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methyl-sulfonium-iodide and alpha-hydroxy-beta-methiol-butyric acid will serveas substitutes for methionine, but the interrelationship seems to be rather specificbecause neither ethionine nor methyl-N-methionine work. Cystine and othersulphur-containing substances are likewise unable to neutralize the norleucineeffect by themselves.The inhibitory action of norleucine seems to be associated in some way with

sulphur metabolism by this organism, since less methionine is required to neutral-ize norleucine in the presence of cystine, and it interferes with the anaerobicproduction of H2S from cystine. However, it is possible that norleucine alsointerferes with the ability of methionine to serve as a methylating agent.

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGYAl-10 Food and Dairy BacteriologyAl 1-22 Fermentation, Sanitary, Lake and Marine

Bacteriology

Al. A Proposed Standard Method for the Bacteriological Examination of 80-40Mesh Edible Gelatin. MuRRAY P. HoRWOOD,1 Massachusetts Instituteof Technology, Cambridge, Mass., and Atlantic Gelatin Co., Inc.,Woburn, Mass.

The lack of official standard methods for the bacteriological examination offine ground edible gelatin and the use of varied methods in different laboratoriesstimulated this investigation. As a result of a previous study conducted by thesame group of laboratories (six in number) and published in 1943, it was con-cluded that the bacteriological examination of fine ground gelatin should bemade on one per cent solutions. This study inquired into the effect of time ofincubation, temperature of incubation and culture medium on the total count.A temperature of 35-370C. was compared with 28-30'C; a 48-hour period ofincubation was compared with 72 hours; and nutrient agar was compared withtryptone glucose extract agar.

Although only 11 samples of gelatin were examined in this study, each of thesix participating laboratories examined five one-gram portions of each sample.Counts were made from duplicate plates on each medium and at each tempera-ture after 48 and 72 hours. The same method of examination and the samedehydrated media were used in the participating laboratories.

In spite of all efforts to obtain thoroughly uniform samples and identicalmethods of examination, variations in total count amounted to 200-500 per cent.Low count gelatins however yielded more nearly uniform results in the variouslaboratories. Tryptone glucose agar yielded higher total counts and larger, moredistinct colonies than nutrient agar under identical conditions. Similarly, 35-370C gave higher counts than 28-30'C. Although the 72 hour count is higherthan the 48 hour count, it is not appreciably so, and the longer period of incuba-tion is not warranted to determine bacterial quality.

It is recommended therefore that the bacteriological examination of fine ground

1 Referee, The Technical Committee of the Edible Gelatin Manufacturers' ResearchSociety of America.

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edible gelatin be made on one per cent solutions, on tryptone glucose agar, at35-370C after 48 hours.

A2. Heat Activation Inducing Germination in the Spores of Thermophilic AerobicBacteria. HAROLD R. CURRAN AND FRED R. EVANS, U. S. Departmentof Agriculture, Bureau of Dairy Industry, Agricultural Research Admin-istration, Washington, D. C.

A study of 10 facultative thermophilic cultures isolated from spoiled com-mercially-canned evaporated milk revealed that nearly all would respond topreincubation heat. In the absence of such treatment a large proportion of thepotentially viable spores did not germinate.Washed spores were seeded into various substrates and, excepting the controls,

were heated at 950C, usually for 10 minutes. The heated and control suspensionswere subseeded into glucose nutrient-agar plates which were incubated at selectedtemperatures and counted after 48 hours.The proportion of spores which responded to a preincubation heat stimulus

was found to be dependent upon the amount of heat, the nature of the heatingmedium, the temperature at which the spores were formed, and the temperatureat which they were subcultured. At 95CC the heat-activating process waspractically complete within 10 minutes. The heating mediums arranged in theorder of their effectiveness upon heat activation were: Glucose or lactose (0.5 percent) > peptone (0.5 per cent) > milk > glucose nutrient-agar > beef-extract(0.3 per cent) > glucose nutrient-broth > distilled water > NaCl (0.5 per cent).Spores formed and subcultured at 300°450C were more susceptible to heatactivation than were those formed at 300°45oC and subcultured at 48o-600C.When spores were formed at high temperatures (550-60'C) and subculturedwithout preliminary heating at 30°, 370 and 4500, only a small proportion of thepotentially viable spores germinated. When the spores were formed at the hightemperatures and subcultured with preliminary heating most of the viable sporesgerminated promptly.

These findings have a bearing on certain commercial canning practices and onthe enumeration of thermophilic spores.

A3. Comparative Resistance of Desiccated and Wet Micrococci Heated under Mloistand Dry Conditions. J. YESAIR, E. J. CAMERON AND C. W. BOHRER,Bacteriological Department, National Canners Association, Washing-ton, D. C.

Heat resistance tests in thermal death time tubes were made on moist and driedmicrococci in broth and in moist fat; and on dried micrococci in dry air, and inanhydrous fat. To check a possible effect of reduced air volume on bacterialresistance, capillary tubes, in addition to the thermal death time tubes, wereused in testing the dry bacteria under dry conditions.The results indicated that, in the normal moist state, micrococci were killed

in 30-45 minutes at 550C. Dried micrococci were only slightly more resistantunder moist conditions (killed 45-60 minutes at 550C), but their resistance wasenhanced in moist fat (killed 15-20 minutes at 1000C). In dry air the resistance

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of dry bacteria was marked (killed 135-150 minutes at 1100C). The resistanceof dry bacteria in dry fat approached that of dry sterilization (killed 135-150minutes at 100'C).Dry micrococci (500 million) heated under dry conditions in thermal death

time tubes (volume 3.85 ml) had a resistance of 220-230 minutes at 110'C, buttheir resistance in capillary tubes (volume 0.04 ml) was only 10-15 minutes at1100C. This latter resistance was the same whether the sealed capillary tubeswere immersed directly in the heating medium or enclosed in thermal death timetubes. If the capillary tubes were evacuated or if their volumes were increased,the resistance of the micrococci also increased. The highest capillary resistance(150-160 minutes at 110'C) in capillary tubes was observed when these tubeswere left open in the sealed thermal death time tubes.

A4. Home Canning. I. Survey of Bacteriological and Other Factors Responsiblefor the Spoilage of Home Canned Foods. R. G. TISCHER AND W. B.ESSELEN, JR., Department of Food Technology, Massachusetts StateCollege, Amherst, Mass.

To obtain information on causes of spoilage in home canned foods 293 jars ofspoiled and sound home canned foods put up in 1942, in Massachusetts, by 270families were collected. These families canned a total of 67,632 jars and reportedspoilage of 1.96 per cent. Approximately half of the jars received were spoiledand the remainder were considered to be sound. Each jar was examined bac-teriologically and for physical defects. Of 133 spoiled jars 101 appeared to havebeen understerilized, 22 had been improperly sealed and 7 had no obvious defects.Fifty-one of 146 "sound" jars contained viable organisms which might causespoilage under certain conditions. Four jars of tomatoes showed evidence ofputrefactive spoilage, caused apparently by a preliminary growth of mold whichcreated a favorable environment for the growth of a putrefactive anaerobe presentin the product.

Of 19 putrefactive anaerobes isolated from spoiled jars the spores of four.isolated, respectively, from asparagus, lima beans, and snap beans, survived heat-ing for 30 minutes at 230'F. In phosphate buffer their "F" and "z" values werebut slightly below those of Cameron's putrefactive anaerobe No. 3679. Theseorganisms had a survival time of from 320 to 820 minutes at 212'F.As represented by this investigation it would appear that 80-85 per cent of

home canning spoilage is due to under processing and 15-20 per cent to faultysealing. The boiling water bath method of processing is not adequate to destroycertain types of bacteria encountered in home canning. While a pressure can-ning apparatus, correctly used is satisfactory for processing, understerilizationmay result if it is mis-used.

A5. Home Canning. II. Determination of Process Times for Home CannedFoods. W. B. ESSELEN, JR., AND R. G. TISCHER, Department of FoodTechnology, Massachusetts State College, Amherst, Mass.

Theoretical process times for home canned asparagus, beets, carrots, corn,snap beans, spinach and squash were determined according to accepted methods.

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Calculated process times, under controlled conditions, for these products inpoint jars, at processing temperatures of 212TF, 240TF, and 250'F were derived.Process times based on the heat resistance of Cameron's putrefactive anaerobeNo. 3679 were compared with those based on four heat resistant putrefactiveanaerobes isolated from home canned foods. The theoretical process times forvegetables processed in a pressure canner at 240'F were found to be somewhatshorter than process times recommended at present. Process times at 212TFin a boiling water bath ranged from 5' to 12 hours and show that the processtimes recommended at present are inadequate to destroy certain types of bacteriaencountered in home canning.When the pressure canning apparatus is used the relatively long come-up time

plus the slow air cooling period contribute significantly to the sterilizing value ofthe process. Eighteen to 35 per cent of the sterilization occurs during cooling.In the boiling water method 90-98 per cent of the lethal value of the process mustoccur during the actual process as the effect of the come-up and cooling period iscomparatively small.The information obtained indicates that home canning process times recom-

mended at present may be more severe than are necessary and that in many casesthey might be reduced. Before any general recommendations are made moreexperimental work must be done on home canning process times and a carefulconsideration given to the many variables encountered.

A6. Studies on the Bacteriology of Stored Dried Egg Powder. STANLEY E. HART-SELL, Department of Biology, Purdue University, Lafayette, Ind.

Spray-dried egg powder (both compressed and loose-pack) was stored at 0WC,10'C, 20'C and 37.50C under varying conditions of packaging. After 1, 2 and3 months, total counts were made according to Standard Methods. Incubationof plates was at 320C and 37.5"C. Yeast-water agar was used simultaneouslywith the Standard Methods medium. The genera of bacteria which survivedthe storage conditions were determined.

There appears to be no influence of packaging or compression on the bacterialcontent of dried eggs. Progressively higher temperatures of storage decreasethe total bacterial count more rapidly than lower temperatures. Yeast-wateragar is superior to Standard Methods agar for obtaining total bacterial countsand for the isolation of bacteria present in stored dried egg powder, irrespectiveof the temperature of incubation. Consistently higher counts were obtainedwhen the plates were incubated at 320C than at 37.50C. Similar results wereobtained with a limited series of samples of fresh dried egg powder. Thirtydifferent species of bacteria were detected in 86 isolations which were identifiedas follows: Bacillus-7; Micrococcus-8; Flavobacterium-7; Bacterium-3;Sarcina-2; Achromobacter-1; Actinomyces-1; Leuconostoc-1.

A7. The Occurrence and Possible Significance of Salmonella Organisms in CanadianDried Egg Powder. N. E. GIBBONS AND R. L. MOORE, Division ofApplied Biology, National Research Council, Ottawa, Canada.

Interest in the occurrence of pathogenic organisms in dried egg powder, led to

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the examination of samples from 380 carlots, produced in Canada during 1943,for members of the Salmonella group. Eight types were isolated from 28 lots.The number present was usually small. -Based on most probable numbers, in13 of 21 samples examined there was less than one organism per gram; in seven,between 1 and 10; and in one, 54 per gram. The distribution within carrots wasspotty.

During drying in a laboratory spray drier there is a 99.9 per cent reduction inthe Salmonella count and a 65-80 per cent reduction in the total viable count.On storage the number decreases rapidly. In powder originally containing

30 organisms per gram, there was a 100% reduction after 3 days at 370C, a 99 percent reduction in 4 weeks at 15.60C, and a 98 per cent reduction in 8 weeks at7.20C. With powder containing 40,000 per gram there was a 75 per cent reduc-tion after 3 months at -1.1CC.

Reconstituted powder inoculated with 8,000 to 2,000,000 Salmonella organismsper gram was cooked as scrambled egg, omelet and sponge cake. The organismscould not be detected in the cooked product.

If the reconstituted egg is properly kept and cooked, there should be littledanger from powders containing as few Salmonella organisms as reported.

A8. The Streptococcal Flora of the Non-mastitic Udder. J. J. REID, M. A. FAR-RELL, E. A. KEYES, AND J. F. SHIGLEY, Department of Bacteriology,The Pennsylvania State College, State College, Pa.

The monthly examination of 1200 lactating cattle over a period of two yearshas shown that at some time during the period most of the animals have shedstreptococci in the milk as demonstrated by the Breed smear of incubated,sodium azide, brilliant-green treated milk. A few animals, however, consistentlyshowed no streptococci by the routine tests and showed no clinical evidence ofudder trouble.

Twenty-five of the mastitis-free animals which had never been found to beshedding streptococci were selected for special study. These were chosen fromtwo herds in which mastitis has not been a problem. Forty ml samples weretaken from each animal and the samples were centrifuged immediately. Thesupernatant liquid was removed and 5 ml of veal infusion glucose broth contain-ing brilliant green and sodium azide were added to the sediment. Following anincubation period of 16 hours at 370C smears were made and examined forstreptococci and blood agar streaks were made. In this manner the presence ofstreptococci was demonstrated in each case and isolations from the blood plateswere made for pure culture study. Beta hemolytic streptococci of LancefieldGroup D were found present in the milk of all of these animals and in 22 of the25 animals streptococci of other Lancefield groups were demonstrated.

A9. Contribution of Streptococcus uberis to the Plate Count of Milk. H. W.SEELEY, JR., E. 0. ANDERSON AND W. N. PLASTRIDGE, Storrs Agricul-tural Experiment Station, Storrs, Conn.

A study of the contribution of Streptococcus uberis infection of the bovine udder

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to the total bacterial content of milk was undertaken because of questions fre-quently asked concerning the effect of bovine mastitis on milk quality.

Standard plating methods were employed, with the exception that a selectivemedium (crystal violet, mannitol, inulin, sorbitol, blood agar) was used in placeof standard agar.The complete milking of 23 S. uberis infected quarters was obtained with a

sterile milking unit. The S. uberis population of the samples ranged from 30 to44,000 colonies. The logarithmic average was 3000. No correlation wasobserved between the plate counts and leucocyte counts.

Fifteen strains of S. uberis were inoculated into flasks of milk which were heldat different temperatures for a period of 60 hours. Samples were removed atintervals and tested for pH and bacterial content. All cultures multipliedrapidly at 60'F, 70TF and 970F. Ten cultures multiplied to some extent at50TF. A decrease in the population average of all cultures was noted at 40'F.Measurable changes in the pH did not occur in cultures held at 40'For 500F.The results obtained indicate that the S. uberis content of milk immediately

after it is drawn usually has little influence on the plate count of high-count milk,but may contribute a large share of the flora of low-count milk, and that coolingmilk to a temperature below 50TF will prevent further changes in milk qualitydue to S. uberis.

AlO. Effect of Wilting on the Fermentation of Alfalfa Silage. R. W. STONE,J. J. REID, P. S. WILLIAMS AND S. I. BECEDEL, The Pennsylvania StateCollege, State College, Pa.

As previous work has shown that wilting alfalfa to 60-65 per cent moisturebefore raking materially increases the amount of reducing sugar in the plant andtherefore aids fermentation, a study of alfalfa silage was made to determine theeffect of further wilting treatment. During the summer of 1943, seven silageswere put up with no treatment other than wilting, ranging in moisture contentfrom 30 to 65 per cent. Four additional silages were prepared from the sameforage with ground corn and other preservatives.

All the silages fermented satisfactorily and produced a palatable product.The rate of fermentation and total bacterial count was roughly proportional tothe amount of moisture present. All the wilted silages were relatively high inreducing sugar, and those wilted to 50 per cent and below contained more, on amoisture free basis, than those wilted to 65 per cent. The pH necessary forpreservation appeared also to be related to the moisture present, the less moisturethe higher the final pH. Members of the genus Lactobacillus were pre-dominant in all silages. In those containing 60 per cent moisture and above,no other organisms were present in significant numbers. Below 60 per centmoisture yeasts were common and in two instances increased to numberscomparable to the lactobacilli. In the drier silages some spore-forming rodswere found.

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All. Microbiological Studies on Beer Cooperage. JOHN B. REHM, MARIE SOMMERAND VERNON ELLERBUSCH, Anheuser-Busch, Inc., St. Louis, Mo.

Since draught beer is unpasteurized, sanitation of the packages is of specialsignificance in relation to the keeping quality of the product. This investigationreports results obtained in the routine microbiological examination of almost athousand barrels of various types (wood, laminated wood, aluminum and stain-less steel). Studies made at various stages during the washing, pitching andrinsing processes are summarized, and counts on beer held at various tempera-tures in high and low count containers are discussed. Barrels are rinsed withsterile distilled water which is then plated in glucose agar at 28CC and 370C.Stained films, prepared from representative colonies, are employed to determinethe predominating types of microorganisms present.Wooden barrels are found to be practically sterile when new, but become

increasingly difficult to sterilize after repeated use. Microorganisms apparentlypenetrate into the wood when the barrels are empty, especially during the timebarrels are being returned when temperatures, oxygen requirements, pH, etc.,are apt to be favorable for microbial multiplication. These organisms may enterthe beer when the package is refilled if the pitching is faulty. Aluminum andstainless steel barrels are practically sterile when cleaned and rinsed properly.Age of the container has little, if any, influence in the sanitation of metal pack-ages. Keeping quality of beer is definitely effected if the original bacterial countof the barrel is high, especially at higher storage temperatures.

Predominating organisms are usually yeasts or gram-negative short bacilli.

A 12. A Study of Cultural Methods for the Quantitative Determination of BacterialPopulations in Distillery Mashes. J. C. GAREY, L. A. RIrTSCHOF,L. STONE, AND C. S. BORUFF, Hiram Walker and Sons, Inc., Peoria,Ill.

Bacterial counts conducted by petri plate methods fail to demonstrate thetrue number of bacteria present if the samples examined contain large numbersof yeasts or a predominating flora of lactic acid bacteria or both. When tubecounts were used, the colony count was higher and more representative ofthe true number of bacteria present than when the plate count method was used.

Of several culture media tried, a 40 per cent tomato juice, 1 per cent Difcoyeast extract, 1 per cent glucose, mineral salts, 1.5 per cent agar made up indistilled water and adjusted to pH 7.0, was found satisfactory. Clarified tomatojuice was prepared from commercially canned tomatoes by treatment with oneper cent Filter-Cel. The mineral salts added consisted of: 0.05 per cent each ofmono- and dibasic potassium phosphate, 0.02 per cent magnesium sulfate hepta-hydrate, 0.001 per cent each of sodium chloride, ferrous sulfate heptahydrateand manganous sulfate tetrahydrate.The tube counts were conducted by adding appropriate dilutions to tubes of

melted and cooled (450C) tomato juice agar, mixing and allowing the agar tosolidify. Two per cent agar solution was added as a seal to inhibit growth ofyeasts and prevent "blowing" when gas-forming bacteria were present.The tube count method described above has been adopted for routine use to

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replace the petri plate method for counting bacteria in various phases of dis-tillery operations. Quantitative data were presented to substantiate the advan-tages of the tube count method over the petri plate method.

A13. Bacteriophage in a Butyl Fermentation Plant. E. McCoY, L. E. Mc-DANIEL, AND J. C. SYLVESTER, Department of Agricultural Bacteriology,University of Wisconsin, Madison 6, Wis.

A bacteriophage outbreak Qccurred in a butyl fermentation plant during testof a new process. The unusual feature was the appearance of four phage strainsover a little more than a year's time, during which a single species of the butylclostridium was used in the plant. The several phages were distinguished bytheir specificity. A strain resistant to the original phage A was susceptible tothe later-occurring phages B, C, and D. One resistant to phage C, however,was found resistant also to A and B, whereas all phages attacked the originalparent culture. Investigations were therefore in order to test whether the phageswere separate entities or mixtures. One line of evidence was found in propa-gating each upon its homologous culture strain; i.e., that upon which it hadappeared in the plant. By a sufficiently long series of passages "pure line"phages were obtained. Serological analyses of these phages confirmed theirseparate identity, and cross-neutralization tests of them in the "pure line"antiphage sera and in the sera for the original plant phages A, B, C and Drevealed interrelationships of the plant phages. Four serologically differentphage factors were demonstrated; phage A was proved a single entity but B, C,and D were mixtures of the remaining three factors. Thus their specificitiesand yet the resistance of certain bacterial strains, subjected only to one of theplant phages yet found resistant to others, became understandable.

A14. Variation in Aerobacillus polymyxa and Its Bearing on the Development ofan Industrial Butylene Glycol Fermentation. G. A. LEDINGHAM ANDR. Y. STANIER, Division of Applied Biology, National Research Council,Ottawa, Ontario, Canada.

Aerobacillus polymyxa is an extremely variable organism. Even on first isola-tion from enrichment cultures, a number of markedly different colony types canbe obtained, and these often give rise to further variants in subcultures. At thesame time, strains which are excellent butylene glycol producers on first testsapparently become poorer over the course of a few months. Consequently adetailed analysis of the fermentative properties of the variants has been under-taken. Some seventy cultures, representing the variants isolated from twentystrains of A. polymyxa, were tested for butylene glycol and ethanol productionfrom 10 and 15 per cent whole wheat mashes. The results showed that variantsof a single strain could differ greatly in their fermentative abilities. For example,one variant of a particular strain produced 2.50 per cent butylene glycol and1.50 per cent ethanol from a 15 per cent mash in 3 days, whereas another variantproduced only 1.25 pir cent butylene glycol and 0.67 per cent ethanol under thesame conditions. Such fluctuations would mean the difference between successand failure in an industrial process.

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Another factor of great practical importance, the filterability of the mash afterfermentation, is also variable. Some variants yield homogeneous mashes theconsistency of mayonnaise, from which it is almost impossible to obtain anyliquid; others yield mashes in which the solids aggregate to form a compact"head" or sediment, leaving a clear liquid which filters easily and rapidly. Highbutylene glycol production and easy filterability are not always correlated, sothat strain selection involves consideration of both these factors.

A15. Bacteriophage of Aerobacillus polymyxa in Relation to the Butylene GlycolFermentation. H. KATZNELSON AND A. G. LOCHHEAD, Division ofBacteriology and Dairy Research, Science Service, Department of Agri-culture, Ottawa, Canada.

Bacteriophages against various strains of Aerobacillus polymyxa, employed inthe butylene glycol fermentation, have been isolated from soil and have beenshown to stop, almost completely, fermentation of wheat mash by susceptiblestrains. However, resistant strains, singly, or in a mixture of three, gave satis-factory yields of butylene glycol in the presence of active phage. The phageswere separated into groups on the basis of plaque morphology. They were foundto be very sensitive to heat, being inactivated by a temperature of 550C for thirtyminutes, and were shown to be transmitted through the bacterial spore. Of 50Aerobacillus strains examined, 8 carried phage. It is suggested that underordinary factory conditions, proper sterilization of equipment and materials,aseptic technique, and use of carefully selected non-lysogenic cultures are neces-sary precautions.

A16. Relative Resistance of Eberthella typhosa and Escherichia coli to Chlorine andChloramine. ELSIE WATTIE AND C. T. BUTTERFIELD, Department ofWater & Sanitation Investigations, U. S. Public Health Service, Cincin-nati 2, Ohio.

Using chlorine-free, chlorine-demand-free water buffered at the desired pHconcentrations, 6.0, 6.5, 7.0, 7.8, 8.5, 9.8 and 10.7, observations have been madeon the rate of death of several strains of E. typhosa and E. coli when exposed tochlorine and chloramine in various concentrations. Titrations of the chlorinesolutions added were made by the acid-starch-iodide method with determinationsof residual chlorine by the orthotolidin test and by the flash test for free chlorine.Bacteriological examinations were made at the start and after 1, 3, 5, 10, 20, 40and 60 minutes of exposure. In addition with chloramines and at the higherpH ranges bacteriological tests were necessary at hourly intervals up to six hours.Bacteria surviving just prior to the time of 100 per cent kill were examined todetermine their characteristics.The results indicate that at the lower pH ranges with concentrations of free

chlorine of 0.05 p.p.m. or less E. typhosa is slightly more resistant than E. coli.With greater concentrations of chlorine and at higher pH ranges this sensitivityis reversed. The results indicate in addition that at normal pH ranges whenchlorine is the disinfecting agent approximately 60 times as much residual chlo-rine, as chloramine, is required to produce the same extent of kill as free chlorine

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in the same interval of time, and that to secure the same extent of kill withchloramine in the same concentration as free chlorine requires approximately20 to 30 times as long an interval for chloramine.

A17. The Influence of Hydrogen Ion Concentration on the Bactericidal Action ofOzone and Chlorine. WINSLOW WHITNEY SMITH AND RICHARD E.BODKIN, Department of Bacteriology, University of Southern California,Los Angeles, Calif.

Recent comparative studies have shown that ozone will quickly kill thepoliomyelitis virus and the cysts of Entamoeba histolytica at low concentrations,whereas chlorine, at higher concentrations, fails to destroy these pathogens.These studies suggested a comparison of the lethal action of ozone and chlorine

at various hydrogen ion concentrations. This work, to date, indicates thatozone, over a wide pH range, is many times as effective as chlorine. At a tem-perature of 27.50C, acting upon a dosage of approximately 800,000 bacteria perml, ozone was not markedly influenced by pH changes. At pH 5 and 6, litersamples through which ozone was bubbled were sterile in 5 minutes. At pH 7,8 and 9, the death time was 71 minutes. The ozone residual varied from 0.13to 0.2 parts per million.As opposed to this relative constancy of ozone, chlorine, as has been reported

many times, was highly sensitive to pH changes. The residual concentrationof chlorine required to sterilize as rapidly as the stream of ozone, varied from2.7 parts per million at pH 5, to 7.9 parts per million at pH 8. 0

A18. The Temperature Coefficient of the Bactericidal Action of Chlorine. ADAMAE AMES AND WINSLOW WHITNEY SMITH, Department of Bacteriology,University of Southern California, Los Angeles, Calif.

The widespread use of chlorine by the armed forces for the purification ofdrinking water under a wide variety of field conditions raises the question of theinfluence of temperature on the rate of action of the disinfectant. A study wasmade of the influence of temperature on the ability of chlorine to destroy largedoses of Escherichia coli in the presence of 0.25 per cent organic nitrogen. It wasshown that the action of chlorine is markedly affected by the temperature atwhich it is used. The difference in rate is sufficient to warrant reconsiderationof methods of water purification in use in Arctic temperatures.The temperature coefficient for the bactericidal action of chlorine under the

conditions of these experiments is 1.5 in the range from 400 to 300, 2.0 from300 to 200, and 3.0 from 200 to 100. More than nine times as long is requiredfor a given dose of chlorine to disinfect water at 80 as at 400. Day and nighttemperatures in the desert exceed this range.

A19. Bacteriological Studies of Swimming Pool Waters. ROBERT M. SCOTT,VERNON L. WALKER AND EUGENE S. CLARK, Division of Sanitary Engi-neering, State Department of Public Health, Springfield, Ill.

To determine if bacteriological analyses could be better related to the sanitary

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control of pools, four separate studies were made: (1) Analyses for coliforms,370C plate counts, and streptococci were made at an outdoor pool within fiveminutes after the collection of samples in accordance with Standard Methodsexcept that glucose broth was used for streptococci. Coliforms and streptococcipersisted in spite of an indicated residual chlorine of about 0.7 p.p.m. (2) Froma children' pool samples were similarly collected but in addition duplicatesamples were held for 24 hours in thiosulphated bottles. The holdover samplesshowed much higher plate counts and lower numbers of coliforms and strepto-cocci. (3) An outdoor pool water was tested for coliforms, streptococci, andNeisseria catarrhalis at the pool side and duplicate samples were taken in thiosul-phated bottles and held for about 24 hours before analysis. With residual chlorinetests showing 0.6 p.p.m., pool side samples showed streptococci and some coli-forms. In holdover samples, the plate counts increased greatly and the testsfor coliforms and streptococci were nearly all negative. The N. catarrhalisresults could not be correlated with the other bacterial analyses nor with theswimming load. (4) An indoor pool water was studied as above and the poolside analyses showed coliforms throughout the sampling period, but streptococcionly while swimmers were in the pool. Holdover samples in thiosulphatedbottles showed greatly increased plate counts with a decrease of coliform, in55 per cent of the samples.

These studies indicate grave difficulty in the interpretation of bacteriologicalanalyses of shipped samples of water. Samples enroute to the laboratory over-right may become freed from pollutional bacteria whether plain or thiosulphatedbottles are used. Streptococci and coliform tests at the pool-side appeared to beindicative of the bathing load. The procedure used for N. catarrhalis did notgive any promise as a control test. Pool side samples indicated that normallyhigh residual chlorine tests did not eliminate streptococci and coliforms.

A20. Populations of Heterotrophic Bacteria in Two Sediment Layers of WesternLake Erie. OWEN B. WEEKS, Franz Theodore Stone Laboratory, TheOhio State University, Put-in-Bay, Ohio.

Samples of sediment were obtained from one station in Western Lake Erie atapproximately monthly intervals from November, 1941 to June, 1943. In thebeginning the upper 2 centimeters of each of 10 cores were studied; later separateconsideration was given to a composite of surface scrapings from 3 Petersendredge samples. The heterotrophic bacteria counted were those which grew,under either aerobic or anerobic conditions, on Henrici's sodium caseinate agarafter 3 weeks of incubation at 2300. Five-replicates of the appropriate dilutionwere counted and the average count of all samples was used in calculating thestanding population. The following counts, expressed as number per gram ofoven dry sediment, show the range of the results that were obtained. In coresamples aerobic counts varied from 2,628,000 to 7,731,000 and anaerobic countsfrom 180,000 to 331,000. In surface scrapings aerobic counts ranged between6,780,000 and 18,289,000 and anaerobic counts between 115,000 and 289,000.

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SOCIETY OF AMERICAN BACTERIOLOGISTS

Fluctuations occurred simultaneously in the aerobic populations of both layers.Increases followed periods of phytoplankton mama and those periods of highturbidity of lake water which were caused by river discharge. Such increaseswere not directly related to increases in temperature. The numbers of anaerobicbacteria showed only slight change.

Actinomycetes of the genera Streptomyces Waksman and Henrici and Micro-monospora 10rskov accounted for about 12 per cent of the aerobic population ineach layer. Chromogenic bacteria comprised nearly 24 per cent of the aerobicpopulation in cores and surface scrapings.

AS1. Hydrocarbon Production by Sulfate-Reducing Bacteria. GREGORY J. JAN-KOWSKI AND CLAUDE E. ZOBELL, Scripps Institution of Oceanographyof the University of California, La Jolla, Calif.

Species of Desulfovibrio are widely distributed in marine sediments both recentand ancient. Their presence in oil-bearing sands together with the depletion ofthe sulfate content of many brines in which such bacteria are found suggests thatthey may have been active in situ. The ability of sulfate-reducing bacteria tosynthesize hydrocarbons from certain fatty acids has been demonstrated bylaboratory experiments.The sulfate reducers have been cultivated in sea water enriched with fatty

acids as the only source of organic carbon. After three week's incubation at260C appreciable quantities of oil-like, ether-soluble, non-saponifiable materialhave been extracted from cultures utilizing n-capric acid (Eastman No. 1200).Only faint traces of the extract were recovered from uninoculated controlssimilarly treated. The oil-like extract has been identified as a mixture of ali-phatic hydrocarbons ranging from Clo to C2;. The experiment has been repeatedwith acetic, propionic, butyric, stearic and lactic acids neutralized to pH 7.0with sodium hydroxide. The yield of oil-like substances has been increased byextracting the cultures with carbon tetrachloride. The assimilation of fattyacids by the sulfate reducers is promoted by the presence of a layer of ignitedsand in the bottom of the culture bottles.

A22. Assimilation, of Petroleum Hydrocarbons by Sulfate-Reducing Bacteria.G. DAVID NOVELLI AND CLAUDE E. ZOBELL, Scripps Institution ofOceanography of the University of California, La Jolla, Calif.

In the absence of other types of utilizable organic matter, certain species ofDesulfovibrio are able to assimilate aliphatic hydrocarbons of large molecularweight. Decane is attacked feebly, tetradecane somewhat more energetically,and higher hydrocarbons such as eicosane, docosane, hentricontane, paraffin oil,and paraffin wax are utilized quite readily. Neither aromatic hydrocarbons suchas benzene, xylene, anthracene, naphthalene, and cyclohexane nor aliphatichydrocarbons of lower molecular weight than decane support the growth ofsulfate reducers, thereby substantiating thermodynamic predictions. H2S-production, cell multiplication, and observed changes in the hydrocarbons havebeen used as criteria of hydrocarbon assimilation. Enrichment and pure cul-

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8FORTY-FIFTH GENERAL MEETING

tures have been successful in glass-stoppered bottles filled with mineral solutionfrom which oxygen is excluded by heating to the boiling point prior to inoculation.The hydrocarbons are adsorbed on ignited sand to provide for uniform distribu-tion and large surface areas.

Part of the hydrocarbon is oxidized, part is utilized for the synthesis of bac-terial cell substance, and part is converted into hydrocarbons of lower molecularweight. The effects of bacteria on crude oil are being investigated under theauspices of the American Petroleum Institute.

MEDICAL BACTERIOLOGY, IMMUNOLOGY ANDCOMPARATIVE PATHOLOGY

Ml -12 Disinfectants and ChemotherapyN13-25 Laboratory ProceduresM26-40 VirusesM41-55 Streptococci and Unclassified Studies

Ml. p-Aminobenzoic Acid Synthesis by Neisseria gonorrhoeae in Relation toClinical and Cultural Sulfonamide Resistance. MAURICE LANDY ANDRUTH B. GERSTUNG, Division of Bafteriology, Army Medical School,Washington 12, D. C.

Previous studies in this laboratory have demonstrated that increased p-amino-benzoic acid (PAB) production is associated with the development of sulfonamideresistance in Staphylococcus aureus. However, all other pathogens examinedwere readily made resistant to sulfonamides without detectable increase in theirPAB production; hence S. aureus is the only organism at present known to exhibitsuch an association.

In order to determine whether PAB is concerned in the development ofsulfonamide resistance in the gonococcus, 175 cultures were studied which hadbeen isolated from individuals resistant to treatment with sulfathiazole as wellas from those promptly cured. A synthetic PAB-free culture medium wasdevised which supported good growth of all strains of Neisseria gonorrhoeaeisolated. Cultures were grown in this medium and PAB production measuredby the Acetobacter suboxydans microbiological assay. Simultaneously the resist-ance of the gonococcus cultures to sulfathiazole was measured in vitro, using aninhibitor-free medium.

Al strains of N. gonorrhoeae examined in this study synthesized PAB in readilydemonstrable amounts. Clinical and cultural data from 53 cases yieldingreadily, and 51 cases resistant, to treatment by sulfonamides showed a definiterelationship between clinical response, PAB synthesis in vitro and sulfonamidesensitivity. With but few exceptions, individuals promptly cured have yieldedsulfonamide sensitive cultures with low PAB synthesis, while clinically resistantindividuals have yielded sulfonamide resistant cultures with high PAB synthesis.On the basis of this evidence, it is suggested that PAB synthesis by N. gonornhoeaeis related to its sulfonamide resistance.

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