THIRTY-EIGHTH ANNUAL MEETING

Pathogenic, motile, aerobic sporulating bacilli isolated from a con-taminated blood culture and from air and soil were tentatively identifiedas Bacillus siamensis (Siribaed). A comparative study of this Bacillussiamensis group with type cultures of aerobic spore-formers such asBacillus cereus, Bacillus subtilis, Bacillus megatherium and others showedBacillus siamensis to be culturally indistinguishable from Bacilluscereus. Although the Bacillus siamensis cultures were pathogenic whenfirst isolated, loss of pathogenicity occurred until their virulence wascomparable to that exhibited by Bacillus cereus after rapid subcultureof this species on blood agar. It is, therefore, concluded that Bacillussiamensis is identical with Bacillus cereus. Since Bacillus cereus is oneof the most common of the aerobic spore-formers in soil, it is probablethat many of the "motile, anthrax-like" and "pathogenic subtilis"bacilli isolated by earlier workers were likewise Bacillus cereus.

AGRICULTURAL AND INDUSTRIAL BACTERIOLOGYAt. The Anaerobic Bacteria of the Soil-a Tillable Field. IvAN C.

HALL, University of Colorado School of Medicine and Hos-pitals, Denver.

Present knowledge of the anaerobic bacteria of the soil has beendeveloped along two distinct lines by workers interested in the soil fromwidely differing viewpoints. On one hand soil bacteriologists undertookto solve the complicated problems of decay in the soil, fermentation,putrefaction, nitrogen fixation, oxidation, reduction, and general micro-bic ecology in the interest of soil fertility, while on the other, medicalbacteriologists studied the anaerobic flora of the soil in relation tomalignant oedema, gaseous gangrene, and tetanus caused by woundinfections and in relation to botulism caused by eating improperlyprocessed foods contaminated with soil.The early observations of Mitscherlich (1850) on decay of potatoes,

the discovery of Amylobacter by Trecul (1865), and van Tieghem's(1877-1879) studies on the decomposition of cellulose laid the back-ground for the later work of Beijerink (1896-1902) on Granulobacter,and of Winogradsky (1893- ), Omeliansky (1895-1916), and otherson Clostridium pastorianium. There is still much confusion as to theexact taxonomic position of the anaerobic bacilli concerned in the decom-position of cellulose in the soil and in the fixation of nitrogen; newstudies are needed to redefine the important species involved in theseprocesses.The taxonomic status of the anaerobic bacilli of the soil concerned

76

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

in the production of disease seems to be much better established, butmuch remains to be learned as to their distribution in many parts of theworld and in all kinds of soil environments, with special reference to theapplication of the fecal and telluric theories of origin.

Beginning with Koch's experimental production of malignant oedemaby inoculation of soil suspensions in 1876 it is possible to list the isolationof many pathogenic and non-pathogenic anaerobes from the soil. Butmost of these records were incidental to other studies and there is adefinite opportunity now for further comprehensive surveys similar tothose reported by Meyer and his associates in reference to Bacillusbotulinus, by Zeissler on the anaerobic flora of the German war fronts,and by Sasaki in Japan.The bacteriological analysis of soil samples presents most difficult

technical problems owing to the large number of variable chemicalfactors and the almost constant presence of aerobic sporulating bacilliwhich greatly complicate the isolation of the anaerobes. But thepresent state of bacteriological art and science is such as to encouragenew attacks upon these problems.

A2. Evidence of Localization of Microorganisms about Plant Roots.ROBERT L. STARKEY, Department of Soil Microbiology, Agri-cultural Experiment Station, New Brunswick, N. J.

The Rossi-Cholodny contact slide method was used to obtain informa-tion concerning the influences of root systems of plants upon the micro-organisms in the soil. Microscope slides (50 x 75 mm.) were buriedvertically in the soil. Seeds or seedlings were planted about one to threeinches above the slides. During plant development, roots passed overthe surfaces of the slides and adhered to them.- Organisms growingabout the roots and soil particles also became fixed to the slides andcould be distinguished after being stained. The limited number ofobservations so far made demonstrate that various microorganismsdevelop about plant roots in greater numbers than in the soil mass freefrom root growth. Bacteria and actinomycetes were found in massesin contact with the roots; smaller aggregates and isolated cells werefound elsewhere on the slides. Short rods and coccoid bacterial cellspredominated; thin rods and Azotobacter-like cells were occasionallyencountered.

AS. Fungous Mycelia in the Soil. CHARLES THOM AND MARIE BETZNERMORROW, Bureau of Plant Industry, Washington, D. C.

77

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

Soil fungi may possibly be divided into two sections: (1) Thosecapable of living normally in relation to soil organic matter in thechemical sense, i.e., to residual products of decomposition; and (2)those concerned in primary decomposition, i.e., the breakdown ofplant and animal remains toward those residual products.The distinction is mainly on paper. Certain mushrooms, and such

organisms as Actinomyces, Penicillium luteum, Zygorrhynchus andpossibly Trichoderma, appear to be quite able to cause in vitro the decom-position of the residual products regarded by the chemist as "humus."They probably account for part, at least, of the slow but continuousevolution of carbon dioxide observed from soil otherwise practicallystatic, but they also grow beautifully in culture as causes of primarydecomposition if given the chance.The majority of soil fungi are directly related to the general disin-

tegration of plant remains in and on the surface of the soil. The relationof a root-rot organism to the host root system is therefore sketched asan example of the relationship of soil fungi to organic remains.

A4. Respiratory Enzyme Systems in the Root Nodule Bacteria. P. W.WILSON, Biochemical Laboratory, University of Cambridgeand Department of Agricultural Bacteriology, University ofWisconsin.

Development of methods for use of non-proliferating cells, i.e., the so-called "resting cells," constitutes a notable advance in technique forstudy of bacterial enzyme systems, especially those concerned withrespiration. Mass suspensions from agar or liquid cultures are washedfree from nutrients and resuspended in phosphate buffer solutions, andhydrogen transfers from various substrates are followed in micro-respirometers, Thunberg tubes or by micro-chemical determinations.Free oxygen, dyes (methylene blue), inorganic compounds, as KNOS,etc., may be employed as hydrogen acceptors. Among the advantagesof the technique are: (1) Respiratory activities are separated from thoseassociated with growth. (2) Kinetics of reactions may be determinedwhich allows comparison of rates of reactions instead of total effects.(3) Single reactions may often be studied rather than the complex ofreactions occurring with growing cells. (4) Employment of specificinhibitors allows some separation of related reactions. (5) Resultsare referred to a comparable basis (as QO2) eliminating effects whicharise from differences in total or rate of growth. (6) Experiments areshort-time (1 to 2 hours) which eliminates, in part, differences in metab-

78

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

olism associated with cells in various stages of development and allowscomparison of these differences.

These cited advantages will be illustrated by a consideration of someof the researches of the Cambridge University school, especially thoseconcerned with the hydrogen enzymes of Escherichia coli and with the"Stickland reaction."

Before detailed studies of the various respiratory enzyme systems in agiven species may be undertaken, examination of the chief respirationfunctions of the organism is necessary in order to determine the factorswhich influence the reactions. Unless these characteristics are defined,results may be misinterpreted, since they will represent a complex ofcompeting influences rather than the single reaction under considera-tion. The type of preliminary investigations necessary will be illus-trated by discussion of studies made with Rhizobium trifolii. Whenthis organism is grown on the laboratory media usually employed, thebacteria are low in nitrogen, high in gum, and have a low rate of oxygenconsumption (QO2 on glucose, from 1 to 5). Moreover the endogenousrespiration, i.e., respiration in absence of added substrate, is 50 to 70per cent of that in the presence of glucose. If the organisms are grownon an agar substrate containing yeast extract plus mineral salts but nocarbohydrate, the cells are practically free from gum, contain 10 per centnitrogen, and possess relatively high respiratory activity (QO2 on glucose,50); also the endogenous respiration of these cells is only 10 to 20 percent of that in the presence of glucose.The influence of the following factors on the respiration of Rhizobium

trifolii grown on this yeast extract medium was investigated: (1) Tem-perature, (2) pH, (3) concentration of phosphate in buffer solution, (4)concentration of substrate, (5) age of culture, (6) composition of mediumwith respect to yeast extract and carbohydrate, (7) concentration ofsuspension of organisms, and (8) stability of dehydrogenases. Inaddition, the rate of oxygen uptake on 21 substrates, including carbo-hydtates, polyhydric alcohols and salts of organic acids, was deter-mined and compared with activation of these substrates using methyleneblue as the hydrogen acceptor. The results of these studies will bepresented and briefly discussed.

A5. Studies in the Mechanism of Symbiotic Nitrogen Fixation: Hydrogenas a Specific Inhibitor. W. W. UMBREIT AND P. W. WILSON,University of Wisconsin.

Physical-chemical studies on the pN2 function of symbiotic nitrogenfixation supply evidences that commercial hydrogen may act as a

79

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

specific inhibitor for the fixation reaction in red clover inoculated withefficient strains of Rhizobium trifolii. Establishment of this finding isextremely important for elucidation of the mechanism of the process,since not only does it throw light on the nature of possible enzymereactions involved but also provides a convenient tool for future re-search on this problem. Because of this importance, numerous re-searches have been carried on for the past three years directed towardthe answer of the two crucial questions:

1. Is the inhibition noted in the development of plants grown inatmospheres containing hydrogen specific for the fixation re-action, or is it on the general growth of the plant?

2. Does the inhibition arise from the hydrogen itself or from animpurity in the gas used?

Data concerned with the first question have been discussed previously(Wilson: Trans. Second Microbiol. Cong., London, 1936); these supportthe view that the inhibition is specific for the fixation process. Experi-ments reported in this paper deal with: (1) attempts to locate the effec-tive impurity in commercial sources of hydrogen; (2) effect of hydrogenfrom different sources. All results to date indicate that the observedinhibition is an effect of hydrogen as such and not due to an accompany-ing impurity.

A6. Adsorbed Calcium on Colloidal Clay and an Accessory Growth Factorin Laboratory Production of Rhizobium Cultures. Wm. A.ALBRECHT AND THOs. M. McCALLA, University of Missouri.

Calcium adsorbed on colloidal clay which also carried other nutrientcations, was found responsible for trafisforming abnormal forms ofRhizobium cultures to the normal, and for improving their inoculatingability. Substitution of barium for calcium on the clay transformed thenormal colonies to the abnormal yellow, orange or red forms with lowinoculating power. The colloidal clay medium grew the organism atacidities as high as pH 5.0 and delivered calcium more effectively thanthe agar medium with a calcium carbonate suspension. These resultspoint to the need for generous amounts of calcium in the nutrition ofRhizobium to make it an effective inoculator as well as in the legumeplant to make the symbiotic relationship between these two mosteffective for nodulation and nitrogen fixation.A search for an accessory growth factor more effective than yeast in

laboratory cultures led to that present in kraut juice, a part of thestimulative effect of which is the result of the available nitrogen content.

80

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

Studies on the nature of the responsible factor show it soluble in ethylalcohol, dilute acetic acid, and water, but slightly soluble in methylalcohol and insoluble in petroleum ether and pyridine. It does notseem to be adsorbed on Fuller's earth. It passes through the collodionbag during dialysis, but is inactivated or destroyed on electrodialysisfor complete chloride removal. Partial electrodialysis at low voltageto a clear solution and parchment dialysis are not detrimental.The behavior of the growth factor suggests a molecule of colloidal

dimensions but of small magnitude and an activity associated with theoxygen consumption by the organism. A colloidal clay suspensionwith kraut juice and sugar additions was found the most effectivemedium for rapid multiplication of effective Rhizobium cultures. Theresponse by the Rhizobium as measured by turbidity developmentsrelated to quantities of the growth factor suggests the consideration ofmicroorganisms as rapid aids in the assay of substances for their contentsof such factors.

A7. Attempts to Differentiate the Species of Rhizobium by PhysiologicalMethods. 0. A. BUSHNELL AND W. B. SARLES, Department ofAgricultural Bacteriology, University of Wisconsin, Madison.

In order to determine whether or not there exist any demonstrablephysiological differences among the several species of root-nodulebacteria, representative strains of each of the common Rhizobiumspecies were tested for their ability to grow and to bring about'mheasur-able changes in a number of different culture media in which the sourcesof carbon or of fixed nitrogen were varied.The sugars arabinose, cellobiose, galactose, sucrose, and xylose were

used as carbon sources in a liquid medium in which sodium nitrate wasthe nitrogen source; urea and nine different ammonium salts of in-organic and organic acids: ammonium nitrate, chloride, sulfate, phos-phate, acetate, citrate, lactate, oxalate, and tartrate, were employedas nitrogen sources in a liquid medium in which mannitol was the sourceof carbon.

In all of these tests changes in the reaction of the culture media weretoo variable to be dependable: variations among the strains of one specieswere often as great as were the differences that were noted betweenspecies. In general, however, two large physiological groups of rhizobiawere differentiated by both the sugars and the ammonium salts: theone, characterized by the formation of an acid reaction in these media,was made up of the alfalfa, clover, pea, bean, and Dalea strains; the

81

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

other, characterized by the formation of an alkaline reaction, includedmost of the strains from the soybean-cowpea-lupine group. Otherthan this two-fold separation there were found, in these tests, no phys-iological characteristics which could be used to distinguish any onespecies of Rhizobium from the others.

A8. Nitrogen Transformations in Certain Colorado Soils. HERBERT W.REUSZER, Colorado Agricultural Experiment Station, FortCollins.

In an effort to determine whether appreciable increases in nitrogencontent could be brought about as a result of non-symbiotic biologicalfixation, 12 Colorado soils were incubated in the laboratory for a periodof 120 days. The soils were kept at a temperature of 280C. and a mois-ture content of 20 per cent. Two pots of each soil were incubateduntreated and two with one per cent cellulose (ground filter paper)added. At intervals of 30 days duplicate samples from each pot wereanalyzed for total nitrogen by the Kjeldahl-Gunning method, modifiedto include nitrate nitrogen. The latter form of nitrogen was also deter-mined separately.

Rather large quantities of nitrate accumulated in all soils to which nocellulose had been added. It would seem that in most cases this accu-mulation was sufficiently large to indicate that the nitrogen-fixing organ-isms secure their nitrogen from the soil rather than from the atmosphere.Few of the variations detected in total nitrogen content of the soilswere found to be statistically significant. There was no consistentincrease in total nitrogen with increasing length of incubation. Noconsistent differences could be detected in nitrogen content between soilswith and those without added cellulose.The data were treated statistically by the analysis of variance method

of Fisher. In the final averages for nitrogen content of soils with andwithout cellulose, where 128 Kjeldahl analyses entered into each aver-age, a difference of 0.0015 per cent nitrogen was required for significance.In the averages for each of four periods, where 64 analyses were in-cluded, a difference of 0.0021 per cent was required. Where 32 analysesentered into the averages for each of eight soils, a difference of 0.003per cent nitrogen was required for significance. Even with the largenumber of analyses entering into these results, the differences requiredfor significance were larger than those frequently taken as indicatingnitrogen fixation for smaller numbers of samples.

82

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

A9. Some Factors Affecting the Preparation and Use of Silica-Gel Mediafor the Growth of Non-Symbiotic Nitrogen-Fixing Bacteria.HAROLD W. BATCHELOR, Ohio Agricultural Experiment Station,Wooster.

A preliminary survey of the effects of fertility practices on the numberof naturally occurring Azotobacter and Clostridium butyricum coloniesin plot soils has been previously reported. Preliminary reports havebeen made on the effects of rates and lengths of time of shaking a soilsuspension and of its settling on the apparent populations of Azotobacterin soil.A medium more satisfactory for the growth of Azotobacter than the one

previously used has been developed together with a satisfactory mediumfor enumerating the populations of Clostridium butyricum in soil. Theeffects of sodium and potassium silicates singly and in mixtures, ofdifferent sources of nitrogen in different concentrations, of the anions(-SO4), (-Cl), and (- P04) singly and in mixtures, of different sourcesof organic matter for energy, and of the quantity of medium used in thepetri dishes have been studied. Electrometric titrations of representa-tive silicate and acid mixtures which show the limitations of the use ofthe media, and the compositions of the media finally adopted arereported.

A10. The Purification of Sewage by Bacteria in Pure Culture. C. T.BUTTERFIELD, U. S. Public Health Service, Stream PollutionInvestigations, Cincinnati, Ohio.

In investigating the activated sludge process of sewage treatment,an entirely biological process, the oxidation, adsorption and synthesisphases of purification are being considered in a combined study by thelaboratory staff. The present report deals with the oxidation of sewageby massed growths of pure cultures of bacteria. Predominant bacteriafrom good activated sludge were isolated in pure culture. Growingthese pure cultures in sterilized natural sewage and in sterile syntheticsewage, massed cultures (pure culture "activated sludges") were de-veloped by the fill and draw method of feeding. Dividing each of thesepure massed cultures into two aliquot portions (one for a control andone for testing its oxidative properties on a substrate feed, either steri-lized natural sewage or sterile synthetic sewage), the oxidizing efficiencyof the massed cultures under aeration was determined. At the sametime, the 5-day biochemical oxygen demand of the substrate feeds whichvaried from 142 to 345 p.p.m., was determined by the usual excess-

83

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

oxygen method. The results indicate that these massed cultures (pureculture "activated sludges") can oxidize in 5 hours from 38 to 62per cent of the 5-day biochemical oxygen demand of the substratefeeds.

All. The Effect of Trickling Filters on the Bacterial Count of Sewage.H. 0. HALVORSON AND Louis J. BIERMAN, Department ofBacteriology and Immunology, University of Minnesota.

Our studies indicate that there is no correlation between the abilityof a trickling filter to reduce the oxygen demand of sewage and itsability to reduce the bacterial count. In ordinary trickling filters, thebacterial count is generally reduced from 60 to 90 per cent. The re-duction in count, however, is a function of the number of organismspresent in the raw sewage. If the waste applied to the filter containsfew organisms, there may be a larger number present in the effluentthan in the influent. The bacterial flora of the effluent appears to bemore or less constant and independent of the flora of the influent.To get a reduction in bacterial count, it is necessary that the sewageapplied to the filter contain a large number of organisms. Since this isusually the case, trickling filters as a rule produce effluents with fewerorganisms than are found in the influents. Slides placed in the effluentof a trickling filter showed a wide variety of organisms. There appearsto be no correlation between the morphological characteristics of a floraand the efficiency of the filter.

A12. The Distribution of Heterotrophic Bacteria in the Bottom Deposit.of Lakes. ELIZABETH McCoy AND ARTsuR T. HENRICI,University of Wisconsin and University of Minnesota.

Fifty-six profile samples of bottom deposits have been collected from13 lakes of glacial origin in Minnesota and Wisconsin. These lakesshow a wide range in type and productivity. The bottom depositsincluded sand, marl, various types of black ooze (gyttja) and brownooze (dy).

Three hundred and twenty-two plate counts of bacteria have beenmade from varying levels of these samples. From the mud-watersurface these have ranged from 6000 to 500,000,000 per cubic centi-meter (mean, 370,600). The plate counts show a marked decrease withdepth in the mud. Counts from the 30 to 33 cm. levels ranged from120 to 275,000 per cubic centimeter (mean 21,000). When the countsare plotted against depth, curves of individual samples show much

84

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

variation and irregularity, though there is a general tendency to droprapidly in the first 10 to 12 cm., more slowly beyond. Statistical treat-ment of the entire series of counts indicates that a logarithmic curvewill best fit the data. Such a curve is similar to a survivorship curvefrom a disinfection experiment, and is interpreted as indicating thatbacterial activity at the bottoms of lakes is carried on almostexclusively at the mud-water surface, the bacteria dying below thislevel.The means of the counts from the upper 18 cm. from profundal

stations show a fair correspondence with the productivity of the lakes,being highest (609,300) in the very eutrophic Lake Mendota, andlowest (2160) in the very oligotrophic Crystal Lake. Counts from lit-toral stations are much higher than those from profundal ones if theshoreward zone is occupied by aquatic plants, lower in the case of asandy beach.

A1s. Viability of Bacteria in Sea Water. SELMAN A. WAKSMAN ANDMARGARET HOTCHKISS, Woods Hole Oceanographic Institu-tion, New Jersey Agricultural Experiment Station and NewYork Medical College.

Certain factors are responsible for the low numbers of bacteria usu-ally found in natural sea water. The addition of a culture of an agar-liquefying marine bacterium to fresh sea water resulted in its rapiddestruction; the same culture added to water sterilized by heat or byfiltration through a Berkefeld survived for a considerable period of time.The destruction of the bacteria in the fresh sea water was not accom-panied by a reduction in the consumption of oxygen. The survivingbacteria in the sterile water consumed less oxygen during a certainperiod of incubation than the rapidly reduced numbers of bacteria in thefresh water. The agents responsible for the destruction of the bacteriawere thus shown to be biological in nature. Protozoa and other marineanimals belonging to the animal plankton were found extensively in thefresh water where reduction in bacterial numbers took place. Theseorganisms are believed to be at least partly responsible for the reductionin the bacteria in the sea water. The limited numbers of bacteria undernatural conditions in the sea can thus be explained by a state of equilib-rium between bacterial multiplication and bacterial destruction by theanimal members of the plankton. A change in this equilibrium can bebrought about by a change in the food supply and environmental con-ditions, such as temperature and aeration.

85

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

A14. The Influence of Solid Surface Upon the Physiological Activitiesof Bacteria in Sea Water. CLAUDE E. ZoBELL, Scripps Insti-tution of Oceanography, University of California, La Jolla.

During the storage of raw sea water which initially contains only afew bacteria per milliliter the bacterial population may increase tomillions per milliliter. The densest populations appear in the smallestreceptacles in which the water is stored. This is attributed to the bene-ficial effect of solid surface on bacterial activity. Increasing the solidsurface with glass beads, glass wool, silica sand or inert colloids tendsto increase the population up to a level where available nutrientsbecome a limiting factor.

Oxygen consumption, denitrification, ammonification and the fer-mentation of soluble carbohydrates are also favored by solid surfacealthough the rate of these activities is not proportional to the bacterialpopulations. There is little or no chemical evidence of such activitiesuntil the bacterial population is actually decreasing and the rate con-tinues to accelerate after the phase of readjustment in the growth curveis reached. This indicates that there are many biochemically activebacteria which are not demonstrated by plating procedures. Directmicroscopic observations by the Henrici technique reveal that thereare more bacteria tenaciously attached to glass surfaces than are foundin the water.

Solid surfaces are believed to enhance the physiological activitiesof bacteria in several ways: (1) They may concentrate the dilute nu-trients and exo-enzymes by adsorption or otherwise. (2) The inter-stices at the tangent of the bacterial cell and the solid surface mayserve as concentration foci which retard the diffusion of exo-enzymesand metabolites away from the cell thereby favoring both digestionand absorption of foodstuffs. (3) These interstices may aid the produc-tion of optimal oxidation-reduction or other physico-chemical con-ditions. (4) Many marine bacteria are known to be obligate periphyteswhich are active only on a solid surface.

A15. Conditions Controlling the Marine Bacterial Population and ItsActivity in the Sea. CHARLES E. RENT, Woods Hole Ocean-ographic Institution and Harvard Biological Institution.

It is striking that the bacterial population of the sea is much lowerthan that of fresh waters, soils, fermenting juices, or of other sitesbearing decomposing organic matter. When the limitations of con-ventional counting methods are compensated for, it still appears that

86

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

the bacterial flora of the sea, even that peculiarly marine, is a suppressedpopulation. Whenever sea water is stored alterations take place thatpermit a level of activity comparable to that in other very dilute media.These storage experiments have indicated some of the factors responsi-ble for this unexpectedly low level of bacterial life in the ocean.

Concentrations of essential nutrients are much lower in the seathan in media with which bacteriologists are familiar, and the demandsof otherforms for particulate organic matter further depresses this alreadylow content. Aside from shallows, shelf fringes and a thin superficiallayer, the sea's greater volume exists at temperatures near freezing towhich the specialized marine bacteria are not uniquely adapted. Itappears, too, that a predatory nannoplankton, favored by lower tem-peratures, is active in grazing off the bacteria.

Particulate substrates, necessary for favorable development of po-tentially large attached populations, tend to settle and carry largenumbers of bacteria into the mud during sedimentation, where theymay or may not be active.

Detailed consideration of chemical and hydrographic processes in thesea is necessary for evaluating the function of bacteria in the ocean'sliving economy, and it is necessary that laboratory experiments bescaled with appreciation of these processes.

A16. Direct Microscopic Evidence of an Indigenous Bacterial Flora inGreat Salt Lake. W. WHITNEY SMITH AND CLAUDE E. ZOBELL,University of Utah and Scripps Institution of Oceanography.

When chemically clean, sterile glass slides are suspended in GreatSalt Lake, water of which contains 336 grams of salt per liter, bacteriaattach themselves thereto. After 12 hours many attached cells can bediscerned and some appear as micro-colonies. The longer the slides areleft in the lake the larger and more numerous become the colonies.Controls show that this does not occur with lake water inoculated withbacteria from other sources and that killed bacteria do not become at-tached to slides.

A17. A Comparison of MacConkey's Bile Salt Lactose Broth and Stand-ard Lactose Broth as Presumptive Test Media for Use in WaterAnalysis. MICHAEL A. FARRELL, The Pennsylvania StateCollege.

Raghavachari (1936) as a result of an extensive study of variousAmerican presumptive test media with MacConkey's broth on Indian

87

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

water supplies, found that MacConkey's medium was the most efficientpresumptive test medium examined. In view of these results he recom-mends that the Standard Methods Committee include MacConkey'sbroth in the comparative study they are making of presumptive testmedia.

Earlier work by the author had indicated that MacConkey's mediumwas inferior to standard lactose broth. In view of Raghavachari'sresult this problem was further investigated. The relative efficiencyof standard lactose broth, MacConkey's medium and MacConkey'smedium as modified by Raghavachari was tested on 17 pure culturesof the coli-aerogenes group (using the method of Butterfield and Hos-kins) and 94 surface and well water samples. The results obtainedindicated that standard lactose broth was much more efficient in thedetection of small numbers of pure cultures of the coli-aerogenes groupthan either of the MacConkey broths.

Standard lactose broth was also found to be more efficient than eitherof these two other presumptive test media in testing 94 water samples.

A18. The Bacteriostatic Action of Brilliant Green in Solid Media onMembers of the Colon-Aerogenes Group and Their Intermediates.ARNOLD E. HOOK AND E. R. HITCHNER, Department of Bac-teriology, University of Maine, Orono.

A 1:1000 aqueous solution of brilliant green was added to nutrientagar (Difco), pH 7.0, in sufficient amounts to give the following con-centrations in thousands: 1:25, 1:33, 1:50, 1:100, 1:200, 1:330, 1:400,1:660, and 1:833. Duplicate plates of each concentration were inocu-lated by streaking with a standard 2 mm. loopful of a 1:10, 1:100,and 1:1000 dilution of a 24 hour-old broth culture of the variouscultures and the inhibitory effect was observed after varying periods ofincubation at 370C.

All cultures exhibited a greater dye tolerance when the larger inoculawere used. The Aerobacter strains grew at a much higher dye concen-tration than the Escherichia strains. Within the genus the variouscultures of both Escherichia and Aerobacter exhibited marked differ-ences in dye tolerance. These differences were more marked in repre-sentatives of the Escherichia group. Upon prolonged incubation manycultures produced visible growth at a dye concentration which inhibiteddevelopment during a shorter incubation period.A preliminary study with a few representative strains of Escherichia

coli and Aerobacter aerogenes showed that when a 1:10 dilution of a 24-hour-old culture of the various strains was streaked on nutrient agar

88

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

containing a 1:200,000 concentration of the dye, all Escherichia culturesshowed no visible growth after 48 hours of incubation while all Aero-bacter strains grew rapidly. Further tests with 103 cultures, com-prising 19 Aerobacter strains, 61 Escherichia strains, and 23 inter-mediates, differentiated by the M. R., V. P., indol and the citrate andcellobiose utilization tests, confirmed the results from the preliminarystudy with the Escherichia and Aerobacter strains. The results with theintermediates were irregular, since there was no definite relationshipbetween any specific test and the dye tolerance.The authors suggest the above procedure as a further means of

differentiating Escherichia and Aerobacter strains.

A19. A Bacteriological Survey of a Swimming Pool Treated with Silver.W. L. MALLMANN, Department of Bacteriology, MichiganState College, East Lansing.

A bacteriological survey was made of a swimming pool treated withsilver, introduced by the Katadyn process. All samples were col-lected in sodium-thiosulphate-treated sample bottles. Serial sampleswere collected during the actual period of bathing. Tests were madefor total bacterial counts, streptococcus indices, and coli indices. Thedata show that silver is slower than chlorine in its bactericidal activity,hence bathing loads must be proportionately much lower. Three non-pathogenic bacteria that would grow in the presence of silver werefound in the pool water.

A20. Optical Activity of Lactic Acid Produced by Lactobacillus acid-ophilus and Lactobacillus bulgaricus. LENORE M. KOPELOFF,NICHOLAS KOPELOFF, J. L. ETCHELLS AND E. POSSELT, De-partment of Bacteriology, New York State Psychiatric In-stitute and Hospital, New York.

The optical activity of the lactic acids produced by single strainsof R and S forms of Lactobacillus acidophilus and Lactobacillus bulgaricuswere studied according to the chemical methods of Pederson, Peterson,and Fred (1926). It was found that:

1. The R form of L. acidophilus produced inactive lactic acid. TheR form of L. bulgaricus produced inactive lactic acid in the first sixfractions, while the seventh yielded the dextrorotatory enantiomorph.The latter represented one-sixth of the total zinc salt.

2. The S forms of both L. acidophilus and L. bulgaricus produceddextrorotatory lactic acid.

89

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

3. It is suggested that some of the inconsistencies reported in theliterature might be due to the use of cultures containing various com-binations of R, S, and intermediate forms.

A21. The Gas-Producing Species of the Genus Lactobacillus. CARL S.PEDERSON, New York State Agricultural Experiment Station,Geneva.

The 19 gas-producing species of the genus Lactobacillus listed in thepresent (Fourth) edition of Bergey's Manual bear the same relationshipto the non-gas-producing species of the genus Lactobacillus as does thegenus Leuconostoc to the genus Streptococcus. Strains of only tenof these are available at present although isolations of strains similarto others have been made. Two additional species have been describedrecently. This group of 19 species is comparable in a general way tothe two species included by Orla-Jensen in the genus Betabacterium.A study of the above mentioned cultures as well as several hundred

additional cultures representing the various species have shown somedifferences between individual strains, but only a more or less naturalvariation when the entire group is considered. It is difficult to findany one character which can be used to divide the group into differentspecies. There is little justification for the large number of specieslisted in the group at present. It is doubtful whether the entire groupshould be divided into more than four or five types which may beregarded as species.

A22. Surface Microflora of Limburger Cheese. C. D. KELLY, NewYork Agricultural Experiment Station, Geneva.

The typical limburger ripening is carried out largely by the enzymesof microorganisms growing on the surface of the cheese. In a studypreliminary to an investigation of these organisms smears were made ofthe cheese in 14 New York State cheese factories by pressing micro-scopic slides on the outside of the cheese. These smears revealed themicrobiological changes taking place from day to day.

For the first 24 hours the cheeses were found to have few organismspresent and these were mostly budding yeasts, cocci and rods in aboutequal numbers. From the second or third day the budding yeastswere found to increase rapidly until they were present in large massesat four to five days. After the sixth day short slender rods appearedgrowing with the yeasts. These bacteria increased rapidly until atabout eight days they were found in masses covering the cheese. From

90

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

ten to 18 days the yeasts decreased and on the older cheeses wereseldom observed. A preliminary examination of isolated cultures of theslender rods indicated that this organism is Bacterium linens Weigmann.

Since these two types of microorganisms were found to the exclusionof all others on the better cheeses, it may be supposed that they areresponsible for the surface ripening of this type of cheese.

Oospora lactis was found on some of the cheeses and where thisorganism was present in large numbers the surface of the cheese had awrinkled appearance.

A28. Factors Affecting the Activity of Swiss Cheese Starter Cultures.PAUL R. ELLIKER AND WILLIAM C. FRAZIER, Department ofAgricultural Bacteriology, University of Wisconsin, Madison.

To determine the combined effect of the age of sterile reconstitutedskim milk used and of incubation time and temperature of Lactobacillushelveticus (39aW), cultures were carried at both 370C. and 400C. infreshly prepared milk as well as in milk which had been allowed tostand at room temperature for seven days before use. The cultureswere transferred daily and incubated for 12, 14 and 16 hours, respec-tively, after which they were kept in the icebox until the next transfer.The object of this procedure was to obtain cultures at 370C. which werejust as mature from the standpoint of acid produced as certain of thecultures grown at 400C.When the fresh medium was used, the activity of all of the cultures

during a number of successive transfers steadily increased up to a cer-tain point. Moreover their activity was decidedly greater than whenthey were grown in old milk. Steaming just before use apparently didnot make old milk as favorable a medium as freshly autoclaved milk.To judge the activity of a culture, not only was rate of acid develop-ment determined, but the culture was also inoculated into a new lotof milk, heated in this milk at 600C. for 30 minutes, then incubated ata temperature just below the maximum for the organism and tested forrate and amount of acid development and growth. When grown infresh milk, cultures which had been carried at 370C. were more activeafter heat-shocking than those which had been grown at 400C. Onthe other hand when the older and apparently poorer medium wasused, cultures which had been grown at 400C. were more active follow-ing the heat treatment than those at 370C.

These preliminary results indicate that a culture should reach acertain stage of maturity within a definite time if it is to be active

91

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

following the heat treatment. In a favorable medium this maturitymay be obtained by carrying the culture at a temperature of 370C.,but in a poorer medium a slightly higher temperature may be pref-erable.

A24. A Bio-Physical Study of Oospora lactis. J. R. KuRTZ, L. B.SCHWEIGER AND E. H. PARFirr, Purdue University, Lafayette,Indiana.

Morphological and physical characteristics of varieties of Oosporalactis isolated from butter have been studied from single cell isolations.Differences in the varieties isolated have been found in microscopicappearance. The hydrogen ion concentration of the substrate withinthe pH limits of 6 to 3 influences the rate of growth as measured by thedry weight of the mycelium and spore count. Distinct differences asto optimum growth temperature in the varieties isolated were found asmeasured by spore count. Several varieties were found to have higherthermal death points than the typical Oospora lactis.

A25. The Action of Air under Pressure in the Oxidation of Acetylmethyl-carbinol to Diacetyl in Butter Cultures. C. R. BREWER, M.B. MICHAELIAN, C. H. WERKMAN,- AND B. W. HAMMER,Sections of Bacteriology and Dairy Industry, Iowa AgriculturalExperiment Station, Ames.

Previous work by Michaelian and Hammer (Ia. Ex. Sta. Bul. 205,1936) has shown that the diacetyl content of skim milk cultures of thecitric acid fermenting streptococci can be regularly increased by bub-bling oxygen through the freshly acidified cultures.

In the present work, similar increases up to several hundred per centin the diacetyl content of both pure cultures of citric acid fermentingstreptococci and butter cultures were obtained by bubbling air throughthe cultures under pressure. Slight increases in the acetylmethyl-carbinol content of the cultures were also found. Pressures up to 60pounds per square inch were used.

It was found that the effect of pressure alone (without aeration) onthe oxidation of acetyimethylcarbinol to diacetyl was negligible. Cul-tures saturated with oxygen failed to yield increases in diacetyl contentover controls held at atmospheric pressure.

Experimental churnings of cream to which the cultures aerated underpressure were added, showed a consistent improvement in flavor andaroma over controls grown without aeration at atmospheric pressure.

92

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

A26. Reaction of Escherichia-Aerobacter from Milk on Eijkman Medium.M. T. BARTRAM AND L. A. BLACK, Department of Bacteriology,University of Maryland, College Park.

Four hundred and fifty-four strains of coli-aerogenes organisms wereinoculated from 24-hour agar slants into Perry and Hajna's modifica-tion of Eijkman medium and incubated at 460C. (temperature of themedia). The cultures had been isolated from milk three months totwo years previous to the testing and included five species of Aerobac-ter, five species of Citrobacter and 14 species of Escherichia.

Of the 91 strains of Aerobacter, 87 or 95.5 per cent were negative (ab-sence of gas within 48 hours), 127 or 98.5 per cent of the 129 inter-mediate strains were negative and 15 or 6.7 per cent of the 224 Es-cherichia strains were negative.

Three of the 45 strains of Aerobacter hibernicum and one of 24 strainsof Aerobacter cloacae produced gas. One of 87 Escherichia communior,one of 23 Escherichia paragruenthali, one of 16 Escherichia formica, sixof 15 Escherichia enterica, one of 13 Eacherichia gruenthali, two of fourEscherichia pseudocoloides, one of two Escherichia neapolitana, and thesingle strains of Escherichia anaerogenes and Escherichia leporus failedto produce gas in 48 hours.None of the strains of Aerobacter aerogenes, Aerobacter oxytocum or

Aerobacter levans were positive while gas was formed by all strains ofEscherichia coli, Escherichia vesculiformans, Escherichia anindolica andEscherichia pseudocoscorba.

A27. Productivity of Media Used in the Isolation of Escherichia-Aero-bacter from Milk. M. T. BARTRAM AND L. A. BLACK, Depart-ment of Bacteriology, University of Maryland, College Park.

The method described by Butterfield and Hoskins for determiningthe comparative productivity of media for coli-aerogenes was modifiedby using 10 instead of 15 tubes of each medium and by planting twodilutions instead of three. With solid media two dilutions, selected toyield 30 to 300 colonies per plate, were plated into each of the trialmedia in triplicate. In order to simulate conditions in previous ex-periments in which milk was used as the inoculum, one cubic centi-meter of sterile whole milk was also added to each tube or plate.The media employed were made so that the addition of the milk

reduced the concentrations to the usual value. Two strains each ofEscherichia, Aerobacter and intermediates recently isolated from milkwere used. The dilutions were so made that one cubic centimeter

93

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

quantities were inoculated in each case and in all instances the mediawere inoculated alternately.On the basis of the results obtained the liquid media were placed in

the following order with the most productive first: (1) methylene-bluebrom-cresol purple (2) fuchsin lactose (3) brilliant-green bile and (4)formate-ricinoleate. The Eijkman medium gave negative results inall tests. The solid media ranked as follows: (1) neutral-red bile (2)violet-red bile (3) Endo (4) brilliant-green lactose bile (5) lactose tauro-cholate (6) desoxycholate and (7) trypaflavine agar.

A28. Public Drinking Glass Sanitation in a Southern City. SETH T.WALTON, H. M. MORTON, AND MARY T. DAvIs, City HealthCenter, Charlotte, North Carolina.

In a bacteriological survey of public drinking glasses and rinsewaters made in Charlotte, N. C., 252 glasses and 117 samples of rinsewater were examined for the presence of total bacteria, hemolyzingbacteria, streptococci, organisms of the colon-aerogenes group and ofVincent's Angina. Soda fountains, cafes, and beer saloons were in-cluded in the survey.

Samples were collected at the establishment in sterile containersand transferred to the laboratory for examination. Counts in rinsewaters ranged up to millions per cubic centimeter, including thousandsof coli colonies. On glasses many thousands of bacteria per glass wererecorded with positive tests for the colon-aerogenes group. Hemolyticstreptococci and Vincent's organisms were frequently present.As a result of the survey we were enabled to get a city ordinance

passed recently requiring sterilization by chlorine, but to date no studieshave been made to determine the efficacy of this method of sterilization.

A29. Reduction of Elemental Sulfur by Some Autotrophic and Hetero-trophic Microoganisms. ROBERT L. STARKEY, Department ofSoil Microbiology, Agricultural Experiment Station, NewBrunswick, N. J.

The sulfur material precipitated by the bacterium Thiobacillusthioparus during its oxidation of thiosulfate, has been examined for thepresence of sulfide, since it has been claimed that such material is apolysulfide. The results of gravimetric, volumetric, and calorimetricdeterminations were negative. Likewise no sulfide was found in cul-tures of another sulfur bacterium, Thiobacillus thiooxidans. Duringthe growth of these two bacteria in inorganic media, sulfide was evolved

94

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

in small amounts as indicated by darkening of lead acetate test paperssuspended over the culture solutions. In similar media containing ele-mental sulfur, sulfide was evolved by various heterotrophic organismsincluding bacteria, actinomycetes, and filamentous fungi. The resultsindicate that the sulfide formed by the autotrophic bacteria is a productof hydrogenation of elemental sulfur. This reaction points to the pres-ence of active -SH groups in the sulfur bacteria and appears to havethe same significance as hydrogenation of sulfur by heterotrophic micro-organisms and other living cells. It is considered unlikely that ele-mental sulfur undergoes hydrogenation preceding its entrance into thecells of Thiobacillus thiooxidans which oxidize the sulfur to sulfate.

ASO. Cellulose Decomposition by a Bacterial Culture from the IntestinalTract of Termites. P. A. TETRAULT AND W. L. WEIs, PurdueUniversity, Indiana.

Cellulose-decomposing bacteria were obtained from the gut of thecommon termite, Riticulitermes flavipes. The culture was enriched inKhouvine's nitrate cellulose medium to which 20 per cent yeast waterwas added. Isolations were made on this same medium without car-bonate but with 0.8 per cent agar added and the solidified agar wascovered with sterile vaseline. Colonies were picked into the enrich-ment medium and incubated at 370C.

This purified culture contained two distinct morphological types:one, a long slender rod, occurring singly and in pairs measuring three-tenths microns by six to eight microns; the other was a large thickrod, in chains of three to six. It measured eight-tenths micron by twoto four microns.Two per cent cellulose was digested in from six to eight days. The

products were acetic, lactic and butyric acids, in the order of greatestyield. Ethyl alcohol was produced in very minor quantities. A traceof some reducing substance was always present. The gas was mostlycarbon dioxide.

A31. Bacteria and the Nitrogen Metabolism of Termites. ROBERT A,GREENE AND EDWARD L. BREAZEALE, Arizona State Labora-tory, Tucson.

Bacteria were isolated from an unidentified species of Kalotermes,which, when inoculated into a nitrogen-free mannitol solution, fixedquantities of nitrogen varying from 0.4 to 1.3 mgm. The conditionsin the intestinal tract of termites, because of the wide C:N ratio should

95

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

favor the fixation of gaseous nitrogen. Since the nitrogen requirementsof termites are low and the average nitrogen content of the termitesstudied was 0.862 mgm., it seems probable that microorganisms mayplay an important rOle in the nitrogen metabolism of termites.

A32. Digestion of Corn-Cobs by Bacteria. P. A. TETRAULT AND J.HURWITZ, Purdue University, Indiana.

The bacterial culture used in this work was obtained originally fromthe gut of a termite. It was enriched in Khouvine's nitrate cellulosemedia to which 20 per cent yeast water was added. Purification wasaccomplished by (1) the dilution method, (2) pasteurization and (3)growing at 450C. The resulting culture was very active and coulddigest corn-cobs.

Best results were obtained when the cobs were immersed in waterand autoclaved at 15 pounds for 45 minutes. The water was filteredoff and the cobs were dried at 1050 for 48 hours. These treated cobswere incorporated into the medium and inoculated with an active cul-ture. The fermentation at 370 was complete in six to eight days. Theproducts acetic, butyric and lactic acids were recovered. Ethyl alcoholwas not found.

ASS. Effect and Efficiency of Germicides and Fumigants on Microdrgan-isms Associated with the Baking Industry. GERALD K. ASHBY,C. C. HEDGES AND E. H. GIBBONS, Departments of Chemistryand Biology, Agricultural and Mechanical College of Texas,College Station.

In a study to determine the effect of germicides and fumigants onbacteria that produce "rope" in bread, five species of the genus Bacillu8,proven to produce "rope" in bread, were used. Spores of these organ-isms, suspended in veal infusion, wheat flour suspension, and in saline,were exposed to: hypochlorites; formaldehyde; acetic acid; and phenol.Spores of three of these species were also exposed to hypochlorite sprayand to formaldehyde gas.

Five per cent phenol was found to be ineffective. In disinfectantsolutions containing wheat flour or saline, hypochlorites were veryeffective while formaldehyde and acetic acid were not. Not only hypo-chlorites, but also the other disinfectants were ineffective in solutionscontaining veal infusion.As fumigants, the hypochlorites seemed to be ineffective while form-

aldehyde, if used in strong enough concentrations, was very effective.

96

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

For a study of the protective action of wheat flour and of fat infumigation, contaminated specimens of wood, web belting, and of metalwere heavily coated with wheat flour and with melted shortening. Noprotective action of the wheat flour could be demonstrated, but short-ening afforded the test organisms considerable protection against thefumigants.From a comparison of the relative strengths of the germicides as

used in solution and as fumigants, it appears that hypochlorites maybe effective in spray form if strong enough solutions can be used with-out causing corrosion of the materials sprayed.

A84. The Saprophytic Digestion of Heat-Sterilized Animal Hair andKeratose. L. S. STUART, Bureau of Chemistry and Soils, U. S.Department of Agriculture.

The most common fungi that developed on the hair of salted heavyhides were Aspergillus glaucus and Chaetomium globosum. Studies weremade to determine the ability of these organisms to digest heat-sterilizedanimal hair and keratose.

Chaetomium globosum was found to utilize heat-sterilized animalhair as a sole source of carbon and nitrogen, and to bring about a re-duction of the sulphur groups of keratose simultaneously with anincrease in free COOH groups.

Aspergillus glaucus did not utilize heat-sterilized animal hair as asole source of carbon and nitrogen, but did attack hair after it had beenacted upon by bacteria or in the presence of glucose.

Results suggest that the digestion of animal hair keratin by micro-organisms depends upon their ability to reduce the disulfide linkage.

AS5. Materials Manufactured by Micro6rganisms. J. R. SANBORN,Research Division, International Paper Company, Glens Falls,New York.

Synthesis by microorganisms of industrially useful gummy materialsoffers potential commercial possibilities not yet evaluated. Varioustypes of manufactured products such as semi-transparent sheets, ad-hesives, plastics, possible food ingredients and feedstuffs, add to theindustrial significance of these processes.Two groups of economically useful gum-formers are under investiga-

tion. The first, represented by Oicium and Mucor, grows abundantlyin potato or cereal mashes containing glucose; other types, such asTrichoderma lignorum, appear to grow best in mineral solutions con-taining sucrose.

JOURNAL OF BACTERIOLOGY, VOL. 33, No. I

97

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

A preferred species of Oidium grows in rank profusion in starchymedia containing glucose, mannose, glycerol, or ethyl alcohol. Addi-tional sources of nitrogen such as ammonium nitrate, peptone, or gela-tin, do not cause appreciable increases in yields of Oidium gum. Whilemineral solutions containing utilizable carbohydrates are generallyunsatisfactory for commercial cultivation of Oidium, potato decoction(Fritz) provides an excellent basis for large scale production. Slopsfrom starch manufacture present significant though somewhat lessfavorable possibilities.

Growths allowed to develop undisturbed produce larger total yieldsof gum than those obtained by removal of successive crops.

AMW. The Action of Microfganisms on Fatb. L. B. JENSEN, Swift andCompany, Chicago, Ill.

A study is being made of: (a) Culture methods to demonstrate lipo-clastic actions of microorgansm, (b) kinds of microbial actions on fats,(c) diffusion of chromogenic substances of microorganisms into fats,(d) action of bacterial lipases and oxidases on fats stored at 320F. forone year, and (e) action of microorganism on pure fats.

Observations, of growth on Eijkman's medium, Lieske's emulsionagar, Turner's cottonseed oil-Nile blue agar, cocoanut oil agar,palmoil emulsion agar, and other media of this type point out lipoclasticactions in some instances. If mineral oil is substituted for digestibleoils in these emulsion agars to which fatty acid staining dyes are added,colonies are often observed similar to those considered lipoclastic onvegetable or animal fat emulsion agars. This phenomenon appears tobe due to bacteria concentrated in the interfacial trap in the oil-wateremulsion agar and affecting dye.The data, then, cannot be obtained from emulsion agar media,

but must be obtained by inoculating washed microbes into commer-cial fat. After incubation, inoculated fats are tested: (1) By anaccelerated-rancidity test for critical peroxide value, (2) for free fattyacid, (3) by aldehyde and ketone tests, (4) by the Kreis test, and (5)by organoleptic test. These tests show that bacteria may induce:(a) Oxidative rancidity (lipase-peroxidase formers), (b) hydrolysis withhigh free fatty acid (lipase formers), (c) tallowiness (oxidizers) in beefand mutton fats, and (d) flavor reversion and production of flavoradjuvants (organoleptic).

Fat-soluble pigments of various microdrganims cause "pink" fatsand purple "stamping ink" discoloration by oxidation-reductionmechanisms.

980

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

Killing fats, cutting fats, sweet pickle fats, and mixed killing andcutting fats, inoculated with oxidase-lipase bacteria, after storage forone year at 32TF., show peroxide values in milliequivalents per kilogramof 1.4 to 2.4 (controls) to 12 to 50 (rancid, inoculated samples). Purefats, as free as possible from moisture, apparently do not support anytype of bacterial, mold, or yeast growth.

A37. Observations on Methods for the Study of Lipolysis by Microorgan-isms. H. F. LONG AND B. W. HAMMER, Iowa State College,Ames.

The ability of a microorganism to hydrolyze fat is an important bio-chemical character and one that is of value in the identification ofspecies.

Various investigators have suggested procedures which differ intheir usefulness for detecting lipolysis by organisms. The addition ofNile blue sulfate and dispersed fat to a medium is a simple procedureand gives a clear cut reaction, but the inhibitory effect of the dye isa disadvantage in some instances. However, because of the inhibitionof many non-lipolytic organisms, especially cocci, the Nile blue sulfatetechnic facilitates the enumeration and isolation of lipolytic specieswhen they are present in small numbers as compared to the totalnumbers. When a medium containing dispersed fat is flooded withNile blue sulfate after incubation, the results are easily read and totalas well as lipolytic counts can be obtained; but the picking of lipolyticcolonies is complicated by the washing of organisms over the surfaceof the medium. The lower simple triglycerides (e.g., tripropionin ortributyrin) are more easily hydrolyzed than natural fats; thereforethe results obtained with them may be misleading. The natural-fattechnic, in which lipolysis is detected by a change in the opacity of thefat, is valuable, because in addition to permitting total and lipolyticcounts on the plates the lipolytic organisms are easily isolated.

A38. Reproduction of Yeast Cells in Various Nutrient Solutions. (Withmotion pictures.) CHARLES N. FREY, The Fleischmann Lab-oratories, New York City.

This film shows the reproduction of yeast under normal conditions.Growth and general characteristics under certain abnormal conditionsare also shown. The lack of proper nitrogenous material, deficienciesin phosphorus, magnesium, and potassium were investigated. Theeffect of a number of factors influencing growth and reproduction ofyeast has been studied. *

99

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

A$9. Effect of Various Factors on the Vitamin B1 Content of Yeast.P. L. PAVCEK, W. H. PETERSON, AND C. A. ELVEHJEM, De-partments of Agricultural Chemistry and Bacteriology, Uni-versity of Wisconsin, Madison.

An apparatus in which kilogram batches of yeast can be grownaseptically has been devised. About 60 such batches have been grown,with variations in the type of medium, strain of yeast, and certainconditions of growth (pH, temperature, aeration, etc.). Three types ofmedia (grain wort, molasses-salts, glucose-salts) and six types of yeast(two bakers' yeasts (Saccharomyces cerevisiae), one brewers' yeast,Saccharomyces logos, Willia anomala, and Torula galactose) were used.The yields of dry yeast, based on sugar fermented, in the wort mediumranged from 24 per cent (bakers' yeast B) to 40 per cent (bakers' yeastA); in the molasses medium from 28 per cent (Saccharomyces logos)to 43 per cent (brewers' yeast); and in the glucose-salts medium from12 per cent (Willia anomala) to 29 per cent (brewers' yeast). The effectof omitting aeration, raising the pH (6.0 instead of 4.3) and of loweringthe temperature (200 instead of 300) was studied for one of the bakers'yeasts. All of these changes decreased the yield, but lack of aerationproduced the most marked effect.The vitamin B1 content of the various batches of yeast was deter-

mined by biological assay with one-day-old chicks as test animals.Bakers' yeast contained the largest amount of vitamin when grown onwort medium, approximately 10 I. U. (International Units) per gram ofdry yeast; on molasses-salts the figure was 5 I. U., and on syntheticmedium the vitamin content decreased to 3 I. U. (Commercial bakers'yeast assayed 5 to 13 I. U.). When brewers' yeast, which ordinarilycontains about 50 I. U., was propagated on the above three media, thevitamin content became about the same as that for bakers' yeast on thecorresponding media. Other strains of yeast also assayed about thesame as bakers' yeast.From these data it appears that composition of the medium is the

most important factor in determining the vitamin B1 content of yeast.

A40. Bacterial Dissimilation. C. H. WERKMAN, R. W. STONE ANDH. G. WOOD, Department of Bacteriology, Iowa State College,Ames.

Bacterial dissimilation of glucose is characterized by phosphorylationin the initial phase. The evidence supports phosphorylation by theliving cell. This assimilatory stage in the initial phase of the general

100

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS1

phenomenon of dissimilation has been shown with: Clostridium, Aero-bacter, Citrobacter, Escherichia, Lactobacillus, Propionibacterium, Bacil-lus and Serratia, among others. The hexose phosphate is probablyan equilibrium mixture of the mono- and di-esters. Additional evi-dence is needed.

Re-evaluation of the r6le of methylglyoxal, generally accepted as thekey intermediary in bacterial metabolism is now necessitated by ourisolation of phosphoglyceric acid from the dissimilation of glucoseand hexose diphosphate by a wide variety of microorganisms: (fromhexose diphosphate + glucose) Propionibacterium shermanii, Pro-pionibacterium arabinosum, Propionibacterium pentosaceum, Lacto-bacillus pentoaceticus (heterofermentative), Lactobacillus plantarum(homofermentative), Escherichia coli, Citrobacter freundii, Aerobacteraerogenes, Bacillus subtilis, and Serratia marcescens.

Phosphoglyceric acid was isolated in the presence or absence of addedacetaldehyde, when pyruvic acid replaced acetaldehyde as a hydrogenacceptor, or small yields occurred in the absence of toluene.

Phosphoglyceric is transformed into pyruvic + phosphoric acid.Conversion is slow and details of the intricate mechanism await explana-tion; the behavior of adenylic acid appears to be important. Pyruvicacid has been isolated as an intermediary with Propionibacterium,Escherichia, Aerobacter, Lactobacillus, Clostridium and Citrobacter.The final phases of dissimilation involve delicately balanced oxida-

tion-reduction equilibria or easily diverted hydrogen transferences.

A41. Phosphorylation and First Stages in Glucose Breakdown by Pro-pionic Acid Bacteria. R. W. STONE, H. G. WOOD AND C. H.WERKMAN. Department of Bacteriology, Iowa State College,Ames.

Suspensions of propionic acid bacteria cause phosphorylation ofglucose in the presence of the dephosphorylation-inhibiting substanceNaF. This takes place either with or without the presence of toluene.The phosphate ester, phosphoglyceric acid, has been isolated from suchfermentation mixtures. A small amount of this ester can be isolatedwithout any NaF present, but toluene or some other poison is necessary.The amount of phosphate uptake in the presence of such poisons asNaF is many times greater than the equivalent of phosphoglyceric acidobtained. This suggests that most of the phosphate is retained in oneor more hexose or triose esters, presumably precursors of phospho-glyceric acid. The roles of phosphorylation, methylglyoxal, and phos-phoglyceric acid in the propionic fermentation are discussed.

101

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

A42. The Final Oxidation-Reduction Phases of the Propionic Dissimilartion. H. G. WOOD, R. W. STONE AND C. H. WERKMAN,Department of Bacteriology, Iowa State College, Ames.

Pyruvic, lactic, acetic and succinic acids are proposed as inter-mediates in the dissimilation of glucose by propionic acid bacteria.All have been isolated by special fixation methods and are utilized bythe organisms. A scheme of dissimilation is presented involving aconversion of the hexosephosphate into phosphoglyceric acid which inturn yields pyruvic acid. Pyruvic acid is oxidized to acetic acid andC02 and reduced to propionic acid through lactic. Succinic acid isprobably formed from acetic acid and in turn is dissimilated to pro-pionic acid and C00. The assimilation of C02 to a fermentable un-known compound has been established in glycerol dissimilation. Ther6le of methylglyoxal is discussed.

A43. Mechanism of the Formation of Isopropyl Alcohol by Clostridiumbutylicum. A. F. LANGLYKKE AND E. B. FRED, Departmentsof Agricultural Bacteriology and Chemistry, University ofWisconsin.

In the fermentation of glucose by a strain of Clostridium butylicumacetone accumulated before the production of isopropyl alcohol wasnoted. This suggests that isopropyl alcohol is formed through thereduction of acetone. When added to the fermentation, acetone wasreduced to isopropyl alcohol.The addition of pyruvic acid to the fermentation did not result in

an accumulation of the corresponding reduction product, lactic acid,but gave products normal to the fermentation. Acetylmethylcarbinolwas quantitatively reduced to 2,3-butylene glycol, and acetaldehydewas reduced to ethyl alcohol.The addition of compounds which serve as hydrogen acceptors

always caused increased synthesis of isopropyl alcohol and acetone fromthe carbohydrate and decreased formation of butyl alcohol and butyricacid. The explanation proposed is that the utilization of hydrogen bythe foreign compounds causes a decrease in the hydrogen available forthe usual reductive processes. Consequently, those compounds, iso-propyl alcohol and acetone, which arise through decarboxylation re-actions are produced in greater proportion than those, butyl alcoholand butyric acid, which require hydrogenation reactions.

A44. The Fermentation Products of Clostridium thermosaccharolyticum.N. 0. SJOLANDER, ELIZABETH MCCOY AND L. S. MCCLUNG,University of Wisconsin and University of California.

102

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

Clostridium thermosaccharolyticum has been described by McClungas an anaerobic thermophile, which produces acid and gas from a largenumber of carbohydrates. More definite information on the productsof fermentation is desirable, because of the importance of this organismin spoilage of canned food.

Various basal media were tried and maximum utilization of glucosewas obtained in 2 per cent malt sprouts and in 1 per cent tryptone withasbestos, both containing an excess of calcium carbonate. In thesemedia, glucose in concentration of 2 per cent could be completelyutilized.The products of the dissimilation of glucose were carbon dioxide,

hydrogen, acetic acid, butyric acid, and lactic acid. The lactic acidwas identified by preparation of the para-phenyl-phenacyl ester and thezinc salt, which had three molecules of water of crystallization cor-responding to that of inactive zinc lactate. In a typical experimentin which all products were determined, 100 millimols of glucose yielded238 millimols of hydrogen, 175 millimols of carbon dioxide, 60 millimolsof butyric acid, 48 millimols of acetic acid, and 26 millimols of lacticacid. In this fermentation the weight balance was 103.4 per cent; thecarbon balance was 98.0 per cent; and the oxidation-reduction balancewas 1.008. This ratio of products is apparently influenced by condi-tions of temperature and oxygen tension. On xylose this organismformed the same products in somewhat different proportions.

A45. Some Characteristics of an Organism Causing Spoilage in FortifiedSweet Wines. H. C. DOUGLAS AND L. S. MCCLUNG, Universityof California, Berkeley.

In 1933 a peculiar type of spoilage of sweet wines, not previouslyseen in California wines, was observed by W. V. Cruess. The spoilage,which has been serious, has been found most frequently in muscatel,sherry, and angelica wines, and occasionally in port, Tokay and Malaga.It is characterized by an extensive flocculent albumin-like sediment.Microscopic examination of such material has revealed long intertwinedfilaments of the uniform width of about one micron. These filamentsappeared segmented but no branching has been observed. The major-ity of the filaments are Gram-negative, although Gram-positive segmentshave been observed. Spores have not been demonstrated. Attemptsto cultivate the organism in all of the usual laboratory media withvarious conditions of incubation have met with complete failure.Growth has resulted only with the use of sweet wine diluted with an

103

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

THIRTY-EIGHTH ANNUAL MEETING

equal volume of water and sterilized by steaming. Dilution tubes ofagar of this medium have revealed that anaerobic conditions are es-sential for growth. The optimum pH range in the liquid medium hasbeen found to be 4.1 to 4.3 and the optimum temperature 20 to 250C.Chemical analyses have shown that on prolonged incubation the vola-tile and fixed acids of the wine are increased and the reducing sugarsare destroyed. Slight gas formation has been noted in some instancesand the pH of the wine has decreased. Although the majority of thereports of this spoilage has been in bottled wine positive cultures havebeen obtained from storage vats indicating that in some instances thewine is contaminated in the winery before bottling. Investigation ofcontrol measures has shown that either pasteurization or sulfur dioxidemay be successfully applied.

Additional physiological characteristics of this organism are beingstudied in an effort to determine the systematic position and the rela-tionship to previous types responsible for spoilage of wine.

A46. Proteolysi8 by Mold Enzymes. JULIUS BERGER, M. J. JOHNSONAND W. H. PETERSON, Departments of Agricultural Chemistryand Agricultural Bacteriology, University of Wisconsin,Madison.

The extent of proteolysis attained by enzyme mixtures from animalsources has been shown by other workers to be about 85 to 90 per centof the amount possible by acid hydrolysis. A preliminary experimenthad shown that gelatin was hydrolyzed by mold enzymes only to theextent of 60 to 70 per cent. In order to determine whether this wasdue to the nature of the protein alone or to the absence of certain en-zymes in the proteolytic enzyme complex of molds, other proteins werehydrolyzed by mold enzyme preparations. Lactalbumin, egg albumin,edestin, casein, gliadin and zein.were hydrolyzed by a dialyzed enzymepreparation from AspergiUus parasiticus to the extent of approximately85 per cent after 20 to 30 days' incubation.

Gelatin solutions ranging in concentration from 0.5 to 16 per centwere subjected to the action of Aspergillus alliaceus enzyme. It wasfound that the lower concentrations of protein were hydrolyzed morerapidly and to a greater extent than the higher concentrations.

It was also found that when Aspergillu alliaceus gelatin hydrolysateswere further incubated after addition of hog erepsin or Aspergillus para-sitic=s enzyme, only very slight further hydrolysis took place.

104

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SOCIETY OF AMERICAN BACTERIOLOGISTS

A47. The Variability of Crop Weight Determinations in Liquid MoldCultures. KARL J. DEMETER AND MAx LOEWENECK, Bac-teriology Department, South German Research Institute forDairying, Weihenstephan-Munich. (Read by title.)

It has been shown by us and by others that the growth of parallelcultures of molds (penicillia) in liquid media is very unequal. Thisis true, if in all culture flasks the conditions for growth are strictly thesame, including the amount of spores used for inoculation.

Experiments were conducted by culturing penicillin in 16-17parallel culture flasks simultaneously, under the same conditions, andanalyzing the crop weight of one set after a certain period of days andof the second set one day later.The differences found in the crop weight of the parallel cultures were

considerable, e.g., (values in milligrams): Crop weights of Penicilliumcandidum from seven day and eight day cultures showed mean devia-tions of :1:86 and :1:56, respectively, and coefficients of variation of 79and 25, respectively. Crop weights of Penicillium camemberti fromsix day and seven day cultures showed mean deviations of :4:54 and420, respectively, and coefficients of variation of 38 and 7, respectively.Since many crop weight determinations by other workers with liquid

media have been based usually on not more than one or two parallelcultures, it is clear that such values are accidental and not fit for com-parative investigations on the physiology of the nutrition of fungi. Itis interesting to note how decidedly the results improve, if the culturesare only one day older.

That the results could be influenced readily was shown by otherexperiments in which the cultures had been lightly shaken once on thefirst and once on the fourth day after inoculation. The crop weightswere lowered to one-third to two-thirds of the values for the unshakencultures.To surmount these difficulties experiments were started with solid

media, which were, however, not so solid as to check loosening of themycelia. A synthetic one per cent agar medium proved satisfactory.Using a certain technique it was easy to get off the mycelium quanti-tatively and free it from the adhering agar particles. The first setof experiments with a mean crop weight of 256 mgm. resulted in amean deviation of only 10 mgm. with a coefficient of variation ofonly 4.

105

on March 20, 2020 by guest

http://jb.asm.org/

Dow

nloaded from