitates an accurate and rapid count. Where the number of leucocytes is not great the entire field can be counted in a short time. Where there is a great number of leucocytes a few squares or sets of squares in different parts of the ruled surface will give approximately the number. 412 MILK AND MILK PRODUCTS There are occasionally a few variations desirable from these rules, but it may be well to state that the details have been pretty carefully and thoroughly worked over and compared, and it is seldom that short cuts can be made if correct results are desired. The time and speed of centrifuging are placed as low as possible for accurate work. When there is 4 c.c. or more of sediment, it is necessary to use more of the methylene blue for staining, as there will be too great a number of leu- cocytes to make a satisfactory count in the counting Chamber, it is better to add water until there are 2 c.c, or sometimes even more in the sedi- mentation tube. This method of counting, while long in explaining is in reality short and simple in appUcation. Moreover, it is based on accurate measure- ments in every detail, and the results are correspondingly reUable. (From Report of Committees of the Laboratory Section, American PubKc Health Assn. Am. J. Pub. Hyg., 6 (1910). Pathogenic Bacteria in Milk Bacillus tuberculosis. The tubercle bacillus is recognized as one of the most important pathogens in relation to the milk question. While it may gain entrance from a tubercular person handHng the milk, it is generally admitted that a tubercular cow is usually the source. Human tuberculosis is probably transmitted directly from human to human but may be transmitted by bovines, especially those with udder infec- tions. Griffith (1913) reports the discharge of virulent tubercle baciUi in the milk of a heifer which had been vaccinated when four days old with human tubercle baciUi. Other experiments by Smit (1908), Coquot (1908), Hessler (1909), indicate that there is slight possibility of the tubercle bacilU being discharged into milk unless there are open lesions. Smit found few tubercle bacilli in milk from tuberculous animals with sound udders but where open tuberculosis existed the bacilli could gain entrance to the milk from all channels which com- municate with the exterior. This subject has been well summed up by Schroeder (1907). Delepine's Method for Determining Tubercle Bacilli in Milk (from Savage, 1914). CoUect two tubes of milk containing 40 c.c. and cen- trifugalize for fifteen minutes at 3000 r.p.m. Decant or draw off the cream and milk by means of a pipette leaving about 2 c.c. of milk and residue in each tube. Examine microscopically and inject into guinea pigs. TUBERCLE BACILLI IN MILK 413 Campbell's Method for the Detection of Tubercle Bacilli in Milk. Ten c.c. of a thoroughly mixed sample were placed in each of three centrifuge tubes by means of a sterile pipette. These should be cen- trifuged for thirty minutes at about 1200 r.p.m. The tubes are then removed from the centrifuge and the cream removed by means of a large sterile platinum loop. Three c.c. of sterile water should be added to this cream to bring the volume up for inoculation. The milk remaining in the centrifuge should be drawn down to 1 c.c. These 1 c.c. portions should be transferred to a sterile test tube. Slides may be made from these samples. Three c.c. of the cream emulsion and 3 c.c. of the milk emulsion should be injected subcutaneously into guinea pigs. The pigs should be observed closely for from six to ten weeks and, if not dead before, should be autopsied. Anderson's Method. Mix 50 c.c. of the milk with 50 c.c. of sterile water and centrifuge for one hour at 2000 r.p.m. Inject 4 c.c. of the sediment into a guinea pig. If the guinea pig dies, a careful autopsy should be made. If death does not occur by the end of two months, test with tuberculin, chloroform and perform a careful autopsy. All suspicious lesions should be cultured and cultural and morphological studies made. Besson's Method for Isolation of Bacteritim tuberculosis from Milk. Allow the fresh milk to stand for twenty-four hours and examine the deposit. Centrifuge and use the precipitate for making a microscopical examination. Coagulate 200 c.c. of the milk with a little citric acid and filter. Dissolve the precipitate on the filter in a solution of sodium phosphate (Na2HP04+12H20) and pour the liquid into a large test tube. Add a few cubic centimeters of ether and, after shaking for about te^ minutes, pour off the ether which will carry the fat with it. Centrifuge the aqueous fluid and examine microscopically the sediment. Beattie and Lewis (1913) have isolated acid fast bacilli from milk which were totally different from tubercle bacilli. They emphasize the futility of using microscopic methods instead of animal inoculation. The prevalence of tubercle bacilli in market milk has received the attention of investigators in practically all countries. There is an element of error in comparing closely data from the different investi- gations since a different number of samples were used. This may ex- plain the high incidence of tubercle bacilli which has been reported by some investigators. Table XLIII gives the data which have been se- cured by some of the investigators on market milk. Microspira Cholerae. Basenai (1895) reported that there was no germicidal action of milk on Microspira choleroe and that the organisms 414 MILK AND MILK PRODUCTS could retain their vitality in milk for thirty-eight days. Some of the cells even retained their vitality after the milk had curdled. These statements are interesting when compared to the results secured by Laser (1891) in butter. Table XLIII INCIDENCE OF B. TUBERCULOSIS IN MARKET MILK (Quoted from Parker 1917) Date 1899 1904 1898 1897 1900 1893 1900 1900 1900 1898 1900 1898 1898 1908 1905 1906 1908 X 1/ v/ 1* 1909 1909 1910 1910 Place, Investigator. England Germany Germany Liverpool Liverpool London Petrograd Kiew Krakow Naples Berlin Berlin Schev. Gueund. . Konigsburg Leipsic Rotterdam Rotterdam Washington Louisville New York Philadelphia . . . . Chicago Kochester Macfayden . Muller. . . . Bcatty. . . . Delepine. . Hope Klein .... Scharbekow Pawlowsky . Bmwid. . . . Marconi.. . Petri Reik Ott Jaeger. . . . Eber Smit omit. ...... Anderson. . , Field Hess Campbell. . Tonney . . . , Ooler Samples ]^A.<.irninod. 1596 272 12 228 100 SO 51 60 04 56 27 100 210 567 l'jh4 223 119 105 130 237 Nu rubor Positive. 17 97 27 ZlJU 12 4 1 2 7 9 17 27 7 22 14 15 40 18 15 30 Percentage Positive. 22 1 6 2 10.0 17.6 5.2 7.0 5.0 2 3.3 50 14.0 30.3 11.1 7.0 10.5 2.8 6.7 16.2 13.8 10.5 12.6 Bacillus Diphtheriae. Diphtheria is a typical milk-borne disease. Marshall (1907) has reported the isolation of the organisms from milk. Trask (1912) has given a good resum6 of such epidemics. Pasteueization Pasteurization is the heating of a food substance for a time below the boiling-point. It is now almost exclusively applied to the heating of milk in order to make it a safer food. It is an old idea having been used by Scheele (Mcintosh, 1901), in 1782, to preserve vinegar. In 1831, Appert preserved various animal i- -^ \ _, THEORY OF PASTEURIZATION 415 \. and vegetable products l)y heating in the closed containers. Pasteur (1879) applied heat to bottled beer to make it keep. In recognition of Pasteur's researches, which revealed the causes of deterioration in fer- mented liquors and the means of preserving them, the term ^' pas- teurization is now used. Soxhlet (1886) proposed pasteurization in the home for baby feeding. There has been much confusion between the terms '' pasteurized '' and ^' sterilized " as applied to milk. Rosenau, /• Fig. 68.— Complete Milk Plant. (New York Milk Committee.) (o) Milk Clarifier; (b) Pasteurizing and Holding Plants; (c) Milk Cooler; {d) Storage Tank for Cold Milk; (e) Bottle Filling and Capping Machine. to avoid this has suggested that each bottle indicate the time and tem- perature of heating. Theory of Pasteurization. This involves the use of moist heat as a disinfectant. As discussed in the chapter on steriHzation and disin- fection, bacteria die according to the monomolecular law and this indicates that the killing is a time process. The bacteria do not die at once as soon as the heat is applied. The same law must obtain in the killing of bacteria by pasteurization. From this it would seem 416 MILK AND MILK PRODUCTS that the ^^ continuous ^' process where heat is applied over a longer time would be more satisfactory. Gable (1915) has summed the advantages which might be raised against pasteurization as follows: In the light of recent knowledge some of these are now untenable. OBJECTIONS 1. Increased cost. a. By additional apparatus required, b. By fuel required for heating. c. By ice or ammonia required for cooling. d. By increased labor of handling. 2. Conceals inferiority. a. By encouraging carelessness. b. Discouraging proper care. 3. Interferes with cream rising and whipping. 4, -Modifies taste. 5. Less wholesome. a. Acid less as the lactic acid bacteria are destroyed. b. Spores forms of bacteria are not destroyed. c. Injurious by-products of bacteria are not destroyed. d. Milk is chemically changed. e. Is less easily digested. /. Produces scurvy and rachitis. ADVANTAGES 1. Economic. a. Makes milk keep sweet longer. 6. By saving milk which otherwise would spoil. c. By saving bills for sickness and milk-borne epidemics 2. More wholesome. a. Pathogenic bacteria are destroyed. b. Infant mortality less. c. Bacteria are decreased in numbers. The methods of pasteurization are two in number, the '' flash " and " continuous '' processes. In the continuous process the milk is heated for thirty minutes at 60° C; the flash process requires a higher temper- ature (80° C. to 90° C.) for a shorter time (1-5 minutes), according PASTEURIZATION 417 to the theory of disinfection and since disinfection is a time process the continuous process allows a greater reduction of bacteria. Effect of Pasteurization on Bacteria. This has been given much study in order to refute the argument against pasteurization that pasteurized milk putrifies rather than sours. Ayers and Johnson (1913) Kaw Milk Acid Coagulating Group Acid Group Inert Group Alkali Group Peptonizing Group w Milk Pasteurized for 80 Minutes at A. y (52 8 C (H5°P) (160° F) (170° F) 9SSB Ss (180" F) (1"0"F) (200" F) Fig. 69.— The Hypothetical Relation of the Bacterial Groups in Raw and Pasteur- ized Milk. (After Ayers and Johnson.) studied the bacteria in pasteurized milk by means of their milk-tube method. They used the holder process with a temperature of 62.8"* C. for thirty minutes in most of their experiments. Their results are graphically summarized in Fig. 69 from which it will be seen that pas- teurization increased the percentage of acid-forming bacteria in the milk. Higher temperatures of pasteurization caused a different relation to 418 MILK AND MILK PRODUCTS exist between the groups of bacteria in milk. In an earlier paper (1910), these same authors found that pasteurized milk soured about the same as a clean, raw milk. Weigmann et al. (1916) found that, in milk which had been heated from 60° C. to 63° C, for thu^ty minutes, lactic acid bacteria were present m a much larger proportion to remaining organ- Milk Pasteurized 30 Minutes Fig. 70.— The Hypothetical Relation of Bacteria in Raw and Pasteurized Milk (After Ayers and Johnson, 1913.) isms than in law milk. The same was also found in milk which had been heated for ten to twenty minutes. Souring, however, was much delayed over that in raw milk, which the authors attribute to attenuation of the lactic acid organisms. With regard to the resistance of Bacillus colon to heat, Ayers and Johnson (1915) state that this organism would PASTEURIZATION 419 not be expected, from their results, to survive 65.6*^ C. for thirty min- utes. Erratic results were secured and it is possible that some strains of Bacillus colon may be found which will survive pasteurization under the above conditions. Gage and Stoughton (1906) found that m 55 per cent of thermal death-pomt determinations made with B, coh, the cultures withstood 80° C. for five minutes. Russell and Hastings (1902) report the characteristics of a micrococcus from milk which withstood a temperature of 76° C. for ten minutes. Different individual resistance, however, was found among the cells. Ford and Pryor (1914) claim that milk heated to any tempciature between 65° C. and 100° C. and kept between 22° and 37° C. will be decomposed by spore-formmg bac- teria and consequently may be dangerous. They recommend the boiling of milk and storage on ice since heated milk is liable to decompose. Pathogenic Bacteria. The effect of the pasteuiization process on pathogens has been limited almost entirely to Bacillus tuberculosis. Since this organism is especially important in relation to milk hygiene, many attempts have been made to determine its thermal death point in milk Rosenau has prepared table XLIV, which icviews concisely some of the work which has been done. The work of Smith (1899) and of Russell and Hastings (1904) has been especially convincing. The former of these investigators found that when tubercle bacilli were suspended in milk they were destroyed in from fifteen to twenty minutes at 60° C; the greater portion being killed in 5-10 minutes. The latter tried short periods of pasteurization and found that a temperature of 160° F. (71.1° C.) or above for one minute destroyed the virulence of bovine tubercle bacilli so that guinea pigs were not killed when inoculated with from 2 to 5 mgs. Others have confirmed this work and these temperatures are now accepted in the con- tinuous process. The milkman pasteurizing milk is between two fires. He must avoid a cooked taste in the milk which interests the housewife and must kill all pathogenic bacteria which are the chief interest of the sanitarian. On the relation of pasteurization to other pathogenic bacteria in milk, our knowledge is very meager. Hesse (1894) stated that raw cows' milk was not a good medium for cholera spirilli and that they died in twelve hours at room temperature and more quickly at higher temperatures. The acidity and other bacteria in the milk killed them. Fig, 69 prepared by North and taken from the report of the Commission on Milk Standards gives some data concerning B, typhij streptococci and Bact, diphtheria. 420 MILK AND MILK PRODUCTB Table XLIV SHOWING THE THERMAL DEATH POINT OF THE TUBERCLE BACILLUS AS FOUND BY VARIOUS INVESTIGATORS (After Rosenau) Investigator Martin, 1882 May, 1883 Sormanij 1884 Schill and Fisher, 1884 Voelsch, 1887 Yersin, 1888 Bitter, 1890. Bang, 1881. Bonhoff, 1892 Gancher and Ledeux- Lebard, 1892 Forster, 1892 , DeMan, 1893 . Schroeder, 1894 Woodhead, 1895. Marshall, 1899 . . Th. Smith, 1899. . Morgenroth, 1900 Kobrak, 1900 . . Beck, 1900 Galtier, 1900 Russell and Hastings, 1900 Herr, 1901 Hesse, 1901 Levy and Bruns, 1901 Barthel and Stenstrom, 1901 Bang, 1902 Tjden, 1903 Rullmann, 1903 Barthel and Stenstrom, 1904 Killed at By cooking Boiling, 5 minutes 60° 60^ 68° 70° 60° 10 minutes, no spores. 10 minutes, with spores. 20 minutes. 5 minutes (enfeebles). 5 minutes (sometimes enfeebles). 80°, sometimes kills. 85°, always kills. 60°, 20 niinutes 60°, 70°, 60°, 95°, 55°, 60°, 60°, 50°, 60°, 60°, 70°, 70°, 68°, 60°, 50°, 100' 5 minutes (attenuates). 1 minute (kills). 6 hours momentary 4 hours 1 hour 15 minutes. 15 hours. 8 hours. 45 minutes 45 minutes. 2] minutes. 28 minutes 15 to 20 minutes 3 hours 4 hours. *, 3 hours 60° 65 60 , 65°, 15 minutes. , 20 minutes. °, 15 minutes. °, 20 minutes. Russell and Hastings, 1904 Zelenski, 1906. Rosenau, 1907 85°, 1 to 2 minutes. 65°, 30 minutes. 80°, 1 minute (uncoagulated) 71°, 1 minute. 60°, 20 minutes. Not Killed at 80° C. 90° for 10 minutes. 100°. 100° boiling twice. 50°, 60 minutes. 55°, 3 hours. 60°, 45 minutes. 80°, momentary. 60°, 1 minute. 90°, results contradic- tory. 60°, 10 minutes. 70°, 10 minutes. 100°, momentary 100°. 80°, 30 minutes. 85°, 6 minutes. 80°, 5 seconds. 70°, 15 minutes. 60°, 15 minutes. 60°, 30 minutes. 80°, 1 minute (coagu- lated). 76°, 20 minutes. PASTEURIZATION 421 20' 30' '40' Time in Minutes ' Fig. 71. — Indicating the Relation between the Time and Temperature and Other Factors Involved in the Pasteurization of Milk. (After North.) 422 MILK AND MILK PRODUCTS Pathogenic Streptococci. Much evidence has accumulated to show that septic sore throat may be caused by streptococci which originate in milk from cows suffering from mastitis. Cows suffering from garget are regarded as of special importance in this connection. Capps and Downs (1914) found a dairy farm where mastitis was present in the cows and where the milkers had sore throat. The milk from this dairy was delivered to a dairy company which did not pasteurize its milk end consequently an extensive epidemic of septic sore throat resulted. Krumwiede and Valentine (1915) during the investigation of an epidemic regarded the infection as of human rather than of bovine origin. They suggest that '^ in tracing the source of an epidemic the effort should be toward finding cases of sore throat among those engaged in producing the milk, not mastitis in the cow alone. If human streptococci are found in mastitis, they are most Kkely secondary agents in an already existing inflammation due to bovine strains. . The streptococci in dif- ferent epidemics differ culturally and those similar culturally differ in their immunity reactions. Cultural similarity of strains from man and cattle is insufficient to prove their identity. Cultural identit}^ in every detail or immunological identity is essential." Rosenow and Moon (1915) studied an epidemic of septic sore throat and, after tracing it to milk, isolated virulent streptococci from the milk. These showed selective preference for joints, muscles, gall bladder, etc., and resembled certain rheumatic strains morphologically and culturally. These investigators regard virulent streptococci in milk as possible sources of those which cause, rheumatism and other chronic infections. Rosenow and Hess (1917) also traced another epidemic of this disease to the milk from three cows. Winslow and Hubbard (1916) reported a contact epidemic of this infection. Smaller epidemics have been studied by Overman (1914), Henika and Thompson (1917) and others. Smillie (1917) has stated that the streptococci of septic sore throat resemble those of scarlet fever and that discharges from such patients may infect the milk and cause septic sore throat. Ayres and Johnson (1914) studied the possibility of streptococci surviving pasteurization. Out of 139 cultures isolated from cow feces, the mouth of the cow and milk, 64.03 per cent of them survived a pasteurization temperature of 140° F. Thirty-three and seven hundredths per cent survived at 145° F. At 165° F. none were able to survive. These data might indicate that the streptococci which were pathogenic for man could under certain con- ditions pass through the pasteurization process. Allen (1916, 1917) has shown that raw milk as compared with pas- teurized milk exerts a powerful suppressing influence on the multipli- BACTERIOLOGY OF BUTTER 423 cation of certain bacteria. The pasteurized milk was found to be more favorable to the attack of the gas forming colon bacilHs and B. aerogenes, Allen emphasizes the point that pasteurized milk, although it is safer for human consumption, should be handled with much care since heating has decreased its resistance to many detrimental changes. Ayers and Johnson after a study of this question reported that th-e bacterial increase in clean raw milk and pasteurized milk was about the same when both were stored under the same conditions. Bacteriology of Butter The bacteriology of butter may be considered from two viewpoints — the use of bacteria to bring about desired changes and the dissemination disease-producing bacteria. Bacteria in Cream and Cream Ripening. According to present practice cream is pasteurized and ripened before being made into butter. The pasteurization process removes all of the extraneous bacteria and thus prevents spontaneous changes which are hable to produce unfav- orable flavors and tastes. After pasteurization, the cream is inoculated with a culture known to produce a desired flavor and allowed to ripen. Certain distinct improvements were introduced into the butter industry by ripening of the cream. (1) A better yield of butter is secured from sour cream. (2) The flavor of the butter is kept constant from batch to batch. (3) Sour cream churns more easily than sweet cream. Peiser (1916) has stated that the greater proportion of bacteria in ripened cream are removed during churning by the buttermilk. Butter contains about one-tenth as many as are in the cream. Flavor of Butter. This is dependent upon a large number of factors and opinion is still divided with regard to which is the most important. That bacteria are important in changing the flavor of butter after it has been made is improbable. Conn has attempted to make a distinc- tion between " fiavor.^^ and " aroma '' but such a division is probably quite artificial. Rancidity is a term which covers a multitude of abnormalities in the taste of butter. Guthrie (1917) in his interesting paper has given a review of the literature. In his experiments, he attempted to determine whether rancidity was due to chemical cow enzyme or biological changes. He found that none of these factors were important and believes that rancidity as defined by butter judges is rarely found. No marked change in the iodine number was caused by high temperatures, light or an^. 424 MILK AND MILK PRODUCTS The undesirable flavors in butter may be due to absorbed odors or to products formed in the butter after manufacture. Dyer (1916) showed that the undesirable flavors dcvelopmg in butter held in cold storage at a temperature of 0° C. were not due to an oxidation of the fat itself. Gas analyses indicated that the quantity of buttermilk left in 30 26 u 20 f 18 to o o §12 16 10 i 8 V \ \ v\ \N \ \V\ _^ «- — V ..^ \ ^^ N v^ "^ < I : ^ ' , 5 i 5 r i 3 9 -lO'^F. 10>, 32"F. Months in Storage Fig. 72.— Diagram Showing Decrease in Bacterial Content of Butter Stored at Dif- ferent Temperatures (After MoMer, Washburn and Rogers, 1909.) the butter has a relation to the quantity of carbon dioxide formed. Rancidity is probably the algebraic sum of a number of factors. Milk in the fresh condition is a very suitable medium for bacteria. Northrup (1911) studied the longevity of J5. typhi in sour milk. Before these organisms were destroyed Badermm ladis amdi had to produce TUBERCLE BACILLI IN BUTTER 425 80*^ acid in milk and 28° acid in whey. Bacillus hulgaricus had to pro- duce 208° acid in milk and 60° acid in whey. She quotes the work of other investigators as Bassenge, Behla and others that lactic acid is toxic for Bacillus typhosus. Kruniwiede and Noble (1915) report that the typhoid bacillus is killed in sour cream by acids and that the destruc- tion is proportional to the amount of acid and number of bacilli. Wash- burn (1908), in artificially infected milk found that B. typhi suffered no diminution in numbers up to twenty days. They had practically dis- appeared after forty-three days. Potter (1910) concluded that Bacillus hulgaricus and Bacterium lactis aaidi exerted no appreciable repressing effect on Bacillus typhi and that these organisms may not be depended upon to make milk, which has been contaminated, safe. Table XLV VIABILITY OF TYPHOID BACILLI IN SOUR CREAM (Krumwiede and Noble) Days 2 7 8 9 10 11 Reaction * Number of typhoid bacilli. . . 1 0% 392,000 2 2% 65,000,000 5% 300,000,000 • 113,000,000 181,000 10% 400 * Number of cubic centimeters of N alkali needed to neutralize 100 c c of cream Molds in Butter. Thorn and Shaw (1915) found that mold in butter usually took three forms: first, orange yellow spots produced by Oidium lactis; secondly, dirty green spots produced by cladosporium alternaria, thirdly, green spots which are caused by penicilHum. A salt content of 2.5 per cent prevented the development of the above fungi and, therefore, their presence indicates a low salting. The pres- ence of much curd was stated to allow a more vigorous growth of mold. Hastings (1916) has recommended that butter tubs and liners be placed in boilmg water heated to 150° to kill the mold spores. Tubercle Bacilli in Butter. While the literature on the spread of this disease by butter is not extensive, there are several important investigations on the question. Rosenau, Frost and Bryant (1914) examined twenty-five samples of Boston market butter and found tuber- cle bacilli in two of them, B, coli in six and streptococci in fourteen. Hill (1911, 1913) has reported the spread of typhoid fever and diph- theria by butter. M. Mohler, Washburn and Rogers (1909) found that, contrary to prevailing opinion, tubercle bacilli were not devitalized by 426 MILK AND MILK PRODUCTS cold storage; salt was reported to have no effect on these organisms in butter. Salted butter was found to retain its virulence for six months. Schroeder and Cotton (1908) demonstrated the presence of tubercle bacilli in butter which had been kept for one hundred and sixty days. These data indicate that some butters may contain tubercle bacilli even after comparative long storage periods. Again, the value of pasteurization of dairy products is emphasized. Savage's Method for Tubercle Bacilli in Butter. To detect tubercle bacilli in butter the inoculation n^ethod is the only satisfactory one. .The butter is placed in centrifugal tubes which are stood in warm water at 42° C. until the butter is completely melted. The material is cen- trifugalized when liquid, and the sediment inoculated into guinea pigs as described under milk. It is difficult to keep the butter liquid during the centrifugalization. Bacillus typhosus in Butter. Washburn (1908) studied the lon- gevity of B. typhi in butter which was artificially infected. In this experiment, the organisms were found 150 days after the preparation of the butter. Boyd (1917) reported an epidemic of typhoid fever from butter. He regards the pasteurization of the products before ripening as a wise procedure to prevent disease and undesirable fermentations. Washburn's data indicate that butter may contain virulent typhoid bacilli for 151 days. Probably the conditions under which the data are obtained greatly influence the results. Sergey's Method for Typhoid Bacilli in Butter. Transfer 5 gms. of butter by means of a sterile scalpel to a test tube containing ordinary nutrient bouillon. After incubation for several days, streak plates of Endo's medium, Drigalski-Conradi agar and LoefRer's malachite green agar. Incubate these plates for from 24-48 hours and transfer the suspicious colonies to agar slants and a melted tube of dextrose agar. After incubation only those cultures should be retained for further study which show no fermentation in dextrose agar. Morphological studies should then be made together with cultural studies in lactose, sarbite, rafSnose, dextrose, saccharose, dulcite, adonite and inulin broths. Observation has shown that Bacillus typhosus produces a shght acidity in lactose broth and a more definite acidity and coagulation in the sorbite medium. Determination of Bacteria in Butter, Lohnis Method. For the sterile weighing of butter, cheese and similar substances, put a number of filter papers, 9 cm. and 7 cm. in diameter, into two Petri dishes respectively and sterilize in the air oven. By means of sterilized forceps, place one of the larger papers on the scale pan and then one of the 1 Li iiJiiKOijiii HAvC/Ujl-ji Ixs jDuJ-ili'X'C TC^( Table XL VI INCIDENCE OF TUBERCLE BACILLI IN MARKET BUTTER (After Briscoe and MacNeal, 1911) Author, Brusaferro. Roth. . , ObermuUer Schuchardt ObermuUer Groning. . , . Himesch . . Rabinowitch Rabinowitch Petri Hormon and Morgenroth. . Rabinowitch. . . Rabinowitch. . . Rabinowitch. . . Rabinowitch. . . ObermuUer . . . Korn Ascher Jager Coggi Weissenfield . . . Grassberger. . . . Herbert Herbert ....... Herbert Herbert Abenhausen. . . Hellstrdm ..... Bomhoff Pawlowsky. . . . Tobler Lorenz Markl. ..,,.... Herr and Beninde Aujeszky Thu Teichert Reitz Eber. Briscoe and * MacNeal Eber Rosenau et al. . Marphiottx Date 1890 1894 1895 1896 1897 1897 1897 1897 1897 1897 1897 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1899 1900 1900 1900 1900 1901 1901 1901 1901 1902 1902 1904 1906 1908 1911 1912 1914 1917 Place. Turin Zurich Berlin IMarburg Berlin Hamburg Wien Berlin Philadelphia. . Berlin Berlin. . . . Berlin Berlin Berlin Berlin Berlin Freiburg . . . . Konifrsberg , . Konigsberg . . Milan Bonn Wien Tubingen Wurtteruberg . Berlin Munchen Marburg Helsingfors. . . Marburg Kiew Zurich Dorpat Wien Breslau. , . . Budapest. . . Christiania . Rosen Stuttgart. . . Leipsie Urbana, 111 Boston Samples Ex- amined. 9 20 13 42 14 17 ? 30 50 102 10 15 ? 15 19 10 17 27 3 100 32 10.0 43 58 20 5 39 8 28 23 12 30 43 52 17 16 40 94 150 21 25 I Per Samples ' Cent Posi- Posi- tive. j tive. 1 2 8 14 8 33 3 2 f 15 4 4 2 1 12 3 1 •1 2 6 3 12 8 18 2 2 U 1 10 61 100 47 P 32 3 30 13 3 87 2 100 40 23 5 7 4 33 3 12 Remarks. 9 I 12 5 4 3 16. 7 11. 1 17 30. 8 5 12 33 2 15. 6 9 4 24 Microsconic method. 16 tested, 2 lost. First series. Second &eries- Third series Fourth series. Pseudo tuberculosis 5 per. Pseudo tuberculosis 8 per. Pseudo tuberculosis 4 p«r. 12 samples, 4 lost. 39 samples, 11 lost. Two wore doubtful. Butter from 88 dairies. Creamery butter. 52 per cent of the samples contained acid-fast ba- cilli. 428 MILK AND MILK PRODUCTS fomaller papers on top of it. After taring, with sterilized knife or spoon, place the substance to be weighed on the upper filter paper. Then transfer the material along with the small filter paper, to the first dilu- tion flask. Make the 'dilutions according to the quantity of butter to be examined and pour plates. The water used for dilutions should be previously warmed to 40° C. so that the butter (1 gm. in the first tubes or flasks) may be readily emulsified. Determination of Bacteria in Butter (Schneider's Method). To examine butter directly for the number of bacteria and other contamina- tions, place 1 gm. of the butter into 10 c.c. of ether and shake until all of the butter fat is dissolved. Pour the solution into a special cen- trifuge tube and centrifuge for five minutes. Wash the contents of the 1 c.c. end into 10 c.c. of ether and again shake and centrifugaUze. Pour off the ether and add 2 c.c. of a 2 per cent solution of sodium hydroxide and shake until the casein is dissolved. The sodium hydroxide emul- sifies the small amount of fat which is present. Examine the emulsion for bacteria and body cells by means of the haemocytometer. Conn's Method for Analysis of Butter. Weigh out upon accurate <3hemical scales 5 gms. of freshly made butter. Place this in a sterile mortar, with 9/5 c.c. of sterile water. Rub the water and the butter together thoroughly, so as to distribute the bacteria as uniformly as possible through the water. This mixing should be continued for some time, for, at best, many of the bacteria will remain clinging to the fat. Dilute this mixture to 10,000 times and make a series of agar or litmus gelatin plates. Incubate and count as usual. Cheese Cheese may be classified into two general types, rennin curd and acid curd. In the former type, the enzyme rennin is added to the milk which forms calcium paracaseinate from the calcium caseinate. The acid curd cheeses are made from curd secured by the natural or artificial souring of milk. Cheese Ripening. In this process develops the characteristics which, in part, separate one type of cheese from another. Many theo- ries have been held in the past with regard to this process. Duclaux (1880), Freudenreich (1897), Babcock and Russell (1898), Gorini (1904) and others, have studied the problem and given different theories. The following factors were at times considered the important ones: 1. Enzymes which originated from the cow. 2. Enzymes of bacterial origin. 3. Pepsin which is added with rennin. BACTERIOLOGY OF CHEESE 429 Bacteriology of Cheddar Cheese. Harding and Priicha (1908) studied more than 300 pure cultures of bacteria from cheddar cheese by means of the group number on the classification card of the Society of Amer- ican Bacteriologists. These finally reduced to 33 groups. Ten of these groups disappeared at once; 9 others were found in but single cheeses; the remaining 14 groups were indicated as the most important members of the cheese floia. Badermm lacits acidi included 4 of these 14 groups and was the only species always found and it practically included over 99 per cent of the total bacterial content. Hastings, Evans and Hart (1912) conducted a similar study. They state that the Bactenu7n lacks acidi groups is an important one and sum up the role of these organisms as follows: 1. They favor the curdhng process. 2. They favor the expulsion of the whey. 3. They pei^mit of the fusing of the curd particles. 4. They activate the pepsin in the rennin extract. 5. They have a protective action against the putrefactive bacteria. They state that^the development of B. lactis acidi is followed by the Bacillus bulgaricus group. These same authors in a later paper (Evans, Hastings and Hart, 1914) found the following four groups and assumed that they must function in the ripening process: 1. Bacterium lactis acidi. 2. B. casei. 3. Streptococcus. 4. Micrococcus. When the Bacterium lactis acidi gi^oup was used alone as starters, in pasteurized milk cheese, no cheddar flavor was obtained. Com- binations of the Streptococcus and B. lactis acidi groups when added to pasteurized milk improved the character of the cheese. Hart, Hastings, Fhnt and Evans (1914) isolated coccus forms of bacteria from cheddar cheese and cultured them in sterile milk. Large amounts of volatile acids were found on analysis of the culture. These acids were produced from either citric acid lactose or protein because the milk contained no fat. One strain of streptococcus formed large amounts of alcohols and esters which contribute to the flavor of the cheese. Suzuki, Hastings and Hart (1910) isolated and identified vola- tile acids during the ripening of cheddar cheese. Alcohol was formed probably from lactose fermentation. The agencies operative in the production of volatile acids and esters are not defined. Harding and Prucha (1908) analyzed different cheeses and demon- 430 IMILK AND MILK PRODUCTS strated that the number of bacteria decreases rather rapidly. Fresh cheeses were found to contain large numbers of bacteria. These decreased so that at the end of thirty or forty days there were less than 10,000,000 bacteria per gram. These authors state that a cheddar cheese commercially ripe usually contains some millions of living bac- Commercial score Bacteria content '■'N*,, **•**•. 10 20 30 40 50 60 70 80 90 100 110 320 130 Days Fig, 73.~Bacteria Content and Commercial Score of Cheddar Cheese. (After Harding and Prucha, 1903.) teria per gram. Fig. 73 shows the number of bacteria that were found in a cheese at various ages. The relation of cheese to the spread of disease is an interesting ques- tion which has received some attention from investigators. Tuber- culosis has received the greater part of the attention. Mohler, Wash- BACTERIA IN CHEESE 431 burn and Doane (1909) have mentioned and reviewed some of the work which was carried on previous to their publication. Hormann and Morgenroth (1898) upon examination of 15 samples of cottage cheese found 3 of which contained tubercle bacilli. Rabinowitch (1907) reported 3 positive samples out of 5 of the same product. Three sam- ples of soft cheese purchased from the Berne markets by Harrison (1900) were found to produce tuberculosis when injected into guinea pigs. Heim (1889) and Galtier (1887) infected milk before making cheese. Heim reported tubercle baciUi in the cheese for fourteen days but none after four weeks. Galtier found tubercle bacilli in cheese which was two months and ten days old. He beUeves that such milk products may disseminate the disease among human beings, Mohler, Washburn and Doane (1911) prepared a cheese from infected milk and, by inoculation into guinea pigs, produced generaUzed tuberculosis from samples 220 days old. Injections of emulsions 260 days old caused slight lesions. Schroeder and Brett (1918) carried out an extensive piece of woi'k which has greatly enriched our knowledge. They pur- chased 256 samples of cheese from the Washington market and sub- jected them to examinations for the presence of tubercle bacilli by means of guinea-pig inoculation. Nineteen or 7.42 per cent contained tubercle bacilli. Cheddar cheese was examined to the extent of 59 specimens; no tubercle bacilli were found. None were found in 32 specimens of Neufchatel cheese. Eighteen out of 131 samples of cream cheese were infectei They regard the danger of eating ripened cheese as very slight since the bacilU would die during the ripening process. According to Mohler's work, this would depend on the length of the ripening period. Rowland (1895) inoculated cheese and butter with M. cholerm and B, typhi. After a few days no living organisms could be found, which is regarded as reassuring by this author. He used cheddar, Dutch and American cheeses and both fresh and salt butter. Cheese Poisoning. This type of food poisoning is said to be rather common. Vaughn named the poisonous substance in cheese tyrotoxi- con which he regarded as a ptomaine. Newman (1902) and Lepierre (1894) confirmed his work. Spica (1910) recently isolated a poisonous substance from cheese. Levin (1917) studied this subject and found by means of agglutination reactions that a member of the colon group was involved in a case of cheese poisoning, which he investigated. No tyrotoxicon could be found. Determination of the Ntunber of Bacteria in Cheese. (Harding and Prucha's Method.) Remove a square inch of the rind by means of a sterile knife. Draw out a plug about 4 ins. long with a carefully ilamed 432 MILK AND MILK PRODUCTS cheese trycr. Cut a number of thin slices from different parts of this plug and weigh out 1 gm. of these slices on a flamed copper foil using an analytical balance. This sample should be triturated in a flamed mortar with 10 gms. of sterile granulated sugar or finely ground ster- ilized quartz. Care should be taken to have the room free from dust and the instruments which come in contact with the cheese, sterile. Dilute this freshly ground cheese to 30 c.c. with sterile water. An aliquot portion should be transferred to a water blank. Plate out in litmus whey gelatin in dilutions of from 1000 to 1,000,000 or there- abouts. Incubate for ten days at room temperature. Lohnis' Method. One gm. of cheese taken under aseptic conditions and weighed according to the method described in another place is ground in a sterile mortar with' 10 c.c. of sterile tap water, and some sterile quartz sand or glass powder added if necessary. The mixture is put into a dry, sterile 1000 c.c. flask, which is then filled to the mark with sterile water. After continuous shaking for several minutes, further dilutions with 9 c.c. of water are made in test tubes. Usually dilutions of 1 to 10,000 to 1 to 100,000 are sufficient. This is plated out in plain agar and gelatin. Cultures for anaerobic incubation should also be made. Harrison's Method. Remove a plug of cheese with a sterile cork barer and transfer to a sterile mortar. Triturate this with sterile water of bouillon. This is injected to a 500 gm. guinea pig. A hole is made in the skin with a large needle. Through this by means of a pipette the cheese emulsion is blown in. Animals should be kept under observa- tion and immediately after death should be examined. Smears should be made from the diseased organs and stained from the Ziel Nielsen method. Determination of Tubercle Bacilli in Cheese. (Mohler's et al. Method.) Rub portions of the cheese in a mortar with physiological salt solution. After grinding strain the liquid through a layer of absorb- ent cotton and the equivalent of 2 gms. of cheese should be injected beneath the skin of each guinea pig. Also feed portions of the cheese to other pigs and keep for observations. Fermented Milks These are milks in which the lactose has been changed to lactic acid, alcohol or carbon dioxide by the action of fungi. An extensive literature of the subject, together with a discussion has been prepared by Rogers (1916). CONDENSED MILK 433 Condensed Milk Milk has been condensed for a long time. In 1835, Newton took out a patent in England. Later, in 1849, Harsford added lactose to con- densed milk. The nomenclature of concentrated milks is not set. Condensed milk often refers to concentrated milks to which a carbohydrate, sucrose or lactose has been added. The term evaporated milk is often restricted to the plain concentrated milk to which no carbohydrates have been added. 'Again, these may be referred to as condensed sweetened milk and condensed unsweetened milks. The following definition for sweet- ened condensed milk has been accepted by the Association of American Dairy, Food and Drug Officials, August 7, 1916, and by the Association of Official Agricultural Chemists, November 22, 1916. " Sweetened condensed milk, sweetened evaporated milk, sweetened concentrated milk is the product resulting from the evaporation of a considerable portion of the water from the whole fresh, clean, lacteal secretion ob- tained by the complete milking of one or more healthy cows, properly fed and kept, excluding that obtained within fifteen days before and ten days after calving, to which sugar (sucrose) has been added. It contains not less than 28.0 per cent of total milk solids and not less than 8.0 per cent of milk fat.'' (U. S. Agriculture, Food Inspection Decision 158). Condensed milk is not sterile. The data from many investigations are on record in support of this. There seems to be no more bacteria or body cells in condensed milk than in raw market milk. Some of the processes of condensation remove these. The centrifugal sepa- rators are, doubtless, responsible for much of this reduction. The bac- teria which do pass through the condensation processes lie dormant on account of insufficient moisture and too concentrated solution. Some are present in the spore stage. This is probably true in those cases where members of the Bacillus subUlis group such as Bacillus subtilis and Bacillus mesentericus have been isolated. Thayer (1912) exam- ined condensed milk which spoiled from twelve to twenty-four hours after condensing and found Bacillus subtilis. To obviate this difficulty, this author recommended a sterilization temperature of 125° C. for fifteen or twenty minutes. Hammar (1915) isolated an organism which merely coagulated the canned milk. The name Bacillus coagulans was given to the organism. The coagulum was firm and had a sweetish taste. There was no indication of putrefaction. Andrews (1913) found 434 MILK AND MILK PRODUCTS that there were not enough pus cells in the milk which he examined to cause suspicion. The following sums up his opinion: The presence, in reasonable number of the bacteria commonly found in fresh milk — Bacillus coli, streptococci, a few staphylococci, and B, enteritidis sporo- genes— k comparatively unobjectionable. A large proportion of the bacteria of milk seem to be destroyed in the process of condensation. It was found that con- densed milk was almost a differential medium for staphylococci. From his work it would seem that if a few Staphylococcus pyogenes aureus were present in the milk after condensation many more might be found when the can was opened. Efficient pasteurization before condensation should greatly reduce the pos- sibility of the staphylococci getting into the final product. Park et al. (1915) found that evaporated milk contained fewer bacteria than condensed milk. This may have been due to the higher temperature at which it is prepared. Kossowicz (1908) found that condensed milk was not bacteria free. He found Bacillus fluorescens UquefacienSj Bacillus prodigiosus and Bacillus sinapwagus to be present. Savage (1914) reports several investigations in England, the data from which do not differ materially from those secured in this country. The very interesting experiment of Delepine with regard to the effect of the condensation processes on Bacterium tuberculosis is mentioned. The tubercle bacillus, even though heavily seeded into milks, was killed and could not be demonstrated in the final condensed milk. Delepine (1915) has studied the effect of preserving and drying milk on the virulence of the tubercle bacillus. In the manufacture of sweet- ened milk, the mixed raw milk had a bacterial content of 38,000,000 which was reduced by the treatment to less than fifty in one case. The finished sweetened product failed to produce lesions when inoculated into and fed to rabbits and guinea pigs. Ice Cream The term is a general one and covers many types of similar sub- stances. Those interested in the history of this food should look up the paper by Washburn (1910). The bacteriology of ice cream has, of late, received much attention since it became necessary to estabUsh some bacterial standards. Wiley (1912) and his co-workers studied the ice cream manufactured in the District of Columbia and found rather large numbers of bacteria. Many of them contained streptococci. Esten and Mason (1915) studied the effect of storage on the number and kinds of bacteria. The ICE CREAM BACTERIA 435 cream was frozen in the usual type of freezer and stored in quart bricks. The method of analysis is given in another place. They found that there was Uttle change in the number of bacteria as shown by litmus lactose gelatin plates when cream was kept frozen for a month. Ayers and Johnson (1915) undertook an investigation on ice cream with the following objects: 1. To determine the number of bacteria in commercial ice cream during the summer and winter seasons. 2. To determine what groups of bacteria are found in commercial ice cream. 3. To determine the relative value of different methods for the deter- mination of Bacillus coli in ice cream. They found that the average number of bacteria in ice cream is lower during the winter months. A sum^iary of their results is given in the following table: Item. Average number of bacteria per cubic centimeter . Maximum number Minimum number Summer Series (94 Samples). "W inter Series (91 Samples). 37,859,807 510,000,000 120,000 10,388,222 114,000,000 13,000 Studies by means of the milk-tube method showed that the bacterial groups bore about the same relation to each other in the average summer and winter samples. During the summer months the averages based on 71 samples were as follows: Acid coagulating 49.82 Acid forming 20 . 72 Inert group 13 ,98 Alkali forming groups 1 .86 Peptonizing group 13 . 62 The averages for the winter samples are: Acid coagulating group 30.84 Acid forming group. 38 .03 Inert group 4 .81 Alkali, forming group 5 .42 Peptonizing group 20 . 90 The peptonizing group is of most significance since it is these bac- teria which cause poisoning from ice cream. 436 MILK AND MILK PRODUCTS Ayers and Johnson (1917) in a later paper report the results from a study of some phases of ice-cream analysis. It was found that bacteria are evenly distributed through a can of ice cream and that storage does not tend to cause an uneven distribution. These were factors which had been suspected of causing difficulties in the bacterial examination of ice cream. This was later confirmed by Heinemann and Gardan (1917). As freezing progressed clusters of bacteria were broken up and this tended to increase the count. No marked difference was found in the counts on samples from different parts of the same can. Hammar and Goss (1917) analyzed ice cream and water sherbets. They reported very few organisms in the water sherbets compared with the ice cream. Ice creams, other than vanilla, were found to contain from 130,000 to 40,850,000 bacteria per cubic centimeter. In general there was no increase in the number of bacteria during storage and gen- erally there was a reduction. With some samples increases were re- ported. Table XL VII contains a few analyses reported by Hammar and Goss. Table XL VII SHOWING THE EFFECT OF STORAGE ON THE NUMBER OF BACTERIA IN ICE CREAM (Hammer and Goss, 1917) Mix Freeze 1 day 2 ' 3 ' 4 ' old. 236,000 735,000 360,000 310,000 260,000 32,800,000 30,850,000 7,750,000 4,450,000 2,435,000 1,150,000 120,000 146,000 137,000 216,000 152,000 300,000 139,000 156,000 172,500,000 271,000,000 157,000,000 128,000,000 52,000,000 34,000,000 31,000,000 110,000,000 170,000,000 194,000,000 216,000,000 102,000,000 39,000,000 54,000,000 120,000,000 140,000,000 70,000,000 71,000,000 5 ' 6 ' 310,000 41,000,000 7 ' 8 ' 61,000,000 9 ' 36,000,000 15,000,000 10 ' 11 ' 12 ' 1 No special diflBculties appear in the bacterial analysis of ice cream. Secure a brick of the cream or a pint or quart container if it is not avail- able in brick form. As soon as it has reached the laboratory unwrap ICE CREAM BACTERIA 437 and remove samples from the interior of the mass of ice cream. This may be done by scraping off the exterior with a sterile spatula and by means of a sterile scoop transfer portions to sterile flasks. Heat these on a water bath at 40° C, with occasional stirring until melted. Dilute with sterile pipettes and plate out using the standard media. Bacillus Typhosus in Ice Cream. Several severe epidemics of typhoid fever have been caused by ice cream. Mitchell (1915) found that these bacteria could live for 12 to 39 days in ice cream and that ice cream could thus become an important epidemiological factor in the spread of the disease. Gumming (1917) reported an epidemic of typhoid fever caused by a carrier who participated in the preparation of the cream. Lumsden (1917) has given the characteristics of epi- demies caused by ice cream. The difficulty of securing accurate data in such epidemics is mentioned. Waterman (1917) has reported a small outbreak of typhoid fever which probably came from ice cream; the typhoid history of the people involved is rather striking. Bolten (1918) reported experiments on freezing B. typhi in cream. At the end of a month the number of bacteria is reported to have been reduced to about one-twentieth of the original number. At the end of about 45 days after freezing in brine, some of the bacilli were found to be alive. As the author points out, ice cream may be a potent factor in the dis- semination of typhoid fever. Determination of the Number of Bacteria in Ice Cream (Esten and Mason). Samples are taken with steriHzed knives, the outer layer being cut off before the sample is taken out. Allow the cream to melt at room temperature in the same manner as milk samples. Plate out on litmus lactose gelatin; incubate for 7 days at 21 ^^ C. and coxmt. Make four plates from each sample and report the results as an average of the four plates. By using litmus lactose gelatin the number of acid- forming bacteria and the number of Uquefying bacteria may be deter- mined. Savage's Method for Bacterial Analysis of Ice Cream. The ice cream should be collected in a sterile vessel, e.g., a wide-mouthed sterile bottle with glass stopper — and packed in ice if it cannot be examined at once. To examine, melt the ice cream by placing for fifteen to twenty min- utes in the 22° C. incubator then treat as a milk sample. The degree of dilution and the methods of examination are similar to those used in the examination of milk. Determination of Streptococci in Ice Cream (Wiley et al.). The method for the detection of streptococci in ice cream has been used by 438 MILK AND MILK PRODUCTS Wiley and his co-workers as follows: The melted saraple should be centrifuged for half an hour in a Stewart lactocrite with a speed of approximately 3000 r.p.m. This apparatus, which consists of a jBat aluminum pan holding twenty tubes of 1 c.c. capacity and stoppered at the outer end with a specially constructed rubber plug, causes the sediment not only to be thrown to the end of the tube but drives it against the rubber plug with such a force that it is almost quantitatively adherent to the plug. Accordingly, if one removes the rubber stopper and by rubbing on a glass slide and over an area of known surface attaches the sediment, one can obtain, on staining and examining the film microscopically, an approximation of the number of organisms and leucocytes in 1 c.c. of the liquid. Because of the debris in the ice cream, which ordinarily renders the usual method of centrifuging milk and cream samples quite impracticable, the above method was resorted to so far as the detection of the presence of streptococci was concerned, it was found eminently satisfactory. Davis (1914), in connection with investigations of septic sore throat decided that all of the streptococci concerned were of the hemolytic variety. These were found to remain alive in ice cream for eighteen days with little diminution in numbers. He concludes that ice cream is a suitable medium for the growth and dissemination of dangerous streptococci. The Food and Drugs Act of the United States Department of Agriculture attempts to promote the production of pure. clean foods. With certain foods, as with ice cream, a satisfactory method of labora- tory control has not been worked out, and to this end more attention must be given to the conditions of manufacture. The Commission on Milk Standards has proposed the following score card by which the ice cream plants may be rated. It is then assumed that there is some direct relation to the quality of ice cream and the conditions under which it is produced. ICE CREAM 439 SCORE CARD FOR ICE-CREAM MANUFACTURING PLANTS Location Above ground 5 Free from contaminating surroundings (no score if bad) ... 3 Protected from street dust 3 Not connected with any other room 2 No other business in same establishment 2 Construction Well Hghted (natural) 2 Well ventilated 1 Thoroughly screened ^ 2 Water-closet does not open directly into establishment. . . 2 Separate room for washing utensils 2 Floor: Smooth, water-tight well drained 4 Walls and ceiling: Smooth and tight 2 Equipment Steam at all times 5 Hot water at all times (no credit unless running hot water) . 3 Sterilizer for utensils 3 Connections for sterilizing apparatus 2 Pasteurizer: Holding machine 4 Automatic recording device 1 Refrigeration: Mechanical (proper ice box, 1) 2 Freezer: Type, connections, etc 2 Sanitary piping 2 Wash basins and towels ample 1 Utensils: Condition ' 1 Ample for the service 2 Racks for 1 Employees: Health certificates for : 1 Clean suits provided 1 Methods Freedom from flies 2 Protection of material: ' Before manufacture 3 During manufacture 3 After manufacture . . * 3 Utensils and apparatus sterilized (washed in hot water, 1) 3 CleanMness : Floors 3 Windows 1 Apparatus 3 Walls and ceiling 1 Utensils 3 Employees 3 Character of materials used: Milk and cream, Grade A (Grade B, 4; Grade C, 1) . . . 6 Condensed milk, eggs, etc 2 Thickeners, none used 1 Artificial coloring, none 1 Degree of refrigeration of final product 2 Total Perfect. 15 15 Allow. 30 40 100 440 MILK AND MILK PHODUCTS BIBLIOGRAPHY, Abenhausen. 1900. Investigation on the Presence of Tubercle Bacilli in Marburg Butter and Margarine. Dissertation, Marburg, 1900. Allen, P. W. 1916. Comparison of the Rate of MultipHcation of Bacteria in Raw Milk with the Rate in Pasteurized Milk. Jour, of Infectious Dis- eases, 19 (1916), 721-728. Allen, P. W. 1917. Comparisons of the Rate of Gas Production by Certain Bacteria in Raw and Pasteurized Milk. Jour. Infectious Diseases, 21 (1917), 219-225. Andeews, F. W. 1913. The Cytology and Bacteriology of Condensed Milk. Jour. Path, and Bact., 18, 169-178. Exp. Sta. Rec, 1913. Anon. 1912. Tubercle BaciUi and-Uncooked Food. Vt. Bd. Health Bull. 12, 58-60. AscHER, N. 1899. Untersuchungen von Butter und Milch auf Tuberkel- baciUen. Zeit. f. Hyg., 32, 329. Ayers, S H. and Johnson, W. T. 1910. The Bacteriology of Commercially Pasteurized and Raw Market Milk. U. S. Dept. of Ag., Bureau of Animal Industry, Bull. 126. Ayers, S. H. and Johnson, W. T. 1913. A Study of the Bacteria which Sur- vive Pasteurization. U. S, Dept. of Ag., Bureau of Animal Industry, Bull. 161. Ayers, S. H. and Johnson, W. T. 1915. A Bacteriological Study of Retail Ice Cream. U. S. Dept. of Agr. Bull. 303. Ayers, S. H. and Johnson, W. T. 1915. Abihty of Colon Bacilli to Survive Pasteurization. Jour. Agr. Research, 3, 401-410. Aters, S. H, and Johnson, W. T. 1917. The Determination of Bacteria in Ice Cream. U. S. Dept. Agr. Bull. 563. Ayers, S, H., Cook, L. B. and Clemmer, P. B. 1918. The Four Essential Factors in the Production of Milk of Low Bacterial Content. U. S. Depart- ment of Agriculture Bulletin 642. Babcock,'S. M. and Russell, H. L. 1897. Unorganized Ferments of Milk; a New Factor in the Ripening of Cheese. Wise. Ag. Exp. Sta. 14th Annual Report, 1897, 161-193. Baer, A. C 1916. Ice Cream Making. Agr. Exp. Station, Univ. of Wis. Bull 262. Balazs, A. 1913. About the Detection of Boiled and Raw Milk. Abst. in Chem. Ztg., 37, 554. Bang, B. 1891. Experimentelle Untersuchungen uber Tuberculose Milch. Deut Zeit. f. Thiermed, 17, 1. Basenar, F. 1895. The Behavior of Cholera Bacilli in Raw Milk. Arch. Hyg., 23 (1895), 170-183. BIBLIOGRAPHY 441 Beattie, J. M. and Lewis, F. C. 1913. Acid Fast Bacilli in Milk. Jour. Path, and Bact., 18, 122-123, Abstract. Bergey, D. H. 1911. The Isolation of Bacillus Typhosus from Butter. Jour. Med. Res., 25 (1911-12), 231-33. BoLTEN, J. 1918. Effect of Freezing on the Organisms of Typhoid Fever and Diphtheria. Pubhc Health Reports, 33, 163-166. BoswoRTH, A. W. 1913. The Action of Rennin on Casein. N. Y. Agr. Exp. Station Tech. Bull. 31. BoYCE. 1900. Results Quoted from Aunett Laucet, 1900, p. 160. Boyd, M. F. 1917. Butter as a Vehicle of Infection in Typhoid. Jour. Amer. Med. Assn., 69, 2030-2032. Brainerd, W. K. Clean and Sanitary Milk. Virginia Ag. Exp. Sta. Bull. 185. Breed, R. S. 1911. The Determination of the Number of Bacteria in Milk by Direct Microscopical Examination. Cent. Bakt. Abt. II, 30, 337-340. Breed, R. S. 1913. 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In milk and its relation to the public health, pp 249-312. WiNSLow, C. E. A. and Hubbard, L. W- 1915. A Contact Outbreak of Septic Sore Throat in Westchester County (N. Y.). Health News^ N. Y. State Depi. Health, 9, 266-270 v_;jtlAJr X Jcjix -X.il EXAMINATION OF EGGS Eggs have always been an important factor in the diet. As the density of population increased, especially in urban centers, the problem of securing fresh eggs became more and more acute. This was ren- dered more intricate by the fact that the greatest part of the annual lay is secured during the months of March, April, May and June. This makes it necessary to hold over, either by cold storage or preservation, the abundance of these months against the want of the others. It has become of much importance to determine what factors are important in the keeping of this food product. A knowledge of the physiology of the hen which is concerned with the development of the egg is important in this connection. This has been well described by Benjamin (1915) from whose publication the following has been adapted. The oviduct of the hen is located in the rear part of the body cavity. The yolk is the first part to develop and this takes place in the ovary which may contain a large number of minute yolks. Each is contained in a sac, or follicle through which it secures its nourishment while developing. After development the yolk en- closed in its vitelhn membrane escapes from the yolk sac and descends through the oviduct. If fertilization occurs, it takes place soon after the egg has entered the oviduct and before any albumen is deposited about it. About 40 per cent of the albumen is said to be laid down as the yolk passes through*^the upper half of the oviduct. After passage through, this albumen-forming region, it reaches the isthmus where the shell membranes are added with about 10 to 20 per cent more albumen. The uterus is then reached, where the remainder of the shell is added. During its passage through the vagina, just before its expulsion from the cloaca, it probably receives the outer gelatinous coating on the shell. The structure of the egg and shell is given in Figs. 75 and 76. The chemical composition of any food substance is important in determining the type of composition which may take place. This is especially true with a substance like eggs. The analysis quoted in Table XL VIII is taken from the paper of Benjamin (1915). 451 V" 452 EXAMINATION OF EGGS .■ptTT'P' *"J^'W«^^pT,JTnj'- Ij ovary, with minute ovules; 2-3, yolk sacs; 4, suture line; 5, empty yolk sac; 7, funnel opening into oviduct; 8, yolk m oviduct; 9, albumen-secreting region; 10, albumen being secreted; 11, yolk passing through oviduct; 12, germinal disk; 13, isthmus; 14, uterus; 15, large intestines; 17, cloaca. On the right-hand side of the figure are shown, from the top downwrd: Complete egg- yolk of egg incubated for sixteen hours; completed egg in uterus--(l) isthmus, (2) glands of uterus, (3) complete egg, (4) vagina, (8) cloaca, / STRUCTURE OF EGGS 453 Fig. 75. — Structure of an Egg. (After Benjamin, 1914.) A, outer shell; B, outer shell membrane; C, inner shell membrane; D, air cell; E, outer thin portion of albumen; F,G, inner dense portion of albumen; H, vitelline membrane; i, ehalaza; J, thin film of white yolk inside of vitelline membrane: K, layers of yellow yolk separated by thm layers of white yolk; L, germinal disk; M, central part of yolk filled with white yolk; iv, slender tube connecting center of yolk with region of germinal disk. --^ jTiG, 76._Shell Membranes of an Egg Highly Magnified. (After Benjamin, 1914.) A inner shell membrane. Note the fine cellular structure. The cells are bound together by many intertwining fibers. B, outer shell membrane. This is much coarser m structure than the inner membrane. The fibers are very transparent. Table XLVIII COMPOSITION OF HENS' EGGS Water Fat Protein Shell and shell membranes Yolk, Percentage. 46-52 30-35 14-16 White of Egg, Percentage. 80-88 Traces 10-13 Whole Egg, Percentage. 70-76 9-14 10-15 9-12 454 EXAMINATION OF EGGS From Table XLVIII it is seen that the whole egg usually contains over 10 per cent of fat. Nearly all of this fat is in the yolk, and obvi- ously the fowl must have a surplus of fat before the yolks can begin to develop. The eggs of ducks, geese, turkeys, guinea fowls, and other birds vary to some extent from the analysis noted above, but the dif- ferences are slight. The detailed analysis of hens' eggs which follows is taken from Simon^s textbook of physiological chemistry: Analysis of shell (9-11 per cent of whole egg). Percentage. Calcium carbonate 90 . 00 Magnesium carbonate Small amount Calcium and magnesium phosphate Small amounts Water 1.00 Analysis of albumen (60.5 per cent of whole egg). Percentage. Water 80.00-86.68 Solids 13.32-20.00 Albumins 11.50-12.27 Extractives . 38- . 77 Glucose 0. 10- 0.50 Fats and soaps Traces Mineral salts . 30- . 66 Lecithins and cholesterin Traces The mineral ash of the albumen has been found by Poleck and Weber to consist of: Percentage. Sodium (NaaO) 32.56-32.93 Potassium (K2O) 27 . 66-28 .45 Calcium (CaO) 1 , 74-2,290 Magnesium (MgO) 1 . 60- 3 . 17 Iron (Fe203) 0.44- 0.55 Chlorine. (CI) 23 84-28.56 Phosphoric acid (P2O2) 3. 16- 4.83 Carbonic acid (CO2).. , 9.67-11.60 Sulphuric acid (SO3) 1 .32- 2.63 Silicic acid (SiOa) 0.28- 0.49 Fluorine (Fl) , Traces Analysis of yolk (29 per cent of whole egg). GRADING EGGS 455 According to Gautier we have the following: Percentage, Water 47.19-51.49 SoHds 48.51-42.81 Fats (olein, palmitin, and stearin) 21 . 30-22 . 84 Vitellin and other albumins 15 . 63-15 . 76 Lecithins 8.43-10.72 Cholesterin 0.44- 1.75 Cerebrin . 30 Mineral salts 3.33- 0.36 Coloring matter ] . n f;^Q /-S.T r..^i,.. U, Duo Glucose J Poleck and Weber also give the following as the analysis of the min- eral salts of the yolk: Percent 8.£e Sodium (Na20) 5. 12- 6.57 Potassium (K2O) 8.05- 8.93 Calcium (CaO) 12 . 21-13 . 28 Magnesium (MgO) 2.07- 2. 11 Iron (Fe203) 1. 19- 1 .45 Phosphoric acid, free (P2O3) 5 . 72 Phosphoric acid, combined 63 . 81-66 . 70 Silicic acid (Si02) 0.55- 1.40 Chlorine Traces According to Simon, the shell is made up of an organic matrix of the nature of keratin. This matrix is largely impregnated with lime salts. The pigments of the shell are said to be derived from the common pigment of blood. The shell membranes are also composed largely of keratin, with a small amount of mineral salts, principally calcium phos- phate. The white of the egg is supposed to consist of compartments which are divided by thin membranes and which contain the liquid albumen. These membranes are continuous with the chalazse and the shell membranes. The yolk consists largely of spherical cells, most of which are filled with fat. The bacteriology of eggs has received considerable attention and much interesting and useful data are available. As far as is known eggs have not been important in spreading pathogenic bacteria. Grading of Eggs. This is essentially a commercial problem. Under some conditions, the grading of eggs is very diflicult. However, in practice they are graded by the candling process and observations with 456 EXAMINATION OP EGGS regard to their internal condition and appearance, are made. Frazier (1916) and Stiles and Bates (1912) describe the different grades of eggs. Firsts and Extras. This class includes all freshly laid, sound, whole, clean shell, medium and good-sized eggs. The term '' fresh " is used to indicate an egg with practically no age. Before the candle such an egg is characterized by a very small air cell at the large end, absence of defects, transparence and a slow-moving yolk. They appear perfectly homogeneous. Some fresh eggs are laid with defects as '' liver spots '' '' meat spots," foreign material, etc. The appearance of the opened egg gives much information to the microbiologist. The yolk should stand up with a definite shape on account of the strong vitelline membrane about it. The albumen should be transparent and firm. The contents should have no odor and should be practically free from a large number of bacteria. Table XLIX COMMERCIAL SAMPLES OF LEAKING EGGS BROKEN IN THE CANDLING ROOM (Pennington) Gas- Bacteria per produc- Ammonial Gram on Plain ing bac- Liquefy- Nitrogen Wght Sample No. Source Date of Col- Agar Incubated teria per ing or- ganism (Folm). Mois- ture of Sam- lection Gram m Lac- per Gram ples, Lbs 20° C. 37« C tose Bile Wet Basis Dry Basis 4214 F-l May 1 4,300,000 2,100,000 10,000 1,400,000 0017 0054 69 74 420 4224 F~l May 2 3,800,000 1.800,000 100,000 430,000 0016 0054 70 35 4243 F-1 May 3 1,600,000 950,000 10,000 650,000 0017 0062 72 55 4370 F-l May 23 25,000,000 6,300,000 100,000 12,000,000 0020 0067 70 09 150 Seconds. In this grade are classed all eggs not included under the first, except the third grade mentioned below. Stiles and Bates report the following divisions: (a) Undersized. If not for their small size these eggs would be classed as firsts. They conform to the standards mentioned above for a first-grade product. (6) Checks and Cracks. Eggs, the shells of which have become broken by careless handling but which have intact shell membranes. GRADING EGGS 457 (c) Leakers. Eggs whose shells and shell membranes are suffi- ciently broken to permit a portion of their contents to escape. {d) Dirties. Eggs, the shells of which have become soiled from un- clean nests, etc. (e) Weak Eggs. In this grade are placed all eggs in which the albumen has become weak or watery, due to high or varying tempera- ture. Leakers and dirties present the greatest problem to food bacteriol- ogists since they allow the ingress of bacteria. The leakers are either sold for local consumption or are broken for drying. Table XLIX pre- sents some data on commercial samples of leaking eggs secured by Pennington et al. (1914). Dirties appear during wet weather and sometimes during hot weather when moisture from the hen's body allows more dirt to adhere to the shell. Many of these are used for local consumption or for drying since they do not keep well. In the examination of eggs with dirty shells Pennington (1914) secured widely divergent results. The minimum number of bacteria was 400 and the maximum number 1,600,000 per gram at 20° C. The number of 5. coli varied from 10 to 10,000 in six samples. It has been reported by many investigators that dirty eggs do not keep well. Often these eggs are washed but this process reduces the keeping quality since it removes the delicate membrane or film of mucus on the surface of the shell. Spots. All eggs which show a spot before the candle are put into this class. These may be due to bacterial, meat, blood or mold spots. The term " spot-rot '' is applied to a heated egg caused by decom- position of the dead germ. Molds often develop in eggs and the hyphse extend toward the air cell. To prevent this eggs must be stored in a dry place. Kossowic. found that old eggs were more susceptible to mold invasion than are fresh eggs; this is somewhat to be expected. Postolka (1916) confirmed this opinion. Marked growth of molds took place after either natural or artificial infection in the testacea but rarely in the yolk. Natural infection was caused by Penidllium glaucum and Cladosponum herbarum. Under experimental conditions almost any mold will attack and penetrate the egg. Postolka states that with even great infection with molds, no putrefaction takes place. Meat spots are caused by broken-down tissue of the hen's oviduct. In the candling process they appear as small floating bodies and are usually near the shell. Blood clots result from injuries to 458 EXAiMINATION OF EGGFi the oviduct which causes blood to bo laid down with the albumen. Bloody eggs are not used by the average housewife and they cause a great loss. Rots. Different kinds of rots are known since bacteria develops in eggs in different ways. The fertile egg is much more liable to rot than is the sterile egg. Benjamin (1915) describes the following rots: White rots are common and often called watery rots, sour, or addled eggs. They represent the first stages in bacterial decomposition. Such eggs have an enlarged cell before the candle and a mixed interior. When opened they are usually a light yellow in color and watery. Mixed rots represent a more advanced stage of decomposition and are charac- terized by thin interiors; in the open condition they give off an odor of hydrogen sulphide and sourness. The yolk is rarely intact. Black rots are eggs in which the contents are very dark and may be easily shaken about the shell. When opened the odor is much hke that of hydrogen sulphide. In appearance the contents are mixed and very watery. Kuhl (1914) from the examination of large numbers of eggs con- cluded that for trade purposes the following would be a good classifica- tion of commercial eggs: fresh eggs, those up to eight or ten days old; eggs, those not over four weeks old; cooking eggs, any offered for sale which are not spoiled. Factors Influencing Bacterial Content of Eggs. A study of this side of the question involves two points, first the entrance of bacteria before the shell is put down and, secondly the contamination after laying, due to improper handling, etc. While the first point has been studied, the data are not convincing. Mauer (1911), in quoting the work of Per- not (1909) and Conradi states that the infection of the yolks even in the normal ovary is possible. Horowitz (1902), Zimmermann (1878), Abel (1895), Cao (1908), Draer (1895), McClintock (1894), Poppe (1910) and others, maintain that the oviduct is not sterile. Contrary to this are the data of Horowitz (1902) and Rettger (1913). This stage in our knowledge points out that more work is needed on this subject. Obviously, under abnormal conditions, the oviduct may be infected. This has been found to be true with Bacillus pullorum and the white diarrhoea of chicks. Bushnell and Mauer (1914) point out that there are factors which lower the vitality of the hen and render her unable to resisi) invading bacteria. Hadley and Caldwell (1916) think that the preponderance of yolk infections indicates that bacteria are present in the ovaries of the hen. BACTERIA IN EGGF^ 459 PermeabUity of the Shell. The shell about the egg may be regarded as a semi-permeability membrane and, therefore, not resistant to bac- teria. Wittich (1851) reported the infection of eggs by molds. Wilm (1895) succeeded in infecting eggs with cholera vibrios. When the eggs were covered with a broth culture, the organisms passed through the shell in from fifteen to sixteen hours, Golokow (1896), Piorow^ski (1895), Lange (1907), and Poppe (1910) demonstrated the same thing with other bacteria both pathogenic and non-pathogenic. Opposed to this work is that of Mauer (1911), Hoppert (1912), Sachs-Mxike (1907), Zorkendorfer (1894) and Schlegel (1904). Kossowicz (1913) showed that B. proteus penetrated the shell very easily. The above indicates the ability of bacteria to penetrate the shells of eggs and this emphasizes the necessity of storing eggs under sanitary conditions. Bacteria in Egg White. Available data on this subject are con- flicting. Many of those who have found no bacteria in egg white have assumed that this comp Dnent of the egg possesses a bactericidal action. Wurtz (1890), Rettger and Sperry (1912), SchoU (1893), Horowitz (1902), Turro (1902), Laschtschenko (1909), and Riezicka (1912) pro- duce data indicating the same result. Sperry (1913) showed that cold storage eggs exhibited the same action. Bainbridge (1911), Poppe (1910), Hoppert (1912), Mauer (1911), Laschtschenko (1909) found no germicidal action. Mauer was especially interested in B, coli. Rettger and Sperry employed B, putrificus and B. edematis maligni aB the best organisms. Egg white in test tubes was heavily inoculated with the test organisms and incubated under conditions favorable for the devel- opment of the organisms. In the yolk which was treated in the same manner evident putrefaction set in while in the egg white the bacteria were destroyed for microscopic examination failed to demonstrate the presence of the cells. Hadley and Caldwell (1916) in their excellent report on eggs reported data which confirm that of Rettger and Sperry. Bacteria in the Egg Yolk. Practically all of the investigators are agreed that the yolk contains the greater number of the bacteria. Had- ley and Caldwell found 8.7 per cent of 2520 eggs infected in the yolks. They quote from other authors: Mauer is said to have found 18.1 per cent of the yolks to be infected. Bushneli and Mauer raised this to 23.7 per cent. Rettger's data is corrected to show a yolk infec- tion of 9.9 per cent covering examinations over a period of three years. Qualitative studies of the bacteria in eggs have been reported by several investigators. Rettger (1913) gives the following bacteria: 460 EXAMINATION OF EGGS Fresh Eggs. Staphylococcus, usually aurusu or albus Subtilis growths usually B. mesentericus or B. ramosus B. coli and closely related organisms Proteus group Streptococcus Micrococcus (tetragenus, etc.) Streptothrix Diptheroid bacillus Putrefactive anaerobes B. fluorescens Mold B. mocosus Mixed Number of Times Found. 74 60 43 30 14 9 6 5 5 2 4 3 2 257 Hadley and Caldwell studied 40 different strains isolated from eggs. Among them were found 11 cocci, 28 rods, and 1 spirillum. No strep- tococci were reported. They observed no member of the hemorrhagic, septicemia, intestinal, proteus, colon, enteritidis, typhoid dysentery nor diphtheria groups. Bacterium pullorum was not observed in 2520 eggs. No anaerobic bacteria were sought. That eggs in the fresh condition contain bacteria is still an open one. As pointed out by Hadley, it is difficult to explain the presence of bac- teria by contamination during plating. Hadley and Caldwell noticed different types of bacteria on the control plates than on the egg plates. The percentage of infected eggs found by different investigators is given below: Name. Rettger Rettger (10 c.c. samples) Bushnell and Mauer .... Hadley and Caldwell Mauer Examined. Per cent Infected. 3510 9.5 647 3.86 2759 23.70 2520 8.70 600 18.10 Enzymes in Eggs. RuUman (1915) found catalase in eggs which were bacteria free. The amount had no relation to the age and was about equal in the yolks and whites. In putrid eggs the amount was so large that the sample had to be diluted. Pennington and Roberston (1912) studied this question using eggs of known history. They experi- CANDLING EGGH 461 mented to determine the presence of pepsin, trypsin, lipase, catalase, and reductase. Lipase content increases from a little in a fresh egg to a large amount in a stale egg. The catalase content of a fresh egg was found to be variable. Dried Eggs. The production of desiccated eggs has become a well- estabUshed industry. Most of our knowledge with regard to its con- trol rests on the work of Pennington (1916). The methods of manu- facture and their bacteriological control are carefully gone over. From their data the following facts were established. For breaking pur- poses reputable firms use all under-sized or over-sized, dirty, cracked or Fig. 77. — Types of Candling Devices. (After Benjamin.) The one at the left is made by arranging an ordinary oil lamp inside of a pasteboard box. one at the right is a metal device which is placed on a lamp in place of the chimney. The shrunken eggs. Such eggs should be candled, broken and dried under chilled conditions. The lowest count on flaky dried eggs was 65,000 per gram and the highest count was 20,000,000 per gram. The average count for 48 samples was 3,600,000. The number of B. coli varied from to 1,000,000. About three times as many bacteria were found in the dried product as in the liquid egg. Ross (1914) examined 248 samples of dried eggs which had been stored at different temperatures. He gave the following conclusions to his work, " The desiccated egg loses a large percentage of the bacteria originally present if stored for a relatively short period. A more rapid diminution of bacteria results if the storage takes place at higher temperatures. This author states 462 EXAMINATION OF EGGS cj ^ CI fee " 03 m « '-s .-: '^ o ^ o " -M g O O . (._ ^' >» « g M S -^ «3 ^ gel s3J3o Ji^aj d Lxj *> oj ?-< -a X! o 2 ai a>*2 dt ^ ij2»-'-d1:^n S'T! ■♦^ c d -d jia d d UJ to nj CANDLING EGGS 463 that a product prepared even from spots and worse, might satisfy the ordinary bacterial tests if stored for a period of a few months. Frozen Eggs. Stiles and Bates (1912) prepared frozen eggs in the laboratory from second-grade eggs. Such a product had generally a bacterial content of 1,000,000 per gram. Frozen eggs prepared from spots, rots and blood rings gave a bacterial content of from 4,000,000 to 1,000,000,000 per gram with a relatively high content of B, coli and streptococcus. Pennington (1916) reported a careful study of this sub- ject and pointed out many features in the sanitary control of the in- dustry. Candling of Eggs. The control of the commercial egg industry by chemical or bacterial analyses is very impracticable. Pennington (1909) has pointed out that each egg is a package by itself and an analysis of it would in no way indicate the condition of the lot. This has made it necessary for those engaged in food control to look for other methods for determining the fitness of eggs for consimaption. Candling has developed to satisfy this need. Frazier (1917) states that this consists in ascertaining the character of an egg by allowing light to penetrate the contents. Different contrivances are used to do this. An electric light yields the most satisfactory results since it is constant in inten- sity and quality. Many cheap types of candling devices are used. Benjamin has described two common types. These are shown in Fig. 77. The accurate use of the candling apparatus demands some experience before reliable results may be obtained. The colored plates will be of much assistance. They are taken by permission from the bulletin by Professor Benjamin. Pennington et al. (1918) have recently pubHshed instructions for candling eggs. Those wishing a complete classified description of eggs before the candle and outside of the shell will be rewarded by reading this bulletin. Bacterial Examination of Eggs. Much discussion has passed back and forth over the methods of egg analysis. The early methods were probably faulty as Hadley has said and data from the earlier work must be accepted with some reservation. Hadley has discussed the methods of analysis and any one interested in that subject should consult his work. Stiles' Method. The eggs should be washed in a solution of bi- chloride of mercury (1/1000) or 5 per cent phenol for a few minutes after which they should be dried with sterile cotton and placed with the large end uppermost in a small beaker. The air space is then scorched with a gas flame for a few seconds. An opening should be made immediately into the cavity with sterile forceps, a sufficient 464 EXAMINATION OF EGGS T3 a O 5 I ^ I id 3 .- "=3 Ph 5 ;« « §3 ^ '^ >, -^ .^ w ^ w na i r Ha- I— 1 53 (S !^ o rr> S 2 S n-^ « <^ . o § >, g g < I -« S -S S fl e g -^^ ^ -23 g g ft -S ^ ^ ^'^ -^ *; tJ -^ ■5 § ^ g CO UJ I- < CO BACTERIAL ANALYSIS OF EGGS 465 amount of the shell being removed without rupturing the membrane below. When this is accomplished the latter should be broken with a hot platinum spatula and with a sterile pipette 0.5 c.c. of the white of the egg quickly removed and placed in the necessary Petri dishes for cultures. The remaining egg white is then decanted, leaving the un- broken yolk in the shell. With another sterile pipette, the yolk sac is ruptured and suitable portions of its contents removed for study. While this procedure guards against contamination, the breaking up of the respective layers of the egg when out of this shell is difficult and sometimes the inability to do so interferes seriously with the obtaining of quantitative results. With the eggs which have been in cold storage for considerable periods a separation of the whites and the yolks is not possible. Bushnell and Mauer's Method, The egg should be cleaned with brush and soap and immersed for ten minutes in a 1 to 500 solution of corrosive subhmate. It is transferred with sterile crucible forceps to a small conical graduate, acute pole uppermost. The corrosive subli- mate is removed and the egg dried by washing it first with alcohol and then with ether. The acute pole is scorched to kill spores, etc., that might remain. The egg is then immediately removed from the graduate by the operator's holding it by the blunt pole, turning the acute pole down. The hands of the operator should have been thor- oughly greased with vaseline to avoid contamination of the flasks by bacteria which might drop off the hands while handling the eggs. With sharp, stout forceps, which have been sterilized in the flame, a hole about | cm. in diameter is made into the acute pole. Holding the egg with the acute end down, and making the stab from below prevents contamination from above. The shell aroxmd the hole is flamed briskly and the egg is put with the acute pole upon the neck of a tall 300 c.c. Erlenmeyer flask containing 100 c.c. of sterile bouillon. The blunt end of the egg is now heated with a Bunsen flame, while a close watch is kept on the hole. The heating expands the air in the air space and this expels the contents of the egg. As soon as about half of the albu- men has run into the flask the heating is interrupted. The cotton plug is quickly removed from a sterile flask, the neck of the flask is flamed and the egg is transferred from the first to the second flask. Some- times it is necessary to invert the egg, as soon as the heating is discon- tinued, to prevent all of the albumin from running into the first flask. In this case it often happens that a little of the egg content runs down the outside of the shell, where it may become contaminated. To prevent such material from getting into the next flask, it is cemented to the 466 EXAMINATION OI? EGGS ■^ < -I Q. CO BACTERIAL EXAMINATION OF EGGS 467 shell by being heated in the flame. The expulsion of the albumin into the second flask should be done slowly and watched closely. As soon as the yolk appears in the hole the heating is inteiTupted and the egg is tilted from one side to the other to allow the rest of the albmnen to run out. In the same manner the yolk is expelled in two portions. The success of this method depends largely on the size of the hole. If this is too small, it is hard to separate the white from the yolk; if it is too large, it is difficult to expel the yolk in two separate portions. Some- times the yolk obstructs the hole before all of the albumen is obtained. If the yolk does not retract after cooUng, the egg is inverted for a moment. Often the viteUine membrane will not rupture, and the yolk will come out in one piece. This can be prevented by puncturing the membrane with a sterile platinum needle. In this manner four flasks are obtained from each egg, two of them containing albumin and two of them containing yolk. The flasks are repeatedly shaken to mix the contents well. It is of advantage to have tall flasks because the con- tents can be mixed more easily without wetting the cotton plug. Two flasks, one with albumin and one with yolk, are incubated at 38"^ C. for forty-eight hours, and the other flasks are incubated at 20° C. for five days. After this period of development subcultures on agar slants are made to determine if growth has taken place. Rettger's Method for the Bacterial Examination of Eggs. The egg to be examined is placed small end up, in an egg cup or holder. The upper half of the egg is flamed with a Bunsen burner, the cup being turned constantly so that every part of the upper half of the shell is brought into brief contact with the flame. While the egg is held in one hand the upper end of the shell is removed with sterile scissors, leaving an opening about 1 in. in diameter. The white is poured out, care being taken to prevent it from running down the side of the shell. At this point the edge of the opening is flamed after which the entire yolk is poured out into a wide-mouthed flask, or better still, a large tube especially designed for this work by Rettger. Previous to intro- ducing the yolk, definite amounts of nutrient bouillon are placed in the flasks (25-50 c.c.) or tubes (25 c.c.) which are plugged with cotton and sterilized. The yolk and bouillon should be thoroughly mixed. In case the test tube is used this process is greatly facilitated by the pres- ence of a small glass rod about 1| in. in length. The tubes should then be placed in the 37*^ C. incubator and after seventy-two hours' incuba- tion agar streaks made from them. These should be incubated for from twenty-four to forty-eight hours. 468 EXAMINATION OF EGGS to LU H < BACTERIAL ANALYSIS OF EGGS 469 Hadley and Caldwell's Method. The egg shell should be thor- oughly disinfected before any opening is made. This may be accom- pHshed by first washing the egg, if soiled, with soap and water and a wad of cotton, then immersing for ten minutes in 1 : 5000 mercuric chloride solution, containing either citric acid or ammonium chloride to increase the penetrating power. The egg may then be plunged into 95 per cent alcohol, removed, drained and ignited to dry the sur- face. These operations can be carried on in a suitably constructed wire rack in which the eggs can await examination, being meanwhile protected from air contamination. Next, as a final precaution, the egg should be well fiamed at the end opposite the air space until a very thin layer (1-2 mm.) of the albumen lying close to the shell is coagulated. The amount of heating required can be ascertained by experience. After this, by means of sterile forceps, a hole about 2 cm. in diameter is made at the flamed end and the white poured into a tube or flask containing 25 c.c. of the desired medium. Next, the opening in the shell is enlarged to about 3 cm. The yolk is gently allowed to run out of the shell onto a circle of sterile filter paper (kept in a small pile under the bell jar). The paper is then so inclined that the yolk rolls about until the white is entirely removed by the paper, after which the yolk is rolled off the edge of the paper into a tube or flask containing at least 25 c.c. of culture medium, an amount some- what greater than the volume of the average yolk. Great care must, of course, b» taken not to rupture the yolk membrane before the yolk is poured into the tube. The yolk may then be broken by means of a sterile glass rod and mixed with the broth. The preparation is now ready for incubation which may be carried on for forty-eight hours at 37° C, followed by forty-eight hours at 20° C. The white may be mixed with the broth and grown in the same manner. At the end of the period of incubation the tubes should be again mixed by rotation to insure distribution of the bacteria, and a small amount on a straight needle transferred from each to ordinary tubes contain- ing broth. The broth tubes are incubated at 37° and at 20° C, as were the original tubes and examined for growth at the end of four days. In- case growth appears plate cultures are made.* * In addition to the points of technique mentioned above, minor details can per- haps best be worked out by the individual investigator. For instance, cotton plugs in the large tubes are unwieldy. They may be replaced by close-fitting glass covers, coming weU down over the tops of the egg tubes. The breaking of the yolk and the mixing with the broth may be accomplished by means of broken glass placed in the tubes with the broth, rather than by means of glass rods, a procedure which may favor contamination from the air. Wire cages or racks of various sizes and 470 EXAPvIlNATION OF EGGS •^ w o . *^ " ft o ce 13 ^ H J -^ I >: . W ^ -^ ^ ^ ^ ^ 43 -a '^ s 5 a <^ !25 o £-^'^^ •"""^ o •• ,^ ^ -rf< '^ d 5 3+= ^ .^ o .2 ° ° S rTi iM ^ ^ O ^ O (3) P_| O c! "S C aj "n fH fTJ •■ ■!-» CO ~ 1. ' -*-> O P t-i 0) S 'o ^ £ > +» 13 < BIBLIOGRAPHY 471 It should be borne in mind that the recommendations made above are suitable only for fresh eggs in which it is desired to examine the yolk and white separately. In old market eggs, or preserved eggs in which the yolk is weakened, and in decomposed eggs in which the yolk and white have become mixed, different methods would be required; and here, as in the bacteriological examination of other egg products, the technique must be evolved ih accordance with the particular aim of the investigation. BIBLIOGRAPHY Abel, R. and Draee, A. 1895. Das Huhnerei als Kulturraedium fur Cholera Vibrionen. Zeit. f. Hyg., 19, 61. Artault, T. 1893. Recherches bacteriologiques, mycologiques, zoologiques et m6dicales sur Toeuf de poule. These Paris. Cent. Bakt., 1894, 16, 461. Artault, T. 1893. Le bacille pyocyanique dans un oeuf de poule. Cpmpt. Rend. soc. bioL, series 9*, 5, 78. Artaxjlt, T. 1895. Tuberculose provoqu^e chez lapins par des injections de contenu d'oeufs de poule. Comp. rend. soc. bioL ser. 10, 2, 683. Bainbridge, F. a. 1911. The Action of Certain Bacteria on Proteins, Journal of Hygiene,. 11, 341. Barthelmy, M. a. De rincubation des oeufs d'une poule atteinte du cholera des poules. Conxpt. Rend. Acad. Sciences, 96, 1322. Bechamp, a. and Eustache, G. 1877. Sur Falteration des oeufs provoqu^e par des moississures venus de Fext^rieur. Comp. Rend. Acad. Sci., S^, 854. Beckwith, T. D. and Horton, G. D. 1914. Is Poor Hatching of Normal Eggs Due to the Presence of Microorganisms within the Egg? Science N. S., 40, 240. Benjamin, E. D. 1914. The Interior Quahty of Market Eggs. New York Agricultural Exp. Station Bull. 353. Cornell Univ., Ithaca. Bornaxjd, M. 1914. Recherches sur les parasites vegetaux des oeufs des poules. Cent. Bakt. Ref., 63, 43. BusHNELL, L. D. and Mauer, 0. 1914. Some Factors Influencing the Bacterial Content and Keeping Quality of Eggs. Kansas Ag. Ex. Sta. Bull. 201. Cao, G. 1908. Sur la permeabilita delle uova ai microorganismi. BuU. Inst. Pasteur, 6, 472. shapes may be used for the immersion of the eggs in the disinfecting solution, in the alcohol and during ignition. Methods for the detection of anaerobic microorganisms probably do not present unusual difficulties. The method of Hesse, involving the use of a layer of sterile olive or paraffin oil over the surface of the mixture is probably the best. In this case samples may be withdrawn by means of a sterile pipette. 472 EXAMINATION OF EGGS 5 S? ^<^«?^a2 So lJ a oo'^S— « "tju UJ H < Q. (M (0 10 0) BIBLIOGRAPHY 473 DoNiTZ. 1895. Ueber das Verhalten der Choleravibrionen Hiilmerei. Zeit. Hyg.,20,3L Frazier, D. J. 1917. Candling Eggs. American Food Journal, 12, 39. Gayon, U. 1873. Sur Falt^ration spontan^e des oeufs. Comp. Rend. Acad. Sci., 76, 232. Gayon, U. 1875. Recherches sur Talt^ration spontan^e des oeufs. Ann. Sci. I'Ecole Norm. Sup^rieur, Series 2, 4, 205. GoLOKOW. 1896. Ueber das Eindringen von Cholera Vibrionen im Huhnerei. Baumgarten's Jahresbericht, 12, 583. Grigoeiew, a. W. 1894. Vergleichende Studien tiber die Zersetzung des Htihnereiweisses durch Vibrionen. Arch. Hyg., 21, 142. Hadley, p. B. and Caldwell, D. W. 1916. The Bacterial Infection of Eggs. Rhode Island Ag. Exp. Sta. Bull. 164. Hammerl, H. 1894. Ueber die im rohen Eiren durch das Wachstum von Cholera Vibrionen hervor gerufen Veranderung. Zeit. Hyg., 18, 153. HopPERT, M. J. Factors Relating to the Bacteria Content of Eggs. Thesis University of Wisconsin. Horowitz, A. 1902, Contribution k F6tude des moyens de defense de Forgan- isms contre Finvasion microbienne; recherche sur Foviducte de la poule de le blanc d^oeuf . Th^se Paris. Baumgarten's Jahresbericht. 19, 984. HuBPPE, F. 1888. Ueber die Verwendung von Eiern zn Kulturwecken. Cent. Bakt., 2, 80. Kempner, W. 1894. Ueber Schwefelwasserstoffbildung der Choleravibrionen im Huhnerei. Arch. Hyg., 21, 317. Kossowicz, a. 1913. Die Zersetzung und Haltbarmachung der Eier. Wies- baden. J. F. Bergman. Kuhl, H. 1914. The Relation between Spoiling and the Age of Eggs. Hyg. Rundschau, 24, 253-259. Lamson, G. H. 1909. Infection and Preservation of Eggs. Storrs Ag. Exp. Sta. Bull. 55. Lange, R. 1907. Ueber das Eindringen von Bakterien in das Huhnerei durch die Eischale. Arch. Hyg., 62, 201. Laschtschenko, p. 1907. Ueber die keimtotende und entwicklungshemmende Wirkung von Htihnereiweiss. Zeit. Hyg., 64, 419. LiNNOssiER ET Lemoine. 1910. Sur la toxicite normale des aliments albu- minoides frais. Influence de la conservation. Comp. Rend. soc. bioL, 68, 671. McClintock. 1894. Bacteria in Eggs. Modern Medicine and Bact. Rev., 3, 144. Matter, 0. 1911. Bacteriological Studies on Eggs. Kansas Ag. Exp. Sta. Bull. 180. Menini, G. 1908. Richerche intomo alia penetrazione dei bacteria nelle uova di gallina. Lo. Sperimentale, 61, 711. Mosler, F. 1864. Mykologische Studien am Huhnerei. Arch. Path. Anat. Physiol Virchow., 29, 510. 474 EXAMINATION OF EGGS >> n 0) s bO ps (ij X! m S XI «JJ 1^ fi >> m c8 u 03 o O 01 o O 0) pS o bO >> (-1 ^ <1 3 s ;3 1—1 c3 o jQ ■^ 'c3 >> Pi na" w >!. t? cu o a? 13 CI o k; c o w < E3 O >> o o < |2 d c3 a fin fl a ^ O O :2 0) -(-3 o * bO bO > cu 1 1 O bO cu o • +3 o 03 o U OS 0) ■4-> o 00 Cu o o o rH :2; o M 0) o t-t 15.00 EXAMINATION OF CANNED FOODS 511 Baker states that the analyses of probably 100 samples of gas from sound cans has never shown any oxygen. He attributes this to the oxidation of tin and iron salts, and combination with nascent hydrogen. Springers. Bigelow (1914) defines a springer as a ^' can whose ends are more or less bulged, owing to pressure from hydrogen generated as a result of the chemical action of the contents on the metal of the con- tainer or because the can was overfilled or insufficiently exhausted." Cans with bulged ends may be caused by expansion due to a rise in temperature. These will soon return to normal shape when the tem- perature is lowered. Baker (1912) states that the gases in the head spaces of springers are never more than three; carbon dioxide, nitrogen and hydrogen. Often hydrogen is absent and oxygen is rarely found. Flat Sour, This is a general term and may be used to cover many abnormahties of canned goods. Flat sours contain no gas and since there is nothing to indicate any abnormal condition when looking at a can, their discovery is delayed until the can is opened. Examination of Canned Foobs The examination of canned foods is accompanied with danger unless the examiner has had experience in interpreting his data, both chem- ical and bacteriological. Bigelow (1917) has pointed out this side of the question very strikingly. The methods which are used are quite similar to those used in other branches of bacteriology. The objects of the examination may be varied. Quite often it is to determine the sterility of certain foods in order to check up the process of manufacture; or the quality of raw materials may be the object. The examination of canned foods presents the same difficulty that Pennington has men- tioned with regard to the examination of eggs: the examination of one can or one egg may not yield information with regard to other cans or eggs. Each must be regarded as a sealed package and when once opened may be brought back to the original condition with a Httle difficulty. The bacteriological examination of canned foods has been fully discussed by Bitting and Bitting (1917). Some of the discussion below has been taken from that paper. The first step in the examination is to observe the appearance of the can. Rusty cans suggest the possibility of perforations. The cans should be without dents and with slightly concave or flat ends. In certain cases bulged ends may be due to springers, which will go down 512 FOOD PRESERVATION a little after the can is cooled. Bitting points out that incubation at 37° C. and at from 50 to 55° C. for fifteen days should precede the bac- terial examination. The second step, is the opening of the can, which must be carried out under very carefully controlled conditions. The can should be thoroughly steriUzed about the place for opening either by flaming or covering with alcohol and allowing it to burn off. The can may be opened by cutting around the top with a sterile can opener or a hole put through the top by a sterile awl. The contents for examination may then be removed by means of a sterile loop or a sterile pipette. When the hole is punched into the can a flame should be played on the spot so that no bacteria will be sucked into the can with the air. The can should be entirely opened, later as soon as possible, in order to deter- mine the appearance of the contents. The third step, is concerned with the actual microscopical examina- tion. This should be direct as well as cultural. The direct methods of examination have been suflaciently discussed elsewhere in this book. The cultural methods require special media such as corn, agar, tomato bouillon, etc., depending upon the substances which are being exam- ined. Such media create for the organisms which are searched for, an environment as much as possible like the normal. Both aerobic and anaerobic methods of culture should be used. It is often advisable to incubate the cultures in a vacuum, to come more nearly to the conditions inside of the can. Organisms in Canned Foods, These have not received very extended study. Much of our knowledge must come from analogy and there is some danger in reasoning thus. In relatively few instances has time been taken to identify the various varieties of microorganisms. Bur- gess (1912) isolated and identified about 12 organisms from " flat sour ^' corn. Among others, he found B, coli, B. acidi lacticij Ps. syncyaneaj B. megatherium and J5. vulgatus. The first work on the bacteriology of canned foods in this country was done by Russell (1895). He exam- ined swelled canned peas. He isolated two organisms, one of which was a copious gas producer. Prescott (and Underwood, 1897, 1898, 1900), gave this subject quite a little attention. They did much to estabhsh the close relationship of bacteria to the spoilage of canned foods. The importance of the canned food industry should stimulate the examination of these foods. The author has found spore-forming facultative anaerobes to be common. In a few cases vegetative cells have been isolated. In one case mushrooms packed in brine of the following composition — 5 lbs. EXAMINATION OF CANNED GOODS 513 salt, 1 lb. citric acid, 12| gals, of water, developed swelled cans. Bac- teria with the following group numbers were isolated. Bacillus 111.2232033 Bacillus 111.2232032 Bacillus 111.2222022 Bacillus 222.1112031 Bacillus 222. 1212032 The growth of such organisms in the finished products probably indicates insufficient sterilization. The gas-forming bacteria as indi- cated by the above group numbers are members of the colon group. These, undoubtedly, got onto the mushrooms during growth, since they are usually grown on heavily manured areas. Examination of Cans for Leaks. Several ingenious methods have been proposed. 1. One which is widely used is to tap the end of the can. A sound can with a vacuum will give a characteristic ring while one which is a leaker with no vacuum will give a dead sound. The examination of a few cans of both types will show the difference. 2. The suspected cans may also be examined for leaks by plunging them into hot water. If leaks are present, bubbles will be given off. 3. The Meade tester may be used. This subjects the suspected can under water to a vacuum. If the can is a leaker, a stream of bubbles will be given off from it. Storage of Food in Tin Cans. It has become almost an instinct for people to immediately take food out of a tin can after it has been opened. When the housewife is asked why she does this, various reasons are given. One which has often been given is that ^' ptomaine " poisoning will result. Bigelow (1918) has given some definite information on this subject. He regards the storage of food in the tin cans after part has been removed as no more dangerous than storage in containers made of other material, since it is becoming more and more known that food '' poisoning " is really an infection. Bigelow determined the tin content in progressive samples from the same tin of food and it is inter- esting to note that after fermentation had set in (pineapple) larger amounts of tin were dissolved. Bigelow considers that the only reasons which make it advisable to remove food from opened cans are appear- ance and economy of space secured by putting the food into a smaller container, 514 FOOD PKESEEVATION Tomato Products The canning of tomato products is often beset with many pitfalls for the canner. The chemical constitution of the tomato renders it liable to attack from microorganisms. The following table is prepared from some data reported by Bigelow and Fitzgerald (1914). Sample No. 893 701 1048 1051 Table LI COMPOSITION OF TOMATO PULP (Bigelow and Fitzgerald) Description of Sample. Peeled tomatoes not concentrated Trimming stock not concentrated Whole tomatoes, Whole tomatoes. Specific Gravity. 68° F_ 68 203° F. 203 Total Solids. Per Cent. 4.47 4.53 5.57 6.55 Salt Free Solids.^ Per Cent. 4.42 5.53 6.43 Insoluble Solids Per Cent 0.56 0.50 0.75 Salt Free Soluble Solids. Per Cent. 3.86 3.90 5.68 Sample No, 893 701 1048 1051 Description of Sample. Peeled tomatoes not concentrated Trimming stock not concentrated , Whole tomatoes. Whole tomatoes. Sugar. Per Cent. 2.26 2.36 3.55 3.96 Acid as Citric. Per Cent, 0.40 0.30 0.35 0.37 Salt. Per Cent. 0.05 0.10 0.04 0.12 Undeter- mined. Per Cent 1.20 1.04 0.51 1.35 Salt Per Cent of Total Solids. 1.2 0.7 1.80 Insoluble Solids. Per Cent of Total Solids. 12.50 11.00 20.10 11.50 Ketchup. This is one of the most important tomato products and one which has received much attention with regard to the enforce- ment of the Food and Drugs Act. It is made in the following way: Clean, ripe tomatoes are thoroughly washed and pulped. The skins, seeds, etc., are removed after which the pulp is concentrated. To this are then added the condiments to taste. The ketchup is then sieved and bottled. After the bottles are sealed, they may be processed to insure sterility. Much trouble has been experienced in the control of this industry from the standpoint of the Food and Drugs Act. Undoubtedly, in Yearbook U. S. Dept. of Agriculture, 1911. PLATE 9 N Fig. 1. — Spores and Fragments of Filaments of Mold from Decaying Sweet Pepper (X150) The same or an allied species is one cause of " dry rot " in tomatoes. Fig. 2. — Yeasts and Spherical Bacteria from Decaying Tomatoes (X500). The oval bodies are the yeasts, some in budding stage; the bacteria appear as small spheres, or pairs of spheres. ■-..'%. Fig. 3. — Rod-shaped Bacteria from Tomato Pulp, Common in Bad Ketchups (X500). To face page 515. EXAMINATION OF CANNED PRODUCTS 515 former times, many unscrupulous manufacturers used spoiled fruit, and by-products from the tomato canning industry for making ketchup Satisfactory methods for separatmg good and bad ketchups have been hard to find. In general, a bad or low-grade ketchup is one which has been made from decayed or partly decayed fruit. The methods which are now used arc microscopical and center around determining the num- bers of bacteria, yeasts and molds. Bitting (1909) used superficial methods for studying spoilage of tomato ketchup. He found that ketchup prepaied from whole ripe stock had a low content of microor- ganisms and in that prepared from decayed fruit large numbeis of micro- orgamsms were present. Howard (1911) proposed a method for the examination of ketchup which has been in use since then probably because no one has proposed a better one to leplace it. This method is based on examining the tomato product for yeasts, molds and bacteria, and it is thus assumed that these microorganisms indicate that decayed fruit was used or that the method of manufacture is faulty. This latter possibility was found to be the case m an instance quoted by Howard (1917). A manufac- turer was using ripe fresh tomatoes and still secured high counts in the final product. Howard's Method for the Microanalysis of Tomato Products AND Interpretation op the Results Apparatus Required. The outfit used is as follows : A good compound microscope giving magnifications of approyi- mately 90, 180, and 500 diameters. This is accomplished by the use of a 16 mm. (two-thirds of an inch) objective and an 8 mm. (one-third of an inch) objective, together with a medium (X6 compensating) and also a high-power ocular (X18 compensating). A Thoma-Zeiss blood- counting cell,* a 50-c.c. graduated cylinder, and ordinary slides and cover glasses complete the apparatus required. It is impractical to use objectives of a higher power 'than those mentioned, because of their short working distance, which makes their use with the counting cell inpossible. Estimatioii of Molds. A drop of the product to be examined is placed on a microscope slide and a cover glass is placed over it and * This IS a cell named after the designer of the f onn of rulings used, and consists of a slide with a disk ruled m ^^ mm squares, so arranged that when the cover is m place the film of liquid under examination is -^ mm deep They were originally intended for counting corpuscles in the blood and are obtainable from practically all manufacturers of microscopic accessories. 516 FOOD PRESERVATION pressed down till a film of the product about 0.1 mm. thick is obtained. After some experience this can be done fanly well. A jSlm much thicker than this is too dense to be examined successfully, while a much thinner film necessitates pressing the liquids out, which gives a very uneven- appearing preparation. When a satisfactory mount has been obtained, it is placed under the microscope and examined. The power used is about 90 diameters, and such that the area of substance actually exam- ined m each field of view is approximately 1.5 sq. mm. A field IS examined for the presence or absence of mold filaments, the result noted, and the slide moved so as to bring an entirely new field into view. This is repeated till approximately 50 fields have been examined, and the percentage of fields showing molds present is then calculated. Our experience has demonstrated that for home- made ketchups this is practically zero, and with some manufactured ketchup it is as low as from 2 to 5 per cent, while for carelessly made products it may be 100 per cent; that is, every field would show the presence of mold. Investigations under factory conditions clearly indicate that with only reasonable care the proportion of fields having molds can be kept below 25 per cent. A specimen in which 60 per cent of the fields have molds is in more than twice as bad a condition as one containing 30 per cent. After the percentage reaches 30 to 40 per cent it will be found that some of the fields frequently have more than one filament or clump of mold, and the number of such fragments might be counted, but in this laboratory this usually is not done. A Thoma-Zeiss counting cell with a center disk of 0.75 in. instead of 0.25 in., as usually furnished, would give a regular depth of hquid and would be more exact than the method described, but this must be specially manufactured, not being fisted in any of the catalogues of microscopic suppHes, and the method as given is suflBciently accurate for the purpose. When the number of fragments of mold per cubic centimeter is estimated, it has been found to range from virtually zero to over 20,000. There is no excuse for a manufac- turer allowing such conditions to prevail that his ketchup shows more than 2000 per cubic centimeter, while some manufacturers by careful handling hold it down to 150, Estimation, of Yeasts and Spores. Though the spores referred to are those coming from molds and correspond to seeds in more highly developed plants, it is frequently very difficult to differentiate some of them with certainty from some yeasts without making cultures, which is obviously impossible in a product that has been sterilized by heat. For this reason the yeasts and spores have been reported together, and Yearbook U. S. Dept. of Agricultuie, 1911. PLATE 10 Fig. 1. — Normal Tomato Ketchup, Showing Cells and Granular Contents (X200). Fig. 2.— Mold Filament from Ketchup Made 'rom Partially Decayed Stock (X150). To face page 516. EXAMINATION OF TOMATO PRODUCTS 517 if there seemed to be a larger percentage of the latter, mention was made of that fact. To make a comit 10 c.c. of the product is thoroughly mixed with 20 c.c. of water, and, after being allowed to rest for a moment to permit the very coarsest particles to settle out, a small drop is placed on the central disk of the Thoma-Zeiss counting cell and then covered with a glass. Care must be exercised to have the slide perfectly clean, so that, when the cover glass is put in place, a series of Newton's rings ^ results from the perfect contact of the glass surfaces; and furthermore, the drop should be of such size as not to overrun the moat around the central disk and creep in xmderneath the cover glass, thus interfering with the contact. With the magnification of 180, it has been the practice in this laboratory to count the number of yeasts and spores on one-half of the ruled squares on the disk. With the dilution used this calculates back to a volume equal to one-sixtieth of a cubic millimeter in the original sample, and reports are made on that basis rather than on the number in a cubic centimeter, because the former number is more readily grasped by the mind and affords a simpler notation. To obtain the mmibers per cubic centimeter the count made is simply multiplied by 60,000. It has been found in practice that the number of yeasts and spores varies, for one-sixtieth of a cubic millimeter, from practically none in homemade and first-class commercial ketchups up to 100 or 200, and in one sample the number was as high as 1200. Laboratory experiments show that, when the number of yeasts in raw pulp reaches from 30 to 35 in one-sixtieth of a cubic millimeter the spoilage may frequently be detectable by an expert by odor or taste, and from experiments made under proper factory conditions, it seems perfectly feasible to keep the number in commercial ketchups below 25. Estimation of Bacteria. The bacteria are estimated from the same mounted sample as that used for the yeasts and spores. A power of about 500, obtained by using a high-power ocular, is employed in this case, and because of the greater number present a smaller area is counted over. Usually the number in several areas, each consisting of five of the small-sized squares, is counted and the number of organisms per cubic centimeter is calculated by multiplying the average number in these areas by 2,400,000. Thus far it has proved impracticable to count the micrococci present, as they are likely to be confused with other bodies * These are rainbow-colored rings produced at the point of contact when polished plates of glass are pressed against each other. 518 FOOD PEESERVATION frequently present in such products, such as particles of clay, etc. A comparison of this method with the ordinary cultural methods on samples in which the organisms had not been killed has almost invari- ably shown that the one used gives too low instead of too high results. In some cases it was found to give not more than one-third of the entire number present. The estimates of the laboratory on this point may, therefore, be considered, very conservative. As regards the limits which may be expected in the examination of ketchups for bacteria, it might be stated that some manufactured samples as well as good, clean products made by household methods, have been examined and the count found to be so low when estimated by this method that the numbers present were reported as negligible. In other words, it was found that for the areas counted over the number of bacteria averaged less than one — that is, less than 2,400,000 per cubic centimeter. It is unusual, however, for the final number per cubic centimeter to be less than from 2,000,000 to 10,000,000 organisms. Contrasted with this number as a minimum, it has been found that the number has occasionally exceeded 300,000,000 per cubic centimeter. Such a number as this would indicate extremely bad conditions and carelessness in handling, as the studies of factory conditions have shown that there is little excuse for the number ever exceeding 25,000,000 per cubic centimeter. While experiments have also shown that although the effect produced by the bacteria on the product varies with different species, it is true that their presence can frequently be detected in the raw pulp by odor or taste when the number exceeds 25,000,000 per cubic centimeter and sometimes when the count is as low as 10,000,000. To one who has not been initiated into the mysteries of the micro- scope the presence of such a number of bacteria in a food product seems inexcusable. It must be remembered in this connection that the most of these are probably nonpathogenic forms, and many occur naturally on the skins of the fruits. It does not seem just to set a standard so high as to virtually prohibit the manufacture of the product under commercial conditions; rather the idea is to set a limit that the manufacturer can attain if due care is exercised and which will insure a cleanly product. It is, however, perfectly possible to make a cleanly, wholesome product commercially even though the number of bacteria exceed that in the homemade article. The allowable limits for the bacterial content of tomato pulp vary according to the concentration. The number, however, should be low enough so that when the amount of concentrating necessary for its conversion into ketchup has been accomplished the final product TOMATO PRODUCTS 519 will still be within permissible limits (25,000,000 per cubic centimeter). Thus, for a pulp which must be concentrated one-half the bacterial counts should not exceed about half the limits stated above for the ketchup itself, i.e., it should not be more than 12,500,000 per cubic centimeter. The same general rule should also apply to the content of molds and of yeasts. To insure a sound product, free from decay or any filthy material, many factors must be carefully watched, for not infrequently over- sight in one particular has been found to have undone the good effects of the care exercised in all other ways. Thus it is possible for the washing of the fruit to be ideal and the sorting out or removing of the decayed portions beyond criticism, and yet a delay in making up the pulp into the final product may allow an amount of decomposi- tion to occur which offsets the care previously exercised. It has been a matter of surprise to some manufacturers to find with what rapidity some of these organisms increase. In one factory where this point was tested, the bacterial content in a batch of tomato trimming juice was found to be about 7,000,000 per cubic centimeter when taken from the peehng tables, and after standing at room temperature for five hours it had increased to 84,000,000. This was a twelvefold increase in a length of time which was less than half the working day for some of the factories visited. At the end of five days the number had increased to nearly 3,000,000,000 per cubic centimeter. Thus it is seen that delay in manufacture is very liable to result disastrously. Such facts as these serve to emphasize the great importance of absolute cleanliness in every detail about factories of this kind. Dirty floors and ceilings and apparatus left with residues of tomato product clinging to them are most fruitful sources for the contamination of new batches of the product. To clean such an establishment properly it is almost imperative that machinery and woodwork be washed by means of live steam used lavishly at frequent intervals. To leave buckets, tables, conveyors, or any other part of the equipment or floors overnight without cleansing them, as was the practice in some fac- tories, is reprehensible and tends to contaminate the product and lead to spoilage and loss. There are some objections to this method. Prescott, Burrage and Philbrick (1917) have stated that the method is grossly inadequate. They pointed out the following facts which render the method open .to criticism. 1. Low magnification for examining the unstained mounts. 2. No account of coccus forms is taken. 3. Personal equation is given too much importance. 4. Directions are too indefinite. Bitting and 520 FOOD PRESERVATION Bitting (1915) have also stated that the method is unsatisfactory. Indefinite directions are especially mentioned. Vincent (1918) has maintained the above objections. Bitting and Bitting have pointed out the practical impossibility of concentrating tomato pulp to the consistence of tomato paste and having it pass the standard of 25,000,- 000. The inadequacies of the procedure are soon apparent to anyone who attempted to use the method. The difficulty of distinguishing between particles of organic matter and bacteria makes'the results very uncertain. These criticisms caused a restatement of the procedure. More definite details have been given by Howard (1917) as follows: Howard's Revised Method for the Microanalysis ojf Tomato Products Apparatus, (a) Compound Microscope. Equipped with apochro- matic objectives and compensating oculars, giving magnifications of approximately 90, 180 and 500 diameters. These magnifications can be obtained by the use of 16 and 8 mm. Zeiss apochromatic objectives with X6 and X18 Zeiss compensating oculars, or their equivalents, such as the Spencer 16 and 8 mm. apochromatic objectives with Spencer XIO and X20 compensating oculars, the draw tube of the microscope being adjusted as directed below. (&) Thoma-Zeiss Blood-counting Cell."^ (c) Howard Mounting Cell. Constructed like a blood-counting cell but with the inner disk (which need not be ruled) about 19 mm. in diameter. Molds (Tentative). Clean the Howard cell so that the Newton's rings are produced between the slide and the cover glass. Remove the cover and place by means of a knife blade or scalpel, a small drop of the sample of the central disk; spread the drop evenly over the disk and cover with the cover glass so as to give an even spread to the material. It is of the utmost importance that the drop be mixed thoroughly and spread evenly; otherwise the insoluble matter and the molds are most abundant at the center of the drop. Squeezing out of the more liquid portions around the margin must be avoided. In a satisfactory mount, Newton's rings should be apparent when finally mounted and none of the liquid should be drawn across the moat and under the cover glass. « * Comment by the authors. In using these cells the plane parallel cover glasses furnished with them should be used instead of the ordinary microscope cover glasses. Since the latter are subject to curvatures that introduce errors in the thickness of the mounts. YEASTS AND SPORES 521 Place the slide under the microscope and examine with a magnifi- cation of about 90 diameters and with such adjustment that each field of view represents approximately 1.5 sq. nrni. of area on the mount.* This area is of vital importance and may be obtained by adjusting the draw tube to the proper length as determined by actual measure- ment of the field, a 16 mm. Zeiss apochromatic objective with a Zeiss X6 compensating ocular or a Spencer 16 mm. apochromatic objective with a Spencer XIO ocular, or their equivalents, being used to obtain the proper magnification. Observe each field as to the presence or absence of mold filaments and note the result as positive or negative. Examine at least 50 fields, prepared from two or more mounts. No field should be considered pos- itive unless the aggregate length of the filaments present exceeds approx- imately one-sixth of the diameter of the field. Calculate the propor- tion of positive fields from the results of the examination of all the observed fields and report as percentage of fields containing mold filaments. Yeasts and Spores (Tentative). Fill a graduated cyHnder with water to the 20 c.c. mark, and then add the sample till the level of the mixture reaches the 30 cc. mark. Close the graduate, or pour the contents into an Erlenmeyer flask, and shake the mixture vigorotisly fifteen to twenty seconds. To facilitate thorough mixing the mixture should not fill more than three-fourths of the container in which the shaking is performed. For tomato sauce or pastes or products running very high in the number of organisms, or of heavy consistency, 80 c.c. of water should be used with 10 c.c. or 10 gms. of the sample. In the case of exceptionally thick or dry pastes it may be necessaiy to make an even greater dilution. Pour the mixture into a beaker. Thoroughly clean the Thoma-Zeiss counting* cell so as to give good Newton's rings. Stir thoroughly the contents of the beaker with a scalpel or knife blade, and then, after allowing to stand three to five seconds, remove a small drop and place upon the central disk of the Thoma-Zeiss counting cell and cover imme- diately with the cover glass, observing the same precautions in mounting the sample as given above. Allow the slide to stand not less than ten minutes before beginning to make the count. Make the count * Comment by the authors. In order to have an area of 1.5 sq. mm. the diameter of the microscopic field should be 1.382 mm. This is determined by using a stage micrometer and adjusting the length of the microscope draw tube. Obviously after the proper draw tube length has been secured the adjustment should be noted and always used in making the mold counts. 522 FOOD PRESERVATION with a magnification of about 180 to obtain which the following com- binations, or their equivalents, should be employed: 8 mm. Zeiss apochromatic objective with X6 Zeiss compensating ocular, or an 8 mm. Spencer apochromatic objective with XlO Spencer compensating ocular with draw tube not extended. Count the number of yeasts and spores * on one-half of the ruled squares on the disk (this amounts to counting the number in eight of the blocks, each of which contains twenty-five of the small ruled squares). The total number thus' 'obtained equals the number of organisms in 1/60 c.nim. if a dilution of one part of the sample with two parts of water is used. If a dilution of one part of the sample with eight parts of water is used the number must be multiplied by three. In making the counts the analyst should avoid counting an organism twice when it rests on a boundary line between two adjacent squares. Bacteria (Tentative). Estimate the bacteria from the mounted sample used above, but allow the sample to stand not less than fifteen minutes after mounting before counting, f Employ a magnification of about 500, which may be obtained by the use of an 8 mm. Zeiss apochro- matic objective with an X18 Zeiss compensating ocular with draw tube not extended, or an 8 mm. Spencer apochromatic objective with an X20 Speitcer compensating ocular with a tube length of 190, or their equiva- lents. Count and record the number of bacteria in a small area consist- ing of five of the small-sized squares. Move the slide to another por- tion of the field and count the number on another similar area. Count five such areas, preferably one from near each corner of the ruled portion of the slide and one from near the center. Determine the average num- ber of bacteria per area and multiply by 2,400,000, which gives the num- ber of bacteria per cubic centimeter. If a dilution of one part of the sample with eight parts of water instead of one part of the sample with two parts of water is used in making up the sample, then the total count obtained as above must be multiplied by 7,200,000. Omit the micro- cocci type of bacteria in making the count. Explanation of Calculations. Fig. 80 has been prepared to make somewhat clearer the explanation of the areas denoting the yeast and spore and bacterial counts. The light lines in the figure show the arrangement of rulings on the entire slide. The squares (A, A^ etc.), and rectangles (B, B, etc.) designated in the figure by the heavy lines indicate the portions used for the yeast and spore and for the bacterial * Comment by authors. The organisms counted as '* yeasts and spores " are the yeast cells, and yeast and mold spores, not bacteria spores. j- The estimation of yeast, molds and bacteria are made from the same motmt. CALCULATIONS 523 counts, respectively. The eight large squares, A, A, etc., are tKe squares used for yeast and spore counts. Each of these squares has twenty-five of the small squares. The sum of the organisms counted in the eight squares marked A, A, etc., is the number in 1/60 c.mm. if a dilution of one part of product to two parts of water is used. Yeast and Spore Count The ruled square on the slide is 1 mm. on each side and the cell is 1/10 mm. deep. The volume of the ruled part is, therefore 1/10 c.mm. The ruled area is divided into sixteen large squares and the number of organisms is counted in eight of these, which is equivalent to 1/2 of 1/10 c.mm., or 1/20 c.mm. If a dilution of one A / \— —A . A A — 1/ A _ — 4r- .- __ „_ — &J; — ^ - , — B--r" P ^ - u-."3r . hf-- ■-•"—• -- -"— — ^a !■' _ A -— / \~~— —A L A n, f^'i£" ^r-"" - _ - , B-'' \ " '^ -B Fig. 80. — ^Diagram of Thoma Rulings. (After Howard, 1917.) One millimeter divided by lines into twenty spaces in each direction each space equalling ^ mm. To facilitate counting, every fifth space ih subdivided by a line through the middle. part of the product to two parts of water is used 1/3 of 1/20 c.mm.; or 1/60 c.mm. as representing the actual amount of original stock in which organisms are counted, is obtained. Bacterial Count. The rectangles, 5, 5, etc., each including five of the smallest squares, represent the areas used in making the bacterial count. Similar rectangles of equal area might be selected, the object being to count five such areas well distributed over the ruled portion of the sUde. The average number of bacteria counted on five rectangles, such as jB, 5, etc., multiplied by 2.4 million, equals the number of 524 FOOD PEESERVATION bacteria per cubic centimeter. In calculating the bacteria, it is observed that there aie 400(20X20) small squares on the slide. The number of bacteria in rectangles (B, B, etc.), each containing five of these small squares, are counted and an average made. This average represents the bacteria in 1/80 of the total ruled area. Since the cell is 1/10 mm. deep, the volume represented by the organisms counted is 1/80X1/10 or 1/800 c.mm. With the usual dilution of one part of product to two parts of water the actual volume in which the number of organisms is determined is 1/3 of 1/800 cm. or 1/2400 c.mm. or 1/2,400,000 c.c. Howard (1917) made a study of the relation between the number of yeasts and spores, mold counts, and bacteria (rods) and the amount of rot in the tomatoes. With regard to yeasts and spores a count of twenty was represented by 1 per cent decay. From this pomt the rate of increase was lower. A count of thirty-five yeasts and spores indicated, according to Howard about 4 per cent of rot. Fig. 81 devised by How- ard shows the " Zone of Possible Yeast and Spore Counts.'^ From this chart it is possible to determine the relation between a count and the per cent of rot which it represents. The relation between the number of bacteria and the percentage of rot is shown in Fig. 82. Howard thinks that below 15,000,000 little is indicated with regard to the amount of rot. Above this " and up to 20 per cent of rot, the ratio of increase as shown on this chart is about 20,000,000 for each per cent of rot." Howard states that a low bac- terial count does not always indicate a sound stock but a high number of bacteria always indicates bad stock. Fig. 83, devised by Howard, shows the relation between the mold count and the percentage of rot. Prom this chart it is possible to calculate the percentage of decay from a mold count. The following is quoted from Howard. ^' Attention should be called to the fact that beyond 20 per cent of rot the chart is plotted on the basis of the assump- tion that 100 per cent rot would give a mold count of 100 per cent of the fields. As a matter of fact, the mold count reaches this maximum of 100 per cent of the fields with less than this amount of rot. In the study of the relation between the rot and count under factory conditions, 20 per cent of rot was the highest on which the full data was secured. From to J per ce'nt of rot the mold count rises rapidly. Beyond J per cent the rate of rise gradually decreases, until after 20 per cent of rot the rate of increase is slow. On the whole the zone was higher on factory than on laboratory samples. The chart shows that a count of 60 per cent molds represents a rot content of not less than about 4 per cent. It is interesting that the mold count of twenty-five, which was YEAST AND SPOKES 625 oooooooo o ooooo ooooooo *rarao09/|; jod q.unoo ajpdg ^ue ^SBax o 526 FOOD PRESERVATION 4 6 8 10 12 14 10 18 20 22 24 26 28 30 32 34 36 38 40 Per cent of Rot Fig. 82. — Percentage by Weight of Rot and Bacterial Count. (After Howard and Stevenson, 1917.) The line showing the tipper limit of '* Zone of Possible Bacteria Counta " beyond 20 per cent of rot IS based upon the count of a sample having 100 per cent of rot, 100 fields with molds, 22 yeast and spores per ^^ ^ O ^-3 o ^ «4-< o Si 03 bJO,^ •>H c3 • .9 ■e t^ ^ 9. Oi CO fe» ^ ^ r-i »Q , L. A. 1916. Normal* Fermentation of Sauerkraut. Absts. Bact., 1, 108. Round, L. A. 1917. The Bacteriology of Sauerkraut, a Further Study. Abstracts of Bacteriology, 1, 50. RuATA, A. Q. 1918. Action of Cold on Microorganisms. Annali dTgiene Rome, 28; Jour. Amer. Med. Assoc, 70, 1125 (1918); Amer. Jour. Pub. Health, 8 (1918), 399. Sadler, W. 1918. The Bacteriology of Swelled Canned Sardines. Amer. Jour. Pub. Health, 8, 216-220. Sedgwick, W. T., Hamilton, H. W. and Funk, F. J. 1917, Experimental Studies on the Effects of Various Solutions upon the Viability of Bacteria at Low Temperatures. Abstracts of Bact., 1, 49. Seele, W. 1913. Cases of Food Poisoning Noted in the German Army During the Last Two Years. Inaug. Dissert. Univ. Berlin. Hyg, Rund., 24, 531, 532. Serkowski, S. and Tomczek, P. 1911. The Influence of Common Salt on the Bacteria which Cause Meat Poisoning. Zeit. Untersuch. Nahr. Genussmitel, 21, 211-216. Stadler, E. 1899. Ueber die Einwirkung von Kochsalz auf Bakterien die bei den sog. Fleischvergiftungen eine Rolle spielen. Arch. Hyg., 35, 40-82. Thom, C. 1914. The Salt Factor in the Mold Ripened Cheeses. Btorrs Agric. Exp. Sta. Bull. 79, 387-394. Thom, C. and Shaw, R. H. 1915. Moldiness in Butter. Jour. Ag. Research, 3, 301-310. Weichel, a. 1910. The Effect of Salt upon Bacteria of the Ptomaine Group. Arb. kaiserl. Gesundh., 34, 247-265. Chem. Abstracts, 4, 3261 Weigmann, H. 1913. Germ Content of Salt. Jahresbericht. Vers. Stat. Molkw. Landw. Kammer. Schleswig-Holstein, 1913, 9, 10. Wilbur, R. L. and Ophijls, W. 1914. Botulism. A report of food poisoning apparently due to eating of canned string beans with a report of a fatal case. Arch. Intern, Med., 589. CHAPTER X V EPIDEMIOLOGY No attempt will be made here to present a complete discussion of the subject, but merely a few remarks about epidemics and the method of investigating them. Boudreau (1914) has defined an epidemic^ as an unusual number of cases of a communicable disease, arising within a short period, in a limited area, and traceable to a common source or sources. The food bacteriologist may often find it necessary to inves- tigate the cause of an epidemic either to protect an innocent article of food or to check the spread of infection. It is by no means an easy task in some epidemics to pick out the real cause; in others, by using a logical procedure, the epidemiologist may select with great accuracy the focus of the infection and see the reward of his efforts by the immediate decrease of the number of cases. He must be on his guard for any and all information which may help him towards his goal. Different methods have been used in the past by epidemiologists. It used to be customary for him to use indirect methods in securing his data. If called to a community where there was an unusual amount of disease, he would go equipped with sample bottles for the collection of specimens of milk and water and would spend much time in the early part of his investigation in inspecting water supplies, methods of sewage treatment, etc. The mode of attack is now quite different and marks one of the distinct advances which preventive medicine has made. The present practice in investigating epidemics has been well stated by Hill (1912). A certain amount of information is always necessary with regard to the characteristics of the disease and some of the different methods of spreading. Certain characteristics have been worked out for epidemics caused by food, water, carriers, etc. These are by no means constant but serve in a general way along with such other general data such as, population of the community, endemicity of the disease in the community, general appearance, etc. If this preliminary, general study is well made the epidemiologist may save much time when he comes to analyze his other data because suggested causes of the infec- tion may be quickly eliminated. 540 WATER-BORNE EPIDEMICS 541 Milk-borne Epidemics. The character of this article and the type of consumers gives a milk-borne epidemic of infection rather marked characteristics. Newman (1904) has mentioned the following: (a) There is a special incidence of the disease upon the track of the implicated milk supply. It is localized to such area. (6) Better class of houses and persons suffer most. (c) Milk drinkers are chiefly affected and they suffer most who are large consumers of raw milk. (d) Women and children suffer most and frequently adults suffer proportionately more than children. (e) Incubation periods are shortened. (/) There is a sudden onset and rapid decline. (g) Multiple cases in one house occur simultaneously. (h) CHnically the attacks of the disease are often mild. Contact infectivity is reduced and the mortality rate is lower than usual. The above characteristics as outlined by Newman (1904) for milk- borne epidemics also fit other food epidemics. Sawyer (1914, 1915) reported investigations of two typhoid epidemics, one caused by water and the other caused by food. Fig. 84 shows the incidence of cases for both epidemics. These curves show very well the types which are secured for food and water-borne epidemics of typhoid fever. Water-borne Epidemics. Such epidemics of disease have been common in the past and many such epidemics are described in the liter- ature. The important characteristics of such epidemics may be de- scribed as follows: (a) Water-borne epidemics may be preceded by a period of dysen- tery. (6) There is usually a slow onset of the epidemic and a rapid decline. (c) The cases are evenly distributed over the city if the city is served by a municipal supply. (d) The larger water-borne epidemics have occurred in the spring. Epidemics caused by other foods will show, in general, other char- acteristics. If the typhoid fever is caused by oysters, it will be con- fined to that part of the year when oysters are usually eaten. Further- more, the great majority of the cases will be primary cases — those who have partaken of the oysters. To detect carriers may require much inferential evidence and the elimination of other suggested causes. After this has been done, the indication that a carrier is concerned must be supported by bacteriological examinations of the urine and feces of the suspected individual 542 EPIDEMIOLOGY O O -^ CC C'l tH O O o o >^ ID .c3 Ij o -, -9 ^ o II S-3 CD m ;2 CO 1*^ |P*"^ |Mi^ fomi^ ym^ 0? XO "^ CO CM 1-4 O WATER-BORNE TYPHOID FEVER 543 544 EPIDEMIOLOGY Determination of the Origin of an Epidemic. In the majority of epidemics, the foci may be discovered with a fair degree of accuracy. Hill (1912) has given the detailed procedm'e and part of his paper is reproduced below. On account of the standing of this authority in this jGield, the author feels justified in quoting rather extensively from his work. In the last analysis, the epidemiologist must be a many-sided man; he must be somewhat of a bacteriologist, psychologist, and detective. He must have the intuition to separate the essential from the non-essential data. Then, somewhat like the lawyer, he should have studied reported epidemics in order to determine how others have been investigated. " To illustrate the general principles, let us suppose notification be received that a typhoid fever outbreak exists in a far-off community. The public health detective packs his grip and goes. He knows no details; he has never heard of this particular community before; he has not even any general information about the character of the country; he enters the community with no preconceived ideas. But he does know how typhoid fever originates and how it spreads. Water, milk, food, flies, and fingers are the routes — typhoid cases or typhoid carriers the source. His duties are to find both; and to find them, not as a scien- tific amusement, or as a matter of record; not to furnish food for spec- ulation — above all not to make a show of doing something — but to stop the outbreak; and then to advise measures to prevent recurrence. '^ The public health detective on entering the community affected by typhoid fever does not first examine the water supply, the milk sup- ply, the sewage disposal system, the markets, the back alleys, the dairies or anything else. He goes directly to the bedsides of the patients. Of course he must obtain the names and addresses of the patients from someone — from the local health officer, if he has them; from the attending physicians, if the health officer has no list; from the lay citi- zens themselves, if no one else is immediately available. The more complete the list, the faster he can work, because then he is not com- pelled to hunt up the cases personally. Bnt if there be no list, he begins making one himself. His intention is to see Just as many patients as he can, for each furnishes evidence and he wants it all. But he knows that it is not always necessary at this stage to see absolutely all the patients, so long as he sees the majority. '^ Reaching the patient's * bedside, his investigation begins. Auto- * If the patient is a child or delirious or not strong enough for an interview, or speaks only some foreign tongue, the relatives, friends or assoczate,s must supply the information. ORIGIN OF EPIDEMICS 545 matically, almost mechanically, he decides whether or not the patient has typhoid fever or not. Satisfied on that point, his first question is not, ^ Tell me all the different water supplies you have used, or all the sources of milk you have used.' The first question is, ' When did you first show the earliest symptoms of the disease?' Why? Because this date once fixed, the date at which infection entered the patient's mouth is fixed also, i.e., a date between one and three weeks previous to the date of earliest symptoms.* Remember that at this stage the detective may not have even an inkling as to which of the usual factors, water, milk, food, flies or fingers, is involved. Still less can he guess which particular water supply, milk supply, etc., of the many possible ones, may be the guilty one. But the answer to this question reduces possible routes to those used by this patient — not at anytime — but during a specific period, i.e., from one to three weeks preceding his date of earliest symptoms. " Not yet, however, are the milk and water questions offered. The second question is, ' Where were you during that period? ' Why? Because if the patient were not in the community during that period, he could not have contracted his infection within it, and does not belong to the outbreak under examination at all but to some other,- He is, in brief, an ' imported case ' and while, of course, he is to be supervised lest he spread his infection to others, he cannot help to locate the source of the main outbreak — unless perchance he he himself that source, i.e., the introducer to the community of the original infection. If he be an imported case he is noted for further reference and the detective goes to another patient. If not, the questions continue. But not yet is water or milk or flies mentioned. The third question is ' Were you associated during your period of infection with any then known typhoid case? ' Why? Because such association, especially if intimate, makes it more than probable that the case under examination received his infection from the preceding case, rather than from any general route and that he is, therefore, a ' secondary ' case. If he had such associations, this is noted for further reference and the investi- gator passes on to another bedside. If not, the questions continue and now, at last, take up milk, water, food, etc., but, of course, only so far as to determine those used by the patient during his infection period. '' Then the investigator passes to the next patient. What has he learned so far? Nothing much yet. But he has narrowed the possible * The occasional exceptions do not affect the validity of this statement as a practical working rule. 546 EPIDEMIOLOQy routes of infection to certain water supplies, certain milk supplies, certain food supplies, etc., i.e., those used by the first patient during a certain period, and he has done this in thirty minutes — in scarcely the time it takes for the old-style investigator to get his bottles ready to collect his first water sample. " At the bedside of the second patient, the same inquiiies in the same order are made. If this second patient be an imported case, or a secondary case, he also is merely noted for future reference. If he be a primary, however, the origins of his drinking water, milk, food, etc., during his infection period are also ascertained. Perhaps he coincides with the first patient in every detail of alimentary supplies, in history and associations. If so, nothing much has been added to the detective's knowledge. But more than likely, dissimilarities have developed. Since the responsible water supply, milk supply, etc., must be one of those water supplies, milk supplies, etc., used m common by primary cases all those not common to both of these primary cases may be dropped from consideration (except in rare instances of multiple routes). Thus, if both have used the same water, water from that oiigin remains as a possibility. But if the water supplies have been different, water is eliminated from the question entirely. If the milk supplies are identical, milk remains as a possible route of infection; if not, milk is eliminated from the question entirely. ^^ In brief, provided the information obtained be rcHable, and it is a part of the public health detective's training to distinguish at a glance truth from falsehood, the honestly mistaken, or forgetful, or stupid repHes from the reliable ones^ — and above all never to beUeve anything (to the extent of recording it) unless it is checked, confirmed and estab- lished as a fact, the modern investigator has in one hour narrowed his investigation to a point which the old-style investigator often would not reach for weeks. " And so from patient to patient the inquiry proceeds. In the course of the day the investigator has seen perhaps thirty patients. The tabulation (probably already made in his own mind) shows, say, three imported cases, five secondaries, two uncertain or indefinite. The remaining primary cases show in common, say, one water supply only, the milk, etc., varying; or one milk supply only, the water, etc., vary- ing; or no connection except attendance at some one social function. " Going straight to the route thus indicated, the public health detective quickly confirms the indications of his results. He knows that the route indicated must be the guilty one for only that route can account for all the cases. He concentrates on that route until ORIGIN OF EPIDEMICS o47 the evidence is complete — ^when and how that route became infected, when and by what sub-routes the infection was distributed, why it infected the patients found and not others, etc. ^' In this illustration I have assumed complete ignorance on the part of the epidemiologist as to everything connected with the com- munity he is investigating, except what he finds by cross-examining the patients. As a matter of fact, every epidemiologist, however much a stranger to the particular community he enters, begins to learn about it from the moment he enters it. ^^ Thus almost unconsciously he notes the size of the town and com- pares it with the number of cases reported as existing; if it is summer time he almost automatically notes the presence or absence of open toilets in the back yards, of manure piles and of garbage cans — all bearing upon fly infection. If it is winter time or the community' be well sewered, he does not even consider flies. If the cases are grouped in one quarter of the town, while the public water supply extends all over it, he tentatively eliminates the water supply, before he asks a question. If good surface drainage and a sandy soil exist, or driven wells are chiefly in vogue, he tentatively eliminates well water — even before he registers at the hotel. '' This is not and cannot be a complete synopsis of all the combina- tions of circumstances which the epidemiologist meets. It is intended to illustrate his methods and to show why they are incredibly rapid and incredibly accurate — ^how they ehminate speculation and guarantee a correct solution — which means of course the achievement of the great end, the finding of proper measures for suppression. " As soon as the route is indicated, he must go to that route, and establish beyond peradvcnture that it was in truth responsible. A water supply cannot convey typhoid if typhoid fever discharges have not entered it. There is no object in attributing an outbreak to fly infection from toilets into which typhoid feces have not been dis- charged at such a time as to account for the cases. A milk supply, not handled at some point by an infected person, nor adulterated at some time with infected extraneous matter cannot convey typhoid. Whatever his results, they cannot be true unless they are consistent — they should not be accepted unless they are provable — and proved. '' If the public health detective is familiar with the community where the outbreak occurs, including its water supplies, its milk sup- plies, the sociological relationships of its people, etc., etc., he can often tentatively determine the cause of the outbreak by a mere inspection of the names and addresses of primary cases, especially if plotted on 548 EPIDEMIOLOGY a map of the community, taking into account also the time of year, and other general points. But such deductions, while often wonderfully reliable, can never be as conclusive and satisfactory as are the results of an investigation by even a total stranger, if the investigation be con- ducted as above described/' The data received from each patient should be recorded on a case card. Many different cards have been described in the literature. The one given below has been used by the Ilhnois State Water Survey. Others have been described by Hill (1912), Boudreau (1914), Ferguson (191 ) and Hansen (1914). Suggested Form for Securing Information Relative to Typhoid Fever Cases by the Local Health Authorities. Record No TYPHOID FEVER RECORD 1. Name of Patient 2. Residence, Street and Number 3. Age 4. Sex 5. Give Occupation and Place of Business 6. Name of Physician 1, Date when Patient Took to Bed 8. Date of First Symptoms 9. Date of Physician's First Visit 10. Date of Leaving Bed on Recovery. 11. Date of Relapse 12. Date of Death * 13. Was Widal Test Made? Date Positive or Negative 14. Was Spleen Enlarged and Were Rose Spots Present? 15. Was Patient Absent from City During the Two Months Previous to Illness? If so, state where he visited and between what dates 16. If Patient Changed Residence During Two Months Previous to Illness, Give Former Address and Date of Change 17. Character of Residence (private house, boarding house, apartment, hotel, etc.) 18. State Source of Drinking Water. At Home, at Place of Business, Else- where. . . CASE CARD 549 19. State Source of Milk Supply and Name of Dealer. Was Milk Habitually Drunk? Was it Occasionally Used, as in Tea or Coffee or in Ice Cream? 20. Were Raw Oysters, Lettuce or Celery Eaten Raw within Three Weeks before Illness? 21. State from Whom Bread was Obtained 22. Did Patient Fish or Bathe in Neighboring Streams? If so, State at what Point or Points 23. Did Patient use any Pubhc Baths or Swimming Pools? If so, give Dates 24. Give Number of Persons Living in Same House or Apartment Also Give Number in Whole Building in Case of Hotel, Apartment House, etc 25. Give Names of Other Cases in Same Building and Dates when they Went to Bed 26. Give Names and Approximate Dates for Cases among Business Associates 27. Give Names and Approximate Dates for Cases among Friends (Include Schoolmates) 28. Did Patient Attend any Social Gatherings where Food was Served within Two Months before Illness? If so, Gives Dates 29. Describe Sanitary Condition of Premises. Note Especially Outdoor Privies, Cesspools, Wells and Refuse Piles 30. Is House Well Screened? 31. Had Patient a Separate Room? 32. Had Patient a Trained Nurse or was Nursing done by Member of the Family or Household? 33. Where Stools and Uriae Disinfected? If so, Dev^cribe How this was Carried Out and Note Length of Time Continued Remarks. To Include any Facts, Statements or Comments not Included in the Foregoing that May Throw Light on the Source and Mode of Infec- tion Information Given bv Information Obtained by Date of Obtaining Information. 550 EPIDEMIOLOGY APPENDIX I. FORMS FOR TYPHOID INVESTIGATIONS Suggested Form for Physicians' Report or Typhoid Fever Typhoid Fever Name Street and Number Sex , Age Place of Business Occupation Physician Date of Taking to Bed Date of First Symptons Date of Physician's First Visit Was Widal Test Made? Positive or Negative Was Patient Out of Town within Thirty Days? Between what Dates Are There Other Cases in House? If So, How Many State Source of Drinking Water Give Name of Milk Dealer Remarks The preparation of a spot map is very often an aid in studying epi- demics. If the cases are about evenly distributed over a city it is a fair indication that the water supply is to blame. If the cases are grouped in several localities, it may indicate that a milk supply or a carrier is to blame. Fig. 84 shows such a map which was prepared by Bartow (1912) for a water-borne epidemic. BIBLIOGRAPHY Bartow, E. Discussion to Paper by Hill (1912). BoxJDREAXj, F. G. 1914. The Mode of Procedure in the Study of Epidemics. Ohio State Board of Health Bull 4, 1277-1286. Feeguson, H. F. 1916. Epidemic of Typhoid Fever Caused by Polluted Water Supply at Old Salem Chautauqua. Illinois State Water Survey Bulletin No. 13, 272-286. Hansen, P. 1914. A Study of Typhoid Fever in Rockford, Illinois, in the Late Summer and Fall of 1913. Illinois State Water Survey Bull No 11 384-430, BIBLIOGRAPHY 551 Hill, H. W. 1912. The Detailed Procedure to be Followed in an Epidem- iological Determination of the Origin of a Typhoid Outbreak. Proceedings Illinois Water Supply Assn, 1912, 96-105. JoBDAN, E. 0. and Ikons, E. E. 1912. The Rockford (111.) Typhoid Epi- demic. Jour. Inf. Diseases, 11, 21-43. Sawyek, W. a. 1914. Ninety-three Persons Infected by Typhoid Carrier at a Public Dinner. Jour. Amer. Med. Assn., 63, 1537-1542. Sawyer, W. A. 1915. A Water-borne Typhoid Fever Epidemic. California State Board of Health, 10, 303-311. APPENDIX Indicator Solutions. The following table is taken from Hawk's Physiological Chemistry and will be foxmd useful in bacterial work. The amounts of the indicator solutions and their strengths to be used in the determination of hydrogen ion concentrations in 10 c.c. portions of unknown solution are indicated below: INDICATOR SOLUTIONS Drops. Preparation of Solution. 1. 2. 8 Alizarin yellow R (p-nitro- benzine-azo-salicylic acid) Azolitmin (litmus) Cochineal 10-5 0.1 gm. in 1000 c.c. water. Aqueous solution. Alcoholic solution. 4. 5 2.5-dinitro-liydrochmone . . Mauvein 5-2 8-1 5-3 4-2 8-1 20-10 20-3 20-3 15-6 10-3 10-5 5-3 10-4 1 gm. to 1000 c.c. alcohol. 0.5 gm. to 1000 c.c, 0.1 recrystallized salt to 1000 c.c. water. Saturated solution in 50 per cent alcohol. 0.5 gm, in 1000 c.c. water. 0.1 gm. in 500 c.c. alcohol and 500 c.c. water. 0.4 gm. to 60 c.c. alcohol, 940 c.c. water, 0.5 gm. to 500 c.c. water, 500 c.c. alcohol. 0.4 gm. in 400 c.c. alcohol, 600 c.c. water. 0.4 gm. to 500 c.c. alcohol, 500 c.c. water. 0.1 gm. to 1000 c.c. water. Of recrystallized salt, 0.1 gm to 1000 c.c. water. 0.1 gm. to 1000 c.c. water. 6. 7, Methyl orange Methyl red 8 Methyl violet 9. 10 Neutral red p-nitrophenol 11, Phenolphthalein 12 Rosolic acid 13. 14 Thymolphthalein Tropaeolin 15. 16 Tropaeolin 00 . , Tropaeolin 000 In an extensive study of the application of H-ion determination to bacteriological work,. Clark and Lubs (1917) have pointed out the error in determining reaction by titration. They used the electro- metric method and by means of it prepared a series of new indicators 553 554 APPENDIX which will be valuable in future bacteriological work. They are given in Table LII. Table LII LIST OF INDICATORS, PREPARED BY CLARK AND LUBS, 1918 Chemical Name. Thymol sulphon phthalein (acid range) Tetra biomo phenol sulphon phthalein Ortho carboxy benzine azo di methyl aniline Ortho carboxy "benzine a^io di propyl aniline Di bromo ortho cresol sulphon phtha- lein Di bromo thymol sulphon phthalein . Phenol sulphon phthalein Ortho cresol sulphon phthalein Thymol sulphon phthalein (see above). Ortho cresol phthalein Concen- Common Name, tration, Per Cent. Thymol blue 0.04 Brom phenol blue 0.04 Methyl red 0,02 Propyl red 0.02 Brom cresol purple 0.04 Brom thymol blue . 0.04 Phenol red 0.02 Cresol red 0.02 Thymol blue 0.04 Cresol phthalein. . . 0.02 Color Change. Red-yeliow Yellow-blue Red-yellow Red-yellow Yellow-purple Yellow-blue Yellow-red Yellow-red Yellow-blue Colorless-red Range 1.2-2.8 3.0-4.6 4.4-6.0 4.8-6.4 5.2-6.8 6.0-7.6 6.8-8.4 7.2-8.8 8.0-9.6 8.2-9.8 Preparation of Standard Reagents A standard solution contains a known amount of reacting substance and is to be distinguished from a normal solution. A normal solution contains the hydrogen equivalent of the active constituent in grams per liter. This is that amount which brings into the reaction LOOS gms. of hydrogen, 8 gms. of oxygen, etc., or the equivalent. This definition has so many distinct advantages that it is now generally accepted. The following are some of the standard solutions used in bacteriology: N Sodium Hydroxide, Weigh out 40.06 or better about 45 gms. of sodium hydroxide and dissolve in 1 liter of boiled cooled distilled water. Standardize against normal hydrochloric acid. It is best to allow the two solutions to stand at the same temperature for several hours or overnight. The titration should be repeated several times. Calcula- tion is as follows: If 50 c.c.NaOH - 52.6 c.c. NHCl the NaOH is too strong by 2.6 c.c. per every 50 c.c. 50 : 2,6 = 1000 :x a: = ?:^^^15^ = SO.c.c, 50 APPENDIX 555 ■* '* 1 »-t o tH 1-H e« *-i ^ ec 1 1 I— t o o T~« iH S M c» 1 1 ^ o o T-i 1— f t. 1*4 rH 1-1 1 1 1— 1 o o ,_^ I— t o o 1 1 tH o o r-l t-l 1 1 o O l-» tH CP 09 HH «D 1 o 1 o l-H »-» 1— t ua Ok ^ XO 1 O 1 o H» lP-4 r-* P? < o H xfi 1 1 o o ^ r-» m M CO 1 1 o O iH 1—1 e* « t*4 » C3 ?t3 '3 o < ^ i S £ ^ T ■!■ I T t t O o o J I 4 I I I 4 4 a ^ 6: a t i1 I t t t t iJ3 ^ 'P I? ■^ o 1 I i § •i- 4 I I ■!- 4 CD 4 I i i 4 83 t "I cS i s t o t t t t t t t t -9 o. t t t o O o t t o o ■4-3 O o O t •S a o i .s -5 ft "3 a e^ i -!■ o t n3 i I -i 4 o I o 1 T3^ S 2 'CS'o 13 n3 o .S 1 o 'a I I o o bo d o I t t g' Pm ts a 03 a a 1 a GO >1 '3 M cm CO C3C? S3 Je ^ fl o S CO o •§ ^ 556 APPENDIX This amount of water should be added, after which the solution will be exactly normal. It should be checked, however, against the N HCl. From this the N/20 NaOH may be prepared by diluting 50 c.c. of the N NaOH to a liter. N Potassium Hydroxide. Dissolve 56 gms, of KOH in 1000 c.c. of distilled water and standardize against N HCl as was done with N NaOH. N Sodium Carbonate. Ignite the bicarbonate NaHCOs to redness and dissolve 53 gms. in a liter of water. Standardize against normal acid and dilute accordingly with distilled water. N Sulphuric Acid. Follow the procedure which has been given else- where for N HCL This may be standardized against N NaOH or grav- imetrically by precipitating with BaOH. N Hydrochloric Acid. This should contain 36.46 gms. of HCl in a liter of water. The pure concentrated HCl may be diluted to a specific gravity of 1,020. According to the table on page 559, it will be seen that this solution is too strong, having about 4.13 gms. of HCl in a liter. To be made exactly normal, it should be titrated against pure Na2C03. This is obtaiaed by heating the NaHCOs being careful not to allow it to fuse. This is then put into a weighing bottle from which it is weighed into Erlenmeyer flasks for titrating. Dissolve in boiled distilled water and titrate with the HCl solution using methyl orange as the indicator. Calculations are made as follows, assuming for example that 1.9864 gms. of Na2C03 neutraUzed 37.28 c.c. of the HCl solution. If our solution had been exactly normal 1 liter would have neu- tralized Na2C03 106.00 ^^ ..^ ^^ _ — — = — — -53 gj^Qg, of NasCOa 53: 1000 =.1.9864': a: a;=37.48. 37.28 c.c. of the HCl solution were required and it is evident that this solution is too strong and that for each 37.28 c.c. of the solution, 37.48 -37.28 = .20 c.c. of water must be added. The amount of water to be added to each Kter of the HCl solution to make it exactly normal is computed as follows: 37.28: .20 = 1000 \z a; = amount of distilled water to be added. N Nitric Acid. See preparation of N HCl. N Oxalic Acid. Dissolve 47 gms. of pure dry oxahc acid in 1000 c.c. of distilled water. Standardize against N Na2C03. APPENDIX 557 N/10 Potassium Permanganate. Dissolve 3.163 gms in a liter of water and standardize against ferrous ammonium sulphate, oxalic acid or pure iron. 1 c.c. = .0056 gm. Fe 1 c.c. = . 0008 gm. 0. . 0072 gm. FeO . 0063 gm. H2C4O4 . 2H2O. .008 gm. Fe203. N/10 Iodine, Dissolve 12.685 or 12.8 gms. of pure iodine and 18 gms. of potassium iodide in a liter of distilled water. Standardize against N/10 Na2S203. 1 c.c.= .0158 gm. Na2S203, = .00495 gm. AS2O3, = .00575 gm. AS2O5. N/10 Sodium Thiosulphate. Dissolve 24.9 gm. of the cr;^stals in 1000 c.c. of water and standardize against N/10 iodine. N/10 Sodium Chloride. Dissolve 5.9 of the salt in 1000 c.c. of water. Standardize gravimetrically by precipitating as AgCl. 1 c.c. = . 0058 gm.NaCl, -.0035 gm. CI, -.OlOSgm. Ag. N/10 Silver Nitrate. Dissolve 16 . 966 gms. of AgNOs in a liter of water and standardize by precipitating as AgCl. The preparation of standard silver nitrate for water analysis is outlined in another place. Preparation of Antifonnin. Dissolve 720 gms. of chloride of lime and 260 gms. of sodium carbonate in 2500 c.c. of water. Allow to stand and filter. By means of N/10 sodium thiosulphate determine the amount of available chlorine and adjust so that it will contain about 5.6 per cent. Add 7.5 per cent of sodium hydroxide, filter and store for future use. Azolitmin Solution. Add 1 per cent of Kahlbaum's azoHtmin to distilled water and boil for five minutes. Adjust the reaction, if necessary, with NaOH. It should give a distinct blue plate when it is diluted in the Petri dish with the medium. Benedict's Reagent for the Quantitative Estimation of Dextrose. Copper sulphate (crystaUized) 18.00 gms. Sodium carbonate (anhydrous) 100.00 gms. Sodium citrate 200 . 00 gms. Potassium thiocyanate. 125.00 gms. Potassium ferrocyanide (5 per cent solution). 5 . 00 c.c. Distilled water to make 1 liter. 558 APPENDIX Dissolve the carbonate, citrate and thiocyanate in 800 c.c. of water and filter if necessary. Dissolve exactly 18 gms. of copper sulphate in 100 c.c. of distilled water. Add this to the jfirst solution with constant stirring after which add the 5 c.c. of potassium ferrocyanide and dilute to a Hter. Diluting Fluid for Leucocytes. Prepare a 0.5 per cent solution of acetic acid and tinge slightly with gentian violet. Hayem's Diluting Fluid for Erythrocytes. Sodium sulphate (Na2S04) 5 gms. Mercuric chloride (HgCl2) 0.5 gm. Sodium chloride (NaCl) 1.0 gms. Distilled water 200 c.c. Litmus or Azolitmin Solution. The standard solution of litmus shall be a 2 per cent solution of reagent litmus in water. Crush the Ktmus in a mortar with a little water and, after diluting to the mark, boil for five minutes. Distribute in flasks or tubes and sterilize. This solution generally needs no correction but may be adjusted with dilute NaOH or HCl if necessary. Millon's Reagent. Dissolve 30 c.c. of mercury in 570 c.c. of con- centrated nitric acid and dilute with two parts of water. Molisch Reagent. Dissolve 15 gms. of a-naphthol in 95 per cent alcohol. Nylander's Reagent. Boil 4 gms. of potassium hydrogen tartrate and 2 gms. of bismuth subnitrate in 100 c.c. of 10 per cent KOH. When cool filter. Phenylhydrazine Mixture. Thoroughly mix two parts of sodium acetate and one part of phenylhydrazine hydrochloride, Seliwanoflf^s Reagent. Dissolve .05 gm. resorcinol in 100 c.c. hi dilute HCL Toisson's Diluting Fluid for Erythrocytes. Sodium sulphate (Na2S04) 8 gms. Sodium chloride (NaCl) 1 gm. Glycerol 30 c.c. Distilled water 160 c.c. Color with gentian violet. Uffelmann's Reagent. Add ferric chloride to phenol until a bluish- violet color results. APPENDIX 559 SPECIFIC GRAVITIES OF HYDROCHLORIC, NITRIC, AND SULPHURIC . ACIDS (After G. Lunge) Specific Gravity Peh Cent by Weight. . 15° 40 • (Vacuo) . HCl. HNO3. H2SO4. 1.000 0.16 0.10 0.09 1.005 1.15 1.00 0.83 1.010 2.14 1.90 1.57 1.015 3.12 2.80 2.30 1.020 4.13 3.70 3.03 1.025 5.15 4.60 3.76 1.030 6.15 5.50 4.49 1.035 7.15 6.38 5.23 1.040 8.16 7.26 5.96 1.045 9.16 8.13 ♦ 6.67 1.050 10.17 8.99 7.37 1.055 11.18 9.84 8.07 1.060 12.19 10.68 8.77 1.065 13.19 11.51 9.47 1.070 14.17 12.33 10.19 1.075 15.16 13.15 10.90 1.080 16.15 13.95 11.60 1.085 17.13 14.74 12.30 1.090 18.11 15.43 12.99 1.095 19.06 16.32 13.67 1.100 20.01 17.11 14.35 1.105 20.97 17.89 15.03 1.110 21.92 18.67 15.71 1.115 22.86 19.45 16.36 1.120 23.82 20.23 17.01 1.125 24.78 21.00 17.66 1.130 25.75 21.77 18.31 1.135 26.70 22.54 18.96 1.140 27.66 23.31 19.61 1.145 28.61 24.08 20.26 1.150 29.57 24.84 20.91 1.155 30.55 25.60 21.55 1.160 31.52 26.36 22.19 1.165 32.49 27.12 22.83 1.170 33.46 27.88 23.47 1.175 34.42 28.63 24.12 1.180 35.39 29.38 24.76 1.185 36.31 30.13 25.40 1.190 37.23 30.88 26.04 1.195 38.16 31.62 26.68 1.200 39.11 32.36 27.32 1.205 33.09 27.95 1.210 33.82 28.58 1.215 34.55 29.21 1.220 35.28 29.84 Specific Gravity (Vacuo). 1.235 1.240 1.245 1.250 1 255 1.260 1.265 1.270 1.275 1 . Zhu 1.285 1.290 1.295 1.300 1.305 1.310 1.315 1.320 1.325 1.330 1.335 1.340 1.345 1.350 1.355 1.360 1.365 1.370 1.376 1.380 1.385 1,390 1.395 1.400 1.405 1.410 1.415 1.420 1.425 1.430 1.435 1.440 1.445 Per Cent by Weight. HCL HKO3 HsSO^ 36.03 30.48 36.78 31.11 37.53 31.70 38 29 32.28 39.05 32.86 39.82 33.43 40.58 34.00 41.34 34.57 42.10 35.14 42.87 35.71 43.04 36.29 44.41 36.87 45.18 37.45 45.95 38.03 46.72 38.61 47.49 39.19 48.26 39.77 49.07 40.35 49.89 40.93 50.71 41.50 51.53 42.08 52.37 42.66 53.22 43.20 54.07 43.74 54.93 44.28 55.79 44.82 56.66 45.35 57.57 45.88 58.48 46.41 59 39 46.94 60.30 47.47 61.27 48.00 62.24 48.53 63.23 49.06 64.25 49.59 65.30 50.11 66.40 50.63 67.50 51.15 68.63 51.66 69.80 52.15 70.98 52.63 72.17 53.11 73.39 53.59 74.68 54.07 75.98 54.55 From Reed's Manual of Bacteriology, Ginn & Co. 560 APPENDIX SPECIFIC GRAVITIES OF HYDROCHLORIC, NITRIC, AND SULPHURIC ACIDS. — Continued Speeifie Gravity Peb Cent by Weight. Gravity , 15° at 40 • (Vacuo). Per Cent by Weigth. at ^o- (Vacuo) . HCl. HNO3. H2SO4 HCl. HNO3. H2SO4. 1 450 77.28 78.60 79.98 81.42 82.90 84.45 86.05 87.70 89.60 91.60 94.09 96.39 98.10 99.07 99.67 55.03 55.50 55.97 66.43 56.90 57.35 57.83 58.28 58.74 59.22 59.70 60.18 60.65 61.12 61.59 62.06 62.53 63.00 63.43 63.85 64.26 64.67 65.08 65.49 65.90 66.30 66.71 67.13 67.59 68.05 68.51 68.97 69.43 69.89 70.32 70.74 71.16 71.57 71.99 72.40 72.82 73.23 73.64 74.07 1.670 1.675 1.680 1.685 1.690 1.695 1.700 1.705 1.710 1.715 1.720 1.725 1.730 1.735 1.740 1,745 1.750 1.755 1.760 1.765 1.770 1.775 1.780 1.785 1.790 1.795 1.800 1.805 1.810 1.815 1.820 1.825 1.830 1.835 1.840 1,8405 1.8410 1.8415 1.8410 1,8405 1.8400 1.8395 1.8390 1.8385 74.51 1 455 74.97 1.460 75.42 1.465 75.86 1.470 76.30 1.475 76.73 1.480 77.17 1.485 77.60 1.490 78.04 1.495 78.48 1.500 1.505 78.92 79.36 1.510 79.80 1.515 80.24 1.520 ......... 80.68 1.525 81,12 1.530 • 81,56 1.535 82.00 1.540 82.44 1.545 82.88 1.550 83.32 1.555 83.90 1.560 84.50 1.565 85.10 1.570 85.70 1.575 86.30 1.580 86.90 1.585 87.60 1.590 88.30 1.595 89.05 1.600 90 05 1.605 91.00 1.610 92 10 1.615 1.620 ««y Er Eu P Gd Ga Ge Gl Au He Ho XT Jtl In I Ir Fe Kr Pb Lu Mg Mn Atomic Weight. 120.2 39.88 74 96 137.37 208.0 11.0 79.92 112.40 132.81 40.07 12.00 140.25 35.46 52.0 58.97 93.5 63.57 162.5 167.7 152.0 19.0 157.3 69,9 72.5 9.1 197.2 3.99 163.5 1.008 114.8 126.92 193.1 55,84 82.92 139.0 207.10 6.94 174.0 24.32 54.93 200.9 Name. Molybdenum . . . Neodymium . . . . , Neon Nickel Niton Nitrogen , Osmium Oxygen Palladium Phosphorus Platinum Potassium Praseodymium . . Radium Rhodium Rubidium Ruthenium Samarium Scandium Selenium Silicon Silver Sodium Strontium Sulphur Tantalum Tellurium Terbium Thallium Thorium Thulium Tin Titanium Tungsten Uranium Vanadium Xenon Ytterbium (Neoytterbium) Ytterium Zinc. Zirconium Symbol Mo Nd Ne Ni Nt N Os O Pd P Pt K Pr Ra Rh Rb Ru Sm Sc Se Si Ag Na Sr S Ta Te Tb Tl Th Tm Sn Ti W U V Yb Yt Zr Atoinic Weight- 96.0 144.3 20.2 58.68 222.4 14.01 190.9 16.00 106.7 31.04 195.2 39.10 140.6 226.4 102.9 85.45 101.7 150.4 44.1 79.2 28.3 107.88 23.00 87.63 32.07 181.5 127.5 159.2 204.0 232.4 168.5 119.0 48.1 184.0 238.5 51.0 130.2 172.0 89.0 65.37 90.6 * Compiled by the International Committee on Atomic Weights consisting of F. W. Clarke, W. Ostwald, T. E. Thorpe, and G. Urbain. 564 APPENDIX SOLUBILITY OF OXYGEN IN WATER (Winkler, Berichte, 22, 1772.) (1889) Tempera- ture. Oxygen. Temperature. Oxygen. Temperature. Oxygen. 14.70 9 11.58 18 9.56 1 14.28 10 11.31 19 9.37 2 13.88 11 11.05 20 9.19 3 13.50 12 10.80 21 9 01 4 13.14 13 10.57 22 8.84 5 12.80 14 10.35 23 8.67 6 12.47 15 10.14 24 8 51 7 12.16 16 9.94 25 8.35 8 11.86 17 9.75 CIRCUMFERENCES AND AREAS OF CIRCLES Formula for Area Formula for Diameter ^=^ = .7853981)2 4 D = 2a/~--1 -128379 VZ Diameter. Circumference. Area. Diameter. Circumference. Area. i .39270 .012 7 21.991 38.485 I .78540 .049 8 25.133 50.265 f 1.1781 .110 9 28.274 ■ 63.617 i 1.5708 .196 10 31.416 78.54 f 1.9635 .307 20 62.832 314.16 1 2.3562 .442 30 94.248 706.86 i 2.7489 .601 40 125.664 1256.6 1 3.1416 .785 50 157.080 1963.5 2 6.2832 3.142 60 188.496 2827.4 3 9.4248 7.069 70 219.911 3848.5 4 12.566 12.566 80 251.328 5026.5 5 15.708 19.635 90 282.744 6361,7 6 18.805 28.274 100 314.160 7854.0 APPENDIX 565 TABLE SHOWING EQUIVALENTS OF PRESSURE AND HEAD OF WATER Head in Feet and Equivalent Pressrue in Pounds 5 TO 60 Feet. Feet Head. 5 10 15 20 25 30 35 40 45 50 60 Pounds Press. 2.17 4 33 6.50 8.66 10.83 12.99 15.16 17.32 19.49 21.65 26.09 70 TO 180 Feet. Feet Head. 70 80 90 100 110 120 130 140 150 160 180 Pounds Press. 30.3 34.6 39.0 43 3 47.6 52.0 56.3 60.6 65.0 69.2 78.0 200 TO 1000 Feet. Feet Head. 200 250 300 350 400 500 600 700 700 900 1000 Pounds Press. 86.6 108.2 129.9 151.5 173.2 216.5 259.8 303.1 346.4 389.7 433.0 Pressure in Pounds and Equivalent Head in Feet 5 TO 60 Pounds. 70 TO 170 POTTNDS. 180 TO 500 Pounds. Pounds Press. Feet Head. Pounds Press. Feet Head. Pounds Press. Feet Head. 5 11.5 70 161.6 180 415.6 10 23.0 80 184.7 190 438.9 15 34.6 90 207.8 200 461.7 20 46.2 100 230.9 225 519.5 25 57.7 110 253.9 250 577.2 30 69.3 120 277.0 275 643.0 35 80.8 130 300.1 300 692.7 40 92.3 140 323.2 325 750.4 45 103.9 15Q 346.3 350 808.1 50 115.4 160 369.4 400 922.6 60 138.5 170 392.5 500 1154.5 566 APPENDIX U. S. GALLONS TO LITERS CUBIC FEET TO LITERS (Metric) (Metric) Gallons. Liters. 1 Gallons. Liters. Cu.ft. 1 Liters. Cu. ft. Liters. 1 3.78544 51 193.05744 1 28.3166 51 1444.1466 2 7.57088 52 196.84288 2 56.6332 52 1472.4632 3 11.35632 53 200.62832 3 84.9498 53 1500.7798 4 15.14176 54 204.41376 4 113 2664 54 1529.0964 5 18.92720 55 208.19920 5 141.5830 55 1557.4130 6 22.71264 56 211 98464 6 169,8996 56 1585.7296 7 26.49808 57 215.77008 7 198.2162 57 1614.0462 8 30.28352 58 219.55552 8 226.5328 58 1642,3628 9 34.06896 59 223.34096 9 254.8494 59 1670.6794 10 37.85440 60 227.12G40 10 283.1660 60 1698 9960 11 41.63984 61 230.91184 11 311.4826 61 1727.3126 12 45.42528 62 234.69728 12 339.7992 62 1755.6292 13 49.21072 63 238.48272 13 368.1158 63 1783.9458 14 52.99616 64 242.26816 14 396.4324 64 1812.2624 15 56.78160 65 246.05360 15 424.7490 65 1840.5790 16 60.56704 66 249.83904 16 453.0656 66 1868.8956 17 64.35248 67 253.62448 17 481.3822 67 1897.2122 18 68.13792 68 257.40992 18 509.6988 68 1925.5288 19 71.92336 69 261 . 19536 19 538.0154 69 1953.8454 20 75.70880 70 264.98080 20 566.3320 70 1982.1620 21 79.49424 71 268.76624 21 594.6486 71 2010.4786 22 83.27968 72 272.55168 22 622.9652 72 2038.7952 23 87.06512 73 276.33712 23 651.2818 73 2067.1118 24 90.85056 74 280.12256 24 679.5984 74 2095.4284 25 94.63600 75 283.90800 25 707.9150 75 2123.7450 26 98.42144 76 286.69344 26 736.2316 76 2152.0616 27 102.20688 77 291.47888 27 764.5482 77 2180.3782 28 105.99232 78 295 26432 28 792.8648 78 2208. C348 29 109.77776 79 299 04976 29 821.1814 79 2237.0114 30 113 56320 80 302.83520 30 849.4980 80 2265.3280 31 117 34864 81 306.62064 31 877.8146 81 2293.6446 32 121.13408 82 310.40608 32 906.1312 82 2321.9612 33 124.91952 83 314.19152 33 934.4478 83 2350.2778 34 128.70496 84 317.97696 34 962.7644 84 2378.5944 35 132.49040 85 321.76240 35 991.0810 85 2406.9110 36 136.27584 86 325.54784 36 1019.3976 86 2435.2276 37 140.06128 87 329.33328 37 1047*. 7142 87 2463.5442 38 143.84672 88 333.11872 38 1076.0308 88 2491.8608 39 147.63216 89 336.90416 39 1104.3474 89 2520.1774 40 151.41760 90 340.68960 40 1132,6640 90 2548.4940 41 155.20304 91 344.47504 41 1160,9806 91 2576.8106 42 158.98848 92 348.26048 42 1189.2972 92 2605.1272 43 162.77392 93 352,04592 43 1217.6138 93 2633.4438 44 166.55936 94 355.83136 44 1245.9304 94 2661.7604 45 170.34480 95 359.61680 45 1274.2470 95 2690.0770 46 174.13024 96 363.40224 46 . L302.5636 96 2718.3936 47 177.91568 97 367.18768 47 ■ L330.8802 97 2746.7102 48 181.70112 98 370.97312 48 ■ 1359.1968 98 2775.0268 49 185,48656 99 374.75856 49 1387.5134 99 2803.3434 50 189.27200 100 378.54400 50 ; 1415.8300 100 2831.6600 APPENDIX 567 USEFUL FACTORS FOR WATER Based on Weights at 62° F. (Standard Temperature) U. S. gallons X 8 . 3356 = pounds '' '' X .13388 = cubic feet X231. = cubic inches '' X ,83356 = English gallons '' '' X 3.78544 = Liters '' '' X . 020985 = Kokus (Japan) '' '' X .30815 =Bedeps (Russia) English gallons X 10. = pounds " '' X .160372 = cubic feet '^ X 277. 12 = cubic inches '' '' X 1.1997 =U.S. gallons " '' X 4.5413 = Liters '' X . 025175 = Kokus (Japan) '' X .36969 =Bedeps (Russia) Liters X 2.202 '' X .035302 '' X61.023 '' X .26417 = pounds -cubic feet = cubic inches =U. S. gallons = English gallons . 0055435 =Kokus (Japan) .081405 -Bedeps (Russia) KokusX 397.22028 = pounds X 6.3703 = cubic feet XI 1008. 00 -cubic inches X 47.6535 =U.S, gallons X 39 . 722028 = English gallons X 180.39 = Liters X 14.6847 =Bedeps (Russia) BedepsX 27.05 *' X .43381 '' X7;49.618 '' X 3.24512 '' X 2.705 " X 12.2843 = pounds = cubic feet = cubic inches = U. S. gallons = English gallons = Liters ' X . 0680983 = Kokus (Japan) 568 APPENDIX Cubic feet of water X 62 . 355 X 1728. 00 (( it £( (e ct i( (( it t( it (( It X X X X X —pounds = cubic inches 7 4805 =U. S. gallons 6 2355 = English gallons 28 317 = Liters . 15698 =Kokus (Japan) 2 3052=Bedeps (Russia) Pounds of water X .016037 '' '' X27.712 '' '^ X .11997 it s^ it it ^ U it X it it y^ - cubic feet = cubic inches =U. S. gallons = English gallons = Liters . 0025175 -Kokus (Japan) .036969 -Bedeps (Russia) .1 .45413 SURVEYOR'S MEASURE 7.92 inches ==llink 25 links =lrod 4 rods ==1 chain 10 square chains or 160 square rods = l acre 640 acres = 1 square mile 36 square miles (6 miles sq.) = 1 township CUBIC MEASURE 1728 cubic inches 128 cubic feet 27 cubic feet 40 cubic feet 2150.42 cubic inches 268.8 cubic inches 1 cubic foot = 1 cubic foot = 1 cord (wood) = 1 cubic yard ■ 1 ton (shipping) - 1 standard bushel ■ 1 standard gallon •about four-fifths of a bushel APPENDIX 569 METRIC EQUIVALENTS LiNEAE MeaSTJEE 1 centimeter = 0.3937 inch 1 inch = 2.54 centimeters 1 decimeter =3.937 inches =0.328 feet 1 foot =3.048 decimeters 1 meter = 39.37 inches = 1.0936 yards 1 yard = 0.9144 meter. 1 dekameter = 1.9884 rods 1 rod =0.5029 dekameter 1 kilometer =0.62137 mile 1 mile =1.6093 kilometers Squaee Meastjee 1 square centimeter =0.1550 square inch 1 square decimeter =0.1076 square foot 1 square meter =1.96 square yards 1 are =3.954 square rods 1 hektar =2.47 acres 1 square kilometer =0.386 square mile 1 square inch 1 square foot 1 square yard 1 square rod 1 acre 1 square mile Measuee of Volume 1 cubic centimeter =0.061 cubic inch 1 cubic decimeter =0.0353 cubic feet 1 cubic meter 1 stere 1 liter 1 dekaliter 1 hektoliter 1 cubic inch 1 cubic foot 1 cubic yard 1 cord 1 quart dry _ 1 1.308 cubic yards " \ 0.2759 cord 0.908 quart dry 1.0567 quart hquid 1 quart liquid 2.6417 gallons 1 gallon .135 pecks 1 peck =2.8375 bushels 1 bushel =6.452 sq. centimeters ==9.2903 sq. decimeters =0.8361 sq. meter =0.2529 are =0.4047 hektar =2.59 square kilometers = 16.39 cubic centimeters 28.317 cubic decimeters =0.7646 cubic meter =3.624 steres = 1.101 liters = 0.9463 liter 0.3785 dekaliter = 0.881 dekaliter 0.3524 hektoliter 1 gram =0.0527 ounce 1 kilogram =2.2046 lbs, 1 metric ton =1.1023 English ton Weights 1 oxmce=28.85 grams 1 lb. = 0.4536 kilogram 1 English ton = 0.9072 metric ton. Approximate Meteic Equivalents 1 decimeter 1 meter 1 kilometer 1 hektar =4 inches = 1.1 yards = f of mile =2| acres 1 stere, or cubic meter = i of a cord 1 liter _ 1 1.06 quart liquid 1 0.9 quart dry 1 hektoliter =2| bushels 1 kilogram =2i pounds 1 metric ton =2200 pounds 570 APPENDIX Equivalents of grams or c.c. in apothecary's weight. (From Merck's Materia Medica) : The following are approximate equivalents. 0.001 gm. ( or c.c. = -^ gm. or min. 0.3 gm. of c.c. == 5 grn. or min. 0.003 '' ff _ 1 a ■~ so 0.5 ' = 8 '' '' 0.004 " ti _ 1 ic "" 15 0.6 ' =10 '' '' 0.008 '' a __ 1 it — s 0.8 i =12 <^ '< 0.01 " " = i " '* 1 ' =15 '^ '' 0.015 '' — 4 4 ' = 1 dr. or f3-dr. 0.03 '' — 3 15 ^ = 4 ^' <« 0.05 '' "~ 4. 30 * = 1 oz, or fl-oz. 0.6 u ^1 a 120 ' =4 << <« 0,1 '' =1J '' 237 ' = 8 '' '' 0.2 '< =3 '' '' 475 * == 1 lb. or pint 0.25 '' ii =4 '< 950 * = 2 '' ^^ COMPARISON OF THERMOMETER SCALES Centi- grade. Reaumur. Fahren- heit. Centi- grade. Reaumur. Fahren- heit. Centi- grade. Reaura.ur Fahren- heit. -30 -24.0 -22 .t) 14 11.2 57.2 5S 46.4 136.4 -28 -22.4 -18.4 16 12 8 60.8 60 48.0 140 -26 -20.8 -14.8 18 14.4 64.4 62 49.6 143 6 -24 -19.2 -11.2 20 16,0 68.0 64 51 2 147.2 -22 -17.6 - 7.6 22 17.6 71.6 66 52 8 150 8 -20 -16.0 - 4.0 24 19.2 75.2 68 54.4 154.4 -18 -14.4 - 0.4 26 20.8 78.8 70 56.0 158 -16 -12.8 3.2 28 22.4 82.4 72 57.6 161.6 -14 -11.2 6.8 30 24.0 86.0 74 59.2 165.2 -12 ~ 9.6 10.4 32 25.6 89.6 76 60.8 168.8 -10 - 8.0 14.0 34 27.2 93 2 78 62.4 172.4 - 8 - 6.4 17.6 36 28.8 96.8 80 64.0 176.0 - 6 - 4.8 21.2 38 30.4 100,4 82 65.6 179.6 - 4 - 3.2 24.8 40 32.0 104,0 84 67.2 183.2 - 2 - 1.6 28.4 42 33.6 107.6 86 68.8 186.8 0.0 32.0 44 35.2 111,2 88 70.4 190,4 2 1.6 35.6 46 36.8 114.8 90 72.0 194.0 4 3.2 39.2 48 38.4 118.4 92 73,6 197.6 6 4.8 42.8 50 40.0 122,0 94 75.2 201.2 8 6.4 46.4 52 41.6 125.6 96 76.8 204.8 10 8.0 50.0 54 43.2 129.2 98 78.4 208.4 12 9.6 53.6 56 44.8 132.8 100 80.0 212.0 APPENDIX 571 CONVERSION OF PARTS PER MILLION TO GRAINS PER UNITED STATES AND IMPERIAL GALLONS Parts per Miilion. Grains per Imperial Gallon. Grains per United States Gallon. Parts per Million. Grains per Imperial Gallon. Grains per United States Gallon. 1 .0700 .0583 40 2.8000 2.3327 2 .1400 .1166 50 3.5000 2.9129 3 .2100 .1749 60 4.2000* 3.4990 4 .2800 .2332 70 4.9000 4.0822 5 .3500 .2915 80 5.6000 4.6654 6 .4200 .3499 90 6.3000 5.2486 7 .4900 .4082 100 7.0000 5.8318 8 .5600 .4665 200 14.0000 11.6630 9 .6300 .5248 300 21.0000 17.4950 10 .7000 .5831 400 28.0000 23.3270 20 1.4000 1.1663 500 35.0000 29.1290 30 2.1000 1.7495 * Other values may be computed from the above table. Sp. Gr. B "f"130 for Baume lighter than water. 140 Sp. Gr. for Baum^ Ughter than water. Sp. Gr. = 145 154-^* for Baum6 heavier than water. £°-145 145 Sp. Gr. for Baum^ heavier than water. 572 APPENDIX SPECIFIC GRAVITIES IN DEGREES BAUME Liquids Lighter than Watek Degrees Baume. Specific Gravity. Degrees Baum6. Specific Gravity. Degrees Baum4. Specific Gravity. 10 1.000 15 0.966 20 0.933 11 0.993 16 0.959 21 0.927 12 0.986 17 0.952 22 0.921 13 0.979 18 0.946 23 0.915 14 0.972 19 0.940 24 0.909 25 0.903 30 0.875 35 0.849 26 0.897 31 0.870 36 0.843 27 0.892 32 0.864 37 0.838 28 0.886 33 0.859 38 0.833 29 0.881 34 0.854 39 0.828 40 0.824 48 0.787 5S 0.745 41 0.819 50 0.778 60 0.737 42 0.814 52 0.769 65 0.718 43 0.805 54 0.761 70 0.700 46 0.796 56 0.753 75 0,683 APPENDIX 573 SPECIFIC GRAVITIES IN DEGREES BAUME AND TWADDLE Liquids Heavier than Water Hydeometer Reading Degkees. Twaddle. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 17 IS 20 21 23 27 28 29 30 31 32 33 34 Baum^. .0 .7 2!l 3.4 4.1 4.7 5.4 6.0 6.7 8^0 8.7 9.4 10.0 10.6 11.2 11.9 12.4 13.0 13.6 14.2 14.9 16.0 16.5 17.1 17.7 18.3 18.8 19.3 19.8 20.3 20,9 35 21.4 36 ^2.0 37 22.5 38 23.0 39 23.5 40 24.0 41 .24.5 42 25.0 Specific Gravity. 1.000 1.005 1 010 1.015 1.020 1.025 1.030 1.035 1.040 1.045 1.050 1.055 1.060 1 065 1.070 1 075 1.080 1.085 1 090 1 095 1.100 1.105 1.110 1.115 1.120 125 130 135 140 145 1.150 1.155 1.160 1.165 1.170 1.175 1.180 1.185 1.190 1.195 1.200 1.205 1.210 Hydbometer Reading Degrees Twaddle. 43 45 46 47 48 49 50 51 52 53 54 bb 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 Baumd. 25.5 26.0 26.4 26.9 27.4 27.9 28.4 28.8 29.3 29.7 30.2 30.6 31.1 31.5 32.0 32.4 32.8 33.3 33.7 34.2 34.6 35.0 35.4 35.8 36.6- 37.0 37.4 37.8 38.2 38.6 39.0 39.5 39.8 40.1 40.5 40.8 4116 42.0 42.3 42.7 43.1 Specific Gravity. 1.215 1.220 1.225 1.230 1.235 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1.285 1.290 1.295 1.300 1.305 1.310 X . OxO 1.320 X . Oil^O 1.330 1.335 340 345 350 355 360 365 370 375 380 1.385 1.390 1.395 1.400 1.405 1.410 1.415 1.420 1.425 574 APPENDIX SPKCIFIC GRAVITIES IN DEGREES BAUME AND TWADDLE.- {Continued) Liquids Heavier than Water Hydeometek Hydeometer Reading Degrees. Specific Gravity. Reading Degrees. Specific Gravity. Twaddle. Bauin6. Twaddle. Baum6. 86 43.4 1.430 129 56.6 1.645 87 43.8 1.435 130 56.9 1.650 88 44.1 1.440 131 57.1 1.655 89 44.4 1.445 132 57.4 1.660 90 44.8 1.450 133 57.7 1.665 91 45.1 1.455 134 57.9 1.670 92 45.4 1,460 135 58.2 1.675 93 45 8 1.465 136 58.4 1.680 94 46 1 1.470 137 58.7 1.685 95 46.4 1.475 138 58.9 1.690 96 46.7 1.480 139 59.2 1.695 97 47 1 1.485 140 59.5 1.700 98 47.4 1.490 141 59.7 1.705 99 47.8 1.495 142 60.0 1.710 100 48.1 1.500 143 60.2 1.715 101 48.4 1.505 144 60.4 1.720 102 48.7 1.510 145 60.6 1.725 103 49.0 1.515 146 60.9 1.730 104 49.4 1.520 147 61.1 1.735 105 49.7 1.525 148 61.4 1.740 ' 106 50.0 1.530 149 61.6 1.745 107 50.3 1.535 150 61.8 1.750 108 50,6 1.540 151 62.1 1.755 109 50.9 1.545 152 62.3 1.760 110 51,2 1.550 153 62.5 1.765 111 51.5 1.555 154 62.8 1.770 112 51.8 1.560 355 63.0 1.775 113 52.1 1.565 156 63.2 1.780 114 52.4 1.570 157 63.5 1.785 115 52.7 1.575 158 63.7 1.790 116 53.0 1.580 159 64.0 1 795 117 53.3 1.585 160 64.2 1.800 118 53.6 1.590 161 64.4 1.805 119 53.9 1.595 162 64.6 1.810 120 54.1 1.600 163 64.8 1.815 121 54.4 1.605 164 65.0 ' 1 820 122 54.7 1.610 165 65.2 1.825 123 55.0 1,615 166 65.5 1.830 124 55.2 1.620 167 65.7 1,835 125 55,5 1.625 168 65.9 1.840 126 55.8 1.630 169 66.1 1.845 127 56.0 1.635 170 . 66.3 * 1.850 128 56.3 1.640 171 66.5 1.855 APPENDIX 575 Chlorine Data Chlorine [x^<»>pos = green] a greenish yellow gas, easily compressed to a liquid, was discovered in 1774 by Scheele, a Swedish Chemist. Atomic weight 35.45, molecular weight 70.9, vapor density 35.8. Liquefies at-33.6^ C. (-28.5° R), solidifies at - 102° C. (-151.36° F.). Under pressure of 6 atmospheres liquefies at 0° C, (32° F.). 100 90 80 ?70 XI 1 60 gso g bo ^ g40 iso 3 ■g 3 20 I BlO -10 -20 -30 -40 ^ ^ "^ -30 ,^ "^ «a y y^ -20| ^/^ /" a y / A / p / k 1 /\ f' CURVE SHOWING PRESSURE 40 s / f CHLORINE GAS AT VARIOUS TEMPERATURES ■'" ' B / r =20 1 ) 2a ^ 3 4 5 6 Prei 7 jsure 8 :nPoi [) 1( erSq 10 a; uarel ,0 IS Inch JO IJ !0 V ,0 1! iO 11 10 r / / 7^0 WEIGHTS OF CHLORINE Datum. Gaseous Chlorine. Liquid Chlorine. Specific gravity Weight of 1 liter Weight of 1 cu. ft. Weight of 1 gal. 2.49 (air = l) 3.167 gms. 0.198 lb. 0.026 lb. 1.44 (water = 1) 1440 gms. 89.752 lbs. 11.999 lbs. One volume of liquid chlorine is .equivalent to 444.4 volumes of chlorine gas. 676 APPENDIX SOLUBILITY OF CHLORINE Temperature Solubility Ratio by Volume Pounds of Chlorine Soluble C " F o in 1,000,000 Gals of Water. 10 30 32 50 88 1 5 3 1 8 20,000 40,000 24,000 One part per million =a8 34 lbs per million gallons of water =*0 058 grain per gallon One gram per gallon -17 12 parts per million = 142.86 lbs. per million gallons of water ft^ Chlorine —Pounds Per 24 Hours. r^ 0,30.350.40.450.5 0.6 0-7 0.80.9i.ol.li.2l.3i.4l 5 2.0 2.5 3.03 ;h.o4.55.o <'.0 7.0 S.0 9.0 10 ^ui^ lijjilijjj|jji]yu!,iijj'iinliijjliiiiljiji)iiiilit'il^^^^ ffi* y^ iiiii'i iiiijiiii 55 5045 40 85 30 25 20 15 %sl2 n 10 gio 8-0 7.0 <5''0 6.0*^5 i;© 3.5 z^q 2ls ^ ™- — -e- Seconds Per Pulsation Chlorine -Poimds Per 24 Hours'. 20 19 18 17 16 15 U 13 12 11 ip 9 8 T 6 3.0 2.0 Water - MIUloii Galtons Per 24 HotiW. Fig 86. — Chlorine Data for the Treatment of Water Supplies. (Wallace-Tiernan Co.)' See also data on another page. APPENDIX 577 Transportation of Bacteriological Specimens. The preparation and shipment of specimens foi^ bacteriological examinationis a very impor- tant procedm-e. Unfortunately the bacteriologist and 'chemist may not have much to do with this step in the examination of any substance; usually, he is expected to handle the specimen after it has been delivered to his laboratory. At times, he may be quite justified in refusing to handle certain specimens which are sent because it would be dangerous to himself or to those with whom he comes in contact. Since much of the material for bacteriological examination is sent through the United States mails it is important that he have a knowledge of the postal rules and regulations which concern these shipments. Hasseltine (1918)* has given a good resume of the subject. The following quota- tion is cited by this author from the Postal Regulations issued by the Post Office Department. Sections 472 and 473 read as follows: Sec. 472. All kinds of poison, and all articles and compositions containing poison, and all poisonous animals, insects and reptiles, and explosives of all kinds, and inflammable materials and infernal machines, and mechanical, chemical, or other devices or compositions, which may ignite or explode, and all disease germs or scabs, and all other natural or artificial articles, compositions or materials of whatsoever kind which may kill, or in any wise hurt, harm or injure another, or damage, deface, or otherwise injure the mails or other property, whether sealed as first- class matter or not, are hereby declared to be non-mailable matter, and shall not be conveyed in the mails or delivered from any post oflS.ce or station thereof, nor by any letter carrier; but the Postmaster General may permit the transmission in the mails, imder such rules and regula- tions as he shall prescribe as to preparation and packing, of any articles hereinbefore described which are not outwardly or of their own force dangerous or injurious to life, health or property. (The rest of this section is not concerned with shipment of bacteriological materials.) Sec. 473. 1. Specimens of diseased tissues may be admitted to the inail for transmission to United States, State, municipal, or other labora- tories in possession of permits referred to in paragraph 3 of this section only when inclosed in mailing cases constructed in accordance with this regulation: Provided, That bacteriologic or pathologic specimens of plague and cholera shall under no circumstances be admitted to the mails. 2. Liquid cultures, or cultures of microorganisms in media that are ♦Hasseltine, H. E. 1918. Public Laboratory Specimens, Their Preparation and Shipment. Public Health Reports 32, 2016-2032. Reprint from the same No. 438-1918. 578 APPENDIX fluid at the ordinary temperature (below 45° C. or 113^ F.), are unmail- able. Such specimens may be sent in media that remain solid at ordi- nary temperature. 3. No package containing diseased tissue shall be delivered to any representative of any of said laboratories until a permit shall have first been issued by the Postmaster General, certifying that said institution has been found to be entitled, in accordance with the requirements of this regulation, to receive such specimens. 4„ (a) Specimens of tubercular sputum (whether disinfected with carbolic acid or not disinfected) shall be transmitted in a solid glass vial with a mouth not less than 1 in. in diameter and capacity of not more than 2 oz., closed by a cork stopper or by metallic screw top protected by a rubber or felt washer. Specimens of diphtheria, typhoid, or other infectious or communicable diseases or diseased tissue shall be placed in a test tube made of tough glass, not over f in. in diameter and not over 7| ins. in length, closed with a stopper of rubber or cotton and sealed with paraffine or covered with a tightly fitting rubber cap. (6) The glass vial or test tube shall then be placed in a cylindrical tin box, with soldered joints, closed by a metal screw cover with a rubber or felt washer. The vial or test tube in this tin box shall be completely and evenly surrounded by absorbent cotton, closely packed. (c) The tin box, with its contents, must then be inclosed in a closely fitting metal, wooden, of papier-m^che block or tube, at least -5^ in. thick in its thinnest part, of sufficient strength to resist rough handling and support the weight of the mails piled in bags. This last tube shall be tightly closed with a screw-top cover with sufficient screw threads to require at least one and one-half full turns before it will come off, and fitted with a felt or rubber washer. (See Fig. 8.) 5. Specimens of blood dried on glass microscopic sHdes for the diag- nosis of malaria or typhoid fever by the Widal test may be sent in any strong mailing case which is not liable to breakage or loss of the specimen in transit/. 6. Upon the outside of every package of diseased tissues admitted to the mails shall be written or printed the words ^' Specimen for bac- teriological examination. This package to be pouched with letter mail. APPENDIX 579 Frost's Plate Counter. Each of the larger squares represents an area of 1 sq cm. The figures 60 and 40 itidicate the number of square centimeters m the circles bounded by the respective lines Each sector is one-tenth the area of its circle. Department of Bacteriology, University of Ilhnois. 580 APPENDIX ■■■■■I I Frost's Plate Counter. Each of the larger squares represents an area of 1 sq. cm. The figures 60 and 40 indicate the number of square centimeters in the circles bounded by the respective lines. Each sector is one- tenth the area of its circle. Department of Bacteriology, University of Illinois. INDEX Acetaldehyde reaction for ethyl alcohol, 194 Acid fast staining, theory of, 87 , Ziehl-Niehlsen method, 86 Acidity, degrees recognized in milk, 114 — degrees recognized in clear media, 115 Aciduric bacteria, 236 Action of bacteria on carbohydrates, 193 — ~ — glucosamine, 191 — . polypeptides, 190 proteins, 189 bile on bacteria, 234 certain bacteria on proteins, 367 Adjusted milks, defined, 374 Adulteration of compressed yeast with brewer's yeast, 220 Advantages of ''bleach" in water treat- menti, otuo — — pasteurization of milk, 416 Aerobic respiration, 241 Aeroscope, 264 Agar, 43 — colonies, study of, 110 — for Ps. radicicola, 50 — gelatin medium (North), 51 — medium, plain, 48 — stroke, ^tudy of, 110 Age of milk, 377 Agglutination of bacteria, 320 Alanin, 184 Albuminoid- ammonia, determination in water, 282 Alcohol as a disinfectant, 140 — table, 561 Alcoholic fermentation, 194 Alkaline blood agar, 70 — hemoglobin agar, 70 — methylene blue, 90 Alkalinity, determination in water, 285 Allen's microscopic method of milk analysis, 404 Alwood fermentation valve, 220, 221 Analysis of mud deposits, 352 sewage sludge^ 352 Anderson's method for tubercle bacilli in milk, 413 Amino acids in gelatin, 41 proteins, 184 Ammonia production by pure cultures, 116 Ammonium chloride in bread, 218 Anaerobic methods, 17 — • respiration, 242 Analysis of Liebig's meat extract, 41 AniHne gentian violet, 90 Anti-protein sera, 491 Antiseptic values, 143 Albargin, disinfectant, 135 Aperture, numerical, 28 Aqueous alcoholic stains, 81 — stains, preparation, 81 Argentamin, disinfectant, 135 Argentose, 135 Arginine, bacterial decomposition, 244 — , chemical structure, 244 Arnold steam sterilizer, 126 Arnold's test for heated milk, 372 Ascomycetes, 202 Asepsis in food preservation, 495 Ash in milk, 368 Aspaftic acid, 186 Atomic weights, 563 Autoclave, 12§ Available chlorine, 136 — oxygen in water, 344 Ayer's milk tube method, 408 B Basidiomycetes, 202 Babcock test for fat in milk, ^9 581 582 INDEX Bacillus antliracis, isolation from water, 322 — colon index, 314 test in water analysis, 299 — diphtherise in milk, 414 ~~ dysenteriae, isolation from water, 320 — enteritidis m water, 297 — tuberculosis in feces, 258 ~~~ *~~~" -■^-* mixiv, 'xx.iCi water, 323 — typhosus in butter, 426 cream, 437 feces, 254 isolation from water, 317 ^ Carnot and Ralle's method, 256 , Holt-Harris-Teague method, 255 , Kendall and Day's method, 255 , Lumsden and Stimson's meth- od, 254 , Morishama and Teague's method, 257 , Teague and Clurman's method, 257 , longevity in water, 343 Bacteria and life, 233 — , Eberle-Klein method, 248 — in baked bread, 224 cream and cream ripening, 423 egg white, 459 yolk. 459 feces, 238 fresh eggs, 460 self-rismg bread, 223 the stomach, 230 — , MacNeaFs procedure for Winterberg method, 247 — , MatiU and Hawk's procedure, 252 — , Steel's modification of the Strass- burger method, 250 Bacterial count in milk, significance, 385 water analysis, 290 — examination of butter, 423 eggs, 463 ice, 337 milk, 375 — groups in ice cream, 435 raw and pasteurized milk, 417 — v^, chemical examination of water, 271 Bacteriological specimens, transporta- tion, 577 Bacteriology of butter, 423 Cheddar cheese, 429 ice, 336 Balaz's test for boiled milk, 373 Barn conditions in milk production, 377 Barsiekow's medium, 59 Baskets, various types of, 7, 8 Bean agar, 50 Seattle's method for anaerobic bacteria, 22 Beer wort, 57 Beijerinck's medium for nitrate reduc- tion, 67 Benedict's method for estimation of dextrose, 197 reducing sugars, 196 Benzoic acid, detection in milk, 371 Bergey's method for typhoid bacilli in butter, 426 Besson's method for tubercle bacilli in milk, 413 Bile, germicidal action, 234 — salt broth, 46 Biochemical oxygen demand of sewage, 350 Bismuth compounds in disinfection, 135 Blanching, 507 Blood agar, 69 , alkaline, 70 for streptococci, 69 — films, staining, 87 — serum, 69 Boiling in sterilization, 126 Boric acid in disinfection, 139 milk, determination, 371 Bottcher's counting chamber, 10 — moist chamber, 76 Botulism, 533 Bread making, 222 — medium, 59 Breed's capillary pipettes, 397 Breed smears in milk analysis, 397 Bromine as a disinfectant, 134 — reaction for tryptophane, 192 — water test for skatol, 247 Brown's fermep,tation tube, 1 Buchner's ana^obic methods, 17 INDEX 583 Bushnell and Mauer's method for egg analysis, 465 Butter flavor, 423 Cadaverine, 529 Calcium compounds in disinfection, 135 — oxide, 138 Calibration of micrometer oculars, 36 Campbell's method for tubercle bacilli m milk, 413 Candling eggs, 461, 463 Canned meats, 481 Canning, 506 Cannon's hydrolized casein medium, 63 Capaldi's egg medium, 58 Capsules, determination on bacteria, 109 Capsule stain, Huntoon's method, 84 , Muir's method, 83 , Welch's method, 84 Carbohydrate agar, 48 — broths, 44 Carbohydrates, action of bacteria on, 193 — , classification, 193 Carbol fuchsin, 90 Carbonated broth, 47 Carbon in disinfection, 140 Carnot and Halle's method for typhoid bacilli in feces, 256 Carriers and food infection, 530 Carrot infusion medium, 60 Case history of botulism, 535 Casein agar after Ayers, 70 — medium, 63 Causes of food poisoning, 533 Cellulose agar, 61 Certificates for chemical examination of water, 276, 277 Characteristics of a good indicator oi pollution, 294 anaerobic water bacteria, 298 fermentation, 240 milk-borne epidemics, 541 water-borne epidemics, 541 Cheddar cheese, bacteriology, 429 Cheese bacteriology, 428 — poisoning, 431 — ripening, 428 Chemical analyses of ice, 337 — and bacterial examination of water, 271 — changes in milk from souring, 366 — constituents of milk, 363 — examination of milk, 367 — standards for milk, 373 Chicago drainage canal and Illinois River, 341 Chicken broth medium, 46 Chinese ink preparations, 78 Cholera red reaction for M, cholerm in water, 322 Chlorine data, 575 Chloramine, 139 — m water treatment, 326 Chloride of lime in disinfection, 135 Chlorine as a disinfectant, 132 — , determination in water, 280 Cholera, M. microspira in feces, 257 Chopped meats, 480 Chromatic aberration, 29 Classification of water bacteria, 193, 286 proteins, 182 yeasts, 213 Cleaning apparatus, 6 — hemocytometers, 16 — solution, 6 Coagulation coefficient of disinfectants, 171 — of milk by rennin, 365 Coarse adjustment on microscope, 25 Cohn's classification of bacteria, 94 — solution, preparation, 63 , characteristics of bacteria in, 112 Cold storage, 505 Cole's method for microscopic agglutina- tion, 321 Collection of milk samples, 393 water samples, 273 Colloidal silver in disinfection, 135 Color, determination in water, 278 — reactions of proteins, 191 Coloring matter in milk, 371 Combined acidity of the stomach, 233 Comparison of thermometer scales, 670 Composition of hens* eggs, 453 milk serum, 365 tomato pulp, 514 584 INDEX Completed test for B. coli in water, 302 Compounds in goats' and human milk, 364 Compressed yeast, 216 , microscopic examination, 220 — ■ — , outline of manufacture, 217 Concave slides, types, 77 Concentrated solutions in food prepara- tion, 498 Condensed milk, 433 Conjugated proteins, 183 Conn's method for bacteria in butter, 428 Constituents of media, 39 Cophn's jars for staining, 80 Copper salts in disinfection, 139 Corn agar medium, 50 Cover glasses, sizes and thicknesses, 5 Cresol as a disinfectant, 141 Cystine, decomposition, 245 — , structure, 185 Czapek's solution, 66 D Dairy score card, 380 Dakin-Carrel solution in disinfection, 137 Dark ground illumination, 78 Deaminization, 243 De Bary's classification of bacteria, 95 Decarboxylation, 243 Decoction of dried fruits, 59 Delepine's method for tubercle bacilli in mUk, 412 Derived proteins, 183 Descriptive chart, 100 Description of microorganisms, 101 Detection of foreign proteins, 490 Determination of the origin of epidemics, 544 Development of the Descriptive Chart, 100 Dextrose potassium phosphate broth, 47 Diagnosis of food poisoning, 535 Dialysis sterilization, 128 Dialyzed milk, 72 Diet and the intestinal bacterial flora, 235 Differential staining of blood films, 87 Dilution bottles, 2 — pipettes for blood counts, 15 Dilutions, 10 Direct V8. plate count on milk, 396 Dirt in milk, 384 Disadvantages of ''bleach" in water treatment, 325 Disinfectants, standardization, 144 Disinfection, 131 — vs. periodic system, 133 Dissolved oxygen in water, 344 Doane-Buckley method for leucocytes in milk, 410 Dolt's agar medium, 50 — medium, 65 Dorset's egg medium, 58 Dough raising power of bread yeasts, 220 Dox's solution for fungi, 65 Draw tube on microscope, 27 Dried eggs, 46 Drigalski and Conradi's medium, 62 Dry heat sterilization, 121 Drying in food preservation, 504 Dunham's solution, 47 Durham's fermentation tube, 1 E Effect of baking on microorganisms in bread, 224 low temperatures on bacteria, 504 pasteurization on bacteria in milk, 417 pressure in food preservation, 506 storage on ice cream bacteria, 436 yeast on flavor of bread, 222 Efficiencies of peptone, 40 Efficiency of bread yeasts, 220 Egg-meat mixture for putrefaction, 58 Ehrenberg's work on classification of bacteria, 94 Ehrlich's reaction for indol, 115 Elements in inorganic compounds, 187 — of ''quality" in milk, 381 Eisner's potato medium, 60 Endo's agar medium, Kendall's method, 52 , Hygienic Labotatory method, 53 — ' — — , Levine's method, 54 — medium for isolating 0. typhosus from water, 318 INDEX 585 Endospore, observation of, 109 Enzymes, 118 — in eggs, 460 Eosin-brilliant green agar medium, 54 methylene blue agar medium, 54 Epidemics of paratyphoid fever, 532 Errors in staining fiagella, 85 Erythrocytes, enumeration, 13 Esten and Mason's method for bacteria in ice cream, 437 Estimation of bacteria in tomato prod- ucts, 517 dextrose (AUihn's method), 196 molds in tomato products, 515 Ethyl acetate reaction for alcohol, 195 — alcohol, aldehyde reaction, 194 — ■ — , iodoform reaction, 194 Evaluation of bleaching powder, 328 Examination of bread, 224 canned foods, 511 clams, 489 Hamburger steak, 483 meat, 482 molds, 212 oysters, 485 sausage, 483 shell fish, 483 tin cans for leaks, 513 water for available chlorine, 329 Factors influencing bacteria in milk, 375 eggs, 458 disinfection, 133 Fasting and intestinal bacteria, 236 Fats, 197 — in milk, 368 Fecal and non-fecal B. coli, 308 FehHng's solution, preparation, 195 — test for reducing sugars, 195 Fermentation in food preservation, 497 the intestinal tract, 240 — of sugars by streptococci, 295 — reactions of pure cultures, 117 — tubes, types, 1 Fermented milks, 433 Fermenting powers of yeasts, determina- tion of, 220 Fermi's culture fluid, 68 Fernbach's antitoxin flask, 5 Ferric chloride test for phenol, 247 Ferrous sulphate in disinfection, 139 Filtration through liquids, 128 — sterilization, 127 Fine adjustment of the microscope, 26 Fiagella staining, 86 Flaming sterilization, 122 Flasks, types of, 5 Flat sours in canning, 511 Flavor of butter, 423 Flours used in bread making, 222 Fluorescein for tracing water pollution, 323 Food-borne epidemics of disease, 542 Food poisoning, 528 — preservation, 494 Formaldehyde in disinfection, 142 Forms of growth, 102 Frankel and Voges' solution, 68 Frankel's solution, 64 Free ammonia in water, determination, Freudenreich's flask, 5 Frost and Ravenel's test for heated milk, 372 Frost's microscopic method for milk analysis, 401 — plate counter, 579, 580 Frozen eggs, 463 Fructification in molds, 203 Fuller and Johnson's classification of water bacteria, 289 Fungi, 202 — imperfect!, 202 G Gases in canned foods, 510 Gastric analysis, 233 — juice, germicidal action, 229 Gedding's method for M. cholerce in feces, 257 C^elatin, preparation and composition, 42,43 — agar, 56 — , colonies, study of. 111 — stab, study of, 111 586 INDEX Gerber's test for milk fat, 369 Germicidal action of saliva, 228 gastric juice, 229 Giblet broth, 46 Giltay and Aberson's solution, 67 Glossary of terms, 105 Glucosamines, bacterial action on, 191 Glucose formate broth, 47 agar, 49 Glutamic acid, 186 Glycerol in disinfection, 142 — potato broth, 60 Glycocoll, 184 Grades on milk, 391 — milk by bacterial count, 387 Grading eggs, 455 Gram positive and negative bacteria, 83 — stain, Nicoll's modification, 83 , theory of, 81 Graves' modification of the Durham fermentation tube, 2 Griess method for nitrites, 116 Groups, bacteriological, 99 -of bacteria in intestines, 236 XX Halogens as disinfectants, 134 Ham souring, 499 Hanging blocks, 77 — drops, 77, 78 Harding and Prucha's method for bac- teria in cheese, 431 Haricot decoction, 58 Harrison's haethod for bacteria in cheese, 432 Hay infusion, 57 Hazen theorem, 269 Heated milk, determination of, 371 Hemocytometer rulings, 14 — chamber, 12 Heese agar medium, 51 -andNiedner'sag;ar,50 High-pressure steam sterilization, 122 Hiss agar-gelatin medium, 51 — medium, 56 Histidine, 187 Holt-Harris-Teague method for B, typho- sws in feces, 255 Hot air oven sterilization, 122 Houston's method for isloating strepto- cocci from feces, 296 Huntoon's method for staining capsules, 84 Hydrogen sulphid formation, 116 Hydrolysis of carbohydrates during sterilization, 44 Hyphse, structure of, 212 Ice and typhoid fever, 339 — cream, 434 — — plant score card, 439 Identification of bacteria by agglutina- tion, 321 Illuminating power of the microscope, 29 Illumination of the microscope, 31 Incidence of tubercle bacilli in market butter, 427 milk, 414 Incineration in sterilization, 122 Inclination joint on the microscope, 25 Indol from tryptophane, 244 — , test for, 115 Indicator solutions, 553 Indicators of pollution, 294 Infection of food by carriers, 530 Influence of salt on bacteria, 499 Initial contamination of milk, 375 Inorganic disinfectants, 133 — elements in organic compounds, 187 Inspissator, 127 Instructions for collecting water samples, 274 Intermittent sterilization, 127 Interpretation of bacterial tests on milk, 387 results in water analysis, 315 Intervals before analysis of water samples, 273 Intestinal bacteria, effect of protein diet, 236 water drinking, 237 — flora, classification, 238 Invigoration of cultures, 108 Iodine absorption number of fats, 198 — in disinfection, 134 JLlN JJJcjJV. 587 lodoformogen, 134 Iodoform reaction for acetone, 195 ethyl alcohol, 194 lodo-hemol, 134 lodol, 134 lodomuth, 134 Isolation of B. ententtdzs sporogenes from water, 299 B. typhosus from water, 317 pure cultures, 10 Isoleucine, 185 Jeffer's counting plate, 9 Jensen's system of classification of bac- teria, 97 Jones' anaerobic method, 23 Jordan's classification of water bacteria, 290 •— non-protein medium, 66 Kendall and Day's procedure for J5. typhosus in feces, 255 Ketchup, 514 Key to Aspergillus molds, 210 genera of budding fungi, 215 true yeasts, 214 penicillia, 207 Koch's concave slide, 76 — culture flask, 5 Koettstorfer number of fats, 199 KoUe culture flask, 5 Kusserow's method for determining fermenting power of yeasts, 221 Kuster's anaerobic method, 18 Labarraque's solution, 137 Lactic acid fermentation, 193 in the stomach, 233 Lactose bile in water analysis, 311 Lafar's counting plate, 9 Leavening agent in self-rising bread, 223 Leeuwenhoeck's work on bacteria, 94 Leffman-Beam test for milk fat, 369 Lehmann and Neumann's work on classification, 97 Lellendahl's staining dish, 80 Lentz's anaerobic apparatus, 18 Leucine, structure, 185 — , enumeration, 15 Leucocytes in milk, 408 Levine's eosin-methylene blue agar, 54 Liebermann's reaction, 192 Liebig's meat extract, 41 Life cycles of bacteria, 99 Light, sterilization, 128 Lime in disinfection, 136 Lipman-Brown medium, 66 Liquid chlorine, addition to water, 331 Lister culture flask, 5 Lister's work on milk, 95 Litmus gelatin, 56 — lactose agar, 48 -milk, preparation, 71 , cultural characteristics in, 115 — whey, 71 Lohnis' method for bacteria in butter, 426 cheese, 432 Loss of virulence on culture media, 118 Low temperature, effect on bacteria, 504 Lugol's iodine solution, 90 Lumsden and Stimson's method for B. typhosus in feces, 254 Lymphocytes, staining of, 89 Lysine, decomposition, 245 — , structure, 186 Lysol in disinfection, 141 M MacKenzie's culture flask, 68 McLeod's anaerobic method, 18 Magnifying power of microscope, 29 Magnification tables, microscopes, 33, 34 Malachite green medium, 52 Malt extract medium, 68 Mayer's culture fluid, 62 Meat extract, composition of, 40, 41 , partition of nitrogen in, 42 — infusion, preparation, 40 — juice (infusion), 41 Medium for fungi, 65 588 INDEX Meissl's method for determiniBg fer- menting power of fungi, 220 Metals, action of on bacteria, 133 Methods of adding ''bleach" to water, 327 water treatment, 324 — for study of yeasts, 218 Methyl red test in water bacteriology, 307 Micro-analysis of tomato products, 515 Micrometer, ocular, 436 Micrometry, 34 Microscope, 23 Microscopic agglutination, 321 — examination of gastric contents, 233 — method for milk analysis, 395 Microspira cholerm, in feces, 257 — 1 milk, 413 water, 322 Migula's classification of bacteria, 95 Milk agar, preparation of, 72 — borne epidemics, 541 — medium, characters of bacteria in, 112 , preparation of, 71 — of lime, 138 — proteins, 365 — samples, 396 — standards, 392 Millon's reaction, 191 Mills-Reincke phenomenon, 268 Modified aeroscope, 265 Mohler's method for bacteria in cheese, 432 Moist chamber preparations of yeasts, 219 — heat sterilization, 122 Molds in butter, 425 — , structure, 203 Moore's staining dish, 80 Miquel culture flask, 5 Mordants, 81 Morishama and Teague's method for B. typhosus in feces, 257 Motility, 109 Mucor, 205 — mucedo, 204 Muir's method for staining capsules, 83 Miiller's classification of bacteria, 94 Mycoderma in bread yeast, 221 Naegeli's medium, 63 Naples jar for staining, 80 Neisser's method for spore staining, 85 Nesslerization, 281 Neutral red agai^j 50 bile salt aglir, 49 broth, 46 NicolFs modification of Gram's stain, 83 Nitrate broth, 45 Nitrates, determination in water, 283 Nitrite formation, 116 — , test for, 116 Nitrites, determination in water, 283 Nitrogen-free medium for bacteria, 65 — , significance in water analysis, 280 Nitroprusside reaction for acetone, 195 Nitrosoindol test, 115 North's agar-gelatin medium, 51 Novy's anaerobic method, 23 Number of bacteria in feces, 238 Numerical aperture, 28 Nutrient broth, preparation, 44 , characteristics of bacteria in, 110 — gelatin, 55 Nylander's test for reducing sugars, 196 O Objections to pasteurization, 416 Objectives on microscope, 27 Oculars on microscope, 31 Odor, determination of, in water, 279 Oidium lactis, 205 Omelianski and Winogradski's medium, 67 Opinions on bacterial count in milk, 389 Organic acids in the stomach, 233 Organic halogens, test for, 189 — nitrogen, test for, 187 — sulphur, test for, 188 Origin of bacteria in meat, 479 epidemics, 544 Ox-bile medium, 59 Oxygen consumed, determination of, in water, 284 — relations, study of, 110 — solution in water, 345 INDEX 589 Paratyphoid fever, 531 Parietti's solution, 62 Partially confirmed test for B, coli in water, 301 Pasteur's medium, 66 Pasteurization of milk, 414 Pathogenic bacteria in river watar, 343 and pasteurization of milk, 419 — streptococci in milk, 422 Pathogenicity of pure cultures, to ani- mals, 117 , to plants, 118 Pea-flour extract, 57 Penicillium, 207 Peptone, 40 — sucrose solution (Buchanan), i57 Permeability of egg shells, 459 Petri dishes, types of, 2, 3 Pettenkofer's experiment on cholera, 230 Phenol in disinfection, 141 — from tyrosine, 243 Phenylamin, 185 Phenyl-hydrazine reaction, 196 Phycomycetes, 202 Physiological method for dilutions, 219 Pickling in food preservation, 503 Piorowski's culture flask, 5 Plain milk medium, 71 Plan of modern corn packing plant, 508 Plate vs. microscopic count oi^ milk, 396 Platinum wires, 7 Polynuclear neutrophilic leucocytes, 89 Polypeptids, bacterial action on, 190 Potato agar, 61 — gelatin, 60 ' — infusion, 60 — mash, 60 — slant, study of, 111 -^starch jelly, 111 Pouring plates, 9 Prazmowski's culture flask, 63 Precipitin test for proteins, 491 Preparation of common stains, 90 standard reagents, 554 Preservation of food by asepsis, 495 by concentrated solutions, 498 Preservatives in milk, 371 ^"^ Pressure and head of water, 565 Presumptive test for B. coli, 301 Procedure for B. coli identification at Cincinnati, 313 studying water bacteria, 287 Process of canning, 507 Processing canned foods, 509 Proline, 187 Properties of ice, 336 Proskauer and Beck's culture medium, 68 Protargol in disinfection, 135 Proteins, bacterial action on, 189 — , color reactions of, 191 — , m milk, 371 — , precipitation reactions, 192 — , structure of, 184 •■ Prune decoction, 57 Pseudo yeasts, 213 Ptomaines, 529 Pure cultures, invigoration of, 108 , isolation of, 10 — — , methods of study, 108 , transferring, 10 — water, 271 Putrefaction in the intestines, 243 Putrescine, 529 Qualitative tests for elements in pro- teins, 189 — estimation of fecal indol, 246 ''Quality" in milk, 379 Quinone in disinfection, 143 R Rack for fermentation tubes, 1 Raisin gelatin, 56 Rating of oysters, 487 Raulin's solution for molds, 66 Reaction in milk cultures, 114 Reagents, preparation of, 554 Red corpuscles, 13 Reddening of cod, 503 Redfield's medium, 59 Reed's methods for leucocyte in milk, 409 Reichert-Meissl number of fats, 199 590 INDEX Relation between bacteria and dirt in milk, 384 — of microscopic to plate count, 407 pressure to temperature, 122 the 37° to the 20° C. count, 291 water filtration to typhoid fever, 270 Relative stability of streams, 347 Reliability of bacteria tests on milk, 386 Reporting milk analysis, 395 Required oxygen in water, 345 Residue, determination of, in water, 279 Rettger's aeroscope, 264 — method for egg analysis, 467 Revised method for tomato products, 520 Rhizopus, 205 Rice milk medium, 72 Ropy bread, 224 Roszahey's counting flask, 9 Routine milk analysis, 393 — steps in bacterial water analysis, 305 Roux culture flask, 5 RusselFs medium, preparation of, 52 , use in water analysis, 318 Sabourand's agar for yeasts, 51 Salicylic acid in milk, detection, 371 Saliva, germicidal action of, 228 Salt in food preservation, 498 Sample bottles, 5 Sampling of milk, 367 Sanitary inspection in water hygiene, 269 Saponification number of fats, 199 Sausage bacteriology, 481 Savage's method for bacteria in ice cream, 437 leucocytes in milk, 409 tubercle bacilli in butter, 426 Scheme for identifying yeasts, 219 Schneider's method for bacteria in butter, 428 Score card, 380 Sediment in milk, 384 Self-improvement of streams, 340 Self-rising bread, 223 Seliwanoff's reaction for ketoses, 195 Separation of B. coli and streptococci in water, 297 Serine, 185 Serum agar, 70 Serum bouillon, 47 Sewage streptococci, 296 Shell membrane of eggs, 453 Significance of bacterial count in milk, 385 Silicate jelly, 117 Silver citrate, 135 — , colloidal in disinfection, 135 — salts as disinfectants, 135 Sizes and capacities of tin cans, 510 Slaked lime, 138 Slides for mounting bacteria, 5 Slimy bread, 224 Smear, preparation, 80 Smillie's method for anaerobic bacteria, 20 Smith's fermentation tube, 1 Smoking in food preservation, 497 Soil extract agar, medium, 49 gelatin medium, b^ Solubility of oxygen in water, 564 Sources of water supply, 269 Souring of milk, 365 Specific gravities of common acids, 559 — gravity of milk, 368 Spherical aberration, 29 Spore staining, 85 Springers in canning, 511 Spronck's peptone yeast extract, 57 Stability in streams, 347 Stabilized gentain violet, 91 Staining, methods, 81 — , theory, 79 Standard aeroscope, 264 — loops, 7 Standardization of the microscope, 398 Starch agar, 60, 62 — jelly, 61 Steam sterilization, theory of, 124 Steel's method for fecal bacteria, 252 Sterilization methods, 121 Stewart's counting apparatus, 11 Stiles' method for egg examination, 463 Stokes' method for leucocytes in milk, 409 INDEX 691 Storage of food in tin cans, 513 Storch's method for heated milk, 371 Strassburger method for fecal bacteria, 250 Streaming steam sterilization, 126 Strength of yeasts, 220 Streptococci in water, 297 Structure of eggs, 453 hyphse, 212 Structure of molds, 202 Substage on the microscope, 27 Sugar in food preservation, 503 Sullivan's culture fluid, 64 Swells in canning, 510 Synthetic agar, 49 Tsague and Clurman's eosin-brilliant green agar, 54 method for B. typhosus in feces, 257 Travis' method for ikf. cholerce in feces, 258 Temperature of milk, 378 — relations of bacteria, 112 Theory of pasteurization, 415 staining, 79 Thermal death point, 118 Thymol, disinfectant, 141 Tin cans, 509 Tobacco smoke, effect on mouth bac- teria, 228 Toleration of bacteria to acids and alkalis, 118 Torrey's anaerobic method, 19 Total acidity of the stomach, 233 Toxicity coefficient of disinfectants, 175 Toxin of BadlluB hotulinuSf 533 Tracing pollution in water, 323 Transporting bacteriological specimens in the U. S. mail, 677 Treasury Department Standard for water examination, 311 True yeasts, 213 Trypsin broth, 45 Trjrptagar, 49 Tryptophane, bromine reaction, 192 — broth, 47 Tryptophane, decomposition of, 244 — , structure, 186 Tubercle bacilli in butter, 425 Turbidity, determination in water, 278 Typhoid bacilli m ice, 338 — death rate in American cities, 271 — fever case record, 548 from oysters, 487 in Pittsburgh, 268 Tyrosine, Decomposition, 243 — , structure, 185 Ultra-violet light, action on microor- ganisms, 130 Urine medium, 58 Uschinsky's medium, characteristics of bacteria in, 112 Use of the microscope, 24 Utensils influencing milk production, 377 Valine, 185 Van Delden's solution, 64 Van Ermengen's procedure for staining flagella, 86 Vegetable bouillon, 46 Vegetative cells, 109 Ventilation tube, 220 Vincent's method for tomato products, 528 Voges-Proskauer reaction in water anal- ysis, 307 W Ward's classification of water bacteria^ 288 Water bacteria, classification, 286 Water-borne epidemics of disease, 541 — drinking and bacteria in intestines, 237 Welch's method for staining capsules, 84 Whey agar, 71 — gelatin, 55 White corpuscles, enumeration, 15 Wiley's method for streptococci in ice cream, 437 592 INDEX Wine medium, 57 Winogradski's medium for nitrite for- mation, 67 Winterberg method for fecal bacteria, 247 Working distance of the microscope, 30 Wolffhiigel's counting apparatus, 9 Wording reports on water analysis, 317 Wort gelatin, 56 Wright's stain, preparation, 88 — anaerobic method, 18 — procedure for staining blood films, 87 X Xantho-proteic reaction, 191 X-ray fluorescence, bactericidal prop- erties, 129 Yeast foods, 216 — , genera of true, 214 — in bread making, 222 — in feces, 258 Yeasts, classification of, 213 Zichl-Niehlsen method of acid staining, 87 Zinc chloride in disinfection, 139 Zinsser's anaerobic method, 19 fast