»E. 59th St.. N.Y.I Columbia ^iiibtrsittp l^tUrmtt IGtbrarg PRACTICAL SANITARY SCIENCE Digitized by tine Internet Arciiive in 2010 witii funding from Open Knowledge Commons http://www.archive.org/details/practicalsanitarOOsomm PRACTICAL SANITARY SCIENCE A HANDBOOK FOR THE PUBLIC HEALTH LABORATORY BY DAVID SOMMERVILLE B.A., M.Sc, M.D., M.R.C.P. (Lond.), D.P.H. (Camb.), F.C.S. ASSISTANT-PROFESSOR OF HYGIENE AND PUBLIC HEALTH, WITH CHARGE OF THE LABORATORIES OF HYGIENIC CHEMISTRY AND PHYSICS, UNIVERSITY OF LONDON KINg's COLLEGE; EXAMINER IN BACTERIOLOGY AND CHEMISTRY, B.SC. (pUBLIC HEALTH), UNIVERSITY OF GLASGOW J EXAMINER IN D.P.H., UNIVERSITY OF ABERDEEN; MEDICAL OFFICER OF HEALTH, harrow; late DEMONSTRATOR OF PHYSIOLOGY IN THE MEDICAL SCHOOL OF ST. Thomas's hospital SECOND EDITION NEW YORK WILLIAM WOOD & COMPANY MDCCCCXV \ r^ f >"^ K A 4 X6' PREFACE TO THE SECOND EDITION The arrangement in chapters has been altered, and con- siderable additional matter has been added. The introduction to qualitative chemical analysis has been discarded in order to prevent increase in size of the book. Some less frequently occurring operations are outlined in a brief appendix. D. S. University of London King's College, November, 1914. PREFACE TO THE FIRST EDITION This little book is a brief summary of the course of practical lecture-demonstrations given to the D.P.H. class at King's College, London. Its intention is to put in the hands of students working in laboratories of Public Health a short outline of the more important matters — chemical, ph3'sical, etc. — discussed at practical examinations in Sanitar}' Science. The methods described are few, but it is hoped they will be found reliable. It is felt that where a large field must be cultivated in a limited time, it is better to use a few tools which have been well tried. Whilst going through the work, the student will do well to constantly refer to elementary up-to-date textbooks in the subjects of experimental physics, systematic organic and inorganic chemistry, analytical chemistr}-, geology, and bacteriolog3^ Further, it will be necessary for him at the outset to bear in mind that no amount of theoretical reading can be made a substitute for the laborious and constant use of the test-tube, microscope, etc., which must take place at the benches. A short account of the preparation of the standard solutions referred to in the work, and a few brief notes on the general chemical reactions of the more commonly occurring metals and acids, are set out in an appendix. D. S. King's College, November, 1905. CONTENTS CHAPTER PA(;E I. GENERAL OBSERVATIONS UPON POTABLE WATERS IN RELA- . TION TO THEIR SOURCE, AND METHODS OF EXAMINATION ADOPTED FOR SAFEGUARDING THEIR PURITY - - I II. THE PHYSICAL EXAMINATION OF WATER - - - 7 III. THE CHEMICAL EXAMINATION OF WATER - - -12 IV. ORGANIC MATTER IN WATER - - - - 38 V. OXIDIZED NITROGEN NITRITES AND NITRATES - - 5I VI. GASES IN WATER WATER SEDIMENT INTERPRETATION OF RESULTS OF CHEMICAL ANALYSES - - - - 60 VII. THE BACTERIOLOGY OF WATER EXAMPLES OF WATERS FROM VARIOUS SOURCES - - - - - - 83 VIII. SEWAGE EFFLUENTS - - - - - -97 IX. SOIL -------- 105 X. AIR -------- 118 XI. FOODSTUFFS: MILK BUTTER CHEESE CEREALS — BREAD MEAT ALCOHOLIC BEVERAGES LIME AND LEMON JUICES VINEGAR MUSTARD PEPPER — SUGAR TEA — COFFEE COCOA ------- i^g XII. DISINFECTANTS - - - - - - 287 APPENDIX - - - - - - - 313 INDEX - - , _ - - _ _ 221 LIST OF ILLUSTRATIONS FIG. I. 2. 3. 23- 24. 25- 20. 27- 28. 29. 31- 32. 33- 34- 35- 3^- 37- 3«- 39- 40. 41. 42. 43- 44. 45- 46. 47- 48. Geological fault, etc. - Curve of ground water Diagrammatic scheme of organic pollution under- going purification Thresh's apparatus - 22. Objects found in water sediments - 69, 70, 71, 73. 74. 75. 76. 77 Adeney's apparatus - Barometer and vernier scales - - . - Hempel's gas burette and absorption pipette - Apparatus used in milk analysis ... Apparatus used in milk analysis . - . Apparatus used in butter analysis . . - Granules of wheat starch - Granules of barlej* Granules of rye Granules of rice Granules of oat Granules of maize Granules of sago Granules of tapioca - Granules of pea Granules of haricot bean - Granules of arrowroot Granules of potato Vibrio tritici Bruchus pisi - - - Acarus farinae - Penicillium glaucum - Aspergillus glaucus - Mucor mucedo - Peronospora - - - Ustilago segetum PAGE FIG 4 49. 5 50- 40 5t- 61 52. 72. 53- .78 54- 103 55- 121 56. 131 57- I 58 58. 59. 160 00. 61. 1S6 62. 215 63- 215 64. 216 65. 216 66. 217 217 67. 218 68. 218 69. 219 70. 219 220 71- 220 72. 221 73- 221 74- 221 75- 222 76. 222 77- 222 222 78. 223 79 Tilletia caries (Uredo foe- tida) - . . . Wheat-stem infected with puccinia - . - Portion of Fig. 50 more highly magnified - Teleutospores - - . .iEcidium berberidis - Gonidiospores and teleuto- spore - - - - Ergot in rye - - . Sclerotium - bearing stro- mata - - - . Stroma containing asco- carps - - - - Ascocarp containing asci - Ascus containing ascospores Head of cysticercus - Taenia solium - - - Trichina spiralis Head of Distoma hepaticum Ascarus lumbricoidcs Oxyuris vermicularis Apparatus used in estima- tion of alcohol Cells of cuticle of mustard - Black pepper - - - Cuticle of tea-leaf Idioblasts in section of tea- leaf - - - - Tea-leaf - - - - Elder-leaf - - - - Willow-leaf - - . Sloe-leaf - - - - Cuticle of tobacco-leaf Coffee-berry ... Ground coffee, showing cell 3 of testa - - - - Lacteal vessels of chicory - Dotted vessels of chicorv - PRACTICAL SANITARY SCIENCE CHAPTER I GENERAL OBSERVATIONS UPON POTABLE WATERS IN RELATION TO THEIR SOURCE, AND METHODS OF EXAMINATION ADOPTED FOR SAFEGUARDING THEIR PURITY Water is often the vehicle of infectious diseases, poisonous metalhc salts, and a large number of undesirable materials — animal, vege- table, and mineral. When we consider how drinking waters are obtained, and how liable they are to contamination at all points from source to final distribution, it will be readily admitted that every potable water should be the object of the most careful, intelhgent, and constant concern. The primary object of a water analysis for public health purposes is to ascertain whether or not it contains sewage, as in the organic matter contributed by sewage are found the organisms of infectious disease, such as Bacillus typhosus, Vibrio cholercB asiaticce, etc. All other information is of very secondary import compared with this. The detection of or- ganic filth, whether of animal or vegetable origin, and of harmful inorganic matters, when in small quantities, is often a work of no little difficulty. In certain cases where a small amount of sewage containing pathogenic micro-organisms finds its way into a water- supply, no chemical analysis, however delicate, can furnish evidence of the pollution. So also in other cases the most exact bacterio- logical examination may wholly fail to discover a dangerous water. The well-informed analyst will not pin his faith to one method of examination to the partial or total exclusion of others, but will welcome all reliable methods that can assist in throwing light on his search. I 2 PRACTICAL SAXITAJRY SCIENCE At present four mithods of examination are utilized — viz.. Physical, Chemical, Biological, Bacteriological — each of which has its place and its limits. The physical examination may detect pollution so gross that further inquiry is unnecessary. The chemical analysis can render no information concerning liability to contamination, and is useless in detecting small quanti - ties of sewage. A systematic chemical analysis is of value in demon- strating variations in character produced, for example, by the lowering of the level of well waters, by change in rainfall, action on lead, iron, and zinc, in pipes, mains, cisterns, boilers, etc. Wher^' the estimation of sahne constituents must be determined for health purposes, manufacturing and engineering purposes, etc., th*; chemical method alone is of value. Here it may be stated as a general principle that waters most suitable for domestic purposes are also most suitable for manufacturing and engineering purposes. Acid waters corrode boilers, so do waters containing marked quantities of MgCL and CaCl.,, as these chlorides at high tempera- tures decompose, forming HCl, which at once attacks the iron. CaS04, being insoluble, is deposited as a crust. CaCOg and MgCO;} together with salts . of Fe render water unsuitable for tanning, dyeing, paper-making, and other industries, owing to their great insolubilit3^ whereby particles are left in the fabrics. Neutral and alkaline (Na^COg) waters are best suited for boilers. Special chemical analyses are required in dealing with medicinal waters. By careful and systematic study of the lower forms of animal and vegetable life, much information may be acquired as to the source and mode of entry of surface waters into water-supplies. Such biological examination has not had in this country the atten- tion it deserves. Where the question of infective micro-organisms in water arises, which to the sanitarian is of all questions the most important, the bacteriological examination only can afford positive evidence. The examination of the source of a water-supply is of the hrst import, and should never be omitted. Personal inspection of the catchment area, all streams arising therefrom, and all feeders of such streams, should be made in situ, and the relations of these GENERAL OBSERVATIONS UPON POTABLE WATERS 3 to possible sources of pollution carefully noted. When the gathering ground has been thoroughly investigated, attention should be turned to the storage reservoirs, and finally the efficiency of filtra- tion should be bacteriologically tested. Such examination pre- supposes an intimate knowledge of the entire area set apart for collection, which should be protected from all possibility of con- tamination from manured soil, house drainage, and storm waters. A good working knowledge of the geology of the district is essential, and every student of water analysis should intimately cultivate the solid and drift maps of the Ordnance Survey. The following brief table gives an outhne of the more important strata in this country, detailed descriptions of which will be found in any textbook of geology. Post-tertiary deposits : Alluvium, sands, gravels, boulder clay. Tertiary deposits: Sands of the Eastern Enghsh counties. Bagshot sands (upper, middle, and lower). London clay. Secondary deposits: Chalk. Greensands — upper and lower — with gault lying between., Weald clay. Purbeck marble. Kimmeridge clay. Oolite. Lias. New red sandstones. Primary deposits: Coal, ironstones. Limestone. Old red sandstones. Shales and slates. Crystalline rocks. Shallow wells sunk in the post-tertiary sands and gravels are very liable to pollution. The Bagshot sands yield a fairly soft water. The London clay is an impervious stratum, and the waters rest- ing immediately on it are generally hard. 4 PRACTICAL SANITARY SCIENCE The chalk formations of England, which are extensive, yield both hard and soft waters. The hardness is mostly temporary. Fissures make it possible for pollution to readily get access to these waters. The greensands, especially the lower, bear waters rich in calcium and iron salts. Oolites produce waters almost identical with those of the chalk. The magnesium limestones (dolomite) and new red sandstones give origin to much hardness, of which a large portion is per- manent. Slates and igneous rocks, being practically insoluble, 3'ield waters destitute of saline matters, and are consequently very soft. Impermeahle Stratum. . Water hearing S>tratanv ImpermeabLe Stratum. . Fault . Fig. The drainage area of a well depends upon the depth of the well, the porosity of the soil and subsoil, direction of flow of ground water, and the daily depression produced by pumping. It may be con- sidered as the base of a cone whose apex is the water-level in the well. Even with a good knowledge of the geology of the catchment area and districts through which the water passes, the analj'st is subject to pitfalls at all points. Strata may contain caverns and fissures which lodge pollution in the most unlikely positions. Geological faults account for unexpected positions of springs. Where a water-bearing, permeable stratum intervenes between two impermeable strata, and a fault occurs, the imprisoned fluid may become subject to such pressure that it escapes at the surface with tremendous force. GENERAL OBSERVATIONS UPON POTABLE WATERS 5 It is to be noted that the curve of the ground water near the well is steep, but rapidly shades off into the horizontal. It is obvious that with different types of soil the form of this curve changes as the surface water in the well is lowered. The drainage area increases in direct proportion to the porosity. This area should be protected from all forms of organic pollution, including cultivated soils, and it has been laid down as a minimum requirement that it should have a radius of twenty times the maximum depression of the water through pumping — e.g., if the depression in the well be 5 feet, the area should have a radius of 100 feet, etc. Outside this cone it is considered that filtration is so slow that purification is complete. A wide margin, however, Drainage Area Fig. 2. should be allowed in the drainage area to meet the effects of in- creased rainfall, possible faults in the brickwork of the well, and other factors, so that wells supplying drinking waters should be removed widely from all sources of drainage, farmyard manure, etc. A slight acquaintance with the situations of many rural wells in this country must call forth unqualified condemnation. There is no doubt that many epidemics of typhoid fever have their origin in the waters of these wells. It is a matter of little difficulty to determine whether or not leakage from the immediate surroundings takes place into a well, and an alkaline solution of fluorescin, an emulsion of Bacillus prodigiosus, or a concentrated solution of NaCl, poured around its mouth and thoroughly washed into the soil, will afford the necessary evidence within a limited time. Peat which lies for the most part on igneous rocks imparts to 6 PRACTICAL SANITARY SCIEXCE water certain organic acids capable ot dissolving metals. Wherever possible such waters should be cut out of a supply. If this cannot be done the acids should be neutralized before the waters pass to the consumer. Rivers and streams from which water-supplies are procured should be scrupulously preserved from the entrance of pollution, with a special view to the exclusion of infective bacteria. All river water should be sedimented and filtered before use, and the efftciency of filtration should be constantly tested by bacteriological examina- tion. All forms of animal and vegetable life should be excluded from service reservoirs, cisterns, mains, etc. It is well known that certain low vegetable forms, especially when dead, give origin to offensive odours. Water moves in a cycle. Evaporation produces clouds, which return to the earth as rain. This rain, according to the nature of the soil, subsoil, and rocks, pursues various paths. If it fall on impervious granite it runs off in large quantity; a part may be evaporated, and this will occur to the greatest degree during dr3^ hot, and windy w^eather. If it fall on sandy soil a large proportion percolates, and the more porous and deep the sand, the more rapidly and deeply the water sinks into the earth. When it meets with an impermeable stratum its further course is directed by the slope and contour of this stratum. Should the latter take the form of a basin the w^ater will accumulate until it overflows the lip of the basin, forming a spring at a point where the stratum outcrops. Again, if the stratum form an inclined plane, as on the sides of a river vallev, the water will flow along the plane to its outlet at the lowest point. Such pure waters may be intercepted before reach- ing polluted rivers by sinking wells at the bases of the hills forming the sides of the river valleys. Ihe upper surface of this mass of moving ground water is indicated by the level of the water in super- ficial wells. This surface is not necessarily horizontal. It is in constant motion, travelhng towards the outflow, and the rate of movement is governed by the porosity of the soil, slope, nature of outlet, etc. An intimate knowledge of the entire history of a water will often be necessary to an intelligent comprehension of certain analytical data. CHAPTER II THE PHYSICAL EXAMINATION OF WATER The physical examination comprises a determination of the tur- bidity, colour, odour, and taste.. Turbidity. — Pure waters are free from visible particles in suspension: the slightest degree of opacity should render a water suspicious. On the other hand, the most transparent and brilliant waters may contain the most pronounced pollution. Turbidity may be produced by access of the contents of cesspools, drains, manure heaps, and surface refuse of all types, especially after rains, when it forms often the worst kind of pollution. It may be pro- duced by particles of clay, iron, chalk, etc., when a chemical and microscopical examination may be necessary to disclose the nature of the matter in suspension. Waters containing iron very often deepen in opacity during the first day or two after collection, owing to the formation of persalts of that metal, which are highly insoluble. Such opacity immediately disappears on the addition of a small quantity of dilute HCl. Estimation of Turbidity. — Place the Winchester on a white porcelain tile in a good north light, and examine it carefuUy with the naked eye. Much information regarding opacity, sedi- ment, etc., may thus be gained by a practised eye. The sample may be described as brilliant (aeration good), clear, slightly turbid or opalescent, turbid, markedly turbid. To estimate the quantity of matter in suspension, filter lOO c.c. through a hard filter and evaporate in a platinum dish to dryness. The difference between the weight of the residue dried at ioo° C. and that of loo c.c. of the unfiltered sample similarly treated will represent the desired result. Or, where a centrifugal machine is available, by means of small tubes the sediment may be read off quantitatively on a gradu- 7 8 PRACTICAL SANITARY' SCIENCE ated scale. The amount of light permitted to pass through a cohimn of opalescent water mounted in a glass cylinder can be matched by the illumination of a polarized light ray passing through a second similar glass cylinder containing no water; the degree of rotation of the Nicol of the eyepiece expresses the degree of turbidity. Coloup. — Uncontaminated rain water presents a pale blue tint in the ' two-foot ' tube. Yellow tints point to organic matter, brownish-red suggest a peaty origin, and reddish-yellow indicate iron. Any appreciable shade of yellow or brown will excite sus- picion, and lead to a careful search for the cause. Colour tables have been formulated for the use of water analysts, but do not seriously assist a trained eye. Clean thoroughly and fill the ' two-foot ' tube ; place it on the tile ; look down through the column, noting the tint of colour, which may range from a pale sky-blue to a 3^ellow or brown. As to colour, for all ordinary purposes the naked-eye inspection is sufficient, but if for any reason great accuracy is required a tinto- meter may be used. Two hollow glass wedges containing respec- tively dilute solutions of CUSO4, and a mixture of ferric and cobalt chlorides slightly acidified, are made to slide over each other in front of an empty tube, so that any desired combination of blue and brown tints can be obtained. Alongside is placed a similar tube filled with the sample, and the wedges are arranged so that on looking down upon a white surface the colours exactly match. The prisms are graduated in millimetres, and the results are expressed in terms of millimetres of blue and brown. Water may be variously coloured by algae and other vegetable organisms. Crcnothrix polyspora (rich in iron) colours it red or reddish-brown, and decomposing accumulations of the dead or- ganism may produce serious nuisance. Green and blue algse produce their respective tints, and peat, according to its concentration, all shades of brown. Odour. — Drinking water should be free from all odour. Dis- solved gases may be liberated by slightly warming the water, say to a temperature of 37° C. In the case of peaty waters it has been found at times that even after the most careful filtration a slight odour still attaches to the water. For many reasons peaty waters THE PHYSICAL EXAMINATION OF WATER 9 do not furnish good supplies, and where other sources are available should be passed over. River waters usually have a faint smell, due to a variety of causes, most often, perhaps, to vegetable organisms, some of which^ — e.g., the well-known sewage fungus — are associated with the production of HgS. The dead and decomposing remains of plants and animals furnish a variety of odours, not only in river waters, but in cisterns, reser- voirs, and mains. Of late years attention has been called to distinct species of lowly vegetable forms which produce disagreeable odours in water. It is customary to obtain the sample of water for physical exam- ination from the vessel containing that for the chemical examina- tion, and something may now be said respecting the mode of collect- ing such samples. The so-called ' Winchester ' quart bottle has long been used for this purpose, and when made of colourless glass answers the purpose admirably. The bottle should be thoroughly cleansed by rinsing with dilute HCl, and afterwards removing the last trace of acid with distilled water. Where it has to be sent by rail, etc., it is packed in a tightly-fitting wicker case or wooden box fitted with padlock. Before, filhng, the bottle should be rinsed with a portion of the sample. A little air space should be left under the stopper to avoid cracking of the neck through rise of tempera- ture. Procedure in collection will vary according to the object of the examination.. If it is desired to ascertain whether, for example, lead is dissolved by a water in the house-pipes, it will be necessary to collect the first runnings from the taps in the morning. When a bacteriological examination is required, a special method must be pursued, which will be described later. Where the sample is to be taken from a river, cistern, etc., the bottle, prepared as above, is usually immersed some little distance below the surface, where the stopper is removed and the bottle filled. A small portion is poured out in order to procure the air space mentioned, and the stopper inserted. Various forms of apparatus have been devised for collect- ing samples under different conditions, but the circumstances will in all cases suggest the mode of procedure, if it be kept in mind that a fair sample of the water as it is usually found is the object desired. u> PRACTICAL SANITARY SCIENCE A correct record of the sampling process, etc., should be made on the spot, and attached as a label — 1. Date, time, and place of taking sample. 2. Depth below surface, state of water-level — high. low, or average. 3. Particulars of rainfall and of geological strata of district. 4. Depth of water-level below ground-level. 5. Description of surroundings, possible sources oi pollution, such as sewers, cesspits, cemeteries, etc. In many cases it is well for the analyst to supply his own collect- ing-bottle, with instructions for taking the sample and filling up the label. The sender should be made to understand that the specimen must be of the same nature exactly as that actually consumed, and that it is desired to ascertain the maximum degree of ])o]lution that may at any time obtain. All such particulars, as also the results of the analysis, should be transcribed into a book and preserved for future reference. Estimation of Odour. — Place 250 c.c. in a stoppered flask, and heat to 37° C. in an air-bath. Remove the stopper and smell. It is generally sufficient to shake the sample well in the cold, rapidly remove the stopper, and smell. The variety of odours is inhnite. Many odours are produced by organisms, either as products of their life-history or of their death and putrefaction. Beggiatoa, Chara, and certain species of Crenothrix produce an offensive odour of H.,S. It is believed that Beggiatoa during its life-cycle reduces sulphates, and produces under favourable circumstances large quantities of H.,S. Crenothrix, moreover, often produces abundance of colour, varying from brown to red. Tabellaria, Meridion, and certain diatoms, as also the protozoon Cryptomonas, furnish a dis- tinctl}' aromatic odour. A lishy odour is produced by Volvox and the protozoa O^cnodinium, Bursaria, and Uroglena. A grassy odour accompanies Rivularia, Anabsena, and Caelosphserium. Taste. — Pure rain water well aerated has a fairly distinctive taste, more easily appreciated than described. So also have peaty waters, sea water, and chalybeate waters. The taste of a particular sample may be, however, everything to be desired, whilst the water is the foulest of the foul. Taste, however, is of little service to the analyst, and not always to be recommended. Iron is about the THE PHYSICAL EXAMINATION OF WATER ii only ingredient that can in this way be detected in very small quan- tities, being recognisable to the amount of 0-5 part per 100,000. Potable waters have been classified as — Wholesome.— {1) Spring water; (2) deep- well water; {3) upland surface water. Suspicious.— {^) Stored rain water; (5) surface water from culti- vated land. Dangerous.— [6) River water polluted with sewage ; (7) shallow- well water. CHAPTER III THE CHEMICAL EXAMINATION OF WATER It will be well for the student from the lirst to ht up his own appa- ratus, and make his own standard solutions. He must learn to use properly the chemical balance, and a special demonstration is devoted to the mechanism, methods of adjusting and using this all-important instrument. Before commencing to weigh, he should see that the balance is accurately levelled, and that the index moves without effort over the whole field of the graduated scale, and comes to rest at zero. All weights, basins, etc., should be transferred to and from the scale-pans only when these are supported. It is customary to use three rows of weights, grammes (brass), deci- grammes and centigrammes (platinum), and milligrammes (plati- num). A rider of platinum applied to the beam also reads milli- grammes. The right-hand pan should be used only for weights, and these should be placed methodically in three rows in front of the operator. By this means the total reading is most easily obtained and checked. Immediately on finishing a weighing all weights should be trans- ferred to the box, with the forceps used for the purpose, and the box and balance carefully closed. The standard solutions in use in water analysis are of two types : 1. Normal, decinormal, centinormal, etc. 2. Standards of such strength that a litre contains the equivalent of a gramme, or submultiple of a gramme, of the substance to be estimated. A standard solution is said to be normal when one litre contains the equivalent weight in grammes of an element, acid, alkali, or salt. The molecular weight of HCl is 36-35; therefore 36-35 grammes HCl per litre = normal HCl, written N.HCl. THE CHEMICAL EXAMINATION OF WATER 13 In like manner N.NaOH= 40 grammes per litre. Since the term ' equivalent ' signifies the weight in grammes of the substance under consideration, which is chemically equivalent to 1 gramme of H, normal H2S04= =49 grammes per litre. A decinormal solution {—) is one-tenth the strength of a normal — thus 3~j- NaOH=4 grammes per litre — and a seminormal (^) and centinormal (y^jj) are respectively one-half and one-hundredth the strength of the normal — viz., 20 grammes and 0-4 gramme per litre respectively. In tribasic acids one-third of the molecular weight in grammes per litre constitutes a normal solution, and so on for acids of higher basicity. The terms ' normal,' ' decinormal,' etc., are used sometimes with a different meaning. Permanganate of potassium, as we shall see presently, in acid solution is reduced by many substances, accord- ing to the equation KgMn.Pg = KgO + 2MnO -t- O5, in which 2 gramme molecules of KMn04 correspond to 5 gramme molecules of oxygen or to 10 gramme molecules of hydrogen. Accordingly, in order to put permanganate of potassium on a hydrogen basis, a 1 1 X- ■ J . X ■ 316-3 /2KMn04\ r normal solutionis made to contain - — - = 31' 03 grammes 10 V 10 / ^ per litre. In the same way K2Cr207. which in acid solution parts with O3, requires for a normal solution -^ — ( ^a^ ^ ) = 49'^ grammes per litre. The second type of standard solution used is constructed so that a minimum amount of calculation suffices in estimating results. Since it is customary to represent the various items of the analysis as parts by weight per 100,000 of the water, and since i c.c. of water weighs I gramme (1,000 milligrammes), 100 c.c. of water will weigh 100,000 milligrammes. It is therefore convenient, when possible, to work on 100 c.c. of the water sample throughout the various estimations, and to use a standard solution that will give readings directty in the above terms. Suppose we wish to estimate the quantity of CI in a water, we use a solution of AgNOg of such strength that i c.c. is equivalent 14 PRACTICAL SANITARY SCIENCE to I milligramme CI. To make this solution we refer to the molec- ular weight of AgNOg, and the atomic weight of CI. AgNOa-t- XaCl-AgCl + NaNOg. 170 grammes AgNOg precipitate 35-35 grammes CI. .-. dividing by 35-35, we find that 4-8 grammes AgNO., precipitate I gramme CI. If, then, we dissolve 4-8 grammes AgXO.j in i litre of water we obtain a solution i c.c. of which precipitates i milligramme of CI, and working with 100 c.c. of water, the number of c.c. of the silver nitrate solution used indicates the number of milligrammes of CI in 100,000 milhgrammes of the water, which is parts per 100,000. Again, in estimating XH3 a standard solution of XH4CI is pre- pared and used in the same way. One molecule of XH4CI contains i molecule of XH^. 53*35 grammes ,, contain 17 grammes and 3-14 ,, ,, ,, I gramme Therefore a litre containing 3-14 grammes XH4CI will contain I gramme NH3, and consequently i c.t. contains i milligramme. It is found convenient to dilute this 100 times, so that i c.c.= o-oi milligramme XH3. Standard solutions should be stored in bottles in such manner that both internal and external evaporation are impossible. In the first case, where the bottle is not quite full, pure water will evaporate and condense on the upper portions of the vessel; in the second, evaporation will take place into the atmosphere. The loss of water will naturally depend on the substance dissolved, the tem- perature, the age of the solution, and the frequency with which it is used. A rough estimate may often be made of the probable amount of change in strength by noting the date of preparation, which should always be found on the label. Some standards undergo chemical change by the action of light, and should therefore be kept in the dark. In reading a burette, arrange it so that the lower convex line of the meniscus is in the same horizontal plane with the eye; the THE CHEMICAL EXAMINATION OF WATER 75 division of the scale cut by the lowest point of this convex line is the reading. In measuring small quantities of liquids much time may be saved by using a few plain 10 c.c. pipettes graduated to tenths of a c.c, and for quantities under a c.c. a i c.c. pipette graduated to hundredths. These can be easily and rapidly cleaned, and as easily and rapidly manipulated, and may often take the place of burettes. In weighing platinum and porcelain basins, crucibles, etc., it is very necessary to see that they are quite dry. To insure this, especially after heating, they should be placed for ten minutes in a desiccator immediately before going to the balance. It is also necessary to be certain that all such vessels are thoroughly clean. Accurate notes of all operations, measurements, weights, etc., should be made in the bench notebook, and considered as much a part of the work as the operations themselves. Without this notebook it is impos- sible to get on with analytical chemistry. Where possible it is well to write down the chemical equations representing decompositions. When in doubt in this matter refer to a work on chemistry. All colour matches are best made in glass cylinders standing on a white ground, as the operator faces a north light. The Reaction of Water. — This is an important item, and should form the first step in the routine chemical examination. In addition to the use of red and blue litmus-papers, it is often well to use a more delicate indicator, such as phenolphthalein, and to esti- mate the amount of acidity (when acid) in 100 c.c. by titrating with -^0 NaOH, or of alkalinity (when alkaline) with ^ H2SO4. An acid water dissolves lead, iron, and zinc; it also fixes ammonia, and so prevents its being distilled off. Some hold that neutral waters and those possessing very slight temporary hardness are capable of dissolving lead. It should be remembered, however, that sodium carbonate when present prevents this action. Houston has corre- lated the acidity and plumbo-solvency of a large number of moor- land waters. He causes the sample to percolate upwards through a column of specially prepared lead shot at a uniform rate. He then collects successive 50 c.c.'s, and estimates the amount of lead in each. The following figures are taken from a report to the L.G.B.: I6 PRACTICAL SAXITARY SCIENCE Acidity. Pl.UMBO-Soi.VENXY, Number of c.c. Mg ms. of I'b in N Na.iCOj required to neutralise 100 c.c. of the Water after " loo c.c. of the Water. Filiration through Lead Shot. 0-2 0-28 0-3 0-25 0-4 0-4 0-5 0-66 0-6 0-92 0-8 1-55 0-95 2-66 1-5 2-8 17 5-6 2-2 8-6 Some waters not acid, and failing to dissolve lead, exert an ' erosive ' action, forming an insoluble film of oxyhydrate upon the lead, which after a time may become detached, and produce a degree of opacity. Chlorides in Water. — Free CI rarel}' occurs in water-supplies. Certain manufacturing effluents ma3' on occasion contain small quantities of free CI, but the quantit}^ is so small and the occurrence so rare that this form of CI may be practically ignored. The great bulk of CI in drinking water is found as NaCl. All soils and sub- soils contain this salt in large amounts. The water-bearing strata are rich in chlorides, especially NaCl, and consequently rain water (which itself may contain as much as 0-5 part per 100,000 NaCl), as it percolates from the surface to the impermeable stratum on which it rests, dissolves these in considerable quantities. CaClg and MgClg are found in certain strata— chalk and limestone — in much smaller quantities, but MgCU abounds in sea water, and in large quantity is distinctive of it. Wells, reservoirs, etc., to which sea water can obtain access will yield waters rich in ^MgCl,. Sources of water subject to much evaporation, especiall}' if situated near the sea, exhibit large quantities of chlorides. The total CI in sea water approaches 2,000 parts per 100,000, and if this figure be kept in memory it will explain the large estimations often found some considerable distance from the littoral. During the passage of water through the soil, subsoil, and strata, CI is not likely to be diminished as are the organic matter and bacteria. When we have accounted for all the CI contributed by rain water, THE CHEMICAL EXAMINATION OF WATER 17 sea water, soil, subsoil, and strata, and trade effluents from chemical works, paper factories, etc., there may remain a surplus furnished by organic pollution of animal origin. This surplus is of some import to the analyst, as indicating sewage; but before it is returned as such all the possible sources of origin just mentioned must be rigidly excluded. Vegetable organic matter does not yield this surplus CI. Attempts have been made in U.S.A. to estimate and permanently record the CI due to the natural causes named, so that sewage pollution may be readily detected. Maps have been con- structed and points furnishing equal quantities of CI joined by lines named ' isochlors.' In districts remote from the sea, and centres of population and land cultivation, such maps may be more or less reliable, but in this country they would be useless. MHiilst it is true that animal pollution contains much CI (urine about i per cent, chlorides), and that soils, strata, etc., in certain districts yield fairly constant quantities, still there are variations in many localities in these natural sources, and it is only where large quantities of sew- age have gained access to waters that we can rely on the surplus CI as evidence of this accession. In the case of small amounts of sewage this surplus CI figure is of little if any value. But in a water analysis the most important information lies very often not so much in the exact amount of a particular constituent as in the fact that its presence points to past pollution, and consequently to the possibility and even probability of a recurrence of such pollution. In this light CI and nitrates play an important role. These afford unmis- takable evidence of previous contamination; they are the distinct and unchangeable indications of previous pollution, but as to whether recent or remote they indicate nothing. Hence the neces- sity for further and different forms of examination. As to the amounts of chlorides that should condemn waters, it is difficult to speak, since there is such infinite variety in the quantities contained in different soils and strata. MgClg and CaClg render waters hard, so that more than 4 or 5 parts of either or both of these per 100,000 will cause a large destruction of soap, and these figures will in most cases form the limit for domestic waters. NaCl may go up to perhaps 50 parts per 100,000; above this it imparts a taste, and the water consequently will not be fit for drinking. i8 PRACTICAL SAXITARY SCIENCE Estimation of CI. Apparatus and Reagents Required. A white porcelain basin capable of holding 250 c.c. A glass stirring-rod. A burette charged with standard solution of AgNOa, of which 1 c.c. is equivalent to i milligramnie CI (4"8 grammes AgNO., to a litre of water). A 5 per cent, solution of KoCrOj. Place 100 c.c. of the water in the porcelain dish. Add I c.c. of the K2Cr04 solution, and stir. J\un in from the burette drop bj^ drop the silver nitrate solution until the pale ^-ellow colour remains permanently orange. Take the reading. The rationale of the process is as follows: AgNOg, when added to a solution of chlorides, forms AgCl, a white curdy precipitate insoluble in HNO3, soluble in NH^HO. Without a special indicator it would be impossible to determine when the whole of this white precipitate had been formed — when the whole of the CI had been deposited. K2Cr04 is also acted on by AgNOg, and Ag2Cr04 formed, which is red. But so long as any chloride remains ununited with Ag, the silver chromate is decomposed and AgCl formed; hence the dis- appearance of the red colour on stirring. Immediately the whole of the CI is precipitated as AgCl the red silver chromate remains. The reactions are represented by the equations — AgNOg + NaCU AgCl + NaNOg. 2AgX03 + K.,Cr04 = Ag2Cr04 + 2KXO3. 2NaCl + Ag2Cr04=2AgCl + Na2Cr04. It is obvious that the K2Cr04 should be free from CI. Acidity in the water will dissolve Ag2Cr04 ; hence if a water is even slightly acid it must be neutrahzed. Freshly precipitated CaCOg is the best alkali to use, and it should be used only to the point of neutralization. If too little K2Cr04 is used the CI reading will be too high, and if too much be used it is difficult to determine the end; i c.c, accord- ingly, is found a suitable quantity when the solution is of the above THE CHEMICAL EXAMINATION OF WATER 19 strength. It will be noticed that as the titration proceeds the red AggCrO^ disappears more slowly on stirring, until finally it ceases to disappear. This is explained by the, continuous decrease in the original chloride. Whilst abundance of this undecomposed chloride remains in solution, the Ag2Cr04 is rapidly robbed of its Ag and the red colour discharged; but as the chloride diminishes and the end approaches, the decomposition of the Ag2Cr04 becomes slower and slower, until at the end of the reaction it ceases, and the red Ag2Cr04 permanently remains. Since the colour-change from pale yellow to red is somewhat difficult to detect in daylight (it is more easily perceived by gas- light), a flat glass cell whose plates are | inch apart should be filled ^dth chromate solution of the same tint as that of the contents of the basin, and interposed between the eye and the basin during titration, when the appearance of the red silver chromate becomes strikingly manifest. The effect is to neutralize the yellow and to cause the appearance of the basin to be the same as if it were filled with pure water. In working with turmeric, cochineal, etc., cells should be used filled with corresponding solutions of turmeric, cochineal, etc. The number of c.c. of silver nitrate run in represents the number of parts of CI per 100,000. 100 c.c. water = 100,000 milhgrammes, I c.c. AgN03= I milhgramme CI; .■. the number of c.c. AgN03 used= number of parts CI per 100,000 water. Some operators subtract -i c.c. from the AgNOg figure as the quantity required to form the slight permanent orange colour. Others add a small measured quantity of the water sample from a burette until the permanent orange tint departs, and reckon half of this with the AgNOg reading. It is well always to do tw^o careful estimations, and take the mean. When once an idea of the quantity of CI present is obtained, two careful estimations can be performed very rapidly. A control basin containing 100 c.c. of the same water and i c.c. of K2Cr04 may assist ill determining the end reaction. Where small quantities of CI are to be estimated, 250 c.c. or 500 c.c. 20 PRACTICAL SANITARY SCIENCE of the water may be concentrated by evaporation to lOO c.c. Alkaline silicates, nitrates, and phosphates slightly affect the CI estimation, but not to such a degree as to require correction. Chlorine is sometimes returned in terms of sodium chloride This figure is found bv multiplying the CI return by - — ~ . WHiere 35"35 CaClo, or i\IgClo, or both, enter into the problem, corrections have to be made in accordance with the respective molecular weights and the quantities of each present. In chalk and red sandstone waters 3 parts of CI per 100,000 may occasion no suspicions of sewage, and 4 or 5 parts may be passed, unless organic pollution is indicated by other items of the analysis. Pure surface waters seldom contain more than i part per 100,000, whilst deep greensand waters may give rise to 15 to 20 parts per 100,000, and still be absolutely pure. The following are a few examples of the CI figures for different waters : Parts per 100,000. A well in St. Pancras ----- 4-5 Lambeth water-supply - - - - - i -g ., ,. - - - - - 2-0 Southwark water-supply - _ . . - 1.85 A well in Devonshire - - - - - 3-1 Thames water at Waterloo Bridge - - - lo^-z Deep well near Hindhead _ _ . - ii2'3 Sample of rain water taken from rain gauge in Herts - 0-3 Hardness. The hardness (soap-precipitating power) of a water exerts little influence on health, but from an economic point of view is of some importance. A soap is a chemical salt formed by the union of an inorganic base with one or more fatty acids. Sodium and potassium soaps are soluble in water, and when shaken with it form a dense froth or lather. Calcium and mag- nesium soaps are insoluble in water, and fail to form a lather. Hence, if a solution of a soluble soap be added to water containing calcium or magnesium salts, these last will be completely precipitated in the form of insoluble calcium or magnesium soaps before a lather is produced. Accordingly, by using a standard soap solution, an THE CHEMICAL EXAMINATION OF WATER 21 approximate estimate of the quantity of such soap-precipitating bodies in a water can be made. The total quantity of such bodies, as measured by the standard soap solution, constitutes the total hardness. Other bodies than calcium and magnesium salts are occasionally present in water, which act in a similar manner on soap. If much sodium chloride be present, it will precipitate soap from its solution in an unaltered state. CaCOg and MgCOg, especially the first, have by far the greatest share in rendering waters hard. These salts are formed in solution in the soil as bicarbonates [Ca(HC03)2 and Mg(HC03)2] by COg dissolved in rain water. On boiling such waters, CO2 escapes, and insoluble carbonates separate out as a precipitate — Ca(HC03)2-^CaC03 + CO2 + HgO. The addition of slaked lime to water containing the bicarbonates of the alkaline earths results in the precipitation of the lime added and the bicarbonates thus : Ca(HC03)2 + Ca(OH)2= 2CaC03 + 2H20. (Clark's process.) If now the boiled water be filtered, made up to its original volume with distilled water, and again titrated with standard soap solution, the permanent hardness is obtained. The difference between the total and the permanent hardness is the temporary hardness. The soap test has been made to measure the quantity of CaCOg and other salts which produce hardness, but this is not accurate quantitative analysis. It should be ciearly understood that the chemical action is multiple and indefinite, and altogether different from that which usually takes place, when in quantitative analj'sis we titrate one definite compound against another. All that can be claimed for the soap process is that it indicates the amount of soap-destroying bodies present in a given water, but fails to form a measure for any in particular. The following compounds produce hardness: CaCOg, MgCOg, CO2 in solution, CaS04, MgS04, FeoOg, and other Peroxides, zinc salts, Si02, A\^ (OH)e, chlorides, nitrates, phosphates, and free mineral and organic acids. The temporary hardness, which is got rid of by boiling, is fcr 22 PRACTICAL SANITARY SCIENCE the most part produced by CaCO^ and MgCOg, held in sokition by COo. After these come small quantities of CaS04 and MgS04, which are also thrown out immediately C0.> is driven off, but the great bulk of these sulphates remains in solution. Lasth', in a few cases minute quantities of oxides of Fe, silica, and alumina are deposited. Phosphate of Ca, if present in appreciable quantity, may, under certain conditions, be deposited in very small amounts. On cooling some of the precipitated MgC03, and to a less degree CaCOg, CaS04, and Ca3(P04)., will redissolve and go to form per- manent hardness. MgCOg destro^'S nearly 50 per cent, more soap than CaCOg, but is found in potable waters in very much less quantity. Estimation of Hardness. — Prepare a standard solution of calcium chloride in the following manner: Weigh accuratel}^ 0-2 gramme pure calcite (CaCOg), and dissolve it in dilute HCl, taking care to keep the vessel covered so as to avoid loss by spirting. Evap- orate this solution to dr3mess on the water-bath. Add water, and again evaporate to dryness, and repeat these processes in order to remove all free hydrochloric acid. Now dissolve the residue of neutral CaClo in water and make up to a litre. One c.c. = the equivalent of 0-2 milligramme CaCOg. In other words, this solution possesses hardness = 20 parts per 100,000. Prepare a standard soap solution by dissolving about 13 grammes of Castile soap in a litre of equal parts methylated spirit and water. Stand in a cool place for some hours, and filter. The titration and dilution of this soap solution is carried out as follows : Make up 50 c.c. of the calcium chloride solution to 100 c.c. with distilled water (10 parts hardness per 100,000), and place in a stoppered bottle of 250 c.c. capacity. Run in from a burette, i c.c. at a time, the soap solution. Close the bottle, and shake vigorously for a short period until a lather remains on the surface as an un- broken layer for five minutes. Towards the end of this operation the amount of soap solution added should be lessened, and finally should not exceed i c.c. As the end is reached, the sound and shock produced by shaking becomes much more gentle. The student should carefully prepare a number of similar lathers by shaking 100 c.c. distilled water in a similar bottle, and note exactly the amount of soap solution required. This quantity THE CHEMICAL EXAMINATION OF WATER 23 will be found to be about i c.c. of the finished standard soap solution. In the present case the quantity of soap solution used should be II c.c. (10 c.c. to precipitate the equivalent of 10 milligrammes of CaCOg, and i c.c. to produce the lather). Suppose, however, that 9 c.c. soap solution be found sufficient to produce the characteristic lather, it is evident that the solution must be diluted with aqueous spirit in the proportion of 9 to 11. Dilute, therefore, 900 c.c, or thereabouts, of the original litre to the volume ~ c.c, and 9 keep the remainder for fortifying the standard, as in time it loses strength, especially when, on keeping, it becomes turbid. Label the solution Standard Soap i c.c.= i miUigramme CaCOg. Should the soap solution prove too weak, it must have additional soap added and be put through the same process of standardization imtil found correct. A standard soap solution may be prepared in another way: Dissolve 80 grammes chemically pure oleic acid in alcohol, add a few drops phenolphthalein and a strong solution of KOH in alcohol, until the oleic acid is neutralized and saponification therefore com- plete (the liquid retains the faintest purple colour) ; then titrate with the calcium chloride solution, and dilute to standard strength. The following is an example of the determination of the hardness of a sample of a London (New River) water: Take 100 c.c. of the water in a 200-c.c. stoppered bottle. Fill a 50-cc burette mounted on a stand with standard soap solution (i c.c.= i milhgramme CaCOg). Run in the soap solution i c.c at a time, shaking vigorously after each addition, until a permanent lather remains unbroken for five minutes when the bottle is laid on its side. As the end of the reaction approaches, the hard metallic sound at first heard on shaking gives place to a dull thud, the froth which previously disappeared almost instantaneously remains, and adheres in specks to the sides of the bottle. Twenty-one c.c of standard soap solution were required in this case to complete the titration. Subtracting i c.c. used in producing the lather, we find that 20 c.c were precipitated by the 100 c.c of water. But each c.c.= i milligramme CaCOg ; .-. 20 c.c.= 20 milligrammes CaCOg, 24 PRACTICAL SANITARY SCIENCE and 100 c.c. of this, water contains 20 milligrammes of soap-precipi- tating substances, or a ' total ' hardness equal to 20 parts per 100,000. In waters containing magnesium salts the lather is slowly produced, and of a dirty, granular appearance, very unlike the light frothy condition seen in hard waters destitute of Mg salts. To obtain the ' permanent ' hardness in the above example, place 100 c.c. in a small beaker on a porcelain ring over a Bunsen flame, and boil for fifteen minutes, or till one-third of the volume has evaporated. Filter into a clean 100-c.c. flask, and make up to the mark with distilled water. Transfer to the stoppered bottle and determine the hardness as above: this is ' permanent hardness.' 13-5 c.c. of the soap solution were required to lather the 100 c.c. of water prepared as described. 13-5 c.c — I c.c.= 12-5 c.c, or 12-5 parts permanent hardness per 100,000. The 'temporary hardness ' = difference between 'total' and ' permanent ' hardness. 20— 12-5 =7*5 parts temporary hardness per 100,000. Hard waters, whilst palatable, cause waste of soaps, and fail somewhat in cooking vegetables, meats, etc., and in making infu- sions of tea and coffee. They are unsuitable for boilers, in that a deposit forms on the interiors which by reason of its low conductivity of heat wastes fuel, and from its divergent coefficient of expansion may lead to explosions. This deposit or crust will consist of bodies representing both temporary and permanent hardness. Carbonates of Ca and Mg will fall out first, and be followed by their sulphates, together with salts of iron, silica, and alumina. In this country the hardest waters arise from the chalk, dolomite, and new red sandstone strata, carbonates of Ca and Mg forming by far the largest proportions of soap-destroying compounds. Where the hardness exceeds 20 parts per 100,000, it is well in performing the estimation to dilute the sample with an equal bulk of distilled water. The total hardness of a potable water should not exceed 25 to 30 parts per 100,000. Waters whose hardness falls below 10 parts are considered soft, whilst those containing 20 to 30 parts are hard, and upwards of 30 parts very liard. THE CHEMICAL EXAMINATION OF WATER 25 Clark's scale of degrees represents hardness as grains per gallon (parts per 70,000). It is universally agreed that a good water should contain less than 10 parts per 100,000 of permanent hardness, and of this little should be due to magnesium salts. Temporary hardness can be easily got rid of; not so permanent. In Clark's process, as noted above, slaked lime is used for softe ning — in other words, for combining with the COg in ^:olution, thereby causing insoluble carbonates to separate out which were previously held in solution by the CO^. Care should be taken that no excee;s of lime is added. CaCOg, H2O, C02 + Ca(OH)2=2CaC03 + 2H20. Softening of permanent hardness may be effected by the use of NagCOg : CaS04 + NagCOg = Na2S04 + CaCOg. Clark's method does not yield accurate results if a large quantity of Mg salts is present. These salts do not materially affect the pro- cess now to be described. Estimation of Hardness by Standard Acid. — Determine first the temporary hardness by titrating the calcium and magnesium salts which form it with ~j^ H2SO4, using methyl orange (the sodium salt of a colour acid which is not interfered with by CO2) as indi- cator. Add to I litre of the water, or less if it be very hard, 4 or 5 drops of methjd orange solution, and run in —^ H2SO4 from a burette until the colour changes pink. Calculate the weight of CaCOg from the number of c.c. of acid used and convert this into parts per 100,000. Example. — 500 c.c. water required 9 c.c. decinormal sulphuric acid. I c.c. Y^ H2S04= I c.c. ^ CaC03= 0-005 gramme CaCOg; .'. 500 c.c. water = 0-005 x 9 grammes CaCOg; .•. 100 c.c. water= o-ooi X 9 ,, = 0-009 " ^ =9 milligrammes CaCOg. Hence the temporary hardness in terms of CaCOg =9 parts per 100,000. Permanent Hardness. — To 250 c.c. water add excess -^ NaaCOg, say, 50 c.c, and boil for half an hour. Should Mg salts be present, 26 PRACTICAL SAXITARY SCIENCE evaporate to dryness and extract the residue with water. Filter. Wash the precipitate with boiled distilled water. Cool, and make up the filtrate to 250 c.c. Titrate, say, one-fifth of this with ■^^ H2SO4, using methyl orange as indicator. Calculate from the number of c.c. acid used the weight of NaoCOg engaged in precipi- tating the salts forming hardness, and from this the permanent hardness in terms of CaCOg, in parts per 100,000. Example. — 50 c.c. taken from the 250 c.c. cold filtrate required %-^ c.c. ^^ H2SO4 for neutralization. 250 c.c. require %'d> x 5 = 44 c.c. J^, H^SOj. 50-44= 6 c.c. yN. NaaCOg used = 6 c.c. yN_ CaCOg = 0-005 >< 6 grammes CaCOg in 250 c.c. water 0-030 = — ^ ,, ,, 100 c.c. 2-5 = 0-012 or 12 parts per 100,000. Temporary hardness, g. Permanent hardness, 12. Total hardness, 21. Water containing Na2C03 is alkaline in reaction and contains no permanent hardness. Since boiling fails to interfere with NagCOg, this salt can be estimated in the filtrate from the carbonates of Ca and I\Ig precipitated by boiling in the above process, for the deter- mination of permanent hardness. The number of c.c. -^ H2SO4 used X 0-0053= weight of Na^COg. Rain water is the softest of all natural waters, and hardness in- creases in the following order: Upland surface water, river water, spring water, deep-well water, shallow-well water. Calcium salts react quickly in the double decomposition with soaps; magnesium salts react more slowly. Hence, w-here mag- nesium salts are present in quantity, a more prolonged shaking is necessary in producing the characteristic lather. Where it is desirable to estimate the quantity of Mg present in a sample, the ordinary methods of quantitative analysis must be employed. In using the soap test, hard waters should be diluted so that not more than 16 c.c. of the standard soap solution is required to complete the reaction. THE CHEMICAL EXAMINATION OF WATER 27 Solid Residue. The total solids in waters vary greatly in extent, ranging from 2 to 3 parts in rain water to over 3,000 parts in sea water per 100, oco. From the purely health point of view, perhaps little information will be derived from an estimation of the total solids. Occasions, however, arise in which it may be desirable to estimate the total sohds, and also the quantities of certain constituents, such as Ca and Mg salts. Where these latter exist in large quantity in the form of sulphates, it is found that the waters are unfit for drinking, from their action on the alimentary tract. The incineration of the dry residue affords a check on some of the other portions of the analysis dealing with organic matter. On the whole, any useful information that can be obtained will, for the most part, centre round the quantities of Ca and Mg salts present, especially the sulphates. Chalk waters contain little sulphates; hmestone waters hold chiefly CaS04, at times to the extent of 15 to 20 parts per 100,000; magnesium limestone (dolomite) generally contains much less CaS04 and considerable MgS04. Sulphates in water are chiefly derived from strata; a very small amount is due to the oxidation of S in organic matter; a small quantity in the rain water of large towns has its origin in the solution of the oxides of S found in the atmosphere; whilst in exceptional cases an appreciable portion is due to the oxidation of metallic sulphides. Phosphates in marked quantities indicate organic pollution, especially urine. The phosphates of the alkalies are those chiefl}^ found in water. But, in that certain geological beds and organic matter of purely vegetable origin contain phosphates, no very direct information is obtained from their estimation, and it is usually unnecessary to go beyond a qualitative examination. In a few instances silica may require to be estimated: this com- pound lessens the plumbo-solvency of water. In clear waters the solids are all in solution; in turbid waters they are partly in solution and partly in suspension. It is cus- tomary to estimate the solids in solution, but as this requires complete sedimentation it may be advisable in cases where time is limited to perform the estimation on the sample after thoroughly 28 PRACTICAL SAXITARY SCIENCE shaking. The method adopted, however, should be stated on the report. Measure out loo c.c. of the water, and place in a clean platinum basin on a water-bath, 25 c.c. at a time, as evaporation proceeds. When dr}', transfer the basin, after carefully wiping the outside, to an air-bath at ^y° C. for half an hour. Remove to a desiccator for ten minutes, and weigh. By drying at this low temperature no water of crystallization is lost, and no decomposition takes place. Further drying should be effected, if necessary, until a constant weight is obtained. This weight, less that of the dish, represents the total solids. \\"\i\\ platinum-tipped tongs hold the dish over a Bunsen flame until thorough incineration is effected. After cooling in the desic- cator, weigh again to obtain the non-volatile solids. The difference between this last and the previous weight represents the volatile solids. The degree of charring (organic matter) which occurs during incineration should be noted; also the smell — odour of burnt sugar indicates vegetable matter, burnt horn animal substance. Ca. — Where it is deemed necessary to estimate the quantity of Ca salts, Mg salts, or both, 500 c.c. of the sample should be evaporated down to 200 c.c, and the Ca removed by precipitation with (NH4)H0, NH^Cl, and [^li^)X.^i. The precipitate of CaC204, when thoroughly washed, dried, ignited, and weighed, represents the Ca as CaCOg, 56 per cent, of which is Ca. The weight of the crucible and ash of filter-paper must be accurately known and accounted for. It is well to let the beaker or other vessel containing the mixture of precipitate and fluid stand for some hours in a warm place, b}^ which filtration is rendered much more easy and thorough. The student may be reminded that the addition of NH4CI holds Mg salts in solution. Mg*. — Concentrate the filtrate down to one-fifth its bulk or less. Add shght excess of sodium phosphate, and stand aside in a warm place for some hours. Filter, wash the precipitate well with dilute (NH4)H0, dry, ignite, and weigh as MgoP207 (magnesium pyro- phosphate). The Mg forms t^V of this weight. Phosphates. — It is rarely necessary to estimate phosphates. Where, however, required, proceed as follows : Evaporate 200 c.c. of the water to dryness. Moisten the residue THE CHEMICAL EXAMINATION OF WATER 29 with a few drops of pure HNO.5 and evaporate again to dryness, in order to render insoluble any silica that may be present. Dis- solve in dilute HNO3 and filter. Add ammonium molybdate in slight excess; keep, if possible, in a warm place over night, and filter. Wash the precipitate well with hot water, and dissolve in ammonia. Add a few drops NH4CI and shght excess of MgClg, and filter. Wash the precipitate thoroughly with dilute am.monia. Dry, ignite, and weigh the MgaPgO^. The phosphates returned in the form of P2O5 will be represented by yy^ of this weight. It is hardly necessary to say that a qualitative test for phosphates should be carefully performed before entering on the more lengthy quantitative estimation. For this test concentrate by evapora- tion a quantity of the water — say 200 c.c. — to one-tenth its bulk. To 10 c.c. in a test-tube add a drop or two of HNO3, i or 2 c.c. solution of ammonium molybdate, and heat to a temperature somewhat below boiling, for several minutes if necessary. A green- ish-3'ellow coloration indicates traces of phosphates, a canary-yellow colour an appreciable amount, and a yellow precipitate larger quantities. Silica. — ^This compound generally exists in water, either as soluble silicates of the alkalies, or as insoluble silicate of alumina. Evaporate 300 c.c. of the water to dryness after acidulating with HCl. Treat the residue with strong HCl, and transfer by washing to a filter with boiling water. Dry, ignite, and repeat the fore- going treatment with acid and boihng water three or four times. Finally dry, ignite, and weigh as SiOa- Sulphates are readily detected by concentrating to about one- tenth, and adding to the warmed sample a drop of HCl and a few drops of BaCla in solution, when a white insoluble precipitate of BaS04 is formed and rapidly sinks to the bottom of the test-tube. The insolubility of this precipitate should always be tested with sufficient strong nitric acid. Estimation of Sulphates. — A measured quantity of the water is heated to boiling in a beaker ; a few drops of HCl are added, and sufficient hot solution of BaClg to precipitate the whole of the sulphates run in. Time is given to the precipitate to settle, and a little more of the BaClg allowed to fall into the supernatant clear solution. If no turbidity is produced the reaction is complete; 30 PRACTICAL SANITARY SCIEXCE but if even the slightest turbidity occur more BaClg must be added, and the mixture again allowed to settle, until the addition of a drop of BaCU produces no turbidity. The white precipitate is collected on a filter-paper, the weight of whose ash is known, well dried, ignited in a cmcible, and weighed as BaS04. The SO4 is returned as tr-yW of this weight. Alkaline phosphates, sulphates, and chlorides may indicate animal organic matter, especially urine, but it is often difficult to attribute to these salts a source in recent pollution, as all are found in strata free from organic matter. Where marked excess is found, the composition of the geological strata accurately known, and where frequent analysis of pure waters from the same strata are made, an increase of any or all may be attributed to organic pollu- tion. But it should be remembered that slight variation in amount of these salts is met with from time to time in waters arising in certain strata, where contamination is out of the question. Nitrites, nitrates, and poisonous metals, when present, will be found in the dry residue forming the total solids. The metals are most easily detected in this residue. Poisonous Metals. There are only a few metals whose compounds are found in water-supplies. Lead and copper are the chief; occasionally iron and zinc occur; and ver}- rarely chromium and tin. A qualitative examination should be performed in all cases for each of these metals; and where a possibility of other metallic- compounds derived from mines, industrial wastes, etc., exists, a further careful investigation is necessar}-. Lead. — Waters possessing an acid reaction, such as those derived from peaty moorlands, in which organic acids (ulmic, geic, etc.) are formed by certain micro-organisms, dissolve lead. The primary action of water on lead is an oxidation. In alkaline and strictly neutral waters the coating of oxide remains intact, but in acid waters it dissolves. Hard waters containing abundant carbonates form an insoluble ox^xarbonate ; hence hard waters lack the property of dissolving lead. Houston distinguishes between the solvent action of acid waters, and the ' erosive ' action of neutral waters containing dissolved oxygen. Acid waters should be cut THE CHEMICAL EXAMINATION OF WATER 31 out of public supplies, if they cannot be passed through chalk, limestone, etc., so as to be completely neutralized. Four parts of CaCOg or MgCOg per 100,000 are necessary to eliminate plumbo- solvency. The effects of acid moorland waters on lead have been only too clearly seen in certain districts of Yorkshire and Lancashire, where the inhabitants have suffered from anaemia, constipation, colic, wrist-drop, depression, gout, renal disease, and other classical effects of lead-poisoning. The lead is dissolved out of materials of joints, block-tin pipes, house pipes, cisterns, etc. Whilst carbonates and sulphates in water diminish pkimbo- solvency, nitrates favour it, as lead nitrate is the most soluble salt of the metal. A rise in temperature up to 48° to 50" C. increases plumbo-solvency. Compounds of lead and copper in acid solution are precipitated as sulphides by H2S. [Pb, it should be remembered, is partially precipitated from strong solutions by HCl as chloride.] Copper is not precipitated by HCl or soluble chlorides. The precipitated sulphides of Pb and Cu are insoluble in (NH4)2S and KOH. Strongly acid solutions of these metals are not pre- cipitated completely until suitably diluted with water. Lead sulphide (PbS), produced by adding HgS water, or bj^ passing H2S gas, is black, insoluble in KOH, KCN, and (NH4)2S, but soluble in boiling dilute HNO3; it is changed by boihng strong HNO3 into white insoluble PbSO^. Solutions of lead salts on addition of excess of dilute H2SO4 give white PbS04. K2Cr04 produces a yellow precipitate (PbCr04) soluble in KOH, insoluble in acetic acid. KI precipitates yellow lead iodide (Pbl2), more insoluble in water than the chloride. Quantitative Estimation. — Prepare a standard solution of Pb(C2H302)2,3H^O, containing O'oooi gramme Pb per c.c. The molecular weight of lead acetate is 379, of which 207 parts are Pb. Accordingly -, or 1-831, grammes of the salt contam I gramme Pb. Dissolve therefore, with the aid of a little free acetic acid, one-tenth of this quantity — i.e., 0-1831 in a litre of 32 PRACTICAL SANITARY SCIENCE water — and each c.c. will contain ooooi Pb. To loo c.c. of the water in a Nessler glass standing on a white tile add a few c.c. dilute acetic acid and sufficient HoS solution to precipitate all the lead. To 100 c.c. of distilled water in a similar Nessler glass add the same amounts of acetic acid and HoS solution, and run in from a burette or pipette the standard lead acetate solution until the depth of tint in the two Nesslers is exactly the same. Perform a second experi- ment, in which the whole volume of the standard lead acetate solution is added to the acidified distilled water at once, and then the HoS solution added and well mixed. The weight of Pb present in milligrammes per lOO c.c. (parts per 100,000) = 0-1 milligramme (o-oooi gramme) xthe number of c.c. of standard lead solution used. It may be necessary to dilute the water sample, in which case a careful record of the amount of dilution must be made, and taken into account in calculating the result. Or it may be necessary to evaporate 500 c.c, or a litre, down to 100 c.c, and to use this for the estimation. Concentration ma}' be necessary also in the quali- tative examination. Not more than 0-025 ps-^t Pb per 100,000 may be present in water without producing an effect when the water is drunk; 0-095 per 100,000 has proved fatal, and 0-050 is dangerous. In a word, all drinking water should be free from lead, as its poisoning action is increased through accumulation in the body. Copper — Qualitative Examination. — There are two classes of copper salts — cupric and cuprous. Cupric salts are blue or bluish-green, and when freed from water of crystallization become pale or lose colour. Cuprous salts are usually white or colourless; they yield red CU2O when mixed with KOH, and white CugL when mixed with KI solution. CuO is black; Cu.,0 red. A little dilute NH4OH added to solutions of copper salts produces a greenish-blue precipitate. More NH4OH dissolves the precipitate, forming an intensely blue liquid. KOH forms a pale blue precipitate, which when heated becomes black. H2SO4 produces no precipitate ; difference from lead. K4Fe(CN)g produces a reddish-brown precipitate, Cu2Fe(CN)f., insoluble in acetic acid. THE CHEMICAL EXAMINATION OF WATER 33 HgS throws down a brownish-black precipitate of CuS insoluble in KOH, (NH4)2S, in boiling dilute H2SO4; soluble in boih'ng HNO., and in KCN solution. Quantitative Estimation. — Prepare "a standard solution of copper sulphate containing 0-3929 gramme CuS04,5H20 per litre. One c.c. of this solution = 0-000 1 gramme Cu. Dilute or concentrate the water if necessary, and place 100 c.c. in a Nessler glass as in the case of Pb. Add a few c.c. decinormal acetic acid and a few drops K4Fe{CN)g solution; a reddish-brown tint is produced. Match the intensity of this colour in a similar Nessler glass by mixing the necessary volume of standard copper solution with 100 c.c. distilled water and the same quantities of decinormal acetic acid and K4Fe(CN)g as were added to the glass containing the sample. The number of c.c. of standard copper solution x o-i gives the weight of Cu in the water in parts per 100,000, as in the case of Pb. Not more than o-i part per 100,000 Cu is permissible in potable water. Tin. — There are two classes of tin salts — stannous and stannic. 1. Pass HgS into a solution of stannous salt acidified with HCl: a dark brown precipitate soluble in KOH and yellow ammonium sul- phide forms on heating ; reprecipitated by HCl from the KOH solu- tion as brown SnS, and from the ammonium sulphide solution as yellow SnSg. [Note SnS is insoluble in colourless ammonium sulphide.] 2. Add HgClg to acidified solution of a stannous salt: a white precipitate, Idg^Cl^; turns grey on boiling if the Sn salt is in excess through formation of metallic mercury and stannic chloride — Hg2C1.2 + SnCl.2 = Hga + SnCl4. 3. Add to the acidified stannous salt a drop of Br-water and a little AuClg! purple precipitate. ' Purple of Cassius.' Stannic salts in acidified solution : 1. HjS: yellow precipitate of SnS.2, soluble in both yellow and colourless ammonium sulphide; soluble in KOH on heating; re- precipitated b}^ HCl as yellow SnSg from both solutions. 2. HgClg: no precipitate. 3. AuClg : no precipitate. Quantitative Estimation of Tin [Stannous or Stannic).- — In a measured quantity of water, concentrated if necessary to a small 3 34 PRACTICAL SANITARY SCIENCE bulk, precipitate the Sn as sulphide. Stand in a warm place till the smell of H.,S has nearly- disappeared. Filter. Wash well. Dry. Ignite in the air into SnO.,. Weigh, and calculate the Sn. [Inciner- ate the filter-paper apart from the precipitate, and add tlie ash to the crucible containing the SnOo-] No tin should be present in drinking water. Iron. — Ferruginous waters are found in mountain limestone, chalk, Bagshot sands, and greensands. They are generally opal- escent, and slightly 3'ellow in colour. The metal occurs as a bicarbonate which is readily converted into an insoluble carbonate, and also oxidized into the w^ell-known ' rust ' — hydrated ferric oxide, FcaO^, H-iO. Ferrous salts decompose nitrates, absorbing and producing nitrites, which in turn are further reduced to XH3. This reducing action accounts for the free NH3 often found in pure waters derived from the greensands and other strata. Chalybeate waters may be quite clear when drawn, but as oxidation of the Fe proceeds they become turbid and more or less brown. The insoluble and highly oxidized particles dissolve on the addition of a little dilute acid. Such turbidity may have its origin in iron pipes, cisterns, etc., in addition to strata. Two classes of iron salts exist : ferrous, in which Fe is divalent, and ferric, in which it is trivalent. They may be readily distinguished b}' the three reagents, potassium ferrocyanide, K4Fe(CN)g, potassium ferricyanide, K3Fe(CN)fi (a solution always being made from the crys- tals immediately before use), and potassium sulpho-cyanide, KCNS. Reagent. Ferrous Compound. Ferric Compound. K4Fe(CN)6 - K3Fe(CN)6 - KCNS - Light blue precipitate, be- coming dark blue on oxi- dation by the air, HNO3, or Br. Dark blue precipitate; Turnbull's blue insoluble in HCl. No red colour. Dark Prussian blue, in- soluble in HCl; turned brown by KOH. No precipitate. Blood-red colour (no ])re- cipitate). Colour de- stroyed by dropping a few drops into a solution of HgClg. THE CHEMICAL EXAMINATION OF WATER 35 Sulphuretted hydrogen passed through a solution of a ferric salt reduces it to the ferrous state, with deposition of S. H^S gives no precipitate with a ferrous salt in acid solution. (NH4)2S precipitates from a ferrous salt black ferrous sulphide. This reagent reduces a ferric salt to the ferrous state, and then precipitates ferrous sulphide with S. Except in connection with greensands, ferrous iron is rarely, met with in water work, ferric salts alone being found. (NHJaOH produces with ferric salts a reddish-brown, flocculent precipitant, FealOH)^, insoluble in KOH, soluble in HCl. (NH4)2S precipitates black FeS soluble in HCl, insoluble in KOH. These tests with the above reactions, produced by K4Fe(CN)g and KCNS are sufficient to identify ferric compounds in water. Quantitative Estimation. — If the quantity of iron is small, it may be estimated colorimetrically like lead and copper, in which case prepare a standard solution of iron alum, Fe(NH4)(S04)2.i2H20, by dissolving o-86i gramme in a litre of distilled water. This solu- tion contains o-oooi gramme Fe per c.c. If necessary, evaporate half a litre of the water to 100 c.c. Place this in a Nessler glass^ and add i c.c. of dilute K4Fe(CN)g solution. Match the colour of the liquid in this glass by adding to 100 c.c. distilled water in a similar Nessler the same amount of K4Fe(CN)g and the requisite quantity of the standard iron solution. It is well to add a drop or two of nitric acid free from iron to each of the Nesslers. If the quantity of iron is too great for colorimetric estimation, acidify a litre of the water with HCl, and evaporate to dryness. Complete the drying in the air-bath at 150° C. Moisten with HCl, add water, and heat. Filter off any insoluble silica (which may be washed, ignited, and weighed). To the filtrate add a few drops pure HNO3, and boil. Then add a little (NH4)C1 solution and a shght excess of NH4OH, and allow the precipitate of ferric hydroxide to settle. Filter this off; ignite and weigh as Fe^Og. [Should it be necessary to estimate Ca, which is now contained in the filtrate, add excess of ammonium oxalate, allow to settle, filter off, and ignite the calcium oxalate. Weigh as CaO.] Not more than o-i part Fe per 100,000 should be present in a domestic water. A distinct chalybeate taste is produced by 0*3 part per 100,000. 36 PRACTICAL SANITARY SCIENCE Chromium. — Evaporate a litre of the water to be tested to dryness, and fuse the ash with solid potassium nitrate and sodium carbonate to produce yellow KoCr04, which, in neutral solution, produces a red precipitate with AgNOg (soluble in ammonia and dilute nitric acid), and in. solution in acetic acid gives a yellow precipitate with lead acetate insoluble in dilute acetic acid. A few c.c. of a largely concentrated sample may be dropped on a thin layer of ether which has been floated on a dilute solution of H2O2 acidi- fied with H2SO4. Upon slight agitation the blue colour which forms in the lower solution passes to the ether. In chromates (yellow or red in colour) Cr exists in combination with oxygen, acting as an acid radicle. Cr also forms a set of salts in which it acts as a metallic radicle. These are green or violet in colour, but pass through oxidation into chromates. Conversely, chromates pass by reduction into green chromic compounds. Acidify a chromate with HCl, add Zn, and warm; the yellow chromate passes into a green chromic salt. (NHJOH and KOH in small quantity produce a pale bluish- green or purple precipitate of Cr2(0H),j, more or less soluble in excess of the precipitant. Quantitative Estimation. — A chromate is first transformed by a reducing agent into a chromic salt. A solution of the chromic salt is then precipitated by NH4OH in presence of NH4CI, and the resulting hydrate converted by ignition into CraOg, and weighed. From the weight of CroOg the amount of Cr is calculated. No chromium should be present in a drinking water. Zinc. — Concentrate the water. (NH4)2S produces a white, flocculent, gelatinous precipitate, which often appears yellow owing to excess of yellow ammonium poh'sulphide, (NH4)2Sa. This reaction is characteristic, as zinc sulphide is the only white sulphide capable of being precipitated. Zn is only partly precipitated from neutral solution by HjS, but by adding sufficient NaOH, NH4OH, or sodium acetate, the whole of the metal may be precipitated by this reagent. Solution of NH4OH gives a white precipitate of Zn, (0H)2, readily soluble in excess of ammonia. K4Fe(CN)g produces a white gelatinous precipitate of zinc ferro- cyanide. THE CHEMICAL EXAMINATION OF WATER 37 Quantitative Estimation of Zn. — Prepare a standard solution of ZnS04,7H20. In 287 parts of this salt there are 65 of Zn, or in 4-4 parts I of Zn. Dissolve 4-4 grammes of the crystals in a htre of water (each c.c. = o-ooi gramme Zn). Use this standard solution volumetrically, as in the case of Fe, precipitating the Zn with K4Fe(CN)p. Avoid much excess of the ferrocyanide. This may be effected by placing a drop of the mixture on a white tile in contact with a drop of a saturated solution of uranium acetate, when a brown colour appears immediately free ferrocyanide is present. Gravimetric Estimation. — Heat a measured quantity of the con- centrated water to boiling, and add slight excess of a solution of NagCOg. Boil again, and allow the precipitate to settle. Wash several times by decantation with boiling water; transfer the pre- cipitate to a filter, and finish the washing thereon. When finished the wash-water shows no alkalinity to litmus and gives no precipi- tate with BaClg. Dry the precipitate, and carefully transfer it to a porcelain cruci- ble. Heat to redness. Wet the filter-paper with strong ammonium nitrate solution, and dry it; incinerate it in the flame in a coil of platinum wire, and let the ashes fall into the crucible. The flame should not enter the interior of the crucible during ignition, lest reduction of the ZnO take place. Cool and weigh. Calculate Zn from ZnO. Carbonate of Zn, ZnCOg, is found in certain mineral waters in quantities varying from q-ooi to 0-005 parts per 100,000. As much as 10 parts per 100,000 ZnS04 have been detected in such waters. Zinc may be introduced by galvanized iron tanks or pipes. It should not be found in a drinking water. CHAPTER IV ORGANIC MATTER IN WATER As vegetable organic matter has little significance from the sanitary point of view, attention is almost entirely directed to animal matter in the form of sewage. It is not proved that animal organic matter per se in the quantities found even in dilute sewage is hurtful to health; its importance lies rather in the fact that pathogenic microbes, especially those of intestinal origin, accompany' it. Wherever, then, faecal matters in quantity large or small are met with danger exists. The complex remains of dead animals and plants are slowly changed to simple inorganic compounds in the superficial laj^ers of the soil under the action of manifold ferments, the products of micro-organisms in association with favourable quantities of heat, moisture, and oxygen. The sum total of these changes is spoken of as an oxidation, since the end products are oxides of carbon, nitrogen, etc. ; but there is no doubt that as in the case of the various fermentations which take place in the alimentary canal of animals, known collectively as digestion, reductions frequently alternate with oxidations. There is some evidence to show that these fer- ments, metabolic products of aerobic and anaerobic bacteria, act along certain lines which are intimately correlated. The specific action of one enzyme furnishes the necessary conditions for the opposed functions of a succeeding enzyme. Whilst an accurate qualitative or quantitative estimation of organic matter in a potable water is impossible, still there are certain chemical tests of value in directing us towards the source of the organic matter, which source maj' ultimately be discovered by other means. A rough differentiation of animal from vegetable matter may be 38 ORGANIC MATTER IN WATER 39 effected by a consideration of the ratio of ' organic carbon ' to ' organic nitrogen,' which ratio forms the basis of Frankland's well-known method of estimating organic matter. The process is only suitable for experienced chemists and laboratories equipped with apparatus for gas analysis. But in skilled hands it is simple and direct. A measured volume of water is carefully evaporated to dryness; the residue is introduced into a hard glass tube along with some oxide of copper, and the tube is heated in a furnace until combustion of the organic matter is complete. The gaseous products of combustion — carbon dioxide, nitric oxide, and nitrogen — are severally collected and weighed, as ' organic carbon ' and ' organic nitrogen.' If in surface waters the proportion of organic carbon to organic nitrogen be as low as 3:1' the organic matter may be considered as of animal origin, while if it be as high as 8 : I it is chiefly vegetable. In certain fresh peaty waters the ratio of C : N has been found as high as 12 : i. In fresh sewage the proportion of C : N may be 2 : i. Frankland held that the smaller the proportion of organic carbon and organic nitrogen in a water, and of these constituents the larger the proportion of C : N, other things being equal, the better is the quality of the water. The fermentation of dead organic matter, known as ' putrefaction,' is effected by many types of micro-organisms. Dead proteins are hydrolysed to proteoses; these to peptones; peptones to amino-acids; finally amino-acids are split, evolving ammonia. If we follow this ammonia as it escapes, say, from, a dung-heap in solution into the soil, we shall find that in the presence of the ' nitrous ' organisms nitrous acid is formed, which in contact with the bases of the soil rapidly becomes nitrites. Later, through the activities of the ' nitric ' group of micro-organisms, nitric acid is generated, which speedily becomes nitrates. These various stages in the oxidation or purification of nitrogenous matters stand out as chemical landmarks, and present considerable information to the water analyst. As carbohydrates and fats are much less complex bodies contain- ing C, H, and only, their decomposition and oxidation are much more simple: carbon is burnt to COg, and H to HoO. These changes in nitrogenous matter ma}^ be studied directly. 40 PRACTICAL SANITARY SCIENCE If, for example, A be a source of organic pollution, say a manure- heap, on the surface of the ground, and B, C, D, and E wells at increasing distances from it, analysis will show that the water in B contains abundance of NH., ; nitrification has not yet taken place. At C the oxidation processes have advanced to the stage of nitrous acid; this water will contain less ammonia and some nitrites. The water from D has travelled farther, encountering more nitrifying organisms, with the result that ammonia has disappeared, and nitrites and nitrates are found. At E purification is complete — the whole of the N is oxidized to nitric acid; hence this water contains no NHg, no nitrites, but onh' nitrates. The opportunities for purification offered between A and B are not sufficient to carry the oxidation changes beyond the stage of NH.,; whereas the journey from A to E is of such length that the entire A B C C Nitrite *"" Nitrate Nitrite Nitrate Fig. 3. changes have been completed. At intermediate points are observed intermediate stages in the purification. From the consideration of a single instance of this kind, no con- clusions as to the distance a well must be removed from a source of contamination in order to be safe can be drawn, since the factors in the problem of safety are numerous and variable. The distance between A and E, if the water in E is to be completely purified, would require to be much greater if the slope from A to E be con- siderable, or E would need to be much deeper. On the other hand, if the slope of the ground water descended from E to A, it is possible that the water of B may be free from all organic matter. The porosity of the soil, conditions of heat and moisture necessary to vigorous growth of purifying organisms, direction of slope of ground water, geological features of subsoil and underlying strata, rainfall, and a number of other factors, all influence this question of safe distance of well waters from foci of contamination. Each case must be worked out on its ow-n merits; and here the chemical ORGANIC MATTER TN WATER 41 examination renders useful service. A sample of water from E may be pure to-day — that is, contain no organic matter as such, no NH3, no nitrites, but only nitrates ; to-morrow, owing to increased rainfall, whereby more organic matter than usual is washed into the soil, or to some other condition by which the powers of the soil for purification are lessened, this same water may contain, besides nitrates, nitrites, NH3, and even undecomposed organic matter. It should ever be borne in mind that the machinery by which organic matter is purified in the soil is liable at any point to break down, and in too many instances although just sufficient for the work is near breaking-point. The question, therefore, should be, not how near to a focus of contamination may it be safe to procure water, but rather how far from the focus is it possible to acquire it. Chemical analysis, if frequently and regularly performed, will, in most cases, discover such breakdown in the purification machinery, although a single analysis, unaccompanied by further information as to source and surroundings, may be quite useless. It is the comparative information regarding a water acquired by systematic and repeated analyses that is of value. The student should note that the nearer the nitrogenous organic matter of domestic sewage stands to the stage of raw proteins the worse, as it is in this stage that pathogenic bacteria are found in their most toxic and vigorous condition; and that, conversely, the farther from this stage such matter stands the less dangerous it is. When organic matter reaches the stage of nitrates no patho- genic germs will live in it. From the standpoint of infection, fresh faecal matter and urine are the most dangerous of all forms of organic matter. It is not possible in water analysis to separate and estimate raw proteins, proteoses, peptones, and amino-acids. The next stage, that of NH3, lends itself to ready estimation. When this ' free and saline ' ammonia, as it is called, is removed, the remaining organic matter, which consists of the nitrogenous complexes constituting the antecedent stages, can be rapidly oxidized by the aid of a powerful oxidizer and heat (^\'anklyn's process) into ammonia, and estimated as ' albuminoid ' ammonia. This figure, inasmuch as it measures those portions of the nitrog- enous organic matter likely to contain pathogenic micro-organisms. 42 PRACTICAL SAXITARY SCIEXCE is obvioush' the most important determination connected \vith this portion of tlie subject. Estimation of 'Free and Saline' NRj. — Prepare a standard solution of (XHjjCl, i c.c. of which = o-oi milhgramme NH3. 53-5 grammes NH4CI contain 17 grammes NH3. 3-14 ,, ,, ,, I gramme NH3. Dissolve 3-14 dr}' anhydrous NHjCl in i Htre ammonia-free distilled water. One c.c. of this solution =1 milligramme NH3. This is too strong. Dihite 10 c.c. of it to a litre; i c.c. now=o-oi milligramme XH3. The process depends on the fact that when the water is distilled with a little sodium carbonate all the ammonia in the water, free or combined, passes over in the first portions of the distillate, and may be estimated by Nessler's solution. Prepare Nessler's solution. Dissolve 62'5 grammes KI in about 250 c.c. distilled water. Set aside a few c.c. of this solution. Now add to the larger portion saturated mercuric chloride solution till precipitated mercuric iodide cea,ses to dissolve on stirring. Add the reserved KI so as to redissolve the precipitate, and again add cautiously sufficient mercuric chloride solution to produce a shght permanent precipitate. Dissolve 150 grammes KOH in about 300 c.c. water; cool; add gradually to the above solution, and make up with HoO to a litre. A brown precipitate settles out on standing, and the supernatant fluid is clear and of a pale greenish-yellow colour. It is ready for use as soon as it is perfect!}' clear. It should be decanted without stirring up the sediment. Keep in bottles closed with well-fitting rubber stoppers. This solution is rendered sensitive from time to time by the addition of a little more HgCU solution ; its sensitiveness depends on its being saturated with HgCU. Sodium Carbonate. — Heat anhydrous NaaCOg to redness, taking care not to fuse it ; transfer to a mortar, and grind to a fine powder. Store in a clean, dry, wide-mouthed, stoppered bottle. Ammonia-free water is prepared bj' distilling ordinary water in the presence of NaaCOg or H.,S04, and rejecting the first portions of the distillate until there is no trace of colour produced on Nesslerising 50 c.c. of it. ORGANIC MATTER IN WATER 43 A preliminary test may be made in order to ascertain what quantity of the water-sample should be distilled in order to make an exact determination of the ammonia. Place two Nessler glasses on a white tile; add 50 c.c. of the sample to one, and 50 c.c. am- monia-free distilled water to the other. To the ammonia-free water add 0-5 c.c. of the dilute standard NH4CI solution. To both Nesslers now add 2 c.c. Nessler's reagent, and stir. If on standing five minutes the intensity of colour in both cylinders is the same, 500 c.c. of the water may be used for distillation. If the intensity of the colour of the sample is much greater, dilution is necessary prior to distillation, otherwise the quantity of ammonia in the first 50 c.c. distillate will be too large to match. Arrange a distilhng-flask, condenser, and Bunsen burner. Pour into the flask 500 c.c. of the water (or water sufficiently diluted); add some prepared sodium carbonate, and if the water is acid a little more than usual (the least acidity fixes NH3). Receive in Nessler glasses 150 c.c. distillate in three lots of 50 c.c. each. The boiling should be briskly effected; it is generally useful to place a piece of pumice in the flask to prevent bumping. As each Nessler glass is filled it should be Nesslerised or covered until Nesslerisation is accomplished. Nesslerisation is one of a number of colorimetric methods of volumetric analysis in which the amount of a substance is esti- mated by adding to it a second body capable of forming a char- acteristic colour with it. The same conditions are accurately fulfilled in a similar vessel, using distilled water and such quantity of the substance sought, in standard solution, as will match the colour of the first when the same quantity of the second body is added. In order that shght differences in tint may be appreciated and matched, it is necessary to work with dilute solutions of the body to be estimated and the standard reagent ; hence the necessity at times of diluting the water under examination. Having collected the three 50 c.c.'s of distillate, Nesslerise each separately. Stand the Nessler glass on a white tile in a good north light, and by its side place a second Nessler glass of similar shape containing distilled ammonia-free water, and that quantity of standard solution of (NHJCl deemed necessary to match the first. Into each deliver 2 c.c. of Nessler's reagent, and carefully mix. In 44 PRACTICAL SANITARY SCIENCE a few minutes the yellow colour will have fully developed, and its depth can be gauged by looking down through the column. Should there be some discrepancy in the tints, rapidly add to another Nessler glass containing distilled ammonia-free water a little more or a little less of the standard solution, as the case may be, until an exact match is produced. In all such colorimetric work every condition should be exactly similar in the two cases — length of time reagents are in contact, order in which reagents are added, shape and size of containing vessels, etc. The standard solution of XH4CI must be added to the second Nessler glass before the Nessler's reagent, as this occurred in the Nessler glass containing the distillate. If the standard solution be added after the Nessler reagent an opacity is likely to form which prevents to some degree an exact match being made. Several trials may be necessary before an accurate result is reached. The second and third 50 c.c. of the distillate are treated in the same way, and the sum of the results in terms of c.c. of the standard solution noted. \\'anklyn found that the whole of the free and sahne NH3 was contained in 150 c.c. distillate, and that the first 50 c.c. contained three-fourths of the total. Nesslerise the second distillate first, and note whether more then 1-5 c.c. of the standard NH4CI solution is required to match it. if so, the first distillate must be diluted before Nesslerisation, other- wise the colour will be too intense to be accurately matched. Example. First Nessler glass matched by 3-00 c.c. XH4CI (ic.c. = o-oi milligramme XH3) Second ,, ,, ,, ,, 0-75 Third ,, ,, ,, ,, 0-25 Total XH3=4'Oo ,, ,, ,, ,, But each c.c. standard NH4Cl = o-oi milligramme NH3; .■ . 4 c.c. = 0*04 And in 500 c.c. of the water under examination there is 0*04 milligramme NH3. In 100 c.c. there will be o-ooS milligramme XH3, or, since 100 c.c. water = 100,000 milligrammes, this water contains free and saline XH3 to the extent of O'OoS part per 100,000. Estimation of 'Albuminoid' NH3.— \Miilst the Nesslerisation of the free and saline NH3 is going on, 50 c.c. of alkaline potassium permanganate (composed of 200 grammes KOH, 8 grammes per- ORGANIC MATTER IN WATER 45 manganate, a litre of water) should be boiled, so as to expel any ammonia that it may contain, and to heat the liquid in order to prevent cracking the retort when pouring it in. Ihis is a strongly oxidizing reagent, and rapidly converts undecomposed organic matter into NH3. By this moist combustion process a degree of oxidation is effected in the course of half an hour or so in the laboratory that would require weeks or months by the natural processes outside. When the alkaUne permanganate is ready, the cork of the retort is removed and the hot solution poured in. This portion of the distillation should be carried out more slowly, as organic matter is slowly decomposed, and the distillate should be collected as long as any NH3 comes over. No relation exists between the number of the Nessler glasses collected and the total NH3, as in the case of the free and saUne portion. Moreover, the second Nessler may contain as much NH3 at times as the first. The student should fit up his apparatus himself, and see that all connections are water-tight and gas-tight, as the case may be. Corks should be carefully bored and made to fit flasks and con- denser tubes, and indiarubber corks are preferable to wood. The distilling-flask should be thoroughly cleansed with weak acid and rinsed out with distilled water until all traces of acid have dis- appeared. It is well to distil some pure ammonia-free water through the condenser in order to get rid of any traces of NH3 that it may contain before starting the distillation of a sample. A large and constant stream of water running through the condenser is necessary throughout the entire process. A long-stem funnel is to be used for delivering water, etc., into the retort, and this is especially necessary for the introduction of the hot alkaline per- manganate, so that none of the reagent may enter the central tube of the condenser and foul the distillate. Seeing that the atmosphere of an ordinary chemical laboratory contains quantities of NH3, it is well to have a separate room for water analysis. In very rare instances a potable water ma}^ not yield the entire free and saline NH3 to the first 150 c.c. of the distillate. In such cases it will be necessary to distil over and Nesslerise a fourth or fifth 50 c.c. 46 PRACTICAL SANITARY SCIENCE It is possible that the ' saline ' ammonia exists in water in con- junction with some acid, which, on being boiled in the presence of cai"bonates, yields up the ammonia in the form of (NH4)oC03. In the second part of the process, the distillation of the albu- minoid ammonia may require to be carried to a point at which the volume of fluid in the flask becomes dangerously small; this should never be allowed, but ammonia-free distilled water should be added to the flask as required, so that the volume may be kept up. With regard to the amounts of ' free and saline ' and ' albuminoid ' ammonia which may be allowed in different potable waters, there is some little difterence of opinion. All observers agree that the two ammonias must be considered together, and most agree that in drinking waters if the ' albuminoid ' reach 0-005 part per 100,000 the ' free and saline ' should not be more. If the ' albuminoid ' be small — say less than 0"002 part per 100,000 — the ' free and saline ' may be allowed to slightly exxeed 0-005. Much 'albuminoid ' and little ' free and saline ' ammonia indicate vegetable matter; whereas much ' free and saline ' and little ' albu- minoid ' indicate animal matter. These indications must not be too literally rehed upon. As a general rule, it ma}^ be stated that where a water has been contaminated with sewage the high ' free and saline ' ammonia figure will be supplemented by an increase in chlorides, phosphates, and oxidized nitrogen. Whilst accepting the principle that animal pollution is indicated by a relatively larger figure for ' free and saline ' ammonia than for ' albuminoid,' and that vegetable matter produces much ' albuminoid ' ammonia, with little or no ' free,' it must be borne in mind that these relations are liable to be upset. Peat}^ waters, whilst producing ' albuminoid ' ammonia in quantity, should not produce any ' free ' ; still, there are peatj^ waters met with at times which give rise to a small quantity of ' free ' ammonia, although no animal matter can be traced. Good spring waters rarely contain ' albuminoid ' ammonia above 0-002 part per 100,000. Upland surface waters, as a whole, should not produce ' free and saline ' ammonia beyond o'ooi part per 100,000. The degree of initial dilution necessary to produce the best colour-tint for matching on Xesslerisation can only be discovered ORGANIC MATTER IN WATER 47 by experience, and here, as in all matters practical, the student should ever appeal to experiment. Scores of waters must be patiently worked out in complete detail before he can expect to acquire even an elementary knowledge of the subject. Much ' free and saline ' ammonia in the absence of ' albuminoid ' may be accounted for by the water passing through strata rich in ammonium salts, portions of which are carried away in solution ; water-bearing strata containing nitrates and subsalts of iron afford ' free and saline ' ammonia by the reduction of the nitrates through the intermediate phase of nitrous acid to NH3; rain water falling through the atmospheres of towns abounding in ammoniacal fumes will yield appreciable quantities of ' free ' ammonia, and at times small quantities of ' albuminoid ' also from the organic matter in suspension in the air. It may be noted that, although the Wanklyn process does not decompose urea, the most important and abundant nitrogenous constituent of urine, nor recover NH3 from a few other bodies in sewage, still it is of the greatest value in dealing with the con- tamination of water by organic matter, from the comparative results afforded, so long as the determinations are carried out under similar conditions. Oxidizable Org'anic Matter in Water. Forchammer applied to water analysis his knowledge of the ex- perimental fact that organic matter in the presence of an acid can rob KaMugOg of a portion of its oxygen. This process was slightly modified by Tidy, and is usually known in this country in connection with his name. It is not a reliable test of either the quality or quantity of organic matter present, but, in that pure waters absorb practically no O from permanganates of potassium, and foul waters a great deal, the process has some value as corroborative evidence of the presence of organic matter. It should be noted that other bodies beside organic matter, such as ferrous salts, nitrites, sul- phides, etc., abstract from KaMngOg, and when these are present they must be accounted for before drawing a conclusion as to the amount of organic matter dealt with. The quantity of absorbed 48 PRACTICAL SANITARY SCIENCE varies with the time of contact, the temperature, and, to less extent, with the acidit}-, and hght admitted during digestion. Potassium permanganate in contact with organic matter and H0SO4 furnishes 5 atoms of and colourless sulphates of manganese and potassium. KaMuoOg + 3H.,S04 = 2MnS04 + K0SO4 + sHoO + 5O. If sufficient acid be not added, the liydrated peroxide falls as an opaque brown precipitate, and only 3 atoms of O are set free. KjIMnoOg + H2SO4 + 3H20= 2Mn{OH)4 + K2SO4+ 3O. During the digestion the reaction should be carefully watched, to see that the fluid remains transparent throughout. If much organic matter be present, it may be necessary to add further quan- tities of permanganate from time to time. Various times and temperatures have been employed in this process for digesting the sample of water with the acid and per- manganate, some anah'Sts recommending four hours at 80° F., others three hours, two hours, or fifteen minutes, at higher and lower temperatures. In a laboratory where an incubator is kept at blood-heat (37° C.) it is convenient to use it, and three hours is a sufficient length of time. In examinations two hours at room- temperature may be found most convenient. Prepare a standard solution of potassium permanganate (i c.c.= O'l milligramme of available O) by dissolving 0-395 gramme of the pure crystal in a litre of distilled water. Make a fresh 10 per cent, solution of KI, and a fresh solution of sodium thiosulphate, of about I gramme to a litre of water. Lastly, prepare a boiled I per cent, solution of starch, and test its delicacy with water con- taining the merest trace of free iodine. Clean two Erlenmeyer flasks (capacity 150 c.c. or less), and into one measure 100 c.c. of the water sample. Mark it ' Sample ' with a wax pencil. Into the second, marked ' Control,' measure 100 c.c. distilled water. Now carefully pipette into each 10 c.c. of the standard solution of KoMn^Og, and with another pipette run into each 10 c.c. of a 25 per cent, solution of pure H2SO4. Stopper and set aside in an air oven or incubator, as the case may be, at 37° C. for a period of three hours. Should the amount of organic matter in the water be large, the whole of the permanganate ma}' be de- ORGANIC MATTER IN WATER 49 composed and become colourless; in such a case a second 10 c.c. of the standard solution is added, and should this be decolourized a third, and so on. Account of the further additions will be taken in the calculation at the end of the experiment. When the time allowed has expired, and a portion of the per- manganate remains undecomposed, as demonstrated by the red tint still to be seen, a few drops of the KI solution are added to the flask containing the water sample, when free iodine is liberated in quantity proportional to the amount of undecomposed KgMngOs remaining. A very few drops of the KI solution will contain an excess of iodine. This liberated I — the measurer of the undecom- posed KaMugOg left in the Erlenmeyer — is made to oxidize thio- sulphate run into it from a burette, the end reaction being definitely ascertained in the presence of a few c.c. of the boiled starch solution by the disappearance of the blue colour of the iodide of starch. The same procedure exactly is carried out with the control, and here, as no KaMngOg has been decomposed, but the whole of the 10 c.c. remains intact, we obtain a figure in terms of c.c. of thio- sulphate solution which represents this amount, or i miUigramme available O. The following equations represent the liberation of free I and its subsequent oxidation of sodium thiosulphate to sodium tetra- thionate : KaMuaOg + loKI + 8HoS04= 6K2SO4 + 2MnS04 + SH^O + 5I2. I2 + aNagSgOg^^ 2NaI + Na2S40g. Example. — The intact 10 c.c. standard solution of permanganate in distilled water liberated iodine equivalent to 27 c.c. of the thio- sulphate solution. The undecomposed portion of the 10 c.c. of standard permanganate in the water sample liberated iodine equiva- lent to 23-2 c.c. of thiosulphate. From this it is plain that the amount of permanganate solution decomposed by the organic matter (assuming that no nitrites, sulphides, etc., were present) is represented by 27-23-2 c.c. thiosulphate. But 10 c.c. standard permanganate or i milligramme 0= 27 c.c. thiosulphate; .•.27 : 27-23-2 : : i milhgramme : x\ (27-23-2) XI x= ^-' '^—^ = 0-14. 27 50 PRACTICAL SAXITARY SCIENCE There is, therefore, in lOO c.c. of this water organie matter capable of absorbing from pemianganatc of potassium O to the extent of 0-14 milHgramme, or 0-14 part per 100,000, under the conditions of time and temperature employed. If it be desired to obtain some indication of the nature of the reducing substances, two samples of the water may be treated with the standard permanganate, one at 37° C. for fifteen minutes, and the other for three hours at the same temperature. Nitrites, ferrous salts, and sulphuretted hydrogen effect reduction almost imme- diately, whilst a relatively large amount of ordinar}' organic matter reduces the reagent only after a considerable time. The O absorbed from permanganate is higher as a rule in upland surface waters than in waters from other sources; and whilst no strict standards can be insisted on, it may be stated generally that in upland surface samples of great purity this figure in parts per 100,000 (time three hours, temperature 37° C.) will not exceed o-i, in waters of medium purity 0-3, and in waters of doubtful purity 0*4. The corresponding figures for other sources will not exceed 0-05, 0-15, and 0-2 CHAPTER V OXIDIZED NITROGEN— NITRITES AND NITRATES During the early stages of putrefaction of organic matter much free N escapes in gaseous form, and the rest unites with H to form NHg. As has been already stated, certain bacteria in the soil and elsewhere convert NH3 into HNO2, which latter combines with various bases to form nitrites. Of the so-called ' nitrous ' organisms several species have been studied, one of which is the Nitrosomonas of Winogradsky. Nitrites, therefore, represent chemicallj' the intermediate stage in the process of oxidation or purification. Under certain conditions, to be presently mentioned, they also represent an intermediate stage in the reduction of nitrates to NHg. The presence of nitrites in a water indicates more remote contamination in point of time or space, or of both, than does NH3. In like manner ' nitric ' organisms, such as the Nitrobacier (Wino- gradsky), transform HNOg into HNO3, which readily becomes nitrates. This class of bacteria has no action on NH3, and the previous class is unable to carry the oxidation of NH3 further than HNO2, so that two distinct and independent types of organism are necessary to the complete oxidation of NH3. It will be readily seen that the detection and estimation of nitrites and nitrates are of considerable importance in the investiga- tion of the problem of organic pollution. The presence of nitrates alone in a water indicates previous pollution that has been oxidized and rendered harmless. But if the quantity of nitrates be great, purified sewage may be sus- pected, which, through a breakdown at any moment in the machinery of purification, may become most dangerous sewage. Moreover, in view of the fact that sewage effluents contain almost as man}- micro- organisms as crude sewage, no effluent, however high its degree of 51 52 PRACTICAL SAXITARY SCIENCE purification may be chemically, should ever be allowed to come in contact with drinking water. In all waters possessing a high nitrate figure this possibility of the presence of purified sewage should be borne in mind. When nitrates, which form the end of the purification of organic matter, occur alone it is obvious that no indication of the date of the previous pollution is given. In determining the true significance of nitrates in potable waters it is necessary to consider (i) whether they arise from geological strata (chalk, lias, oolite, sandstones) through which the water has percolated, in which case the evidence of organic pollution supplied by the other steps of the analj^sis — such as the ' free and saline ' NH3, ' albuminoid ' NH3, O absorbed from permanganate of potas- sium, etc. — will be negative; (2) whether they are due to purified sewage, in which case the quantity will be much too great, as also that of CI; (3) whether they represent a small amount of organic matter that has undergone complete oxidation, and is to be con- sidered harmless. In this case the quantity will be small — in rain and upland surface waters not exceeding o-i part per 100,000 — and all the other items of the analysis employed to discover organic matter will afford negative evidence. In the few cases where strata alone contribute soluble nitrates the quantity will rarely exceed 0-5 part per 100,000, but no figures can be laid down as an accurate standard, and each case must be worked out in connection with the rest of the analytical data. In a few instances strata containing nitrates (in particular the lower greensand) contain also reducing minerals, such as proto- salts of iron, which reduce HNO3 to HNOg, and the latter to NH3. The same reduction can be effected by denitrifying micro-organisms. Free XH3, due to reduction of nitrates and nitrites, will be identified by the absence of organic NH3, and all other evidence of organic pollution. Nitrites are very unstable, and in the presence of available O rapidlv become nitrates. In the early stages of the oxidation of large quantities of animal organic matter they are mostly found in company with NHg, but a foul water may at a particular moment fail to furnish any nitrites. They are significant of recent con- tamination, except in those cases just mentioned, where they are OXIDIZED NITROGEN— NITRITES AND NITRATES 53 due to the reduction of nitrates in strata. Nitrites, then, which in deep-well waters may be merely the products of reduction of nitrates by iron in strata, iron pipes, etc., and consequently quite harmless, will in shallow wells and surface supplies condemn the water. All the inorganic N — apart from strata — found in nitrates, nitrites, free and sahne NH3, after deducting that present in rain water, may be regarded as due to previous sewage contamination. Detection and Estimation of Nitrites. Potassium Iodide and Starch.— To lo c.c. of the water in a test-tube add i c.c. of a clear and boiled i per cent, starch solution and a drop of KI solution. Mix and add a little dilute H2SO4, when immediately a blue colour is produced if nitrites be present in con- siderable amount. On standing, nitrates give this reaction also Pure sulphuric acid should be used, and it is found that, owing to the instability of KI, Znl gives better results. This test can be made a quantitative colorimetric one by operating on 100 c.c. of the water in a Nessler glass, and in a second Nessler 100 c.c. of a mixture of distilled water and the amount of a standard nitrite necessary to form a colour match. When the proportion of nitrites in a sample is I in 10,000,000, the blue colour is formed in a few minutes; when I in 100,000,000, in twelve hours; and when i in 1,000,000,000, in forty-eight hours. Lintner's soluble starch should be used. Griess's Method. — Make a 5 per cent, solution of meta- phenyleije-diamine in water. Decolourize with animal charcoal, and render slightly acid with H2SO4. Much acid must not be used. To 100 c.c. of the water to be tested in a Nessler glass add I c.c. of the reagent, cover, and set aside in a warm place for twenty minutes. A yellow to orange colour is produced, according to the quantity of nitrites present. The reagent should be made at the time of use. When metaphenylene-diamine (diamido-benzol) reacts with nitrous acid, triamido-azo-benzol (Bismark brown) is produced; hence the colour. 2C6H4(NH2)2 + HN02= C6H4(NH2)N.C6H3(NH2)oN + 2H0O. By using a standard solution of potassium nitrite, the colour produced in the 100 c.c. of water may be matched in the same 54 PRACTICAL SAXITARY SCIEXCE quantity of distilled water. A series of trials must be made, in which the reagent is added to the contents of the two cylinders at the same moment, and the cylinders covered and set aside in a warm place for twenty minutes. The standard nitrite is prepared thus: Dissolve 0-406 gramme of AgNO^ in boiling water; add shght excess of KCl. Silver chloride is formed, and gradually falls to the bottom. ]\Iake up to a litre and allow to settle. When clear, decant off the supernatant fluid, and dilute each 100 c.c. up to a litre. It should be kept in the dark and in small bottles filled to the stopper, so as to protect it from the air. I c.c. = 0-01 milligramme NgOg. , ,^„ /mol. wt. NO., \ = 0-000 ,, N0o=( — -, — ^ XT ^" X O'Oi )■ - Vmol.wt.NoOg / -, /mol. wt. Np \ = 0-0037 " N,= l — , — . ^. „ -xo-oi ). ^' - \mol.wt.N2O3 / Detection and Estimation of Nitrates. Brucine Test. — To 10 c.c. of the water in a test-tube add i c.c, of a saturated solution of brucine, and shake. Incline the test-tube and pour down the side 2 c.c. of pure H^SO.j. Carefully bring the test-tube to the vertical against a white ground. A pink zone is foiTned at the junction of the acid and supernatant mixture, which lasts for a few seconds, and then changes to brownish yellow. When nitrates are in large quantity, the colour changes very rapidly. Where the reaction is doubtful, a fresh layer of the mixture can be brought in contact with the acid by imparting to the test-tube a slight centrifugal motion. Or, 10 c.c. of the water ma)^ be evaporated to dryness in a platinum dish, a drop of pure H2SO4 added, and a small crj^stal of brucine dropped on the contents, when a pink colour will appear, even where the quantity of nitrates is so small as o-oi part per 100,000. Diphenylamine Test. — Mix about 10 milligrammes of diphenyl- amine with i c.c. pure H.2SO4 in a porcelain basin, and carefully run I c.c. of the water over the mixture. A blue colour develops in the presence of nitrates ; the depth of the tint is roughly propor- tional to the amount of nitric acid. This reaction is not simulated by any other constituent of potable waters. OXIDIZED NITROGEN—NITRITES AND NITRATES 55 Crum's Quantitative Method. — This method consists in shaking up the residue obtained from the concentration of a measured quantity of the water with metalHc mercury and pure H2SO4, when nitric oxide is produced, which is afterwards conducted to a gas analysis apparatus and measured. It requires some experience in collecting and measuring gases, but in the hands of a skilled operator is one of the most exact methods known. The nitric oxide produced represents the N of nitrites and nitrates. To obtain the N due to nitrates alone, that obtained for nitrites by Griess's method is subtracted from the total. This method may be used for the estimation of nitrous and nitric N in sewage effluents. Process of Estimation. — Evaporate to dryness in a dish 100 c.c. of the water. Add a small quantity 25 per cent. H2SO4. Heat the dish to remove CO2 from any carbonate present, and if the volume of the liquid exceeds 2 c.c. evaporate down to that volume. Fill the nitrometer with Hg, and pour the contents of the dish into the cup of the nitrometer, rinsing out with a very small quantity of the dilute H2SO4. Now run the liquid through the stopcock, taking care that no air enters. Run through also about twice the volume of pure concentrated H2SO4, and shake so as to cause part of the Hg to mix with the hot liquid. In a short time NO will be liberated. Continue the shaking till gas ceases to come off (five to ten minutes). Cool to the temperature of the air. x\djust mercury levels, and take the reading. Note atmospheric temperature and pressure, and calculate weight of N in volume of NO obtained. An estimation gave 2 c.c. NO; temperature 18° C; pressure 758 millimetres. ^^^S32^=,.87NOatN.T.P. 291 X 760 ' As NO contains half its volume of N, and weight of i c.c. H = ^ ^ ^o j-u ■ 1,1. r XT • ^1- xT^ 1-87 X o-oooo8o X 14 0-000089 gramme, the weight of N m the N0= — - = 0-001165 gramme^ 1-165 part per 100,000 N in the water. From this subtract the weight of nitrous N found by Griess's method; the remainder is that due to nitrates. CoppeP-Zinc Couple Method.— This method estimates nitrous and nitric N as NH3. In calculating the nitric N, it is plain that from the amount of NH3 obtained in the process deduction must 56 PRACTICAL SAXITARY SCIENCE be made for original NH., in the water as well as that derived from nitrites. ^^'hen zinc is immersed in CuSOj solution, a spongy deposit of Cu is precipitated upon it, and in this condition it is capable of bringing about various decompositions in which H is liberated. The H is occluded by the spongy copper, and when thus occluded reduces nitrates to nitrites, and nitrites to ammonia. The reaction is hastened by the presence of traces of NaCl and other salts, rise of temperature, and any condition which increases the electrolytic action of the couple. Take a piece of clean zinc-foil and cover it with 3 per cent. CUSO4 solution until a copious, firmly-adherent coating of black spongy Cu has been deposited. This deposition should not be pushed too far, otherwise the Cu will be so easily detached that it cannot be washed. When sufficient deposit has accumulated, the CUSO4 solution is removed and the couple carefully washed with distilled water, when it is ready for use. A clean, wide-mouthed, stoppered bottle is selected and washed out with some of the water to be tested. The coated foil is inserted, and a measured quantity — say 100 c.c. — of the water poured in so that the foil is completely covered. The bottle is tightly stoppered and set aside in a warm place for some hours. If the bottle be properly closed, the tempera- ture may be raised to 28° or 30° C. without fear of losing NH3. When it is desirable to hasten the reaction, a little NaCl may be added to the water (o-i gramme to 100 c.c), or COg ma}' be passed through the water before it is placed in the bottle. In calcareous waters lime may be removed by the addition of some pure oxalic acid previous to digestion with the couple. Nitrous acid remains in the solution until the reaction is complete, so that it is necessary to test a small quantity of the water from time to time by Griess's reagent for the presence of this body. Metaphenylene-diamine easily detects i part of nitrous acid in 10,000,000 of water. When the last trace of nitrous acid has disappeared, the water is poured off the couple into a clean, stoppered bottle, and if turbid allowed to subside. A portion of the clear fluid, diluted if necessary according to the degree of concentration of the nitrates in the water, is trans- ferred to a Nessler glass and the NHg estimated in the usual manner. In the case of coloured waters, or those containing magnesium and OXIDIZED NITROGEN— N ITU ITKS AND NITRATES 57 other salts that interfere with the Nessler reagent, a measured quantity of the water poured off the couple should i)c put in a retort, a httle NagCOg added, and Nesslerisation performed on the distillate. It has been found that about half a square decimetre of zinc-foil should be used for each 100 c.c. of a water containing 5 or less parts of nitric acid per 100,000. A larger proportion of foil should be used for waters richer in nitrates and for sewage effluents. The couple, if carefully washed after use, may be used for at least three estimations. It is convenient in most laboratories to digest overnight. Where accurate results are required, and in the hands of the inexperienced, it is advisable to distil the water removed from the couple and estimate the ammonia in the dis- tillate. From the total N found as NH3 deduct that due to inorganic NH3 found by Wanklyn's process, and that due to nitrites found by Griess's process; the remainder is the N due to nitrates in the water. Sprengel's Phenol Method.^ — This is a much less accurate method (error of under-estimation), but can be performed in a limited time. It estimates the N of nitrates alone, and is chiefly appHcable to waters containing small quantities of nitric acid. When phenol sulphonic acid reacts with nitric acid, picric acid (trinitro-phenol) is formed. C6H3(OH)H2S03 + 3HN03= 2H2O + H2SO4+ QH^NOaJgOH, and the ammonium salt of picric acid being yellow, this body lends itself to quantitative colorimetric estimation. C6H2(N02)30H + NH40H= C6H2(N02)30NH4 + H2O. The solutions required are: Standard potassium nitrate, containing 0-7215 gramme KNO3 in a litre of water. One c.c. of this solution=o-i milligramme N. A dilution of 100 c.c. to a litre should be made for the analysis. One c.c. will then contain o-oi milligramme nitric N. Phenol Sulphonic Acid. — ^The phenol sulphonic acid used should be the pure disulphonic acid (CgH3(OH)H2S03), which, with HNO3, gives, according to Kekule, picric acid even in the cold. Three grammes pure phenol and 37 grammes (20*1 c.c.) pure H2SO4, specific gravity 1-84, are mixed in a beaker and heated for six hours 58 PRACTICAL SANITARY SCIENCE on a water bath at ioo° C. Should the acid thus formed crys- tallize out on standing, it may be brought into solution bv reheating for a short time. Process. — Place lo c.c. of the water in a porcelain basin on a water bath and in a similar basin lo c.c. of the standard nitrate; when just dry, add to each i c.c. of the phenol sulphonic acid and allow to remain for a few minutes on the bath. Now transfer to two Nessler glasses the contents of the dishes, and wash out with 25 per cent, ammonia solution the last trace of material from the dishes; add further ammonia to the Nesslers until all effervescence ceases and a small excess of ammonia is found in each. Spirting ma}' be prevented by washing out the contents of the basins into the Nessler glasses with a small quantity of distilled water, and adding the ammonia solution afterwards. Make up to 100 c.c. in both cases with distilled water, and let stand for fifteen minutes. In performing the estimation, take a third Nessler glass and pipette into it from the more deeply-coloured cylinder (which is generally that containing the standard nitrate) a quantity deemed necessary to match the tint of the cylinder containing the water sample. Make up to 100 c.c. with distilled water, and compare the tints on a white tile. An exact match can be effected in a few trials. Suppose that 20 c.c. from the standard soliition match the water sample, the latter contains y%°„ of the N as nitrates contained in the standard. Each c.c. of the standard contains o'oi milli- gramme N. .•. 10 c.c. = 0-1 milligramme N, but y-^"jj of o-i = o-02; .'. 10 c.c. water under examination =0-02 milligramme N; • ■. 100 ,, ., ,, ,, =0-2 or this water contains 0-2 part nitric N per 100,000. In the case of very good waters 20, 50 or more c.c. should be evaporated to dryness as above, and only 5 c.c. of the standard nitrate taken. The amount of sulphonic acid used, so long as there is enough, is of little import. In comparing the colours, the best results are obtained when the intensity of the colour does not exceed that pro- duced by I c.c. of a water containing about 0-05 part N per 100,000. The colour produced by o-i part per 100,000 is difficult to match accurately. The loss of N during evaporation is less when the OXIDIZED NlTROGEN—NITIirrES AND NITRATES 59 evaporation is made to take place rapidly in an op(,'n dish at 100'' C. Slow evaporation at a lower temperature causes more loss, and the dry residues, if further heated, lose N. Chlorine does not interfere if present in less quantity than 2 parts per 100,000. If it exceed 7 parts, it should be removed before evaporation by Ag2S04. This process does not estimate the N as nitrite, as the action of nitrous acid results in the formation of nitroso-phenol, CeH4(N0)0H, which is colourless in dilute solutions. CHAPTER VI GASES IN WATER— WATER SEDIMENT— INTERPRETATION OF RESULTS OF CHEMICAL ANALYSES Water dissolves gases in quantities depending on temperature, pressure, and solubility of the gas. The principal gases found in potable waters are N, 0, COo, and occasionally CHj, HoS, and NH3. Of these O and CO2 are alone worthj^ of estimation. As organic matter in water throughout all its stages of change lays hold of dissolved oxj'gen, the presence or absence of this gas may afford valuable infomiation regarding such organic material. These remarks apply equally to sewage effluents. From a hygienic point of view the subject of gas extraction from waters is not sufficiently important to warrant the expenditure of time and labour inseparable from accurate gasometric work. Nor is the information gained, even when the work is most exactly performed, of constant or certain value. Estimation of Dissolved in Water (Thresh). When sulphuric acid is added to a mixture of KI and a nitrite, iodine is set free. If be carefully excluded, this free iodine rapidly reaches its maximum, and remains constant. But if O be admitted, the amount of iodine liberated varies with the time of exposure, and has no relation to the amount of nitrite present. Thresh concludes that the NO produced acts as a carrier of O, forming N2O3, which liberates more iodine and is again trans- formed into NO, and that this action continues as long as any free dissolved O remains in the water, 2NaNOo + 2H.,S04 + 2KI = K,S04 + NaoS04 + 2HNO2 + 2HI, 2HI + 2HN0.= I, + 2H,0 + 2NO. 2X0 + d=N.,0,- N2O3 + 2HI = 2Nd + 12 + H2O. L, + 2Na2S203 =- 2NaI + Na2S406. 60 GASES IN WATER 6l In the above reactions it will be noted that free nitrous acid is first formed, and that this liberates I. If now the total amount of I liberated be determined, and the Fig. 4. amount of I theoreticalh^ hberated by the nitrite be calculated, the difference will represent the I liberated b}^ the O dissolved in the water. The estimation is carried out as follows (see Fig. 4) : 62 PRACTICAL SAXITARY SCIENCE Into a wide-mouthed glass bottle A, of 500 c.c. capacity, is fitted an indiarubber cork, with four perforations. The stem of a separ- ator funnel B, holding about 300 c.c. of the water, is pushed through the cork. Through another perforation is run a piece of glass tube attached b}' rubber tubing to the lower end of a 100 c.c. burette C, graduated to tenths of a c.c. Through the remaining two per- forations are run pieces of glass tubing bent at right angles, one D, connected by rubber with a gas-tap, and the other E, by similar tubing with a short piece of glass tube thrust through an india- rubber cork, which fits the top of the separator funnel. The funnel B is filled with water to the top, and the glass stopper inserted, displacing a small quantity of water. The contents are accuratel}^ measured once for all, and the capacity of the funnel noted. The funnel is now filled with the w'ater to be examined. The burette C is charged with thiosulphate (i c.c. = 0*25 milligramme 0), made by dissolving 775 grammes of crystalline sodium thiosulphate in a litre of distilled water. [2NaoS203.5 H.O + 1, - 2NaI + NaoS^Og- " ^ " i;=o. 496 = 16 31 = I 775 = 0-25]. Having thoroughly cleaned and dried the bottle A, the cork is inserted and the tube connected with the lower end of the burette C, fixed in position. The funnel B is filled up to the top, and the stopper inserted; the stopper is now taken out and i c.c. of a solu- tion of sodium nitrite and potassium iodide (sodium nitrite 0*5 gramme, KI 20 grammes, distilled HoO 100 c.c.) poured in from a I c.c. pipette. From a second i c.c. pipette is run in i c.c. H2SO4 {25 per cent.). The higher specific gravity of the nitrite mixture and of the HoSOj solution causes these to sink rapidly to the bottom of B, and when the stopper is replaced a negligible quantity, if any, of the reagents just added is lost in the small amount of water which overflows; in this way the entry of air is excluded. The funnel is inverted a few times, so as to effect a uniform admixture, and its nozzle pushed through the cork. The tube D is joined up with a gas-tap, and gas rapidly passed through the bottle. When GASES IN WATER 63 all the air has been expelled the gas may be lighted at the end of E, where it will burn quietly. The stopper of B is removed, and having rapidly extinguished the flame at the end of E, the cork of the latter is fixed in B, after which the tap is turned, and the mixture of water and nitrite solutions is discharged into A. The tap of B is now turned off, the cork at the end of E removed, and the gas relighted and turned down to a small flame. Thiosulphate is then run in slowly from C until the brown colour produced by the liberated iodine is nearly removed. About 3 c.c. of a fresh starch solution is poured into B, and i c.c. of this carefully run through the tap into A, in order to definitely fix the end of the reaction. As the blue colour returns in most instances after a few seconds, it is well to wait for a little and add a further drop or two of thio- sulphate to complete the decolorization. The amount of thiosulphate used will represent : (i) The I (and accordingly its equivalent as 0) liberated by the nitrite in the reagent. (2) The I (and its equivalent O) liberated by the nitrite, if any, originally in the water. (3) The O dissolved in the reagents. (4) The dissolved in the water sample. The value of (4) can obviously be determined by subtracting the sum of the values (i), (2), and (3) from the total. The values of (i) and (3) can be easily determined by making a blank experiment, using five times the amounts of nitrite-iodide solution, sulphuric acid, and distilled water in lieu of thiosulphate, as it may be assumed that the oxygen in distilled water is equal to that in thiosulphate. The number of c.c. of thiosulphate solution required divided by 5 gives the joint values of (i) and (3). In order to estimate (2) the nitrous acid in the sample must be very carefully determined, and as 94 parts by weight of this are equiva- lent to 16 of O, the calculation is easily made [2HN02- — >0]. For a given piece of apparatus, the values of (i) and (3) having been once determined, it is unnecessary to repeat the process, granted that the same quantities of reagents are always used. In (2) the nitrous acid may be estimated by Griess's method. A simpler method for the estimation of dissolved in water is that of Winkler : 64 PRACTICAL SAX IT A RY SCIENCE In this method manganous hydrate serves as the oxygen carrier, and enables it to hberate its equivalent of iodine, which is then titrated in the usual way. In collecting the sample of water, care must be taken to avoid agitating it and exposing it for any length of time to the air. It is transferred with similar precautions by syphoning to a stoppered bottle of known capacity — say 250 c.c. One c.c. of strong manganous chloride solution (40 grammes MnClo.HoO to 100 c.c.) and 2 c.c. of a solution containing 33 per cent. KOH and 10 per cent. KI are introduced by a pipette with long stem which carries its contents to the bottom, thus displacing 3 c.c. of water from the top. The bottle, which must be full of liquid, is now closed with the stopper without including any air-bubble, and the liquids are mixed b}' several times inverting the bottle. The manganous hydroxide precipitate which forms will be more or less discoloured by higher hydroxide, according to the proportion of O which was dissolved in the water sample. As the oxidation of the manganous hvdroxide is not immediate, and the result is influenced by light, the bottle is put aside in a dark cupboard for fifteen minutes; 5 c.c. strong HCl aie added, which cause the precipitate to disappear, and leaves the liquid coloured with dissolved iodine. The iodine is titrated with standard thiosulphate, of which the oxygen value should be known, so as to give the amount of oxygen directly. If 250 c.c. of water be used, it will be convenient to use a solution of thiosulphate of 775 grammes to the litre [i c.c. = 0-25 milligramme O], as then each c.c. thus used can be read as i milligramme O dissolved per litre of water. It is usual, however, to determine the amount of thiosulphate required by the same volume of fully aerated pure water of similar character, or of distilled water, and then to calculate the percentage of the possible amount of oxygen present in the palluted water directly from the amounts of thiosulphate which equal volumes of the two samples require. The manganous chloride must be free from iron, and all the reagents must be free from nitrites. It has been objected that iodometric methods are inapplic- able to waters containing much organic matter, as this ma}' absorb iodine — but this objection does not appear to be well founded. GASES IN WATER 65 Ordinary tap water at room temperature contains about 7 c.c. O dissolved per litre, or by weight about i part per 100,000. 2MnCl2 + 4KOH - 4KCI f 2Mn(0H),. 2Mn(OH)2 + H20 + 0=2Mn(OH)3. 2Mn(0H)3 + 6HC1= 2MnCl3 + 6H2O. 2MnCL + 2KI = 2MnC]o + 2KCI + L. CO2 in Water. Carbon dioxide may exist in solution in water in the free state, as a bicarbonate, or as a carbonate. Estimation of Total CO2 (free CO2, CO2 in bicarbonates, CO2 in carbonates). — Solutions and apparatus required: BaCl2 solution, 10 per cent. H2SO4, baryta- water, flask of capacity about 300 c.c, fitted with a perforated bung through which three holes are bored, the first carrying a funnel tube provided with a stopcock, to hold H2SO4 ; the second carrying a glass tube almost to the bottom of the flask, and connected outside to a bottle containing baryta-water; and the third carrying a glass tube connected with a CaClg tube and a weighed potash bulb containing 50 per cent, solution of KOH. Process. — Measure into the flask 200 c.c. of the water to be examined, and 50 c.c. baryta- water, together with 5 c.c. BaClg. Shake, and allow to settle for twenty-four hours. Decant off as much of the clear fluid as possible without disturbing the sediment. Should there be a scum on the surface, rapidly run the fluid through a filter-paper, and drop the filter into the flask. Replace the bung and run in slowly the H2SO4, which decomposes the carbonates. The Ba(0H)2 has previously precipitated as carbonates all the free COg and that existing as bicarbonate. The total COo evolved by the action of H2SO4 is absorbed by the KOH in the bulb. \^'eigh the bulb, and difference in weight represents this COo. When during the experiment the CO, ceases to come off, the flask should be gently heated in order to assist the evolution, and air drawn through in order that all the CO2 may reach the KOH. Estimation of Free CO2. — Measure into a porcelain basin 100 c.c. of the water; add a few drops of phenolphthalein, and run in from a burette a solution of ^^ NagCOg until a faint red colour is 66 PRACTICAL SAXTTARY SCIENCE developed. The sodium carbonate forms %vith tlie CO, sodium bicarbonate (NaHCO.,), and immediately all the CCX is used up further carbonate turns the indicator red. The amount of NaoCO., used measures the quantity of COo present. Na.,CO.j + CO. + H.,0= 2XaHC03. io6 grammes Xa-^CO^ neutralize 44 grammes CO,. (^ NagCOg contains per c.c. 0-00265 gramme.) I c.c. of the sodium carbonate therefore neutralizes j^^j^ x 0-00265 gramme C02= o-ooii gramme COo. If in an estimation it is found that 3 c.c. -j^ NagCOg are required to neutrahze the CO., in 100 c.c. water, the amount of CO., in this water will be 3 x o-ooii gramme = 0-003 gramme = 3 milligrammes^ 3 parts per 100,000. Estimation of Free COo and CO. as Bicarbonate. — If to 100 c.c. of the water a little BaCl. be added to precipitate carbonates, sulphates, and phosphates of any alkalies which might be present, and which would precipitate barium from baryta-water; and, further, if a little saturated ammonium chloride be added to prevent the precipitation of magnesia (IMgCOg would precipitate BaCO., from Ba(0H)2), the CO. existing free and as bicarbonate ma}^ be neutraUzed by excess of Ba(0H)2; ^^id the loss in alkalinity of the measured excess of Ba(0H)2 solution used may be estimated by titration with standard oxalic acid (as carried out in Pettenkofer's method of estimating CO. in the air, cf. p. 133), and converted into terms of CO2. The CO2 due to bicarbonates is equal to the figure found for this estimation less that for free CO2. Estimation of CO. as Carbonates and Bicarbonates. — To 100 c.c. of the water add a few drops of phenolphthalein, which immediately becomes red from the action of the carbonates (phenolphthalein remains colourless in the presence of bicarbonates). Run in standard oxalic acid, i c.c.= i milligramme COg, until the indicator loses colour. This measures the carbonates. Now boil the water for fifteen minutes, and run in further standard oxalic acid until the phenolphthalein, which in the meantime has become coloured, again becomes colourless. The first addition of acid converts the carbonates into bi- carbonates: hence colourless phenolphthalein. WATER SEDIMENT 67 Boiling converts the bicarbonatcs (original and converted) into carbonates: hence red phenolphthalein. The second addition of acid measures the CO2 after boiling. Twice this figure represents the COg as bicarbonatcs (original and converted) before boiling. If from this last figure be subtracted the first figure found — ^viz., the CO, as carbonates — the remainder is the amount of COg as original bicarbonatcs. One hundred c.c. of a water required 3 c.c. oxalic acid, which means that it contains 3 parts CO2, as carbonates, per 100,000. When boiled and titrated further 4 c.c. of acid were required. Total bicarbonatcs therefore equal 4x2=8 milligrammes. Lastly, 8-3 = 5; therefore the original bicarbonatcs in the water amounted to 5 parts per 100,000. Or these estimations may be carried out without heat by titrating with a standard acid and two indicators — phenolphthalein and methyl orange. When the phenolphthalein has become colourless (end of carbonates estimation) methyl orange is added, and addition of acid continued until the indicator proclaims the presence of free acid (end of bicarbonatcs estimation). Methyl orange is sensitive to bicarbonatcs. Sulphuretted hydrogen in water may be estimated, by titrating a measured quantity with -^ I. I2 + H2S=2HI + S. A drop or two of boiled starch solution is used to fix the end-point. [i c.c. J^ 1 = 0-34 niilhgramme HgS.] WATER SEDIMENT. The biological examination of a water sediment may throw much light on the problem of its origin and the nature and mode of its contamination. A |-inch and ^-inch objective of the ordinar}^ English microscopes furnish good fields for this work. The number of possible organic forms — animal and vegetable- — that may con- taminate a water is so great that no expert could be expected to recognise all. But in the search for sewage pollution a number of unmistakable objects may be seen that will clinch the diagnosis. The micro-chemical examination of mineral particles, such as iron 68 PRACTICAL SAXITARY SCIEXCE compounds, carbonates, oxalates, etc., is in certain cases of some import; but the investigation of animal and vegetable matter is much more likely to lead to positive evidence of sewage and other organic forms of pollution. In this chapter a few, and only a few, general remarks will be made on the biological examination, and the student will do well to consult such works as Cooke's * British Desmids,' ' Fresh-water Algae,' by the same author, Whipple's ' Microscopy of Drinking Water,' and other writers on the Infusoria, Rotifera, Fungi, etc. A necessarily limited number of illustrations are given, but it is hoped that these will be sufficient to introduce the beginner to the microscopic study of water sediments, which in every examination should be faithfully carried out. Much has been written on methods of procuring the sediment. Where a centrifugal machine is at hand it is most satisfactory to use it, and where none can be had the ordinary conical urine glass suffices in ever}' respect. In using the latter, the water should stand overnight. The clear fluid is carefully syphoned or poured awav, and the sediment at the bottom is removed by a fine pipette, and dropped in single drops on a series of microscopic slides. Some workers use well-slides. Should there be a scum on the surface of the water in the conical glass, this is removed separately and trans- ferred in like manner to slides. Cover-glasses are applied, and the slides carefully examined, first by the low and afterwards b}- the higher objective. Certain biological forms inhabit only foul water, and disappear when it becomes purified. Where a supph' usuall}' satisfactory develops colour, turbidity, or odour, a microscopical examination alone mav elucidate the causes. A satisfactory water should be free from all suspended matter, and especially from all living and dead animal and vegetable matter. Certain animal and vegetable growths may occur in storage reservoirs and cisterns through the admission of light to the water: plants containing chlorophyll (green algae, diatoms, etc.) grow in light. The different seasons bear different forms and amounts of animal and vegetable life, therefore a systematic microscopical examination is necessary. Vegetable growths may take place at dead ends in mains. Much dead organic tnatter will be found in the form of unrecognisable debris, but WATER SEDIMENT fjrj Fig. 5. I. Wood cells. 6. Particles of sand. 2. Cotton fibre. 7- Paramoecium. 3- Linen fibre. 8. Amoeba. 4- Hemp fibre. 9. Encysted Infusorian 5- Algal zoospore. 10. Algal zoospore. Fig. 6. 1. Fresh-water Hydra. 2. Scale of insect. 3. Egg of Taenia solium. 4. Egg of Trichocephalus dispar. 5. Egg of Ascarus lumbricoides. 6. Paramoecium. 7. Amoeba. 8. Wool fibre. 9. Euplotes Charon. 10. Diatoms. PRACTICAL SAXITARY SCIEXCE 1. Pleurococcus (Algae). 2. Amoeba (Protozoa). 3. An Infusorian. 4 and 5. Diatoms (Algae). 6. A Desmid (Algae). Fig. 7. 7. Hair of insect. 8. Vegetable tissue. 9. Fibre of wool. 10. Ulothrix (Algae) Fig 1. Anguillulae (Xematoda) 2. Ulothrix. 3. Zoogloea of micrococci. 4. Anabena. 5. Cryptomonas. 6. Chara fragilis. 7. Diatom (Synedra). 8. Uroglena. 9. A Desmid (Cosmarium). 10. Encysted Infusorian. WATER SEDIMENT 71 1. Vorticella. 2. Spirogeira. 3. Sphgerotilus natans. 4. Beggiatoa. 5. Daphnia. Fig. 9. 6. Crenothrix polyspora. 7. Volvox globator. 8. Tabellaria. 9. Species of NostocJ 10. Melosira. Fig. 10. 1. Rotifer (Annuloida) . 2. Paramoecium (Protozoa). 3. Animal spine. 4. Wing scale of an insect. 5. Vegetable debris. 6. Crystals of calcium sulphate. 7. Algal filaments. 8. Not identified. g. Bacteria. 10. Diatom. 72 PRACTICAL SAX IT A RY SCIENCE amongst it much that is recognisable, as epithelium, striped muscle, cotton, silk, and linen fibres, starch granules, dotted vegetable ducts, wool,, hair, ova of intestinal worms, and numerous other bodies, all distinctive of sewage. It will thus be seen that a knowledge of the fauna and flora of water will enable workers to recognise certain organisms, alive or dead, which produce odours in water, others which live only in pure waters, and whose presence excludes gross pollution, and those which live in polluted waters, and consequently point to sewage or other contamination. Fislw odours, according Fig. II. 1. Oscillatoria. 2. Small Infusorian. 3. Free-swimming Vorticella. 4. Cotton fibres. 5. Navicula (Diatom). 6. Confervoid Alga (Synura uvella). 7. A Heliozoon. 8. Egg of an Entozoon. 9. Pith cells partially covered with vegetable debris, [o. Wood of a Conifer. to Whipple, are produced by Endorina, Volvox, Pandorina, and other Chlorophyceae, Uroglena, Bursaria, and other Protozoa. Aromatic odours are created by numerous diatoms — Tabellaria, Meridion, Diatoma, etc. — and Protozoa. Grassy odours are pro- duced by Rivularia, Anabsena, Cselosphaerium, and other Cyano- phycese. WATER SEDIMENT 73 In river water and unfiltered supplies possessing odours the organisms are likely to be found in the supply; whilst in filtered waters they mostly grow on the filters. The foul odour and reddish colour of the Cheltenham water some years ago was shown to be due to a species of Crenothrix growing in the reservoirs and on the filters. In deep-well and spring waters any low forms, animal or vegetable, indicate insufficient protection from light, such as storage in uncovered reservoirs. The so-called sewage fungus, Beggiatoa alba, including Carchesium Lachmanni, and other forms, occurs in Fig. 12. Leptomitus lacteus (from impure river) . Carchesium Lachmanni (from water polluted with sewage) . Conferva bombycina(pond water) . Fresh- water Alga (Lyngyba). Bursaria gastris. 6. Hydrodictyon (fresh-water Alga) 7. Sand particles. 8. Algal lilament. 9. Hypha of fungus (sporing). 10. Encysted Protozoon. 11. Water bear. effluents from sewage-farms and bacteria-beds. Beggiatoa also occurs in river beds and stagnant waters containing H.2S. Wino- gradsky holds that it does not produce the S which it contains in the dried state, but that this S is derived from the HgS by other means. Cohn states that it produces S from sulphates and albuminous bodies. The organisms forming the slimy superficial layer (Schlammdecke) 74 PRACTICAL SAXITARY SCIEXCE Fig. 13. — -Beggiatoa Alba. I'lG. 14. — Daphxia Pulex. WATER SEDIMENT 75 Fig. 15. — ^VORTICELLA. Motile. Resting. Fig. 16. — Amceba Coli. 76 PRACTICAL SAXITARY SCIEXCE Fig. 17. — Leicestershire Wool. Fig. iS. — Chinese Silk. WATER SEDIMENT 77 Fig. 19. — Flax Fibres. %!■ 4 Fig. 20. — Hemp Fibres. 78 PRACTICAL SAXITARY SCIEXCE Fig. 21. — Jute Fibres. A/ Fig. 22. — Cotton Fibres. INTERPRETATION OF RESULTS 79 of a sand-filter are innumerable, and vary with the source of the water and other factors. When a sand-filter is first set to work, it acts merely as a strainer. In the course of a few days a slimy organic layer consisting of green and blue algce, fungi, zoogloea masses of bacteria, diatoms, and a multitude of other organisms, makes its appearance, and true filtra- tion then commences. The source of the water, season of the year, etc., determine the presence of specific forms. Certain green algse are produced in the spring, blue algae in the summer, and their colouring matter may be liberated at any time and remain on the surface long after the organisms have died. The matter obtained as sediment from a centrifugal machine, conical glass, or surface scum, when examined microscopically, may be found to contain (i) living animal forms, (2) dead animal forms, (3) living vegetable forms, (4) dead vegetable forms, {5) mineral detritus, and (6) unrecognisable debris, requiring micro-chemical and other methods of investigation. The differentiation of some lowly animal and vegetable organisms is frequently a matter of no little difficulty, but careful search should be made for these, as their presence has special significance. INTERPRETATION OF RESULTS OF CHEMICAL ANALYSES. As previously indicated, judgment should be exercised at all times in expressing an opinion on a water without a personal inspection of the source, etc. ; but many instances will arise in which no doubt can exist as to the foulness of the sample. Positive results in the search for sewage contamination are much more easily dealt with than negative. The liability to such pollution should ever be kept before the mind of the analyst. Deep springs and wells for the most part afford the purest waters. Upland surface waters may be also quite pure. But subsoil waters and waters from cultivated lands, as also most river waters, are rarely free from pollution. Waters collected from the surfaces of the more impervious rocks destitute of animal and vegetable life, are extremely pure. These rarely contain any appreciable NH3, and rarely more than i part chlorine, o-i part nitric N, 5 parts hardness, and 10 parts total 8o PRACTICAL SAXITARY SCIENCE solids per 100,000. Waters collected from rocks covered with peat will present high figures for organic ammonia, and O absorbed by organic matter, and their acidity will be great. Such waters are plumbo-solvent, and should be neutralized before distribution to the consumer. Waters from mountain limestone are moderately hard, with high total solids and neutral or faint alkaline reaction. The mineral residue is chiefly composed of carbonate and sulphate of calcium and magnesium. Great variety in composition is found amongst waters originating in the lias, magnesium limestone, red sandstone, and oolite; total solids may range from 10 to 15 parts; total hardness 10 to 15; chlorine i to 2; and nitric N o-i to 0-2. Alluvial strata furnish waters of high total solids (50 to 100) ; and waters from cultivated soils vary within very wide limits in total solids, hardness, chlorine, and nitric N. Hard waters are derived from the chalk, limestone, magnesian limestone, oolite, and dolomite. Chalk waters are mostty bright, transparent, and charged with CO.2. \Mien the CO2 is driven off, these waters are almost univer- sally alkaline, although before boiling the reaction to litmus may be distinctly acid. Chlorine varies from 2 to 3 parts, nitric nitrogen from 0-2 to 0-4, total hardness 15 to 30 (the hardness is chiefly tem- porar}^ and may be nearly all due to carbonates of Ca), and total solids from 25 to 50 parts. Waters from oolite closely resemble those from chalk, with the exception that they contain a little more permanent hardness. Limestone waters contain more total solids and more permanent hardness (due principally to calcium and magnesium sulphate). Waters from dolomite strata occupy an intermediate position be- tween chalk and limestone waters in point of hardness and total solids. Greensands, in that they frequently contain much nitrates and variable quantities of ferrous iron, furnish, through the reduction of the nitrates bj' the iron, quantities of free NH3. The intermediate stage of nitrites ma\^ be occasionally demonstrated. The lower greensand furnishes water collected at great depth — often many feet below the chalk — and accordingly the total soJids are high, often 80 to 100 parts per 100,000. Hardness is very variable, and much is permanent. Chlorine may run to 10 or 12 parts per 100,000, and nitric X to as much as 0-5 or o-6. These waters are very free from INTERPRETATION OF RESULTS 8i organic matter. Where water is procured from lias clays much permanent hardness may be expected (CaS04 and MgvSO^), 20 parts or more, and total solids may range from 200 to 300. A water containing over 30 parts of total hardness may be con- sidered unsuitable for domestic purposes, unless it can be largely softened. Waters containing more than 20 parts of permanent hardness are not suitable for washing and cooking. Deep wells, if sufficiently steined, are for the most part pure. Very occasionally a well in the chalk may tap a hidden reservoir of unpurilied sewage which has leaked through fissures from a cess- pool. Sewage derives the bulk of its CI from urine, which contains, as above mentioned, about i per cent, chlorides, but although it con- tains this large amount of CI, it is obvious that deadly pollution by sewage may occur in such small amounts as are wholly incapable of detection by chemical methods ; the chlorine figure, therefore, will be chiefly of diagnostic value in those cases where the soil, subsoil, and water-bearing strata are of constant composition and beyond the reach of contamination by cultivated land. If after a series of analyses the CI figure is found fairly constant, a particular rise of 0*5 to I part per 100,000 may justly arouse a suspicion of sewage pollution. Considering the varieties in source and surrounding conditions from source to distribution, it is quite impossible to erect standards of purity for waters in this country. An inspection of the source and surroundings is of the utmost importance in all cases. In considering the ' free and saline ' NH3, the merest trace should be considered of import if not suspicious, except in those cases where reduction of nitrates has taken place, such as occurs in the green- sands. As previously stated, if the ' albuminoid ' ammonia be very small (less than 0-002), the ' free and saline ' may be allowed to exceed slightly 0-005. In peaty waters, where the ' albuminoid ' ammonia may reach o-oi, the ' free and saline ' should be negligible. In a deep-well water the O absorbed from permanganate in 3 hours at 37° C. should not exceed o-oi or 0-02. In a peaty water free from animal pollution this figure may exceed -i . In passing judgment on river waters, analyses, in addition to 6 S2 PRACTICAL SAX IT A RY SCIENCE inspection, should be made of all tributaries, lest evidence of present or past pollution be overlooked. The search for poisonous metals should" be carefully carried out, and when any of these is found a quantitative estimation should in\-ariably be made. Lead to the extent of 0-025 P'^rt per 100,000 is sufticient to condemn a potable water. Present or recent sewage pollution may be mt)re or less accuratel}' differentiated from past and remote, in that, whilst high CI and nitrate ligures obtain in both, in the present or recent contamination there will be marked free and organic XH3, whereas in the past and remote little or no free or organic NH3 will be found. Further animal pollution may be more or less accurately differentiated from vegetable by contrasting the two ammonias, oxygen absorbed b}' Tidy's process, CI, and nitrates. All these figures are high in cases of marked animal pollution; whilst in A-egetable pollution free NH3 is low, organic NH3 high, CI and nitrates are low, and in the last two no increase if the water is drawn from below the surface. Where much vegetable matter exists the water is usually coloured, as in the various peaty waters, and the solid residue chars on ignition. Sulphates and phosphates occur in larger quantities in water polluted with animal matter than in those contaminated with \-egctable material. Little has been said of nitrites, because, although the}^ are easily foimed by oxidizing and reducing agents, they are rarely present in natural waters. They are found in purifying sewage, but, unfortunately, as they may be formed from other sources than ammonia (such, e.g., as nitrates in contact with iron, zinc, and lead pipes or cisterns), it is not alwaj^s possible to locate their origin. The faintest trace, however, of nitrites should condemn a water, except in the single instance of a pure water containing nitrites undergoing reduction b}^ metallic or other inorganic compounds, and not by organic matter. The solids impart different properties to waters according to their composition, so that no strict limit can be set to their amount. Sulphates should not exist in larger quantity than S parts SO2 per 100,000. Magnesium salts, especially MgSOj, should be very small, if at all present, in a good water. And per- haps, all forms of mineral matter considered, the total figure should not nearly reach 100. CHAPTER VII THE BACTERIOLOGY OF WATER The student who works with a microscope should be familiar with the elementary mathematics of the instrument ; he should understand the principles which underlie the formation, magnification, and brightness of images. The following matters require special atten- tion: (i) The conditions which produce an aplanatic image as expressed in Abbe's sine law — in other words, the conditions which ex- clude spherical aberration and coma. (2) The angular and numerical aperture of a lens and their relations to the refractive indices of glass, air, cedar-wood oil, etc. (3) The meaning of resolution as applied to lenses and the factors determining its limits. (4) The definition of lenses. (5) Methods of excluding chromatic aberration. (6) The flatness of images. (7) The theory of the Huygenian eye- piece, (8) Methods of illumination including oblique or dark ground illumination. (9) The simple relations between objectives (high and low power), Abbe condenser, mirror (plane and concave), diaphragm, and source of light. The student approaching the bacteriology of water is assumed to have a good bench knowledge of — 1. The preparation and examination of the hanging drop with a view to determination of motility, immotility, and Brownian move- ment. 2. The preparation and staining by a simple stain of a smear or section with the object of discovering the morphology of the micro- organism or micro-organisms under examination — coccus, bacillus, vibrio. 3. The preparation of a Gram specimen. He ought to be able to definitely state whether his specimen is positive or negative. 4. The few special stains — (?.g., Ziehl Neelsen's, used for Bacillus 83 84 PRACTICAL SANITARY SCIENCE iiiberciilosis and acid-fast bacteria; Neisser's, used for the Klebs- Loffler bacillus, etc. 5. The preparation of and results obtained by the various fermen- tation media in common use, especially those for intestinal bacteria. 6. The methods employed in carrying out immunity reactions between micro-organisms and blood serum. The bacteriological examination of water as a routine procedure seeks (i) to measure the extent to which it has been polluted b}^ sewage; or (2) to determine the degree of completeness of purifica- tion processes; or (3) to detect the presence of definite disease- producing organisms, such as B. typhosus, etc. Since the number of definite pathogenic organisms compared with the total number of bacteria in water is very small, and since competition may have wholly eliminated the disease-producers by the time the water reaches the laboratory, the search under head (3) becomes so un- satisfactory that it is but rarely attempted. The search under head (2) is most serviceable in determining the efficiency of sedi- mentation and filtration of large quantities of water. The micro- organisms characteristic of sewage generally styled ' indicator ' organisms — viz., B. coli, streptococci, and B. enteritidis sporogenes — when estimated quantitatively determine with considerable accuracy the degree of sewage pollution remaining at any stage in the puri- fication of a water-supply, and to the expert in charge this piece of bacteriological evidence is of the first moment. But the search under head (i) is that most widel3^ engaged in. B. Coli. Of the three indicator organisms above named, B. coli is by far the most important ; so universally is this recognised that the bulk of bacteriological examinations of water is limited to a quantitative determination of this organism alone. We have in the B. coli group bacteria extremely numerous in excreta and sewage, but which do not occur in air, soil, or water unless these have been in contact with sewage. It is difficult to define the characters of the group. All its members are non-sporing short bacilli. Gram negative, motile, although motility is not always seen, fermenters of glucose and THE BACTERIOLOGY OF WATER 85 lactose with production of acid and gas, and fail to liquify gelatin in fourteen days. Attempts have been made in recent years to differentiate the strains of B. colt found in human excreta from those of the domestic and other animals. At present it is impossible to distinguish B. coli isolated from water as belonging to any species of animal. Whether or not B. coli of intestinal origin can be definitely separated from B. coli of soil, etc., is a matter of much difference of opinion. The broad landmarks that separate the fermentation reactions of B. coli from those of B. typhosus and B. enteritidis (Gartner) necessarily disappear when varieties of B. coli are to be distinguished. Under favourable conditions B. coli may persist for considerable periods outside the intestinal tract which is its natural habitat; but under ordinary conditions it disappears rapidly from soil, water, etc. This last statement is vindicated by the self- purification of rivers from B. coli carried into them by sewage, and by experimentally applying sewage to soil, water, etc., and determin- ing the dates at which B. coli can no longer be found. Whether it be safe to rely on fine distinctions in f ennentative reactions and on pathogenic and agglutination properties as means for separating B. coli of recent intestinal origin — the type most clearly indicative of danger — from organisms that have persisted in water, soil, etc., after typhoid bacilli or cholera vibrios have perished, is a question which all water investigators have to face, and until it can be definitely answered — and that time is not yet — it would appear to be safer to regard all forms of B. coli as possible indicators of sewage. Houston some years ago worked out a set of tests represented by the symbol ' flaginac ' to assist in distinguishing B. coli of intestinal origin — viz. : Greenish fluorescence in neutral red broth = fl. Acid and gas in lactose peptone media = ag. Indol in broth or peptone water = in. Acidity and clotting in litmus milk = ac. Later he modified his procedure somewhat and adopted the follow- ing three tests for B. coli, using in each case portions of water measur- ing 100 c.c, TO c.c, I c.c, o-i c.c, o-oi c.c, and o-ooi c.c. : I. Presumptive. — Gaseous fermentation of a bile salt glucose peptone medium. 86 PRACTICAL SANITARY SCIENCE 2. Confirmatory. — Isolation of a coli-likc microbe forming gas either in a lactose or glucose medium. 3. Typical. — Isolation of a coli-like organism forming indol in peptone water and gas in a lactose medium. Houston was the first to use the above and lesser dilutions with the object of placing Public Health bacteriology on a combined qualitative and quantitative basis. He used the words ' flaginac ' and ' typical ' only as an indication that specified tests have been carried out, and did not claim that ' flaginac ' or ' typical ' B. coli are onl}' found in human excremental matter. Technique of the Search for ' Flaginac ' B. Coli. — Remove bottle of water for examination from its case and gently shake it. Remove cork and flame mouth. Sow 100 c.c. of the water into 50 c.c. MacConkey's fluid, triple strength, in a Durham's tube. Sow 10 c.c. into 10 c.c. MacConkey double strength- Sow i c.c. into 10 c.c. MacConke}' ordinary strength . After forty-eight hours incubation at 37° C. note presence or absence of gas. If gas is found, dilute a loopful of the culture in 10 c.c. sterile water and spread two loopsful of the dilution on a surface culture of MacConkey's tauro-chloate- lactose agar for isolation. Examine after forty-eight hours for coli- like colonies. If such be found sow one or two in a tube of liquefied glucose-gelatin and incubate at 20° C. If gas be formed liquefy the gelatin and use it for sowing neutral red broth, peptone water, and litmus milk. Examine for fluorescence, indol, and acid, and clot respectively. An organism giving all these reactions is said to be ' flaginac ' or ' typical ' B. coli. Streptococcus Group. There does not appear to be any uniform classification of strepto- cocci. Morphology, pigment production, agglutination tests, patho- genicity, and production of acid in sugars have all been recommended as bases for classification. But for water examination attempts to differentiate isolated streptococci have been up to the present wholly unsuccessful. Technique of Search for Streptococci. — Sow i c.c. of the water into ID c.c. of ordinary broth. Incubate at 37° C. After forty-eight hours examine the deposit microscopically for streptococci. THE BACTERIOLOGY OE WAT Eli 87 B. Enteritidis Sporogenes (Klein). This bacillus possesses distinctive characters. It is fairly large — 2 to 4 // long by o-8/^ broad; it is motile; it spores near the ends of the rods ; it is Gram positive ; it grows anaerobically in milk, producing a characteristic coagulum of casein and a transparent or turbid and acid whey, whilst gas is formed in quantity. The contents of the incubated milk tube smell of butyric acid. When a c.c. of the whey containing numbers of bacilli is injected into the groin of a guinea-pig the animal dies within twenty-four hours, and post- mortem examination reveals an extensive gangrenous slough at the seat of inoculation. These post-mortem appearances, together with the changes in the milk, identify the organism. As B. enteritidis sporogenes is a sporing organism with prolonged powers of resistance it can hardly be regarded as indicative of recent excretal pollution. Indeed, opinion is far from united concerning its value as an indicator of sewage pollution. Technique of Search for B. Enteritidis Sporogenes.- — Sow 10 c.c. of the water into 50 c.c. of milk, taking care to pass the pipette well below the cream. Sow i c.c. into 10 c.c. milk. Heat the tubes to 80° C. for fifteen minutes, and then incubate in an anaerobic appara- tus at 37° C. The typical ' enteritidis ' change consists in formation of gas, odour of butyric acid, separation of curd, and tearing of same by gas. It is impossible to set up rigid bacterial standards for waters. But the source being known general indications can readily be offered as to what should be expected of a good water. Since the more recent the excremental pollution the greater the number and the older the pollution the less the number of B. coli present, the bacterio- logical potentialities of a sewage-contaminated water would appear to be best expressed in terms of the number of B. coli found. For deep wells and springs a more restricted standard will be demanded than for shallow wells, rivers, upland surface waters, etc. For deep wells and springs it may be required that B. coli and streptococci be absent from 100 c.c, and that B. enteritidis spor- ogenes be absent from a litre ; that the growth on gelatin at 22 degrees does not exceed fifty organisms per c.c, whilst that on agar at 37 degrees does not exceed five per c.c 88 PRACTICAL SANITARY SCIENCE In shallow wells, rivers, upland surface waters, etc., this standard may be relaxed to one-tenth — viz., absence of B. coli and strepto- cocci from 10 c.c, and of B. enteritidis sporogenes from lOO c.c. ; gelatin growth not to exceed 500 per c.c, and agar not more than 50 per c.c. Sea water is regarded as polluted by most observers when it contains B. coli in i c.c. Houston states that no sample of sea water remote from pollution contains B. coli or spores of Enteritidis sporogenes in 100 c.c. Whilst no absolute standards can be fixed, it may, perhaps, be stated in a general way that samples in which B. coli is present in 10 c.c, but absent in i c.c, are to be regarded as suspicious. Techniqiie — Collection of Sample. — i . A white glass bottle, capacity 200-500 c.c, is sterilized and plugged with sterile cotton-wool. 2. Before filling flame the mouth and remove the plug; fill quickly, and insert a new cork which has just been passed through a flame till slightl}' carbonized. Cut off cork level with mouth and seal with wax. Cover with a rubber cap. In taking water from a river, submerge bottle some distance from bank with mouth upstream; from a tap, let run to waste before filling; from well, lower under same conditions as bucket is lowered, or fill from bucket, or use a Miquel flask. As organisms rapidly multiply in water at ordinary temperatures the sample should be kept at 0° C. until examination is commenced. Special boxes containing ice are prepared for this purpose. The label should specify (i) Reasons for examination (epidemic, etc.); (2) source of water; (3) particulars concerning recent rains, snow, pollution, etc ; purposes for which water is required (drinking, cooking, lavatories, etc) ; (4) atmospheric temperature; (5) day and hour of collection. Enumeration of Organisms. — Prepare a few 10 c.c. pipettes plugged at the upper end with wool, and sterilize them ; also a drop pipette (20 drops = I c.c). Sterilize some conical flasks plugged with wool. Liquefy a few tubes of gelatin in a water-bath, and prepare some sterile distilled water. Measure 9 c.c. sterile water into a flask, taking the necessary precautions to avoid all contamination; to this add i c.c. of the THE BACTERIOLOGY OF WATER 89 water under investigation, and mix. The mixture is a dilution of I in 10. Flame the mouth of a conical flask; remove the plug; introduce with the drop pipette 2 drops of the i in 10 dilution. Flame the mouth of a gelatin tube, remove the plug, and quickly pour the contents into the conical flask; mix, and stand the flask on a cold horizontal surface — ice in hot weather. A gelatin plate has been made containing o-oi c.c. of the water. Incubate this at 20° to 22° C. Examine the flask daily for appearance of colonies, and make counts until the gelatin is completely liquefied. Suppose by the fifth day there are 90 colonies, and on the sixth the plate is completely liquefied, record is made that " the water contains 9,000 (100 x 90) aerobic micro-organisms per c.c, liquefac- tion of the gelatin having finished the count on the sixth day." Enumeration may be carried out with pipettes (made in France) which deliver about 50 drops to the c.c. The exact number of drops per c.c. is marked on the stem. In the same manner inoculate melted agar at 40° C. and pour plates; incubate at 37° C. for three days; count. Qualitative Examination. — Sow i drop of the water in a tube of melted gelatin or agar; mix; sow 2 loopfuls of the mixture into a second tube of gelatin or agar; mix; sow 2 loopfuls of the last mixture into a third gelatin or agar tube; pour plates in Petri dishes with these mixtures. The plates are carefully observed daity, and subcultures sown in other media for the identification of a particular colony. Many saprophytes in water, although incapable of causing infections, may, like Proteus vulgaris and Micrococcus prodigiosus, produce soluble toxins which injuriously affect man and the lower animals ; others may cause a nuisance bj^ producing in dead organic matter foul-smelling gases. The detection of pathogenic species, such as Bacillus typhosus, is generally a matter of some labour. When the student has gained facility in technique, he should conscientiousl}^ work out the various sugar reactions, growths on special media, and the tinctorial and morphological characters of this and a few other pathogenic fonns, such as B. pyocyaneus, Friedlander's bacillus, and the micro- 90 PRACTICAL SANITARY SCIENCE organisms of suppuration. Detailed descriptions of methods must be sought in systematic works on pathological bacteriology- The various items of the analysis are recorded in some such form as this: Sample of water from — ■ — Date Labelled Brief particulars of source — Physical characters : Turbidity Colour Odour Reaction - Free and saline NH.^ parts per 100,000. Albuminoid NH3 '- - CI Nitrous N Nitric N Hardness (total) ■■ (permanent)- (temporary) _ O absorbed at 37° C in three hours. Metals Solids (total). (volatile) - (fixed) ._ Appearance on ignition Microscopic examination of sediment- Bacteriological Examination WATERS FROM VARIOUS SOURCES 91 EXAMPLES OF WATERS FROM VARIOUS SOURCES Results Expressed as Parts per ioo.ooo. No. I. A Pure Water. No. 2. Rain Water Collected on Grass Land. Physical characters - _ Excellent Good Reaction - - - - Faint alkaline Faint alkaline Free and saline NH3 - - o-ooi 0-012 Organic NH3 - O'OOI Nil CI - - - - - I-20O 0-200 Nitrous N - - - Nil Nil Nitric N - - - - O'OIO o-oio Hardness (total) - 8-500 0-600 (permanent) - 3-000 0-600 (temporary) - 5-500 Nil absorbed at 37° C. in three hours 0'0i3 0-002 Metals (Zn, Pb, Fe, Cu) - Nil Nil Solids (total) - ii'5oo 2-500 ,, (volatile) - - 2-500 I-OOO „ (fixed) - - g-ooo 1-500 Appearance on ignition - Nil Nil Microscopic examination of sedi- ment - - - - Nil Nil Bacteriological examination 20 non - liquefying Not performed saprophytes per c.c. Intestinal organisms absent No. 3. Foul. No. 4. Chalk Water from Deep Well. Physical characters - _ Excellent Excellent Reaction - - - - Alkaline Alkaline Free and saline NH3 - 0-030 0-005 Organic NH3 - O-02O 0-006 CI - - - - - 5-000 4-500 Nitrous N - - 0-050 Nil Nitric N - - - - 0-600 0-300 Hardness (total) - 20-000 22-000 (permanent) - 12-000 I2-000 (temporary) - 8-000 lO-OOO absorbed at 37° in three hours 0-150 0-060 Metals (Zn, Pb, Fe, Cu) - Nil Nil Solids (total) - - 30-500 38-000 (volatile) - 10-500 I2-000 ,, (fixed) - - 20-000 26-000 Appearance on ignition - Marked charring charring Microscopic examination of sedi- ment - - - - Objects indicating sewage pollution Nil Bacteriological examination B. coli found in Not performed 50 c.c. 92 PRACTICAL SANITARY SCIENCE Sample No. 2, although it possesses a high ' free ' NH3 figure, is good. Rain water in towns is general!)' impure; it is slightly acid from SO,, and contains NHg. Sample No. 3 has had a small amount of untreated sewage admitted to it. Sample No. 4 is an average chalk water with low total solids. This figure may be allowed to go up to 200 or over. The hardness of chalk waters varies considerably. Sample No. 5. — The saline NH3, chlorine, and nitrates are high, and nitrites are present. These items in general point to animal No. 5. No. 6. Deep-well Water from Deep-well Water from the Lower Greensand. Chalk near the Sea. Physical characters - - Good Saline taste, green- ish colour, no odour Reaction - - - - Alkaline Alkaline Free and saline XH3 - - 0-035 Nil Organic XHo - o-ooi 0-003 CI - - - - - 12*250 115-000 Nitrous N - - 0-020 Nil Nitric N - - 0-320 i-ooo Hardness (total) - 16-000 47-000 (permanent) - 10-000 — (temporary) - 6-000 — absorbed at 37° C. in three hours 0-020 0-035 Metals (Zn, Pb, Fe, Cu) - Nil Nil Sohds (total) - 105-000 260-500 (volatile) - 20-000 35-500 „ (fixed) - - 85-000 225-000 Appearance on ignition - Nil Slight darkening Microscopic examination of sedi- ment . - - - Nil Mineral particles Bacteriological examination Excellent Excellent pollution; that they are not due to this cause here is shown b}' the low organic XH3 and absorbed. Reduction of nitrates by iron salts is going on, as demonstrated b}' the high saline NH3 and presence of nitrites. Sample No. 6 is contaminated by sea water. Before contamina- tion CI was 3, and total hardness 20. Much. MgClj is present, and the water is unfit for domestic use. Sample No. 7 contains much acid, and could not be allowed to traverse lead pipes. Its organic XH3 and absorbed are not so high as in many peaty waters. WATERS FROM VARIOUS SOURCES y;< Physical characters - - - Reaction - - - . . Free and saline NH3 - - - Organic NHo - - - _ CI - -" - Nitrous N - - _ . Nitric N - - - - - Hardness (total) _ . _ ,, (permanent) (temporary) O absorbed at 37° C. in three hours Metals (Zn, Pb, Fe, Cu) Solids (total) - - - . (volatile) - - - ,, (fixed) - - - - Appearance on ignition Microscopic examination of sedi- ment - - - - - Bacteriological examination No. 7. Surface Water, Peaty. No. 3. Surface Water, not Peaty. Colour brownish; slight taste Acid Almost colourless, no taste Neutral O'OOI 0-002 0-030 0-003 0-600 NU 0-800 Nil O-OIO 3-000 0-030 3-000 3-000 Nil 2-500 0-500 0-150 Nil 0-050 Nil 9-000 4-000 2-000 I -000 7-000 Charring 3-000 Faint darkening Vegetable debris No intestinal or- Nil No intestinal or- ganisms ganisms Samples Nos. 9 and 10 were taken from the same house. The analysis of 10, carried out a month after that of 9, shows some slight variations, which are to be expected, when it is remembered that the composition of river water varies, with its varying powers of Physical characters - - - Reaction - - - - . Free and saline NH3 - - - Organic NHo - - - . CI ----- - Nitrous N - Nitric N - - - - - Hardness (total) - - - ,, (permanent) ,, (temporary) O absorbed at 37° C. in three hours Metals (Zn, Fe, Pb, Cu) Solids (total) - - - _ (volatile) - - - . ,, (fixed) - - - . Appearance on ignition Microscopic examination of sedi- ment - - - - . Bacteriological examination No. 9. New River Water from the Lea. No. 10. New River Water from the Lea. Excellent Slightly alkaline Nil Excellent SUghtly alkaline O-OOI 0-002 1-900 Nil o-i6o 0-003 I -8600 Nil 0-2I0 20-500 21-500 11-500 9-000 13-500 8-000 0-017 Nil 32-600 I0-200 0-023 Nil 28-560 10-000 22-400 Nil 18-560 Nil Nil Nil 94 PRACTICAL SAXITARY SCIENCE self-purification, with the nature of the strata in which its springs of origin occur, and of the strata over which it flows, and with the No. 11. No. 12. Peaty Water. Well Water. Physical characters - . Colour light-brown Excellent Reaction - - - - Acid Neutral Free and saline NH3 - - 0-005 Nil Organic NHo - 0-026 o-ooi CI - - - - 1-500 3-800 Nitrous N - - Nil Nil Nitric N - - - - 0-220 0-362 Hardness (total) - 3-500 26-000 (permanent) - 3-500 12-300 (temporary) - Nil 14-000 absorbed at 37° C. m three hours 0-146 0-008 Metals (Zn, Fe, Pb, Cu) - Nil Nil Solids (total) - 12-300 38-260 ,, ^ volatile) - - S-300 8-500 „ (fixed) - 4-000 29-760 Appearance on ignition - Charring Nil Microscopic examination of sedi- ment - . - - Vegetable d6bris Nil Bacteriological examination Negative Nil nature of the soils and subsoils of its basin, especially in regard to cultivation, density of population, and the presence of sewage and industrial waste. Physical characters - - - Reaction ----- Free and saline NH3 - - - Organic NH3 - - _ - CI ----- - Nitrous N - Nitric N - - - - - Hardness (total) - - - (permanent) (temporary) O absorbed at 37° C. in three hours Metals (Zn, Fe, Pb, Cu) Solids (total) - - - - ,, (volatile) - - - - ., (fixed) - - - - Appearance on ignition Microscopic examination of sedi- ment ----- Bacteriological examination No. 13. Lambeth Supply. No. 14. Chelsea Supply. Excellent Excellent Faint alkaline Neutral Nil Nil 0-005 0'002 1-850 Nil I -740 Nil 0-086 0-009 18-600 18-400 S-500 8-400 lO-IOO 10-000 0-043 Nil 0-023 Nil 26-400 6-800 26-720 6-400 ig-600 20-320 Nil Nil Nil Nil Nil Nil WATERS FROM VARIOUS SOURCES 05 Sample No. ii is plumbo-solvent, and is slightly polluted with animal matter, in that free and saline NHg, CI, and nitric N, arc too high for a peaty water. Sample No. 12 is a pure water from a deep well in Kent. Samples Nos. 13 and 14 are fair specimens of filtered Thames water. In the ' nil ' returns of the bacteriological and sediment examina- tions, it is to be understood that nothing was found indicative of animal pollution. A 'Dorset Water-Supply. Three samples selected from a series of thirteen investigated at the same time by the writer, and showing the changes effected by treatment with chalk and filtration. These waters, derived from Bagshot sands, covered with peat, are acid and ferruginous. (A) (B) (C) Before Treatment After Treatment A fhpr P'iltrr) tinn with Chalk. with Chalk. ^A.ILr:r r llli dLlUil. Physical characters — Colour, smell, turbidity Good Good Excellent Chemical reaction Acid Acid Acid Acidity = ) 0-500 0-350 2-190 HClj Free and saline NH3 O'OiS 0-022 O-OOI Organic NH3 - - - - o-oi6 o-oio O-OOI absorbed in three hours at 37° C. . - - - 0-040 0-020 Nil Total solids - - - - 12-300 11-500 11-500 Hardness (total) - - - 2-500 2-500 2-700 (temporary) — — (permanent) 2-500 2-500 2-700 Chlorine ----- 2-500 2-000 2-500 Nitric N - O-IOO O-IOO O-OIO Iron in solution _ _ . 0-325 0-220 O-IIO As the filtered water still contains acidity, it may not be passed through lead pipes. Sea water contains in 100,000 parts nearly 2,000 parts CI, and between 3,000 and 4,000 parts total solids. Hardness ranges between 500 and 600 parts. Lime and magnesia together form about 240 parts, and the ratio of the first to the second is about I :6. 96 PRACTICAL SANITARY SCIENCE The following is an estimation : In icx5,ooo pts Free and saline XH3 - . . . . o-oo6 , Total solids ------- 3,380-000 Lime -------- 35-000 Magnesia -------- 205-000 Silicia -------- 0-450 Hardness -------- 580-000 Chlorine -------- 1,875-000 The table below represents the comparative figures for the principal chemical constituents of a well-filtered river water, delivered to a town of some 40,000 inhabitants, and the sewage of the same town before treatment : Water. Sewage. Free and saline NH3 - - - Nil 6-800 Organic NH3 - - - - 0-002 2-000 absorbed in two hours at So° F. 0-020 4-080 Chlorine ----- 1-850 1 1 -800 Nitric X - - - - - 0-120 Nil Total solids- - - - - 28-500 160-000 CHAPTER VIII SEWAGE EFFLUENTS An average sample of the day's working should in all cases be obtained, and the analysis performed forthwith. It has been usual to estimate the ' free and saline ' and ' albu- minoid ' NHg, absorbed from permanganate, total solids, solids in solution, suspended matter, oxidized N, and CI. The physical characters may be noted, and incubation at 37° C. for forty-eight hours may be effected in order to determine the presence or absence of further fermentation, as indicated by odour. The analysis is frequently required for the determination of the degree of purification at a particular stage, or the comparative value of a certain method of sewage treatment. It is usual to record the purification as percentages of the figure for albuminoid NHg. If, for example, before treatment the albuminoid figure is -6 part per 100,000, and after treatment 0-15, it is clear that purification has taken place to the amount of 0-45 part per 100,000, or 75 per cent, of the original albuminoid NH3 has been oxidized. The ammonias are estimated as described under water, but a large dilution of the effluent is necessary; 10 c.c. may be made up to 1,000 c.c. with distilled water, and in some instances 5 c.c. in the same volume will be convenient. The absorbed from permanganate is estimated by Tidy's process, and care should be taken that sufficient permanganate is added from time to time, and that the flask is frequently shaken. A convenient dilution is 10 c.c. in a litre. In the working out of this process it should be noted that various bodies besides organic matter absorb from permanganate, such as nitrites, sulphites, sulphides, sulpho-cyanates, numerous d3^es, and vaiious coal-tar products. 97 7 98 PRACTICAL SAXITARY SCIENCE Total solids are estimated by evaporating lOO c.c. of the sample in a platinum dish on a water-bath. When dry, the dish is trans- ferred to an air-bath, and dehydration continued. It is then passed through a desiccator, and weighed. The difference between this weight and that of the dish represents the ' total solids.' The solids in suspension are found by passing loo c.c. of the sample through two folds of lilter-paper, whereby the solids in solution alone pass through. The filtrate is evaporated to dryness, further delu'drated, desiccated, and weighed. The result represents the ' solids in solution.' The difference between this weight and that of the total solids represents the ' solids in suspension.' In estimating nitrous N, dilute the liquid with distilled water (free from nitrite) to a convenient strength. Take lOO c.c. in a Xessler glass, as described under water, add i c.c. metaphenylene- diamine and i c.c. HoSOj (i in j). Match by treating in a similar manner a standard solution of potassium nitrite made up to lOO c.c. Stand for twenty minutes before comparing. The nitric N is estimated by Crum's method or by the copper- zinc couple. A convenient dilution must be made. Where time is an item, as in examinations, the less accurate phenol sulphonic acid method may be used. If raw sewage is to be anal3'zed, weaker dilutions must be used — 5 c.c. or less in a litre. In the distillations carried out in Wanklyn's process the volume of the boiling fluid should never be allowed to fall below 150 c.c. Hot, ammonia-free distilled water when necessary should be added. Griess's test should be promptly performed, and if the fluid is not transparent it should be filtered before adding the reagents. Estimation of the total X by Kjeldahl's method is a much more accurate index of the organic pollution than that by Wanklyn's process for albuminoid ammonia. The latter usually gives less than half the N figure obtained by the former. With a little practice Kjeldahl's method can be carried out rapidly and accurately as follows: In a Kjeldahl flask put 10 c.c. sewage effluent and i c.c. H2SO4, and evaporate on a water-bath to half the biilk. When cool, add about 10 c.c. oil of vitriol, and about 10 grammes sulphate or bisul- phate of potassium (to raise the boiling-point). Digest under a hood in a draught-chamber. Continue the digestion until the solu- SEWAGE EFFLUENTS 'yj tion is a pale, transparent yellow colour — i.e., initil ;ill the C lias been completely oxicliz;ed. Cool and wash out into ;i. distilling- flask. Make up to 500 c.c. with ammonia-free distilled water; add excess KOH and a piece of ignited pumice-stone, and distil over nearly 350 c.c. Nesslerise the ammonia collected, and subtract from the result the amount of free and saline ammonia previously estimated. The difference is the NHg due to organic nitrogen. The reagents used should be free from NHg. Digestion with concentrated H2SO4 converts the N into (XH4)oS04. Subsequent addition of excess of KOH decomposes (NH4)oS04, with liberation of NHg, which is distilled over. Instead of Nesslerising, the NHg can be received in excess of standard acid, and the unsaturated acid finally titrated with alkali : the amount of acid saturated is equivalent to the XHg from which the N is at once calculated. The purification of sewage has little influence on the amount of its chlorine, which in average samples reaches 10 or 11 parts per 100,000. A sewage effluent should be colourless and without odour. The albuminoid NHg should not exceed o-i to 0-15 part per 100,000, nor the absorbed in four hours at 2y° C i to 1-5 parts. CI and free and saline NHg are unimportant. The pouring of crude sewage or badly purified effluents into rivers of limited volume will cause deoxidation of the water, with con- sequent injury to fish and other forms of aquatic life, putrefac- tion of organic matter with resulting nuisance, growth of sewage fungus, disposition of suspended matters, etc. In order to determine the condition of contaminated streams in respect, of odour, development of grey algse, accumulation of putre- fying sewage solids, and injury to fish life, the Sewage Commissioners {1898, still sitting) have confined their attention mainly to three tests : 1. The amount of ammoniacal N. 2. The amount of absorbed from permanganate in four hours. 3. The amount of dissolved taken up in five days. Whilst the ammoniacal N may be considered as the most delicate chemical index of recent sewage pollution, it is not equally reliable in demonstrating the character of the pollution as indicated by the effect which the sewage produces on the stream. The nuisance- producing power of a sewage or effluent is broadly proportional to its power of deoxygenating the water of the stream, and tests based on 100 PRACTICAL SAXITARY SCIENCE tlie rate and degree of absorption of O are the most trustworthy for determining whether or not nuisance is likely to occur in a stream. The five days' test represents naturally the actual process by which the more readily oxidizable constituents of the polluting matter absorb the dissolved in the ri\-er-water, and shows smaller differ- ences in quality of water. The pennanganate process may give, approximately, the same figure for a water polluted with tank liquor and for a water polluted with filter eftluent, while the five days' dissolved test will give a liigher figure for the water polluted with tank liquor, thus indicating differences in kind as well as in degree of pollution. The Commissioners conclude that if loo.ooo c.c. of river water do not take up more than 0-4 gramme dissolved in five days, the river will be free from signs of pollution; but that if it takes up a higher figure it will most probably show signs of pollution. This number 0-4 they term the limiting figure, and regard it as the best foundation on which to construct a scheme of standards. As results will be found to vary according to temperature, they adopt the temperature of 65^ F. (18-3° C), and in order to provide a wide margin of safety the dr}- weather flow of the ri\-er. It will be seen that the amount of dissolved O taken up in five da\^s by a mixture of river water and sewage depends — (i) on the am.ount taken up by the sewage liquor; (2) on the amount taken up by the river water ; (3) on the proportion in which the two liquids are mixed. If a; = parts of dissolved taken up per 100,000 by sewage; y=parts of dissolved taken up per 100,000 by river water above outfall ; z = dilution (proportion of ri\-er water to sewage) ; then — ^ = 0-4. Thus, if an effluent which takes up o'l part dissolved in five days be discharged into ten times its volume of water, we get — ;»;-l-(o-i X 10) 10 + 1 ^ x + i =4-4 A'' -3-4 SEWAGE EEFIJJENTS loi — that is, in this case, the effluent may be allowed to take up 3-4 parts dissolved per 100,000 in five days, which figure would be the standard for this particular discharge. The most important local condition is the degree of dilution afforded by a river to the contaminating discharge. It is advised that a standard effluent should not contain more than 3 parts suspended solids per 100,000, and that samples which satisfy this test must also be considered in relation to the five days' test. The latter is fixed at 2 parts per 100,000. An effluent which takes up 2 parts per 100,000 dissolved in five days will need some dilution if nuisance is to be avoided. The minimum degree of dilution required for safety can be found from the formula: 2 + (o-2 xz) ^^ =0-4, It. is considered safe to assume that the majority of effluents are diluted by more than eight times their volume of river water. It is recommended, therefore, that an effluent should not contain more than 3 parts suspended matter per 100,000, and that, including its suspended matter, it should not take up more than 2 parts dis- solved per 100,000 in five days at 18-3° C. It is suggested that this be considered the normal standard for effluents. An effluent is considered satisfactory that contains less than 3 parts per 100,000 suspended solids, and which, after filtration, does not absorb in parts per 100,000 more than 0-5 dissolved in twenty- four hours, or 1*0 part in forty-eight hours, or 1-5 parts in five days. Adeney's method of determining the rate of absorption of dis- solved O by polluted waters is described in detail in the Fifth Report of the Royal Commission on Sewage Disposal. The Report (Cd. 4,278, 1908) states that effluents which are de- rived from strong original liquids may often contain large amounts of organic matter in solution, and yet not take up dissolved oxygen rapidly from water or cause injury to the streams into which they are discharged. Such effluents, judged by the empirical tests hitherto in common use, might be regarded as polluting liquids. The effect of an effluent on a stream does not depend on the absolute amount of organic matter in it, but on the nature and condition of 102 PRACTICAL SAXITARY SCIENCE that organic matter, and the important thing to ascertain is the extent to wliich the original organic matter has undergone fermenta- tion. Dunbar has sliown that after a certain percentage purification tlie residual organic matter in certain sewages is so altered as to be non-putrescible. To determine the rate of absorption of dissolved 0, it is only necessary to ascertain by a volumetric process the amount of dis- solved O in the effluent when fresh, and in a portion of the same effluent after it has been kept for a definite period of time — two to live days. The difference between the two estimations will give the amount of absorbed during the time of keeping, and the rate of absorption may be taken to be uniform, at least for the first two days of obser\-ation. If a knowledge of the attendant changes wliich take place during the various stages of the fermentation be required, it will be neces- sary, in addition to estimation of the dissolved O, to determine the NH.J and HNOo and HNO.j before, during, and after period of fermentation. For most practical purposes it is only necessary to determine the rate and total absorption of ox\'gen and the character of the fermentation, whether a carbon or nitrogen one. The first can be done by estimating the loss of in the atmosphere of the flask con- taining the polluted water, and the second by ascertaining whether nitrites and nitrates have been formed or not. In most cases, how- ever, as Adeney shows, even this will be found unnecessary, as the completion of the carbon-oxidation stage of fermentation will be indicated by the cessation of the absorption of oxygen which occurs during the interval of rest which takes place before the commence- ment of the nitrogen-oxidation stage. The Process. — A measured quantitv of the polluted. water (loo- 250 c.c, according to amount of polluting matters contained) is decanted into B, into which a little freshly precipitated magnesium hydrate has been previously placed for the purpose of fixing the ("Oo in the water. A similar volume of distilled watei is poured into A. Similar volumes of air are thus left in the two bottles. These volumes should be sufficiently large (capacity, a litre or more) to c nsure much more in B than can possibly be used. Corks, connecting-tube, and stopcocks are fitted. A slight rise of SE VVA GE EFFL (JEN J. S 103 capillary water will occur in the portion of the connecting-tube in A. The height of this capillary column is marked with a diamond or file; the mark serves as an. index for subsequent measurement. With both stopcocks open the two bottles are immersed in a water- bath for a few minutes to allow of their contents assuming a common temperature. Both stopcocks are then closed, and the temperature of the bath and the height of the barometer are noted. The bottles are taken out of the water-bath and dried. When completely dry the corks are coated with shellac varnish to prevent Fig. 23 diffusion of air through them. The apparatus is then put in a mechanical shaker, which keeps the contents in gentle motion. As is absorbed by the polluted water in B, the pressure of the atmosphere is reduced relatively to that of the atmosphere in A, which is unaffected by the distilled water. Accordingly, water from A will rise in the connecting-tube in proportion to the volume of O absorbed by the polluted water from the atmosphere in B. This volume of can be measured at anj'' time by attaching by means of a flexible tube a burette containing distilled water at the temperature of the laboratory. As the water from the burette is 104 PRACTICAL SAXITARY SCIENCE cautiously allowed to flow into B the water in the comiecting-tube will gradually sink back to the index, at which instant the stopcock to B is closed. The reading on the burette is equal to the volume of O, which has been absorbed from the atmosphere of B at the tem- perature and pressure obtaining at the commencement of the ex- periment. The distilled water bottle A acts as a reference pressure bottle. If a comparatively rapid absorption of occurs during the first hour or two and this is followed by a slower and regular absorption, it may safely be taken to be due to the polluted water being de-aerated to start with, and possibly also to the presence of easily and directly oxidizable substances in it; the subsequent slower and regular absorption being due to indirect oxidation accompanying the fer- mentation of the polluting matters. It is very important, as noted above, that sufficient excess of air be always secured, other^vise the operation is open to certain obvious inaccuracies. The replacement of the absorbed by water is equivalent to increasing the pressure of the N in B, which will lead to absorption of N by the polluted water and the distilled water added to it, unless the air in the bottle be in such large excess that the absorbed be only a small fraction of it. Further, with in- sufficient air there may be such a reduction in the store of in the atmosphere of B as to lead to appreciable reduction in the rate of fermentation in the polluted water. Bacteriological Examination of Sewage and Sewage Effluents. — Houston's method of water examination is equally suitable for sewage effluents and sewage when these have been properly diluted. In the estimation of B. coli it may be necessary to work on as small a quantity as o -000001 or even o-oooooooi c.c. of sewage. CHAPTER IX SOIL Analysis of Soils. The analysis of soils is a large subject, and requires for its proper execution a special training in chemistry. For public health pur- poses, however, very few estimations are required, and these are of a simple kind. The powers which a soil possesses for absorbing and retaining moisture are of some importance, but direct examina- tion of the soil and subsoil in position in a given locality will furnish more valuable information than laboratory tests. The capacity for absorbing moisture may be estimated by means of a percolator and burette. A quantity of dried soil (say lOO grammes) is flooded with water for two hours and allowed to drain for four hours. The difference in the reading of the burette before and after the operation gives the number of c.c. of water absorbed by 100 grammes, or the absorption per cent. Perhaps a simpler method is the following : One hundred grammes of dried soil are covered with water in a cylinder. Sufficient time is allowed for saturation, which in the case of clay soils may be several hours. The water is drained off through a muslin filter, and the soil is reweighed. The increase in weight roughly represents the percentage absorption. The determination of the size of the particles of a soil is carried out by using a series of sieves possessing meshes of 2 millimetres, I millimetre, and -5 millimetre respectively. A number of meshes larger than 2 millimetres may be used. One hundred grammes of dried soil are pulverized with the fingers. The larger pebbles, roots, etc., are removed and weighed. The residue is transferred to the 2 -millimetre sieve, and when all has passed that will, the remainder is further rubbed between the fingers, and 105 io6 PRACTICAL SAXITARV SCIEXCE once moic shaken on tlie sieve. What remains on this sieve is weighed. In like manner the amounts left on the other sieves are weighed. Finally, tlie soil wliich passes the 5-millimetre sieve is weighed, and the results are collected as (a) coarse masses removed by hand, (/;) masses kept back by the 2-millimetre sieve, (c) sand retained by the i -millimetre sieve, {d) fine sand retained bj^ the o-5-milIimetre sieve, and (e) fine soil passing the 0-5 -millimetre sieve. The specific heat of soils is determined by a sensitive calorimeter. The specific heat ranges from 0-2 to o-3. and is greatest in peaty soils. The determination of the porosity of a soil is effected by linding the real and apparent specific gravity of the soil, and di\'iding the latter by the former. The real specific gravity is obtained by ])lacing in a 50 c.c. specific gravity bottle 10 grammes of the soil dried at 100° C. to constant weight, rinsing the last particles into the bottle with distilled water, and making up with distilled water to the mark. The whole is weighed at 15° C The weight of the water displaced by the 10 grammes of soil is thus easil}' calculated. The weight of the soil — viz., 10 grammes — divided by the weight of displaced water is the specific gravity. The apparent specific gravity is obtained by filling a 1,000 c.c. cylinder with soil, introduced in small quantities at a time, and thoroughly settled in tlie cylindei b}- tapping from time to time on the bench. When full the cylinder is covered with a glass plate and weighed. The weight of the soil (cylinder full - cylinder empty) divided by 1,000 is the apparent specific gravity. The real specific gravity of a sample was found to be 2*46, and the- apparent 1*36; the porosity is therefore :^f = 0-55, or expressed as a percentage = 55 per cent. Pore volume, or porosity, being the sum total of the interstitial spaces which may be filled with water or air, or both, does not depend on the size of the particles but on their uniformity, or want of uniformity, of size, and on their arrangement. The porosity of a soil composed of unifonn spherical particles the size of peas is the same as that of another composed of particles the size of small shot, and in each case is about one-third of the whole. SOIL 107 Permeability of soil to air depends not on the amcjunt of its pore volume, but on the size of the individual spaces. Permeability diminishes to an extraordinary degree with diminution of the size of the particles. The water retained in soil exists as hygroscopic water adherent by surface attraction to the soil grains, and as capillary water held up in the capillary spaces. The latter constitutes by far the larger portion of the retained moisture. If the texture of a soil be so fine that all spaces are within the limits of capillary magnitude, the maximum water-retaining power is attained. The differentiation of the constituents of soils into sand, clay, and organic matter is of some importance. Sand consists of the coarser particles which rapidly sink in water ; clay of the fine particles which remain for a time in suspension. The estimation of sand and clay may be performed thus: Take 10 grammes of dried soil in a beaker. Moisten the soil with a little distilled water and a few drops of a solution of NH^Cl. When moist add 80 to 100 c.c. distilled water, and stir. Allow to settle for five minutes, and pour off the fluid into a tall cylinder. Another 100 c.c. of water is added, and the soil well stirred. After settling again for five minutes the fluid is poured off into the cylinder. These manipulations are repeated until the overlying fluid is quite clear. The sand is turned on to a filter-paper, well washed, dried, weighed, and recorded as sand. The cylinder is set aside for twenty-four hours, and when fully settled the upper portion of the fluid is run through a filter-paper without disturbing the sediment. When nearly all the water has been drained off, the sediment is stirred and poured on the filter. The last traces of clay are washed from the cylinder, and the entire contents now on the filter are thoroughly washed, dried, weighed, and recorded as clay. Clay soil contains over 30 percent, clay; some brick clays contain 95 per cent. Sandy soils contain as little as i or 2 per cent, clay; a loam contains 10 per cent. In order to determine the amount of organic matter in a soil, take 10 grammes of a dried sample in a platinum dish, and heat it at a temperature a little over 100° C. until a constant weight is obtained. io8 PRACTICAL SAX IT A RY SCIENCE Oxidize over a flame at a low red heat, transfer to desiccator, and weigh. The loss in weiglit gives roughly the amount of organic matter. Lime mav be estimated thus: Dissolve a few grammes of the dried soil in dilute HCl, and dilute the resulting solution to about lOO c.c. with water. Heat, add NH4OH in slight excess, and a solution of ammonium oxalate also in slight excess. Allow the precipitate to settle in a wami place. Pass the clear liquid through a small filter and then bring the precipitate upon it. Wash with hot water and set the filtrate and washings aside. Push the precipi- tate and filter-paper through the funnel into a flask, add some H2SO4, dilute freely, warm to 60° or 70°, and run in ^^ per- manganate until faint pink remains. Each c.c. of f"^ permanganate represents 0*0028 gramme CaO. Magnesia. — Evaporate the filtrate and washings to small bulk on the water-bath, render alkaline with XH4OH, add sodium phosphate, and set aside for eight or ten hours in order that the magnesia may separate out as NHjMgPO^.GH.^O. Wash this precipitate on to a filter with ammonia solution. Dry in hot-air chamber and ignite to form Mg2P207, from which the weight of MgO is easily calculated. Or the ammonio-magnesium phosphate precipitate may be brought upon a filter washed with ammoniacal water in the cold, dissolved in acetic acid, and titrated with standard uranium solution, each c.c. of which represents 0'0028i5 gramme magnesia. The phosphoric acid in soils is determined as follows : (i) Incinerate and digest a weighed quantity of the soil with HCl, evaporate to dr}-ness to render silica insoluble, redigest with acid, filter, and wash. (2) Concentrate the filtrate and washings to small bulk and add excess of ammonium molybdate in nitric acid, stand aside in a warm place for two days, decant the liquid through a filter, wash the pre- cipitate several times by decantation first with dilute HNO3, and afterwards with small amounts of distilled water, then transfer it to the filter and wash free from excess of acid, dissolve the am- monium-phospho-molybdate in ammonia, add magnesium mixture, filter, wash, dry, and ignite the precipitate, ^^'eigh the resulting Mg2P207, from which calculate the P2O5. Or the method described for magnesia may be used, wherein the ammonio-magnesium phosphate precipitate is dissolved in acetic SOIL 109 acid, and titrated with standard uranium acetate or nitrate solution, each c.c. of which equals 0-005 gramme P2O5. The total organic nitrogen of soil is best estimated by Kjcddahl's method. The ammonia resulting from the distillation of the am- monium sulphate with excess of KOH is received in j^ or -^^ H2SO4. At the end of the distillation the standard H2SO4 remaining is titrated with standard alkali, and the ammonia absorbed by the acid calcu- lated. The N forms \y by weight of the NH3. Where total N (including HNO2 and HNO3) is required the oxidized N must first be converted into NH3 by boiling with Al and NaOH, or by the action of the Cu-Zn couple. Clay and humus are the two most important ingredients of soils. The plasticity and adhesiveness of clay, together with the fineness of the particles, serve to hold together various other aggregates of soil. The extreme fineness of the particles of clay causes it to retain water, solids dissolved in water, and gases. If by plastic or colloidal clay be understood the particles of soil under o -01 millimetre diameter which remain suspended in a column of water eight inches high for twenty-four hours, soils may be divided into the following six classes : Very sandy soils containing up to 3 per cent. clay. Sandy ,, ,, ,, 3-10 Sandy loams ,, ,, 10-15 Clay „ „ „ 15-25 Clay soils ,, ,, 25-35 ,, Heavy clays ,, ,, 35-45 ,, ,, and over. Admixture of fine powders, such as Ca(0H)2 and Fe2(OH)6 diminish greatly the adhesiveness of clay, caused by the hydrated silicates. ' Humus ' or ' vegetable mould ' is formed by the decomposition of organic matter, largely cellulose, derived from the roots, stems, and leaves of plants. Its accumulation near the surface is natural, and it distinguishes soil from subsoil. Its production is controlled by moisture, oxygen, temperature, and micro-organisms. With a low temperature and as much water as will shut out air the organisms that transform vegetable tissue into humus are bacteria; but the disinfectant compounds produced soon kill the bacteria, and the process remains henceforth a slow and purely chemical one. In no PRACTICAL SANITARY SCIENCE the solid brown decomposition products formed in peat are found ulmic and apocrcnic acids soluble in caustic and carbonated alkalies, and fonuing insoluble salts with the earths and metals, and ulmin, insoluble in alkalies but after^vards soluble on oxidation. CO., and CH4 are formed in large quantities under these conditions. Pro- longed cultivation of soils tends to production of acids; hence the advantages of calcareous formations. In the presence of earthy carbonate, especially that of lime, which neutralizes acids as formed, moderate degrees of moisture, and free circulation of air, humification proceeds under the influence of moulds instead of bacteria. and H are eliminated as CO., and HoO, and an increase takes place in the percentage of C and X. When humification is complete and oxidation proceeds, the X may rise to high figures, portions being wholly oxidized to nitrates. Humus is highly porous, absorbs water and gases, and is gradually oxidized by bacteria. The measure of this oxidation can be gauged by the amount of CO2 produced. Humus substances are gelatinous when moist, but not markedly adhesi\'e or plastic. The densit)' of humus is about 1-4 ; hence soils rich in humus are light (humus is the lightest ingredient of soil) when compared with clay and sandy soils, and ' light ' in the agricultural sense of being easily tilled. The X of humus does not exist in the form of XH3, as it cannot be set free by treatment in the cold with lime or alkalies. When humus is boiled with lime or alkalies ammonia is slowly evolved for an indefinite time, but the whole of the X is not expelled. Such be- ha\dour, together with its slightly acid reaction, points to humus being of the nature of an amido-compound. Hmnus formed from sugar, cellulose, gums, etc., combines with ammonia as with other bases, and at first the ammonia can be readily expelled from this as from other ammonia salts. But after a time the amidic condition appears to be assumed, as caustic alkalies act but slowly, and are unable to expel the whole of the X. These facts are of importance in nature, as XH3, generated in or taken up by the soil, is in the course of time rendered inert and unavailable for plants until nitrification has been effected. Humin and ulmin found in the deeper layers of peat are in process of time oxidized into humic and idmic acids capable of combining with bases. Further oxidation produces crenic and apocrenic sou. Ill acids, readily soluble in water and capable of uniting with Imsts to form salts. These acids react on decomposable silicates and dissolve them; they also dissolve ferric hydrate. In this way rust-coloured soils are bleached by stagnant water and deprived of much of their mineral plant food. In ordinary soils humus rarely exceeds 5 per cent., in peat and marsh lands it may reach 20 per cent. Humus may be estimated by extracting the soil with dilute ricid to set free the humic bodies from their combinations with lime and magnesia. The residue is then extracted with moderately dilute solutions of ammonia. Evaporation of the ammonia extracts leave the humus as a black lustrous substance [matiere ■noire of Grandeau). As this contains a variable amount of ash it is burnt and the ash is subtracted from the first weight. A determination of the ash of humus gave : Insoluble matter (principally silica) - - 62-6 K,0 - 7-5 Na,0 - 8-1 CaO o-i MgO - 0-3 FeoOg - 3-1 AlA - 3-4 P2O5 - 12-3 SO3 - 0-9 CO, - 17 lOO-O Bacteria of Soil. — Inseparably correlated with humus and carbo- hydrates in soil are varied forms of bacteria. More than 40,000,000 per c.c. have been found. The bulk of soil bacteria reside near the surface, as there alone are to be found the conditions necessary to growth and multiplication. The best foodstuffs appear to be water, proteins, and soluble carbohydrates derived from deca5'ing plants, stable manure, etc. When the decaying matter reaches the stage of humus, only few bacteria remain. The most important functions of soil bacteria are related to putrefaction, nitrification, denitrification, and nitrogen-fixation. Most of the putrefactive bacteria are concerned with the breaking down of complex protein matter, with evolution of NHg, and fonna- 112 PRACTICAL SAXITARY SCIENCE tion of ammonium salts. Common examples of soil organisms of this type are — Bacillus mycoides, B. suhtilis, B. mesentericus vidgatus, Proteus vulgaris, P. zenkcri, Bacillus coli, B. putrificus, B. lactis aero- genes, B. fiuorescens liqiiefaciens, streptococci, etc. In acid soils rich in humus certain fungi such as Peniri Ilium glaucum, Mucor mucedo, and species of Botrytis and Torula accomplish the cleavage of proteins. Nitrification is carried out in two stages: ammonium compounds are oxidized to nitrites by such organisms as Winogradsky included in the genus Nitrosoinoiias curopcBa ; and nitrites are oxidized to nitrates by several forms included in the genus Nitrohacter. The conditions necessaiy to these changes are definite. In addition to nitrifiable material and nitrifying bacteria, a fairly high tempera- ture (24° C), a moderate degree of moisture, free access of oxygen, a base or its carbonate with which the acids formed in the process of oxidation can imite, free CO.,, and darkness are essential. In acid soils nitrification ceases, as also in soils in which the bases have become fully saturated. Carbonates of lime and magnesia are the bases most favourable to nitrification, and excess of these produces no injury. The amounts of carbonates of potash and soda must be strictty limited. The nitrifying organisms are strictly aerobic; in non-porous or water-logged soils they quickly die out. They derive their carbon from CO2, as when cultivated in the presence of carbonates in an atmosphere washed with KOH they fail to develop. Various denitrifying bacteria have recently been studied. One of the most effective organisms is Burri's B. denitrificans, found on the surface of old straw and in fiesh horse-dung. If some fresh hcrse-dung be placed in a close-flask containing KNO.5, nitrogen and carbon dioxide are evolved, and in a few days the nitrate has disappeared. B. hutyricus, which in the absence of easily reducible compounds evolves free nitrogen, reduces nitrates to nitrites, and also forms NH3 by addition of H to N just set free by reduction. B. mycoides forms ammonia from antecedent proteins, and also reduces nitrates to nitrites and ammonia. Reduction may be to nitrites, and no further; it may go on to ammonia; nitrates and nitrites may be reduced with evolution of NO and N2O; and, finally, nitrates and nitrites may be reduced with production of free N. A SOIL 113 large number of bacteria found in faecal matter, water, and soil decompose nitrates with evolution of free N. The reactions between nitrates undergoing denitrification and organic carbon compounds may be represented by the equations: C +2NaN0.j= CO2 +2NaN02 C + 2NaN02 = N2O + NaX03 C+2N20=2N2 + C02, where C represents the oxidizable carbon of the carbon compounds. An important group of soil bacteria is found in connection with the root nodules of leguminous plants. The mode of supply of nitrogen to plants was long a subject of debate. Liebig thought that it was derived from the ammonia in rain water. Boussmgault proved that plants do not take N directly from the air. Lawes and Gilbert confirmed Boussingault's con- clusions. Hellriegel and Wilfarth pointed out that the tubercles on the roots of leguminous plants are produced by bacilli which absorb free N from the air, and pass it over to the host. Beyerinck later separated and described the B. radicicola. The tissues of one of these nodules on microscopic examination are found to contain a number of free motile bacteria, and a niunber of quiescent forms much larger in size. When the nodule has reached full size, the large quiescent bacteria begin to collapse, and part with their nitrogenous substance. Later the shells drop off and carry minute bacteria into the soil, which in due course again become active. The nodules adhere but loosely to the roots. The ease with which they may fall off doubtless accounts for the diffi- culty experienced in transplanting legumes. The nodules above mentioned vary in shape and size, according to the species of leguminous plant to which they are attached, and are caused by the Bacillus or Pseudomonas radicicola (Beyerinck) penetrating the root hairs. On entering root hairs the organism develops and forms a thread-like zooglea technically known as the ' infection thread,' which resembles the hyphaof a fungus, and which excites the neighbouring cells of the rootlet to rapid multiplication and the formation of the nodule. In the infection threads and youngest nodules the organisms are straight rods. In older parts they are branched and curved, and are known as bacteroids which 114 PRACTICAL SANITARY SCIENCE have lost their power of di\ision : Liter tliey are digested by a ])roteo- lytic enzyme secreted b}' the protoplasm of the root. The digested substances pass to the flowers and seeds of the ripening plant. N- fixation reaches a maximum at the time when the plants begin to flower. When the crop is har\-ested, a large sur})lus of nitrogen is left behind in the nodules in the soil. Other bacteria are known to absorb fice N, of which may be mentioned Winogradski's Clostridium pastorianum and Beyerinck's Azotobacter. The nitrogen-fixing powers of soil may be determined by esti- mating the total N in, say, 200 grammes of soil, and repeating the experiment after six weeks' incubation at 20° C in a solution composed of grape sugar 40 grammes, K2HPO4 2 grammes, NaCl 2 grammes, CaCO., 10 grammes, and water 2 litres. The nitrifying and denitrifying powers of soils can be estimated in the same manner by adding known quantities of ammonium salts, nitrites, and nitrates, respective!}', to a suitable inorganic medium containing a soluble carbohydrate. When the surface soil is wetted, moisture may rise toward the surface from the lower layers ; this is probably due to evaporation from below, followed by recondensation by the cool wetted layer. The condition is of some practical interest, inasmuch as cold rain on the surface may raise water from below. The downward percolation of water is most rapid in those soils in which capillary ascent is quickest — i.e., in coarse sand. The rapidity of percolation decreases as the wetted soil column increases in depth; as the wetted column lengthens, the frictional resistance increasingly opposes the effects of the hydrostatic pressure from above until downward movement becomes little more than lateral movement or capillary ascent from below. The frictional resistance has counteracted gravity to such a degree that the capil- lary coefficients of the soil become the governing factors of the water movement. It is often desirable to protect a soil from excessive evaporation in order either to prevent lowering of temperature or to save vegeta- tion in time of drought. The preparation by tilth of a layer of loose dry surface soil is the best means of securing this object. It would appear on first sight that such a soil admits of leady access of air, SOIL 115 and therefore of evaporation; whilst this is true, it is equally true that the coarse particles are incapable of withdrawing moisture from the denser layers beneath in the same manner as a dry sponge is incapable of withdrawing moisture from a wet brick, notwithstanding the fact that a dry brick will readily absorb all the water from the relatively large pores of a wet sponge. The disinfectant action of dry soil and its capacity for absorbing offensive gases have long been known. The decoloriziation by soil of drainage from manure-heaps, dye-works, and tanneries, and the filtration of drinking waters on the large scale by fine sand, are equally familiar. It should not be forgotten, however, that these powers are strictly limited. Dry soils are powerful gas-absorbers, and peat appears to excel all others in this property. The temperature of the soil is derived from the sun's rays, chemical changes in the soil, and from the heat of the earth's interior. The first of these is the chief factor in influencing tem- perature. The more perpendicularly the rays strike the soil the greater the amount of heat received. The colour, composition, moisture, and compactness of the soil influence the temperature. A black surface absorbs heat rays more than a white one. Snow melts more rapidly when covered with soot. Sands and mineral substances in general conduct heat better than water, air, or organic matter. Organic matter is a poor conductor of heat ; hence the more humus a soil contains the more slowly will it respond to the action of the sun. Moisture influences soil tem- perature through the high specific heat of water and through the disappearance- of heat due to evaporation. The specific heat of ordinary dry soils is about one-fifth that of water. The drier a soil, the less the evaporation and the greater its warmth. Veg-etation protects against excessive heating in hot climates, and loss of heat in cold climates. Trees impede wind currents and obstruct the sun's rays, so that less loss of moisture occurs by evaporation. It may be quite calm in the centre of a wood whilst a gale blows outside. A soil in which the ground-water is high — say 5 to 10 feet from the surface — has long been regarded as unfavourable to health. Such a soil renders the atmosphere damp, and appears to conduce Ii6 PRACTICAL SAXITARY SCIENCE to rheumatism and diseases of the respiratory tract. Lowering of the gronnd-\vater le\-el by drainage has largel}- inipro\-ed the health conditions of man\- soils. Soil dampness appears to be connected with pulmonar\" tuberculosis. T\phoid fever, cholera, dysenteries, and other intestinal maladies have been etiologically related by various observers to soil, ground- air, and ground- water : it is probable that each and all of these act as media of conveyance of the specific micro-organisms of these diseases. Newsholrne regards epidemics of diphtheria as intimately related to dry years, and holds that they do not occur when the rainfall is above the average. Malaria is connected with soil conditions in the breeding of the specific mosquitoes. Ankylostomiasis or uncinariasis is intimatel}' connected with the soil, in that the eggs of the parasite Ankylostomnm duodenale escape with the faeces and are deposited in the soil, where they hatch in twenty-four hours. The embryos shed their skin twice, and after a few weeks are ready to infest man. The chief portal of infection is the mouth. Several observers assert that the parasites can reach the intestine through the skin. The various theories which connected goitre with particular con- stituents of the soil, such as metallic sulphides, magnesian limestone, etc., are now practically abandoned. Bactepiologrical Examination of Soil — This examination is of service principally in connection with water-supplies, more especially contamination of water by surface washings. Much work has been done on B. typhosus in soils, and findings have been very varied. Under favourable conditions it appears that this organism can survive for a considerable time. The organisms of tetanus and malignant oedema are widely dis- tributed in cultivated soil. They are isolated anaerobically from small quantities of soil or soil washings in the usual way. Advan- tage is taken of the fact that their spores survive heating at 80° C for a quarter of an hour, when all non-sporing organisms are de- stroyed. These spores are grown on various media over alkaline pyrogallic solution, and the ;growths investigated in the usual manner. SOIL 117 In collecting soil for examination, the depth from which the material is to be recovered having been decided upon, a sterile instru- ment is used for procuring six to twelve specimens, which are mixed in order to produce an average sample. This is carried to the laboratory in a sterile vessel. A gramme is shaken up in 100 c.c. sterile water in a sterile flask, and from this dilutions are made— i c.c. of this solution is trans- ferred to 100 c.c. sterile water in a second flask, etc. Quantitative and qualitative estimations of B. coli, streptococci, and B. enteritidis sporogenes are carried out in these liquid prepara- tions in the same manner as in dealing with water. B. coli is absent from un contaminated soils, or present in very small numbers only. Houston finds that it is not readily isolated even from polluted soils unless the contamination is recent and large in amount. He considers the spores of B. enteritidis sporogenes indicative of contamination, but not necessarily recent. Strepto- cocci are found in minimum quantities of soil recently polluted with sewage. They disappear extremely rapidly. CHAPTER X AIR The air is a mechanical mixture of gases. One hundred volumes contain, roughly, 21 of oxygen, 78 of nitrogen, and i of argon, krypton, heliimi, neon, zeon, and carbon dioxide. A distinguishing property of gases is that a mass of gas introduced into a closed vessel alwaj^s completely fills the vessel, however large. Consider two vessels of eiqual volume connected by a tube carrying a tap, and let one of these vessels be filled with a gas and the other exhausted; on opening the tap, the gas rushes into the exhausted vessel until the same quantity of gas exists in each vessel. Close the tap, and once more exhaust one of the vessels; on opening the tap, the gas expands and again fills equall}' the two vessels. The operation may be repeated indefinitely, and the gas will always exert some pressure on the inside of the containing vessel. The density of a gas, like the densit}' of any other body, is the mass of unit volume, and is sometimes referred to hydrogen and sometimes to air as unity at 0° C, and under a pressure of one standard atmosphere. The only elasticity of which a gas is capable is that of volume or bulk, since it is alone to a change of volume that a gas offers any permanent resistance. If the pressure on volume V of a gas be increased from F toF +p, and as a consequence the volume be reduced from V to V — v, the temperature remaining constant, then the strain produced in V voliune V is v, and per unit volume y, and the corresponding stress is p. Therefore, since the elasticity of a body is the ratio of the V V stress to the strain, the elasticity of the gas is /> -^y, or />- . By compressing air with mercury in a U-tube closed at one end, 118 AIR fil9 Robert Boyle found a series of values for the volume of a given mass of air under different pressures, and he enunciated in 1662 a law known by his name — viz., that (the temperature remaining un- changed) PV= constant. Mariotte fourteen years later enunciated the same law. That all gases have the same coefficient of thermal expansion was first enunciated by Charles. Consider a mass of gas of volume Vq at pressure pQ, and imagine its volume kept constant while its tempera- ture is lowered from 0° C. to - 1°, the pressure p will by Charles's law be given by p=Po{i~at), where a is the coefficient of expansion. If the cooling be continued -1° to a temperature ~^> P=Pq{i~i) = o, i.e., at this temperature the gas would exert no pressure on the walls of the containing vessel. According to the kinetic theory of gases, this can only occur when the velocity of translation of the molecules is 2;ero. This temperature is called the absolute zero. ' Taking a as o-yrr (the mean value for hydrogen between 0° and 100° C), the absolute 2;ero will be -273° C. In order to convert temperatures referred to 0° C. to the corresponding temperatures referred to the absolute zero, it is only necessary to add 273. If T and t represent respectively the absolute and the ordinary tempera- ture, •■ T=^ + 273. By Charles's law — • p=PQ{i+at), and v=Vo{i+ at), substituting for a its numerical value, -PJl - 273' . , and.= -. 120 PRACTICAL SANITARY SCIENCE At any other temperature T^, if when the volume is constant the pressure is />\ and when the pressure is constant the volume is v^ 273 ' 273 .. ^1-^1, ana ^^i-;j-i. or the pressure at constant volume varies directly as the absolute temperature, and the volume at constant pressure varies directly as the absolute temperature. A Barometer is an instrument used for measuring the pressure exerted by the atmosphere. Barometers may be divided into two classes: (i) Those in which the pressure is measured in terms of the height of a column of a liquid; (2) aneroid barometers, in which the pressure is measured b}- the strain produced in the lid of a metal box. Mercury is practically always used in liquid barometers on account of its great density rendering the height of the column supported by the atmosphere a convenient quantity with wliich to work. Further, mercury does not, as does glycerin, absorb moisture from the air ; it has a fairly low freezing-point, and a high boiling-point. The simplest fonn of barometer is the siphon barometer, consisting of a U-tube, the longer limb (86 centimetres) of which is closed while the shorter is open. The tube is filled with mercury; by boiling the mercury any air or moisture adhering to the mercury or bore of the tube is expelled. The distance between the levels of the mercury in the two limbs is the barometric height. When the pressure in- creases, the mercury falls in the open limb and rises in the closed by the same amount, so that the difference of level is double the rise in the closed end or fall in the open. If a scale be attached to either tube, and each inch or centimetre, as the case may be, be marked half an inch or centimetre, the reading at once gives the height of the barometer. In the Fortin barometer the scale is graduated in inches to 0-05, and the vernier usually reads to 0-002 inch. The cistern is closed below by a leather bag protected by a metal sheath, into the bottom of which is fitted a screw for the requisite adjustments. Having AIR taken the temperature by the attached therm.omctcr, the mercury in the cistern is raised or lowered by the screw until the ivory point (fiducial point) or zero of the scale and its reflected image in the mercury are just in contact; the vernier is then moved by the upper milled head until its lower edge just excludes the light from the top of the mercurial column; the reading is then made from the scale and vernier. Verniers are of different lengths, and contain variable numbers of divisions. A I — 3 7 common form is i^ inches long, divided into twenty-five parts, which correspond in length with twent3/-four divisions of the principal scale. A division on the principal scale is there- fore greater than one on the vernier by (~ X i^ inches) — (ttV x li inches) = -^ — - X 1-2 inches 6oo = 0-002 inch. (J To read the vernier adjust its lower edge with the top of the meniscus, when two very small triangles of light will] appear, one on either side. If the lower edge of the vernier correspond with a division of the principal scale, this is the reading; but if not, it is evident that the interval between the surface of the mercury] and the division of the' principal scale next below is equal to the^difference between the lengths of the divisions of the vernier and principal scales (o-oo2 inch) multiplied by the number of vernier divisions which intervene between the lower edge (zero of vernier) and that division which exactly corre- sponds with a division on the principal scale. Suppose in a given example that the lower edge of the vernier cuts the principal scale between 29-15 and 29-2 inches, and when the vernier scale is examined it is found that its thirteenth division 20- 15- 10- 30 S.9 Fig. 24. 122 PRACTICAL SAXITARY SCFEMCE corresponds with a division of the principal scale, the reading will be : 29-15 inches + 13 X 0-002 inches = 29-15 inches + 0-026 inch = 29-176 inches. The Kew barometer, originally invented by Adic for use at sea, has a closed iron cistern, and scale of contracted inches. The tube is of small calibre throughout, in order to lessen the oscillations of the mercury by the ship's motion (known as ' pumping '). A small aperture, covered with leather, in the roof of the cistern, allows atmospheric pressure to exert itself on the contained mercury. Fitzroy's gun barometer is a modification of the Kew. Hooke's wheel barometer is a siphon barometer. On the surface of the mercury in the lower limb is a float carrying a needle indicator, which moves on a graduated circular dial. Various self-recording barographs are on the market, records being obtained mechanically, photographically, and electrically. In order to make an observation of the barometer comparable with other observations taken at other times and places, certain corrections must be applied to it ; some of these refer to an individual instrument, and others to all readings of any instrument. Of the former class there are three — corrections for index error, capacity, and capillarity. Of the latter class there are also three — corrections for temperature, altitude, and gravity. The index error is made by the workman who laid off the scale of the instrument. It is discovered when the instrument is verified at Kew or elsewhere. Correction for capacity depends on the propor- tion borne by the sectional area of the tube to that of the cistern. At one point of the scale the reading is correct ; when the mercury is above that point the correction is additive, when below subtractive. Capillaritv between glass and mercury tends to depress the mercury, and in larger degree the smaller the tube; it is also greater in an ' unboiled ' than in a ' boiled ' tube. All certificates from Kew for ' Kew ' pattern barometers give a correction at each ^ inch, including the above three corrections. Corrections independent of the Special Instrument.— r^m- perature. — If the scale by means of which the height of the column is measured be correct at 0° C, then at all temperatures above 0° the length of the divisions will be too great, since all metals increase in AIR 123 length when heated. Let a be the coefficient of h"near expansion of the metal of which the scale is made, so that unit length of the scale at 0° C. becomes i+at at t° C If ht is the reading at temperature t, then the height as measured with the scale at 0''-' would be greater, since the length of each division of the scale would be less in the ratio of I to I +at, so that the number of divisions corresponding to a given length (length of mercury column) will be increased in the ratio T+ at to i. If Hq be the barometer reading corrected for expansion of the scale, hQ= ]\t{i + at). But h^ is the height of a column of mercury at temperature t, and the problem is to find what the height would be if the temperature were 0° C. If dt be the density of mercury at t°,-dQ the density at 0°, S the coefficient of cubical expansion of mercury, and H the height which the column would have if the mercury stood at 0° C., then i c.c. of mercury at 0° becomes i +S c.c. at 1°, and I + S^ c.c. at t°. Since the mass M of the mercury remains unchanged M= Uo^o= ''^i^i' where Vq and V<= volumes of mass M at tempera- tures 0° and t° respectively. Since S is excessively small, its second and higher powers may be neglected, and j =r—U. Since the height of a column of liquid supported by a given pressure is inversely proportional to the density, B. dt .^ .-. Y{=h^[i-U)^ht{T-+at) {i~8t)=Iit{i-{8~a)t), if 8at^, which is excessively small, be neglected. For mercury, 8 = 0-000182; for brass, a =0-00002. Therefore, for a mercury barometer with a brass scale, the corrected height corresponding to an observed height ht at temperature t° C., is given by H=;^.(i- 0-000162/). Altitude and Gravity. — If g = acceleration of gravity at place of observation, and ^45 that at latitude 45° and at sea-level, /= latitude of observation, and/= height above sea-level, = 1—0-0026 cos 2^ — •0000002/". 124 PRACTICAL SANITARY SCIENCE If Hq be the height under standard conditions corresponding to the same pressure as does H at the place of observation, Hg=Hog'45; orHo= '^ = /i/(i -o-oooi62/)(i — 0-0026 cos b45 2^—0-0000002/). If a bubble of air be passed into the vacuum of a barometer, the mercury falls; if several bubbles be passed in, each produces a de- pression. If instead of air a drop of ether be introduced, the mercury also falls and the ether becomes complete^ vaporized, even at a temperature much below its ordinary boiling-point. If suc- cessive drops of ether be introduced, it will be found after a time that further addition of ether fails to produce further depression, and that the ether does not vaporize, but floats on the top of the mercury. Now, if the space above the mercury be enlarged or diminished by raising or lowering the barometer-tube in the cistern, it will be found that so long as any liquid ether remains, the height of the mercury column is constant, but that the amount of ether which vaporizes varies with the space above the mercur}'. If the temperature be increased, more ether vaporizes, and the mercury column becomes more depressed. The vapour exerts a pressure which partly balances the pressure of the atmosphere. The depression of the mercury measures this vapour pressure. When excess of liquid is present, so that the vapour exerts its maximum pressure, the vapour is said to be saturated. If, on the other hand, more liquid would vaporize on introduction to the vacuum the vapour is said to be unsaturated or superheated. The vapour pressure, or tension of a liquid, depends on temperature only. Xon-saturated vapours obey Boyle's and Charles's laws only approximately, approximation being the more complete the further the vapour is removed from its saturation- point. Altitudes are calculated from barometric readings either (i) by Laplace's formula, or (2) by Apjohn's formula. Laplace's formula is — 0=18,363 (log P-log p) (i+"/ooo)' where D= difference in altitude in metres of the two stations. P= barometric pressure in mm. Hg at lower station. p= ,, ,, ,, ,, higher / = temperature in "C. at lower station. t' = „ „ higher „ AIR 125 Apjohn's formula is: 16,000 (P-/>) / 2t + t' \ ^- P + ^ ""V^ + i.ooo^ where D= difference in altitude in metres of the two stations. P= barometric pressure in mm. Hg at lower station. p= „ ,. „ „ higher „ t = temperature in °C. at lower station. t'= ,, ,, higher Thermometers. — The freezing-point of a thermometer is deter- mined by surrounding the bulb with a mixture of ice and distilled water. The boiling-point is fixed by suspending the instrument in steam issuing from water boiling at a pressure of 760 mm. of Hg. The tube is then calibrated between these two points into 100° in the Centigrade instrument. Errors of Mercury Thermometers. — ^The observed expansion is really the difference between the expansion of the mercury and of the glass surrounding the mercury. As different kinds of glass do not expand exactly alike, thermometers made of different glasses do not completely agree. Owing to the gradual recovery of the glass from the effects of the heating to which it was subjected when the thermometer was made, the zero-point rises, at first rapidly, later slowly. One of the oldest forms of self-registering thermometers provided with a contrivance to mark the highest or lowest temperature ob- taining in a given interval of time, is that of Six, made in the eighteenth century. It consists of a glass tube bent twice at right angles, and furnished with a bulb at each end. The bulbs are filled with spirit, except that a bubble of air is placed in the smaller one. The bends of the tube are occupied by a column of mercury. Two steel pins sealed in glass tubes have hairs attached to them, so that they may retain any position reached by being pushed by the mercury column. A magnet is emplpyed to set these indexes. When the temperature rises, the spirit in the large bulb expands, and pushes the index and column of mercury before it. When the temperature falls, the spirit contracts, and the pressure of the air-bubble in the small bulb drives the column of mercury back, which in turn pushes the minimum index before it as soon as the temperature falls below that at which the instrument was set. The defects of the instru- ment are — it must always be kept in the vertical position, otherwise 126 PRACTICAL SAXITARY SCIEXCE the spirit may pass the mercuny at the bends of the tube. The mercuiy tends to pass beyond the ends of the indexes so that small quantities are retained by them. Modern maximum and minimum thermometers are now always distinct instruments. The student is ad\-ised to study these by personal inspection at the show-rooms of a good meteorological instrument maker. Rutherford's maximum thermometer consists of an ordinary mercury thermometer, with an iron index introduced into the bore (mercury does not wet iron). With rise of temperature the index is pushed before the column of mercury; with fall of temperature the mercurN' at once parts company with the index. The liquid of the minimum thermometer is alcohol, and the index glass (alcohol wets glass) . When the temperature rises, the alcohol flows past the index without mo^'ing it ; when it falls, the index is carried by the retreating surface of the alcohol by capillarity. In estimating the weight of volumes of air and aqueous vapour at \"ar3ang temperatures and pressures, it is necessary to understand aright the meaning of ' densit}-,' ' specific gravity,' and ' relative density.' Density is defined as the mass of unit volume (mass being the amount of matter as measured by inertia) ; specific gravity is the ratio of the weight of a certain volume at a given temperature and pressure to the weight of an equal volume of a standard sub- stance at the same temperature and pressure. vSince the unit volume is I c.c, and the unit mass i gramme, it follows that water is the standard substance whose density is unity. When the density of oxygen is spoken of as i6, it is meant that the specific gravity of oxygen is i6, h\-drogen being taken as the standard; the real densit}' (mass of i c.c. 0) is 0-0014 gramme. It is preferable to use the phrase ' relative density ' of oxygen, etc., and consider it as meaning the same thing as specific gravity when air or hydrogen is the standard. The atomic weights of gaseous elements such as H, N, etc., represent their relative densities, whilst the relative densities of compound gases are represented by half their molecular weights. The relative density of is 16, that of CO2 22, H being the standard. The relative density of air referred to the same standard is 14-47. In hygiene it is customary in calculating the weights, etc., of gases AIR 127 to take air as the standard. The relative density of H, is therefore ; of O, ; of COo, : and of water vapour, —^ . 14-47' ^'14-47 -14-47 ^ 14-47 In the metric system the weight of a litre of H at o^ C and 760 millimetres Hg= 0-0896 gramme. In English measure the weight of a cubic foot of air at 32° F. and 30 inches Hg= 566-86 grains. The pressure of water vapour increases with its temperature until at boiling-point it equals that of the atmosphere (30 inches Hg). The following figures are extracted from a table of vapour tensions : 32° F. =o-i8i inch pressure 33° F.=o-i88 , 34° F. =0-196 , 42° F. =0-267 . 43° F. =0-277 > 44° F. =0-288 , 50° F. =0-361 , 52° F. = 0-388 , 53° F. =0-403 , 54° F. =0-418 , 62° F.-0-556 , 63° F. = 0-576 , 64° F. =0-596 , Example. — Find the weight of a cubic foot of aqueous vapour at 62° F. At this temperature the tension or pressure is 0-556 inch. As the relative density of aqueous vapour (air being the standard) is , or 0-622, it is necessary to find the weight of a cubic foot 14-47 . of dry air at 62° F. and 0-556 inch pressure, and multiply the result by 0-622. The weight of a cubic foot of dry air under these conditions of temperature and pressure will be given by finding the fourth term x of the proportion : 521 : 491 :: 566-86 grains :'.i; 30 : 0-556 - - 491 0-556 ^^ „^ X = "P- X ^^^^ X 566 -86 = 9 -9 grams, 521 30 -i^ V y t) ' i-'S PRACTICAL SAXITARY SCIENCE and this multiplied by 0-622== weight of aqueous vapour= 6*i6 grains. The pressure of aquei)us \'upt)ur is constant for a given tem- perature, whether it is in vacuo or mixed with a gas or gases, and varies directly, as has aliead\^ been stated, as the temperature. Two other types of problem arise in connection with this subject — namel3', (i) finding the weight of a volume of air saturated with vapour at a given temperature and pressure; and (2) finding the weight of a volume of air partially saturated with vapour at a given temperature and pressure. Example i. — Find the weight of a cubic foot of air saturated with aqueous vapour at 62° F. and 30 inches Hg. By the table of vapour tensions it is seen that 62° F. corresponds with 0-556 inch Hg. As the total pressure of air and vapour is 30 inches, the pressure exerted by the air alone must be 30-0-556, or 29-444 inches. The problem, therefore, resolves itself into finding the weight of a cubic foot of dry air at 62° F. and 29-444 inches, and that of a cubic foot of aqueous vapour at 62° F. and 0-556 inch. 491 29-444 — X -^^ X 566-86 = 524-32 grains (weight of dry air). ^x ^- X 566-86 X 0-622= 6-16 grains (weight of aqueous vapour) . . '. the cubic foot of saturated air = 524-32 +6*i6 grains= 530-48 grains. Example 2. — Find the weight of a cubic foot of air partially saturated with aqueous v^apour at 62° F. and 30 inches, dew-point being 50° F. The dew-point is the temperature of complete saturation of the atmosphere. If the atmosphere be raised in temperature, its capacity for holding aqueous vapour will be increased; if lowered, this capacity will be diminished. When the temperature is lowered below the dew-point, vapour is deposited in the fluid form. Vapour tensions in the above table correspond with temperatures of complete saturation or dew-points, hence, in problems of the tyjje A IR 129 under consideration, if the dew-point be not given, it must be found- This may be done directly by such instruments as Daniell's or Regnault's hygrometers, or indirectly by Glaisher's formula, or by Apjohn's formula. In the indirect method the wet and dry bulb thermometer are used. By Glaisher's formula the dew-point= D — G(D — W) where D = temperature of dry bulb, G = Glaisher's factor for reading of dry bulb, and W = temperature of wet bulb. By Apjohn's formula : For temperatures above 32° F. : / d h\ For temperatures below 32° F.: ^"^ V87'^30. \96^30/' Where P = pressure of aqueous vapour at dew-point. p = pressure of aqueous vapour at temperature of wet bulb. d = difference in degrees F. between dry and wet bulbs. h = height of barometer in inches. Returning to the problem, when the dew-point 50° F. has been found, the pressure of the aqueous vapour 0-361 inch is obtained from the table of vapour tensions. The problem is resolved as before into two portions — viz., the weight of dry air at 62° F. and pressure 30 -0-361 inches, and the weight of vapour at 62° F. and pressure 0-361 inch. 4Q1 20-630 ^^ „^ „ . 1^ X -^^ X 566-86= 527-8 grams. 491 0-361 ^^ „^ ^ - . T^ X -^^ — - X 566-86 X 0-622= 3-98 grams. 527-8 + 3-98=531-78 grains. Relative humidity represents the ratio between the weight of aqueous vapour present in a given volmne of air, and the weight of vapour which would be required to saturate the same volume of air under similar conditions of temperature and pressure, and is expressed as a percentage. 9 130 PRACTICAL SANITARY SCIEXCE Consider the last example, in which the temperature is 62° F. and the dew-point 50° F. : Relati\e humidity = pressure at 50° F. X — X — x 566-86 x 0-622 pressure at 50° F. pressure at 62° F. x ^ x _^ x 566-86 x 0-622 pressure at 62° F. 0-361 , , 0-361 X 100 ^ = f.;-Ff:', or expressed as a percentage, .^ — = 64-9 per cent. The composition of the air expired from the lungs contrasted with ordinary air demonstrates the invariable nature of the X and the limits of variation of O and COo. Ordinary Air. Expired Air. O - - - - 20-96 per cent. 16-4 per cent. N - - - - 79-00 ,, 79-0 ,, COo- - - - 0-04 ,, 4-6 The practically uniform composition of the air all over the earth is maintained by variations of temperature leading to variations of voliune and pressure, with resulting air-currents, diffusion of gases, the above-named circulation affected by respiration of animals, transpiration of plants, rain, etc. Oxygen is the most important constituent of the air, in that it is a prime necessity to life. Its quantity is diminished by respiration, putrefaction, combustions of all types, and at high altitudes. The estimation of O may be readily carried out in the following ways : I. The nitric oxide (NO) method. This method, although it has been adversely criticized, yields, in careful hands, excellent results. The reaction is represented b3^ the equation: 2XO + O0-2NO2. The NOg is soluble in water. There is a contraction of three volumes of the mixture for every one volume of 0, therefore one- third of the contraction represents the 0. To a sample of air in a gas burette excess of nitric oxide prepared from Cu turnings and HN0.j is added. The mixture is passed into AIR 131 an absorption pipette charged with water. The ruddy fumes of NO2 are rapidly absorbed, and after passing the gas backwards and forwards a few times the reading becomes constant. One-third of the contraction represents the O. Fig. 25. — Hempel's Gas Burette and Absorption Pipette. 2. Hempel's gas burette and absorption pipette. In the figure the mounted tube— the gas measurer— next to the bulbs is graduated into c.c's. and tenths; the other— the levelling- tube — is plain. The absorption pipette used is a double one, consisting of four 132 PRACTICAL SAXITARY SCIEXCE bulbs; the first and largest contains alkali and pyrogallic acid (dis- solve i6o grammes KOH in 130 c.c. water, producing about 200 c.c. of solution ; in this dissoh'e 10 grammes pyrogallic acid: if these pro- portions are not adhered to, evolution of CO may take place during absorption of O. and cause error) ; the second and fourth are empty; whilst the third contains water to seal off the atmosphere. The reagent absorbs O and COo- The graduated burette is supplied at the upper end with a stopcock and a sliort piece of fine pressure tubing (carrying a screw clip) which connects it with the small manometer U-tube of the bulbs. In order that this piece of tubing may be as short as possible, the bulbs are raised on a block, so that the end of the manometer-tube is near to the burette. The burette and the levelling-tube containing water are connected at their lower ends b}' rubber tubing. In making an estimation, first mark on the ivory slip the height at which the coloured liquid stands in the capillary U-tube, turn the stopcock so that connection is made between the burette and bulbs, then raise the levelling-tube until all the air is driven over out of the burette into the bulbs. Now connect the atmosphere with the burette, and lower the levelling-tube until a definite quantity of the particular atmospliere (say 25 or 50 c.c.) is admitted. Then make connection with the bulbs, and raise the levelling-tube until this quantity of air is dri\-en o\er into the absorption apparatus. Turn the stopcock off, screw down the clip, and unfasten the bulbs from the burette. Shake carefully for ten or fifteen minutes, re- unite with burette, and bring back the air by lowering the levelling- tube. Repeat these manipulations until a constant volume is obtained, when the liquid stands at the original mark in the U-tube and the burette is levelled. The decrease in volume is due to the O and COo absorbed. Deduct the CO2 obtained by Pettenkofer's method, and the remainder represents the O. This volume of O is then reduced to standard temperature and pressure. Since the temperature should not vary during the operation, the burette must not be handled. The absorption reagent in the first bulb, the water in the third, and the water in the burette, should all be saturated with air before commencing the estimation. It is to be noted that the pyrogallic solution will absorb besides other gases, such as H._,S, SOo, HCl, etc. AIR 133 3. Where accurate estimations are required, tlie combustion method of Dumas may be used. A measured volume of air is drawn through KOH to free it from CO2, and thence over ignited spongy copper in a combustion-tube. The copper fixes the O, and the amount of the Jatter is estimated from the difference in weight of the copper and copper oxide. Carbon Dioxide.^ — Carbon dioxide may vary in an atmosphere from 0-2 to 07 or o-8 per cent. The quantity ordinarily found in a pure atmosphere ranges from 0-035 to 0-04 per cent. The atmosphere of London during a fog often contains o-o8 per cent. In a living-room lighted by coal-gas the COg may reach 0-2 per cent., with an appreciable amount of CO. Carbon dioxide arises from (i) animal respiration; (2) combus- tion of all kinds of fuel; (3) organic combustion in the form of putrefaction, fermentation, etc. Its special significance lies in the fact that as a product of respiration, it can be made a fairly accurate measure of the organic impurities which accompany it. Carbon dioxide per se, in the quantities commonly found, may be considered harmless. It is generally agreed that the amount furnished by respiration may not exceed 0-02 per cent. Taking 0-04 per cent, as the average quantity found in the air, o-o6 per cent. (o-02 -f 0-04) will represent the limit of CO2 allowable in any atmo- sphere contaminated by respiration. The number of cubic feet of fresh air required to dilute the CO2 of a room, so that this limit may be preserved, will be found b}^ the formula : cubic feet CO2 added x 100 0-02 The quantity of COo added to the air through respiration is, roughly, o-6 cubic foot per head per hour. Substituting this figure in the formula, it is found that 3,000 cubic feet fresh air per head per hour must be admitted to living-rooms if the CO2 is to be kept within the limits named. The Estimation of CO.^ in the Atmosphere- — Pettenkofer's Method. — When CO2 is shaken up with barj/ta water (Ba(0H)2), insoluble BaCOg is formed, and the alkahnity of the fluid is lessened. Take a 5-litre air-jar, cleansed and filled with water, into the r34 PRACTICAL SAX IT A RY SCIENCE apartnit-nt in wiiich tlie estimation is to be made. Pour out the water so that the air may enter the jar, and stopper carefully. Prepare baryta water by adding about 5 grammes Ba(0H)2 to a htre of distilled water, and accurately estimate, in terms of standard oxalic acid solution, the alkalinity of 25 c.c, using phenol- phthalein as indicator. The acid is prepared by dissolving 2-82 grammes of the crystals in a litre. This solution is of such strength that I c.c. is equivalent to 0-5 c.c. CO., at standard temperature and pressure. Now add 50 c.c. of the clear barium hydrate solution to the contents of the jar, and roll it round the interior for some time. When, in say twenty minutes, the whole of the COo is absorbed and neutralized, take out 25 c.c. of the solution with a pipette and rapidly titrate it with the standard oxalic acid, delivered from a burette. The difference in alkalinity of this and the original 25 c.c. multiplied by 2 is equivalent to the CO2 in the jar in c.c. at N.T.P. Reduce the volume of air in the jar to X.T.P., and calculate the percentage of CO.2 on this. The following is an example: Temperature 15- C, pressure 750 millimetres. Twenty-five c.c. of the freshly prepared Ba(0H).3 were measured by pipette into a porcelain basin, a few drops of phenolphthalein added, and standard oxalic acid run in until the pink colour just disappeared after thorough stirring; 21-5 c.c. of the standard acid were used. Fiftv c.c. Ba(0H)2 were run into the jar, and after complete absorption of the COo had taken place, 25 c.c. were removed and titrated with acid : 19-9 c.c. standard acid were used. 21-5 — 19*9 = 1-6 c.c; and i-6 c.c. x 2= 3-2 c.c. = the total amount of acid equiva- lent to the CO., in the jar. But each c.c. of acid=o-5 c.c. CO2; therefore 3-2 x 0-5= i-6 c.c, the volume of CO2 in the jar, or i-6 c.c in 4,950 c.c. (5,000 c.c. -50 the volume displaced by the Ba(0H)2). The volume of this 4,950 c.c. at 0° C. and 760° millimetres = 4950 X 750 760 X {i +(0-0036 X 15)} (in the C. scale ^|.y or 0-0036= coefficient of expansion of gases per degree) = 4,635 c.c. 1-6 c.c. COo in 4.635 c.c. air= 0-03 per cent. AIR 135 Baryta water is best prepared fresh, but if it must be kept, it should be stored in a vessel shut off from the atmosphere by a hollow tube filled with pumice moistened with KOH. Ba(0H)2 has a slight action on glass, but any error that might arise through liberated alkalies is so infinitesimal that it may be neglected, especially when a jar has been used a few times. Angus Smith's ' household test ' consists in running ^- ounce of clear lime water into a 10 1- ounce bottle. No turbidity will be found so long as the CO2 in the air does not exceed the limit allowed- — viz., o-o6 per cent. Lunge and Zeckendorf's Method.- — A bottle of 70 c.c. capacity and an india-rubber pump of the same capacity are connected so that air can be pumped into the bottle. A weak standard solution of NaOH is prepared and tinted with phenolphthalein, by adding 2 c.c. ^Q NaOH containing i per mille phenolphthalein, to 100 c.c. ammonia-free distilled water. The ball of the pump is squeezed until the bottle is filled with the air to be tested. Ten c.c. of the 5^0 NaOH are now placed in the bottle, and the stopper, through which the delivery tube of the pump and an exit tube pass, inserted. The ball is then gently pressed, causing air to bubble through the liquid, and the bottle is carefully shaken after each addition of air until the colour is discharged. The number of times the ball is emptied indicates the amount of CO2, according to the following table, compiled by Lunge from estimations made by Pettenkofer's method: 2 3 4 5 6 7 8 9 10 II 12 13 14 15 Per Cent. 0-3 16 0-25 17 0-21 18 o-i8 19 0-155 20 0-135 22 0-II5 24 o-io 26 0-09 28 0-087 30 0-083 35 o-o8 40 0-077 48 0-074 Per Cent. 0-071 0.069 0-066 0-064 0-062 0-058 0-054 0-051 0-049 0-048 0-042 0-038 0-030 136 PRACTICAL SANITARY SCIENCE When, from respiration, CO., rises above o"o6 per cent., a certain unpleasant odour is experienced in rooms, due to the accompanying organic exhalations, and when much above this figure headache and even faintness may super^•ene. Volatile fatty acids exhaled from the skin and H^S are responsible for most of these unpleasant odours. A cubic foot of coal-gas yields on combustion o-6 cubic feet COg. It is obvious that when COo is due solely to the combustion of coal-gas the quantity ma}' be allowed to exceed considerably the above-named limit. Carbon Monoxide. — This odourless gas possesses a special affinity for haemoglobin, displaces oxygen from it, and thus destroys the ox\'gen-carrying function of the blood and ultimately life, by cutting short internal respiration. When haemoglobin is saturated to the extent of 30 per cent., symptoms of poisoning set in, and 70 per cent, saturation is fatal. Coal-gas contains by volume about 6 per cent. CO, and when imperfectly burnt leaves small quantities in the flue ; but greater danger attaches to the escape of the gas from ill-con- structed taps and joints. The use of coke, especially in cast-iron stoves, is a fruitful source of CO. As CO^ passes over hot coke it is reduced according to the equation C -i-C02= 2CO. The carbon of the hot cast-iron acts in the same manner, reducing COo to CO. Solid particles of organic matter floating in the atmo- sphere become charred on the exterior of the stove, and this partial oxidation results in the formation of CO. This gas is present in tobacco smoke. The characteristic cherry-red colour of CO-hfemoglobin serves as an excellent test for the presence of carbon monoxide. If a few drops of fresh mammalian blood be diluted with water down to about 2 per cent., and the solution shaken up with CO, the distinc- tive colour is at once formed. If dilution be extended to 0-2 per cent., and HbCO fomied by shaking with the gas, the characteristic spectrum consisting of two bands between D and E occupying nearly the same position as those of HbOa, but differing in that they do not disappear on the addition of reducing agents such as (NH^loS or H.,S, may be readily seen. HbCO may also be distinguished from HbOj by adding to 10 c.c. of the blood solution 10 to 15 c.c. 20 per cent, solution K4Fe(CN)g AIR 137 and 2 c.c. acetic acid (i volume acetic acid + 2 volumes H^O). A reddish-brown = HbCO ; greyish-brown precipitate = HbOg. The estimation of CO in the air may be performed by (i) Haldane's haemoglobin percentage saturation method, or (2) by the cuprous chloride method for large quantities. I. The following is Haldane's account of his method: ' A solution of about i of normal blood to 100 of water is made; also a solution of carmine dissolved with the help of a little ammonia, and diluted till its depth of tint is about the same as that of the blood solution. Two test-tubes of equal diameter (about i inch) are then selected. Into one of these 5 c.c. of the blood solution are measured with a pipette; into the other about an equal quantity is poured. Ordinary lighting gas is then allowed to blow into the second test-tube through a piece of rubber tubing for a few seconds. The test-tube is then quickly closed with the thumb before the gas has time to escape, and the blood solution thoroughly shaken up with the gas for a few seconds. The haemoglobin is thus completely saturated with carbonic oxide, and the solution has now the char- acteristic pink tint. The carmine solution, which has a still pinker tint, is now added from a burette to the 5 c.c. of normal blood solution in the other test-tube until the tints are the same in the two test-tubes. Not only, however, must the tints be equal in quality, but they must also be sensibly equal in depth. If the carmine solution is too strong or too weak, the latter will not be the case, and the solution must be diluted or made stronger accord- ingly. It is usually easiest to make the carmine a little too strong at first, so that on adding both cannine solution and water equality can be established. From the amount of water which is required to be added it is easy to calculate the extent to which the original carmine solution needs to be diluted. The solutions are now ready for use, and the actual analysis is made as follows: 5 c.c. of the solution of normal blood are measured into one of the test-tubes, and a drop of the suspected blood placed in the other test-tube and cautiously diluted with water till its depth of tint is about equal to that of the normal solution. If carbonic oxide be present in the haemoglobin, a difference in quality of the tints of the two solu- tions will now be clearly perceptible. Carmine solution is then added from the burette to the nonnal blood, and water (if neces- 13S PRACTICAL SAXITARY SCIEXCE san') to the abiiDrinal bluod, till the tints arc equal in both quality and depth. The carmine is added by about 0-2 c.c. at a time, the points being noted at which there is just too httle and just too much carmine, and the mean being taken. The solution of normal blood is then saturated with coal-gas, and the addition of carmine to the other test-tube continued until equality is again established and the amount of carmine noted. The percentage saturation with carbonic oxide of the abnormal blood can now be easily calculated, since we know how much cannine solution its saturation represented as compared with what complete saturation represented. ' The method of calculation is illustrated by the following ex- ample: To 5 c.c. of normal blood solution 2-2 c.c. of carmine is required to be added to produce the tint of the blood under examina- tion, and 6-2 c.c. to produce the tint of the same blood fully satu- rated. In the former case the carmine was in the proportion of 2-2 in 7-2, andin the latter of 6-2 in 11-2. The percentage saturation (.v) of the haemoglobin with carbonic oxide is thus given by the following proportion sum : 6-2 2-2 : — : : 100 : x. 1 1 -2 y-2 X is therefore = 55-2. As the compound of carbonic oxide and haemoglobin is, to a slight extent, dissociated when the blood is diluted with water, the value found is a little too low. The cor- rections needed are as follows: Add 0-5 if 30 per cent, saturation be found, i-i if 50 per cent., i-6 if 60 per cent., 2-6 if 70 per cent., 4-4 if 80 per cent., lo-o if go per cent. Thus, in the above example we must add 1-3, so that the true saturation is 56-5 per cent. In comparing the tints, the test-tubes should be held up against the light from a window, but bright light should be avoided as much as possible, as it increases the dissociation. Failing daylight, an incandescent burner, with a chimney of blue glass and an opal globe, may be used as the source of light. ' Haemoglobin brought into intimate contact with air containing 0-07 per cent, of CO will finally reach a state of equilibrium in which it is saturated to an equal extent with CO and oxygen. If the percentage of CO or oxygen in the air be increased or diminished, there will be an exactly corresponding increase or diminution of AIR 139 the relative share of the hiemoglobin which cither gas detains. Air containing 2 x 0-07= 0-14 per cent, of CO will, for instance, produce two-thirds saturation with CO, and one-third saturation with oxygen, and so on. In the living body the proportion of CO taken by the hgemoglobin from respired air containing a given percentage of CO is not so large as outside the body, about i per cent, of CO in the air breathed being necessary to produce half saturation of the haemoglobin. The general law of absorption is, however, much the same, and it follows that there is a certain maximum of satura- tion for each percentage. With less than 0-05 per cent, of CO in the air this maximum does not exceed 33 per cent, saturation, and the corresponding symptoms are scarcely appreciable, except on mus- cular exertion. With more than about 0-2 per cent, the maximum exceeds 60 per cent, saturation. ' The detection and determination of small percentages of CO in the air was formerly a matter of great, and often almost insuper- able, difficulty. I have recently, however, introduced a simple and, I think, very satisfactory method, depending on the already described action of CO on blood solution in presence of air. The sample of air is collected in a clean and dry bottle of about 4 ounces capacity. The cork of the bottle is removed in the laboratory under a 0-5 per cent, solution of blood, and about 5 c.c. of the air allowed to bubble out, a corresponding volume of the blood solution entering. The cork is then replaced, covered with a cloth to keep off the light, and shaken continuously for about ten minutes, when the haemoglobin will have reached the point of saturation corre- sponding to the percentage of CO present. The solution is then poured out into a test-tube, and the saturation is determined with carmine solution in the manner described above. It is evident that as in each case the saturation found corresponds to a definite percentage of CO in the air, it is easy to calculate this percentage. If p be the percentage required, and s the percentage saturation found, p is calculated from the following formula: 5x0-055 100 — s " Thus, if s= 60, p is 0-0825. This method may also be used for the direct determination of carbonic oxide in lighting-gas. The latter 140 PRACTICAL SAXITARY SCIENCE must, liowever, be lirst diluted to ^}j^, (or with carburetted water- gas to jI^) with air. As it is quite easy to make this dilution with perfect accuracy, the method is an exact one, and is not only rapid, but a\-oids the difficulties and sources of error connected with the ordinary method of determination by cu})rous chloride, or by ex- plosion.' 2. The cuprous chloride method. Cuprous chloride is prepared from copper turnings, copper oxide, and strong HCl. and dissolved in distilled water. This solution absorbs CO. The air to be treated is lirst freed from and COg by passage through Hempel's burette. The residue is slowly and repeatedly passed into a second absorption pipette containing cuprous chloride in solution. The bulb containing the copper salt should be large, the time for absorption long, and the transference from burette to pipette and vice versa as often repeated as necessary to procure a constant reading. The loss in volume, assuming that ethylene, acetylene, etc., are absent, represents the CO present. This method is by no means reliable. Ammonia is found in traces in all atmospheres. It is a product of putrefaction, and although in small quantities it seems to be harmless, it should be regarded with suspicion, b}- reason of the noxious bodies which accompany it. It is found in larger quantity in air in immediate contact with peat. It may be collected and estimated by aspirating a know^n volume of air through ammonia- free distilled water, and aftenvards distilling and Xesslerising. Sulphur Dioxide, Ammonium Sulphide, and Sulphuretted Hydrogen are all present in the atmospheres of cities, and are hurtful to health and vegetation. Sulphur dioxide abounds w^here impure coals are consumed, and HgS where organic decomposition takes place. It is stated that 0*06 per cent. H2S in an atmosphere is dangerous to life, and fatal accidents in scw^ers have been attributed to this gas. SO2 may be estimated by aspirating a large and known volume of air through bromine water, and precipitating the H2SO4 thus formed with BaCU- From the weight of the insoluble BaS04 ob- tained the weight of SO., is calculated. Sulphuretted Hydrogen may be detected by exposing to the air AIR 141 strips of filter-paper moistened with lead acetate, and estimated quantitatively by aspirating a known volume of air through a solution of decinormal iodine containing a little starch paste. Im- mediately the blue colour departs the aspiration is stopped. 17 milligrammes HgS correspond with i c.c. ^ij ^^ H2S + l2=2HI+S. Ammonium Sulphide. — The violet colour produced by the inter- action of (NH4)2S and sodium nitro-prusside may be utihzed for matching a standard solution with another containing an unknown quantity of (NH4)2S. Chlorine may be absorbed in 10 per cent. KI solution, and the liberated I estimated with y^ sodium thiosulphate. Bromine may be estimated in the same way: 2C1+2KI=2KC1+I2. 3-5 parts by weight Cl= i2-6 parts I, or 7-9 parts bromine. Nitrous, Nitric, and Hydrochloric Acids may be estimated by absorption of measured quantities of air in water, and employing the methods described in water analysis. Carbon Bisulphide. — The vapour of CS2 found in the air of india- rubber works is estimated by passing it into strong alcoholic potash. This solution is then acidified with acetic acid, and finally neutralized with CaCOg. It is now diluted to twice its volume with water and titrated with standard iodine solution (i-66 milligrammes I per litre) and starch paste. One c.c. 1= i milligramme CSo- The reaction is complete when a faint blue tint appears. Chlorine and bromine are injurious to human beings in dilution of o-i part per 100,000, and the following as noted: Iodine _ _ _ _ _ 0-5 part per 100,000. SOaandHCl - - - - i-o „ H2S and NHg _ _ _ . lo-o parts per 100,000. CO ----- - 20-0 ,, Ozone. — Ozone, an allotropic modification of oxygen, O3, is a gas possessing an odour of phosphorus and an irritating action on the cells of the respiratory and conjunctival mucous membranes. It is produced by electric discharges over the sea, and to a greater extent at night than in the day. It is stated that more ozone is found in 142 PRACTICAL SAXITARY SCIEXCE tlie winter (especially after snowstorms) tluiii in tlie smnnier. It is absent from the air of towns, li\'ing-rooms, and foggy atmospheres. Detection and Esti^nation of Ozone. — Pieces of blotting-paper are soaked in a solution of KI and starch, and dried. These are then suspended in a cage, which protects them from direct sunlight, dust, and rain for twelve or twenty-four hours; where ozone is present, it hberates I, which forms a blue colour with the starch. O3-I-2KI + H2O = 2KOH + I0+O0. It should be remembered that N2O.5, HoOo, and CI act in the same way; that free iodine may be partially volatilized, or in part form iodide or iodate of potassium, instead of blue iodide of starch; and that constant results cannot be expected owing to the variability in the conditions of temperature, light, and moisture. Houzeau's test consists in moistening faintty red litmus-papers with a solution of KI and exposing them to the air. If ozone be present I is liberated, and alkaline KOH is formed, which renders the paper blue. Ammonia and hydrogen peroxide are the only other two gases which could produce this result. As H2O2 is practically never present, NH3 is the only other gas to be con- sidered. If, therefore, a second piece of litmus-paper untreated by KI is exposed at the same time, and if the entire colour is not due to XH3, the difference in the shades of the two papers must be furnished by ozone. The intensity of colour created by ozone acting on papers exposed to the atmosphere may be matched by one of a series of ten papers forming a standard scale. Each pair of papers is exposed to a known quantity of ozone. Measured quantities of air are aspirated over the papers in tubes. If the papers are suspended in the atmo- sphere, wind currents, etc., by bringing unequal quantities of air into contact with them, will vitiate the results. Hydrogen Peroxide.^ — Aspirate 20 to 100 litres of air containing HoO, through 100 c.c. distilled water. To 10 c.c. of the water add I drop of a I per cent, potassium chromate solution, 2 or 3 drops of 25 per cent. H2SO4, and 2 c.c. of ether. Shake gently for some time ; perchromic acid is formed and goes into solution in the ether, rendering it blue. Phosphoretted Hydrogen. — When grades of ferro-silicon rich in silicon (40 to Go per cent.) are exposed to water or damp air, a AIR 143 reaction takes place between calcium j)hosi)liide, Ca.jF^, an impurity, and the water, resulting in the production of PH,;, and sometimes AsHg. The presence of PH3 is detected by aspirating the air containing it over two sets of filter papers — (a) moistened with a solution of AgNOg, and {h) moistened with a solution of Pb(C2H302)2- The nitrate of silver only is darkened, no action taking place between the gas and lead acetate. As HgS darkens both papers, it is well to remove it before testing for PHg; this can be readily accomplished by aspirating the air through a solution of lead acetate. Suspended Matter in the Air. — Sol^id animal, vegetable, and mineral particles float in the atmosphere, and tend to settle on objects as favourablje conditions occur. In factories and work- shops the amount of such matter may be so great as to be positively dangerous to health. Pathogenic micro-organisms adhere to dust and are carried with it. The collection and microscopical examination of dust is effected by aspirating large quantities of air over gelatin, etc., or through water. In the first case the microscope will detect mineral and dead organic matter, and where living bacteria are present these will grow and produce colonies, which can later be subcultured and studied at length. In the second a few drops of the water are evaporated on a slide and the sediment microscopically studied. Pouchet's aeroscope is a simple instrument in which known volumes of air are aspirated over a drop of glycerin on a micro- scopic slide; the intercepted particles are afterwards studied microscopically. The dust in the atmosphere of towns commonly exceeds 10 milli- grammes per cubic metre, or from 10,000 to 200,000 particles per c.c. On the top of a lofty mountain there may be no dust, or only a few particles per c.c. As to the nature of the particles forming dust, it is sufficient to say that they are derived from every conceivable substance with which we have to do that is capable of existing in particulate form. The most important substances, from a health point of view, are solid particles capable of irritating the various internal channels in man, and bacteria and their spores. Sewer air contains less oxygen and more CO2 than that of the 144 PRACTfCAL SAXITARY SCIENCE atmosphere. Ammonia, ammonium sulphide, and various com- pound ammonias emitting f(ttid odours, sulpliuretted hydrogen, and mairsli-gas, are present in ever-varying quantities. Ground air is very rich in COg, especially in the autumn season of the year. Ground air should be excluded from all living-rooms, not only because of its own impurity, but because where it is allowed entrance, other more dangerous gases, such as coal-gas, sewer-gas, etc., may often enter too. A sample of ground air may be collected for examination thus: A hollow, sharp-pointed steel cylinder, with many perforations, is pushed into the soil for a distance of 4 to 6 feet. The upper end of the cylinder is connected with an air-jar, and this in turn with an aspirator. The jar being shut off from the cylinder, is lirst emptied by the aspirator ; connection is then made, and the sample collected. Besides COg, which may reach 5 or 6 per cent., small quantities of XH3, CH4, H2S are usualty found. Qualitative Examination of Air for Noxious Gases in Large Amounts. Where the air of factories, etc., contains noxious gases, qualitative examination is readily performed by aspirating large quantities of the air through pure water or other suitable solvent. Where, how- ever, the gases are in considerable quantities, tests may be applied direct to samples of the air in jars. Occasionally the atmosphere surrounding chemical works, etc., contains such large quantities of CI, HCl, SOo, etc., that this direct method of examination may be adopted. The following gases may be readily recognised by a few simple chemical tests : HCl, CO.,, N2O3. HNO3, H^S, SOo, CI, CO, CS2, NH3, (NH4)2S. I. Having collected a sample in an air-jar, remove the stopper and smell the gas. Replace the stopper quickly. CI has a charac- teristic odour. HCl has a faint odour of chlorine. SOo has a characteristic odour, so also have NH3, (NH4)2S, HgS, CS,. CO2, CO, X2O3, HNO3 have no odours. AIR 145 2. Take the reaction by moistening a red and blue filter-paper with water and rapidly inserting them in the jar, fixing the ends between the neck and the stopper. If doubt exist as to the effect on the litmus-papers, the reaction may again be taken when the gas is dissolved in a small quantity of distilled water. HCl, COo, N2O3, HNO3, SOo are acid. NH3, (NHJgS are alkaline. HgS, CO, CS2 are neutral. CI first reddens blue litmus-paper and afterwards bleaches it. 3. Dissolve the gas in 10 c.c. of water by vigorous shaking, and if the reaction be acid, to 2 or 3 c.c. of the solution add a drop or two of AgNOg solution. A white precipitate indicates [a] HCl. Acidity marked; precipitate marked and soluble in (NH4)H0; insoluble in HNO3. (6) COg. Acidity slight; precipitate slight. Addition of Ba(0H)2 produces turbidity, increased on further addi- tion of a drop or two of (NHJHO. (c) SOg. Odour characteristic; acidity marked; precipitate marked, soluble in HNO3. Two or three c.c. of the solution from the jar added to iodide of starch will decolourize it. If 2 or 3 c.c. of the same solution be heated with a drop of HCl, a granule of Zn,H2S will be formed, which will darken lead acetate paper. [d] No precipitate, HNO3. Peiform the brucine test; also the diphenylamine test. {e) No precipitate, NgOg (now HNOg). Test for nitrous acid with KI, starch, and H2SO4; and perform the meta- phenylene-diamine test. 4. If the reaction be alkaline, the gas is either [a) NH3. Odour characteristic. To 2 or 3 c.c. of the solution from the jar add a drop or two of Nessler's reagent, and the well-known yellow colour is developed. Or [b) (NH4)2S. Odour characteristic. Nessler's reagent causes a t)lack colour when mixed with the solution from the jar. To a few c.c. add a drop or two of sodium nitro- prusside, and a violet colour rapidly appears. 5. If the reaction is neutral, one or other of the following is present : {a) HoS. Odour characteristic. Lead acetate paper is dark- ened. Solutions of salts of iron, lead, and copper pro- duce the dark-coloured sulphides of these metals. 10 146 PRACTICAL SAXITARY SCIENCE [b) CSo. A liquid at ordinary temperatures. Set alight a drop on a porcelain slab, and note the yellow deposit of sulphur left behind. 6. The only gas which lirst reddens blue litmus-paper and then slowly bleaches it is CI. Odour characteristic. Suspend a moist KI pa])er in the jar. Free I will be liberated and darken the paper; later the darkened paper will be bleached. Chlorine added to a mixture of ferrous sulphate and potassium sulpho- cyanide produces a red colour. Note the differences between H^S and (NH4)2S. HoS has a neutral reaction, odour of rotten eggs only, and forms no colour ^\•ith nitro-prusside of sodium. (XH4)2S has an alkaline reaction, odour of rotten eggs and NH3, and produces a violet colour with sodium nitro-prusside. 7. CO is distinguished by absence of odour, no reaction \\itli litmus, and by the characteristic colour and spectrum when shaken with blood. Bacteriolog'y of the Air.^ — The number of micro-organisms in the air is largely determined by the quantit}' of dust particles in it. Bacteria adhere to and are carried by dust particles; the types found in air are for the most part chromogenic saprophytes, yeasts, and spores of moulds. The number varies with the altitude, date, and amount of recent rains, and other factors. Numerical detei'- mination is of ser\-ice as a means of comparing methods of ventila- tion. Gordon, in his report on the ventilation of the House of Commons, 1906, states that in the dust of the chamber there were present per gramme: Streptococci, 10 to 1,000; B. enteritidis sporo- genes, 1,000 to 10,000; B. coli, 1,000 to 10,000; total number of bacteria, 100,000 to 1,000,000. Haldane found the number of bacteria in the air of book-binding workshops per litre 6, cloth factories 11, tailoring workshops 12, ropemaking premises 327. Andrewes has shown in his reports to the Local Govemment Board that in certain circumstances characteristic sewage bacteria are found in the air of drains and sewers. He has carefully studied the characters of the organisms found in drain and sewer air: B. coli oi drain air corresponds in characters with the same organism as AIR 147 found in sewage. He considers that splashing produces droplets so minute as to be carried some distance in the air, and that through these bacteria are conveyed. He concludes that tlie number of faecal bacteria in drain air is largely proportional to the faecal content of the sewage. The original methods of enumerating bacteria in the air designed by Pasteur, Koch, and Hesse are not in use to-day. Modern methods are of two types: (i) Those based on filtration; (2) those based on bubbling air through a suitable liquid. It is well to sow both agar and gelatin plates, as in most instances gelatin liquefies in a short time. Some form of aspirator is used, and where the air passes through a liquid, care must be taken that the air passes slowly and regularly, in order that the bubbles may burst one by one. 1. Filtration Methods. — Petri used a sterile wide tube containing alternate segments of wire gau:^e and fine sand. When aspiration was complete, the sand was mixed with sterile gelatin, and plates poured. Frankland substituted for sand glass-wool or asbestos. After aspiration the filtering medium was shaken up with broth, and with this gelatin plates were sown. But these insoluble filtering media are now displaced by soluble media. Sodium sulphate is fused, powdered, sifted, and introduced into a glass tube, one end of which is drawn out and sealed in the flame, and the other end plugged with wool. The whole is sterilized in the hot-air sterilizer. When about to be used, the pointed end is broken off, and the other plugged and connected to an aspirator. When aspiration is finished the powdered sulphate is dissolved in a measured volume of broth, and plates are sown with known quan- tities of the liquid. A mixture of glass-wool and one-third its weight of cane-sugar is used as a filtering medium in much the same manner. 2. Bubbling Methods.- — Miguel used a Pasteur flask with two side tubulures — one drawn out and sealed, the other plugged with wool. A small measured quantity of water was placed in the flask, and the whole sterilized in the autoclave. Aspiration of air through the water was slowly effected, and when a sufficient quantity had passed through, the sealed tubulure was broken, and measured quantities of the water sown in media, and the latter incubated. Laveran uses two glass tubes connected at the junction of 148 PRACTICAL SAXITARY SCIExXCE their upper and middle thirds by a bridge tube. Each of the up- right tubes is plugged with an india-rubber stopper carrying a pipette which reaches to the bottom of the tube. The pipettes are plugged abo\-e with wool. One pipette is graduated in tenths of a c.c. The tube carrjang the other pipette has a 10 c.c. mark on the glass. Ten c.c. of a i per cent, solution of sugar in water are placed in this tube, and the apparatus is autoclaved. When about to use, remove the plug from the pipette which dips in the sugar solution, and connect the other pipette with an aspirator. The aspirated air bubbles through the solution, into the first tube, through the horizontal connecting-tube, down through the second tube, and passes out through the pipette connected \\'ith the aspirator. When sufhcient air has bubbled through, gently aspirate the sugar solution into the entry pipette to wash it; then run the liquid through the connecting-tube into the second upright tube, and so into the second and graduated pipette ; repeat this several times so as to collect all the bacteria that have been caught on the glass. Now, by the graduated pipette, distribute the liquid into the various culture media. This method is suitable for large volumes of air, and supplies plenty of material for sowing cultures. It is thus one of the best methods for detecting pathogenic bacteria. Suppose 250 litres have been aspirated and 20 colonies have grown on a gelatin plate so\\ti with i c.c. of the sugar solution: 20 X 10 X -^-:~ =Soo = nmnber of aerobic organisms contained in a cubic metre of air (1,000 litres =1 cubic metre). A simple method which may be made by careful manipulation fairly accurate is that of plate exposure : Pour Petri plates of gelatin and agar. When solid, expose them to the air under examination by removing their covers for selected periods — say fifteen to thirtj' minutes. At the end of the period replace covers and incubate. When organisms have developed, count and calculate to units of area and time — say per square foot per minute (area of a Petri dish = iry^ where r is the radius). If necessar}-, the various subcultural methods may be resorted to for the identification of individual species. CHAPTER XI FOODSTUFFS MILK. Since the milk of the cow is used to a much greater extent than that of any other mammal, its composition and properties have been much more thoroughly studied. Its liabiHty to early decom- position and the fact that it forms an excellent culture medium for bacteria render it necessary that the strictest attention should be paid to its production, collection, and distribution: Composition of cow's milk: Per Cent. 8775 Water Proteins Lactose Fat Ash 3-50 4-60 3-40 075 Our knowledge of the pr oteins of milk is still very incomplete The application of ordinary and crude chemical methods to the investigation of vital products necessarily leads to unsatisfactory results. The preparation of pure proteins is a most difficult task, and the probabilities are that in many cases where it is thought that a pure product has been isolated it is contaminated by re- agents. The proteins of the milks of different animals vary^ considerably. On the addition of an acid to cow's milk or goat's milk, a curd or clot composed of casein is formed, and it is believed that in these cases the casein is chemically combined with the phosphates of the alkaline earths. In human milk and the milk of the ass and mare no such clot is produced on the addition of acid. Here it is believed that the protein is not combined with phosphates. Besides casein, a second protein (lactalbumin) is found in all milks. Storch de- 149 150 PRACTICAL SAXITARY SCIEXCE scribes a miico-protoin which he holds forms a gelatinous envelope round the fat globules. Amylolytic and proteolytic ferments are said to occur in milk. The jirotein molecule is highly complex, as e\-idenced by its indiffusibility. Through the action of enzymes in the presence of acids and alkalies these complex bodies are hydrolysed, passing through various intermediate stages (varieties of albumoses) into diffusible peptones. Further hydrolysis produces amino-acids. The number of proteins in the milk of the cow has been variously stated. Duclaux maintains that there is only one — casein, existing in two forms, coagulable casein and non-coagulable casein. Ham- marsten describes two- — casein, corresponding to Duclaux's coagu- lable casein, and lactalbumin, corresponding to Duclaux's non- coagulable casein. This observer admits that lactalbumin has the properties of a true albumin, and closely resembles serum albumin; but holds that, owing to differences in certain physical constants, it is a distinct body. Hammarsten's casein and Halliburton's caseinogen are doubtless the same body. Sebelein describes a globulin in milk. Casein. — \\'hen pure, this is a white, non-crystalline, odourless, and tasteless substance, insoluble in water, weak acids, alcohol, and ether. It is soluble in stronger acids and weak alkalies. It appears to possess a peculiar affinity for calcium phosphate as it is almost, if not quite, impossible to free it from this salt. Casein contains less sulphur than either globulin or albumin, but much more phos- phorus. In solution in weak alkalies it is Isevo-rotatory on polarized light. Bechamp holds that it is a weak dibasic acid, forming two types of salts, and his view is confirmed by S51dner. This body is readily prepared by diluting milk about five times and adding acetic acid until the solution contains o-i per cent. The precipitate formed carries down the fat with it. This precipitate is well washed on a filter, dried by pressure, and dissolved in the least excess of am- monia. By this means the fat rises to the surface and the under- lying solution can be siphoned off. It is again precipitated by acetic acid, washed, dried, and redissolved in ammonia. After three or four such precipitations the casein is rubbed up with alcohol in a mortar. The alcohol is poured off, and the residue treated in the same manner with ether. It is afterwards extracted MILK 151 with ether in a Soxhlet apparatus to remove the fat. The treat- ment with alcohol and ether is repeated a number of times. It is finally dried at 100° C. If dried whilst containing water, it forms a hard horny mass. Soldner showed that two lime compounds exist (CaO and 2CaO) to one molecule of casein (C^7oH268N42SP05i). Lactalbumin. — This protein coagulates at 70° C, although tlu; precipitation is never complete. Like other albumins it is not pre- cipitated by saturating its solutions with MgSO^. It is precipitated like other albumins, by saturating its solution with Na2S04. Its rotatory power is [a]^ = - 67-5°. It can be prepared by saturating milk with MgS04, filtering, and adding to the filtrate acetic acid until 0*25 per cent, of the solution is reached, when lactalbumin is precipitated. It is redissolved in water, again saturated with MgS04, and reprecipitated with the same strength of acetic acid. This treatment is repeated three or four times. The solution of lactalbumin is next dialyzed to remove salts. When the salts have been got rid of, the solution is precipi- tated with alcohol and ether, and dried at a low temperature. The result is a tasteless white powder completely soluble in water. Lactogflobulin. — This protein is not coagulated by rennet, but is coagulated by heat and neutral sulphates. It occurs in very small quantities in milk, and it is doubtful whether it is distinct from serum globulin. Muco-Protein of Storch. — This body is insoluble in dilute am- monia and weak hydrochloric acid. It is partially soluble in the hydroxides of potassium and sodium, undergoing at the time of mixture considerable increase in bulk. It gives the character- istic protein reactions with the xantho-proteic and Millon's tests. On gently heating with dilute sulphuric acid it yields a reducing sugar. When washed with alcohol and ether and dried at the ordinary temperature of the atmosphere it forms a light grejash powder which is very hygroscopic. It may be prepared by centrifugalizing separated milk and wash- ing the deposit with weak ammonia-water. The resulting mass is then well washed with alcohol and ether, and dried. Or it may be prepared from cream. Storch has, by means of benzene, alcohol, and ether, separated it from butter. 152 PRACTICAL SAXITARY SCIEXCE Milk contains traces of extractives and colouring matters. Its characteristic white appearance is held to be due to the interference of light rays produced by casein in pseudo-solution, a state in which particles exist in the solution not of sufficient size to settle under gravity, but which interfere with the passage of light. These particles can be separated by a current of electricity. There is no sharp line of di\'ision between crystalloids and colloids in solution, substances in pseudo-solution, and bodies in suspension. In milk, fat is in suspension, casein in pseudo-solution, albumin in solution as a colloid, and lactose in solution as a crystalloid. The variety in size of the particles or masses of molecules probably determines tlie presence of one or other of these states in a given case. Lactose. — Lactose (CjoHojOj^HoO) is an aldose, and exhibits the constitution of a galactose-glucoside in that on hydrolysis by acids it produces a mixture of galactose and glucose. The aldehyde group of the galactose has been eliminated in lactose, whilst the glucose remains. Several modifications of milk-sugar are known, distinguishable from each other chiefly by their action on polarized light. Lactose, like other aldoses and ketoses, reduces alkaline solutions of CuSOj, forming cuprous oxide, the well-known Fehling's reaction. Each sugar effects a definite amount of reduction, and this affords an excellent method of distinguishing them. Lactose differs from other sugars in that its osazone forms an anhydride soluble in boiling water. Lactose is hydrolysed by a specific enzyme lactose found in certain torulfe, in some kefir preparations, and in aqueous extract of almonds. Lactose is not hydrolysed by maltose, invertase, or diastase. It easily undergoes lactic and but\^ric acid fermentations. Mineral acids hydrolyse it to glucose and galactose. It reduces Fehling's solution, and exhibits mutarotation. It is manufactured by evaporation of whey, the resulting crystals being purified by re- crystaHization. Fat of Milk.- — The fat of milk consists of a mixture of ethereal salts of glycerol, forming small globules ranging in size from O'OOi millimetre to o-oi millimetre. It is highly probable that there are three separate acid radicles combined with each glycerol group, thus : MILK 153 a compound of the acid radicles of butyrin, olein, and stearin with glyceryl. Milk-fat has the following composition: Per Cent Butyrin • 3-90 Caproin • 3-45 Caprylin 0-50 Caprin . 1-85 Myristin . 20-30 Laurin • 7-50 Stearin 2-00 Palmitin • 25-50 Olein • 35-00 In addition to the above fats, traces of certain extractives, such as urea, lecithin, cholesterin, together with colouring matters, exist in the fat of milk. The vexed question of the presence or absence of a definite mem- brane round the fat globule will not be discussed in this work. It may be stated in a word that Bechamp from his studies of the appearances found on mixing ether with milk and of the behaviour of milk towards certain stains has concluded that an endosmotic membrane exists ; whilst Storch by his observations is led to believe that instead of a definite membrane a muco-protein capsule encloses the fat globule and insensibly shades off into the surrounding fluid. Human milk has the following composition : Per Cent. Water . . 88-2 Fat -- 3-3 Casein i-o Albumin 0-5 Lactose . . 6-8 Ash 0-2 The fat globules are smaller than those of cow's milk, ranging from o-oog to 0-0009 millimetre. Its composition varies much more than that of cow's milk. It contains small quantities of citric acid. It is almost always alkaline. When milk is allowed to stand for a time, a series of well-known 154 PRACTICAL SAXITARY SCIEXCE changes succeed each other. The fat, the hglitest portion, rises to the surface as cream. After a variable period, depending on the temperature, presence of certain micro-organisms, and other factors, the milk becomes acid and separates into solid curd and liquid whey. The principal agent in this reaction is the B. lacticns, which converts lactose into lactic acid. Other micro-organisms, such as the B. hiUyricus, B. coli communis, etc., are also capable of fonning acid, and thereb}^ curdling milk. Rennet is used artificially for bringing about the same change. The curd consists of precipi- tated proteins with entangled fat, and the whey of water, lactose,, and salts. The cream of ordinary milk forms about lo per cent, by volume of the whole. The variations in the composition of milk, even from the same animal, are due to a number of factors, such as the health of the animal, the age — 3'oung animals secrete less milk and a product of poorer quality^ — the time that has elapsed from the last milking, the stage of milking, the breed of the animal, the time that has elapsed since previous parturition, the nature of the food eaten, etc. There are, however, limits to these variations, and all good milks at all times fall within these limits. Fatty solids may range from 2 to 7 per cent., non-fatty solids from 8 to 11 per cent., ash from 0-6 to 0-9 per cent., cream from 2 to 25 per cent., specific gravity from 1-027 to 1-037. But it is rare that the fatty solids fall below 3 per cent., and the non-fatty solids below 8*5 per cent., and these figures are insisted upon by law. Comparative analyses of various milks are represented in the following table: Water. Cow . . . . . . 87-7 Human subject . . . . 88-2 Goat . . . . . . 86-0 Mare 89-8 Ass . . . . . . . . 90-1 Ewe . . . . . . 79-4 In cattle-plague and foot-and-mouth disease marked changes occur in the milk of the animals affected. The quantity is dimin- ished, the curd separates out quickly on heating from a pale blue whey, and blood and pus corpuscles are generally present. In roteins. Fat. Lactose. Ash. 3-5 3-4 4-6 07 1-5 y^ 6-8 0-2 4-3 4-6 4-2 0-7 1-8 i-i 6-9 0-3 1-6 1-2 G-5 0-4 6-7 8-6 4-3 0-9 MIIJx 155 tuberculosis the milk is not markedly affected except in those rare cases in which the udder is extensively diseased. The Analysis of Milk. A chemical analysis is of service from a public health point of view in detecting the removal of fat, the addition of water, or both, and the presence of artificial colouring matters and preservatives. A bacteriological examination is often necessary in the investiga- tion of milk-borne epidemics — such as enteric, diphtheria, etc. — and for the detection of tubercle bacilli; to obtain evidence respecting the healthiness of the udder, etc., of the cow; and to measure the general bacterial content, and especially the degree of contamina- tion from fascal matter. For chemical analysis the milk must be fresh, as after standing for a time the lactose is transformed into lactic acid, and the non- fatty solids consequently diminished. Reaction. — The reaction is mostly alkaline, sometimes ampho- teric when litmus is used as an indicator. This is due to the pres- ence of NaH2P04 and Na2HP04, the first turning blue litmus red and the second red litmus blue. Estimation of Total Acidity — Lactic Acid. — Place loo c.c. of the milk in a beaker, add 5 c.c. of a o-i per cent, phenolphthalein solution, and titrate with ^ NaOH until a faint pink tint appears. It will be found that generally 20 c.c. of ~ NaOH is required; each c.c. of the decinormal alkali represents i degree of acidity. If litmus be used as indicator instead of phenolphthalein, a smaller figure will be obtained, as the salts of milk and carbonic acid are not sensibly acid to litmus-paper; for this reason litmus may be used in roughly determining the quantity of lactic acid. There is no good method for quantitatively estimating lactic acid. When milk is boiled, its acidity is diminished. Specific Gravity. — Specific gravity is the weight of unit volume, and may be determined in two ways: first, by finding the weight of a known volume, and second, by finding the volume of a known weight. The first method may be used by taking the weight of liquid which fills a vessel of known volume — example, specific- gravity bottle, or Sprengel's tube. This method may also be used 156 PRACTICAL SAXITARY SCIENCE b}' immersing a phmimet of known volume in the liquid, and noting the loss of weight due to the displacement of the same volume of liquid — example, Westphal's balance. The second method is applied by immersing a float of known weight in the liquid, and noting the volume immersed, which will be equal to a volume of the liquid of the same weight as that of the float — example, hydro- meters, of which the lactometer is a special form used in testing milk. In using the specific-gravity bottle, which is perhaps the most exact method, care should be taken that the bottle is clean. It is well to observe the ritual of subjecting the bottle to cleansing with weak acid, water, alcohol, and ether on each occasion before use, and to weigh it direct from a desiccator. The bottle is first weighed. It is then filled with milk, the stopper is gently let in, and its hollow channel is filled to the top with the fluid. Any superfluous milk is carefully wiped away with a clean and dr\' duster, and the bottle is again deposited in the desiccator for a short period before weigh- ing a second time. The temperature should remain constant and at 15-5° C. during the entire process. The second weight minus the first is equal to the weight of the milk contained in the bottle. This weight divided by the weight of the same volume of distilled water at the same temperature is the specific gravity. Most specific-gravity bottles have the weight of distilled water which they contain at 15-5° C. marked on their surface, so that it is unnecessary to take this weight. Taking the specific gravity of HoO at 15-5° C. as i-ooo, that of milk is about 1-032. It is obvious that the removal of fat which is the lightest constituent of milk raises the specific gravity, and its addition lowers it. The addition of water also lowers the specific gravity. So therefore a low specific gravity may mean either abun- dant fat or added water. The specific gravity of milk is observed to rise slightly for some hours after milking — e.g., a milk of specific gravity 1-031 when drawn from the cow may in ten hours show a specific gravity of 1*032; this rise is known as ' Recknagel's phenomenon.' If the quantity of cream as measured in a cream-tube reading percentages be the normal 10 per cent, after standing twenty-four hours, and the specific gravity be found low, it is clear that water has been added. MILK 157 - The Westphal balance consists of a graduated swinging arm resting on a knife-edge and a glass plummet suspended from a hook attached to one end of the arm. The other end of the arm is drawn out to a point which when the balance is adjusted and the plummet hangs in air should rest exactly opposite a similar point on the frame. Three riders are used on the graduated arm: their weights are wholly empirical, and indicate hundreds, tens, and units respectively. The milk or other fluid is poured into a glass cylinder; the arm is raised or lowered by means of a screw in the upright support until the plummet is just completely reversed, and the riders are so placed on various divisions of the scale that the points come to rest exactly opposite each other. Supposing that in an estimation the largest (as must be) is suspended from the hook carrying the plummet, the tenth division of the scale, and the tens and units riders rest on the scale divisions 3 and 2 respectively, when the point of the swinging arm comes to rest at zero the specific gravity will be 100 X 10 + 10 X 3 +1 X 2 =1-032. Lactometers, special forms of hydrometers, are less accurate in estimating specific gravities. The specific gravity of milk varies between 1-013 and 1-039. By removal of all the cream from a milk of specific gravity 1-032, the figure is raised to 1-036. On the other hand, by adding 4 per cent, fat to the same milk, the specific gravity is reduced to 1-028. The specific gravity test is not an absolute one, but a useful pre- liminary test. Like most substances, milk alters in specific gravity with change of temperature. It does not share, however, the peculiarity which water possesses of attaining its maximum specific gravity at 4° C. It decreases in specific gravity from freezing- point (-0-5° C.) upwards. Tables of corrections for temperature have been constructed when the determination is made at tempera- tures above or below 15-5° C; but it will be sufficiently exact to add or subtract i degree of specific gravity for every 6 degrees of temperature registered above or below 15-5° C. The Fat. — Of the many methods at present in use for the estima- tion of fat, the following two are to be recommended, and the first is preferable to the second: I. Adams's Process.- — In this gravimetric method the solvent 158 PRACTICAL SAXITARY SCIEXCE used for the extraction of fat is ether, convenient on account of its low boihng-point and heat of volatihzation, its high solvent power for fat, and its miscibilit\- witli water. When milk is dropped on blotting-paper, it spreads out to a much greater degree than when placed on glass or in a dish, and Adam MILK T59 considered that extraction of the fat by etli(;r would accord- ingly be much more complete. After passing through various stages of evolution, the process is now carried out somewhat as follows : A strip of Schleicher and Schiill's fat-free paper is hung up by one end. The other end is held in the fingers so that the surface of the strip is as nearly as possible horiziontal, and 5 c.c. of the sample of milk carefully measured in a pipette are distributed over the paper. The weight of this volume is determined by running into a convenient weighing vessel 5 c.c. of the same sample at the same rate as it was run on to the paper. The paper is allowed to hang until dry, and must be protected from flies and all other disturbing influences. When dry the paper is rolled up into a loose coil of a diameter such that it will easily pass into the Soxhlet extractor (say I inch). A blank coil containing no milk should be dried and rolled up in the same way, and both further dried at 100° C. for an hour. Each coil is then placed in a Soxhlet extractor, arranged in an upright position, and connected with vertical condensers. Small weighed flasks of 150 c.c. capacity containing dry ether in sufficient quantity to fill the extractor well a]>ove the upper portion of the siphon, are attached to the lower end of the Soxhlet apparatus, and the ether is made to boil by immersing the flask in water at 55° to 60° C. Extraction should be continued for two to three hours, although many analysts are satisfied with twelve to eighteen siphonings. The flasks containing ether and dissolved fat are then discon- nected, the ether is driven off by evaporation, and the flasks dried and weighed. The difference in weight represents the fat ; the small amount of extract derived from the paper of the blank experiment is finally subtracted from the weight of fat found for the sample, and the difference represents the fat contained in 5 c.c. The weight of 5 c.c. has been determined; accordingly the percentage of fat is readily calculated. Sour milk may be operated on if the acidity be neutralized by —Q NaOH, using litmus as indicator. It is advisable to put a small piece of blotting-paper in the mouth of the open tube at the top of the condenser so as to limit the i6o PRACTICAL SAXITARY SCIEXCE entrance of moist air which would shghtl\' wet the ether. In driving off the ether from a flask, it is well to lay the flask on its side in one of the openings of the water-bath, and afterwards, when the drj-ing is being completed in an air oven, the flask should be rotated from time to time, and air blown in every fi\'e minutes, to remove ether \-apour. Dr3'' ether is prepared by washing commercial ether with water, shaking the washed ether with calcium chloride, and, after allowing it to stand over calcium chloride for a day or two, distilling. A, Evaporating basin; B, specific-gravity bottle; C, fat flask; D, pipette; E, boiling-tube; F, loo c.c. stoppered cylinder. Sufficiently dry ether may also be obtained for most purposes by distilling the commerical variet}^ and rejecting the first fractions which pass over below 34-3° C, and the last above 34-8° C. If at anv time doubt exists as to the completion of the extraction process, a second weighed flask containing fresh ether should be afiixed, and the process continued for some time. This flask after evaporating the ether and drying at loo'^ C. should not increase in weight. 2. The Werner-Schmidt Method. — The specific gravity of the sample is ascertained or a measured volmne is weighed. Fifteen c.c. MILK i6i are pipetted into a boiling-tube and a like measure of pure hydro- chloric acid added. The mixture is shaken up and gently boiled until the contents appear dark brown in colour. The boiling must not be continued too far, as certain bodies soluble in ether are liable to be formed from the milk-sugar. The process is not suitable for milks containing cane-sugar. Boiling with acid renders the casein soluble, and so eliminates the obstacles which, in the solid condition, it offers to the extraction of fat. When cold, pour the contents of the tube into a graduated and stoppered lOo c.c. cylinder. Wash out the tube with ether, and finally make the column up to 75 c.c. with ether. Invert the cylinder several times, and put aside to settle. Read the height of the ethereal column, including three-fourths of the thin grey layer of casein. Draw off an aliquot part of this column and evaporate ; dry, and weigh the residual fat in a small flask, in the manner described in Adams's process. -Stokes's tube, a specially graduated tube prepared to treat 10 c.c. of milk, is also employed in this country. After completing the boiling with 10 c.c. of HCl, and cooling, ether is added until the surface of the column reaches the 50 c.c. mark. An aliquot portion of this column is afterwards drawn off, evaporated, dried, and the residue weighed as above. The special tube is not to be recom- mended, as the narrowed central portion offers resistance to the free escape of hot air during boiling, and the consequent explosive action frequently causes loss of the contents. This process is much more rapid than that of Adams, and in skilled hands almost as accurate. The student will remember that, owing to the low boiling-point of ether, it should never be added to a hot solution, and will accord- ingly always cool the boiling-tube before adding it. This may be rapidly done by holding it under a water-tap. In drying and weighing the fat it is essential that the last trace of ether vapour be got rid of by blowing dry air into the flask, and by ascertaining that two successive weighings, separated by half an hour's heating at 100° C, are the same. Example. — A milk whose specific gravity is 1-032 is subjected to the Adams process, and the fat collected in a flask weighing II 102 PRACTICAL SAXITARY SCIENCE 16-056 grammes. The weight of the flask and fat is 16-236 grammes. The weight of the fat is therefore o-i8o gramme. 5 c.c. of specific gravity 1-032 =5-16 grammes. If now 5-16 grammes of milk yield o-iS gramme fat, what is the percentage of fat ? 5-16 : 100 : : o-i8 : the percentage. Percentage therefore =3-5 nearly. The same sample subjected to the Werner-Schmidt process yielded practically the same result. 15 c.c. of the milk yielded 0-542 gramme fat; but 15 c.c. of specific gravity 1-032 =15-48 grammes; 15-48 : 100 : : 0-542 : 3-5. 3. There are several forms of centrifugal apparatus used for esti- mating fat, such as the Babcock, Leffmann-Beam, Gerber, etc. The Leffmann-Beam provides small flasks graduated on the neck into eighty divisions — ten divisions corresponding to i per cent, of fat. Run into the flask 15 c.c. milk. Add 3 c.c. of a mixture of equal parts HCl and amyl alcohol; shake and add slowly with agitation 9 c.c. concentrated H.^SOj. Fill up to 2iero with hot mix- ture of equal parts concentrated H0SO4 and water. Place in the centrifuge and rotate for two minutes. If fat and acid liquid are both quite clear, read off the fat column; if fat or acid liquid be cloudy, rotate again. The amyl alcohol assists in the collection of the fat globules: this reagent should be good, otherwise large error may occur, generally in the direction of excess of the truth. Where the operation is carefully carried through with sound reagents results are obtained to within 0-15 per cent, of those got by the Adams's process. The reagents used in the Gerber process are H.,SOj and amyl alcohol. The centrifuge runs on ball bearings, and reaches a velocity of about 2,000 revolutions per minute. Three minutes are sufficient to separate the fat. In the Babcock method H^SOj and boiling water are employed as reagents. The centrifuge revolves at about 1,000 times per MILK 163 minute — hence a longer time is required for completing the separa- tion of fat (seven to eight minutes). These centrifugal methods are used for determining the fat — (i) in condensed milk after it has been diluted to form a 10 per cent, solution ; (2) in cream after it has been diluted five or six times with hot water; (3) in butter and (4) in cheese after mixture with a small quantity of cold or hot water, as suitable, by addition of modified quantities of the reagents, and the necessary rotation. Estimation of Cream.^ — A cream-tube or creamometer provided with a short scale at its upper part, each division of which reads I per cent, of the capacity of the tube up to the highest line (^ero), is filled to the zero with the milk to be tested, and set aside for six, twelve, or twenty-four hours, and the volume of cream measured. A good milk should throw up 10 per cent, of cream in eight hours. The method is by no means accurate, as the same milk under different conditions of setting may show very marked differences in the quantity of cream formed. The estimation of fat in dried milk may be made by the Wernef- Schmidt method or by the Rose-Gottlieb method, preferably the latter. Rose-Gottlieb Method. — Weigh 0-5 gramme milk powder into a stoppered cylinder holding about 50 c.c. Add 5 c.c. water and 0*5 c.c. ammonia (equal parts o-88 ammonia and water). Shake and warm if necessary until solution is obtained. Add 5 c.c. alcohol and shake again until homogeneous. Now add 12-5 c.c. ether and mix very thoroughly; finally add 12-5 c.c. petrolemn ether (boiling below 60° C.) and again mix thoroughly. Settle out and draw off the ether. Repeat the extraction with a mixture of equal parts ether and petroleum ether until the whole of the fat is re- covered. Remove the solvent by distillation, dry, and weigh the fat. Total Solids. — Pipette into a clean, weighed, platinum dish 10 c.c. of the milk, and reweigh to obtain the weight of the milk used. Heat on a water-bath, breaking up occasionally the film that forms on the surface, in order to hasten evaporation. After an hour's drying, the dish is removed to a tray, carrying two or three layers of blotting-paper to remove moisture, and the tray is placed in an air oven at a temperature of 95° C, and provided i64 PRACTICAL SANITARY SCIENCE with sufficient draught; here the drying is completed. It ma^'' require two to three hours in the oven to produce a constant weight. Platinum basins are preferable to porcelain, as the}^ cool much more rapidly, and thus require less time in the desiccator. As milk solids are highly hygroscopic, no time must be lost in conveyance from the desiccator to the balance, nor in the process of weighing. Supposing that the specific gravity of the milk is 1-032, the weight of 10 c.c. will be 10-32 grammes. Further, supposing the difference in the first and second weighings of the dish to be 1-3 grammes, the percentage of total solids will be found from the proportion : 10-32 : 100 : : 1-3 : x ; 100 X 1-3 10-32 ^ From the total solids the ash is obtained by ignition at a low heat over an argand burner. The last trace of dark, separated carbon must disappear and the residue consist of a greyish-white mass before the dish is removed from the flame. Overheating causes loss of NaCl. Cooling, weighing, and percentage calculation are carried out in the usual manner. TheP and S of the milk proteins produce phosphoric and sulphuric acids. Carbonic acid is formed by the combustion of organic carbon. The ash does not truly represent the inorganic consti- tuents. It is computed that at least 8 per cent, of the phosphoric acid arises from the P of the casein. Bases predominate over acids in milk, and unite with proteins to fomi soluble protein salts, and with citric acid to form citrates. Composition of ash: Per Cent. Lime . . . . . . . . 19-3 Phosphoric acid Potash Chlorine Soda Ferric oxide . . Magnesia Carbonic acid Sulphuric acid 28-3 277 13-9 6-7 0-3 27 I'D O-I A probable composition for the salts as they exist in milk has been theoretically calculated: MILK i<>5 Per Cent. NaCl . 10-62 KCl 9-16 KH0PO4 . . . 1277 K2HPO4 . . 9-22 KsfCeH^O,) . . ■ 5-47 MgHPO^ . . . 371 Mg3(CeH,0,) • 4-05 CaHPO^ . . • 7-42 Ca3(P04)2 . . 8-90 CagiCeHgO^)., • 23-55 Lime combined with proteins . • 5-13 ash is materially less than 073 per cent. , watering may be suspected. Solids not Fat. — This item of the analysis is calculated by finding the difference in weight between the total solids and the fat. The solids not fat have been found to vary between 5 and 10 per cent. The law fixes 8-5 as the lower limit for whole milk. In case it is necessary to determine the percentage of proteins in milk, the best method to employ is the following modification of Kjeldahl's method for the estimation of total organic nitrogen, and multiply the result by 6-38. To obtain the total organic nitrogen, weigh 5 grammes of milk into a Kjeldahl flask of about 150 c.c. capacity, and add 20 c.c. pure H2SO4. Place over a small flame in the fume-chamber, and heat till thoroughly charred. Remove the flame, and add 10 grammes bisulphate of potash to raise the boiling- point of the mixture. Place a pear-shaped bulb in the neck of the flask and apply the flame, increasing its siz;e as frothing ceases. The liquid becomes colourless in thirty minutes or thereabouts. Cool, dilute largely with water, and transfer to the distillation-flask pro- vided with perforated cork carrying a dropping funnel with stop- cock, and a wide tube with one or more bulbs blown in it, which are loosely packed with asbestos. One end of the tube is connected with a condenser, and the other is made to dip below the surface of 50 c.c. i\ H2SO4. Through the dropping-funnel pass about 100 c.c. of a 20 per cent, solution of NaOH. Shake well by a rotatory motion. Apply a flame to the distilling-flask and collect about 200 c.c. of the dis- tillate. Take care that the condenser remains throughout quite cold. Titrate with y^ NaOH, using litmus as indicator. Subtract 166 PRACTICAL SAXITARY SCIEXCE the number of c.c. -i^g- XaOH solution used from the 50 c.c. ^c- sulphuric acid, and the remainder represents the acid neutralized by the ammonia distilled over. From this deduct the figure ob- tained in a blank experiment in which all the factors are exactly the same, except that milk is eliminated. Each c.c. of the j\y H2SO4 neutralized by ammonia is equal to 0-0014 gramme of X, which, when multiplied by 6"3S, is equal to the total protein. Colostrum is a term applied to the first milk secreted after par- turition. Hi)udet describes two forms — a viscous, brownish product, and a non- viscous lemon-yellow liquid; the earlier milkings furnish the first, and the later the second; the two co-exist often in the same animal. The fat differs somewhat from that of ordinar\' milk, in that its melting-point is high (42° C.) and its Reichert-WoUny figure low (6 to 7). The most characteristic feature of colostrum is the presence of the corps granuleux of Donne, consisting of cells clustered together like bunches of grapes, and measuring in diameter from 0-005 to 0-025 millimetre. The specific gravity of colostrum averages i-o68. Estimation of Citric Acid in Milk. — Prepare acid nitrate of mer- cur}' by dissoh'ing mercur}" in twice its weight of HXO3 (specific gravity 1-42), and adding an equal volume of water. With this reagent precipitate the proteins of the milk, and filter until the filtrate is clear. Rapid clearing may be effected at this stage by addition of some super-saturated solution of aluminium hvdrate. To a measured volume of the filtrate add dilute caustic soda solution until the neutral point is reached (phenolphthalein as indicator). Filter off the white precipitate of calcium phosphate, calcium citrate, and mercuric nitrate ; wash well with water ; remove from the filter, and suspend in water, to which a little dilute HCl has been added. Pass HoS through the fluid until all the mercury comes down as HgS. Filter again, and boil the filtrate to remove H^S. Add a little calcium chloride and cool. Carefully neutralize a second time with dilute caustic soda, and filter off the calcium phosphate. Concentrate the filtrate to small bulk. This contains the citric acid as calcium citrate. After thorough boiling, filtering, and washing the precipitate with boiling water, ignite it, and add to it excess -j^g- HCl. Titrate back the excess MILK 167 with ~Q NaOH (methyl orange as indicator). Each c.c. -^^^ HC used =0-007 gramme citric acid. Action of Heat on Milk. — When heated to 70" C, the albumin of milk, although not precipitated, is so changed that it is readily precipitated by acids and MgSO^. At 80" C. a further unknown change occurs in certain organic constituents, recognizable by the fact that they cease to evolve a gas from HgOg, and to produce a blue colour with paraphenylenediamine and H2O2. Test for Boiled Milk.—SYvake 5 c.c. milk in a test-tube with i drop of a 2 per cent. H2O2 solution and 2 drops of a 2 per cent, para- phenylenediamine solution. If the milk has not been heated above 80° C, a dark violet or blue colour appears at once; but if it has been boiled or pasteurized to this temperature, no colour appears. At 100° C. calcium citrate is deposited, and a reduction in the rotatory power of milk-sugar takes place. When milk is heated in contact with air a pEotein film, probably an oxidation product, is formed on the surface, which has been variously classified. It has been claimed that boiled milk is more easy of digestion than the raw secretion, and perhaps this is true. The much-dis- cussed question of boiled milk producing symptoms of scurvy has not been settled; there is no reliable evidence from which a con- clusion may be drawn. Lactose.- — An important product of milk in connection wath the artificial feeding of infants is milk-sugar. Its estimation may be carried out as follows : To 50 c.c. distilled water add 6 grammes of milk-sugar finely powdered, and stir with a thermometer for ten seconds; allow to settle for twenty seconds, and read the fall in temperature. Filter, and fill a polariscope tube with the clear filtrate. Take readings every minute until the specific rotatory power begins to diminish. When the polarized solution is kept at a temperature of 15° C, several readings can be obtained which are nearly constant; the mean of these is the initial rotation. After twenty-four hours, polarize again at the same temperature to obtain the normal rota- ^, initial rotation .,,,,. tion. Ihe \ — TT^- — IS the birotation ratio. normal rotation The amount of sugar may be estimated in 100 c.c. of the above 1 68 PRACTICAL SANITARY SCIEXCE solution by dividing the normal rotation reading in angular degrees by i-io6. Principles of Polarimetry. — The vibrations which constitute an ordinary light ray take place in all directions in a plane perpendicu- lar to the line of propagation of the ray. If one looks at an object through a crystal of Iceland spar, two images are seen — the light ray has been split by the crystal into two, the more refracted or ordinary ray, and the less refracted or extraordinary ray. The less refracted or extraordinary ray does not obey the ordinary laws of refraction, but presents an image which moves when the crystal is rotated. Both rays are said to be polarised — i.e., consist now of vibrations in one direction only in the plane perpendicular to the line of propagation of the ray. If a crystal of spar be cut through its obtuse angles, the sections polished and cemented together, and the long sides blackened, a Xicol prism is formed. Such a prism absorbs the ordinary ray by the black sides after it has been totally reflected by the cut surfaces, whilst it allows the extraordinary ray to pass through in a direction parallel to the source of light. A polarimeter consists of two Nicols mounted parallel to each other — one, the polarizer, fixed; the second, the analyijer, movable. If the movable prism is exactly parallel to the fixed, a beam of light will pass through it; if not exactly parallel, but inclined at an angle, less light will pass through; if at right angles, all light will be cut off. If a solution of an optically active substance be interposed be- tween the Xicols set parallel, the quantity of light passing through is diminished, but the original intensity can be recovered by rotating the analyzing prism. The amount of such rotation is equal to the power of rotation of the solution. In all polarimeters the analyzer is mounted on a graduated circle, so that the number of degrees of rotation can be easily measured. The recognition of equal intensity of light in a polarimeter before and after the passage of light through an optically active solution is very difficult, and readings are accordingly far from correct. To overcome this difficulty, Laurent placed behind the polarimeter a quartz plate of such thickness that one of the two light rays pro- duced in it is retarded by half a wave length (and consequently reversed in direction), and of such size tha.t it covered half the field. MILK 169 The ray resulting from the blending of the affected and unaffected rays accordingly emerges in a plane at an angle to the original plane; in other words, the polarized light passing through the quartz plate is rotated through an angle. Two sets of rays of polarized light at an angle to each other will accordingly reach the analyzer. If the analyzer be arranged parallel to the light coming from the covered portion, this half of the field will appear Hght, and the other half dark. On the contrary, if the analyzer be set parallel to the light coming from the uncovered portion, this half of the field will appear light, and the other half dark. By adjustment the analyzer can be placed in a position in which the two halves appear equally illuminated. This position corresponds with the zero of the circular scale and with the zero of the vernier. With Laurent's polarimeter monochromatic sodium light is used, a cell containing potassium bichromate being placed in front of the polarizer to intercept the blue rays. The instrument consists of the following parts: Bichromate cell, polarii^ing prism, quartz plate covering half field, trough to carry solution under examination, circle graduated in degrees, a double vernier attached to analyzing prism and moving on graduated circle, and a telescope to focus edge of quartz plate. When the active substance in solution is placed between the Nicols and equal illumination of the two half fields restored, the vernier will have moved in the direction of the hands of a clock (dextro-rotatory), or in the opposite direction (laevo-rotatory) . The distance traversed measured on the circular scale gives the amount of rotation in degrees. The specific rotatory power [a]^- — that is, the rotation produced by I gramme of the substance dissolved in i c.c. of liquid examined in a tube i decimetre long (rotatory power of 100 per cent, solution) — being known, we can determine the strength of an unknowm solution. For glucose [a]D=+52-5°; lactose + 52-4°; sucrose + 66°; galac- tose + 82°; maltose + 1377°; fructose — 93-8° at 20°' C. The percentage strength of a solution is given by the formula- — ax 100 170 PRACTICAL SAXITARY SCIEXCE where [a]„ =specilic rotation for sodium, or D light, a = observed rotation on the circular scale, c = concentration. 1 = length of tube in decimetres. Polarimetric Estimation of Lactose in Milk.— Put 60 c.c. milk in a 100 c.c. flask; add i c.c. mercuric nitrate (Hg dissolved in twice its weight of HNO3, specific gravity, 1-42 + an equal volume of H.,0), and fill up to the mark with water. Shake thoroughly and filter through a moist filter-paper. When quite clear, determine rotation in polarimeter. ^lake several readings, and take an average. Correct for space occupied by proteins and fat. The volume of the fat in c.c. is found by estimating its weight and multiplying by 1-075, and the volume of the proteins is found by multiplying their weight by o-S. Water equal to the sum of these volumes in c.c. is added to the 100 c.c. The calculation involved by taking 60 c.c. of milk may be avoided by taking a simple multiple of the standard amount of the polari- meter used. Thus, in the case of an instrument adjusted so that 20-56 grammes lactose in 100 c.c. of solution produce 100 degrees on the percentage scale, 61*68 grammes (20-56x3) are weighed, treated with mercuric nitrate solution, and made up to 100 c.c. The volumes of fat and proteins are calculated, and the sum added to the 100 c.c. Finall}' the polarimeter reading divided by 3 will give the percentage of hydrated lactose. Estimation by Fehling-'s Method. — This method depends on the fact that, whilst Fehling's solution may be boiled without change if a small quantity of glucose or other reducing sugar be added at the boiling temperature, a precipitate of cuprous oxide is formed, and that the amount of copper salt reduced is proportional to the quantity of sugar used. Prepare Fehling's solution. Powder and press between blotting-paper to remove moisture crystals of pure copper sulphate (CuS04,5H20) ; weigh 69*28 grammes; dissolve in water; add 0-5 to i c.c. pure H2SO4; dilute with pure water to a litre. Weigh 350 grammes Rochelle salt / CH (OH) -COOK \ I sodium potassium tartrate 1 ) \ CHOH-COONa,4H20/ and dissolve in about 700 c.c. water; weigh 100 grammes NaOH MILK 171 prepared with alcohol; dissolve in aljout 200 c.c. pure water, mix the solutions, and make up to a litre. These two solutions are kept separate, and mixed in equal pro- portions immediately before use. Each c.c. of the mixture should contain 0-03464 gramme cupric sulphate, which corresponds with 0-005 gramme anhydrous grape sugar and 0-006786 gramme lactose. For estimation of lactose, Pavy's modified Fehling process is preferable. Pavy added ammonia to the ordinary Fehling solution to prevent the precipitation of cuprous oxide. The end reaction is fixed by the disappearance of the blue colour in a perfectly clear solution. Mix 120 c.c. ordinary Fehhng (not 100 c.c, because in ammoniacal solution only 5 molecules CuO are reduced by one molecule glucose, instead of 6 CuO) with 300 c.c. ammonia (specific gravity, 0-88); add 100 c.c. 10 per cent. NaOH or 14 per cent. KOH, and make up to a litre, [i c.c. =0-0005 gramme glucose and 0-0006786 gramme lactose.] A little more time should be allowed during titration for the reduction, as Pavy's solution acts more slowly than the ordinary Fehhng, but the operation should be completed within three or four minutes, otherwise the ammonia disappears and CugO is deposited. Weigh 25 grammes milk into a 250 c.c. flask; add h, c.c. of a 30 per cent, solution of acetic acid; shake well, and stand aside for a few minutes. Dilute with about 100 c.c. boiling water, and add 25 c.c. alumina cream; shake and set aside again. Pour the more or less clear liquid through a wet filter, and finally wash out on the filter the entire contents of the flask. Collect the filtrate, w^hich must be perfectly clear, and make up with water to 250 c.c. Transfer a portion of the clear sugar solution to a burette. Raise to the boiling-point in a porcelain dish 10 c.c. of the Pavy- Fehling solution. Run in the sugar solution (drop by drop if possible towards the end) until the last trace of blue disappears. The number of c.c. delivered from the burette =0-006786 gramme lactose. From this calculate the percentage. If the ordinary Fehling method be used, a difficulty is encountered in determining the end-point — i.e., the instant at which the blue colour is completely discharged. Dilution of the sugar solution with very weak NaOH causes the CU2O to separate out. If soda 172 PRACTICAL SAXITARY SCIENCE be used in very large excess, the oxide is kept in solution, as in Pavy's modification. The end-point difficulty is perhaps best met by using Ling's indicator: Dissolve in lo c.c. water at 45° i -5 grammes ammonium thiocyanate and i gramme ferrous anmionium sulphate; cool immediately; add 5 c.c. strong HCl when a brownish-red solu- tion is obtained. The colour is got rid of b\' adding a small quantity of zinc dust. To determine the end-point, remove a drop of the reduced copper solution, and mix it with a drop of the indicator on a white surface; when a red coloration ceases to appear, the reduction is complete. Genuine commercial milk-sugar crystallized from water gives : Not more than 0-05 per cent. ash. Solubility at 15° C. =7-0 grammes per 100 c.c. (with an increase of 0"i gramme per 100 c.c. for each degree of increase of temperature). Fall of temperature, 0-5° C. Birotation ratio, i-6. Amount of milk-sugar, 99-5 to 99-9 per cent. Milk-sugars are adulterated with cane-sugar, maltose, dextrose, and various mineral matters. Cane-sugar can be detected by treating a solution with yeast at 55° C. for six hours. Milk-sugar is unchanged in specific rotatory power, whilst the addition of as little as I per cent, cane-sugar produces a marked change. Maltose is detected b}' a decrease in the birotation ratio; dextrose by an increase in this ratio and a decrease in the fall of temperature. Adulteration of Milk. — The principal adulterations are the addi- tion of water and the abstraction of cream. The estimation of the water added is made from the solids not fat, as these solids, in different samples, do not depart so far from the mean as the fat. The legal limit is 8-5 per cent. Example. — A milk yields 3 per cent, of fat by one of the foregoing processes of estimation, and 11 per cent, of total solids. The sohds not fat amount to 11 —3 =8 per cent. On the assumption, therefore, that 8-5 per cent, represents 100 per cent, pure milk, 8 per cent, will represent 94-1 per cent, pure milk. (8-5 : 8 : : 100 : 94-1). In other words, 5-9 per cent, of water has been added to this sample. MILK 173 It may be urged that, since a few animals produce milk con- taining less than 3 per cent, of fat and less than 8-5 per cent, of solids not fat, it is unfair to the dairyman to enforce these figures as legal limits. But the number of animals in a herd producing milk below these standards is so small in proportion to the whole number, that the mixed milks should in all cases not only reach but surpass the standards. Cane-sugar, starch, dextrin, and other bodies have been added to mask the addition of water by raising the solids not fat; these may be detected by the sweet taste, deficiency in total nitrogen, and by the ash. Starch is denoted by the blue colour formed with iodine. Common salt has been added, and is detected in the ash by increase of CI. Chalk, carbonate and bicarbonate of soda, borax, fluorides, etc., may be found. Of these, borax and boracic acid are used as preservatives. The employment of preservatives — bodies such as boric acid, formalin, etc. — that prevent the growth of micro-organisms has been much discussed. Some hold that it is better to add these bodies than to allow the milk to decompose; whilst others advocate the exclusion of all such reagents. There does not appear to be any experimental evidence to show that small quantities of preservatives like boric acid and its com- pounds exert an injurious effect upon healthy adults, or even upon healthy children; but in the case of weakly infants there is a strong feeling in favour of excluding all such bodies from their food. Salicylic acid holds a somewhat different position. In quantities necessary to prevent the growth of micro-organisms this drug is likely, in certain cases, to produce injurious effects. Besides, it inhibits the action of the digestive en2;ymes of the alimentary tract. Its use as a preservative has been rightly forbidden in France. Formalin, formal, or formol is a 40 per cent, solution of formal- dehyde in water, and in the strength of i per cent, has been much used as a milk preservative. It produces a very decided change in casein, rendering it insoluble in the digestive juices. A patented process exists in Germany for converting casein,' by the action of formalin, into a substance resembling celluloid. This preservative should be rigorously excluded from all foodstuffs. The question of the addition of preservatives to milk has another aspect. All purchasers of milk expect to get a thoroughly fresh article. Any 174 PRACTICAL SAXITARY SCIEXCE procedure allowed the dairyman by which he can retain milk for a number of days puts a premium on the sale of a stale substance. Milk should be consumed on the day on which it is drawn from the animal. Its constitution is such that, apart from sterilization, which can only be legitimately performed by heat, it rapidly decom- poses and becomes unfit for consumption. If dairymen were com- pelled to keep their churns scrupulously clean, and the temperature of the milk during transit sufficiently low, there would be no need for preservatives, and we should hear little of decomposed milk. It is stated that a mixture of boric acid and borax is more effica- cious in preserving milk than either alone, and that 35 grains of this mixture are required to preserve a gallon of milk. Detection of Boric Acid or Borax — The Turmeric Test. — Evap- orate 100 c.c. of the milk, which has been made alkaline with caustic soda (0-5 gramme), to dryness; incinerate. Take up a portion of the ash in water, arid the remainder in weak HCl. Add to each portion a few drops of freshly prepared turmeric solution, and evaporate to dryness. When boric acid or borax is present, the residue assmnes a brownish-pink colour, which changes to dark green on the addition of a solution of sodium bicarbonate. If the watery extract gives no reaction, whilst the acid extract reacts strongly, it may be concluded that borax is present; if the two reactions are of equal intensity, boric acid has been added; and if the reaction produced b}' the acid extract is stronger than that produced by the watery extract, it is probable that a mixture of the two is present. If the ash be moistened with dilute H2SO4, methylated spirit added, and the mixture thorough^ stirred and set on lire, a green border will appear on the flame when boric acid is present. Estimation of Boric Acid. — The following method is recom- mended by R. T. Thompson: To 100 c.c. milk add 2 grammes caustic soda and evaporate to dryness in a platinum dish. Char the residue thoroughly, and heat with 20 c.c. water. Add HCl drop by drop till all but carbon is dissolved. Transfer to a 100 c.c. flask, and add 0"5 gramme dry CaClo- Run in a few drops of phenolphthalein, and then a 10 per cent, solution of caustic soda till a permanent pink colour is perceptible, and finally 25 c.c. lime- water. The phosphoric acid is all precipitated as calcium phos- MILK 175 phate. Make up to 100 c.c, mix, and filter througli a dry filter. To 50 c.c. of the filtrate (representing 50 c.c. of the milk) add normal sulphuric acid till the pink colour is gone, then a few drops of methyl orange, and continue the addition of acid until the yellow is just changed to pink. Next add ^ NaOH till the liquid assumes a yellow tinge, avoiding excess of soda. All acids likely to be present at this stage exist as salts neutral to phenolphthalein, except boric acid, which is neutral to methyl orange, and a little carbonic acid, which latter is expelled by a few minutes' boiling. Cool the solution, add a little more phenolphthalein, and as much glycerine as will form 30 per cent, of the solution, and titrate with ^ NaOH till a permanent pink is produced. Each c.c. of ~ NaOH is equal to 0*0124 gramme crystallizied boric acid ( =0*007 gramme boric anhydride). Phosphoric acid can be separated from boric acid by precipitation as calcium phosphate, if not more than 0-2 per cent, of crystalhzed boric acid be present. It is necessary not to carry the charring further than that required to produce a colourless solution, as excessive heating drives oft boric acid. Richmond and Miller have published a process for estimating boric acid without ashing the milk or removing phosphoric acid: Weigh about 10 c.c. of the milk; add half the bulk of a half per cent, phenolphthalein solution; run in normal NaOH till pink colour appears; boil and titrate back with normal HCl till white, and finally with ~r NaOH, till faintly pink (colour, though faint, is distinct) ; add 30 per cent, of glycerol and continue the titration with ^ NaOH. [A glycerol blank is done and subtracted if necessary]. The number of c.c. y^ NaOH used for the final titration, multiphed by 0-0062, gives the quantity of boric acid contained in the quantity of milk operated upon. Fopmalin.- — Pure H2SO4 and pure formaldehyde give no colour reaction with proteins. Addition of small quantities of oxidizing substances such as hydrogen peroxide, ferric chloride, sodium peroxide, potassium persulphate, etc., produces a characteristic colour. The reaction fails if the quantity of formaldehyde is in- creased beyond a certain limit which is in proportion to the amount of oxidizing reagent used. Rosenheim has shown that the formaldehyde 176 PRACTICAL SAXITARY SCIENCE is oxidized, producing an intermediate oxidation product, wliich then reacts with the protein. He found that the ammonium com- pound of diformaldehyde-peroxide-hydrate, OH.CHoO.O.CH.O.OH, an oxidation product intemiediate between formaldeh3-de and formic acid, reacts with proteins and pure sulpliuric acid, producing the characteristic colour. This is a general reaction for proteins, and depends on the presence of tryptophane (indol-amino-propionic acid). The intensity of the reaction with different proteins varies directly as the amount of tryptophane present in the protein mole- cule, and bodies destitute of tryptophane fail to give the reaction. Hehner's Test. — To 10 c.c. of milk in a test-tube add i drop ferric chloride solution, and dilute the milk to about 30 c.c. To a portion of tliis in another test-tube add concentrated H2SO4, by cautiously pouring it down the side of the tube so as to form a laj'er at the bottom of the milk. A violet-blue ring will be formed at the junction of the liquids. A few c.c. of the milk are curdled by dilute sulphuric acid, and a little Schiff' s reagent (a solution of fuchsin decolourized by sul- phurous acid) added to the filtrate in a test-tube, which is corked and allowed to stand. In a short time a violet-pink colour is produced in the presence of the aldehyde. A further qualitative test: Boil 10 c.c. of milk; add a few drops 25 per cent. HoSO,, ; filter; to filtrate add 5 c.c. o-i per cent, solution of phloroglucin and 5 c.c. 5 per cent. NaOH. A rose-pink colour indicates formalin. Estimation of Formalin. — There is no satisfactory method of estimating formalin. An approximate estimation, which must be made early, as the aldehyde rapidh' disappears, may be carried out by the following method: — Reagents required : a normal solution of H2SO4, a few 100 c.c. bottles, with close-fitting rubber stoppers, and a boiler, in which they may be immersed to the neck, a solution of methyl orange, and an approximately normal solution of ammonia. Place in each bottle 25 c.c. of the ammonia solution, and to half of them add a sample containing 0-5 gramme formalde- hyde. Stopper tightly, place the bottles in the boiler, fill with water to the neck, and boil for one hour. Cool slowly, and titrate with the sulphuric acid, using methyl orange as indicator. The differences in the readings of the blanks and the samples represent MILK 177 the ammonia consumed in normal c.c. Each c.c. = o-o6oi gramme formaldehyde. Any acid that may be present must be accounted for. The following method originally described by Shrewsbury and Knapp is perhaps as satisfactory as any other: An oxidiajing reagent is prepared by adding 0-05 to o-i c.c. pure HNO3 to 100 c.c. con- centrated HCl. Add to 5 c.c. of milk in a test-tube 10 c.c, of the reagent ; shake vigorously, and place in a water-bath at a tempera- ture of 50° C. In about ten minutes the contents of the tube are cooled to room temperature. A violet colour indicates formalde- hyde, and its intensity indicates the amount. The quantitative estimation is effected by setting half a dozen milk tubes containing known quantities of formalin at the same time as the sample, and at the end of the time allowed for the test selecting the match. Salicylic Acid. — Precipitate the proteins from 50 to 100 c.c. of milk by the addition of mercuric nitrate, and filter. Shake up the filtrate with half its volume of a mixture of equal parts ether and petroleum ether, and stand aside until the ether separates out. Pipette off the ether, and evaporate to dryness in a clean flask. Dissolve th,e residue in a few drops of hot water, and add to a portion of the solution a drop of a i per cent, ferric chloride solution ; in the presence of salicylic acid a violet or purple colour is produced. Add to a second portion of the solution a little bromine water: salicylic acid produces a curdy, yellowish precipitate. Evaporate the third portion of the solution to dryness with strong HNO3, and take up the residue in a few drops of water. If salicylic acid be present, a yellow coloration is produced on adding ammonia. It should be borne in mind that carbolic acid and other hydroxy- benzene derivatives act in a somewhat similar manner to salicylic acid. The colour reaction with ferric chloride remains in the presence of alcohol in the case of salicylic acid, but disappears on addition of alcohol in the case of carbolic acid. A further test consists in evaporating a part of the ethereal extract to dryness, placing a minute portion of the residue in the subliming cell, and comparing the crystalline subHmate with one obtained from pure salicyl,ic acid. The melting-point of pure salicylic acid is 155"^ to 156° C. 12 ijS PRACTICAL SANITARY SCIENCE A quantitative estimation may be approximately made by matching the colour produced by ferric cliloride in a standard solu- tion containing 0-05 per cent, salicylic acid in 50 per cent, alcohol. A I per cent, iron alum is recommended instead of ferric chloride. Definite amounts of the salicylic acid solution should be added to a milk filtrate resembling as nearly as possible that of the sample. Benzoic Acid is but very occasionally found in milk. It is detected as follows: Render alkaline with bar\i;a-water 200 c.c. milk, and evaporate down to one-fourth. Mi.x the residue with CaSO^ to form a paste, and dry on the water-bath. Powder, moisten with dilute H0SO4, and extract with cold 50 per cent, alcohol. Neutralize the alcoholic extract with baryta-water, evaporate to small volume, acidulate with dilute H.jSOj, and extract with ether. On e\'aporating the ether, any benzoic acid will be found sufficienth' pure for testing. Make a water^^ solution of the benzoic acid, and add a little sodium acetate; now add a drop or two of ferric cliloride to obtain a reddish- yellow colour. HydPOg'en Peroxide. — HgOo in the presence of organic matter rapidly splits into water and oxygen. If milk to which it has been added be examined before it disappears, it may be detected by addition of paraphen3dene-diamine, when a blue colour is produced. The reaction depends on the presence of an oxidase, which is destroyed by heat; hence if the sample has been heated, it \x\\\ be necessary to add a little fresh milk. Milk free from HaO, de- colourizes Schardinger's reagent (5 c.c. alcohoHc methylene blue, 5 c.c. formaldehyde, 190 c.c. HgO), but milk that has been treated with H2O2 (' Buddeized ') fails to decolourize the reagent, and only regains this power after bacterial fermentation has taken place. Sodium Carbonate. — Ash a weighed portion of the milk. The ash of 5 grammes nomial milk does not contain more alkalinity than that neutralized by three-tenths of a c.c. of j"^^ HCl. Excess of alkalinity over this may be regarded as sodium carbonate. Mix 10 c.c. of milk with 10 c.c. of rectified spirit in a test-tube; add 2 or 3 drops rosolic acid solution (rosolic acid, i gramme; alcohol, 25 c.c; water to a litre). A rose-pink colour indicates sodium carbonate. A preservative named ' mystin ' (a mixture of formaldehyde and mii:k -ijcj sodium nitrite) has been found in milk. This mixture may bo detected by destroying the nitrite in lo c.c. of milk, with a few c.c. of a 2 per cent, solution of urea, and then testing for formaldehyde; or by distilling off the formaldehyde and applying Griess's test for nitrites. Colouping" Matters. — The most commonly occurring colouring matter is the vegetable substance annatto; carrot juice, turmeric, and saffron are also used. These colouring matters are all soluble in alcohol, but not soluble in water. To detect annatto, add to a few c.c. of milk a little bicarbonate of soda, and immerse a strip of white filter-paper over night; a brown stain in the paper indicates this substance. If an alcoholic solution of annatto, saffron, and turmeric be evaporated down to dryness, and a drop of concentrated H2SO4 placed on the residue, a dark blue colour is produced, changing to green in presence of annatto and saffron. In the case of saffron a final reddish-brown is formed. Turmeric produces a violet-red turning brown on the addition of an alkali. Whilst vegetable colouring matters may be regarded as incapable of damaging the digestive organs of man, it is not clear that certain coal-tar dyes are equally innocent. A dilute mineral acid added to milk containing an azio-coal-tar dye gives a pink colour. Soup Milk. — During the lactic fermentation of milk wherein half the lactose may be transformed into lactic acid and volatile bodies, little or no change takes place in the fats. But before estimating these some preliminary treatment of the curdled sample is necessary. A uniform emulsion is made by means of a whisk. The total solids are estimated on a portion of this in the usual way. Fat and Non-Fat Solids. — Weigh 10 grammes into a flat tared platinum basin, carrying a glass rod; add 2 drops (0-5 per cent.) phenolphthalein ; run in decinormal strontia until alkaline, noting the number of c.c. used; evaporate on a water-bath till the consis- tency of dry cheese is reached. Pour 20 c.c. of dr}^ ether over the solids and thoroughly triturate with the glass rod. Decant through a dry weighed filter-paper into a weighing flask. Repeat the ether treatment several times. Distil off ether; dry and weigh residual iat. Transfer the solids completely to a weighing flask ; add the filter- i8o PRACTICAL SAX IT A RY SCIENCE paper which was previously thoroughly washed with ether; dry for three hours at loo^, and weigh; dry for a further two hours and again weigh; drj- for another liour and weigh (last two weights should not differ by more than a milligramme). Deduct 0-00428 gramme for each c.c. of strontia used, also the weight of the filter- paper. Result =non-fat solids. Correction for Alcohol formed from Lactose. — Distil 100 grammes of the milk and neutralize the distillate with -j^g- XaOH (litmus indicator). Redistil the neutralized distillate and calculate the percentage amount of alcohol from an alcohol table. The percentage weight of alcohol, x 4'" = percentage of lactose that has disappeared in formation of alcohol. Correction for Volatile Acids. — Determine the total acidity in 10 grammes of the milk by y^y XaOH (phenolphthalein indicator). Weigh another 10 grammes of the sample in a platinum dish, and add half the quantity of y^^ XaOH necessary to neutralize. Evapor- ate to dr}-ness on a water-bath with frequent stirring; add 20 c.c. boiling water and thoroughly detach solids from dish; now add j""^ XaOH till neutral. Difference between original acidity and acidity of evaporated portion = volatile acidity recorded as acetic acid; and 60 parts acetic acid (CH3COOH) =62 parts original lactose (CO.2-1-H2O). Richmond rightly points out that this correction is inaccurate — COo driven off is calculated as acetic acid; all volatile acids are not driven off ; there is a possibility of lactic acid being volatile, and it may be converted into a lactose. Thorpe makes an ammonia correction: 2 grammes of milk are made up to 100 c.c. with ammonia-free distilled water, and filtered through a carefully washed filter. Ten c.c. of the clear filtrate are similarly made up to 50 c.c. in a Xessler glass, and the ammonia estimated bv standard ammonium chloride (i c.c. =o-oi milligramme XH3) after Xessler's method. Richmond shows good reasons for regarding the ammonia correc- tion as unnecessar}'. The total correction (0-2 to 0-3 per cent, additive) is fairly con- stant in properly sealed samples three to six weeks old. Bacteria in Milk. — Micro-organisms enter milk from the udder, during milking, and during transit and distribution. It is not possible to estimate the total number of bacteria in milk. Perhaps MILK i8i the best count is that which demonstrates the presence of pollution by manure: B. colt, B. enteritidis sporogenes. The methods employed in this work differ in no main principle from those used in connection with water. The utmost care is necessary in the collection of samples. Dilutions are conveniently made in sterile flasks or bottles by placing lO c.c. of milk or of a particular dilution in the vessel which already contains 90 c.c. of sterile distilled water. Estimation of B. Coli. — To a series (better to a double series) of lactose, bile-salt broth tubes are added respectively — i-o, o-i, o-oi, o-QOi, o-oooi, o-ooooi, o-oooooi c.c. (and smaller fractions, if neces- sary, depending on the degree of pollution of the sample.) Record is made of the smallest quantity producing acid and gas in two days at 37° C. B. Enteritidis Sporogenes. — Add i, 1-5, and 2 c.c. of the sample to tubes containing 10 c.c. fresh milk recently sterilized. Add 5, 10, and 20 c.c. to empty sterile tubes. Heat the six tubes for ten minutes at 80° C. Cool promptly and incubate anaerobically for two days at 37° C. Look for the characteristic enteritidis changes. Pathogenic Micro-Organisms — B. Tuberculosis. — Centrifugalize 50 or 100 c.c. of the milk. Examine a portion of the sediment microscopically. Make and fix films on microscopic slides. When thoroughly fixed, wash out all fat with a mixture of equal parts of anhydrous ether and absolute alcohol. Stain by the Ziehl- Neelsen method. Use the remainder of the sediment for inoculating several guinea-pigs subcutaneously on the inner side of the left leg. Evidence of infection may be found at various subsequent dates in enlargement of the popliteal, inguinal, sublumbar, and retro-hepatic lymph glands on left side, and in tubercles in the spleen. Four weeks is an average time for the production of these appearances. When the milk contains large number of B. tuber- culosis, they may be found as early as fifteen days after inoculation ; when few bacilli exist, five to six weeks may be required to give results. It is well to make smears from the enlarged glands and stain with Ziehl-Neelsen's fluid. Inasmuch as certain non-patho- genic acid-fast bacteria presenting morphological characters somewhat similar to B. tuberculosis- — such as Moller's Timothy- iS2 PRACTICAL SANITARY SCIENCE grass bacilli, Rabinowitcli's butter bacillus, the smegma bacillus, mist baziUns, and Johne's bacillus — produce tubercular lesions in the guinea-pig somewhat resembling those produced by B. tubercu- losis, it is not always safe to rely on inoculation. The diagnosis can be established definitely bj^ sowing on glycerin, agar, or other media portions of the pulp of the lymph glands, from which the smears above mentioned are made. All the non-pathogenic organisms will form definite growths in two or three days, whereas B. tuberciUosis will ordinarily require three or four weeks for growth. It is necessary to investigate the cream in all these details, as well as the sediment. The Klebs-Loffler Bacillus. — Sediment and cream arc investigated morphologically and culturally. If organisms resembling the diph- theria bacillus morphologically are found, they must be growm on blood serum, and their virulence must be tested by animal inocula- tion. B. Typhosus. — This organism is almost as difficult to detect in milk as in water. Portions of sediment and cream are applied to those media intended for the differentiation of B. typhosus, such as lactose bile salt neutral red agar, followed by subculture on Conradi and Drigalski's medium, malachite green agar, etc. Streptococci. — In the milk of cow^s suffering from mastitis, enormous numbers of streptococci are found, and when these are inoculated into the teats of goats, they set up an inflammatory reaction. But since they are found in certain numbers in the milk oi healthy cows collected in the most cleanly manner, it is difficult, if not impossible, to estimate their significance. All that can be said at the moment is, that where streptococci exist in milk in large numbers, the indication is to examine the animal for mastitis, ulceration of teats, etc. Streptococci arise from the teats and milk ducts of the udder, in large quantities from manure, in smaller quantities from the air, and may be contributed by filthy vessels, foul water, etc. ; and in those cases where they occur in very large numbers, if no inflammatory condition of the udder be found, it may be assumed that their presence is most likely due to manure. They can be readily demonstrated in the sediment b}' making smears and staining with methylene blue. Quantitative estimation is effected by inoculating glucose neutral MILK 183 red broth with i-o, o-r, o-oi, o-ooi c.c, etc., Jind incuijiitin/^ for two days at 37^ C. Hanging-drop ;i,nd stained ]:)re])arations exliihit definite chains of cocci. Cellular Elements of Milk.^ — These are recovered and studied in the sediment microscopically. Where pus enters milk in large quantities, the fact is at once revealed by a microscopic examina- tion. But whether a few round cells resembling dead leucocytes are to be regarded as evidence of a small quantity of added pus or as normal constituents of certain milk remains an open question. Bacteriological examination of condensed milk, dried milk, and cheese is carried out in the same manner after a thorough emulsion has been made of a definite weight of the substance in sterile water. Koumis. — The original koumis was made by the Tartars from mares' milk, which is rich in lactose, and readily fermented. This stimulating, beverage is now largely made from cow's milk, to which sugar and yeast have been added. It undergoes a multiple fermen- tation — alcoholic, lactic, and proteolytic. Kephir. — This is a fermented milk similar to koumis. The proteolytic fermentation is less pronounced, and the alcoholic and latic fermentations are established by a fungus — kephir grains. Condensed Milks are found in four forms: (i) Condensed whole milk sweetened; (2) condensed whole milk unsweetened; (3) con- densed separated milk sweetened; (4) condensed separated milk unsweetened. The process of condensing unsweetened milk appears to kill all bacteria, and organisms that are found in this variety, according to Gordon, are introduced subsequently from the air. In all the sweetened varieties streptococci, with characters similar to those found in milk, were discovered by the same observer. The chemical and bacteriological examinations of these modifica- tions of milk are worked out on the same lines, as in the case of ordinary milk, condensed milks being first mixed with a definite measured quantity of distilled water. Dried or powdered milk is produced (i) by applying the fluid in a thin stream to the surface of a heated revolving metallic cylinder, or (2) by passing it in the form of a fine spray into a hot-air chamber. It should contain fully 27 per cent, of fat, and about 32 per cent, each of proteins and sugar. 1 84 PRACTICAL SAXITARY SCIENCE The fat is best estimated by Adam's process, and the proteins by Kjeldahl's total organic nitrogen process, using the factor 6-38. Cream is prepared by centrifugalizing milk, and contains 45 to 63 per cent, of fat. Cream is artilicially thickened with gelatin, starch paste, condensed milk, and saccharate of lime. Gelatin mav be detected by drying a weighed quantity and washing out the fat with ether. The residue, when dissolved in boihng water, will contain the gelatin, which sets on cooling. Or mix a weighed quantity of cream with warm water: precipitate proteins and fat with acetic acid; filter; add to the clear filtrate a little strong solu- tion of tannin, when, if gelatin be present, a voluminous precipitate falls out. A control sample of genuine cream should be operated on in the same way. It will give but a shght precipitate. Starch is discovered by the blue colour it forms with a solution of iodine. Calcium saccharate is determined as CaO in the ash. The dicalcium phosphate, tricalcium phosphate, calcium citrate, and lime united with proteins in normal cream, when transformed into CaO, amount to about 22-5 per cent, of the ash. Cane-sugar in cream is detected by the rich red colour produced when to 15 c.c. of cream, O'l gramme of resorcinol, and i c.c. of con- centrated HCl are added, and the mixture raised to the boiling- point. BUTTER. Butter is produced from milk or cream by churning. The agita- tion causes the fat globules to coalesce to form granules of a fine spongy nature. When butter is collected and worked, it assumes a more homogeneous appearance. The mean composition of butter made from ripened cream, accord- ing to Storch, is : Per Cent. Fat . . . . . . . . 82-97 Water Proteins Milk-sugar Ash Salt 1378 0-84 0-39 o-i6 1-86 The composition of different butters varies considerably. If butter be churned at a higher temperature than 13° to 18'' C.^ BUTTER 185 it will contain more water than at medium temperatures. Very low temperatures and rapid (duirninj,^ produce an article; containing too much water. Butter is adulterated with various foreign fats, animal and vege- table, under the name of margarine, which as a rule are little inferior in nutritive qualities to the fat of milk. In the pro- duction of margarine, animal and vegetable fats are melted, filtered through coarse filters, and worked up with milk, to look and smell like pure butter. It is stated that margarine, as prepared for the market, is not quite so digestible as butter. Whether or not this be true, it is illegal to substitute margarine for butter, and the principal object of a butter analysis is to determine the presence or absence of foreign fats. The odour and taste of butter are characteristic, and excellent tests of its purity. By heating it to 25° C. any unpleasant taste that it may possess becomes more apparent. Adulteration. — Foreign fats are the chief item of adulteration. Colouring matters, especially annatto, are employed. Water is worked into butter for the purpose of increasing its weight; but, as the addition of water renders butter liable to decomposition, it is only possible to escape detection in cases where the butter is rapidly disposed of. The addition of pepsin, rennet, etc., -to milk before churning aims at increasing the yield of butter by securing an increase of contained water. The Estimation of Water. — At present butter is allowed to contain 16 per cent, of water. The following two methods readily determine the amount of water; the second is the more accurate. 1. Weigh out 10 grammes of butter into a small platinum or porcelain basin provided with a piece of glass rod. Heat on a sand- bath or over a small flame, and carefully stir until all frothing ceases. It is necessary to regulate the temperature so that the curd is not appreciably browned during the heating, and that there is no loss by spirting. The basin with its contents is cooled in a desiccator and weighed. The loss of weight represents the water in 10 grammes. 2. Fill a small platinum or porcelain basin with pieces of pumice that have been recently washed and ignited. Select portions of butter from three different regions of the sample (water is not always 1 86 PR A C TIC A L SA XITA R \ ' SCIEXCE equally distiilnitcd thri)\iglu)ut tlu' mass of butter), and plaee them in a clean, wide-mouthed, stoppered bottU'. Melt at as low a tem- perature, as possible, and shake vigorously until the mass is solid. Place 5 grammes of this mass in the porcelain basin, and heat in a drying-oven with good draught at ioo° C. for an hour. Cool and weigh. Replace in the oven for a further half-hour, and again cool and weigh. Repeat the heating and weighing until a constant weight is obtained. The difference between the lowest weighing and that of the original butter is taken as water. r.eaker for melting Bottle beaker with funnel Graduated test-tube Platinum crude butter. for filtering butter-fat. for Valenta test. basin. Fig. 28. The following table represents the variations of water in Danish butters : Number of .Samoles. entage of Water. Summer. Winter. 9 to 10 I I 10 ., II 16 8 II ,, 12 136 20 12 ,, 13 335 138 13 „ 14 . . 534 431 14 ,, 15 .- 512 562 15 ,, 16 287 447 16 ,, 17 124 205 17 ,, 18 39 95 18 ,, 19 . . 13 20 Above 19 4 3 Average I 4-03 per cent. 14-41 per cent BUTTER 187 Butters containing 13-5 per cent, of water are said to liave the best flavour. Estimation of Curd and Salt.— The residue from the deter- mination of water is taken and melted at a low temperature. Ether is added, and the whole well stirred and set in a warm place until the ether is quite clear, when the fluid is decanted into a small weighed flask. Fresh ether is poured on the residue, and when clear poured off. This treatment, repeated three or four times, removes the whole of the fat. A little practice and ordinary care will prevent any of the non-fatty solids being poured away with the solvent. The residue is dried in the hot-air oven to constant weight, and represents salt and solids not fat. The Salt. — To estimate the salt, the residue from the last deter- mination is treated with hot water and filtered. The filter with its contents is well washed, and the filtrate, when cold, is titrated with standard nitrate of silver, using a 5 per cent, solution of neutral potassium chromate as indicator. The amount of sodium chloride is easily calculated. The silver nitrate should be standard- ized on pure sodium chloride. Curd. — ^The estimation of the proteins is best effected by Kjeldahl's process for the estimation of total N on the residue left after estimating the fat, and multiplying the N by 6-38. The Fat. — This item is estimated by subtracting the combined weights of water, salt, and solids not fat from 100. As a control the ethereal extract may be evaporated, and the fat residue weighed. Preservatives. — Besides salt, several substances are used as preservatives, such as boric acid, borax, formalin, salic^dates, sulphites, and nitrates. These are all estimated in the watery fluid which separates out underneath the fat on heating the butter. The estimation of boric acid is carried out as follows : Heat 10 grammes of butter in a dish; wash out the melted mass into a separating funnel with about 50 c.c. boiling water; shake thoroughly, and when fat is separated run off the water into a 100 c.c. flask. Repeat this treatment twice, using less than 25 c.c. boiling water each time. When all the washings are collected in the flask and cold, make up to the 100 c.c. mark. Filter through a drj^ Alter, and titrate 50 c.c. as described on p. 175. The titration may be iSS PRACTICAL SANITARY SCIENCE perfomicd on this solution without any trcatnient. as butter is free from phosphates. Formalin cannot be estimated with any degree of exactitude, as it enters into combination with the proteins, so that the uncom- bined formahn alone reacts, and this gives no information as to the amount originally added. To estimate salicylic acid treat 20 grammes of butter with a solution of sodium bicarbonate several times in a separating funnel : salicylic acid is converted into sodium salicylate. Acidify the extract with dilute H2SO4, and extract with ether; evaporate the ether, and to the residue add a little mercuric nitrate, forming a precipitate nearly insoluble in water. Filter the precipitate off, and wash it with water. From the washed precipitate liberate free salic3'lic acid with dilute H.^SOj. Redissolve in ether, evaporate, and dry residue at 100° C. Extract the residue with petroleum ether, and add an equal volume of 95 per cent, alcohol. Titrate with y^ KOH (phenolphthalein indicator), [i c.c. ^^ KOH = 0-0138 gramme salicylic acid.] Processes which depend on the precipitation of proteins and detection of preservatives in the filtered liquid are liable to error owing to the great solubility of salicylic acid and benzoic acid in butter-fat. The extraction of the fat with solvents (ether, alcohol, chloroform, etc.) frequently gives rise to troublesome emulsions. To overcome these troubles Monicr- Williams has devised a method of detecting small quantities of benzoic acid, saccharin, and salicylic acid in cream : Acidify 100 grammes of cream with i c.c. concentrated phosphoric acid; heat with constant stirring either in a porcelain dish on gauze over a Bunsen or on a boiling-water bath in vacuo (temperature should not rise above 120° C.) until all water is expelled. At least 95 per cent, of the salicylic and benzoic acids remain in the fat, and only the merest traces escape in the steam. Filter the clear fat through a dry filter. Allow the fat to cool to 60'' to 70° C; shake with 50 c.c. of 0-5 per cent, sodium bicarbonate previously heated to 60° to 70" C; when separated from the fat filter the alkaline liquid through a wet filter; acidify with i c.c. concentrated HCl; cool, and extract three times with 15 to 20 c.c. ether. Dry combined ether extract with CaClg, and distil off ether. The residue will have a distinctly sweet taste if saccharin be present. Stir the residue on BUTTER 189 a water- bath with i c.c. strong ammonia; evaporate to dryness; add three or four drops of water and a drop of 10 per cent, iron alum solution on a glass rod. The characteristic purple colour appears in presence of salicylic acid, and a buff-coloured precipitate in the presence of benzoic acid. The method is said to detect with cer- tainty in 100 grammes of cream the following quantities of these preservatives occurring singly or all together: 0-0075 per cent, benzoic acid; o-ooi per cent, saccharin; 0-0002 per cent, salicylic acid. Sulphites. — A portion of the watery liquid is distilled with dilute HCl, and the gas evolved is passed into -^^^ I solution, which in turn is titrated with sodium thiosulphate. Sixty-four parts of SO2 are converted into sulphuric acid by 254 parts of I. Or the SO2 gas may be passed into bromine-water, and the H2SO4 formed, estimated as BaS04. Sixty-four parts SOg represent 233-5 parts BaS04. Butter-Fat: Preparation of Fat for Analysis.— A portion of the sample of butter is placed in a beaker and heated at a tem- perature of 45° to 48° C. in an air-oven. In a little time three layers separate out in the beaker. The largest, the butter-fat, containing a few particles of curd in suspension and a few drops of water underneath the surface film on the top; a greyish- white layer, the curd, near the bottom ; and underneath a small quantity of water. If the sample be genuine butter, the melted fat is quite transparent, whereas if mixed with margarine, melted, and re- emulsified, churned at a high temperature, or rancid, it is generally turbid. The fat is poured on a dry filter kept at a temperature above the melting-point of butter, and is now free from the other constituents, except about o-i per cent, of water, and a trace of lactic acid. These manipulations are readily carried out by placing the beaker con- taining the melted butter, and a second beaker (carrying a fiinnel and filter-paper) in which the fat is received, on the top of an air- oven whose inner temperature approaches but does not exceed 50° C. After filtration the fat is rapidl}^ cooled so as to prevent partial solidification, and to obtain a homogeneous mass. Butter-fat contains considerable quantities of the glycerides of the fatty-acid series CnHzn+iCOOH, of low molecular weight. i()o PRACTICAL SAXITARY SCIEXCE The lowest and most important is butyric acid. Acids of the oleic series are also present. The various foreign fats which are admixed with butter, such as beef and mutton fats, lard, cottonseed oil, and other vegetable oils, present several important physical and chemical differ- ences. Physical Properties of Fats : i . Melting-Point. — Fats arc not single substances, but mixtures of different glycerides; the melting-points are therefore not sharp. The melted fat is drawn into a capillary tube i millimetre bore, so as to give a column about I centimetre in length. Not less than a day should elapse before the test, as even pure glycerides of fatty acids that are single chemical entities melt at a much lower temperature if they have been recently melted than that at which they melt if they are kept in the solid state for some time. The capillar}' is attached by a rubber band to the stem of a delicate thermometer, reading tenths of a degree, so that the column of solidified fat is opposite the thermometer bulb. The thermometer and its attached capillary tube are then immersed in water in a test-tube, and the test-tube in turn is immersed in a beaker of water mounted on gauze over a Bunsen burner. The water in the beaker is heated gradually (rise of temperature not to exceed 0-5° C. per minute), and the exact temperature noted at which fusion of the fat occurs: this is the melting-point. The f\a.me is removed, and the temperature noted at which the fat solidifies: this is the solidifying-point. Butter-fat melts at about 33° C. Some foreign fats have melting-points lying very near to that of butter-fat; moreover, artificial butters are made to melt at the same temperature as butter, so this test is of little practical value in distinguishing pm-e butter from margarine. 2. Specific Gravity. — On account of the glycerides of low molecular weight which it contains, butter-fat has a greater density than the fats used to adulterate it. As it is more convenient to take the specific gravity of a fluid than a solid, and as Skalweit found that at, or around, the temperature 38° C. there is the greatest difference between the specific gravities of butter and foreign fats, this temperature is usually adopted for the taking of specific gravities. Fill the pycnomcter with water at 38° C. and weigh it. Remove BUTTER 10 f the water and dry the flask in an air-oven tliroiigli whidi a j^ood current of air passes. Now fill it with fat, and place it in water at 38° C. till the volume is constant. Weigh again rapidly, and the weight of the fat divided by the weight of water gives the specific gravity at 38° C. The limits of specific gravity for pure butter-fat at 38° C. are 0-914 and 0-909. The fats usually added as adulterants have a mean specific gravity of 0-903. The density may also be determined by a hydrometer or by West- phal's balance at 38° C. The presence of glycerides of lower fatty acids raises the specific gravity of a fat; hence rancidity is accompanied by an increase in the specific gravity. A pycnometer with capillary side-tube can be used for estimating the specific gravity of solid fats : the bottle is filled with water and weighed; a weighed amount of the solid fat is introduced and the stopper inserted. The diminution of weight plus the weight of the solid fat gives the amount of water displaced and the volume of the fat. 3. Solidification-Point and litre Test.— When melted fat is cooled, the temperature falls gradually to a variable degree, then rises rapidly to a certain constant temperature, at which it remains steady for a time before it begins to fall again; this maintained temperature is the solidification -point. This test is generally carried out on the fatty acids obtained by saponification of the fats, and is then known as the ' Titre test.' A method of carrying it out is the following: Saponify 75 grammes of the fat in a metal dish with 60 c.c. of 30 per cent. NaOH and 75 c.c. 95 per cent, alcohol, or 120 c.c. water. Evaporate to dryness and dissolve in i litre of water. Boil to remove alcohol. Add 100 c.c. 30 per cent. HgSOj, and heat till clear: the fatty acids are separated. Wash them with hot water till free from soluble acids, and filter through a dry filter on a hot-water funnel. Dry for twenty minutes at 100°, and cool down to within 15° to 20° of the solidification-point. It is important that the fatty acids be thoroughly dried. Pour into a test-tube 100 millimetres long and 25 millimetres in diameter, which is sus- pended by a cork in the mouth of a jar 70 millimetres wide and 150 millimetres high. A thermometer, graduated in tenths of a degree, between 10° and 60°, with a bulb 3 centimetres long by 192 PRACTICAL SANITARY SCIENCE 6 millmutivs diameter, is made to act as a stirrer. The determina- tions should not vary by more than o-i° C. 4. TKe Refractive Index : Oleo-Refractometpy.— The oleo- refractometer measures the rei'ractinn wiiich a ray of hght under- goes in passing through a layer of butter. It is found more con- venient to read the angle of total reflection, as indicated by the sharp colourless border-line which vertically intersects the scale of the instrument between the light and dark sections of the field of view. A few drops of the butter-fat to be tested are poured warm into the prism of the instrument, and the deviation noted at a temperature of 45° C. Pure butter gives a deviation of about 30° to the left. Certain forms of margarine give deviations much less — 15° and 20° — whilst cocoa-nut oil gives over 55°. The butyro - refractometer of Zeiss is a modification of the Abbe refractometer, and gives rapid readings in scale divisions which by reference to a table can be read off as refractive indices . 5. Microscopic Examination. — When examined microscopically butter-fat presents a collection of small round refractile globules, together with a few larger globules fairly uniform in size and in the number present in a single field. Margarine presents a mass of small globules much less distinct in outline and more crowded together. The larger globules occur in relatively greater numbers, and present much more diversity in size. Chemical Methods used in Analysis of Fats : i. The Acid Value- — This is represented by the number of milligrammes of KOH required to neutralize a gramme of the fat. It is accordingly a measure of the degree of hydrolysis of the fat which may be due to rancidity or to ferment action. Five to ten grammes of the fat are dissolved in alcohol and titrated against tenth normal alkali in presence of phenolphthalein or alkali blue, 66 of Meister, Lucius, and Briining. 2. The Saponification Value is a measure of the mean molec- ular weight of the fatty acids entering into the composition of a fat. It is to be noted that in the titration of fatty acids soaps are hydrolysed by water, and accordingly react alkaline; such hydrolysis is prevented if 40 to 50 per cent, of alcohol be present. The saponification value is given by estimating the number of BUTTER 193 milligrammes of KOH neutralized in saponification of i gramme of the fat by the total fatty acids that it contains, whether originally combined with glycerol or other alcohol, or free. Heat 2 grammes of fat with 25 c.c. of alcoholic potash in a Jena flask (glass that does not give off alkali) under a reflux condenser for half an hour. Carry out a blank control with the same volume of alcoholic KOH in a similar flask lest the titre be altered by COg or other agency during heating. When the saponification is complete titrate the alkali in each flask with | HCl and phenolphthalein. The difference between the amounts of acid required by the two flasks gives the amount of alkali neutralized by the fatty acids contained in and liberated during saponification from the amount of fat taken; from this the saponification value is calculated. 3. The Hehner Value. — This is the percentage of fatty acids insoluble in water produced on saponification by a fat. Two or three grammes of the fat are saponified with alcoholic potash. The saponified mass is washed with hot water into a beaker on a steam bath, and acidified with dilute H2SO4. When the subjacent aqueous layer has become clear, the contents are filtered through a weighed filter; it is well to half fill the filter with water before pouring the fatty acids on. The beaker is washed with a jet of hot water, and the acids washed continuously as long as any acid reaction can be detected in the washings. The filter with its funnel are then immersed in cold water, so that the fatty acids solidify; the filter is dried and weighed, or when dry it may be extracted in a Soxhlet apparatus with petroleum ether, the ether evaporated, and the residue dried and weighed. The Hehner value of butter is between 86 and 88, of triolein 95-7, of lard and most oils about 95. 4. The Iodine Value. — This value gives the amount of halogen reckoned as iodine that the unsaturated acids in the fat take up, expressed as a percentage by weight of the fat. Triolein, for example, whose molecular weight is 884, takes up 6 atoms of iodine (6 X 127 = 762), or 86-2 per cent.; oleic acid has an iodine value of 90-1. As saturated acids and their glycerides absorb no halogen, the iodine value is a measure of the amount of unsaturated acids 13 194 PRACTICAL SANITARY SCIENCE present. Acids with unsaturated bonds in more than one phice absorb proportionately more iodine. Determination by the Method of Wijs.— Prepare a titrated sokition ol iodine nionochloridc, a titrated solution of sodium thiosulphate, and a lo per cent, solution of KI. The monochloridc is obtained thus : Weigh 9-4 grammes iodine trichloride into a 300 c.c. flask, pour in 200 c.c. glacial acetic acid, fit the flask with a cork through which passes a CaCL tube ; heat on a water-bath till the contents are dissolved. Weigh 7-2 grammes of iodine, which has been thoroughly pulverized in a mortar, into a second flask; w'ash out the mortar with glacial acetic acid, and heat this flask as the other. Pour the contents of the two flasks into a stoppered litre flask. Any undissolved iodine is fmrther heated with additional acetic acid till all is dissolved and added to the litre flask. This flask is then stoppered and allowed to cool; when cold, the solution is made up to a litre with acetic acid and titrated next day. The strength of the iodine chloride solution is likely to alter a little in the first twenty-lour hours, but after that remains fairly constant for some weeks if care has been taken to exclude all water from the glacial acetic acid. To do the titration pipette into an Erlenmeyer flask exactly 20 c.c. of the iodine chloride solution; add about 10 c.c. of the KI solution and about 300 c.c. of water. Run in a standard sodium thiosulphate solution (24 grammes to a litre standardized by Volhard's method), and finish off with starch solution. From the amount of thiosulphate used the amount of iodine in the measured amount of Wijs's solution is calculated. Estimation of the Iodine Value of the Fat or Fatty Acid.— Weigh into a stoppered flask of 100 to 150 c.c. capacity a quantity of the fat or fatty acid depending on the iodine value of the Wijs's solution used (there should be two or three times as much iodine in the Wijs as the fat can absorb), say 0-2 to 0-5 gramme, and dissolve it in 10 c.c. CCI4; add, say, 25 c.c. Wijs, stopper and stand aside for a couple of hours in the dark. Now pour the contents of the flask into an Erlenmeyer (half to litre size) ; wash out any traces of iodine with 10 c.c. of the KI solution and afterwards with water; the bulk of fluid obtained should be about 300 c.c. Lastly titrate with thiosulphate and calculate the unabsorbed iodine. BUTTER 195 This figure, subtracted from the amount of iodine contained in the Wijs used, gives the iodine absorbed, which is readily calculated into a percentage of the amount of fat taken. 5. The Acetyl Value. — This determination gives the number of milligrammes of caustic potash required to neutralize the acetic acid liberated when i gramme of acetylated fat or fatty acids is saponified. Those fatty acids in a fat or oil which are hydroxy become acetylated when heated with acetic anhydride: RC<^2"^.C00H + CH^'CO/^" RC/^^^^CO.O (,QQj^ ^ CH3COOH. The number of acetyl radicals taken up depends on the number of hydroxy acids present, and the number of hydroxyl groups contained. On saponification, the acetyl groups are split off as acetic acid, and the amount of acetic acid so liberated is a measure of the hydroxyl groups. Heat 5 to 10 grammes of the fat or fatty acids with twice the weight of acetic anhydride in a flask under a reflux condenser for a couple of hours. Transfer the contents to a large beaker, and add I litre of boiling water. Heat for half an hour whilst a stream of CO2 is led through to prevent bumping. Separation is allowed to take place, and the watery layer is syphoned off. Salt may be added if the oily layer does not separate easily. More water is added, and in turn syphoned off till the acetic acid formed from the excess of anhydride has all been removed. Filter through a dry filter in a drying oven to remove water. Weigh 3 to 5 grammes of the acetylated product ; saponify with a known amount of KOH and determine the saponification value. Free the soap from alcohol by evaporation, and estimate the acetic acid as follows : Add an excess of 10 per cent. HgSO^, and distil the liquid in a current of steam. Collect the distillate until 100 c.c. require not more than o-i c.c. of decinormal alkali to neutralize it. More than 600 c.c. of distillate will generally be obtained. Titrate this with decinormal alkali (phenolphthalein indicator), and multiply the number of c.c. used by 5-61; divide the product by the weight of the acetylated product used to get the acetyl value. 6. The Reichert-MeiSSl Value.— This is that usually applied for identification of butter-fat. It is a measure of the amount of lower 196 PRACTICAL SAXITARY SCIENCE fatty acids in a fat which volatilize in a current of steam. The value is expressed b\' the number of c.c. of -^^^ alkali required to neutralize the volatile fatty acids liberated under certain prescribed conditions from 5 grammes of the fat. In this country the Wollny modification is used. Most fats and oils in the fresh state contain only traces of volatile acids or their glycerides, and give values less than one. Cocoa-nut oil gives Reichert-Meissl-Wollny value 5 to 8; butter a notable exception possessing a value 26 to 32, Some porpoise oils are said to reach 60. The estimation is a comparative one, and that only whilst the conditions are accurately observed. Weigh 5 grammes of prepared butter-fat into a flat-bottomed flask of 300 c.c. capacity, having a neck 7 to 8 centimetres long by 2 centimetres wide. Add 2 c.c. NaOH solution prepared by dis- solving 98 per cent. NaOH in an equal weight of water (protected from the action of atmospheric COo) and 10 c.c. 92 per cent, alcohol. Heat the flask on a boiling bath for fifteen minutes under a reflux condenser. Remove the condenser, and drive off the alcohol com- pletety by heating further for half an hour. Add 100 c.c. of water which has been boiled (to remove CO.,). and heat till the soap dis- solves. Add 40 c.c. of normal sulphuric acid and some bits of pumice or porous clay, and connect the flask with a condenser tube 7 millimetres in diameter, surrounded by a water-jacket 35 centi- metres long by means of a bent tube 15 centimetres long from the cork of the flask to the bend of the tube, on the middle of which a bulb, 5 centimetres in diameter, is blown. The flask is heated on an asbestos board, with an opening in its centre 5 centimetres in diameter, by a small flame till the insoluble acids are melted. When fusion is complete the heat is increased, and no c.c. are distilled in about thirty minutes into a graduated flask. Shake the distillate and filter off 100 c.c. into a beaker; add 0-5 c.c. of a i per cent, solution of phenolphthalein in alcohol, and titrate with — soda or baryta. Carr}' out a blank experiment with the same quantities of everything except fat ; the amount of ^^^ alkali required to neutralize the distillate should not exceed 0-2 to 0-3 c.c. The number of c.c. decinormal alkali used, less the blank, multiplied by i-i, gives the Reichert-Meissl-Wollny number. Leffmann and Beam employ 20 c.c. of glycerol instead of the BUTTER 197 alcohol used by Wollny. They heat the fat with glycerol and soda for eight minutes, when the fluid becomes clear and is allowed to cool to about 80° C. Then 90 c.c. of water at about 80° C. and 50 c.c. of a 2'5 per cent. H2SO4 solution are added, and the process is finished as above. In this distillation only a part of the volatile acids distils over [87 per cent, of total volatile acids (according to Richmond) ; 88 per cent, butyric, 88 to 100 per cent, caproic, 24 to 25 per cent, capryllic (according to Jensen)]. Five grammes of pure butter-fat give a number never less than 24, margarine never more than 3. In order to prevent fraud not more than 10 per cent, of butter-fat is permitted in margarines, which will produce a Reichert-Wollny number of 4. Example. — In a mixture of margarine and butter-fat the Reichert- Wollny figure is 16; find the percentage of butter-fat. Taking 3 as the highest possible figure for margarines, and 24 as the lowest for butter-fats, 21 (24-3) will represent 100 per cent. of pure butter-fat. 21 : 16-3 : : 100 : x. 13 X 100 , , x=-^ =62 nearly. 21 ^ This sample, therefore, contains 62 per cent, of butter-fat, and consequently 38 per cent, of margarine. The Polenske Number. — This number represents the volatile fatty acids insoluble in water. It is much used in detecting cocoa- nut oil in butter and other fats. It may be determined with the Reichert-Meissl figure in one weighed portion of the fat. Saponify 5 grammes of prepared fat with 20 grammes of glycerol and 2 c.c. of a 50 per cent. NaOH solution. This requires about five minutes, and is complete when the liquid is quite clear. While still hot add 90 c.c. of boiled water, at first drop by drop, to prevent frothing, and shake till the soap is dissolved. Warm to 50°, and add 50 c.c. dilute H2SO4 (25 c.c. to a litre) and ^ gramme of granu- lated pumice (grains i millimetre in diameter). Connect with dis- tilling apparatus used in the Reichert-Meissl method, and distil over no c.c. in twenty minutes. Cool the flask by immersion in water at 15° C. Stopper it, and invert four or live times. Filter 198 PRACTICAL SANITARY SCIENCE through a dry lilter fitted close to the funnel (loo c.c. of the filtrate may be titrated for the Reichert-Meissl number). Wash the material on the lilter with three 15 c.c. portions of water, each of which have washed out the flask and the condenser. Dissolve the fats on the filter with three 15 c.c. portions of neutral 90 per cent, alcohol. Titrate the united alcoholic washings ^^'ith -j^ barium hydrate, using phenolphthalein as indicator. The number of c.c. used is the Polenske number. Samples of butter possessing Reichort-]\Ieissl figures 25 to 30 will give Polenske numbers of 1-5 to 3. Samples of cocoa-nut oil of Reichert-IMeissl values 6 to 7 will give Polenske figures 16 to 17. Lard and tallow give Reichert and Polenske tigures of about 0-5 each. Valenta's Test. — \'alenta demonstrated the fact that there is a considerable difference in the temperatures at which various fats dissolve without turbidity in acetic acid. Weigh out 275 grammes of butter- fat into a test-tube; add 3 c.c. 99-5 per cent, acetic acid; insert a thermometer, and gently heat with vigorous shaking until the mixture becomes transparent. Now cool down gradually, stirring with the thermometer until the first trace of opacity makes its appearance, generallj^ as a fine tail in the fluid at the extremity of the thermometer's bulb. This is the required temperature. The glycerides of the saturated fatty acids are deposited as the acetic acid cools. The temperature corresponding with pure butter-fats runs from 29° C. to 39° C, and that for margarine falls between 94° C. and 97° C. A standard sample of butter may be tested against a weaker acid giving a temperature of turbidity of, say, 60° C. Margarine then gives 100° C. or over. This is a very good preliminary test for the differentiation of pure butters from margarine. Examination under Polarized Light. — A small particle of butter is placed on a clean microscopic slide, and a cover-glass affixed. The slide is placed on the stage of a microscope provided with crossed nicols, and examined with a coarse objective. In order to shut out light from the upper surface a short black tube is laid on the slide in such a manner that the objective dips into it. When pure butter-fat, which is non-crystalline, is examined, it pre- BUTTER 199 scnts a uniformly dark jicld. On the otlicr hand, when margarine is examined, certain portions of the field arc bright, and crystalline masses are dimly perceived. These may be critically studied by uncrossing the nicols. To Detect Cottonseed Oil in Butter.— To the melted fat add an equal volume of a saturated solution of lead acetate, and a smaller quantity of ammonia, and stir. On standing for a little time the superficial layers turn orange-red. Bechi's Silver Test. — Dissolve i gramme AgNOg in 100 c.c. of 95 per cent, alcohol; add 20 c.c. ether and a drop of HNO3, and thoroughly mix. To 10 c.c, of the sample of fat add 2 c.c. of this reagent. Mix and stand the test-tube in boiling water for fiifteen minutes. The mixture assumes a considerably darker tint, due to reduced silver, in the presence of cottonseed oil. These tests are best performed in the presence of blank tests on pure butter- fat. Halphen's Test. — Mix equal vcrlumes of amyl alcohol and CSg in which I per cent. S has been dissolved. To 5 c.c. of this mixture add an equal volume of the fat in a test-tube, and heat in a bath of boiling saturated brine for fifteen minutes. A deep red or orange colour is produced in the presence of cottonseed oil. In its absence little or no colour is developed. Pyridin in the amyl alcohol appears to be the active reagent. Sesame Oil. — Fats containing this oil give a red colour when heated with stannous chloride on a water-bath. The coloxir is not discharged by moderate dilution with water, thereby differing from the colour produced by turmeric. Baudouin' s Test. — Dissolve o-i gramme cane-sugar in 10 c.c. HCl (specific gravity 1-2). Add to the solution in a test-tube 20 c.c. of the fat and shake thoroughly for a minute. Allow to stand till the oil separates from the aqueous solution. In the presence of i per cent, sesame oil the aqueous solution is coloured deep red. Colouring^ Matters.- — Annatto, turmeric, and some coal-tar pro- ducts have been used to increase the yellow tint of butter. These colouring matters are, for the most part, vegetable, and harmless. If the colouring matter can be extracted with alcohol it is foreign, since the natural colouring matter of butter is not soluble in alcohol. Coal-tar dyes may be fixed on silk or wool by boiling fibres in the alcoholic extract diluted with water and acidified with HCl. 200 PRACTICAL SANITARY SCIENCE If saffron be present, the alcoholic extract will be coloured green by HXO3, and red by HCl and sugar. Tunueric is detected by evaporating the alcoholic extract to dryness, and boiling the residue in a few c.c. of dilute boric acid solution. A strip of filter-paper soaked in the latter and slowly dried becomes cherry red. Addition of a drop of alkali turns the red to olive green. In recent years a number of liquid fats have been hydrogenated by the catal^'tic action of nickel and other catalysts — oleic acid, e.g., becoming stearic acid — Ci8H3402 + H2= CjgHggO.^. The physical change from liquid to solid has enabled manufacturers to incorporate various oils in margarines and butter. Whether such hardened fats are equally digestible and equally nutritious with the natural bodies they now chemically represent remains to be seen. Their appearance has caused considerable trouble to analysts, as many of the ph^-sical and chemical constants have been completely upset. Bacteria in Butter. — Economic bacteria take part in the conver- sion of cream into butter. In Europe and America much butter is made from pasteurized cream, to which ' starters ' (cultures of lactic acid bacteria) are added. By this means the process of butter-making is much better controlled, and results are much more uniform. British butter contains from 1,000,000 to 50,000,000 micro-organisms per gramme. The bulk of these are Bacillus acidi lactici, B. lactis aerogenes, etc., which keep in check the development of unfavourable bacteria, such as B. mesenfericus, B. fluorescens, B. suhtilis, etc., which give origin to evil flavours, bitter taste, and rancidity. The principal pathogenic organism found in butter is the B. tuberculosis. To detect this organism warm a sample of butter to 42° C. Centrifugalize the liquid, and inoculate guinea-pigs with the sediment. It is of importance to note that the butter bacillus of Rabinowitsch and Petri is acid-fast, and morphologically like the tubercle bacillus; and, moreover, when injected intraperi- toneally mixed with butter, it produces similar lesions in guinea- pigs. It is, however, readily distinguished from B. fttberctdosts by its rapid growth on glycerine agar and other ordinary media, forming an abundant dry mass in three or four days. CHEESE CHEESE Cheese consists, for the most part, of proteins and fat. It may be prepared (i) by adding rennet to milk, whereby the casein clots and entangles most of the fat; and (2) by allowing the milk to become sour through the formation of lactic acid, or by the addition of a dilute acid, such as vinegar, when the cheese contains little fat. The characters of different cheeses depend on the kinds of milk used, the methods of preparation employed, and the types of micro-organism admitted to the original milk or to the cheese whilst ripening. During the ripening of cheese a partial digestion of proteins is effected, resulting in the production of the so-called primary products of digestion — albumoses and peptones. Later, secondary products of ripening are found — viz., amido-compounds and ammonia. Whether during these changes fat is increased at the expense of protein, as was once believed, is doubtful. The relative propor- tions of this digestive work carried out respectively by milk enzymes and by enzymes of added bacteria are unknown. The flavour of a particular cheese is due to the micro-organisms growing in it during ripening. The old idea that a particular cheese, such as Stilton, can be made only in one locality is exploded. Magnificent Stiltons are now made in Hampshire by the agency of a ' cheese mould ' carried to that county from Leicester. Soft cheeses, such as Brie and Camembert, are produced by clotting milk with rennet at temperatures below 30° C, and using little pressure. Hard cheeses, like Stilton, Cheddar, Gorgonzola, and Gruyere, are clotted at higher temperatures — 30° to 35° C. — and submitted to greater pressure. Soft cheeses contain much water, and therefore fail to keep long. The nitrogen of the proteins in cheese exists in a variety of forms. Van Slyke found that the 3-86 per cent. N of an American Cheddar was distributed as follows: Water-soluble N 1-46, para- casein-mono-lactate 0-94, paranuclein 0-14, caseoses 0-22, peptones 0'i8, amides 0-79, and ammonia 0-13. A full-cream cheese contains 30 to 35 per cent, butter-fats. Filled cheeses may contain any proportions of foreign fats mixed with butter-fats. 202 PRACTICAL SAXITARY SCIENCE If the fat is considerably less than the protein, the cheese was made from skimmed milk. fat In a whole-milk cheese the ratio ^ r^^, is greater than i 6-37 total N (generally 1-25 to 1-5). In a skimnied-milk cheese this ratio is less than i. The digestibility of cheese in the stomach is less than that of meat, on account of its proteins being covered with fat. Cheese should therefore be well masticated, or, better, thoroughly grated before being used. Its digestion in the small intestine is effected without difficulty. Owing to the small quantity of water con- tained in cheese compared with that in beef, it has a higher nutritive value than the latter. The energy derivable from cheese, as measured b}' calories, is about three times that of beef. More- over, the fact that the protein of cheese is chiefly casein, and accordingh' purin-free, should highly' recommend it as an article of diet to those who are in any wa}- troubled by uric acid. Percentagre Composition of a Few Soft Cheeses : Camembert Brie Stracchino . . Water. Proteins. Fat. Ash. 50-9 i8-6 27-4 3-1 50-0 i8-3 27-6 4-1 39-2 29-3 277 3-8 of a Few Hard Cheeses: Water. Proteins. Fat. Ash. 27-2 36-6 32-0 4-2 30-4 36-1 287 4-8 28-6 35-6 31-8 4-0 32-0 35-1 28-1 4-8 Cheddar Cheshire Stilton Gruyere .... The ripening of cheese has been somewhat differently explained by Freudenreich, Duclaux, and Babcock and Russell. When the curd is thrown down by rennet, it carries with it most of the bacteria of the milk. Freudenreich believes that the lactic acid organisms, which develop early and rapidly, are the chief factors in the process of ripening. Duclaux holds that, since the ripening proceeds after the lactic acid organisms have considerably diminished, the active agents are enzymes secreted by a variety of organisms, which he CHEESE 203 names Tyrothrix. Babcock and Russell ac(:c[)t the view that ripening is effected by an enzyme originally present in milk. Moulds in Cheese. — Green mould [Penicillium glaucum) is found in Roquefort and Gorgonzola. Aspergillus glaucus pro- duces the appearance known as blue mould, whilst red mould is accounted for by the growth of Sporendonema casei. The common mould {Mucor mucedo) is found in more than one variety. Of animal parasites found in cheese, the two most frequently met with are ' the cheese mite ' {Acarus domesficus), and ' cheese maggots ' (larv?e of Piophila casei). Adulteration in Cheese. — The principal adulterations in cheese are the use of skimmed milk for whole milk, and the addition to skimmed milk of foreign fats. Mineral adulterants, such as chromate of lead, used to tint the rind and sulphate of zinc (' cheese spice '), used to prevent gas formation from fermentation, are rarely met with. Estimation of Water in Cheese. — Dry 5 grammes cut into thin slices in an air oven at 100° C. to constant weight. Loss of weight equals water.- Ash. — Ignite the residue from the water determination at a low red heat; cool in a desiccator and weigh. Fat.— Place 50 grammes of cheese in a muslin bag in a beaker on a water-bath; the fat will pass out in. a pure state into the beaker. Perform the Valenta and Reichert-Meissl tests on this (as described under Milk) to detect and estimate amounts of pure butter- fats and foreign fats. The total fat is estimated as follows: Grind 5 grammes of cheese in a mortar with 10 grammes of anhydrous copper sulphate ; place a layer of anhydrous copper sulphate about 2 centimetres thick on the bottom of the receiver of a Soxhlet ; add the ground mixture, and rinse the mortar with a little of the ground sulphate and afterwards with ether. Extract for sixteen hours. Evaporate the ether from the extraction flask, and dry the fat in a steam-chest to constant weight. Werner-Schmidt Method. — Boil 2 grammes of cheese with 5 CO. of water and 10 c.c. of concentrated HCl in a large test-tube, with constant shaking until all but the fat is dissolved. Cool; add 25 c.c. ether, and shake well. When separated, draw off as much 204 PRACTICAL SANITARY SCIENCE as possible of the ether. Extract with four additional portions of ether, and collect the whole in a flask. Distil off the ether and weigh the fat. Proteins. — Treat I gramme of cheese by the Kjeldahl method. N X 6-25 = proteins. Separation and Determination of N Compounds (Van Slyke). Mix 25 grammes of cheese with an equal weight of quartz sand in a mortar, and transfer to a flask; add about 100 c.c. of water at 50^, and keep the temperature at 50° to 55""' for half an hour, shaking frequently the while Decant the liquid through a cotton filter into a 500 c.c. graduated flask. Treat the residue with repeated portions of 100 c.c. of water in the same manner until the water extract amounts to just 500 c.c. Employ aliquot parts of this for the various estimations. Watcr-Soliihle N. — Perfonn the Kjeldahl process on 50 c.c. of the water extract ( =2-5 grammes of cheese). Para-Niiclein N. — To 100 c.c. water extract add 5 c.c. of a i per cent. HCl solution; stand at 50° to 55° till separation is complete, as shown by a clear supernatant liquid. Filter, wash the precipitate with water, and determine the N by Kjeldahl. N as Coagiilahle Protein. — Neutralize the filtrate from the last determination with dilute KOH; heat at 100° till the coagulum, if any, settles out completely. Filter, wash the precipitate, and determine the N in it as above, N as Caseoses. — Treat the filtrate from the preceding with i c.c. of 50 per cent, sulphuric acid, saturated with ZnS04, and warm to 65° to 70° until the caseoses settle out completely. Cool, filter, wash with saturated ZnSOj acidified with H2SO4, and determine the N in the precipitate. N as Amides and Peptones. — Put 100 c.c. of water extract in a 250 c.c. graduated flask, add i gramme NaCl and a 12 per cent, solution of tannin till a drop added to the clear supernatant solution fails to produce further precipitation; dilute to the mark, shake, and pour on a dry filter; determine the N in 50 c.c. of the filtrate = N in amido-acid and ammonia compounds. This minus the ammonia N = amido-X. Peptone N = total N in water extract minus sum of para-nuclein X, coagulable protein X, and X of caseoses, amides, and ammonia. CHEESE 205 A^ as Ammonia. — Distil 100 c.c. of the filtrate from the tannin- salt precipitation into standard acid, and titrate against standard alkali, N as Para-Casein Lactate. — Wash the insoluble residue produced in obtaining the water extract with several portions of a 5 per cent. NaCl solution to form a 500 c.c. salt extract; determine the N in an aliquot part of this salt extract. Determination of Lactose. — Boil 25 grammes of finely divided cheese with two portions of 100 c.c. each water. Pour on filter, wash residue with hot water, make up the watery extract to 250 c.c, and determine the lactose by the Fehling or Pavy-Fehling method. Detection of Foreigrn Fat. — Submit the prepared fat to the Reichert-Meissl method. Detection of Bacillus Tuberculosis. — Rub up portions from the central parts of the cheese with sterile normal saline until a good emulsion is obtained. Strain through sterile absorbent cotton, and inject the equivalent of 2 grammes of cheese into each of two or three guinea-pigs. Lard. — Freshly rendered lard (internal abdominal fat of pig) con- tains no free fatty acids. It is much adulterated with cottonseed oil and beef stearin. It has the following constants : Melting-point, 36° to 45° C; iodine absorption, 50 to 65 per cent.; saponification value, 195 to 197; Zeiss butyro-refractometer at 40° C. =48-8° to 51°; specific gravity at 15-5° C, 0-931 to 0-932. If the iodine value fall outside the above limits, the lard is adulter- ated, but a normal iodine figure is no guarantee of genuineness, as a judicious mixture of cottonseed arachis or other oil, with beef stearin, will give normal values when tested. Infants' Foods.- — The market is flooded with a large number of products of very varying composition. If the milk preparations (condensed, dried, and humanized milks) be grouped as a class, all the other foods contain flours in which the starch is altered or unaltered, or capable of being altered or otherwise during prepara- tion of the food. It is necessary to determine the presence, nature, and amount of unaltered starch, the extent to which the starch is converted during the preparation of the food according to instruc- tions on the label, the presence or absence of diastase in active form, and the nature of the cereal from which the starch is derived. 2o6 PRACTICAL SAXITARY SCIENCE Witli the exception of full-cream dried milks and full-cream con- densed milks, it may be fairly stated that practically all the infants' foods advertised are highly deficient in fat, and many deficient in proteins. The only physiologically suitable food for a young mammal is the milk of its mother or some other animal of the same species. The chemical composition of a foodstuff is no criterion of its nutritional value. Due proportion of protein carbohydrate and fat does not constitute a correct diet. For example, the proteins of different milks \"ary because of the fact that milks have a develop- mental as well as nutritive function: accordingly milks of different species are not interchangeable. It is known that the milk of animals whose chief digestion is gastric (cow, goat, etc.) forms solid clots of casein, \\hilst that of animals whose chief digestion is intestinal (mare, etc.) does not form solid clots, but soft gelatinous masses, which easil}' traverse the stomach and intestine. The digestion of infants is largely intestinal, and human milk is the onl}^ form which in the early days puts no strain on it. It is common clinical knowledge that infants in the first months of life fed on artificial foods containing starch become the subjects of scurv^', atrophy, and gastro-intestinal disorders. The process of digestion is accompanied by the liberation of considerable potential energy-, and in the case of the infant with little energy to lose the digestion of an artificial food, containing in addition to a foreign milk starch only partially converted, there may not be nearly sufficient energy to meet the largely increased call, with the well- kno\vn accompaniments of this failure — grave nutritional dis- turbance, rachitis, scurvy, anaemia, and more than one form of profound gastro-intestinal fermentation. Estimation of Starch: i. Direct Conversion by Acid.— Hemicellulose and all carbohydrates capable of conversion to sugar are included with starch. Wash 2 grammes of the finely divided material on a filter with ether, using lo c.c. four or five times; continue the washing with first 100 c.c. 10 per cent, alcohol, and then with lo c.c. absolute alcohol. Now wash off the contents of the filter into a flask with 150 c.c. water and 20 c.c. HCl (specific gravity, 1-125). Place the flask on a boiling-water bath under a reflux condenser for two hours. CHEESE 207 Cool, neutralize with NaOH, add alumina cream if necessary, mix, make up to | litre, filter, and estimate the dextrose in an aliquot part of the filtrate by the polarimeter or by Fehling's method. Calculate the dextrose figure into starch by multiplying it by o-g. 2. Conversion by Diastase. — By this method starch only is acted on; hence in the presence of other substances it is to be recommended. Starch is first converted into maltose and dextrin, and finally into glucose. Prepare 2 grammes with ether and alcohol as above. Wash off the filter into a beaker and boil for fifteen minutes, or until com- pletely gelatinized, stirring constantly. Cool to 55°, and add sufficient malt extract (10, 15, 20 c.c, according to degree of activity), or better animal diastase, and digest for an hour at 55^. Boil for fifteen minutes, add further animal diastase, replace water lost by evaporation, and digest for another hour, or until when treated with iodine under the microscope no starch appears. Cool, make up to 250 c.c, add 20 c.c. HCl (specific gravity, 1-125), S-^id proceed as in the acid conversion method. Reducing Sugars as Dextrose (Lactose excepted). — ^A cold- water extract is made and titrated with Fehling's solution. Lactose. — Baker and Hulton have shown that an aqueous solu- tion of lactose, unlike maltose, dextrose, cane-sugar, etc., is not fermented by ordinary brewer's yeast; hence small amounts of lactose added to flours, etc., can be estimated by measuring the reducing action on Fehling's solution of the residue after fermen- tation. Boil the aqueous extract with 2 per cent, of citric acid to invert any cane-sugar, thus facilitating fermentation ; neutralize ; cool and add a little cold aqueous extract of diastatic malt. Close the con- taining flask with cotton-wool, and incubate at 27° C. for seventy- two hours. The solution, now destitute of all reducing sugars except lactose, is cleared with alumina cream, filtered, boiled, made up to an appropriate volume, and titrated with Fehling's solution. Ten c.c. of Fehling's solution =0-074 gramme of pure lactose. Cane-Sugar. — A portion of cold water extract is boiled as above with 2 per cent, citric acid, the solution neutralized, and titrated with Fehling's solution. From the value of the invert sugar so 2oS PRACTICAL SANITARY SCIENCE obtained is subtracted the dextrose already found; the difference reduced by 5 per cent, (hydration correction) is the percentage of cane-sugar. Fat. — Fat of dried milks. Owing to the inclusion of fat globules amongst dried proteins the solvent action of ether in the Soxhlet method may be greatly inhibited. The Werner- Schmidt method is in this case more suitable. Proteins. — The N is determined in 0-5 gramme of the food by Kjeldahl's method, and the figure x 6-25. Water. — Five grammes are dried on a water-bath (five hours or longer) to constant weight. Ash. — Five grammes are burnt at a dull red heat in a muffle. If the food bums with difficulty, H.2S04may be added, and a correc- tion made by deducting one-tenth of the weight of the ash. Cellulose (material insoluble in boiling* water and not attacked by diastase). — To 5 grammes of the food freed from fat by ether if necessary add 200 c.c. distilled water, and bring to the boil. Continue the boiling for half an hour. Add some cold extract of malt (15 to 25 c.c. according to degree of activity), and digest at 55° to 60'^ for three or four hours. Filter through a dry tared filter. Wash the residue repeatedly with water at 60° C. until free from all reducing sugar; then with alcohol and ether. Dry for several hours on water-oven and weigh. Transfer filter-paper and residue to a Kjeldahl flask, and determine the protein. Carry out the procedure in duplicate, but in the second estimation determine the ash instead of the protein . The first weight less the protein and ash = cellulose. Saccharifying" Diastase. — Two or three c.c. of a 5 per cent, cold- water extract of the food are allowed to act for an hour at 21° on 100 c.c. of a 2 per cent, soluble starch solution. At the end of the hour the action is stopped by the addition of 10 c.c. /^y NaOH, and the whole made up to 200 c.c. The amount of maltose present is determined by Fehling's solution. A food may be regarded as having a diastatic activity of 100 when 0-2 c.c. of the 5 per cent, solution produces under these conditions sufficient maltose (o-o8 gramme) to reduce completely 10 c.c. Fehling's solution. If double the amount is required, the diastatic power is 50, etc. CEREALS 209 CEREALS. The composition of a few common cereals is given in the following table : Wheat Barley Rye Oats Maise Millet Rice Protein, Fat. J^f^^' Cellulose, Water. Ash. nyclrates. II-O 1-7 71-2 2-2 12-0 1-9 lo-i 1-9 69-5 3-8 12-3 2-4 I0'2 2-3 72-3 2-1 II-O 2-1 ii-o 5-2 57-3 12-0 II-8 27 9-9 5-4 68-9 2-2 12-3 1-3 10-4 3-9 68-3 2-9 12-3 2-2 6-8 1-6 68-1 9-0 10-5 4-0 It will be seen that the proteins vary somewhat in amount in the different cereals. The fat appears in increased quantities in those cereals which grow in high latitudes. The chief carbohydrate is starch: it forms 65 to 70 per cent, of the whole grain. The ash averages about 2 per cent., and is composed principally of lime and phosphoric acid, thus resembling the ash of animal foodstuffs much more than that of vegetables. The high percentage of carbohydrates is an indication that cereals should be mixed with other foods richer in proteins and fat; this physiological require- ment we find almost universally complied with: butter is spread upon bread, and the mixture eaten with cheese. On the whole, cooked cereals are easily digested and absorbed. Wheat-FlouP. — ^Wheat is the most important cereal used in this country. It is consumed to the extent of six bushels per head per annum. The grain of wheat consists of three portions: (i) The bran or outer envelope of cellulose, containing mineral matter, and forming 13-5 per cent, of the grain; (2) the endosperm, con- stituting 85 per cent, of the whole, and consisting of nutritive material for the growth of the embryo; (3) the embryo or young plant, forming 1-5 per cent, of the grain. The bran consists of an outer layer of fibres of cellulose impregnated with salts, a middle layer of pigment cells, and an inner layer of aleurone grains. The endosperm consists of a delicate reticulum of cellulose, in whose meshes are found numerous starch granules. The embryo is com- posed of small cells rich in protein and fat. The milled grain known as flour differs in composition, according 14 2IO PRACTICAL SANITARY SCIENCE to whether the bran or embrj-o, or both, have been largely removed or retained. The reduction of bran to a powder by grinding is a difficult and expensive matter, and as a rule the miller removes it altogether. In roller-milling, the germ is also removed, in order to prevent the fat which it contains becoming rancid. Enzymes present in the germ act upon the starch, converting it into dextrin and sugar, which darken the colour of the bread; so the genu is excluded. This rejection of the bran and germ means the loss of some of the most useful constituents of the wheat; and the recog- nition of this loss has led to a number of patent processes for treating the bran and germ so as to prevent the production of a dark loaf. In the ' Hovis ' process, the fat of the genu is treated with steam, with the object of preventing its becoming rancid. In the ' Frame Food ' process, the bran is boiled with water under pressure, with the object of breaking do^^^l the cellulose, and extracting the bulk of the nitrogenous and mineral constituents. In Smith's patent the genu is partiallj^ cooked by superheated steam, whereby the ferment is killed which transforms the starch of the flour. Accord- ing to the method adopted in milling, some flours contain more bran than others, and some more starches and gluten. Wheat from different countries varies in chemical composition. Ordinary bread is made from a mixture of flours derived from different wheats, and sometimes such a mixture includes different types of milling. The average composition of wheat-flour is: Water • • 13-0 Sugar Ash .. 07 .. 0-8 Fat • 1-5 Protein . . II-O Starch, dextrin, and cellulose . . • • 73-0 Physical Characters of Flour. — Flour should be free from acidity, white in colour, and smooth when rubbed between the fingers. It should be entirely free from fungi and all other parasites. A yellow colour denotes age or femientation. If flour be kept in a damp place, an odour is generated by the growth of moulds and various micro-organisms. Gluten. — The crude protein of flour known as gluten possesses CEREALS 211 a constituent, gliadin, which confers upon dough its characteristic adhesiveness. When dough is thoroughly washed so as to get rid of starch and all other soluble bodies, such as salts, albumin, sugar, etc., gluten remains as a somewhat tough and sticky mass; when this is blown up with a gas, it coheres sufficiently to remain in the form of a sponge. Barley, rice, and oatmeal do not contain gluten, but other forms of protein, which are destitute of this viscid char- acter; hence they cannot be made into bread unless mixed with a sufficient quantity of wheat-flour. Estimation of Gluten. — Place 20 grammes of flour in a basin and stir it into a stiff dough with warm water ; next thoroughly work the dough with the fingers in a fine muslin bag in a stream of running water until all the starch and other soluble materials have been washed away. The absence of starch may be proved by the iodine test. Generally a small quantity of fats and salts (i per cent.) remains. Now spread out the gluten in a weighed dish in a water-oven; dry until a constant weight is obtained. This weight, minus the weight of the dish, represents the gluten. A more delicate and reliable method is the estimation of total nitrogen by Kjeldahl's process, as described previously. The total N X 6-3 gives approximately the gluten. In carrying out the process great care should be taken that no ammonia and no nitrates exist in the reagents used. If the gluten fall below 8 per cent., the flour may be regarded as not pure wheat-flour. Ash. — The ash of wheat-flour consists principally of phosphates of potassium, magnesium, and calcium, together with mixed salts of sodium and iron, and lastly silica. The total quantity should not much exceed i per cent. The estimation should be done in a platinum basin, and a wholly white ash obtained. Ash amounting to 2 per cent, shows the addition of mineral adulterants. Water. — This constituent should not exceed 16 per cent. The adulteration of wheat-flour at present consists essentially in the addition of other flours, as those of rice, maize, pea, and bean. The microscopic appearances of the different starch granules will assist in the detection of such adulterations. Starch Granules. — To estimate the amount of starch in a sub- stance, weigh out a gramme of the dried powdered material, and mix it with 50 c.c. of a 5 per cent. HCl solution in a flask, to which a 212 PRACTICAL SAMTARY SCIE.XCE reflex condenser is attached ; boil for several hours under a hood : the starch is converted into sugar (dextrose). Make the solution slightly alkaline with NaOH solution, and estimate the dextrose by Fehling's method. The result, multiplied by 0-9, gives the quantity of starch in a gramme. Where cellulose is present, the small amount converted into sugar may be ignored. The micro- scopic appearances of manj' starch granules are such as to afford an easy means of recognition. If a mere speck of a particular flour or powdered starch be placed on a microscopic slide, a drop of water added, and a cover-slip applied, the starch granules can be thoroughly studied by low and high powers of the microscope. As in mounting specimens of bacteria, it should be noted that it is almost impossible to apply too little of the material to the slide. The student should observe that in most cases characteristic cells appear, but that many cells may be unrecognizable, as belonging to any particular kind of starch. Where starch granules of different foodstuffs closely resemble each other, it may be quite impossible to decide whether or not slight admixture has been effected. On the other hand, when the granules are dissimilar the slightest admixture is easily detected. If an estimation of the amount of the adulteration be required, a rough average percentage of the foreign granules may be obtained by counting a number of fields, and this estimation may be checked by making a mixture containing the true and foreign ingredients in the proportions observed; such mixture should present the same microscopic appearances as the original. Several trials may be made in this way before the required match is obtained. The student should carefully study the microscopic characters of all starch granules occurring in vegetable foods, and make drawings of them. Bleaching- of Flour and Flour-Imppovers.— With a view to improving the baking qualities of flour, millers resort to bleaching and the addition of ' improvers.' Ozone, halogens, and nitrogen peroxide have been used as bleaching agents. Nitrogen peroxide alone appears to give satisfactory results, and is the only bleacher now used. The gas is produced chemically from nitric acid and ferrous sulphate, or electrically by the combination of the N and O of the air by an electrical sparking discharge. The latter is said CEREALS 213 to be the better method in that the degree of bleaching is more easily controlled, and condensation of acid resulting in staining of the flour less likely to occur. Air charged with nitrogen peroxide and ozone is agitated with the flour in a suitable machine. It is stated that the nitrogen peroxide produced by 3 c.c. of nitric oxide in 3 litres of air will bleach i kilogramme of flour. A watery extract of bleached flour reacts to the nitrite test of Griess. This reaction is not given by ordinary unbleached flour. When bleached flour is baked, one-half to two-thirds of the nitrite disappears, and an increase in nitrates occurs. The whole of the nitrite may disappear from biscuits. Effects of Bleaching. — Bleaching destroys the yellow colouring- matter dissolved in a thin layer of oil which surrounds the individual granules of starch; the iodine value of this oil is lowered. The acidity of flour is increased. It is probable that certain amino-groups in the protein are destroyed. Improvers used. — Water added to the flour in a fine spray; phos- phates, especially calcium phosphate; phosphoric acid; sulphury] chloride. It has been experimentally shown that even traces of nitrites in flour inhibit both proteolytic and amylolytic digestion. The introduction of roller-milling made it possible to utilize any variety of wheat since pulverization of the bran is avoided, and consequently a more complete removal of bran and germ effected. The germ contains no gliadin nor glutenin (these substances unite with water to form gluten); it contains 10 per cent, of albumin, 5 per cent, of globulin, and 3 per cent, of proteose. Its nucleated cells contain a considerable amount of nucleic acid combined with albumin and globulin. Little organic phosphorus accordingly occurs in the endosperm. Wheat contains probably about 2 per cent, of germ, and as the latter possesses at least 30 per cent, of proteins, retention of the germ raises the protein content of flour by o-6 per cent. The greater portion of the phosphorus in bran can be extracted with dilute acid, and it has been shown that the bulk of this phos- phorus occurs in organic combination as a phospho-organic acid, combined with potassium, calcium, and magnesium. Wholemeal or Graham flour is produced by grinding the entire wheat grain; it should contain the whole of the germ. 214 PRACTICAL SAXITARY SCIENCE ' Entire ' wheat flour or line meal is obtained by removing a portion of the bran, and finely grinding the rest; it contains a portion of the germ. ' Straight-rmi ' flour is the whole of the flour produced in the roller-mill. The percentage composition of bread made from samples of these flours is as follows: Protein (NX 5-7). Carbo- hydrates. Fats. Water. Ash. Graham flour • • 9-54 46-10 0-29 42-68 1-39 ' Entire ' . . • • Q-32 4875 0-19 40-97 0-77 ' Straight run ' • • 0-63 51-06 0-04 3877 0-50 Experiments have been made on the nutritional values of different varieties of flours. Young rats have been fed on ' standard ' or ' straight run,' and others of the same age on ' entire.' The first lot throve much better than the second. Again, the same experiment has been carried out with Graham flour and ' entire,' or white flour, with results in favour of the Graham variety. In the milling of rice the cuticle, consisting of pericarp, testa, and nucellus, is frequently removed. A diet consisting exclusively of such rice produces polyneuritis and other changes, constituting a disease known as ' beri-beri.' If the offal (about 10 per cent, of the grain) be returned to the rice, no beri-beri occurs. Or if the offal be extracted by 0-3 per cent. HCl and the extract precipitated by proof spirit, the substances soluble in alcohol (1-6 per cent, of the grain) will equally prevent the disease. It is significant that the precipitate containing 85 per cent, of the phosphorus of the offal is wholly ineffective in preventing the disease. A good rice should not contain less than 0-4 to 0-5 per cent, total phosphorus. The milling of rice deprives it of its cuticle, and leaves it with a dull appearance. To improve its appearance it is ' polished ' in hollow cylinders fitted with revolving rollers covered with sheep-skin. In order to obtain a high polish talc or steatite, in the form of a fine powder, is added to the rice prior to polishing, and for the most part as it passes through the mill. Gypsum, kaolin, and gums have been also used for this purpose. The colour of rice is changed from a cream to a dead white by the addition of blue pigments (generally ultramarine) during milling, and to make it transparent it is treated with arachis and other oils. CEREALS 2i5 I. Granules of Wheat, Barley, and Rye— t^/j^a^— These are (i) large, round, or oval, which do not exhibit concentric striae; n ©4 ^}u -^^ - 'O m Q Fig. 29. — Wheat, x 200. J ^^ Fig. ^o. — Barley, x 200 ■ ^ '^,.' (2) small, ill-defined granules scattered irregularly throughout the field. Intermediate sizes are rare. 2l6 PRACTICAL SAXITARY SCIENCE Barley. — These are (i) large, (2) small, (3) intermediate in size, In a very few are there any markings. o Q ._^. ' Fig. :ti. — Rye. x 200. il Fig, 32. — Rice, x 200. Rye. — ^These are very similar to those of barley, except that in the large granules some show a rayed hilum and cracked edges; CEREALS 217 the large granules are more generally circular and flattened than those of wheat and barley, and somewhat larger. _.,^A)^.«,^,.^ 4?- t' _*3-c,.''^ ' '■ ^.".^ '^^V5^ Fic '• 33-- -Oat. X 200. ' 00 Cs v^ ■ % ... " 0^ t) ■ ■fi 't^ ^<^^^ OO' "k^* ®' .\ W ! P' 3 .0 • ; ^-^ ^•(3 V^ Q - oO \ 1 . O0 b " ' ^.^^o-^ ^' Fig. 39. — Arrowroot, x 200. Fig. 40. — Potato, x 200. Tapioca granules are much smaller, and the hilum is generally placed towards the rounded extremity. CEREALS 221 4. Pea and Bean. — These granules are oval in form, fairly uniform in size, and possess a central linear liilum and faint con- centric strise. Those of the pea present a central longitudinal hilum, sometimes exhibiting cross-striation. The granules of the bean are somewhat larger and broader, and the cross-striation of the central hilum is more marked. V Fig. 41. — -Vibrio Tritici. x 30. Fig. 42. — Bruchus Pisi. Fig. 43. — AcARUS Farin.^. 5. ArroWFOOt and Potato. — The granules of these starches are large, pyriform, and marked distinctly with concentric strise. A circular hilum is found in both, placed at the large extremity in arrowroot, and at the small in potato. The granules of farrow- root do not swell in a solution of KOH, as do those of the potato. PRACTICAL SAX IT A RY SCIENCE Parasites found in Wheat and Flour— Animal: Tylenchns tritici (ear cockle). — In the infected ears of grain are to be seen the larvje of a nematode wonn, occurring as a white powder in dark misshapen grains. Specimens may be mounted directly in Fig. 44. — Penicillium Glaucum. Fig. 45. — Aspergillus Glaucus. M ^h Fig. 46. — MucoR Mucedo. A, Head ; B and C, conjugation; D, spore-bearing hyphse. Fig. 47. — Peronospora. Farrant's solution, or dehydrated and cleared in the ordinary manner, and mounted in Canada balsam. Bruclms pisi and Calandra gran aria are beetles (Coleoptera) which infest grain and pulses. The first attacks the pea, an allied CEREALS 223 species the bean, whilst Calandra is found in grain. The female lays her eggs in the young fruit, and the larvae destroy the internal parts. The C. granaria perforates the husk of the grain and abstracts the contents. . Acanis Farince. — This parasite is found in inferior and damp flour. It may be distinguished from the A. scabei by its legs remaining thick up to their' extremities, whilst in the itch parasite the distal ends of the legs are quite thin. Veg-etable Parasites found in Wheat, Flour, Bread, etc. — Moulds : Penicilliiim glaticum, Aspergillus glaucus, Miicor ^ © ^O o Fig. 48. — UsTiLAGo Segetum. x 250. mucedo, and Peronospora. — These moulds are easily distinguished by the characters of the ends of the spore-bearing hyphffi. In penicillium the last hypha branches into three or four temiinal filaments, which develop round or oval spores in rows in their long axes. In aspergillus the end of the spore-bearing hypha enlarges, and from this pedicle-bearing spores grow out and form a more or less dense head. In mucor the end of the spore-bearing hypha enlarges greatly, and the spores, instead of grow-ing out from the enlargement, as in aspergillus, grow inside a membrane which surrounds the head. When the spores have matured, the membrane ruptures and sets them free. 224 PRACTICAL SAXITARY SCIENCE Peronospora, which caused the Irish potato famine of 1847, first affects the leaves, then travels down the stem, and finally attacks the tubers. The spore-bearing hyphae branch and rebranch, and at the end of a terminal branch a single spore is developed. Ustilago Segeiiim (smut). — The spores of this parasite are found as a black powder infesting ill-developed cars of com, and fall off when the ear is rubbed. Examined microscopically, they are seen to be brown, spherical, free spores. t Fig. 49. — TiLLETiA Caries (Uredo Fcetida). x 250. Tilletia caries (bunt) is another member of the ustilaginse, and is found in the interior of the grain; it may escape detection until the process of milling takes place. The spores are brov^Ti, spherical bodies, generally free, and give to the interior of the affected grain a sooty appearance and foetid odour. These spores germinate in the spring, forming a hypha, the promycelium, which bears promycelial thread-like spores. The next stage in the life-history of this organism is the conjugation of contiguous spores. Two such conjugated spores bud and form an elongated secondary CERE A LS 225 promycclial spore, which, if it find a suitaV^lc host, sends out hyphie and enters the interior of the grain, where a myceHum is developed. After a time the hyphic swell, become dark in colour, and a differ- entiation into spores takes place. As these ripen, the mycelial structure disappears, and leaves the resting spores in the condition from which the cycle commenced. Pticcinia graminis is one of a large number of parasitic species affecting corn in the manner above described. A spore attaches itself to a grain or stem, and sends hyphge into its substance, from Fig. 50. — Wheat Stem infected with Puccinia. which a mycelium and spores are formed within; as a result, the grain ruptures, and the spores appear on the surface as rust. A distinctive feature of puccinia is the double spore attached to a peduncle. It is this form which is found attached to grain or grass in the autumn, as rust, and which, known by the name teleuto- spore, remains quiescent during winter. In spring it germinates, and produces a non-parasitic mycelium. The individual cells of this mycelium produce filaments, known as gonidiophores, which in turn produce spores at their free ends. Distributed by the wind, these latter fall on the leaves of the barberr}', where they 226 PRACTICAL SANITARY SCIENCE germinate and form a dense mycelium in the substance of the leaf, giving rise to swellings which project on its under-surface. Spherical Fig. 51. — Portion of Fig. 50 slightly MORE Highly ^Magnified. Fig. 52. — Teleutospores Fig. 53. — .lEciDiuM Berberidis. Fig. 54. — Gonidiospores (Uredo gonidia) and Teleutospore. structures, termed ascidia, form and develop within themselves spores which are set free on rupture of the wall of the aecidium. CEREALS 227 These spores are carried by the wind to grass plants, to which they attach themselves and develop a mycelium from which grow certain hyphse, bearing single spores (uredogonidia). On rupture of the leaf of the host, the spores arc to be seen as a yellow dust. Again the wind carries the gonidium to another grass, where it germinates, produces hyphse, and repeats the previous process (uredo form). As the autumn approaches, special hyphse produce gonidia, which have a septum perpendicular to the long axis, dividing the spore into two cells — the teleutospore ; this rests Fig. 55. — Ergot in Rye. x 30. through the winter, and commences the cycle once more in the following spring. Claviceps purpurea (ergot) grows in rye, and the mycelial growth (sclerotium) replaces the grain. The ergot masses (or grains) are larger than the rye-grains, and of a deep purple colour. In the spring the sclerotium, which has rested through the winter, ger- minates, and produces long hyphse (stromata), which develop a swelling at the distal end, which latter contains oval receptacles (ascocarps). Attached to the inner end of the ascocarps are asci, containing eight filiform spores (ascospores). The asci rupture 228 PRACTICAL SAXITARY SCIENCE and the spores escape. Carried bj' the wind, the spores alight on the ovarj' of the rye liower and form a niycehuni. On the surface of this mycehum free spores (gonidia) develop, and are surrounded by a viscid substance, known as honey-dew, which attracts insects, % :>"' Fig. 56. — ScLEROTiuM bearing Stromata. X I. Fig. 57.— Strojia containing ascocarps. x 75 by which the spores are carried to other flowers, where the process is repeated. This stage is known as the sphacelia form. As the rye is developed, the mycelial growth increases to such Fig. 58. — AscocARP containing Asci. X 350. Fig. 59. — Ascus containing ASCOSPORES. an extent that the young grain is wholly absorbed, the pericarp is no longer able to contain it, and it projects like a spur from the spike. Ultimately it falls to the ground. The cycle is repeated in the following spring. BREAD 229 The seeds of Lolinm ienmlentum, possessing nareotic properties, may gain access to flour, l)ut rarely produce poisoning. BREAD Bread is chiefly made from wheat-flour. A dough is first formed by mixing the flour with water or other fluid, and a gas, generally CO2, is passed through it. Carbon dioxide is obtained cither by tlie action of yeast on sugar, when this gas and alcohol are formed, or through the liberation of the gas from an alkaline bicarbonate by the action of an organic acid. The dough is sometimes aerated by charging water with air and mixing this with flour under pressure in air-tight chambers ; afterwards the pressure is lowered by opening a trap, when the dough is blown up by the expanding gas, forming ' aerated ' bread. The dough is then cooked in an oven at a tem- perature of 200-205° C. Composition of White Bread : Water Proteins Fat Starch, sugar, dextrin Cellulose . . Ash Composition of Whole Meal : Water Proteins Fat Starch, sugar, dextrin Cellulose Ash .. .. 40-0 6-5 i-o 51-2 0-3 T-O 45-0 6-3 1-2 44-8 1-5 1-2 During the cooking a crust is formed, which should neither be very light nor very dark in colour, and which should crack readily on breaking. The shining appearance of crust is due to the for- mation of dextrin, and its flavour and dark colour to the production of caramel. Two-thirds of the volume of a good loaf is gas. Great whiteness in a loaf, although much desired by the public, is by no means essential from a nutritive point of view, as a very white loaf possesses a maximum of starch and a minimum of protein. 2 3') PRACTICAL SAXITARY SCIEXCE Comparative Composition of Crust and Crumb : Crust. Crumb. Water •• 17-15 44-45 Insoluble protein . . • • 7-30 5-92 Soluble protein ■ • 570 075 Dextrin and sugar . . . . 4-88 3-79 Starch .. 62-58 43-55 Fat .. i-i8 070 Ash I-2I 0-84 By these figures it is seen that there is a much larger proportion of solids, and also more soluble proteins and carbohj'drates, in the crust than in the crumb. The crumb should be elastic in con- sistence, should have a sweet, nutty flavour, and be of a uniform whiteness throughout. As bread grows old, it becomes hard, and it has long been known that reheating softens it. The real explana- tion of the staling of bread does not seem to be known. Bibra holds that in fresh bread there is free water present, which, as staleness supervenes, unites with starch or gluten, and that re- heating sets this water free. He states that the freshness will not return if the bread has lost 30 per cent, of its water. Others hold that the stale condition is produced by the shrinkage which takes place in the fibres forming the walls of the pores. The water vapour formed by the second heating drives these fibres apart again. It should be noted that during the baking of bread a large pro- portion of the fat is lost, amounting to as much as 7 per cent, in some instances, that the proteins are diminished from i to 2 per cent., and the carbohydrates from 3 to 4 per cent. Some of the starch is converted into soluble starch and dextrin to the extent of 8 per cent. The estimation of Water and Mineral Matter in bread is per- formed as in the case of flour. Twenty grammes of the crumb make a convenient quantit}^ with which to work. The Ash is generally greater in weight than that of the flour used, owing to the sodium chloride, baking-powder, etc., added. Any excess of ash above 3 per cent, is generally regarded as due to salts added in order to improve the colour. Silica is estimated by treating the ash with strong HCl and hot distilled water in a platinum dish, then filtering through a Swedish filter-paper, carefully washing the platinum dish with further boil- BREAD 231 ing distilled water, and transferring the washings to the lilter-papcr. When the residue on the filter has been several times washed with boiling distilled water so that all soluble substances have passed through, it is dried in a water oven, transferred to a porcelain crucible, ignited, and weighed as silica. It should not exceed 2 per cent. Acidity. — Soak 5 grammes of bread in 50 c.c. of water for an hour. Filter and titrate the filtrate with y^ NaOH, using phenol - phthalein as indicator. The number of c.c. of decinormal soda used multiplied by 6 equals milligrammes of glacial acetic acid in 5 grammes of bread. The acidity should not exceed o-il per cent. Adulteration. — Formerly Alum was used to whiten inferior flours, but at present it is practically never found. The detection of alum in bread is carried out as follows : Dissolve a small quantity of haematoxylin in alcohol, and to this add a little freshly prepared solution of ammonium carbonate in distilled water. Cubes of crumb are cut from the centre of a loaf, and small quantities of the solution poured upon them, after which they are removed to a water oven and dried at a low temperature. The production of a permanent lavender colour denotes the presence of alum. To a small degree magnesium salts simulate alum in this reaction, but the colour on drying is not so permanent. Silicate of alumina exists normally in flour, but in such small quantities that a 4-pound loaf will not contain more than 6 or 7 grains. The part played by alum when added to inferior flours is that of checking fermentation, which otherwise would lead to the production of glucose, and con- sequently a discoloured bread. It is stated that alum increases the porosity of bread. This adulterant has been found in quantities ranging from 20 grains to 100 grains per 4-pound loaf. Quantitative Estimation. — Reduce ^ pound of the bread to ash, and separate off the silica on a filter by treatment with strong HCl and boiling water in the usual manner. The filtrate contains phosphates of lime and magnesia, iron, and aluminium. To this solution add 5 c.c. of (NH4)H0, which will precipitate all the phosphates, and 20 c.c. of strong acetic acid, which redissolves the phosphates of lime and magnesia. Filter and wash the residue of phosphates of iron and aluminium with boiling water. Dry, 232 PRACTICAL SAXITARY SCIENCE ignite, and weigli. The residue is now dissolved in strong HCl, and diluted to 200 c.c, and the iron estimated colorimetrically. Convert the iron thus found into ferric phosphate by multiplying by 2-7, and subtract this from the weight of phosphates of iron anil aluminium previously obtained; deduct also the weight of the filter ash, and the difference is aluminium phosphate, which may be returned as commercial alum (crystallized ammonium alum) by multiplying by 37. Various cereal and casein preparations have appeared in recent years containing added Phosphorus Compounds in different forms — glycerophosphates, lecithins, and lipolins, etc. In order to measure these added substances it is necessary to estimate the phosphorus. This is easily effected by Neumann's method: Prepare 2 litres of Neumann's molybdate-nitrate solution; dis- solve 75 grammes of ammonium molybdate in 500 c.c. of water, and pour this into 500 c.c. HNO3; add a litre of 50 per cent, ammonium nitrate solution. Prepare mashed hlter-paper for pressure filter. Place 30 grammes of minced filter-paper in a litre of water containing 50 c.c. HCl. Heat on water-bath with shaking for an hour. Filter. Wash with water repeatedly till all acid disappears. Leave in 2 litres of dis- tilled w^ater from which remove portions to pressure filter as required. Decompose 0-5 gramme of the substance with nitric-sulphuric acid; add 60 to 100 c.c. of water and molybdate-nitrate solution in excess until solution remains clear on warming. Filter on pressure filter (about five minutes required). Add more molybdate-nitrate solution till all yellow precipitate is down. Wash the precipitate with water till free from acid (but not too long, as acid may separate out of the precipitate). Wash precipitate, pulp, and disc (used for supporting paper pulp in funnel) into a beaker; add about 300 c.c. of water and excess ., NaOH, and boil for ten minutes or so until NH3 passes off. Titrate back with ^ H2SO4. Boil again to get rid of CO2, and finish the titration with a drop or two of | NaOH. One molecule P2O5 =56 molecules NaOH. I c.c. I NaOH =1-268 milligrammes PoOg. [200 c.c. molybdate-nitrate solution =o-i gramme PoOj]. MEAT 233 MEAT The principal food animals arc cattle, sheep, pigs, goats, horses, the buffalo and reindeer in a few countries, and in Saxony and Italy dogs. Inspection of animals before slaughter is necessary for the detec- tion of infectious diseases — anthrax, glanders, rabies, etc. — and for the discovery of intoxications in which meat and internal organs are but slightly altered. Of the many pathological conditions which affect food animals these are of interest in laboratory work: Infective granulations; a few diseases produced by invisible organisms ; and animal parasites. Infective granulations are found, as tuberculosis, glanders, and actinomycosis. To determine the extent of tuberculosis in slaughtered animals, it is necessary to make a methodical inspection of the hung-up carcase from above downwards. The meat is first examined, and after- wards the lymphatic glands, which receive the lymph from the meat, in the following order: (i) Popliteal, inguinal (superficial and deep), pubic, or supramammary lymph-glands. (2) Iliac and retro- peritoneal lymph-glands. (3) The lymph-glands along the sides of the vertebral column, ribs, and sternum. (4) Prescapular and axillary glands. (5) Pharyngeal and submaxillary lymph-glands. On completion of the examination of the lymphatic glands of the carcase, the internal organs with their lymphatic glands are next examined — viz., the kidneys and renal lymphatics, the spleen, liver, lungs, and the udder in female animals. Lastly the peritoneum and pleurse are systematically inspected. Actinomycosis occurs as small or large tumours delimited from the surrounding tissues by a thick wall of dense connective tissue in the jaws, tongue, skin of head and neck, and much more rarely in the lungs, liver, kidneys, udder, and abdominal wall. Sections and smears containing Bacilhis Uiberculosis are readily prepared and stained with Ziehl-Neelsen's carbol-fuchsin, and counterstained with methylene blue. Glanders must be distinguished from bovine farcy, not trans- missible to man, and produced by a fungus of the genus Discomyces. The ass is more susceptible to glanders than any other animal. 234 PRACTICAL SAXITARY SCIEXCE If a little discharge from the nose be rubbed into a few scarifications on the skin of the forehead, an nedematous swelling rapidly appears, followed b}' ulceration along the lines of the scratches; the tem- perature quickly rises to 40° C. or 41° C. The neighbouring glands swell, a discharge from the nose appears, and the animal dies in a few days. The chocolate-coloured growth of the glanders bacillus {B. mallei) on potato is characteristic. Microscopically B. mallei is a small straight rod (3 to 5 /x), with rounded ends. It is non-motile, non-sporing, and Gram-negative. Sections and smears containing the filaments of actinomyces bovis stain well b}' Gram's method and by carbol-fuchsin. The micro- scopic appearances in both cases are unmistakable. Some difficulty may be experienced in isolating the parasite from pus in artificial culture, as the pj'ogenic organisms overrun the media before the actinomyces has had time to start. Spread pus containing the yellow granules on a couple of gelatin plates, and incubate at 22° C. for two days. Most of the grains will be surrounded b}' colonies of contaminating organisms, but a few will be found here and there discrete and isolated; pick these off with a stout platinum wire, and inoculate three or four coagulated serum slopes, and incubate at 37° C. In live or six days (note time as compared with a possible CcLse of tubercle) colonies of actinomyces begin to grow. Sown in glycerin broth, hemispherical colonies appear in the same time (five to six days), as large as a small pea, and fall to the bottom, leaving the medium clear. On glycerin-agar growth occurs in two days, which later becomes yellowish- white, dry, and wrinkled. On potato in six to seven days small colourless colonies appear, which quickly become grey, yellow, and tinall}' wrinkled and edged with black. The invisible organisms, or so-called fiUrahle viruses, producing diseased conditions in food animals, are those of pleuro-pneumonia, foot-and-mouth disease, rinderpest, horse-sickness, swine-fever, cow- pox, sheep-pox, and bird-plague. Prior to 1898 laborator}^ methods failed utterl}' to throw an\- light on the causative agents operating in these diseases. In that year Nocard and Roux devised a new method of investigation. In pleuro-pneumonia the essential lesion is the distension of the ME A T 235 meshes of the interlobar connective tissue witii mucli clear, amber- coloured fluid. Subcutaneous inoculation of this fluid in another animal reproduces the disease, but the microscope and ordinary methods of cultivation are useless in searching for the micro- organism. Nocard and Roux filled collodion sacs sown with a drop of the fluid from a case of pleuro-pneumonia, and introduced them into the peritoneal cavities of rabbits. In two to three weeks the contents become cloudy. Microscopical examination with a mag- nification of 2,000 diameters show motile retractile points so small that their shape cannot be determined; these cannot be stained. By the twentieth day the virus has produced in the rabbit extreme emaciation, but no lesion; the organs and body fluids arc sterile. The control animals in which similar but sterile sacs are inserted remain healthy. It would appear that rabbits are immune to the organism, but susceptible to the toxin. The contents of the sacs cannot be cultivated on ordinary media. After much experimenta- tion these observers devised a medium on which the organism can be grown. This consists of i part of rabbit's serum and 20 parts of Martin's peptone solution (mix 200 grammes of cleaned and minced pigs' stomachs, 10 grammes of HCl, i litre of water at 50° C. ; heat to boiling to destroy pepsin, and pass through cotton-wool; heat the filtrate to 80° C, and neutralize at this temperature; filter through Chardin paper, and autoclave the filtrate for four or five minutes at 120° C. ; run 10 c.c. of the clear filtrate into test-tubes, and sterilize for twenty minutes at 115° C). Tubes of this medium sown aerobically with a drop of exudate or of the contents of the collodion sacs, and incubated at 37° C, produce a virulent growth resembling in its microscopical and other characters that of the sacs. This disease runs both an acute and chronic course. In the acute form the respiratory symptoms are most marked. The exudate filtered through a Chamberland (F) bougie fails to produce the disease and to give cultures. In experiments with ultramicroscopic viruses it is necessary to use a new and sterilized filter, and not to allow more than two hours for the filtration process, nor a temperature above 20° C. The pressure of filtration should be as low as possible, and the emulsion should be diluted to prevent blocking of the pores of the filter with 236 PRACTICAL SANITARY SCIENCE albuminous matter. Several animals shovild be inoculated with a large volume of the filtrate. Foot-and-mouth disease infects cattle, sheep, goats, and pigs, and is" transmissible to man. Aphthous lymph loses its infectivity when kept for five to six weeks. Loffler, liy mixing such old lymph with fresh lymph, attenuated by heating for hve minutes at 60° C, has been able to produce immunity in oxen. Bird-plague virus has been grown by Marchoux on defibrinated fowl blood spread on glucose-peptone-agar, and incubated at 37° C. Growth occurs in the zone of blood adjoining the surface of the agar. Animal Parasites in Meat. — Three groups of animal parasites may be recognized : I. Parasites not transmissible to man. II. Para- sites which may be transmitted to man by eating meat. III. Para- sites not immediately harmful, but which may become so after a preliminar}.' change of host. 1. Parasites not transmissible to Man — i. The Hair- Follicle Mite in the Skin of Hogs {Demodex phylloides suis). — It is from 0-2 to 0-25 milhmetre long, and produces small swellings of the hair-follicles, greyish-yellow in colour, and containing disinte- grated epithelial cells and dermal oil. 2. Dipterous LarvcB. — Larva of warble fly {CEsirus bovis), 28 x 15 millimetres, found in subcutis, causes considerable loss to cattle- raisers through deterioration of flesh and skins. It is found in the fjesophagus from July to September, in the spinal canal from Sep- tember to January, in the subcutis and skin from January to -May. Other larvae are the Gastrophilns equi and G. nasalis. 3. Numerous Worms which appear in Organs of Food Animals. — (a) All tapeworms except Tccnia echinococcus of the dog, such as Moniezia expansa found in lambs, Drepanido tcenia lanceolata and D. setigera in geese, Davainea tetragona in young fowls, Taenia coemirus, T. marginata, and T. serrata, in the dog. (b) Larval stages of all tapeworms, except Cysticercus bovis, C. celluloses, and Echino- coccus polymorphus, such as Tcenia ccenurus {Coenurus cerebralis), which causes the disease known as ' gid ' in sheep, and Cysticercus tenuicollis (larva of T. marginata) found in sheep, pigs, and cattle. (f) Flukes (Trematodes), such as Distoma hepaticum and D. lanceola- tnm, found in the liver of the sheep. These flat organisms measure as much as 25 x 13 millimetres. They are covered with scale-like MEAT 237 spines on the integument, which irritate the bile-ducts where they arc, located, and cause the thickening of these vessels so characteristic of the condition. They may wander from the liver to the lungs. Their embryonic stages are passed in a free condition in molluscs, mostly water-snails. Apart from the catarrh and cirrhotic condition of bile-ducts produced by these parasites, hsemorrhages occur, and the health of the affected animals may be seriously damaged. (d) Round-worms (Nematodes), with single exception of Trichina spiralis, such as Ascarus, Eustrongylus, Filaria (Schneider's group Polymyaria), Oxyuris, Strongylus (Schneider's Meromyaria), Tri- china spiralis, Trichocephalus, Anguillula (Schneider's Holomyaria). II. Parasites which may be transmitted to Man by eating- Meat. — I. The Beef bladder worm {Cysticercus bovis), which is the larval form of Tcenia saginata of man, known also as T. medio- canellata, consists of a somewhat elongated, roundish bladder located in the interfibrillar connective tissue of the striated musculature, and occasionally in lungs, liver, and brain. The grey transparent bladder consists of a connective-tissue capsule produced by reaction in surrounding tissues, and of the parasite. The latter consists of a scolex and caudal bladder filled with fluid ; the scolex possesses four suckers, but no hooks. The size of the cysticercus varies from that of a pinhead to tliat of a small pea. 2. The Pork bladder worm {Cysticercus celhdoscs) is the larval stage of TcBuia solium. In macroscopic appearances and location between muscle fibres it closely resembles C. bovis. For the rest, the cyst is more transparent, so that the scolex when invaginated into the caudal bladder appears more clearly. The scolex has twenty- two to twenty-eight hooks in a double circle; the hooks are of compressed shape, stout at the base and with slightly curved points. The cysticerci prefer the lumbar and abdominal muscles, pillars of the diaphragm, intercostal and masticatory muscles. 3. Trichina Spiralis. — Hilton investigated calcified trichinse in 1832. Zenker discovered trichinosis in Dresden in i860. After ingestion of trichinous meat, sexually mature trichinse develop in the intestines of certain mammals; the parasite is set free from its capsule b}^ the gastric juice. Males and females copulate, and the females deposit enormous numbers of embryos. Leuchart assumed that the embryos bore their way out of the intestine into the peri- 23S PRACTICAL SAXITARY SCIEXCE toneal and thoracic cavities, and ultimately reach the muscles. Heitzmann argues that this migration cannot possibly take place in the few days that elapse lx;t\veen the swallowing of infected meat and the appearance of embryos in the muscles, and that the embryos are conveyed by the blood-stream, and caught as emboli in the capillaries. Arrived in the muscles, a capsule is formed which in due course becomes calcified. The frequent occurrence of the parasite in rats is explained by the presence of the rat in abattoirs, knackers' yards, etc. Degeneration of trichinae in their capsules frequently takes place. The muscles most likely to contain parasites are those of the tongue and larynx, and the pillars of the diaphragm. Rhabditides (larvpe of strongylidce) may be mistaken for trichinae. III. Parasites not immediately Harmful to Man, but which may become so after a Preliminary Change of Host — I. EcJiinococci.- — {n) Tccnia echinococcns resides as a parasite in the small intestine of the dog, and is the asexual stage of a tapeworm with three to four segments. It is 2 to 6 millimetres long by 0-3 to 0-5 millimetre wide. It possesses a protruding rostellum with twenty-eight to fifty hooks. The last proglottid is 2 millimetres long, and contains mature eggs. The echinococci occur in two chief forms — (a) E. unilociilaris and (6) E. muUilocnlaris. E. nnilocularis forms simple cysts surrounded by connective tissue; in some cases daughter cj'sts are developed from the mother cysts, in other cases not. E. multilocularis forms daughter cysts by constriction from a central mother cyst, which in turn are furnished with the same reproductive power. The daughter cysts do not remain in the mother cyst or inside the organic membrane formed about it, but after constriction become separated from the mother cyst by con- nective tissue. Accordingly, the vesicles attain no great size, but lie in the connective tissue like the epithelia of an acinous gland. The hooks of the multilocular form are somewhat larger than those of the unilocular. The intermediate host is man. The E. unilociilaris occurs in the liver, lungs, and spleen, of the ox, sheep, and pig, and less often in the heart, kidneys, lymph- glands, muscles, and marrow cavities of bones. MEAT 239 The E. muUilocidans occurs in the liver of bovincs, forming tumours of various sizes which exhibit a constant growth. 2. Larvce of Pentastomum Tcenioides. — These are flat white struc- tures, 4 to 5 milhmetres long by i to 1-5 millimetres broad, divided into about eighty segments furnished with backwardly-directed tooth-like spines. Below the mouth there are two slit-like apertures on either side, from each of which the points of two claws protrude. These openings gave origin erroneously to the name Pentastomum (five-mouthed). The embryos are provided with a boring apparatus under the mouth opening, and at the opposite end of the body are several spines which serve for locomotion. Tlie parasites are found in hares, goats, sheep, and more rarely in cattle, under the peritoneum, in the liver, in the mesenteric glands, and in the lungs. Dogs are the chief source of pentastome larvae, and man, through intimate association with the dog, may become infected by ingestion of pentastome eggs. A subdivision of the Protozoa — viz., the Sporozoa — are of some importance in meat inspection. This subdivision consists of the following orders: Coccidia, Myxosporidia, Sarcosporidia, and Hsematosporidia. The Coccidia are parasites of epithelia, and occur in the liver of the rabbit and other animals, and occasionally in the liver of man. C. perforans occurs in the intestinal epithehum of rabbits, sheep, and calves, and causes a catarrhal diarrhoea. Myxosporidia are chiefly parasitic in fish. Sarcosporidia (Miescher's sacs) occur in hogs, mostly in the striated muscles. HcBmatos-poridia. — Theobald Smith's discovery of the organism of Texas fever in cattle conferred an importance on this group in relation to meat inspection, which with the constant discovery of new forms ever increases. The flesh of different animals differs materially in appearance. Veal, mutton, and pork, are lighter in colour than beef. The method of slaughter has something to do with this, as in those cases where free bleeding takes place the flesh is of a lighter hue through loss of hemoglobin. The flesh of young animals, containing as it does less haemoglobin, is also lighter in tint. 240 PRACTICAL SANITARY SCIENCE Fig. 6o. — Head of Cysticercus. x 20. Fig. 61. — TAENIA Solium x 4. There is a widespread feeling in this country, not by any means founded upon knowledge, that the carcase of an animal which has ME A T 241 Fig. 62. — Trichina Spiralis, x 100. Fig. 63. — Head of Distoma Hepaticum. x 4. died of any disease should not be used as food. In certain cases this is obviously correct, but in others there is no evidence to show that the edible parts are in any way deteriorated as food materials. 16 242 PRACTICAL .s\-i.V7 7.-i/eV SCIEXCE Flesh containing infective parasites, and flesh which is in a state of putrefaction, inchiding ' high ' game, should be rigorously excluded from human consumption. Good fresh meat possesses certain well-recognised characters which are easy of detection. It is Arm and somewhat elastic to the touch, pointing to the fact that rigor mortis is well developed. It is dry on section, of a clear red colour and acid reaction. A section through the whole thickness of a joint presents a uniform appearance. The odour of fresh meat may be obtained by running a clean wooden Fig, 64. — AscARUs Lumbricoides. x 7. skew'er down to the bone, and withdrawing and smelling it. The fat is firm, and not too yellow in colour. Old animals and those fed on oil-cakes exhibit fat of a deep yellow colour. The bone-marrow is bright red, and coagulates within twenty-four hours. The lymphatic glands arc of normal size, colour, and consistence. The ash contains a normal quantity of phosphoric acid and salts of potash. When cooked, meat should not lose more than 30 per cent, of its weight, and when dried on a water-bath to constant weight it should not lose more than 75 per cent. MEA T 243 Bad meat may present many evil characters. A deep purple colour points to acute septicemia, pulmonary disease ending in asphyxia, or when found in patches to hypostatic congestion. The odour may be that of advanced putrefaction, or it may be urinous, as in uraemia. There is absence of elasticity in a section when pitted with the finger; some parts are softer than others, and the flesh may be generally sodden and dropsical. The fat is highly coloured, soft, and perhaps hgemorrhagic. The juice expressed from unsound meat is alkaline in reaction from the formation of Fig. 65. — OxYURis Vermicularis. x 20. ammonias. At later stages the meat becomes green, and even black, when no critical examination is required to establish its con- dition. Certain chemical tests have been devised to detect putre- faction in the early stages, but none of them can convey more reliable information than that obtained by well-trained eyes and noses. The carcases of animals that have died of anthrax and allied conditions, pyaemia, and septicaemia, present congested, ecchy- mosed, and haemorrhagic tissues. In all cases where one or other of these diseases is suspected the offal should be seen and carefully examined. 244 PRACTICAL SAXITARY SCIEXCE Preservatives in Meat — Boric Acid. — A portion of finely- divided nuat mcchanioalh- freed from fat is warmed with water acidulated with HCl. The extract is tested with turmeric. Quanti- tative estimation is made from the same extract as under milk. Salicylic Acid. — A portion of meat freed from fat as above is slightly acidified and shaken up with ether; the ether extract is evaporated to dryness, and the residue tested in aqueous solution with ferric chloride. A violet colour indicates sahcylic acid. Formaldehyde. — This preservative may be used as a solution, and as a gas in meat-safes and the holds of vessels carrying chilled meats. Inside safes is placed a receptacle carrying pastilles of polymerized formaldeh3'de — paraformaldehyde or trioxymethylene — which is heated until the paraformaldehyde is depolymerized and simple aldehyde vapour is given off. The meat is left in contact with the vapour for twenty minutes or more. In the holds of vessels formalin is evaporated in the presence of the quarters of dressed meat in the proportion of lo ounces to i,ooo cubic feet of space. Fomialdehyde penetrates the substance of the meat, especially areas not covered bj- fat, to distances extending from 5 to 20 millimetres. The proteins and amino-acids of meat unite with formaldehyde to form methylene-imino compounds, as demonstrated by Schiff. The reaction is reversible, and only proceeds to completion in the presence of excess of formaldehyde: CHa'NHa-COOH + H-CHO -CH, : X-CHo-COOH + H.,0. Amino-acetic acid. Amino-acids composed of basic and acid groups have an ampho- teric reaction; when treated with H'CHO they become acid, and the amount of liberated acid can be readily determined by titration with standard alkali: hence the amount of formaldehyde which enters into the reaction can be detemiined. The colour reactions b}' which formaldehyde can be detected in milk are not applicable to meat, inasmuch as meat gives a violet colour when heated with HCl in the absence of formaldehyde (formation of hsematoporphyrin from Hb). Schryver uses the following test: To 10 c.c. of the water in which a portion of meat has been heated for five minutes in a boiling water- bath, add 2 c.c. of a I per cent, phenylhydrazine hydrochloride MEAT 245 solution. Cool and filter through c(jtton-wool. Add i c.c. of 5 per cent, potassium ferricyanide solution and 4 c.c. of concentrated HCl. A brilliant fuchsin-like colour is formed, which in a few minutes reaches its maximum and lasts for several hours. (The ferricyanide oxidizes the aldehyde condensation product to a body which is a weak base, which forms a scarlet hydrochloride. On dilution with water this body hydrolyses, forming a base which can be extracted with ether to form a yellow solution. If to this last concentrated HCl be added, the base passes back into aqueous solution in the form of the scarlet hydrochloride.) In those cases in which the formaldehyde amounts to about I in 50,000 parts of meat, 10 grammes of minced meat are used with 10 c.c. of water. Where the concentration reaches i part in 5,000 meat, 10 grammes of meat are heated with 100 c.c. of water and 20 c.c. of the phenylhydrazine hydrochloride solution. After filter- ing and cooling, 12 c.c. of the filtrate (as above) are mixed with i c.c. of the ferricyanide and 4 c.c. HCl. By comparing the colour obtained with carefully prepared standards, the amount of formaldehyde in any sample of meat can be determined {see Appendix). Bacterial Food-Poisoningr [cf. L.G.B. Food Reports, No. 18).— Three groups of bacteria appear to take part in outbreaks of food- poisoning — viz., the Gartner group of hQ.c\\\\; Bacillus coli, B. proieus, etc. ; and B. hotulinus. The Gartner group {B. enteritidis, B. snipestifer, B. paratyphosus B, etc.) has been found responsible for many outbreaks of poisoning through eating pork, pork pies, pork sausages, brawm, meat and minced and baked meat, tinned tongue, tinned salmon, veal pies, milk, etc. The B. coli group has been found in milk, meat pies, tinned meat, etc. B. proteus and other putrefactive bacteria are occasionally found in cases of poisoning by sausages, chilled meat, etc. B. hotulinus (studied by Van Ermengem) is occasionally respon- sible for cases of sausage-poisoning. An experimental investigation in the human subject on the influence of boric acid and borax on food, by Dr. Harvey W. Wiley, United States Department of Agriculture, was published in 1904, as Bulletin No. 84, part i. Bureau of Chemistry, Washington; and 246 PRACTICAL SANITARY SCIENCE a further similar investigation on the influence of saHcylic acid and saUcylates was published in 1906, as part 2 of the same bulletin. Wiley's findings on the influence of boric acid and borax were critically reviewed by Professor Oscar Liebreich ; an English trans- lation of Liebreich 's report, dated iqoO, is published by J. and A Churchill. ALCOHOLIC BEVERAGES The alcohols (C„H2„+20) may be regarded as oxygen derivatives of the paraffins. They are colourless and neutral substances pos- sessing neither alkaline nor acid reaction. Those with few carbon atoms are liquid; the higher members of the series are solid. Methyl, ethyl, and propyl alcohols are miscible with water; butyl alcohol dissolves in 12 parts, amyl alcohol from fusel-oil requires 39 parts of water. The relative proportion of oxygen determines the solubility in water; as ox3'gen decreases with increasing molecular weight, the physical characters of the paraffin corre- spondingly predominate. Alcohols resemble water in certain reactions, in others caustic alkalies. They, like water, liberate one atom of H when treated with sodium, and retain as a substitute one atom of the latter. The action of P, Br, etc., on alcohols results in compounds similar in structure to those formed from water : 2H.,0 + Na, = 2H0Na + H.. 2CH40(methyl alcohol) + Na,"= 2CH50Na + H,. H„0 + PCir- 2HCI + POCI3." CH4O + PCI5 = CH,C1 + HCl + POCI3. 3H.,0 + PBr3 = sHJBr + H3PO3. 3CH4O + PBr^ = sCHgBr + H3PO3. The similaritv in constitution between alcohols and caustic alka- lies is seen by the following reactions : NaOH + HCl = NaCl + H.,0. CH4O + HCl - CH3CI + H.,0. NaOH + HoSOj = NaH SO^ + H.,0. CH4O + Ha^SO^ - CH3HSO4 + H.O. It follows, then, that the graphic formula of an alcohol may be constructed in the same manner as that for water and caustic soda: ALCOHOLIC Li EVER AGES 247 H H— 0-H Na— 0— H H— C— 0— H. I H The different alcohols do not behave alike on oxidation. Some form aldehydes, others ketones. This difference in behaviour on oxidation divides them into three groups — primary, secondary, and tertiary alcohols. A primary alcohol has the hydroxyl group linked to an end carbon atom of a straight chain, and contains the group -CHalOH). A secondary alcohol has the hydroxyl group attached to a middle carbon atom of a straight chain, and contains the group .•CH{OH). In a tertiary alcohol the carbon atom attached to the hydroxyl group is linked to three carbon atoms :C(OH). Methyl alcohol, CH3(0H), has a specific gravity 0-812, and boiling-point 66° C. Ethyl alcohol, C2H5(OH), has a specific gravity o-8o6, and boiling-point"78° C. Propyl alcohols, C3H7(OH). Propyl alcohol (primary), CH3CHoCH2(OH), has a specific gravity 0-804, ^^'^ boiling-point 97° C. Propyl alcohol (secondary), CH.,-CH(0H)-CH3, has a specific gravity 0-789, and boiling-point 81° C. Butyl alcohols, C4H9(OH). Butyl alcohol (normal primary), C^-^-CH.j^-CR^-OK, has a specific gravity 0-810, and boiling-point 117° C. Butyl alcohol (normal secondary) C2H5-CH(OH)-CH3, has a boiling-point 100° C. Butyl alcohol (tertiary), (CH3)2C(OH)-CH3, hasaspecificgravity 0-786, and boiling-point 83° C. Amyl alcohols, C5Hu(0H). Normal primary, CoH5-CHo-CH2-CH2(OH), has a specific gravity 0-815, 3-nd boiling-point 138° C. Isobutyl carbinol, (CH3)2-CH-CHo-CH,(OH), has a specific gravity 0-810, and boihng-point 131° C. Secondary butyl carbinol, CH3-CH-(C.,H5)-CH2(OH), has a boihng-point 128° C. Methyl propyl carbinol, CoHj-CH^-CHOH-CHg, has a boiling- point 119° C. Diethyl carbinol, C.2H5-CHOH-C2H5, has a boihng-point 117° C. 248 PRACTICAL SANITARY SCIEXCE The primary alcohols on oxidation lose two atoms of hydrogen and form aldehydes; the latter, on continued oxidation, take up one atom of oxygen, and are converted into acids. Ethj'l alcohol ^aelds acetaldehyde, and then acetic acid: CH3 I CH; 3 I I H— C— 0-,-H + 0=H— C=0 + H,0. I H CH3 CH3 H— C=0 + =H0— C = 0. The secondar\^ alcoliols yield up two atoms of hydrogen in the first stage to form ketones. Further oxidation forms acids con- taining fewer carbon atoms than the ketones. The tertiary alcohols decompose on oxidation, fonuing ketones, or acids containing fewer carbon atoms than the alcohol. The alcohols are found as constituents of man\.' natural products, such as fats, oils, waxes, etc. They are prepared mainly by fermenta- tion. Eth\i, propyl, butyl, and amyl alcohols arc all produced in this way. Methyl alcohol is obtained by the distillation of wood,, and by the destructive distillation of the by-products of the beet- sugar industry. Commercial methyl alcohol contains acetone. When yeast is added to a solution of grape-sugar or cane-sugar, the liquid froths and appears to boil ; the sugar is broken up into ethyl alcohol and carbon dioxide. Pasteur described this as the result of life without oxygen, the yeast cells being able in the absence of free oxygen to use combined oxygen liberated in the decomposition of the sugar or other substance. Many explanations of the phenomenon were offered by observers in a controversy which has lasted for many years. In 1896 Buchner discovered accidentally that yeast-juice (free from cells), to which sugar had been added in order to prevent putrefaction, fermented the sugar; on heating the juice to 50° C. its power of fermentation was destroyed. He concluded that the production of alcoholic fermentation does not require so compli- cated an apparatus as the yeast cell, and that femicntation was effected by a dissolved substance in the cell to which he gave the ALCOHOLIC BEVERAGES 249 name of " zymase." Yeast-juice contains a powerful tryptic enzyme. Zymase when it has acted for some time disappears, and Buchner conchided that it was destroyed by the endotrypsin. When a mixture of alcohol and ether is added to juice, a precipitate is formed which can be dried to an amorphous powder (zymin) of high fermentative activity. The action of living yeast appears to follow the same law as that of most enzymes — viz., the enzyme unites with the fermentable material (substrate or zymolyte), forming a compound which only slowly decomposes, so that it remains in existence for a perceptible interval of time. The rate of fermentation depends on the rate of decomposition of this compound, and hence varies with its con- centration. It has been shown by Harden that the addition of a soluble phosphate to a fermenting mixture of a hexose with yeast- juice or zymin causes the production of an equivalent quantity of carbon dioxide and alcohol, which fact, it is concluded, indicates that a definite chemical reaction occurs in which sugar and phosphate are concerned. An equation can be constructed embodying two molecules of sugar in action in which carbon dioxide and alcohol are equal in weight to half the sugar used, and hexosephosphate and water to the other half : 2CfiHi20e + 2PO4HR2 = 2CO2 + 2C2H6O + 2H2O + CeHioOiCPO^Ra).^. The main difference between fermentation by yeast-juice and by the living cell appears to consist in the rate of decomposition of the hexosephosphate. A comparison of living yeast, Z5^min, and yeast- juice, shows that these form an ascending series with respect to their response to phosphate. Using fructose as the zymolyte, yeast does not respond to phosphate at all, the rate of fermentation by zymin is doubled, and that by yeast- juice increased twentv to forty times. It may be that the balance of enzymes in the living cell is such that the supply of phosphate is maintained at the optimum, and a further supply, consequently, does not alter the rate of fermentation. Although alcohol is the principal constituent by which such beverages affect the nutrition of the body, it must not be forgotten that in many cases ethers, aldehydes, and other bj'-products of 250 PRACTICAL SAXITARY SCIEXCE fermentation, are likewise found. Alcohol to the extent of i per cent, seems to be favourable to a digesting mixture in the stomach ; 10 per cent, slightly retards gastric digestion, and 20 per cent, arrests it. Pancreatic digestion is much more sensitive to alcohol ; but as digestion is not only a chemical process, but greatly in- fluenced by the movements of the stomach and other factors differ- ing widely in different individuals, it is not surprising to find that alcohol has ver\' different effects in its relation to individual cases. It is admitted on all hands that it quickens the activity of stomach movements and secretions. If the retarding influence of alcohol on the chemical part of digestion be weighed against its quickening influence on the flow of gastric juice and on gastric peristalsis, the balance is in favour of its use as a digestive stimulant. In certain conditions of disease these properties are greatly enhanced. Alcohol, unlike water, is freely absorbed b}' the mucous membrane of the stomach, and requires no digestion. It passes into the blood at once. Not only is it rapidly absorbed itself, but it assists the absorption of other bodies. Whilst it passes from the stomach into the blood, water passes from the blood into the stomach: the endosmotic equivalent of alcohol is 4-2, which means that, for every gramme of alcohol passing through an animal membrane in one direction, 4-2 grammes of water pass in the opposite. Alcoholic beverages are all in a broad sense saccharine products, the result of the fermentation of sugar. In fruits sugar exists in the juice, which on exposure to the air ferments: C6Hi.A=2CO., + 2C,,HeO. In grain a preliminary' fermentation takes place — starch is con- verted into sugar: 2CeH,oO,, + H,0 = CeH.o O^ + CeH, A- (starch) (dextrin) (dextrose) C,Hio05 + H.30 = CeHiA (dextrin) (dextrose) Beer. In making beer, barley is steeped in water and spread in layers a few inches deep on floors, where a temperature favourable to germi- nation is maintained. Diastase is formed in the grain. When germination has proceeded sufficiently, the grain is dried on a kiln, ALCOHOLIC BEVERAGES 251 and is known as malt. The malt is mixed witli water at fxP to 65° C, and the diastase rapidly converts the starch into dextrin and maltose. The extract, or wort, is run into copper pans and boiled, with addition of hops. The liquid is now rapidly cooled to 15'' to 17° C. and drawn into vats ; yeast is added, and the maltose alone undergoes fermentation. As this sugar forms only a small portion of the extract, the quantity of alcohol is not large. The addition of glucose to the boiling-pan increases the amount of alcohol. The wort is capable of growing other bacteria than yeast, and if great care is not taken secondary fermentations occur, and pro- duce diseased beers. In brewing, the temperature largely affects the character of fermentation. Slow fermentation, known as ' bottom fermenta- tion,' in which the yeast settles out at the bottom, proceeds at 6° to 8° C. Top fermentation, in which the yeast is carried to the surface, occurs at 16° to 18° C, and is not so easily controlled. The yeast cells in either case feed on the dextrin, maltose, peptones, and amides of the wort. Lager beers contain a low proportion of hops (female flower of Humulus lupuhis) and a high proportion of extract and alcohol. At the proper phase beer is drawn off the yeast and run into casks, where it undergoes a secondary fermentation. Most of the German white beers are produced by quick top fermentation, and have a high percentage of carbon dioxide, being bottled before the second fermentation is complete. Enghsh ale is made by top fermentation of a wort which contains a considerable proportion of hops. The fermentation is checked at an early stage, hence it is rich in sugar. Porter is a dark ale made from brown malt dried at a high tem- perature. It has a large extract, mainly sugar, and may contain caramel. Stout is porter with larger alcohol and extract contents. Detection of Ethyl Alcohol. — Warm 10 c.c. of the fluid under test with a few drops of benzo^d chloride ; add a little NaOH solu- tion; ethyl benzoate is formed with characteristic odour where as little as o-i per cent, alcohol is present. Other alcohols produce ethers with characteristic odours. The Iodoform Test. — ^Warm 10 c.c. of the fluid in a test-tube 252 PRACTICAL SAXITARY SCIENCE with a few drops of strong solution of iodine in KI ; add solution of NaOH till the mixture is nearly decolourized. On standing a precipitate of iodoform (star-shaped or hexagonal tablet crystals) forms where alcohol is present to the extent of o"i per cent. Acetone, lactic acid, and certain aldehydes and ketones, give this reaction, but not pure methyl alcohol, amyl alcohol, or acetic acid. Fig. 66. — Estimation of Alcohol. Estimation of Alcohol.— Expel free CO.y by shaking in a flask or separator funnel and drawing the still liquid away from the froth. Into a 250 to 400 c.c. flask pour 100 c.c. beer; add some tannic acid to prevent frothing: dilute to about 150 c.c. with HoO and distil. All the alcohol will come over in the first 75 c.c. distillate — i.e., three-fourths the original measured volume. In the case of liquors high in alcohol, it is better to distil over about 100 c.c. Make up the distillate to the volume of the liquor originally taken ALCOHOLIC J3 EVER AGES 253 and shake well. Take the specific gra,vity in a pycnometcr. Refer to the alcohol table, and read off the percentage of alcohol by volume or by weight. Tabarie'S Method. — Find the specific gravity of the beer. Evaporate 100 c.c. on a water-bath to one-fourth the volume. Make up to the original volume with distilled water, and find the specific gravity of the dealcoholized fluid. Add i to the original specific gravity, and from the sum subtract the second specific gravity. The difference is the specific gravity corresponding to the alcohol in the liquor. Suppose the specific gravity of the sample to be I'gSgg, and that of the dealcohohzed sample I'OogQ. Then 1-9899— 1-0099 = 0-9800 = 16-24 P^r cent, alcohol by volume. Acidity. — The total acidity is usually expressed in terms of lactic acid. Measure 20 c.c. beer and free it from CO2 by raising it to the boiling-point. Cool, and titrate with y^ NaOH, using litmus as indicator, i c.c. ^^5- NaOH = 0-009 gramme lactic acid. The Fixed Acid expressed as Lactic. — Evaporate 20 c.c. beer to one-fourth its volume, dilute with water to original volume; titrate with -f-ij NaOH as before. Volatile Acid expressed as Acetic. — Distil 100 c.c. beer nearly to dryness. Should the residue in the retort be still acid, add some water and continue the distillation to dryness. Now titrate the distillate with ~ NaOH, each cubic centimetre of which = o-oo6 gramme acetic acid. The normal acidity of beer is due to CO2, acetic, lactic, malic, and other organic acids, and should not exceed in 100 c.c. that neutralized by 30 c.c. ^^ NaOH. The Malt Extract.— To estimate this item with any degree of accuracy, a small quantity must be operated on. Take 5 c.c. or 5 grammes in a large platinum dish so that a thin film is formed on the bottom. Dry for two or three hours on the water-bath, and finish the drying in an air-bath at a temperature somewhat above 100° C. Bitters. — The bitter of hops is readily soluble in ether; the bitters of quassia, aloes, and hop substitutes, are insoluble in ether; whilst many bitters that might be employed are soluble in ether, the absence of a bitter taste from the ether extract demonstrates the absence of hops. In performing the test, evaporate the beer to the consistence of a syrup before extracting with ether. Further, lead 254 PRACTICAL SAXITARY SCIENCE acetate coniplctely precipitates the bitter material of hops, but leaves behind some of the bitters of hop substitutes, which may be recognized on concentrating the filtrate. Aloes. — Dry 200 c.c. beer and treat the residue with ammonia. Filter, cool, and treat the filtrate with HCl. Collect the resin on a filter. This is insoluble in cold water, ether, petroleum ether, and chloroform, but soluble in alcohol. It has a characteristic odour which identifies it. Gentian. — Treat the acid residue with chloroform in the cold: no colour is produced; warm, and a camiine-red colour appears. A small quantity- of the red solution mixed with a drop or two of ferric chloride solution changes to a greenish-brown. Qitassia. — Ouassiin in acid solution is soluble in chloroform, and, when mixed with a little alcoholic solution of ferric chloride, gives a mahoganv-brown coloration. Preservatives in Beer. — Boric acid and salicylic acid are detected in the same manner as described under milk, concentrating the beer if necessar}^ to one-fifth or one-tenth of the bulk. Sac- charin is detected by acidulating a portion with H0SO4, shaking with a mixture of equal volumes of ether and petroleum spirit, evaporating down with a little NaOH solution, and carefully heating for a short time to about 250° C. Salicyhc acid is formed, and this is tested for in the ordinary way. Sulphurous Acid. — To 25 grammes sample in a 200 c.c. flask add 25 c.c. N-KOH. Shake and set aside for twenty minutes. Add 10 c.c. 25 per cent. H2SO4 and a little boiled starch solution. Titrate rapidly with ^^ iodine till a blue colour is produced. One c.c. of the iodine solution =0-00064 gramme SOo. Sulphurous acid is used to regulate the fermentation and to produce a flavour of age. Sodium Chloride. — Where common salt has been added, an allowance not exceeding 50 grains per gallon must be made for the amount of this compound present in the water, malt, and hops used. Ash a suitable quantity of beer, say 100 c.c; exhaust the ash with water; titrate the solution with /^ AgNOg, using neutral potassium chromate as indicator. Arsenic. — In Lancashire in 1900 an outbreak of arsenical poisoning occurred, in which arsenic amounting to --^^j grain per gallon was fre- ALCOHOLIC BEVERAGES 255 quently found, and it was stated in some cases that i grain in a gallon was found. Marsh Test — Preliminary Treatment of Beer. — Place 100 c.c. beer freed from COg by shaking in a porcelain dish; add 20 c.c. pure concentrated HNO3 and 3 c.c. concentrated H2SO4 ; heat in a fume chamber till vigorous frothing occurs; lower the flame and stir till frothing ceases ; boil freely ; continue heating till mass chars and fumes of H2SO4 are given off; pieces of filter-paper may be stirred in till the residue is dry; cool, add 50 c.c. water, and remove masses of char from sides of dish with glass rod ; heat to boiling and filter; use the filtrate in the Marsh apparatus. Marsh Apparatus. — Fit up a generating flask with funnel tube. Attach a U-tube containing pumice moistened with 10 per cent, lead acetate solution to absorb H2S. To this attach a CaCla dr\'ing tube, and a hard glass tube of about 6 millimetres bore drawn out for about 4 centimetres to i millimetre internal diameter; draw out the end to still narrower dimensions; support the tube over a two- or three-burner furnace, wrapping the portion in contact with the flame in wire gauze. Place in the generating flask 20 to 30 grammes arsenic-free stick zinc and a perforated platinum disc to form an electric couple. Run in through the funnel sufficient 20 per cent. H2SO4 to start the reaction and expel air. When aU air has been driven out and danger of explosion has passed, heat the tube to bright redness. When absence of As in the reagents has been settled, add slowly through the funnel the solution of the substance in 20 per cent. H2SO4. When the flow of gas begins to slacken, add some 30 per cent. H2SO4, and later 40 per cent, acid, tiU all As has been ex- pelled. Two or three hours may be required to finish the expul- sion. If no mirror forms in the constriction of the tube in an hour, it may be taken that there is no As present. If more than o-i milligramme As appears to be present, cut off the constriction from the tube and weigh it on a fine balance. Dis- solve the As out with a solution of sodium hypochlorite ; wash the tube with water; dry with alcohol and weigh. The loss of weight is As. If the As is very small in amount, compare the mirror with a series of standard mirrors prepared in the same apparatus from 256 PRACTICAL SAXITARY SCIEXCE quantities of a standard solution of As containing from 0-005 to 0-05 milligramme As^Og. Such standard solution is prepared by dissolving o-i gramme pure As.,0.j in a little pure NaOH solution, acidifying with pure H0SO4, and making up to 100 c.c. with water. Ten c.c. of the latter fluid is further made up to i litre. One c.c- = 0-0 1 milligramme As.,0.}. Reinsch's Test. — Acidify 100 c.c. beer with i c.c. HCl (free from arsenic) ; evaporate to less than half the bulk. Set up two beakers on gauze over Bunsen burners (the second to act as a control). In the first place the prepared beer, and in the second an equal volume of water. To each add 5 c.c. concentrated pure HCl and a strip of bright pure copper-foil 10 millimetres by 5 millimetres. Heat for an hour, replacing from time to time the water lost by evapora- tion. If a deposit forms on the copper, remove it, and wash very carefully with water, alcohol, and ether. Place in a subliming tube and heat over a low flame. The crystals are for the most part regular octahedra, with perhaps a mixture of rectangular prisms. Clarke has made this test quantitative : Dissolve the arsenic from the Cu slip in dilute aqueous solution of potash and H-.O^ in the cold. Then boil, and filter off any CuO. Concentrate the filtrate to a small bulk and wash into a distilling flask with strong arsenic- free HCl ; add some ferrous chloride ; fit the flask with a safety tube and connect with a small worm condenser. Distil down twice with pure strong HCl. Pass H^S into the distillate. A precipitate will form if more than o-i milligramme be present; if less than this quantitv, a 3'ellow colour. As little as o-ooi milligramme arsenic sulphide gives a faint yellow colour, which may be matched by a series of standard colours produced under the same conditions.* Wine. In making wine the juice of the grape is left in open vats where its sugar undergoes spontaneous fermentation. The bloom which co\'ers the outside of the grape contains the necessary yeast, and the natural acidity of the juice, or must, excludes foreign organisms. * See description of Marsh-Berzelius process, Analyst, February, 1902, xxvii., 48, 210. ALCOHOLIC BEVERAGES 257 The relative porportions of protein and sugar influence the character of the wine, as yeast {Saccharomyces ellipsoideus) Hves upon the protein, and sphts the sugar, forming alcohol and other products. If yeast grow in little sugar and much protein, it can maintain its existence until all the sugar is changed; such a wine is said to be dry and acid, like hock. Conversely, if there be much sugar and little protein, the growth of yeast comes to an end before all the sugar is used, and that left behind produces a sweet wine. Intermediate proportions of sugar and proteins produce corre- sponding results. It may be noted, though, that, no matter what the proportions of protein and sugar, fermentation cannot proceed after 16 volumes per cent, of alcohol have appeared in the liquid; this is why a natural wine can never contain more than this pro- portion of alcohol. Sherry and port are fortified wines — that is, containing, as they do, more than 16 per cent, of alcohol, they have the difference added to them. Claret and hock are natural wines. The quality of wine depends on the species of yeast used, the variety of grape, the soil and climatic conditions of growth of the grape, and the mode of its cultivation. The colour of red wines is produced by a pigment {cenocyanin) residing in the skins of the grapes, which is turned red by the acids present. As alcohol is produced, it dissolves out this pigment, and so colours the distillate. Wine, when placed in casks, undergoes important changes: water evaporates more quickly through the woodwork than does the alcohol, and so the alcohol becomes concentrated. Further, some oxidation of the tannic acid takes place; this causes white wines to be somewhat darker in colour, and red wines lighter, through the carrying down of some of their pigments by oxidized tannic acid. Frequently a small amount of yeast enters the cask, and continues the fermentation, thereby increasing the quantity of alcohol. With the lapse of time, some of the alcohol is oxidized into acetic acid, and certain compound ethers are formed. Wine in bottles adds to its contained ethers, although its alcoholic strength rarely, if ever, increases. It is an error to suppose that very old wine contains most alcohol: slow oxidation in the case of wines, as in all other organic compounds, produces degeneration. It is more than probable that no wine improves in quality after a period of ten to fifteen years. 17 25S PRACTICAL SANITARY SCIENCE Fermentation progresses most rapidly at a temperature between 25° and 30° C, but finer bouquet is produced by slower fermenta- tion, and accordingly must is fermented in open vats in cool cellars at 5° to 15° C. till it settles out comparatively clear, care being taken to avoid acetic fermentation. When the first or active fermentation is complete, the wine is drawn off into casks, where it undergoes a second slow fermentation, with deposit of potassium bitartrate and development of the characteristic flavour. The wine is sometimes clarified with gelatin, and sometimes pasteurized, before the final bottling or casking. Volatile ethers predominate in natural wines, fixed ethers in fortified. Sparkling wines, as distinguished from still, are highl}^ charged with COg, either pro- duced naturally by after- fermentation of added sugar (champagne), or artificially b}^ carbonating, as in the case of soda-water. Port wine is rich in tannin, and to certain inferior wines this astringent, together with alum and catechu, is added. Port con- tains a large amount of extracts, which give it a full body, and old port a large proportion of ethers, of which (vmlike sherry) the fixed ethers predominate over the volatile. Sherries, as imported into this country, are all fortified and plastered, and contain from 15 to 25 per cent, of alcohol b}' weight. Old sherry contains a large proportion of volatile ethers, and to this property much of its value as a stimulant must be attributed. Champagne is produced from black grapes, and depends for its character very largely upon the quality of the grapes of a particular vintage. The expressed juice, after sedimentation for twelve hours, is drawn off and fermented; it is then bottled and allowed to undergo secondary fermentation for a couple of 3'ears, during which time much COo is produced, and a deposit. To the wine, which is up till now sour, cane-sugar, which has been dissolved in old cham- pagne, is added in varying quantities. Dry champagnes which find their way to England contain little sugar — not more than i or 2 per cent., whilst sweet chcmipagnes may contain 10 to 15 per cent. Claret is a deep red wine, somewhat acid and astringent; it con- tains little sugar, but considerable quantities of volatile ethers. Its content of alcohol varies from 8 to 12 per cent, by volume. Hock is a white wine containing little sugar, 9 to 12 per cent, by volume alcohol, and is mildly acid. ALCOHOLIC BEVERAGES 259 Plastering is the term applied to the adulteration of the must before fermentation with plaster of Paris or gypsum, wherein objectionable potassium sulphate is left in solution in the wine: CaSOj + 2KHC4HJO6 = H2C4H4O6 + CaC4H40f, + K.SO^. The precipitation of calcium tartrate carries down impurities, the colour is improved, and the fermentation hastened and made more complete; the practice is said to enhance the keeping qualities of the wine. Cane-sugar is added to the must to increase the yield of alcohol. Glucose is used instead of cane-sugar, and introduces unfermentable matter, dextrin, and various mineral salts. Added. Water. — Gautier (' Traite sur la Sophistication et r Analyse des Vins ') has shown that the sum of the weight in grammes of alcohol in 100 c.c. and total acidity (as H2SO4) in a litre varies in pure wines within narrow limits, being rarely below 13 or above 17. If considerably below 13, water may be assumed to have been added. Colouring' Matter in Wine. — Cubes of solid transparent gelatin, | inch square, are immersed in the wine for twenty-four hours, after which they are removed, washed in water, and cut in half. In genuine wines the colouring rnatter will not have pene- trated more than one-sixteenth of an inch, whilst in wines coloured with fuchsin, cochineal, logwood, litmus, indigo, etc., the cubes will be penetrated to the centre. The colouring matter of alkanet root, turned blue b}^ ammonia, is the only foreign matter in general use which slowly penetrates the gelatin. Dilute ammonia dissolves cochineal and logwood out of the gelatin, the cochineal becoming purple and the logwood brown. Estimation of Alcohol.— iVs in beer. Acids. — The acids of wine are chiefly tartaric, malic, and tannic, and certain acids of the fatty series — acetic, formic, etc. — produced during fermentation. Tartaric acid forms with potassium a bi- tartrate. i\.s alcohol increases in wine this salt becomes less soluble, and finally faUs out in the form of a crust, so that the acidity diminishes on keeping. Tannic acid is obtained from the skins and stalks of the grapes used; it diminishes by oxidation on keeping, and in old wines is small in amount. 26o PRACTICAL SANITARY SCIENCE Get rid of CO., by shaking. Heat about 20 c.c. to boiling, and titrate with ^^ NaOH (in white wines and cider use phenol- phthalein as indicator). One c.c. ^xr NaOH = 0-0067 gramme mahc acid, or 0-0075 gramme tartaric acid. This is the total acidity. Volatile Acids. — Place 50 c.c with a little tannin in a distilling flask connected with a condenser. Connect a second distilling flask containing 250 c.c. water with the first by glass tube passing almost to the bottom. Heat both to boiling: then lower the flame under the distilling flask and pass steam through the wine until 200 c.c. distillate come over. Titrate the distillate with -^^ NaOH (indicator phenolphthalein). One c.c. y\ NaOH =0-006 gramme acetic acid. Ethers. — Ethers are produced in wines by the chemical action which takes place between the acids and alcohols. Volatile ethers are obtained from volatile acids, such as acetic, and these, especially acetic ether, predominate in natural wines. Fixed ethers are derived from fixed acids, such as tartaric, and are found in forti- fied wines: they impart to wine its bouquet. CEnanthic ether I part in 50,000 wine imparts the vinous smell and taste to all wines in common. Extract. — Dry 10 grammes to constant weight in a platinum dish: a small amount of glycerin may be lost. Ash. — Ignite the dried residue at a low temperature and weigh. Most natural wines contain i part ash to 10 parts extract. Sug'ars. — The chief sugar of wine is laevulose, of which a natural wine should not contain more than 0-5 per cent. Fortified wines mav contain from 2 to 25 per cent. Extractives found in wine consist of gums and various carbohydrates, and contribute to the taste and so-called body of the wine. Reducing sugars are determined by Fehling's method. Potassium Sulphate. — Acidify 100 c.c. of the wine with HCl; boil and add excess BaCU. Filter, wash well, dry, ignite, weigh as BaS04: calculate the equivalent K0SO4. More than 0-06 gramme indicates plastering. ALCOHOLIC BEVERAGES 261 Spirits. Spirits. — Whisky, brandy, rum, gin, etc. Whisky. — Whisky is made from malt or malt and grain, and distilled in pot-stills or patent-stills. For many years superior claims were made for the pot-still article, but these claims have been destroyed by the report of the recent Royal Commission. In 1905 a London magisterial investigation decided that patent- still spirit alone is not whisky, and that whisky cannot be made from maize ; the above report upsets this view. The pot-still in its simplest form is a pot with a long neck over which the distilled alcohol passes when the wash or fermented mash of grain is boiled. Usually two distillations are carried out in producing Scotch whisky. The patent-still is an arrangement of pipes and chambers through which steam is passed continuously as the wash distils. This is a cheaper process capable of a much greater output. The Commission concluded that it would be no advantage to pro- hibit the use of foreign barley, and it would be too arbitrary to say that Scotch whisky should be made from malt alone, and Irish from a mixture. Maize affects the flavour, but there is no valid reason for excluding it. Patent-still whiskies are less varied than pot-still, but the same effects are produced by both kinds if taken in the same quantity and in the same strength. Pot-still distillers admit the need of blending with patent-still whisky, unless their own spirit can be matured longer; patent-still tones down the pungent taste of the other. Cheap blends contain as little as 10 per cent, of pot-still. As whiskies used in England are usually blends, and as the patent-still is adapted for economical and larger production, and as there is no evidence that the form of still has any relation to the wholesomeness of the spirit, the Commission could not recommend that the term ' whisky ' should be restricted to the pot-still variety. Brandy is determined by the report as a potable spirit made from fermented grape-juice and from no other materials. ' British brandy ' is defined as a compounded spirit prepared by a rectifier or compounder by redistilling duty-paid spirits made from grain with flavouring ingredients, or by adding flavouring materials to 262 PRACTICAL SAXITARY SCIEXCE such .spirits; the nature of the flavouring materials is not dis- closed. True brandy is distilled wine, and was originally procured from a rich Cognac district in France. Its quality varies with the character of the grapes used, the best grapes yielding grande champagne, a genuine liqueur brandy. It is to be feared that little of the brandy sold in this country is so derived. Brandy contains, beside ethyl alcohol, volatile ethers in large amount, an important distinction from whisky. Its percentage of alcohol is about the same as that of whisky. Alcohol. — Estimation by distillation as under Beer. Metallic Impurities. — Pb, Cu, etc. Detection and estimation as under Water. Fusel-Oil. — Fusel-oil is the most important impurity of spirit. It is more injurious than ordinary alcohol, and should not be permitted to exceed o-2 per cent, (i) Shake 20 c.c. of the spirit with 2 c.c. dilute KOH. Evaporate on water-bath to 2 or 3 c.c. Cool and add 5 c.c. strong sulphuric acid. The odours of valerianic and butyric acids will be detected if fusel-oil be present. (2) Distil off four-fifths of the sample, and extract the residue with ether: allow the extract to evaporate spontaneousl}', and treat what is left with H2SO4 and sodium acetate: the odour of pear is emitted. (3) Evaporate 50 c.c. slowh' over a steam-bath; carefully smell the remainder for traces of fusel-oil. (4) Decolourize a portion of the sample with animal charcoal and add a few drops each of h3^dro- chloric acid and colourless aniline-oil. In the presence of fusel-oil a rose tint is produced in the aniline-oil. [Tests for methylated spirit: (i) Odour: (2) a weak solution of sodium nitroprusside (i per cent.) and ammonia, added to a mixture containing methylated spirit, give a red colour within ten or fifteen minutes.] Estimation — Marquardt Method.— Jo 100 c.c. spirit add 20 c.c. -^; NaOH, and saponify by allowing to stand overnight, or by boiling for an hour under a reflux condenser. Distil 90 c.c. ; add 25 c.c. water, and distil an additional 25 c.c. Saturate the distillate with NaCl, and add saturated NaCl solution till specific gravity is i-i. Extract the salt solution four times with CCI4 (recently purified by boihng with sulphuric acid and potassium bichromate, and distilling) ALCOHOLIC BEVERAGES 263 using 40, 30, 20, and 10 c.c, respectively. Wasli the CCI4 extract three times with 50 c.c. portions of a saturated Na('l solution, and twice with the same volumes of saturated sodium sulphate solution. Now boil the tetrachloride for eight hours with 5 c.c. concentrated H2SO4, 5 grammes potassium bichromate, and 45 c.c. water under a reflux condenser. Add 30 c.c. water, and distil till about 20 c.c. remain; add 80 c.c. water, and distil till 5 c.c. are left. Neutralize the distillate to methyl orange; add phenolphthalein, and run in /o NaOH till neutral. One c.c. -^-^ NaOH=o-oo88 gramme amyl alcohol. In the oxidation and second distillation the corks used should be covered with tinfoil. Rose's Method. — Chloroform quickly removes fusel-oil from dilute spirit, and the presence of fusel-oil in chloroform increases the capacity of the latter for dissolving ethylic alcohol. So, there- fore, if chloroform be shaken with dilute ethyl alcohol containing fusel-oil, its volume will be considerably greater than when shaken with the same volume of pure ethyl alcohol. Dilute the spirit to be tested until its specific gravity is 0-9655 at 15° C. (30 per cent, alcohol by volume). If the sample is weaker than this it must be fortified by absolute alcohol (i per cent, error + or - corresponds with o-oigg per cent, by volume of fusel-oil). In the special tube place 20 c.c. chloroform, which at 15° C. reaches the lower division of the scale. Add 100 c.c. alcohol and i c.c. H2SO4, specific gravity 1-2857. Stopper the apparatus and shake a definite number of times, say 150. Let stand for some time, and read the volume of chloroform. Submit pure alcohol of the same strength to the same process, and note the difference in volume of the chloroform. An increase o-oi c.c. (the scale is readable to o-oi c.c.) is equal to 0-006631 per cent, amyl alcohol. Methyl Alcohol — Method of Riche and Bardy. — This method depends on the formation of methyl-anilin-violet. To 10 c.c. sample add 15 grammes iodine and 2 grammes amorphous phos- phorus. Stand in iced water till action has ceased. Distil on a water-bath the methyl and ethyl iodides into 30 c.c. water. Wash with dilute NaOH to remove free iodine. Separate the heavy oily liquid which settles, and mix with 5 c.c. anilin in a flask placed in cold water. After an hour boil and add about 20 c.c. 15 per cent, soda solution. The bases rise to the top as an oily layer; float 264 PRACTICAL SANITARY SCIENCE them up with water and pipette off. Oxidize i c.c. of the oily liquid by heating in a glass tube at 90 °C. for eight or ten hours with 2 parts NaCl, 3 parts Cu(N0.,)2, and 100 parts clean sand. Exhaust with warm alcohol, filter, and make up to 100 c.c. with alcohol. In the case of pure spirits the liquid is red, but in the presence of i per cent, methyl alcohol it is violet. Dilute 5 c.c. of the coloured liquid to 100 c.c. with water, and dilute 5 c.c. of this again to 400 c.c. Heat the liquid in a porcelain dish with some pure white merino wool (free from sulphur) for half an hour. If the spirits be pure the wool will remain white, but if methylated the fibre will become violet. A quantitative estimation can be made by comparing the tint with a set of standards containing known percentages of methylic alcohol. Rum is prepared from molasses, a by-product in the manufac- ture of sugar, but the best varieties are obtained by fermenting the juice of the sugar-cane. One by-product — ethyl butj^rate — confers upon it its characteristic flavour. Like brandy, however, much of the rum sold in this country is made from silent spirit, flavoured with characteristic bj^-products. Gin is prepared by distilling and redistilling a mixture of vyo. and malt. In the last distillation juniper berries, salt, and hops, are added, and the product is run off into cisterns lined with white tiles, whereby colouring matters are prevented entering the spirit. The best gins are distilled in Holland; but much of the gin of commerce is concocted from silent spirit, resins, and juniper berries. The term ' proof-spirit ' is applied to a mixture of 57-06 per cent, by volume of absolute alcohol in water. It has a specific gravity of 919-8 at 15° C. Brandy, whisky, and rum, may be 25 degrees under proof — that is, may contain 75 per cent, of the alcohol found in proof-spirit. Gin may be 35 degrees under proof — that is, may contain 65 per cent, of the alcohol found in proof-spirit. Spirits generally contain 40 to 60 per cent, of alcohol; wines 8 to 16; beers 5 to 7. Acidity of Spirits. — Titrate with decinormal alkali and calculate as acetic acid. One c.c. y alkali = o -006 gramme acetic acid. Esters. — Dilute 250 c.c. of the spirit with 50 c.c, water, and distil 200 c.c. Neutralize 50 c.c. of the distillate with decinormal alkali ALCOHOLIC BEVERAGES 265 (phenolphthalein indicator); add yg- alkali in considerable excess. Boil for an hour under a reflux condenser. Cool and titrate with -^^ alkah. The number of cubic centimetres -/i; alkali used in the saponification multiplied by o' 0088=^ grammes esters calculated as ethyl acetate. Fupfupal. — Prepare a standard furfural solution. Dissolve I gramme redistilled furfural in 100 c.c. 95 per cent, alcohol. Dilute I c.c. of this to 100 c.c. with 50 per cent, alcohol. One c.c. == O'oooi gramme furfural. Dilute 20 c.c. of the above distillate to 50 c.c. with 50 per cent, alcohol free from furfural. Add 2 c.c. colourless anilin and 0-5 c.c. HCl, specific gravity 1-125. Make standards, from which match the tint. Furfural is found in pot-still, but not in patent-still, spirit. Alcohol Table. Sp. Gr. at Per Cent, Per Cent. Sp. Gr. at Per Cent. Vc. Alcohol (Vol.). under Proof. 15° c. Alcohol (Vol.). I'OOO 0-00 100-00 0-973 23-10 0-999 0-66 g8-84 0-972 24-08 o-ggS 1-34 97-66 o-g7i 25-07 0-997 2-12 96-29 o-g70 26-04 0-996 2-86 95-00 o-g6g 26-95 0-995 3-55 93-78 o-g68 27-86 0-994 4-27 92-50 o-g67 28-77 0-993 5-00 91-23 0-966 29-67 0'9g2 5-78 89-87 0-965 30-57 0-99I 6-55 88-50 0-964 31-40 o-ggo 7-32 87-16 0-963 32-19 0-989 8-i8 85-65 0-962 32-98 0-988 9-04 84-15 o-g6i 33-81 0-987 9-86 82-70 0-960 34-54 o-g86 IO-73 81-20 0-959 35-28 o-g85 ii-6i 79-65 0-958 36-04 o-g84 12-49 78-10 0-957 36-70 0-983 13-43 76-46 0-956 37-34 o-g82 14-37 74-82 0-955 38-04 o-g8i 15-30 73-18 0-954 38-75 o-g8o 16-24 71-54 0-953 39-47 o-gyg 17-17 69-90 0-952 40-14 o-gyS 18-25 68-00 0-951 40-79 o-g77 19-28 66-20 0-950 41-32 0-976 20-24 64-53 0-949 41-84 0-975 2i-ig 62-87 0-948 42-40 0-974 22-18 61-13 0-947 42-95 Per Cent. under Proof. 59-52 57-80 56-06 54-37 52-77 51-18 49-60 48-00 46-44 44-97 43-60 42-20 40-74 39-47 38-18 36-83 35-68 34'57 33-32 32-08 30-84 29-66 28-52 27-60 26-67 25-70 24-74 266 PRACTICAL SANITARY SCIENCE Alcohol IdihlQ— continued. Sp. Or. at Per Cent. Per Cent. Sp. Gr. at Per Cent. Per Cent. IS" c. Alcohol (Vol.). •inder Proof. Vc. Alcohol (Vol.). over Proof. 0-946 43-5 CH3. CO. CH.. COOH. Glucose is oxidized in the tissues to glycuronic acid: HoO., effects the same reaction — H„. OH. CHOH. CHOH. CHOH. CHOH. CHO — >COOH. CHOH. CHOH. CHOH. CHOH. CHO. Indol is oxidized to indoxyl: Yijd., brings about the same reac- tion — H ^ / \_COH "SCH -- > I I "^CH 'N \/ N H H And so with other reactions. Such similarity of action is not only interesting from an academic point of view, but also from the practical, as when a mild antiseptic for use in the human subject is to be selected. Hydrogen peroxide is prepared by acting on a peroxide of an alkaline earth by an acid, and other means— BaO^ + H^SO, - HoO, + BaS04. DISINFECTANTS 295 Its action as a disinfectant is somewhat slow. It is said, liow- ever, not to have the same tendency to oxidize dead organic matter as permanganates, whilst it destroys associated bacteria. It has been used to sterilize water and milk. Estimation of H./)o. — This body is sold as containing 5, 10, or 20 volumes O in solution. To 10 c.c. peroxide solution under test add about 30 c.c. H2SO4 (i in 3) in a beaker (the sulphuric acid must be in fairly large excess), and crystals of KI in excess, and, after standing for five minutes, titrate the liberated I with y^ thiosul- phate and starch. Before testing, solutions of peroxide should be diluted to the strength of two volumes O — 2HI + H,A=l2^ 2H2O. I c.c. -^jj thiosulphate = 0-00085 gramme H2O2, or O" 0008 gramme O. Ozone is formed from atmospheric oxygen in a variety of ways : When phosphorus is left in contact with air, it is slowly oxidized and ozone formed. Platinum may be used for its production. Permanganates treated with concentrated H2SO4 yield ozone. The most common and most inexpensive method of procuring it is by means of the silent electric discharge. Electrical ozonizers have been erected in recent years for the sterilization of the water of the Marne, outside Paris, and the results have been reported as good. Various schemes have from time to time been initiated in different countries for the purification of the air of towns, public buildings, and private dwellings, by ozone; but whether advantageous results have accrued from any of these undertakings is highly doubtful. Free chlorine is capable of killing bacteria by combining with and coagulating their protoplasm. Chlorine destroys the offensive odour of HgS, a product of nitrogenous putrefaction, by decom- posing it, with formation of HCl and S — H2S + Cl2= 2HCI + S. But chlorine acts as a germicide for the most part, by combining with the hydrogen of water and liberating nascent oxygen — H20 + Cl2 = 2HCl + 0. The liberated O is the disinfectant. Light increases this reaction. The application of dry chlorine gas in disinfection may be regarded as useless. 296 PRACTICAL SAX IT A RY SCIEXCE In the so-called chloride of lime (;i mixture of CaCl., and Ca(OCl)o) and other hypochlorites, such as chloros, Hermite solution, etc., this halogen is used in considerable quantities. Its action in all these cases is that of an oxidizer. Chloride of lime, or bleaching powder, is produced by passing chlorine over moist lime, and is preferred to the soda and potash compounds in that it can be kept as a dry powder. The hypo- chlorite portion is strongly alkaline, and in the presence of moisture reacts with the CO., of the air to form hypochlorous acid and calcium carbonate — Ca(0Cl)2 + H.O + CO., = CaCOg + 2HCIO. In the act of disinfection, the HCIO sphts into HCl and nascent O. One part of fresh bleaching powder to ten parts of water has been recommended as a disinfectant solution for general work, and i part to 100 of water as a solution for the hands. When solutions of chlorides of the alkalies or alkaline earths are electrolyzed, hypochlorous acid and the corresponding hydrate are formed— MgCl, + 2H.,0 = Mg(OH)o + 2HOCI. Hermite applied this preparation to sanitation. Chlorine and hypochlorites fail as disinfectants when used for materials rich in dead organic matter. Whilst the dead matter is being oxidized, the germs escape. Estimation of CI in Bleaching Powder. — Prepare a decinormal solution of sodium thiosulphate, Na.^SoOg.sHaO. Dissolve 24-827 grammes of the crystals in a litre of H2O. Weigh a gramme of bleaching powder, and grind it thoroughly in a mortar. Add small quantities of water at a time, and rub into a smooth cream. Decant the liquor into a litre flask. Continue to grind the sediment with successive quantities of water until the whole is transferred to the litre flask as a fine emulsion. ]\Iake up to the mark. Take 20 c.c. of the uniform emulsion in a basin; add excess of KI solution, dilute slightly, and acidify with acetic acid. Titrate the liberated I with ^^ thiosulphate and starch. One c.c. YzT thiosulphate = 0-0035 -4 gramme CI. DISINFECTANTS 207 Another method: Prepare -j'^ I, and l^^-^ solution of .'ilk;ilin<; arsenite. Mix commercial rcsubHrncd iodine with half its weight KI, and dissolve in half its weight of water. Precipitate the I with water, and filter through asbestos; wash well to remove KI, and dry over H2SO4. Sublime between two large watch-glasses twice, and finally weigh out 12-7 grammes. Dissolve this in 18 grammes KI (pure) and about 250 c.c. H^O. Make up to a litre. Dissolve 4-95 grammes pure sublimed and powdered ASgOg with 20 grammes pure sodium carbonate in about 25,0 c.c. HoO. Warm and shake occasionally until solution is complete; cool and make up to a litre. Take 20 c.c. of the well-shaken turbid emulsion of bleaching powder in a basin, and run in from a burette ~ arsenious solution in slight excess (a drop fails to produce a blue stain on KI — starch paper). Add some starch and run in ~ I from another burette until a slight blue colour remains. The number of c.c. ~ I required •gives the number of c.c. of arsenious solution that have been added in excess; subtract this from the total added to obtain the number ■of c.c. of -j^ arsenious solution equivalent to the CI in the bleaching powder used — One c.c. y{j arsenious solution = 0-00354 gramme available CI. These methods determine quantitatively chlorinated soda, Hermite solution, chlorine-, bromine-, and iodine- water. Sulphur Dioxide in Solution, and in Sulphite — Estimation in Solution. — Weigh the solution (previously cooled to 5° C. in a freezing mixture) in a stoppered flask; introduce it into a second •stoppered flask, containing excess ^ iodine. Shake thoroughly, and estimate the unchanged iodine with ^ thiosulphate and .c'l" o Tpn SO2 + I, + 2H.,0 = H^SOj + 2HI. Each c.c. of ^ I taking part in the reaction = 0-0032 gramme SO.,. Estimation in Sulphite. — Powder some sulphite finely. Weigh a small quantity in a watch-glass, and introduce it immediately into a measured excess of -^jj I in a beaker. Stir until the reaction is complete, a result only slowly obtained with insoluble sulphites — 298 PRACTICAL SANITARY SCIENCE e.g., calcium sulphite. Estimate the excess iodine. It is well to- do a second determination, using only a slight excess of -^^ I. The SOo is calculated as above. Bromine acts in a similar manner to chlorine by liberating nascent oxygen. Its germicidal power in the free state has been estimated as about equal to that of chlorine, but in combination with organic radicals it is superior. If careful comparative tests be made, how- ever, it will be found that bromine is a more energetic disinfectant than chlorine, and more energetic than can be accounted for by the amount of nascent O liberated. This fact leads to the conclusion that Br acts as a disinfectant in a manner other than by liberating oxygen. Iodine as an oxidizer is feebler than chlorine or bromine, but destroys bacteria more energetically than either by combining with their protoplasm. Matthews found that a solution of iodine in iodide of potassium of a strength of i in i,ooo killed an emulsion of Staphylococcus^ Pyogenes aureus in water in fifteen seconds, whilst iodoform in full dose was without action, Permangranate of potassium, K20,Mn207, when acidified with H0SO4, can yield 5 atoms of oxygen to organic matter: KX>MryJdi + 3H2SO4 = K2SO4 + 2MnS04 + 3H0O + 5O. If insufficient H.^SOj be used, only 3 atoms of O are furnished : K.O.MngO, + H2SO4 + sHgO^ K2SO4 + 2Mn(OH)4 + 3O. Like the other oxidizing disinfectants, its germicidal powers are expended on dead organic matter and inorganic compounds, such as sulphuretted hydrogen, ferrous salts, nitrites, etc., rather than on living bacteria. But for naked bacteria permanganates are power- ful disinfectants. The disinfectant activities of oxidizers are in- creased by the addition of haloid acids. Estimation of Potassium Permanganate. — Prepare y'y oxalic acid by dissolving 6-301 grammes pure crystals in a litre of water. On adding potassium permanganate to a warm solution of oxalic acid and sulphuric acid, the following reaction occurs: SHaC.O^, 2H2O + 3H2SO, + 2KMn04 = 10CO2 + K2SO4 + 2MnS04 + 18H2O. DISINFECTANTS 299 The factors taking part in oxidation may bo written more simply: H2C204'2H20 + = 2CO2 + 3H2O. Place 50 c.c. of the fj^ oxalic acid and a little H2SO4 in a beaker, and dilute with water; heat to 60° C. Gradually run in from a burette the permanganate solution (about 5 grammes to the litre) until a faint permanent pink remains in the liquid after stirring. If the permanganate be added too rapidly, a brown precipitate forms, which is removed with difficulty by adding more sulphuric acid. One c.c. Y^ oxalic acid = 0-003163 gramme potassium perman- ganate. Salts of Mercury. — Of metalHc salts, perchloride of mercury has had, perhaps, a larger application as a disinfectant in medicine and surgery than all the others put together. The metalhc ion in solution unites with the protoplasm of the germ, causing its death. The complex appears to be of the nature of a precipitate rather than a coagulum, as it redissolves in excess of albumin. It is, therefore, necessary to use perchloride of mercury in excess. The salt is highly poisonous. The readiness with which protoplasm is precipitated by its forming albuminate and other protein compounds of mercury militates against it as a disinfectant for sputum rich in albuminoid matters, or for abscess cavities. The precipitated coat of albumin protects the enclosed bacteria from further action ; hence the germs can survive, and on breaking down of the pellicle may migrate and set up infection at a distance. The cyanide and iodide of mercury are both highly germicidal and highly poisonous. Mercury salts interfere with the action of soap. Mercuric chloride is estimated by dissolving it in HCl, and pre- cipitating the sulphide by passing HoS to saturation. The pre- cipitate is allowed to stand for a time, then thrown on a filter and washed, until the washings leave no residue on evaporation. It is then dried at 100° C. and weighed. Hg is calculated from the weight of the HgS. The precipitate may contain free S, in which case it is washed with recently distilled CS,,. Formaldehyde, |t^C:0, is obtained by oxidizing the vapour of methyl alcohol in the air in contact with heated platinum or 300 PRACTICAL SANITARY SCIENCE copper, and receiving the products in water. The lonnahn of com- merce is a 40 per cent, solution of the aldehyde in water and methyl alcohol. On evaporating this solution in vacuo in the presence of a small amount of HjSO^, a crystalline white powder falls out, of undetermined molecular weight (CH.,0)„, and known by the names ' paraformaldehyde ' and ' paraform.' This polymer is volatilized on heating into formaldehyde. Both the liquid and solid forms are used in disinfection. An enormous amount of work has been done on the properties of formaldehyde as a germicide, and everyone is agreed that as such it holds a high position. For application to rooms the solution may be heated, or the solid may be volatilized over a lamp. There can be little doubt that the interaction between formaldehyde and the protoplasm of the germ is of the nature of a coagulation. Its powerful reducing properties remove oxygen from the protoplasm, probabl}' both from liydroxyl groups and from the oxygen united directly to carbon. It is used for the floors, walls and ceilings of rooms as a spray, in the form of vapour produced by an autoclave under pressure, and as the vapour of paraform produced by a lamp. For spray work various strengths of solution have been recommended, ranging from 0-5 per cent, to 2-5 per cent, and higher. Some suggest supple- menting the spra}^ with vapour, more especially where rooms are exceptionally dirty, and unknown organisms like that of smallpox are being dealt with. In the present state of practical disinfection a wide margin of safety should be insisted on. It is possible that in some circumstances the highest concentration recommended fails to sterilize. Estimation of Formaldehyde. — Fonnaldehyde slowly absorbs am- monia to form hexamethylene-tetramine; 180 parts formaldehyde react with 68 of ammonia: 6CH,0 + 4NH3 = (CHoJeN^ + 6H.,0. Place 10 c.c. of the solution to be tested in a flask, and neutralize, if necessary, with ^^ NaOH ; dilute with water, and treat with an excess of standard ammonia solution. It is well to stand over- night. Distil the excess of ammonia b}^ a current of steam into standard acid. Calculate the percentage amount of formaldehyde from the amount of ammonia combined. DISINFEC TAN'I S 30 1 The success which attended the early apphcation of Carbolic Acid as an antiseptic by Pasteur, Lister, and others, attracted attention to coal tars as a source of germicides. By suitable fractional distillation these tars can be separated into — (i) First runnings; (2) light oils; (3) heavy oils; (4) anthracene oils. Carbolic acid is contained for the most part in the light oil fraction ; whereas the heavy oil fraction contains its homologues, especially the cresols. At first acid and alkaline solutions of crude carbolic acid were used as disinfectants, but it was soon found that these were not suitable. Pure watery solutions of cresols were then tried, and likewise abandoned for saponified emulsions. It was discovered that emulsions conferred increased germicidal efficiency on the various active phenolic bodies used, and that side-chain substitution in the benzene ring produced the same result. It was also discovered that metacresol, the least soluble in water, had a higher germicidal power in emulsion than ortho- or para- cresol. The relative solubilities of the three isomers in water are — Per Cent. Orthocresol . . . . . . . . . . 2-5 Metacresol . . . . . . . . . . 0-53 Paracresol . . . . . . . , . . i-8 Two important stages in the evolution of coal-tar disinfectants had now been reached and passed. The emulsion was better than the solution: insolubility in water was of advantage in the same direction. The high germicidal properties of thymol illustrate these princi- ples, containing as it does three side-chains attached to the benzene ring: /CH3 (I) CgHg— C3H7 (4) \0H (3) Its molecular weight is much higher than that of phenol(CgH5.0H). Its solubility in water is about i in 1,100, as against i in 15 for phenol. Koch found that the same germicidal work was performed 302 PRACTICAL SAXITARY SCIENCE on anthrax bacilli by thymol in dilution of i in 80,000 as by phenol in i in 1,250. Estimation of Phenols. — The following method is based on the precipitation of phenol from its aqueous or alcoholic solution by bromine as tribromphenol. Prepare a standard solution consisting of 2-04 grammes sodium bromate and 8-oo grammes potassium bromide in a litre of water. One c.c. of this solution = 0-0012638 gramme phenol. 5KBr + NaBrOg + 6HC1 = 5KCI + NaCl + 3Br., + 3H2O. CgH^OH + 3Br., = QH^OHBrg + 3HB"r. 2KI + Br;=2kBr+L.' 1, + j^^S.p'. - Na^SjOg ; 2XaI. Weigh out a gramme or two of the phenol to be tested in a tared watch-glass, and dissolve in excess of NaOH. Make up to, say, 500 c.c. Take 20 c.c. (one-twenty-fifth of the whole) in a 300 c.c. stoppered flask, and add 25 c.c. of the standard bromide bromate solution. In a second 300 c.c. stoppered flask place 25 c.c. of the standard bromide bromate solution. To each add 5 c.c. pure HCl and shake. Add such a further measured quantity of the standard bromide bromate solution to the phenol flask that, on shaking, the white tribromphenol is left dis- tinctly yellow (excess Br). Shake well and stand for fifteen minutes. Add excess KI to both flasks, and titrate with a solution of thio- sulphate of Na (say 10 grammes to a litre). Example. — 2-i68 grammes phenol required 75 c.c. standard bromide bromate to become yellow. The iodine which the free bromine liberated required i6-8 c.c. thiosulphate. But 25 c.c. bromide bromate in second flask required 51-8 c.c. thiosulphate. Therefore i6-8 c.c. thiosulphate = 8-i8 c.c. bromide bromate solu- tion. Therefore 75 —8'i8 = 66-82 c.c. bromide bromate solution which interacted with phenol. Therefore 66-82x0-0012638x25=: 2-III grammes phenol. 2-i68 : 2-111 :: 100 : 97-4. That is, this sample contains 97-4 per cent, pure phenol. Laubenheimer showed that a i per cent, solution of phenol required ninety minutes to kill a quantity of staphylococci, whereas DISINFECTANTS 303 the same strength of a solution of propyl-phenol, C^// ^.Vr '' did the same work in three minutes. Increase in molecular weight does not always mean increase in germicidal power; because, in the same series of Laubenheimer's experiments, isopropyl-phenol required twelve minutes to kill. Working with m-xylenol, /CH3 (I) CgHg-CH,, (3) \0H (5) S5niimetric, and ^-xylenol, /CH3 (I) CeH3-CH3 (4) \0H (2) he found that the meta-compound was much more powerful than the para- in killing staphylococci. On incorporating an atom of chlorine in meta- and para-cresols, he found that chlor-m-cresol, /CH3 (I) CgHa^OH (3) \C1 (6)1 still retained its advantage, and even increased this advantage over chlor-/)-cresol, /CH3 (I) CeH3-0H (4) \ CI (2) The relative position of the side-chains in the ring is thus shown to be of importance. It may very well be that the disinfection process is assisted by the meeting of suitable side-chain affinities in microbe and disinfectant. Decrease in solubility in water means decrease in toxicity. It is possible, therefore, to apply to the skin and intestinal mucosa ■insoluble phenyloids in concentrations which could not be tolerated in phenol. Henle, working twenty years before Laubenheimer, showed that the germicidal powers of the cresols varied with their boiling- points, the meta-compound, with highest boihng-point, possessing 304 PRACTICAL SANITARY SCIENCE the most intense action, and the ortho-, with the lowest boiHng- point, the least intense action. Working with higher phenols, Sommerville found that the same principle obtains. Using the Rideal-Walker method of estimating germicidal efficiency, and emulsionizing fractions from the same distillate of blast-furnace phenyloids, he found that a fraction boiling at 248° possessed a coefficient three points above that of another fraction boiling at 220°, and five points above that of a third fraction boiling at 207°. Again, it is possible to alter by several points the coefficient of a phenyloid by varying the chemical or physical characters of the emulsion. Changes which make for increased adsorption raise (within limits) the coefficient. Increased viscosit}' in the emulsion lowers (within limits) the coefficient. If a liquid is contained between two parallel plates, and one of these be moved with a constant velocity in its own plane, a certain force is required which depends on the velocity, the surface, and distance, of the two plates, and on the temperature and nature of the liquid. The force required to move a plate of unit surface separated from another plate of the same size by a layer of liquid of unit thickness at unit velocity is known as the viscosity coefficient. Colloidal solutions may be divided into two classes if the increase of viscosity compared with that of the continuous phase (solvent) be made the basis of classification. One class presents a viscosity only slightly higher than that of water (metal and sulphide solu- tions). The other, the organic colloids (albumin, gelatin) presents a marked increase of viscosit3^ In those solutions presenting a low viscosity, the disperse phase is present as solid particles; in those with high viscosity, the disperse phase is liquid. Albumin solu- tions consist of a dilute solution of albumin in which are dispersed globules of a more concentrated solution. Systems of solid particles of microscopic size distributed in a liquid are known as ' suspen- sions ' ; those consisting of two liquid phases are known as ' emul- sions.' The particles in a solution, if sufficiently small, are in constant motion, oscillating round a central position, and also undergoing an irregular translatory motion. Svedberg showed that the ampli- DISINFECTANTS 305 tude of the motion of a particle is directly proportional to the period, and inversely proportional to the viscosity, of the liquid. Perrin showed that this Brownian movement conformed to the principles of the kinetic theory, and that the particles could be treated as large molecules. The stability of the solution is intimately con- nected with the electric charge. The charge can be altered by the addition of electrolytes, and may fall to zero with suitable con- centrations, in which last case the solutions precipitate. It has been long known that the speed of settling of such suspensions can be increased by the addition of electrolytes. In systems of two liquid phases, it can be shown that very small liquid particles approaching ultramicroscopic dimensions possess a high degree of rigidity. Systems of two liquid phases possessing few and widely .separated particles differ in no important respect from systems containing rigid particles; but an important difference appears as the amount of disperse phase per unit volume increases. In the case of rigid spherical particles in contact, the disperse phase may reach a maximum of 74 per cent, of the total volume. If the disperse phase be liquid, the globules may not merely touch one another, but become flattened at the points of contact, from which circumstance it is obvious that there is no limit to the ratio vol. of disperse phase u- u j.- i, v tj. • f — = — i- , which ratio may approach unity. It is total vol. J rr J not possible to prepare emulsions containing such percentages of disperse phase unless the continuous phase is a solution of certain substances, such as soap. Such bodies froth, an indication that the dissolved substance lowers the surface tension of the solvent. The process of emulsification is intimately connected with such lowering of surface tension, or, rather, interfacial tension between the two phases. The stability of. emulsions -varies considerably. They are destroyed by the addition of all substances which destroy the emulsifying agent; thus, "emulsions made with soap solution are destroyed by the addition of an acid which decomposes the soap. In the making of an emulsion, -the two phases are shaken up until the disperse phase is sufficiently finely distributed. In the case of gelatin emulsions and soap emulsions, the behaviour of the solution is not to be explained unless by assuming that it is a system of two 20 3o6 PRACTICAL SANITARY SCIENCE fluid phases; in other words, it consists of globules having a high gelatin content in a continuous phase which is a dilute solution of gelatin. The solvent here may be shifted most readily from one phase to the other. Different behaviour is shown by the albumins. Egg albumin is soluble in water, and does not form a gel. either by cooling or concentration, but it coagulates irreversibly at a temperature of about 60° C. The temperature of coagulation can be changed b}' adding salts, and may be raised to over 100° by the addition of a thiocyanate. In relation to this phenomenon is the change which follows the addition of alkali salts in the cold — the coagulation known as ' salting out.' If at the boundary surface between the phases of a disperse system a change in the concentration of either phase will lead to a decrease of surface tension, this change will occur. The change in concentration is adsorption. It requires work to make or enlarge a surface; when such surface is made, it is the seat of energy. As we have seen above, adsorption plays probably an important role in disinfection. Soap emulsions of coal-tar phenyloids can be con- structed which are eminently suitable for the production of this phenomenon. Such emulsions when compared with suspensions show a decreased size of particle with reduced velocitj' of settlement, increased Brownian movement with increased electric charge, due to the great increase of specific surface. These emulsions provide for a high degree of bombardment of the microbe by the active par- ticles of disinfectant, followed by marked adsorption, both necessary preliminaries to the final chemical action required to kill the organism. In most of the modern better-class disinfectants distilled from tar, and emulsionized in soaps, the active principles are phenyloids. In the raw materials these bodies are mixed with neutral oils, saturated paraffins, unsaturated paraffins (olefines, etc.), pyridines, and a mass of heterogeneous substances. The unsaturated hydrocarbons are washed out with H2SO4, and the phenyloids with NaOH (formation of sodium phenylates). Separation is made in laboratory practice in separator funnels. The addition of a few drops of alcohol sometimes assists the separa- tion. Sodium phenylates are spht with H2SO4, and the free phenyloids DISINFECTANTS 307 recovered. These may be fractionally distilled, and the fractions emulsionized. Hard waters, including sea-water, ' salt out ' soap emulsions in varying degrees. Such waters should be softened before using them for diluting soap emulsions of phenyloids. Gelatin or glue, whilst not forming so good an emulsion as soaps, is not attacked by hard waters to the same degree. To determine the percentage composition of a coal-tar disinfectant in a soap emulsion, fractionally distil 100 grammes of the disin- fectant. Measure the water and weigh the phenyloids. Below 270° C. resin gives no trouble, as any resin spirit present (never more than 5 per cent, in resin soap) is in union with alkali, and resin oils boil between 300° and 400° C. Should a small quantity of neutral oil come over, which rarely happens, it may be separated with the phenolic bodies by washing with soda, and subsequently splitting off the phenyloids with H2SO4. Five grammes of the disinfectant are incinerated, resulting in NagCOg or KgCOg. The residue is lixiviated with water, filtered, titrated with standard HCl, and calculated as NagO or KgO. The weight of the chloride will at once determine whether one is dealing with K or Na. As the residue in the distillation retort consists of anhydrides of fatty acids or resin acids, or of both, of the form R. CO ONa R. COO Na it is plain that in the original disinfectant those anhydrides plus H2O are equivalent to the NagO. Hence 5 grammes disinfectant minus weight of NagO equals fatty acids plus resin in 5 grammes. If the fatty acid and resin figures are required separately, they can be easily worked out from the retort residue by Twitchell's method. In 1903 Rideal and Walker published a method of standardizing disinfectants. The method has since undergone slight modifica- tions, and to-day is carried out as follows: The materials required for the test are a standard nutrient bouillon, standard carbolic acid, dilution of the disinfectant, and the broth culture. The nutrient bouillon is composed of 20 grammes of Liebig's extract of 3o8 PRACTICAL SAXITARY SCIENCE meat, 20 grammes of \\'itte's peptone, 10 grammes of sodium chloride, and i litre of distilled water. This mixture is boiled for thirt}^ minutes, filtered, and neutralized with normal sodium hydrate, using phenolphthalein as indicator. To avoid contamin- ating the broth with phenolphthalein, a small aliquot part, say 10 c.c, should be taken out and titrated with y^^ NaOH; from the result obtained a calculation is made of the amount of nonnal sodium hydrate necessary for the neutralization of the remainder of the broth. When quite neutral, 15 c.c. of N.HCl is added. The broth is then made up to a litre and sterilized. Where 2 or 3 htres are prepared at one time, as is customary, the broth is distributed in 500 c.c. flasks on the following day and again sterilized. With the aid of a small separating fvmnel, 5 c.c. are then run into sterile test-tubes, which, after plugging with sterile cotton-wool, are placed in the steam sterilizer for half an hour. As carbolic acid crystals are frequently contaminated by cresols to such an e.xtent as to make them unreliable for purposes of bacteriological control, their purity should be established by a determination of the solidifying-point on at least 50 c.c. of material with the thermometer in the liquid. The point is very sharp, the thermometer showing a constant temperature for a period of from five to ten minutes. The sohdifying-point of the crystals is 40-5, but anything over 40 may be accepted. A 50 per cent, by weight stock solution is then prepared and standardized by titration with decinormal bromine. From this solution, which keeps indefinitely in stoppered bottles, the various working strengths are made by diluting a comparatively large quantity, such as 100 c.c, to the desired volume; this serves to eliminate the error introduced by measuring out small quantities of strong acid. In preparing dilutions of the disinfectant, a stock solution or emulsion should be prepared in a 100 c.c. stoppered cylinder with sterilized distilled water — 10 per cent, if the coefficient be under r, and I per cent, if over i. Ten c.c. of this stock are used in preparing each of the four dilutions required for the test. Thus, working with a sample having a coefficient under i, if it is desired to prepare a dilution i in 70, 10 c.c. of the 10 per cent, stock solution are diluted with 60 c.c. of distilled water ; and in the case of a preparation having a coefficient over i, where the dilution required is i in 700, 10 c.c. DISINFECTANTS 309 of the I per cent, stock solution should be diluted with 60 c.c. water. • The culture of B. typhosus is incubated for twenty-four hours at 37° C. in Rideal-Walker broth. It is advisable to make a sub- culture every twenty-four hours from the previous twenty-four- hour culture, even if on many days no test is performed ; but, as this tends to attenuate the organism, it should be continued for not longer than one month, when a fresh subculture in broth should be taken from an agar culture one month old. This procedure secures a test culture varying but little from day to day in resistance offered to disinfectants, and renders the selection of the appropriate dilution of carbolic acid easier than if the culture from which the twenty-four- hour growth is obtained were older on one occasion than on another. The apparatus required consists of a test-tube rack, an inoculating needle, test-tubes, and a dropping pipette. The test-tube rack possesses two tiers, the upper having holes for thirty test-tubes in two rows, each row containing three sets of five. The upper tier holds sterilized broth tubes, each of which is numbered with a grease pencil. The lower tier holds the medication-tubes, four containing the postulant disinfectant dilutions, and one the carbolic acid con- trol dilution. This tier is provided with a copper water-bath intended to preserve the temperature of medication within the prescribed limits (15° C. to 18° C). The test-tubes are numbered in rotation; and it will be seen that the first medication- tube is used for inoculating broth-tubes — i, 6, 11, 16, 21, and 26; the second for inoculating, 2, 7, 12, 17, 22, and 27, etc. The needle recommended is a thin aluminium rod carrying a short piece of platinum wire, o-oi8 inch in diameter (26 U.S. gauge), passed through and twisted round an eye in the end of the rod. A loop 3 millimetres internal diameter is formed on the end of the wire. The length of the wire to the end of the loop should be about if inches. A fairly uniform drop can be obtained after a little practice by dipping the needle in the medicated culture, and bring- ing it out with a slight jerk. The test-tubes should be of strong glass, so as to minimize the risk of breakage, and lipped to facilitate the manipulation of plugs. The size recommended is 5 inches by f inch. The cotton-wool plugs for both medication-tubes and broth-tubes 3IO PRACTICAL SAXITARY SCIENCE should be well made, so that they can be withdrawn and replaced without loss of time. The dropping pipette is standardized to deliver o-i c.c. of the broth culture per drop. It is loosely plugged at the top with cotton- wool, and when not in actual use is kept in a sterile test-tube plugged at the mouth with cotton-wool. For greater convenience, the tube should be passed through the centre of the plug, and fastened thereto with wire. In addition to these, one or two of each of the following are required: i, 5, and 10 c.c. pipettes; 100 and 250 c.c. stoppered cylinders, with inverted beakers, to safeguard against dust after removal from sterilizer; wire baskets to receive tubes for incubation or sterilization. All pipettes and cylinders should be standardized. Before commencing the test, it is necessar}^ to ascertain the car- bolic acid control dilution which will give the desired result — i.e., life in two and a half and five minutes. This is done by running a trial test with five dilutions of the carbolic acid only — say i in 80, I in 90.. I in 100, i in no, and i in 120. Five c.c. of the control solution so ascertained are then pipetted into the fifth medication- tube, the other four receiving 5 c.c. of the various dilutions of the disinfectant under test. To save time and apparatus, one pipette can be made to do service at this stage by starting with the phenol solution, and following on with the highest or lowest dilution of the disinfectant, according as the coefficient is below or above i, rinsing out the pipette in each case with the next dilution before measuring off the sample for test. The plug of the culture-tube is now replaced by the culture pipette, which, as explained above, has a plug attached to it with wire, at such a height that, when the plug fits easily into the mouth of the culture-tube, the point of the pipette is halfway down the broth, and clear of the clumps. The first of the five medication- tubes is now inoculated with five drops of the culture — i.e., 0-5 c.c. At intervals of half a minute each of the other medication-tubes is inoculated in turn. By the time the fifth tube has been inoculated, the organism in the first will have been exposed to the action of the disinfectant for two minutes, and after the next half-minute a loopful of the latter is inoculated into the first broth-tube, loopsful from the other medication-tubes being in turn inoculated into their respective broth-tubes at the rate of one everj^ thirty seconds. By DISINFECTANTS 311 the time the fifth broth-tube has been inoculated from the fifth medication-tube, the disinfectant in the first medication-tube will have acted on the test organism for four and a half minutes, and after the next thirty seconds a loopful is introduced into broth-tube 6, and so on. The actual test, therefore, occupies seventeen minutes, and provides for six two-and-a-half-minute periods of contact in each of the five medication-tubes. It is open to the worker to adopt any convenient method of manipulating the tubes and plugs. The following procedure is given for the guidance of the inexperienced: The first medication-tube is taken from the rack, and the contents gently agitated for a second to insure even distribution of the bacilli; the plug having been taken out and grasped by the left little finger, the tube is held between the back of the left forefinger and front of the second. The corresponding broth-tube (No. i) is taken up by the right hand and transferred to the left between the thumb and forefinger, the plug being extracted and held by the little finger of the right hand. The tubes now being in position for inoculation, the needle, which should have been sterilized before the tubes were touched, is intro- duced into the medication-tube, from which a loopful is taken and inoculated into the broth-tube. The needle is sterilized in the flame (placed to the right), and pushed with a movement of the thumb well up between the first and second fingers of the right hand ; the plugs are then replaced, the medication-tube going back to the rack, while the broth-tube is subjected to a gentle agitation and placed in a wire basket on the right of the rack. This basket, containing the thirty inoculation-tubes and test form, giving particulars of the dilutions, etc., is now placed in the incubator, where it is allowed to remain for fort\^-eight hours at blood heat, when the results are read off. A moment's consideration of the manner in which the test has been conducted will suffice to indicate where the results of each subculture should be placed in the table. The following details of a test of a disinfectant marked ' A ' show the form in which the results are set out ; incidentally it shows the degree of refinement to which the test can be carried wdth a little practice and care. The strength or efficiency of the disinfectant under test is ex- pressed in multiples of carbolic acid, and is obtained by dividing 3i; PRACTICAL SAXITARY SCIENCE the dilution of the disinfectant showing Hfo in two and a half and live minutes by the carboHc acid dikition, which of course must show the same result. In the present instance this ' figure of merit,' or Rideal- Walker coefficient, is i6-6. , To avoid annoyance and loss of time caused by aerial contamina- tion of tubes, etc., it is advisable to conduct the test in a room free from draughts; a further safeguard is. provided by spraying or swabbing the floors and benches with an efficient disinfectant solu- tion. Needless to add, all pipettes, etc., must be rigorously sterihzed before use. In this, as in all other arbitrary tests, the need for strict observa- tions of the conditions of the test is imperative. B. Typhosus: Twenty-four Hours' Broth Culture at 37° C. Temperature of medication 15° C. to iS° C. Sample. Time Culture exposed to Action of Disinfectants (Minutes). Subcultures. 2j. X X X X X 5- X X X X 7J- X X 10. X 12^. •5- Period of Incubation. Tempera- ture. A Carbolic acid 1,900 2,000 2,100 2,200 120 48 hours 37° C. .*. Rideal-Walker coefficient :i6-6. APPENDIX Flock manufactured from rags, to be used in upholstery, bedding, etc., must meet the standard of cleanliness laid down by the Local Government Board's Rag Flock Regulations, 191 2 — viz., not to contain more than 30 parts chlorine per 100,000 parts flock, the chlorine to be removed as chlorides with distilled water at a tempera- ture not exceeding 25° C. from not less than 40 grammes of a well- mixed sample of flock. Steep 50 grammes of a mixed sample of flock in | litre of dis- tilled water overnight. Decant the fluid on a filter, and squeeze out the flock thoroughly. Wash the flock with smaller quantities of water (say 100 c.c.) three or four times, squeezing out all the water possible each time, and passing the washings through the same filter. Makeup the filtrate to a litre. Now evaporate 100 c.c. of this ( = 5 grammes flock) to dr^mess with a small quantity of CaO in a platinum dish, and char the residue. When cool, extract with 50 to 100 c.c. distilled water and filter. Add a few drops of potassium chromate to the filtrate, and run in from a burette silver nitrate solution (used in estimation of CI in water), i c.c. of which equals i milligramme CI. Multiply the number of c.c. used by 20 to obtain parts CI per 100,000 flock. Copper Sulphate is used for greening peas and other vegetables : Estimation of Copper. — Ash 10 grammes of the peas or other material. Moisten the ash with concentrated HNO3; add water, and boil. Make strongly alkaline with ammonia, and filter. If no blue colour, copper is absent. If blue, transfer the fluid to a Nessler glass on a white tile, and match the colour against weighed small quantities of copper sulphate converted into ammoniacal solu- tion in the same manner. Or, the copper may be deposited in the metallic state by passing an electric current through the acid solution, in a suitable apparatus, and weighed as Cu. Tin in Canned Food. — See Local Government Board Reports of Inspector of Foods, No. 7; Report of Buchannan and Schny'ver. 313 314 PRACTICAL SANITARY SCIENCE I. Colorimetric Method. — Prepare a solution of stannous chloride containing 0-286 gramme per 100 c.c. Prepare a solution of dinitrodiphenylaminesulphoxide containing 0*2 gramme in 100 c.c. ,^^ NaOH. Mix 10 parts HNO3 (sp. gr. 1-48) with 10 parts HXO;, (sp. gr. 1-4). Cool this mixture with ice, and add I part of thiodiphenylamine (prepared by heating diphenyl- amine with sulphur) in small quantities at a time with constant stirring. Do not allow the temperature to rise above 5° C, and add such small quantities at a time that a hissing sound is hardly perceptible when the solid comes into contact with the liquid mix- ture. The thiodiphenylamine dissolves at the beginning to form a clear solution of red colour, which, before the whole of the amine has been added, commences to thicken, owing to the separation of the nitro-body. After standing for some hours (not more than half a day), suck off the nitro-body on an asbestos filter, and wash first with concentrated HNO3, then with acid of gradually dimin- ished strength, and finally with pure water. Now extract it with hot alcohol in which it is not appreciably soluble. Introduce 10 grammes of the food into a 700 c.c. Kjeldahl ffask- add 10 grammes of potassium sulphate and 10 c.c. concentrated sulphuric acid. Heat over small flame till mixture chars and froths. Add another 10 c.c. H2SO4, and regulate the size of the flame so- that the H2SO4 can be boiled without loss from frothing. Heat till the contents of the flask are quite white. Cool; dilute with water to about 100 c.c. Pass in H2S gas, and let stand in a corked flask overnight. Warm slightly on a water-bath, and filter off the precipitated sulphide and sulphur. Transfer the filter-paper con- taining the precipitate to a test-tube, and boil with 5 c.c. concen- trated HCl to dissolve the sulphide. Filter through a small conical Buchner funnel into a wide-mouthed test-tube, with a side-tube near the top to connect with a pump. Suck as dry as possible, and wash with 2*5 c.c. concentrated HCl. Connect the wide-mouthed test-tube with a CO2 generating apparatus, and pass the gas through a tube which passes through a cork inserted in the mouth of the test- tube, and which reaches nearly to the surface of the liquid. The side-tube serves as an exit for the gas. Whilst still hot, throw into the strongly acid liquid a strip of zinc foil 2 inches long, 0-5 inch wide, and weighing about 075 gramme, and the stannic chloride is reduced to stannous chloride. As soon as the last traces of Zn are dissolved, add 2 c.c. of the reagent by pipette to the hot liquid, the CO2 passing the while. On addition of the reagent, the nitro-body is precipitated. On warming, it passes again into solution in the concentrated acid. Boil the solution for a minute or two, and dilute to 100 c.c. with cold water. Filter the dilute solution by means of a pump from the unchanged nitro-body. The solution usually turns violet during filtration ; the full depth of colour is rapidly attained APPENDIX 315 by addition of a drop of dilute ferric chloride. It is tlien iTiatf;lied with known quantities of the standard tin solution. | 2. Gravimetric Estimation. — Fifty granames of th(; food are in- cinerated in two lots of 25 grammes in two Kjeldahl flasks of about 700 c.c. capacity, using 25 c.c. of H2SO4 previously diluted with 100 c.c. water, and 25 grammes potassium sulphate. When thoroughly charred, another 25 c.c. concentrated H2SO4 are added, and heat continued till contents are white (perhaps four to five hours required). The contents of the two flasks are brought together and diluted to about 600 c.c. H2S gas is passed, and the mixture allowed to stand corked overnight. It is next warmed, and the mixture of sulphide and sulphur filtered through a small filter-paper 7 centimetres in diameter. The precipitate is washed on the filter- paper with warm water. With it are usually mixed bodies other than sulphur and sulphide, such as silica derived from the flask, etc. To separate these, the sulphide is dissolved on the filter-paper in a small quantity (10 to 20 c.c.) of hot 10 per cent. NaOH. From the yellow solution obtained the sulphide is reprecipitated by glacial acetic acid, filtered off, washed with hot water, dried, oxidized, and weighed as oxide of tin. Estimation of Orgranic Matter in Air.— In addition to the microscopic examination of dust and suspended matters in the air described at p. 143, it may be necessary in certain cases to estimate organic matter quantitatively. This may be roughly done thus: Aspirate a measured volume of air through a tube containing a plug of clean glass-wool, and digest the wool in standard potassium permanganate (p. 48) for an hour at 37° C. Titrate the perman- ganate with standard oxalic acid (07875 gramme crystals to a litre). Perform a blank experiment, and deduct the number of c.c. oxalic acid used from that used in the actual estimation. The result is recorded in terms of O absorbed from permanganate, i c.c. oxalic acid = I c.c. permanganate = 0-1 milligramme O. Haldane's Apparatus for Estimating- CO 2 in the Air.— This consists of an air burette (enclosed in a water-jacket with a glass face) with a wide ungraduated and a narrow graduated portion. Its capacity is 20 c.c. from top to bottom of scale. The graduated portion measures 4 inches in length, and is divided into 100 equal parts, each corresponding to one-ten-thousandth part of the capacity of the burette when moist for mercury. Readings are recorded in parts per 10,000 without calculation or corrections. The water in the water-jacket is stirred up occasionally in order to secure uni- formity of temperature. 3i6 PRACTICAL SANITARY SCIENCE In using the apparatus, the air is tirst expelled by a three-way tap from the burette by raising the mercury bulb attached to its lower end. Air is then taken in by lowering tlie bulb till the mercury falls to the zero of the graduated scale. The tap to the absorption pipette (the latter filled to a mark with lo per cent. KOH) is next opened, and the air is driven over and drawn back several times till all CO2 is absorbed as indicated by constant level of Hg. The difference between the first and last readings gives the amount of CO2 in parts per 10,000. Estimation of Formaldehyde in Meat Foods. — See Local Government Board Food Reports, No. *.). Scliry\er points out that in meat foods it is possible that the formaldehyde may be entirely oxidized to COo and HoO by tissue oxidases; that part of the fomi- aldehyde may be polymerized to paraformaldehyde; and that formaldehyde may enter into chemical combination with some of the constituents of the foodstuffs. He has confirmed the statement made by Cervello and Pittini, and by Batelli and Stern, that forni- aldehyde is destro\'ed by tissue oxidases. When fomialdeiiyde solution is distilled, the distillate contains less aldehyde than the original solution, due to polymerization by heat into a non-volatile pohTner. It is therefore not possible to estimate formaldehyde by steam distillation. . Schiff and Sdrensen have shown that formaldehyde reacts with proteins and amino-acids, with formation of methylene-imino com- pounds, and that the reaction is a reversible one, and only proceeds to completion in presence of large excess of formaldehyde : ' (NH2)CH2.COOH + HCHO ^ > CHg :N.CH2.C00H + H.O. Amino-acids, owing to basic and acidic groups, have an ampho- teric reaction, and become acid on treatment with formaldehyde: the number of amino-groups in combination can be accordingly determined by titration with alkali. Conversely, it is possible by titration to estimate the amount of formaldehyde which can enter into combination with anv product. Meats contain rela- tively large quantities of substances which are capable of entering into chemical combination with the aldehyde. The reaction, as already mentioned, will not proceed to full completion except in presence of excess of aldehyde, owing to the reversibility. In addition to these reversible compounds, formaldehyde can combine with proteins to form relatively stable insoluble products, irom which formaldehyde can be eliminated only by prolonged heating with water. Any effective method for estimating formaldehyde in meat must therefore be applicable to estimation of free aldehyde, the poly- merized product, and aldehyde in combination with the meat. APPENDIX 317 The violet colour obtained when milk containing fonnalflehyde is heated with strong HCl in the presence of an oxidizing agent cannot be used to detect aldehyde in meat, as meat gives a violet colour on warming with HCl in the absence of the aldehyde due to the formation of haematoporphyrin from haemoglobin. ■ The following method is recommended : To 10 c.c. of solution containing aldehyde add 2 c.c. of a freshly prepared and filtered i per cent, solution of phenylhydrazine hydro; chloride. To this add i c.c. of a 5 per cent, fresh potassium ferri- cya.nide solution, and 4 c.c of concentrated HCl. In the presence of fonnaldehyde a brilliant fuchsin-like colour is produced, which reaches its full intensity after a few minutes' standing, and keeps without marked deterioration for several hours. The addition of ferricyanide oxidizes the formaldehyde condensa- tion product to a substance which is a weak base, which forms a scarlet hydrochloride. This, on dilution, undergoes hydrolytic dissociation, yielding a base which can be extracted with ether to form a yellow solution. If this latter be shaken with concentrated HCl, the base passes back into aqueous solution in the form of the scarlet hydrochloride. This reaction detects formalin in concentra- tion of I part in 1,000,000. It is quantitatively best applied when the concentration is i in 50,000. From two standard solutions containing respectively i in 10,000 and I in 100,000, it is possible to make a series of dilutions from I part in 1,000,000 upwards to serve as a colour scale when the reaction is quantitatively applied. Methylene-imino derivatives can be readily hydrolysed by cold water; with ammonia, formaldehyde forms a somewhat more stable derivative; and with Witte's peptone, under certain conditions, an insoluble product from which formaldehyde is only eliminated with some difficulty. By modification of the above reaction formaldehyde can be de- tected in all such combinations. If the mixture containing such product be warmed after addition of phenylhydrazine, the aldehyde after scission combines immediately with phenylhydrazine to form a stable condensation product. This reaction, being irreversible, proceeds to completion. On the.addilion of the ferricyanide and HCl, the colour is developed in its full brilliancy. In the same manner, by heating after addition of phenylhydrazine, formaldehyde can be detected when present in its polymerized form. Heat 10 grammes of meat (minced) with distilled water on a boiling-water bath for five minutes. Where the concentration is I part formaldehyde in 50,000 or less, 10 c.c. of water is sufficient. Where the concentrations are higher, larger quantities of water must be employed. To every 10 c.c. of water used add 2 c.c. of a i per cent, phenylhydrazine h^^drochloride solution. Cool and filter from. 3i8 PRACTICAL SANITARY SCIENCE the coagulum through cotton-wool. To 12 c.c. of the filtrate add I c.c 5 per cent, potassium ferricyanide and 4 c.c. concentrated HCl. Compare colour with standards made from the standard formalde- hyde solutions. It has been found that in chilled beef treated by formaldehyde the superficial fat contains distinct quantities of formaldehyde; muscular tissue unprotected by fat is more largely contaminated than other parts. Grilling of meat but slightly diminishes the amount of formalde- hyde, and apparently causes the aldehyde to penetrate farther into the interior. Boiling greatly diminishes it. Roasting gets rid of most of it. Sausages made from meat impregnated with formalde- hyde and cooked in the ordinary way, retain it. A common depth of penetration into muscular tissue is 20 millimetres. Arsenic in Foods. — See Reports of the Royal Commission on Arsenical Poisoning, 1903. Cd. 1869. Minutes of Evidence and Appendices, Vol. II., especially Appendices 16, p. 183; 19, p. 201; 20, p. 206; 21, p. 208; 22, p. 220; 24, p. 230. Estimation of Araehis Oil found as an Adulterant in Olive Oil. — Saponify 5 grammes of the sample with 25 c.c. alcoholic potash solution (8-5 per cent.). Add that quantity of acetic acid which has previously been found by titration to exactty neutralize 25 c.c. of the above alcoholic potash, and cool the vessel in w-ater. Let stand for two hours. Filter off the acids on a filter-paper, and wash them with 70 per cent, alcohol containing i per cent. HCl. Dissolve the acids on the filter with about 40 c.c. boiling alcohol (95 per cent.). Add about 10 c.c. of water to bring down the alcohol to about 20 per cent., and cool down to room temperature. Filter after an hour, and wash the precipitate with 70 per cent, alcohol. Dry the precipitate (arachidic acid) at 100°, and weigh. As ara- chidic acid forms about 5 per cent, of araehis oil, the weight of the oil is readily calculated. Bakingr-Powders. — These preparations consist of an acid and an alkaline constituent, and a third inert body — generally starch — intended to absorb moisture, and thereby prevent premature chemical action. The alkaline constituent is almost always bi- carbonate of soda. The acid constituent may be (i) tartaric acid or an alkaline bitartrate ; (2) acid phosphate of calcium; or (3) an alum. Whilst sodium bicarbonate and tartaric acid are free from calcium sulphate, acid calcium phosphate (used in the manufacture of APPENDIX 319 baking-powder and self-raising flour) always contains more or less of this contamination. Estimation of CaSO^. — {a) Ca: Dissolve 10 grammes of the sample in boihng dilute HCl; add slight excess of ammonia, then slight excess of acetic acid, and filter off any precipitate that may form. To the filtrate add excess of ammonium oxalate : collect the precipitate of calcium oxalate on a filter; wash; dry in an air oven; ignite; cool and weigh as CaO. {b) Sulphate as SO3: Dissolve 10 grammes of the sample in boihng dilute HCl as above; add sHght excess BaClg, and allow the precipitate of BaS04 to settle. Filter; wash the precipitate free from chloride; dry in air oven; incinerate; cool and weigh. The weight of the ash minus the weight of the ash of the filter-paper x 0-3434 = weight of sulphates as SO3 in 10 grammes. [BaS04 = 233; SOg^So; ^ = 0-3434]. Estimation of Available CO^ in Baking-Powder. — ^An exact method is that recommended by Fresenius in which a small quantity (say O'5-i gramme) of the powder is acted upon by water, and the evolved gas absorbed by soda-Hme. When all the gas that will come off is absorbed, the remainder of the COg can be evolved by dilute acid and estimated in the same manner; or a fresh sample may bs operated on by acid, giving the total COg: this figure minus the available CO2 gives the unavailable or residual gas. Estimation of Tartaric Acid. — Wash 5 grammes of the powder into a 500 c.c. flask with 100 c.c. water. Add about 15 c.c. con- centrated HCl, and dilute with water up to the mark. When starch and other insoluble matters have settled out, filter the liquid. To 50 c.c. of the filtrate, corresponding with ^ gramme of the powder, add 10 c.c. of a 30 per cent, solution of carbonate of potash, and boil for half an hour. Filter and wash precipitate. Evaporate filtrate and washings to about 10 c.c. Add 4 c.c. glacial acetic acid whilst stirring vigorously, and 100 c.c. 95 per cent, alcohol, and continue the stirring till the precipitate appears crystalline. Stand until precipitate separates out (several hours may be required) ; decant the liquid through a small filter ; wash the precipitate on to the filter with alcohol; wash out the dish with alcohol, and the precipitate with the same, till free from acetic acid. Now boil precipitate, and filter with water in a beaker. Finally, titrate the liquid with deci- normal alkah (using phenolphthalein as indicator) to obtain the amount of tartaric acid. Lead and Arsenic in Tartaric Acid, Citric Acid, and Cream of Tartar. — See Local Government Board Food Reports, No. 2, 1907. 320 PRACTICAL SANITARY SCIENCE Approximate Atomic Weigrhts Ag ... 10 8-0 I 12.7-0 Al .. 27-0 K 39-0 As .. 75-0 Mg . . 24-0 Ba . . 137-0 Mn . . 55-0 Br 80 -0 N 14-0 C 12-0 Na . . 23-0 Ca .. 40-0 16-0 CI .. 35-5 P 31-0 Cr .. • 52-0 Pb . . 206-0 Cu .. 63-0 S 32-0 Fe .. 56-0 Sn .. 119-0 H i-o Zn . . 65-0 A litre of water saturatetl with air at 10° C. dissolves 8-68 c.c. O at N.T.P. A litre of water saturated with air at 15° C dissolves 6-96 c.c. O at N.T.P. A litre of water saturated \\ith air at 20° C. dissclves 6-28 c.c. O at N.T.P. One hundred grammes of water at 15° C. will dissolve the following amounts expressed in grammes of the salts indicated: BaSOj .. o-oob KBr . . . 38-500 CaS04 .. 0-208 CaClo . . . 40-800 Ba(NO.O., 7-800 NH4Br . . . 44-900 NaHC6.j' 8-800 NaBr . . . 46-500 K2SO, ■. . 9-600 SrBr., . . . 50-300- Na.,SO, 11-900 Mg(N03), . 50-500 KHCO., . . 18-300 BaBr., . . . 51-000. KNO3 ■ . . . . 21-200 Ca(NOo)., • 53-800 NaaCbg . . 22-000 NH4N63" • 55-300- KCl . . . . 25-000 KI . 58-500. NH4CI .. . . 26-500 Nal . 63-500 (NH4),S04 . . . 33-200 Balo . . . 66-900 MgSO^ . . . . 34-000 MgCl, .. , 66-900 NaNOg . . 34-200 Cal./ .. . 67-000 NaCl . . . . 36-100 KXO3 .. . 100-000' The following salts contain the numbers of molecules of water of crystalhzation indicated: BaCl.„2HoO; Na„HP04,i2H,0 Na.,S.,0„5HoO; Pb(aH30.,)o,3H20; ZnSOJ^H.^O; " FeS04.7H'0 CuS04,5H.,d; AlK(S04)o,i2HoO; MgSOj^HoO; H„C.,04,2H.;0 Cu(NH4)„;6HoO; CuCU,2(5:H4).,C1,2H.,0; NaNH;HP04,4H,0 (micro- cosmic "salt); CaCr.,6HoO;" Na.,S04,ioH20; Na,B407,ioH20; (NH4)2(S04)2,6H20; MgS04",K.S04,6"HoO. INDEX AcARUS domesticus, 203 farinas, 221 Acetyl value, 195 Acid, acetic, 224, 252, 253, 259, 260, 264, 268 benzoic, 178 boracic, 174, 187 carbolic, 301 citric, 166, 267, 268, 317 hypochlorous, 296 lactic, 252, 253 malic, 253, 259, 260 oxalic, 134 phosphoric, 268 salicylic, 177, 223 sulphuric, 224, 267, 268 sulphurous, 254 tannic, 257, 258, 259 tartaric, 259, 267 value of fat, 192, Acidity of beer, 253 of bread, 231 of milk, 155 of spirits, 264 of water, 15, 16, 95 of wine, 259, 260 Actinomycosis, 233 Adams's process, 157 Adeney's process, loi Adsorption, 292 Adulteration (see preservatives) of beer, 254 of bread, 231 of butter, 185, 187, 189 of cheese, 203, 205 of cocoa, 285 of coffee, 281 of milk, 172 of mustard, 269 of pepper, 270 of sugar, 274 of tea, 278 of wines, 259 >;Ecidium berberidis, 226 Air, 118 ammonia in, 140, 144, 145 ammonium sulphide in, 140, 141, 144. 145 bacteria in, 146 Air, bromine in, 141 carbon dioxide in, 133, 134, 135, 136 carbon disulphidc in, 141 carbon monoxide in, 136 chlorine in, 141 composition of, 118 humidity of, 129, 130 noxious gases in, 144 oxygen in, 130 ozone in, 141 sewer, 143 sulphur dioxide in, 140 sulphuretted hydrogen in, 140 suspended matter in, 143 Albuminoid (organic) ammonia, 41, 42, 46, 47, 52, 81, 92 Alcohols, 246, 251, 252, 259, 262 amyl alcohols, 247 butyl alcohols, 247 diethyl carbinol, 247 estimation of alcohol, 252 ethyl alcohol, 247, 251, 252, 253 isobutyl carbinol, 247 methyl alcohol, 246, 247 butyl carbinol, 247 propyl alcohols, 247 table, 265 Alkaline permanganate, 44 Alluvium, 3 Aloes, 254 Alum, 231 Ammonia-free water, 42 AmcEba, 69, 70, 75 Anabasna, 10, 72 Anguillulae, 70, 237 Animal parasites, 236 spine, 71 Ankylostomum duodenale, 116 Annatto, 179, 199 Antipyrin, 288 Antiseptic, 287 Apjohn's formula, 129 Arrowroot, 220, 221 Arsenic, 254, 317 estimation of, 255, 256 in foods, 316 Ascarus lumbricoides, 237, 242 Ascocarps, 228 321 3-2^ PRACTICAL SAXITARY SCIENCE Ascosporcs, 2 28 Aspergillus glaucus, 203, 222, 223 Atomic weights, 320 Azotobacter, 114 Babcock method. 162 Bacillus botulinus, 245 butyricus, 112 toli communis, 84, 85, 86, 87, 88, 91. 104, 112, 117, 146, 181, 245. 2S9, 293 denitrilicans, 112 enteritidis sporogenes. 84, 85, 87, 88, 117, 146, iSi, 245 fluorescens, 200 fluorescens liquefaciens, 112 Johnc, 182 Klebs-Loffler, 182 lactis aerogenes, 112 mallei. 234 mesentericus vulgatus, 112 mist bazillus. 182 Holler's. 181 mycoides, 112 oedematis maligni, 116 paratN-phosus B. 245 prodigiosus (micrococcus), 5, 89 proteus vulgaris. 112. 245 proteus zenkeri. 112 putrificus. 112 . pj'ocyaneus. 89 Rabinowitch. 1S2 radicola, 113 smegma, 1S2 subtilis, 112 suipestifer, 245 tetani, 116 tuberculosis, 84, 181, 182. 200, 205, 233, 253 t\-phosus, I, 84, 85. 89, 116. 182, " 2S9, 293 Bacteria in air. 146 in butter. 200 in meat, 233, 245 in milk, 180 Bacterial food-poisoning. 245 Bacteriological examination of water, 2, 6 Bacteriology of water, 83 Bagshot sands, 3 Baking-powders, 318 Barley, 209, 215, 216 Barometers, 120, 122 corrections of, 122 Fortin. 120 Hooke's. 122 Kew, 122 Baudouin's test, 199 Bean, 2ir, 219. 221 Bcch 's test, 199 Beer, 250 acidity of, 253 alcohol in, 252 aloes in. 254 arsenic in, 254 bitters in. 253 boric acid in. 254 gentian in, 254 malt extract in, 253 salic\dic acid in. 254 sodium chloride in. 254 sulphurous acitl in. 254 Beggiatoa alba, 10, 71, 73, 74 Beri-beri, 214 Bicarbonates. 21. 66 Birotation ratio, 167 Bismark brown. 53 Bitters, 253 Bleaching of flour, 212 powder, 296 Boric acid, 244. 254 Boulder clay, 3 Boyle's law, 119 Brandy, 261, 262 Bread, 229 acidity of. 231 adulteration of. 231 alum in. 231 ash of, 230 composition of, 229 silica in, 230 Bromine. 298 Brownian movement, 305. 306 Bruchus pisi. 221 Brucine test. 54 Bursaria gastris, 10, 73 Butter. 184 adulteration of. 185 bacillus, 182 bacteria in, 200 colouring matters in, 199 composition of, 184 cottonseed oil in, 199 curd in, 187 fat. 187. 189 acetyl value of, 195 acid value of, 192 Hehner value of, 193 iodine value of, 193 melting-point of, 190, 194 microscopic examination of, 192 physical properties of. 190 polarized light test, 198 INDEX 323 Butter i'at, Polcnske number, 197 preparation of, 189 refractive index, 192 Reichert-Meissl value, 195, 203 saponification value, 192 solidiiication-point, 191 specific gravity, 190 titre test, 191 Valenta's test, 198, 203 Wijs's test, 194 preservatives in, 187 boric acid, 187 formalin, 187 nitrates, 187 salicylates, 187, 188 sulphites, 187, 189 saffron in, 200 salt in, 187 sesame oil in, 199 starters in, 200 turmeric in, 200 water in, 185 Caelosphaerium, 10, 72 Caffein, 282, 284 Calandra granaria, 222 Calcium, 28 saccharate, 184 sulphate, 319 Cane-sugar, 184, 207, 271 Carbon (organic), 39 Carbon dioxide in baking-powders, 319 in beer, 251 in water, 65 Carbonates, 21 Carchassium Lachmanni, 73 Casein, 150 Catchment area, 2 Catechu, 280 Cellulose, 208 Cereals, 209 Chalk, 3, 4, 31, 80, 92 Chamberland bougie, 235 Champagne, 258 Chara fragilis, 10, 70 Charles's law, 119 Cheese, 201 adulteration of, 203 ash, 203 Brie, 202 Camembert, 202 Cheddar, 201, 202 composition of, 202 fat in, 203 foreign fat in, 205 Cheese, Gorgonzola, 201 Gruyerc, 201 lactose in, 205 moulds in, 203 proteins in, 204 starch in, 206 Stilton, 201, 202 Stracchino, 202 tubercle bacillus in, 205 Tyrothrix in, 203 water in, 203 water-soluble N, 204 Chemical analysis of water, 2, 12 balance, 12 Chicory, 283 Chinese silk, 76 Chloride of lime, 296 Chlorides in water, 16, 18, 19, 20, 30 59, 82 Chlorine, 290, 295, 296, 297, 298 in air, 141 Chocolate, 286 Chromium, 36 Claret, 257, 258 Clark's process, 25 scale, 25 Claviceps purpurea, 227 Clostridium pastorianum, ii4_ Coal, 3 '■"^ Coal-tar, 301 Coccidia, 239 Cocoa, 285 composition of, 285 Coenurus cerebralis, 236 Coffee, 281 composition of, 281 Colloidal meixury, 293 silver, 293 solutions, 304 Colour of water, 8 Colostrum, 166 Condensed milks, 183 Conferva bombycina, 73 Continuous phase, 304 Copper, 32 sulphate, 3^, 313 Copper-zinc couple, 55 Cosmarium, 70 Cotton fibres, 69, 72, 78 Cream, 163 of tartar. 319 Crenothrix, 8, 10, 71, 73 Creolins, 289 Cresols, 301, 308 Crum's method, 55 Crj-^tomonas, 10, 70 Crystalline rocks, 3 324 PRACTICAL SAXITARY SCIENCE Cuprous chloride methocl of esti- . mating COo, 140 Cysticcrcus bovis. 23G ccUulosae, 236, 240 tenuicoUis, 236 Daphnia pulcx, 71, 74 Dangerous water. 11 Decinormal solutions, 12 Demodex phylloidcs suis, 236 Deodorant, 287 Dew-point, 128, 129 Diamido-benzol, 53 Diastase, 152, 208, 250 Diatoms. 69, 70, 71, 72 Dinitrodiphenylaminesulphoxide, 314 Diphen\lamine test, 54 Disinfectants, 287 Disperse phase, 305 Distoma hcpaticum, 236, 241 lanceolatum, 236 Dotted vessels (chicory), 284 Drepanido taenia lanceolata, 236 Echinococcus multilocularis, 238 unilocularis, 238 Egg (Ascarus lumbricoides), 69 (Taenia solium), 69 (Trichocephalus dispar), 69 Elder-leaf, 278 Emulsions, 305, 306 Endorina, 72 Entire flour, 214 Erosive water, 16 Esters in spirits, 264 Ethers in wines, 260 Euplotes charon, 69 Eustrongylus, 237 Fat (butter), 189 (milk), 152, 153, 157 Fault, 4 Fehling's method, 170 Pavj' modification, 171 Filaria, 237 Filtrable viruses, 234 Flax, 77 Flock, 313 Flour-improvers, 212, 213 Formalin, 175, 188, 244, 290, 299, 300, 316 Frankland's method, 39 Free and saline ammonia, 41, 42, 46, 47, 52, 8r, 92 Friedlander's bacillus, 89 Fungi, 68 Furfural, 265 Fusel oil, 262 Gases in water, 60 Gentian. 254 Geology, 3 Gin, 264 Glaisher's formula, 129 Glenodinium, 10 Glucose, 271. 273 Gluten, 210 Gorgonzola cheese, 201 Graham flour, 213 Greensands, 3, 4 Griess's method, 53, 55, 57 Ground water, 5 curve, 5 Gruyere cheese, 201, 202 Haematosporidia, 239 Hair of insect, 70 Haldanc's apparatus for estimating CO., 315 method for estimating CO, 137 Hardness in water, 20, 22, 25 permanent, 24, 25, 26, 81 temporary', 24, 25, 26 total, 24, 26, 81, 92 Hemp fibre, 69. 77 Hempel's gas burette, 131 Hermite solution, 296 Hock, 258 Houzeau's test, 142 Human milk, 149, 153, 154 Humulus lupulus, 251 Humus, 109, no. III Hydra, 69 Hydrochloric acid in air, 141, 144 Hydrodictyon, 73 Hydrogen peroxide in air, 142 as disinfectant, 294, 295 in milk, 178 Igneous rocks, 5 Infant's foods, 205 Infusoria, 69, 70 Interpretation of chemical analysis of water, 79 Iodoform test, 251 Ions, 297 Iron in water, 2, 34, 82 Ironstones, 3 Isochlors, 17 Jute, 78 Kephir, 183 Kimmeridge cla^^ 3 Kjeldahl's method, 98, 1G5, 184, 314, Koumis, 183 INDEX 32 Lactalbumin, 151 Lacteal vessels, 284 Lactic acid, 155, 253 Lactoglobulin, 151 Lactose, 149, 152, 167, 205, 207 Laplace's formula, 124 Lard, 205 Lead in spirits, 262, 317 in water, 30, 32, 82 Leffmann-Beam process, 162 Lemon-juice, 267 Leptomitus lacteus, 73 Lias, 3 Lime-juice, 267 Limestone, 3 Linen, 69 Lolium temulentum, 229 London clay, 3 Lunge and Zeckendorf's estimation of carbon dioxide, 135 Magnesium in water, 28 Maize, 209, 217 Malt extract, 253 Manganous chloride, 64 Marquardt method, 261 Marsh's test, 255 Maximum thermometer, 126 Meat, 253 inspection, 233 parasites (animal) in, 236 preservatives in, 244, 316 tuberculosis in, 233 Melosira, 71 Meridion, 10 Metallic impurities in water, 261 Metaphenylene-diamine, 53, 56 Methyl alcohol, 263 Methyl butyl carbinol, 247 Methylene- imino compounds, 244, 316 Methyl orange, 25 Milk, 149 acidity of, 155 Adams's process, 157 adulteration of, 172 analysis of, 155 annatto in, 179 ash, 164 bacteria in, 181, 182 benzoic acid, 178 boracic acid, 174 casein, 150 cellular elements of, 183 citric acid, 166 colostrum, 166 colouring matters, 179 Milk, comi)ositif;n of, 149 human, 153 condensed, 183 cream, 163, 184 calcium saccharate in, iHj cane-sugar in, 184 gelatin in, 184 starch in, 184 dried, 183 fat, 152, '153. 157 formalin, 175 heated, 167 human, 153 hydrogen peroxide, 178 lactalbumin, 151 lactic acid, 155 lactoglobulin, 151 lactose, 152, 167, 180 muco-protein, 151 mystin, 178 pasteurized, 167 reaction of, 155 Rose-Gottlieb method, 163 salicylic acid, 177 sodium carbonate, 178 solids not fat, 165 sour, 179 specific gravity, 155 streptococci in, 182 total solids, 163 turmeric, 179 Werner-Schmidt method, 160 Westphal balance, 157 Millet, 209 Miniraum thermometer, 126 Moniezia expansa, 236 Moulds, 203 Mucor mucedo, 203, 222 Mucilage cells, 269 Mustard, 269 Mustard oil, 269 Mycoderma aceti, 268 Myxosporidia, 239 Navicula, 72 Nessler's reagent, 42, 57 New red sandstone, 3 Nitrates in water, 51, 52, 53, 54, 57, 82 Nitric acid in air, 141 organisms, 39 Nitrites in water, 51, 52, 53, 82 Nitrobacter, 112 Nitrogen as amides, 204 as ammonia, 205 as caseoses, 204 (organic), 39 326 PRACTICAL SANITARY SCIENCE "Nitrosomonas Europita, 112 Nitrous acid in air, 141 organisms, 39 Nonnal solution, 12 Nostoc, 71 Oat, 209, 217 Odour of water, 8 (Enocyanin, 257 Qistrus bovis, 23(3 Old red sandstone, 3 Oolite, 3 Ordnance survey, 3 Organic carbon, 39 matter in air, 315 in water, 38 nitrogen, 39 Oscillatoria, 72 Oxidizablc organic matter, 47 Oxidized nitrogen, 51 Oxygen absorbetl from permanganate, 47. 50, 90, 91, 92, 93. 94. 95. 96, 97. 99 dissolved in water, Go, 63, 99, 100 Oxyuris vermicularis, 237, 243 Ozone, 295 Pandorina, 72 Paraform, 300 Paramoecium, 69, 71 Parasites in meat, 236 Pasteurized milk, 167 Pavy-Fehling method, 171 solution, 275 Pea, 219 Peat, 5 Penicillium glaucum, 203, 222 Pentastomum taenioides, 239 Pepper, 269 Peronospora, 222 Pettenkofer's method, 133 Phenol, 289, 292, 302 Phenolphthalein, 15, 65, 66 Phenolsulphonic acid, 57 Phenylhydrazine hydrochloride, 317 Phenyloids, 303, 304, 306, 307 Phosphates in water, 27, 28, 30, 82 Phosphoretted hydrogen, 142 Phosphorus compounds, 232 Physical examination of water, 7 Picric acid, 57 Piophila casei, 203 Plastering of wine, 259 Pleurococcus, 70 Plumbo-solvency, 16, 27, 95 Poisonous metals in water, 30 Polarimetry, lOS I'olishetl rice, 214 Porosity of soil, 106 Port wine, 258 Post-tertiary deposits, 3 Potassium sulphate in wine, 260 permanganate, 298 Potato, 220 Pouchct's aeroscope, 143 Primary deposits, 3 Proteus vulgaris, 89 I'uccinia graminis, 225 Purbcck marble, 3 Putrefaction, 39 Qualitative examination of air, 144 Ouassia, 254 Rain, 6 Reaction of water, 13, 91, 92, 93 Reinsch's test, 256 l^clative humidity, 130 Resin acids, 307 Rice, 209 I^ideal- Walker coefticient, 312 method, 304, 307 Rivularia, 10 Rose's method, 263 Rum, 264 Rye, 209 Saccharomyces ellipsoideus, 257 Salicylic acid, 224 Sarcosporidia, 239 Sea-water, 95 Self-registering thermometer, 125 Sesame oil, 199 Sewage, i, 2, 81, 92, 94, 96 effluents, 60 fungus, 9 Shales, 3 Shallow wells, 3 Sherry, 258 Silica, 29, 230 Silk, 76 Six's thermometer, 125 Sloe-leaf, 278 Soaps, 20, 21 Sodium chloride in beer, 254 tetrathionate, 49, 60, 62 thiosulphate, 49, 60, 63 Soil, 105 bacteria in, iii clay, 107 humus, 109 lime, 108 magnesia, 108 INDEX 327 Soil, organic matter, 107 pcrniLeability of, 107 porosity of, 106 sand, 107 specific gravity of, 106 heat of, 106 Soxhlet's apparatus, 159 Sphserotilus natans, 71 Spirits, 261 Spirogeira, 71 Sporsndonema casei, 203 Sprengel's method, 57 Standard ammonium chloride, 42 arsenical mirrors, 255 arsenious acid, 297 bromate bromide, 302 calcium chloride, 22 copper, 33 iron alum, 35 lead, 31 nitrate, 57 nitrite, 54 iodide, 62 oxalic acid, 134, 298 permanganate, 48, 49 silver, 18 soap, 22, 23 solutions, 12, 14 thiosulphate, 296 zinc, 37 Staphylococcus pyogenes aureus, 298, 303 Starch granules, 211 arrowroot, 220 barley, 215 bean, 219 maize, 217 oat, 217 pea, 219 potato, 220 lice, 216 rye, 215 sago, 218 tapioca, 218 wheat, 215 solution, 48 Starters, 200 Stokes's tube, 161 Straight run flour, 214 Streptococci, 86, 112, 182 Strongylus, 237 Substrate, 249 Sugar, 260, 270 Sulphate of calcium in tea, 278 Sulphates in water, 29, 30, 82 Sulphur dioxide, 144, 145, 297 Sulphurous acid, 254 Suspicious water, 1 1 Synedra, 70 Synura uvella, 72 Tabaric's method, 253 Tabellaria, 10 Taenia cocnurus, 236 echinococcus, 236, 238 marginata, 236 saginata, 237, 241 serrata, 236 solium, 237, 240 Tartaric acid, 260, 267, 319 Taste (water), 10 Tea, 275 adulteration of, 2 78 composition of, 277 leaf, 276 tannin in, 280 thein in, 277, 278, 279 Teleutospores, 226 Tertiary deposits, 3 Testa cells (coffee), 283 Theobi'omine, 286 Thermometer, 125 Thiosinamine, 269 Thresh's method, 60 Thymol, 291 Tidy's method, 47 Tilletia caries, 224 Tin, 33, 313 Titre test, 191 Tobacco-leaf, 279 Triamidoazobenzol, 53 Trichina spiralis, 237 Trichocephalus, 237 Trinitrophenol, 57 Turbidity (water), 7 Turmeric, 179, 199, 200, 269 Two-foot tube, 8 Tylenchus tritici, 222 Tyrothrix, 203 Ulothrix, 70 Ultramicroscopic viruses, 235 Upland surface waters, 50, 52 Uredo foetida, 224 Uroglena, 10, 70 Ustilago segetum, 223 Vapour tension table, 127 Vernier, 121 Vibrio cholerse asiatics, i tritici, 221 Vinegar, 268 acetic acid in, 268 mineral acid in, 268 328 PRACTICAL SANITARY SCIENCE Vinegar, nitrogen in, 268 phosphoric acid in, 268 specific gravity of, 268 sulphuric acid in, 268 total solids of, 269 Viscosity, 304 Volvox, 72 Vorticella, 71, 72, 75 Water, i acidity of, 15, i(), 95 albuminoid ammonia in, 41. 42, 4O, 47, 52, 81, 92 algae in, 68, 69, 70, 71, 72, 73, 79 alumina in, 21 anabaena in, 10, 72 Bacillus coli in, 84, 85, 80, 87, 88, 91 bacteriological examination of, 83 bear, 73 biological examination of, 2, G7 calcium in, 28 carbon dioxide in, 65, 66 chalk in, 27, 80 chemical examination of, 2, 12 chlorides in, 16, 18, 19, 20, 30, 59. 8^ chromium in, 36 colour of, 8 copper in, 32 crystallization of, 317 cycle, 6 dangerous, 11 enzymes in, ^S erosive action of, i(j free and saline ammonia in, 41, 42, 46, 47, 52, 81, 92 hardness of, 20, 22, 24, 25, 26, 81, 92 iron in, 2, 34, 82 lead in, 30, 32, 82 magnesium in, 28 nitrates in, 51, 52, 53, 54, ^j, 82 nitrites in, 51, 52, 53, 82 odour of, 8 organic matter in, 38 oxidizable organic matter in, 47 oxygen dissolved in, 60 peaty, 5 phosphates in, 27, 28, 30, 82 physical examination of, 2, 7, 91, 92, 93. 94. 95 plumbo-solvency of, 16, 27, 95 poisonous metals in, 30 pure, 91 Water, rain, 6 reaction of, 13, 91, 92, 93 sediment, 67 silica in, 29 solitl residue of, 27, 92 sulphates in, 29, 30, 82 suspicious, II taste of, 10 tin in, 33 total solids in, 92 turbidity of, 7 wholesome, 11 zinc in, 36, Sz Weald clay, 3 Werner-Schmidt method, 160, 203 Westphal balance, 157 Wheat, 209 flour, 209 ash of, 211 composition of, 210 fat of, 210 gluten of, 210 starch granules of, 211 sugar of, 210 water of, 211 Whisky, 261 acidity of, 264 alcohol of, 262 furfural in, 265 fusel oil in, 262 metallic impurities in, 262 methyl alcohol in, 263 Wholemeal flour, 213 Willow-leaf, 278 Wine, 256 acidity of, 259 alcohol of, 259 ash of, 260 colouring matter in, 259 ethers of, 260 extract of, 260 plastering of, 259 potassium sulphate in, 260 sugars in, 260 water in, 259 Winkler's method, 63 Witte's peptone, 308 Wood cells, 69, 72 Wool, 69, 70, 76 Xylenols, 303 Zinc in water, 36, 82 Zymase, 249 Zj^molyte, 249 Baillicre, Tintlall <5t^ Cox, 3 Henrietta Street, Ccrvent Garden 1^