ap /e3/ /f/2-. CORNEL L UNIV ERSITY THE Wiamn Bftmnary SItbrary FOUNDED BY ROSWELL p. FLOWER for the use of the N. Y. State Veterinary College 1897 This Volume is the Gift of Dr. V. A. Moore 356 QD 10.R3n912"'"""*^'-"'"^ '■Mir y -uy Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000947535 A STUDY OF HYDROGEN SULPHIDE PRODUCTION BY BAC- TERIA AND ITS SIGNIFICANCE IN THE SANITARY EXAMINATION OF WATER. A THESIS PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF CORNELL UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. BY HARRY WESTFALL REDFIELD. O '- "P, ITHACA, N. Y. ' , 1912. . I ^ J FOREWORD. The author desires to express his deepest gratitude to Pro- fessor E. M. Chamot at whose suggestion and under whose direction this work was carried out. To Professor L. M. Dennis, the head of the Department of Chemistry, and to Professor A. W. Browne, the author wishes to acknowledge his Appreciation of their kindly interest in the work. RSI \&>''' ''' Ui. s f; /. /, I ■ ^ (. TABLE OF CONTENTS. PAGE. Foreword II Chapter I. Historical Review of Literature i Schardinger's work 2 Dunham's work , 4 Introduction to Experimentai, Work ,, 6 Chapter II. Spbciai^ Apparatus and Reagents 7 Apparatus 7 Reagents 7 Sewage sample used for inoculations 8 Chapter III. Eeeect on Hydrogen Sui,phide Evoi,ution Pro- duced BY Filtering Peptone Soi,uTioNS 9 Summary 10 Chapter IV. Efeect on Hydrogen' Sulphide Evolution oe Dif- ferent Concentrations of Peptone 11 The triangular diagram 11 Results with peptone and sodium chloride 13 Two component diagrams 13 Peptone in presence of salts other than sodium chloride 15 Effect of the addition of beef extract to the medium 15 Summary 17 ■Chapter V. Effect on Hydrogen Sulphide Evolution Pro- duced BY Different Inorganic Salts 18 Modified use of triangular diagram 18 Check solutions used 19 Results with sodium chloride 20 Results with sodium sulphate 20 Results with potassium chloride 1 20 Results with magnesium chloride 21 Results with calcium chloride 21 Results with ammonium chloride 21 Results with sodium chloride, potassium chloride, ammonium chloride, calcium chloride, magnesium chloride, sodium sul- phate, potassium sulphate, ammonium sulphate, calcium sul- phate, magnesium sulphate, sodium nitrate, potassium nitrate, ammonium nitrate, calcium nitrate and magnesium nitrate 22 Results with primary phosphate 26 Results with secondary phosphates 27 Results with tertiary phosphates 29 Results with various combinations of potassium chloride, sodium chloride and potassium sulphate in conjunction with secondary potassium phosphate and secondary sodium phosphate 30 Results with calcium carbonate 31 Results with primary sodium carbonate 3^ Effect of the inorganic salts contributed by the water under ex- amination 33 Summary 34 Chapter VI. Eppect on Hydrogen Sulphide Evoi^°-.45f« "95° - -75 /» " 3° +.30% " 37>^°-.i5% " 95° - -45% " 3° +.45% " 20° + ,15% " 95° - -3°% " 3° +.75% "20° + .45% " 37>^° + .30% 4° KFFECT OF SALTS ON REACTION. No curves are shown for the initial reaction and the reaction after 24 hours of incubation of media containing potassium chloride, sodium chloride or potassium sulphate for the reason that these showed exactly the same variations with different temperatures as were shown in Curves No. i and No. 2 ; the inorganic salts mentioned having no effect in this respect and the curves for such media being identical with Curves No. i and No. 2. When primary potassium phosphate was present, it was found that while it materially increased the acidity of the media ; almost the same variations were found with different tempera- tures as in Curve No. i except that the curve flattened at the higher temperatures more than was the case when no phosphates were present. Curve No. 3 shows the results for the initial reactions of media made up with 10 cubic centimeters of 30.0 percent peptone, 5 cubic centimeters of boiled, cooled and filtered tap water, .5 grams of primary potassium phosphate and 85 cubic centimeters of artificial sewage. Curve No. 4 shows the results for the reactions of the same combination as in Curve No. 3, after 24 hours incubation. Curve No. 5 is the curve for .5 grams of primary potassium phosphate in 100 cubic centimeters of boiled, cooled and filtered tap water and explains why the curve flattens out at the higher temperatures when primary phosphate is present in media. OPTIMUM REACTION OF MEDIA. It seems unnecessary to discuss in detail the experimental data with regard to any of the inorganic salts tried except potassium chloride, and therefore, the limits for the best concentrations of initial acidity for the other salts tried are given below in tabular form : — "or NaCl, from 0.0% to 2.ofo N. acid best at i.ofo at 20° C " Na^SO,, " ■5 " I.O tt t( II 1, ^ " MgClj, " .5 " I..') (1 (( ; " " I.O " CaCl,, " I.O " 2.0 II II " " 1.5 " NHp, " I.O " 1.5 11 II " " I.O " KjSO,, " •5 " 2.0 11 (1 " " I.O 41 When' peptone, potassium chloride, hydrochloric acid and potassium hydroxide were used in preparing the media, good results were obtained in some two hundred inoculations over a much wider field than with any other combination, as has already been noted ; very good and quick results having been obtained with concentrations of acidity from .5 percent to 3.0 percent inclusive, but the very best results were obtained when the acidity was from i.o percent to 1.5 percent with very little choice between these concentrations, the preference, if any, being for 1.5 percent. This preference, however, was so slight that it is outweighed by the greater simplicity .of leaving the re- action unadjusted at about 1.02 percent, when 10 cubic centi- meters of 30.0 percent peptone is used in 100 cubic centimeters of inoculated media. • If the best concentration of the peptone were such as to give less than i.o percent of acidity when unadjusted, and if an ad- justment were therefore made necessary, it would be advisable to make the reaction 1.5 percent of normal acid when potassium chloride is used in the medium ; but, under the existing circum- stances, 1.02 percent of acidity without adjustment is preferable on account of its greater simplicity. SUMMARY. 1. The temperature at which peptone solutions are titrated has a very decided influence on the reaction when phenol phtha- lein is used as indicator ; the higher the temperature the greater the amount of acidity found. 2. The reaction of a 3.0 percent unadjusted peptone solution, with or without potassium chloride, sodium chloride or potassium sulphate present, was found to be about one half at 3.0°, two thirds at 20.0°, and four fifths at 37.5° of what it was at 95.0°. This is believed to be due to the greater ionization or hydrolysis of peptone solutions at increased temperatures. 3. The reaction of peptone solutions is unaffected by the presence of potassium chloride, sodium chloride or potassium sulphate, but is affected by primary phosphates very materially. 4. The optimum initial reaction of the simple peptone medium, with or without potassium chloride, for hydrogen sulphide pro- duction is from 1.0 percent to 1.5 percent of normal acid, with a slight preference for the higher value. It makes but little difference whether this amount of acidity to phenol phthalein is contributed by an unadjusted peptone solution or whether the peptone is first neutralized with either potassium hydroxide or sodium hydroxide and then made acid with hydrochloric acid. CHAPTER VIII. REI.ATION TO HYDROGEN SULPHIDE EVOLUTION" OF THE FINAL REACTION OF THE CULTURES TO PHENOL PHTHALEIN. In the latter half of the work, titrations of the cultures after incubation, using phenol phthalein, were made, to ascertain whether there was any relation between the amounts of hydrogen sulphide produced and the reactions of the cultures. Titrations were made of the unfiltered and of the filtered media ; and also, at first, titrations were made at 3.0°, 20°, 37)4'^ and 95° Centigrade, just as in the case of obtaining the initial reactions. EFFECT OF TEMPERATURE ON REACTION. The question of titrations at different temperatures may be dismissed by refering to Curve No. 2 presented on Plate XVI at the end of the article, which is typical of all of the results which were obtained at different temperatures and which showed al- most exactly the same variations due to difference in temperature as were shown in the case of the initial reactions. This is in en- tire accord with what might be expected, for the variations in reaction with variations in temperature are due to the peptone, of which only a small amount out of the total present is broken down during the incubation of the cultures, leaving almost as much undecomposed peptone to cause the variations after incu- bation as before. As the same variations in reaction with different temperatures were found after incubation as before, and as 20° Centigrade was chosen as the temperature for making the titrations of initial reaction, the same temperature was naturally chosen for making the titrations of final reaction. DEDUCTIONS DRAWN FROM FINAL REACTION. The observations which follow are more or less fragmentary and incomplete for the reason that these observations were made on inoculations which were parts of runs primarily planned to obtain information on entirely different questions from that now 43 being discussed, but still it is deemed wise to include the data even though the observations are not all logically related, for the reasons : — first, that the amount of acidity finally developed in the cultures is a function of the activity and amount of growth and developement of the bacterial flora present ; second, that it gives a different basis for studying the effect of adding different inorganic salts to the media ; and third, that some really valuable deductions may be drawn from the observations which were made, these deductions being as follows : — 1 . With varying amounts of potassium chloride, sodium chlor- ide and potassium sulphate added separately to the media, those media which received potassium chloride gave the most uniform amounts of increase in acidity, just as potassium chloride induced the most uniform results as regards hydrogen sulphide production. 2. The data for increases in acidity after 24 hours of incuba- tion, with potassium chloride, sodium chloride or potassium sul- phate present in concentrations furnishing equal amounts of basic element, namely, .25 grams, are rather meagre, only three observations having been made with each salt, but the observa- tions were made at different periods of time and on inoculations made with different samples of sewage and hence are worthy of note. When potassium chloride was present, an average in- crease over the original acidity of .72 percent of normal acid was noted ; for potassium sulphate, an average increase of .65 per- cent was noted and for sodium chloride, an average increase of .42 percent was noted, resulting in respective average reactions of 1.77 percent, 1.70 percent and 1.47 percent. This is in ac- cord with the fact that the quickest results as regards hydrogen sulphide production were obtained in the presence of potassium chloride. 3. In 48 hours of incubation, even with amounts of sewage varying from 5 cubic centimeters to 85 cubic centimeters, little variation in the amounts of acidity produced were noted when potassium chloride, sodium chloride or potassium sulphate were present, the final reaction being in all cases close to 3.0 percent of normal acid ; the average for nineteen inoculations in which .25 grams of potassium as potassium chloride was used, being 3.17 percent; for nineteen inoculations in which .25 grams jjf sodium as sodium chloride was used, being 3.03 percent ; and for 44 nineteen inoculations in which .25 grams of potassium as potassium sulphate was used, being 2.72 percent ; showing potassium chloride to be slightly superior to sodium chloride or potassium sulphate in furnishing the best conditions for the general growth and development of the bacteria, just as it fur- nished the best conditions for the activity of that part of the flora which produced hydrogen sulphide. The largest amount of acidity noted was produced in the presence of potassium chloride but this fact is not considered to be of much impor- tance as it was undoubtedly due to the presence of very active organisms in that particular inoculation. As has been before stated, in work of this kind where a mixed flora is being used one must depend upon the averages of large numbers of runs and not upon single observations or groups of observations which are either small in number or all made at one time from one particular sample. 4. With . 25 percent of potassium as potassium chloride present, more acidity was developed in both 24 and 48 hours than was produced when peptone alone without added salt was used. For peptone alone, average increases of .57 percent and 1.91 percent were found for 24 and 48 hours respectively as against .72 per- cent and 2.12 percent when potassium chloride was present, showing that potassium chloride has a beneficial influence upon the general development of the whole bacterial flora which was present in this investigation as well as upon hydrogen sulphide production. 5. In the presence of primary phosphate, the development of acidity was very slight indeed. 6. Tertiary phosphates had an inhibiting effect upon the de- velopment of acidity. 7. The presence of secondary phosphates in some instances led to a greater development of acidity than was the case when pep- tone alone was used. This was a circumstance which was noted with pleasure as it would seem to constitute a justification of their use in so much of the present day media, although it is still maintained that they be omitted from media for hydrogen sulphide production so long as peptone is used in the media. It may be that when we find a synthetic compound to take the place of peptone in the medium, as we hope to do, that we will find with 45 Frouin"^ that some form of phosphate is absolutely indispensable in media which contain no protein ; but when peptone is present, the phosphates do undoubtedly interfere with hydrogen sulphide production. 8. The presence of calcium carbonate had a beneficial effect in inducing the production of a greater increase in acidity in both 24 and 48 hours in those media which were titrated, and hence, we see its justification in some kinds of media even though it did not seem to have any beneficial influence on hydrogen sulphide production. 9. Those cultures to which no sewage was added did not change in reaction after sterilization or after incubation at 2)7 H° even for 72 hours, and hence it may be stated that the applica- tion of heat does not have the effect of breaking down the pep- tone permanently to give a greater amount of acidity to phenol phthalein. ID. Much of the acid-reacting substances is in the sediment produced in the cultures, as the filtrate from such cultures showed a markedly lower acidity than cultures which were unfil- tered but which were of the same initial composition and which had been treated in the same way in all other respects. As the whole culture and not the filtrate is of special interest in this investigation, all of the foregoing deductions are based on unfil- tered cultures. NOTE ON THE SPEED OP EVOLUTION OF HYDROGEN SULPHIDE. During the course of the work, and more especially, while making titrations of some of the media after incubation, it was noted that there would be at times a very rapid evolution of hydrogen sulphide. This never occurred during the first twenty- four hours of incubation, and never, as far as observed, before the cultures had reached an acidity of 3.0 percent. The evolution was so rapid at times that from one-quarter to one-third of the lead acetate papers in the tubes of the culture flasks would be blackened in the half hour or so that it took to titrate a group of fifteen or twenty cultures. It was also noted that the same sort of thing occurred in the incubator in short periods of time. 46 I The indication is that exactly the right conditions must be inaugurated by the bacteria and that then hydrogen sulphide production proceeds rapidly. SUMMARY. 1. The same variations with temperature were noted after inoculation and incubation of cultures as before. 2. A study of the reactions of the media after inoculation and incubation furnishes excellent corroboration of the conclusions already drawn concerning hydrogen sulphide production, on the assumption that the amount of acidity produced is a measure of the activity and of the amount of growth and development of the bacterial flora present. From such a study the following deduc- tions may be made : — (a) With varying amounts of potassium chloride, sodium chloride and potassium sulphate added separately to the media, those media that had received potassium chloride gave the most uniform amounts of increase in acidity. (b) At the time when hydrogen sulphide was first being pro- duced, an average acidity of about 2.0 percent was found, this acidity increasing with increased production of hydrogen sulphide, until an average acidity near 3.0 percent was reached, when most abundant production of hydrogen sulphide was noted. ' (c) Cultures containing potassium chloride gave, on the average, larger increases in acidity in both twenty four and forty eight hour incubations than did cultures containing either sodium chloride or potassium sulphate, and cultures with any of these salts present gave larger increases in acidity than cultures containing no added salt. (d) In the presence of primary phosphates, the development of acidity was very slight. (e) Tertiary phosphates had an inhibiting effect upon the production of acidity. (f) The presence of secondary phosphates in some instances led to a greater development of acidity than was noted with peptone alone. This would seem to justify their use in certain classes of culture media. 3. More acid-reacting material is in the sediment of the cultures than in the soluble part. CHAPTER IX. THE METHOD FOR DETECTING THE BACTERIA PRODUCING HYDROGEN SULPHIDE AS IT HAS BEEN FOUND TO GIVE THE MOST RAPID AND REI^IABIvE RESUIvTS. To make one liter of the culture medium, bring 700 cc. of tap -water to a boil and add 300 grams of Witte peptone and 75 •grams of potassium chloride. Maintain a gentle heat and stir -constantly until as much of the peptone as will do so, has gone into solution. Cool rapidly by immersing the vessel containing the medium in cold water. Make up to one liter with tap water. Transfer to a flask, bring to a boil, plug the flask immediately .and cool again. Place in the ice box for at least twenty four hours. Filter the cold medium through paper and measure 10 cc. portions of the clear filtrate into the desired number of the ■special culture flasks illustrated in Cut i at the end of the article, which have been provided with tiny cotton plugs in the bottom of the tube of the caps. Sterilize the flasks in the autoclave at •one atmosphere, and after they are cool, place in each tube a strip of bibulous paper measuring 25 mm. by 2 mm. which has previously been impregnated with neutral lead acetate and dried. Finally, apply a tight tin foil cap to the top of the tube. This tin foil cap is made by winding a piece of tin foil measuring about 40 mm. by 20 mm. around the top of the tube in the form •of a cylinder, leaving about one half of the cylinder (10 mm.) projecting above the tube. The part which projects is then twisted into a tight twist and the flasks are ready for either storage or inoculation. (That part of the medium not intro- duced into the special culture flasks may be sterilized and kept for future use.) To inoculate, remove cap from flask and pour in the sample lunder examination until the 100 cc. mark is reached, replace the cap, mix thoroughly by rotating the flask and incubate at from 37 to 38 degrees Centigrade. Examine the cultures at the end of 18 hours and as frequently thereafter as possible, as the length of time required for develop- •ment of hydrogen sulphide may be taken as a rough measure of 48 the degree of activity, or of the number, of the putrefactive organisms present, and hence as an indication of the age and ex- tent of the pollution. Besides noting the amount of hydrogen sulphide produced, as measured by the amount of blackening of the lead acetate paper, the degree of turbidity of the solution should be noted, the color of the solution (whether it has been turned greenish or not), the presence and color of any sediment formed, the presence and color of any floating cakes of material, and the presence of gas bubbles on the liquid. The amount of indol present may also be deterniined as corroborative evidence. SUMMARY. The method which has been found to give the most rapid and reliable results is very easy to follow and the medium is simple to make. CHAPTER X. THE SENSITIVENESS AND REI, ^t ^J3 o« ol .s »•« O.JJ a-o g«5 SS" ■iJ l^ fl-,'« total sulphur. ll in u |g "Is 2S 2« ea>, sg§ sS-s S8 fi'-ss 0" Ph ». h a, Peptone, potassium chloride blank 3.0000 .0302 ■0157 51-99 Cultures in 100 cc. flasks. Filtrate after 48 hours incubation 2.5885 .0145 .0103 71.03 13.72 51-99 34-39 Cultures in 2000 cc. flasks. Filtrate after 48 hours incubation. 2.8850 .0180 .0106 58.89 3-83 40.40 32-48 Cultures in 100 cc. flasks. Sediment after 48 hours incubation ___ .0176 .0001 Cultures in 2000 cc. flasks. Sediment after 48 hours incu- bation Cultures in 100 cc. flasks. Filtrate and sediment combined Cultures in 2000 cc. flasks. Filtrate and sediment combined. .0121 .0001 2.6161 2.8971 .0146 .0181 13-13 51-65 3.43 40 07 EFFECT OF AMOUNT OF SURFACE EXPOSED. The fact that the total solids exclusive of potassium chloride, total sulphur and easily oxidized sulphur were all markedly lower in the case of the cultures in the loo cc. flask than in the case of the culture in the 2000 cc. flask was a fact which de- manded explanation. A possible reason was that in the case of 100 cc. flasks there was a greater amount of surface ex- posed to a greater amount of air and that, in consequence, the bacteria were more energetic and therefore decomposed a larger amount of peptone. This idea is at variance with the belief that the organisms producing hydrogen sulphide are anaerobes. In order to test out the validity of this explanation, two Fernbach culture flasks were prepared by introducing into each, 500 cc. from a mixture of 400 cc. of 30. percent peptone solution, 69 200 cc. of lo. percent potassium chloride solution and 3400 cc. of strained artificial sewage ; two flasks of 500 cc. capacity were prepared by introducing into each, 500 cc. of the same mixture ; ten flasks of 100 cc. capacity were prepared by introducing into each 100 cc. of the mixture, while the balance of the mixture was used as a blank. The blank was immediately sterilized to await analysis while the inoculated flasks of various size and shape were placed in the 38° incubator. By means of a suction pump, air was passed through a wash bottle containing 5:1000 mercuric chloride solution to take out any hydrogen sulphide in the air, then through one of the Fernbach flasks and finally through a Fritz » Friedrichs wash bottle containing 2.5 grams of cadmium chloride in water solution. The function of the cadmium chloride wash bottle was to catch and hold as cadmium sulphide all of the hydrogen sulphide which might be produced by the bacteria in the culture and swept over by the current of air. With the aid of screw clamps the speed of the air current was so regulated that one bubble per second passed through the wash bottles. By means of the portable gas generator devised by A. W. Browne and M. J. Brown, Jour. Amer. Chem. Soc, 29, 859 (1907), carbon dioxide gas under constant pressure was gene- rated from calcium carbonate and hydrochloric acid and passed first through a wash bottle containing distilled water, then through the other Fernbach flask and finally through a Fritz Friedrichs wash bottle containing 2.5 grams of cadmium chloride in water solution, wherein any hydrogen sulphide, generated by the bacteria in the culture would be precipitated as cadmium sulphide. The carbon dioxide generator was so regulated that one bubble of gas per second passed through the wash bottle. The Fernbach flasks through which air and carbon dioxide respectively were being passed and the other flasks through which no gas was being passed but which were being allowed to incubate in the usual manner, were all left in the 38° incubator for 48 hours. They were then sterilized by heat. Without filtering off the insoluble material, this having been proved to be unnecessary in the previous set of inoculations, the blank and the various cultures were analysed for total solids, for total sul- phur, for easily oxidized sulphur, for loosely bound sulphur by the Schultz method, and for sulphur as hydrogen sulphide. For making these determinations, except that of sulphur as hydrogen • sulphide, the blank and the various cultures were evaporated separately to small volumes, then each was made up to 250 cc. in a measuring flask and measured portions taken for analysis. For the determination of sulphur as hydrogen sulphide, the contents of the cadmium chloride wash bottles were acidified with strong hydrochloric acid, an excess of N/io iodine added and the excess titrated with N/io sodium thiosulphate. The results are given in Table VII. 70 punoq Xiasooi ^o ^naojaj *S^H oi^T P31-I3A -noD jtiiidins ' paztp -Txo XiiBB3 JO :^n3Daa(l •p3Xoj;s -sp 'iDSt JO aAisnpxa 'spuos I'E^o; JO ' ;n3DJaj •joj psinnoD -3'B jniidins JO tjuaajsj SB jniidins snid jnv[d -ins p33iDB:nBnn jo rang unndins pssjoE^iBan JO SI anqdins punoq jCissdoi iiDjuM inaojaa ■jn-qdxns p3hob;;e -an JO SI anqdins pazip III < JO •g^H SB Jnttd^ns 001 jad sraEJO •jniidins pnnoq ^lasooi JO "DD 001 Jiad suiEJO •jnndins pazipixo ^itsbs JO "DD 001 Jad StIlBJO uniidxns pasiDB^^E q -un JO "OD ooi jad stnBJO ^ O •IDS JO o aATsnpxa sp^os ib^o; ^ jo'*DO OOI jad stuEao ^ t- m u a> "^-^ ■fl < 13 j 2 1 1 [ 1 3 O ■e 3 o (U oj 1 s t— ( 3 1 1 cpl 1 ^ u o ja J3 1 1 u u ci cJ E 6 o o a 3 00 oT g .t o ■4^ s •^ 3 -4J CO 3 ■3 Oh^ "3 "3 w "3 '■3 ^ dl o o u U 71 DISCUSSION OF TABLE VII. The amounts of total solids, exclusive of potassium chloride, remaining in the media after incubation proved that the bacterial action was most energetic in the Fernbach through which air was passed ; next in the cultures having the greatest surface ex- posed i.e. in the loo cc. flasks ; next in the cultures in the 500 cc. flasks ; and was least energetic in the Fernbach flask through which carbon dioxide was passed ; while the amounts of total sulphur remaining, the difference between which and the total sulphur of the blank is a measure of the volatile sulphur com- pounds produced by the bacteria, proved that the production of volatile sulphur compounds by the bacteria was most energetic in the Fernbach through which air was passed ; next in the cultures in 100 cc. flasks ; next in the cultures in 500 cc. flasks ; and was least energetic in the Fernbach flask through which carbon dioxide was passed. Therefore, the orders for general bacterial activity and for hydrogen sulphide production were identical, and in the case of the flora with which this work was done, at least, the organisms producing hydrogen sulphide in largest amounts were aerobes and not anaerobes and for the maximum production of hydrogen sulphide, broad, low flasks giving the maximum surface of culture exposed to air should be used. The form of flask illustrated in Cut i at the end of the article could be improved by making it lower and broader. DETERMINATIONS OF HYDROGEN SULPHIDE. The fact that the amounts of sulphur as hydrogen sulphide found by the method employed were far from being the differences between the total sulphur of the blank and the total sulphurs of the cultures in which sulphur as hydrogen sulphide was de- termined, demanded attention, as it indicated that the method which had been employed for determining hydrogen sulphide was faulty and that the cadmium chloride had failed to stop all of the hydrogen sulphide evolved. It was therefore decided to try an iodine solution instead of the cadmium chloride solution in the wash bottle. For this purpose, air was drawn by suction for 48 hours, first through a wash bottle containing lead acetate solution to remove 72 any gaseous sulphur compounds, then through a Fernbach flask containing 500 cc. of the culture medium, artificial sewage mix- ture already described, said flask being in the 38° incubator, then through what had been calculated to be an excess of N/io iodine solution in a Fritz Friedrichs wash bottle and finally through a N/io sodium thiosulphate solution in a wash bottle. The sodium thiosulphate was used to stop and hold any iodine which might be volatilized. During the period of incubation, the iodine solution was found to fade rapidly, indicating that larger amounts of hydrogen sul- phide than had been expected were being produced, and conse- quently, small amounts of normal iodine solution were, from time to time, added to the iodine solution in the wash bottle. After 48 hours of incubation, the iodine solution and the sodium thiosulphate solution were mixed together and the amount of iodine which had been used up, determined by titration. It was found that an amount of iodine equivalent to an amount of hydrogen sulphide which would be produced by nearly all of the sulphur of the medium being transformed to hydrogen sul- phide, had been reduced. As determinations of total sulphur showed that only about 25 percent of the total sulphur had dis- appeared from the medium the results were evidently impossible. The explanation which is offered is that volatile, unsaturated /NHx organic compounds like indol, C5H4^^pTT />CH and skatol, / NH X ^6^4 ^CHwere liberated from the medium by the action of the bacteria and that these reduced the iodine solution. The use of iodine solution having been proved to be of no value, it was decided to try solutions of cadmium chloride con- taining potassium chloride to prevent colloidal suspension of cadmium sulphide, of lead acetate containing potassium nitrate to prevent colloidal suspension of lead sulphide, of sodium per- oxide and of potassium hydroxide, determining the total sulphur retained as sulphide by the Liebig-Koch method and not by liberating with hydrochloric acid and titrating with iodine and sodium thiosulphate solutions as had been done when cadmium chloride was before used. 73 The Fernbach flasks containing the usual mixture of culture medium and artificial sewage and the Fritz Friederichs wash bottles containing the various solutions were connected in par- allel to the same suction. The blank was immediately sterilized to await analysis and the Fernbachs were incubated at 38° for 72 hours (allowed to incubate 72 hours because the end of 48 hour period came on Sunday), air being sucked through the Fernbach flasks and their wash bottles at as uniform a rate as possible. After 72 hours, each Fernbach was heated to boiling on a water bath for 30 minutes while air was sucked through it and its respective wash bottles. This was to drive out all hydrogen sulphide held in solution in the cultures. The solution from each Fernbach was then made up to 500 cc. (only a few cc. had been lost by evaporation in each case), and total solids, total sul- phur and sulphur as non-volatile .sulphides determined in meas- ured portions of these cultures and of the blank. The deter- mination of sulphur as non-volatile sulphides which has not been yet described was accomplished by placing 300 cc. of each cult- ure in a dfstilling flask and adding 25 cc. of concentrated hydro- chloric acid. A Vigreux tube was attached and the solution was then distilled for 30 minutes under reduced pressure into sodium peroxide solution (5 :6o) contained in awash bottle, the purpose being to decompose the sulphides and to distil the hydrogen sul- phide formed into the sodium peroxide where it would be held as sodium sulphide, in which form the sulphur was determined by the Liebig-Koch method. The results are given in Table VIII. DISCUSSION OF TABLE VIII. As the sum of the total sulphur remaining in the medium plus the sulphur found as hydrogen sulphide in the wash bottles proved to be .0294 grams per 100 cc. when cadmium chloride was used, .0245 when lead acetate was used, .0277 when sodium peroxide was used and .0302 when potassium hydroxide was used, while .0302 grams of total sulphur per 100 cc, on the basis of 3. grams of total solids exclusive of potassium chloride per 100 cc, had been found as the result of a number of de- terminations to be the amount present in the blanks, the indi- cations were that potassium hydroxide stopped and held all of the volatile sulphur compounds evolved while the other sub- stances failed to do so, although cadmium chloride was almost as efficient. 74 -non o;tit pa^jdAnoo jnttdins ibI^o; jo ;n93i3a jniidins iB^b; jo t^nsojaj •p3i^oi:(s -sp '1331 JO SAisnpxs 'spjios iB^o^ JO ;n3D jsa: •joj paianoo -OB jHTidins JO :tn33J3j M CO SB jnqdins snid jntid -[ns p33iDBi;Bnn jo rang 'S^H. SB jniid[ns JO DD 001 J3d SUIBJO ■apjudins 3|ilBioA-uou SB jriiidins jo' 'DO 001 J3d STOBJO -nn JO 'OD 001 jad sraBJO 'IDS JO snisnpxa spites IBHO; JO -00 001 J3d SUXBJO to rD 0^ O^ o § § § o ° ° ° ° q o 00 •^ CO rO 2, -a- lO rO CO s? M i-i o O s o q lO ID 00 CO w m N 0^ ID M O O ■* ID lO ■^ o o o o ifi -UOU OJUl pd)J3An03 inqdins xejoi jo ;a33J3j 'S'^H o;ni p9;jdAao3 jtiiidins xBio; JO )a33j3j ON 'P3:1C0J;S -3p '102[ JO aAisnpxs 'spixos jBioj jo' ;a93i9,i ■joj pd;iino3 -OB jni{d[n5 JO ■\U30J3^ SB jniidins snid jnqd "ins paaiDB^iBnn jo rang *S^H SB Jtiitdins JO 'OD ooi J3d snjBJto •apiqdins aix^BiOA-non sb * jnqd "iris JO 'DO OOI J3d SUIEJO 'unqdins p32[db;}b -nn"jo -DO OOI jad shibjo o o 00 8 o 'IDX JO 3Aisnpx3 spitos XBlOl JO '33 OOI J3d sniBJO o 12 4* 3*13 (U o L o ■OS* IS.s" BO . ^ O u >^ "o83 . «i£.5* „2o.«j ft 3 fi "> "J o 3 o « 2 "* CS O ft ■2 « •§3 r: f 1 ^ a 3 > rt o o CI ^ u C 0^ « 2 ca 2 ". CO cH o « 2 ^ Tj .a ^ _j y « 4) 2 u S3 3 ^ o - 3 i" 3 O . 3 o -d B 3 to -, ■2 S S 2 " .tl O a ,Q •" li 3 V PL, a 5 a o CIS u 5 i-f 77 MERCAPTANS. If mercaptans as well as hydrogen sulphide are formed by the action of the bacteria of sewage, then these mercaptans which boil at temperatures below that of the incubator would pass over into the wash bottles, there to be held as metallic derivatives just as hydrogen sulphide would be held, and subsequently in- cluded in the determination of the total sulphur of the material in the wash bottles by the I/iebig-Koch method. Hence, they would be included under what has been designated sulphur as hydrogen sulphide. The amounts of mercaptans formed, if any, would undoubtedly be very small and consequently no attempt was made to separate and determine them. SUI,PHUR IN ALCOHOL EXTRACT OF PEPTONE AND IN RESIDUE FROM ALCOHOL EXTRACTION. In order to ascertain whether the bacteria present in artificial sewage produce hydrogen sulphide more energetically or less energetically from those portions of peptone which are soluble in alcohol (containing lipoid sulphur) and insoluble in alcohol (containing protein sulphur) than they do from peptone itself, culture media were made, in one of which, the part of peptone insoluble in alcohol was substituted for peptone, and in the other of which, the part of peptone soluble in alcohol was substituted for peptone. The portions of peptone soluble and insoluble in in alcohol which were employed, were obtained in the course of the work described in Chapter XI. HYDROGEN SULPHIDE PRODUCTION FROM THE PART OP PEPTONE INSOLUBLE IN ALCOHOL. Two Fernbach culture flasks were prepared by introducing into each, 500 cc. from a mixture of 150 cc. of a 30. percent solution of the part of peptoneinsoluble in alcohol, 75 cc. of 10. percent potassium chloride solution and 1275 cc. of strained artificial sewage. The remaining 500 cc. was immediately sterilized and kept as a control to be analysed. One of the Fernbachs was attached to a wash bottle containing sodium peroxide solution (15:225) and the other was attached to a wash bottle containing potassium hydroxide solution (20:225) arranged as previously described. One Fernbach flask to act as 78 a check, was prepared by introducing into it 500 cc. from a mixture of 100 cc. of 30. percent peptone solution, 50 cc. of 10. percent potassium chloride solution and 850 cc. of strained arti- ficial sewage. The remaining 500 cc. was immediately sterilized and kept as a blank to be analysed. The Fernbach flask was attached to a wash bottle containing sodium peroxide solution (15:225). The three Fernbachs were incubated for 48 hours at 38°, air in each case, being sucked through a test tube containing the same solution as the respective wash bottle, then through the Fernbach flask, and finally through the wash bottle at as uni- form a rate as possible. After 48 hours of incubation, each Fernbach was heated to boiling on a water bath for 30 minutes while air was sucked through it and its respective wash bottle. The solution from each Fernbach was then made up to 500 cc. (only i cc. to 2 cc. had been lost by evaporation in each case) and total solids, total sulphur, sulphur as non-volatile sulphide and sulphur as hydro- gen sulphide were determined in measured portions. In the de- terminations of sulphur as non- volatile sulphide, the distillates were caught in 12: percent potassium hydroxide solution. The results of the analysis are given in Table X. DISCUSSION OF TABLE X. The fact to be especially noted is that in both of the Fernbachs containing the medium made from the part of peptone insoluble in alcohol, much less hydrogen sulphide was produced than had been produced in media made from peptone itself. That this was not due to a less energetic bacterial flora in the artificial sewage than had been used in previous work, is proved by the fact that in the check Fernbach made up with the regular pep- tone medium and with the same sewage, the amounts of total solids exclusive of potassium chloride, total sulphur, sulphur as non- volatile sulphide and sulphur as hydrogen sulphide ; com- puted to the basis of 3. grams of total solids exclusive o^ potassium chloride per 100 cc. in the blank ; were 2.7836, .0242, .0006 and .0059 grams per 100 cc. respectively, more than twice as much hydrogen sulphide having been produced as in either of the cultures containing the part of peptone insoluble in alcohol. -non o:|,xiT p9)J3Aao3 79 jnqdins ib}6} jo tnssjsj 00 'paKoj^s '3P 'l03 JO 9&ISni3Z3 'SpllOS IH)0) JO ' )Q9319d 'joj p3;ano9 -3v jniidins }0 )a93J9j to d o s a vd SB jnTfdins snid Jtiqd -[ns p93[3E;;Bnn jo luns 'S^H SB Jnnd -ins JO '33 001 J3d sraBJCO i-T •apiqdjns aii:^BiOA-noti sb' jnnd •ins JO '33 001 jad sniBJO •jni[d|ns p33ide;ib -nn JO ■33' 001 jad shibjo *103 JO 3Ai5npx3 sptios JE^O; JO '33 001 J3d SOIBJQ o o _« u '« _5 cti 'to ^ 3
  • o O CO ft* « « 9 3 ° ii 1 s O rt 2 •=1 .i: |<^ 4) o aj "o S -a ►< d ^ O ki 3 ti u o «J fe a a-S g aj . O -M > w -g o u 8o HYDROGBN SULPHIDK PRODUCTION FROM PART OF PEPTONE SOLUBLE IN ALCOHOL. A set of inoculations was made using a culture medium made from the part of peptone soluble in alcohol. Two Fernbach culture flasks were prepared by introducing into each, 500 cc. from a mixture of 150 cc. of a 30. percent aqueous solution of the part of peptone soluble in alcohol, 75 cc. of 10. percent potassium chloride solution and 1275 cc. of strained artificial sewage. The remaining 500 cc. was immedi- ately sterilized and kept as a control to be analyzed. One of the Ferubachs was attached to a wash bottle containing cadmium chloride solution (10 : 225) and the other was attached to a wash bottle containing potassium hydroxide solution (20 : 225) arranged as previously described. Two Fernbach culture flasks to act as checks were also prepared by introducing into each, 500 cc. from a mixture of 150 cc. of 30. percent peptone solution, 75 cc. of 10. percent potassium chloride solution and 1275 cc. of strained artificial sewage. The remaining 500 cc. was immedi- ately sterilized and kept as a blank to be analyzed. One of the check Fernbachs was attached to a wash bottle containing cad- mium chloride solution (10:225) and the other was attached to a wash bottle containing potassium hydroxide solution (20 : 225) arranged as previously described. The four Fernbachs were incubated for 48 hours at 38°, air in each case being sucked through a test tube containing the same solution as the respective wash bottle then through the Fernbach flask and finally through the wash bottle at as uniform a rate as possible. After 48 hours of incubation, each Fernbach was heated tO' boiling on a water bath for 30 minutes while air was sucked through it and its respective wash bottle. The solution from each Fernbach was then made up to 500 cc. (only about 3 cc. had been lost by evaporation in each case) and the total solids, total sulphur, sulphur as non-volatile sulphides and sulphur as hydrogen sulphide were determined in measured portions. The results of analysis are given in Table XI. DISCUSSION OF TABLE XI. The fact to be especially noted is that in both of the Fernbachs containing the medium made from the part of peptone soluble in alcohol, much less sulphur-containing material was broken down and very much less hydrogen sulphide was produced than had been produced in media made from the part of peptone in- soluble in alcohol which in turn had given smaller amounts than -non o;ni psusAnoa aniidxns ibjo^ jo ;n93J3j Jtindins iBid; jo inao jsj 8i -pdiCoiis -3p 'io:!l JO 3Aisnpx3 'spiios iBioi JO ;n3D aaj O rO « uoj p3:;nno3 -OB jnHdjns jo )n33J3^ SB jniidins sn^d jnifd "ins p33i3Bi:iBan jo rang O ■g^H SE jnnd -ins JO 'OD 001 J9d SUIBjg •apiitdins aii?BiOA-non sb" jniid -jtis JO 03 001 JOd StUBJO uniidins pa3ioB;iBun JO 00 001 J3d sniBJO O ' IDS }0 OAisnpxa spi|os o 1b;o5 jo '00 001 jod suiejoi ° ov Tt- o 00 00 o o If 5 X ■oSa ■ti o « SS ti " U ** O 3 £.2 1: otJ o a. u to rt ^ OJ (Jug S !3 .a OJ u i « Cm OJ n u en (J 53 2 33 S S ^ jS (U cl S 13 I" S S ■ s "a !> O o O Ph ^ 2i t; 9 3 u .2 3 I a u " I I CO O P 3 Tj (U 3 5 ^ 3 2 HH S Ji o S M o 3 (U •3 O O V "9 I en ra OJ " -^ 5 P4 XI a M ■goo 82 had been found in media made from peptone itself. That this was not due to a less energetic bacterial flora in the artificial sewage than had been present in previous tests is proven by the fact that in the check Fernbach flasks made up with the regular peptone medium and with the same sewage, the amounts of total solids exclusive of potassium chloride, of total sulphur, of sulphur as non-volatile sulphide and of sulphur as hydrogen sulphide ; computed to the basis of 3. grams of total solids ex- clusive of potassium chloride per 100 cc. in the blank ; were in the cases where cadmium chloride and potassium hydroxide re- spectively had been used in the wash bottles, 2.8092, .0224, .0013, .0072 grams per 100 cc. ; and 2.8430, .0242, .0006, .0064 grams per 100 cc. Hence, it is evident that a medium made with peptone as the sulphur-containing material is a better one to use for the detec- tion of the bacteria producing hydrogen sulphide than are media made with the part of peptone soluble in alcohol or with the part of peptone insoluble in alcohol. CAI,LIBRATION OF BLACKENING OF LEAD ACETATE PAPERS IN NECKS OF CULTURE FLASKS. As it had been repeatedly demonstrated in this piece of work that the amount of hydrogen sulphide produced might be measured by the difference between the amount of total sulphur present in the culture medium before incubation and the amount of total sulphur remaining after incubation, provided that the culture after incubation was boiled to drive out all gas held in solu- tion, a series of determinations was made to ascertain the actual amounts of hydrogen sulphide corresponding to different amounts of blackening of lead acetate paper when the 100 cc. flasks illus- trated in Cut I were used and when strips of lead acetate paper 2 mm. in width, were placed in the tubular part of the caps. For this purpose, a mixture of 250 cc. of 30. percent peptone solution, 125 cc. of 10. percent potassium chloride solution and 2125 cc. of strained artificial sewage was made and 100 cc. por- tions of this mixture were introduced into 20 culture flasks each of which had been provided with a strip of lead acetate paper and a tin foil cap in the usual manner. The remaining 500 cc. was immediately sterilized and held as a blank for analysis. The flasks were placed in the 38° incubator and left until the lead acetate papers began to blacken. As soon as a flask was 83 found in which the paper was blackened for i mm., it was taken out and boiled to sterilize it and to drive off any hydrogen sul- phide held in solution and the total sulphur remaining in the medium was determined by the Liebig-Koch method. As soon as a flask was found in which the paper had blackened for 2 mm., it was removed from the incubator and treated in like manner. In this way, flasks in which the paper was blackened for 3 mm., 4 mm., 5 mm. etc., up to 20 mm., were removed and the contents analyzed for total sulphur. The blank was also analyzed fbr total sulphur. With the single exception of the culture in which the lead acetate paper was blackened for 9 mm. (the abnormal results of which can be explained only on the basis of experimental errors) the amounts of sulphur as hydrogen sulphide show a gradual in- crease with the increasing blackening of the lead acetate paper. The results are given in Table XII, and are also represented graphically in the curve which follows the table. TABLE XII. All results are computed to the basis of 3.0 grams of total solids exclusive of KCl in the 100 cc. blank. Ji^ u a aj ii o ^v l| si is.g §S5l .2198 .0302 1 .2181 .0300 .0002 2 -2177 .0299 .0003 3 . -2175 -0299 -ooos 4 .2170 .0298 .0004 5 .2161 .0297 .0C05 6 -2155 .0296 .0006 7 .2153 .0296 .0006 8 -2147 -0295 .0007 9 .2170 .0298 .0004 10 .2143 -0295 .0007 11 -2139 .0294 .0008 12 .2138 .0294 .0008 13 .2128 .0292 .0010 14 .2124 .0292 .0010 15 .2123 .0292 .0010 16 .2125 .0292 .0010 .0011 .0012 17 .2119 .0291 18 .2113 .0290 19 .2112 .0290 .0012 20 .2111 .0290 .0012 84 / 1 t 1 1 . / J 1 J 1 ./ / / /, / / / ■f/ ; / / ^ y I k ./ .2 .3 .-* .S- .6 .7 .8 7 /.O /./ /.z f.3 J^ILLJGR/iAf^ Pf/f /OOCC. OF -SULPHU/? AS //,>S . 85 SUMMARY. 1 . The percent of sulphur present in the very small part of peptone which is insoluble in water is practically the same as in peptone itself. This explains why, in testing for bacteria which produce hydrogen sulphide, it makes no difference whether the medium is filtered or not. 2. The lyiebig-Koch method for total sulphur and the potassium chlorate-nitric acid method for easily oxidized sulphur are as accurate for culture media made from peptone and potassium chloride as they are for peptone itself. 3. The ratio of total solids exclusive of potassium chloride to total sulphur is very nearly the same in the soluble and insoluble portions of cultures which have been inoculated with artificial sewage, containing bacteria which produce hydrogen sulphide, and incubated at 38° for 48 hours, and hence the indications are that the insoluble material is mostly peptone. 4. More material is broken down by the bacteria and more hydrogen sulphide is produced in flasks having much shrface of the culture exposed to air than in flasks having comparatively little surface exposed. 5. It is noteworthy that about 50. percent more total sulphur was converted into hydrogen sulphide when sterile air was passed over the cultures as when they were exposed to quiescent air ; and about 100. percent more total sulphur was, under otherwise identical conditions, converted into hydrogen sulphide when sterile air was passed over the cultures as when sterile carbon dioxide was passed over them. Therefore, in the case of the flora which was used, at least, the organisms were most active and produced the most hydrogen sulphide when supplied most freely with air. This fact would appear to be of great importance in connection with work directed toward the elimination of odors arising from septic tanks for sewage disposal. 6. From 25. percent to 30. percent of the total sulphur was converted into hydrogen sulphide when cultures were incubated 48 hours with sterile air passing over the culture, and from 50. percent to 60. percent was thus converted when incubated 72 hours. 7. The volumetric iodine method for the determination of hydrogen sulphide is not available in the analysis of the gases 86 given off by bacteria inoculated into the simple peptone medium, because of the interference of volatile unsaturated organic com- pounds. 8. Solutions of cadmium chloride containing potassium chloride to prevent colloidal suspension, of sodium peroxide and of potas- sium hydroxide are available for use in wash bottles through which the gases given off by bacteria may be drawn for the pur- pose of catching and holding the hydrogen sulphide as metallic sulphide in which form the sulphur may be determined by the Iviebig-Koch method. The use of potassium hydroxide is to be preferred. 9. The sum of the sulphur remaining in the cultures after incubation plus the sulphur in gaseous form, which is practically all present as hydrogen sulphide, equals the total sulphur present in the cultures before incubation, when the determinations are made by the methods adopted in this investigation. 10. In media made from the part of peptone soluble in alcohol much less sulphur-containing material was broken down and very much less hydrogen sulphide was produced than was found in the media made from the part of peptone insoluble in alcohol, the ratios being ajjout 3 : 5 and 1:6; while in both of these media, very much less sulphur-containing material was broken down and very much less hydrogen sulphide was produced than in the simple peptone medium, the ratios being respectively about I : 2 and i : 20 in the soluble part of peptone, and 2 : 3 and I : 4 in the insoluble part of peptone. 11. As shown by the percent of total solids exclusive of potas- sium chloride destroyed, and by the percent of total sulphur con- verted into hydrogen sulphide, a larger percent of sulphur-con- taining material than of total peptone was broken down, the ratio being about 3:1. The fact that the bodies of the bacteria were included in the total solids exclusive of potassium chloride, would not materially influence the ratio. 12. A larger percent of easily oxidized sulphur than of total sulphur was converted into hydrogen sulphide by the bacteria, the ratio being about 4:3. It is evident that if a synthetic medium is to be prepared for the detection of the bacteria pro- ducing hydrogen sulphide, work upon which is herewith prom- ised, that the sulphur should be introduced in an easily' oxidized form. 87 13- A larger percent of loosely bound sulphur than of total sulphur was converted into hydrogen sulphide by the bacteria, the ratio being about 3:2. 14. A very slightly greater percent of loosely bound sulphur than of easily oxidized sulphur was converted into hydrogen sulphide by the bacteria, the ratio being about 10:9. There was about twice as much easily oxidized sulphur as there was loosely bound sulphur, both before and after the bacteria had acted up- on the medium. 15. It is possible with a fair degree of accuracy to determine the equivalent of hydrogen sulphide for each millimeter of blackening of the lead acetate paper strips in the caps of the special flasks. CHAPTER XIII. COMPARISON OF THE RAPIDITY OF HYDROGEN SUL- PHIDE PRODUCTION AND OF THE AMOUNTS PRO- DUCED WHEN ARTIFICIAL SEWAGES MADE FROM HUMAN FECES AND FROM THE FECES OF VARIOUS ANIMALS WERE USED. In order to ascertain whether the feces of the domestic animals contribute to sewage, bacteria which produce hydrogen sulphide and in what relative amounts, a comparison was made using different artificial sewages. A number of the flasks illustrated in Cut i were prepared by introducing into each, loo cc. of a solution containing 30. per- cent of peptone and 5. percent of potassium chloride, after which the flasked media were sterilized in the autoclave at three fourths of an atmosphere, lead acetate papers were introduced and the flasks capped with tin foil. Another group of flasks of the same kind were prepared in ex- actly similar manner except that 80 cc. of Cornell University tap water was also introduced into each before sterilization. Artificial sewages were prepared by introducing into 1000 cc. portions of Cornell Universitj' tap water one loop full ( 02 grams) of human feces, one loop full of feces from Cow I, one loop full of feces from Cow II, one loop full of feces from Calf I, one loop full of feces from Horse I, one loop full of feces from Horse II, one loop full of feces from Pig I, one loop full of feces from Pig II, and one loop full of feces from Sheep I respectively. Ninety cubic centimeters of each of the sewages was intro- duced respectively into each of two of the culture flasks con- taining the small volume of medium, and 10 cc. of each of the sewages was introduced respectively into each of two of the culture flasks containing the large volume of medium and then all of the 36 flasks were placed in the 38° incubator. The amounts of hydrogen sulphide produced may be most easily presented in tabular form and are given in Table XIII. 89 TABLE XIII. Millimeters blackened of lead acetate Amount of sewage inocu- papers in lated, and kind. ,01. 1. i. ,■ ■• o , ' 18 hrs. 21 hrs. 24 hrs. 36 hrs. 48 hrs. 10 cc. human ' 0.0 o 4 12 90 cc. human o i 5 all all 10 cc. cow I. o I 2 10 all go cc. cow I. 000 8 all 10 cc. cow II. o o o 12 all 90 cc. cow II. o I 4 all all 10 cc. calf I o I 4 all all 90 CG. calf I 2 5 8 all all 10 ca horse I o o o 14 all 90 cc. horse I 001 6 all 10 cc. horse II. o o o 14 all 90 cc. horse II. 001 5 20 10 cc. pig I o o o 17 all 90 cc. pig I o o 1 15 all 10 cc. pig II. o I 2 16 all 90 cc. pig II. o I 4 all all 10 cc. sheep I 001 8 all go cc. sheep I 000 6 20 DISCUSSION OF TABLE XIII. It should be noted that hydrogen sulphide had been produced in 24 hours by the organisms from the feces of all of the animals tried, namely, man, cow, horse, pig and sheep and that in 36 hours large amounts of hydrogen sulphide had been produced in every case while the solutions were all greenish in color and very turbid, much gas had formed on them and dark green floating cakes and sediment had been formed. ' SUMMARY. The feces of the domestic animals contain bacteria which are capable of producing hydrogen sulphide from the simple peptone medium as rapidly and in as large amounts, as is the case with the bacteria from human feces. CHAPTER XIV. TESTS FOR HYDROGEN SULPHIDE PRODUCTION MADE WITH DIFFERENT STRAINS OF THE COLON ORGANISM ISOLATED FROM HUMAN FECES. For the purpose, 68 flasks of lOO cc. capacity were prepared by introducing into each one, lo cc. of a solution containing 30. percent of peptone and 5. percent of potassium chloride, and 90 cc. of Cornell University tap water. This mixture was quite turbid but when sterilized at three fourths of an atmosphere in an autoclave, the solution in each flask became perfectly clear with a slight sediment at the bottom due to the agglomeration of the colloidal peptone which had made the solution turbid be- fore sterilization. As both dextrose peptone and lactose neutral red cultures of 34 different strains of the colon organism were available, a flask of media was inoculated with a four millimeter loop full of each of the cultures of each of the strains, making 68 in all. The flasks were then provided with strips of lead acetate paper in the tubular part of the caps, were capped tightly with tin foil, and placed in the 38° incubator and observed at intervals after 18 hours. In Table XIV are presented the results of these inoculations, the number of millimeters which the lead acetate paper was blackened on first observing hydrogen sulphide production in each culture being given. For the sake of clearness, the in- creased blackening of the papers is not recorded. After 8 days, no more evidence of any production of hydrogen sulphide was noted even though the cultures were left in the in- cubator for 30 days and a " O " has been placed in the 8 day column against each culture which failed to produce any hydro- gen sulphide. There were in all, 52 of the cultures which produced hydro- gen sulphide in from i to 8 days, while 16 failed to do so. Of the 34 strains, 21 produced hydrogen sulphide from both the dextrose peptone and from the lactose neutral red cultures, 10 produced hydrogen sulphide when inoculated from one culture but not from the other, while 3 strains did not produce hydrogen sulphide when inoculations were made from either culture. 91 TABLE XIV. Millimeters of lead acetate paper blackened on completion of 1 8 hours 24 hours 36 hours 48 hours 4 days 5 days 6 days 7 days. 8 days a' 1 From aextrose peptone culture. From lactose neutral red culture. 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