C. H. SPOTHOLZ, J. H. LITCHFIELD, AND Z. J. ORDAL

Lactic acid fermentation rate; Effect of continuouslycontrolled pH. Ind. Eng. Chem., 42, 1857-1861.

FINN, R. K. AND WILSON, R. E. 1954 Population dynamicsat a continuous propagator for microorganisms. J. Agr.Food Chem., 2, 66-69.

KEMPE, L. L., HALVORSON, H. O., AND PIRET, E. L. 1950Effect of continuously controlled pH on lactic acid fermen-tation. Ind. Eng. Chem., 42, 1852-1857.

LAKATA, G. D. 1954 Apparatus for controlled submergedfermentations. Appl. Microbiol., 2, 2-4.

MAXON, W. D. AND JOHNSON, M. J. 1953 Aeration studies onpropagation of baker's yeast. Ind. Eng. Chem., 45,2554-2560.

McKEE, M. T. 1955 Bacterial culture with controlled pH.Appl. Microbiol., 3, 355-360.

NEISH, A. C. AND BLACKWOOD, A. C. 1951 Dissimilation ofglucose by yeast at poised hydrogen ion concentrations.Can. J. Research, 29, 123-131.

NELSON, N. 1944 A photometric adaptation of the Somogyimethod for the determination of glucose. J. Biol. Chem.,1953, 375-380.

OLSON, B. H. AND JOHNSON, M. J. 1949 Factors producinghigh yeast yields in synthetic media. J. Bacteriol., 57,235-246.

PAN, S. C., ANDREASEN, A. A., AND KOLACHOV, P. 1949

Factors influencing fat synthesis by Rhodotorula gracilis.Arch. Biochem., 23, 419-433.

ROE, J. H. 1955 The determination of sugar in blood andspinal fluid with anthrone reagent. J. Biol. Chem., 212,335-343.

SOMOGYI, M. 1945 A new reagent for the determination ofsuigars. J. Biol. Chem., 160, 61-68.

STEINBERG, M. P. AND ORDAL, Z. 1954a Effect of fermenta-tion variables on rate of fat formation by Rhodotorulagracilis. J. Agr. Food Chem., 2, 873-877.

STEINBERG, M. P. AND ORDAL, Z. J. 1954b Theoretical rateof fat formation by yeasts. Science, 120, 609-610.

WAKSMAN, S. A. 1949 Streptomnycin, p. 100. The Williams &Wilkins Co., Baltimore.

WHITE, J. AND MUNNS, D. J. 1951T Influence of tempera-ture on yeast growth and fermentation. J. Inst. Brewing,57, 280-284.

WHITE, J. AND MUNNS, D. J. 1951b The effect of aeration andother factors on yeast growth and fermentation. Wal-lerstein Labs. Communs., 14, 199-218.

WICKERHAM, L. J. 1946 A critical evaluation of the nitrogenassimilation tests commonly used in the classification ofyeasts. J. Bacteriol., 52, 293-301.

WICKERHAM, L. J. 1948 Carbon assimilation tests for theclassification of yeasts. J. Bacteriol., 56, 363-371.

A Study of Mixed-Spore Culture and Soil-Burial Procedures in

Determining Mildew Resistance of Vinyl-Coated Fabrics'

A. DAVID BASKIN AND ARTHUR M. KAPLAN

Chemicals & Plastics Division, U. S. Army Quartermaster Research & Development Command, Quartermaster Research & DevelopmentCenter, Natick, Massachusetts

Received for publication May 18, 1956 l

Research in these laboratories2 and by others(Abrams, 1948; Stahl and Pessen, 1953) has shownthat fungi and bacteria can deteriorate certain plasticfilms, whether the films are tested alone (unsupportedfilms) or whether they are evaluated as coatings onfabric (supported films). Films may also be deterioratedby loss of plasticizer through volatilization. However,vinyl film plasticized with equal parts of dioctylphthal-ate (DOP) and methyl acetylricinoleate (P4) showedno stiffening after autoclaving for 24 hr, or exposurefor 8 days to oxygen, or exposure for 30 days to highrelative humidity and temperature2.Our studies2 have shown that phthalate and phos-

phate plasticizers are generally resistant to fungal at-tack, with the aliphatic-substituted compounds showing

' Presented at the Twelfth General Meeting, AmericanInstitute of Biological Sciences, East Lansing, Michigan,Sept. 7-9, 1955.

2 Unpublished studies from 1945 through 1953 by L. Boor,Dorothy Beck Daoust, J. V. Harvey, M. Manowitz, and F.Meloro.

a greater degree of resistance than the aromatic.Natural oils, fatty acid derivatives, and certain long-chain dicarboxylates as plasticizers are readily attackedby microflora. Structural modification of the plasticizerlmay make it fungus-resistant, but not necessarily re-sistant to bacteria (and vice versa). This was demon-strated by the response of Aspergillus versicolor and1Pseudomonas aeruginosa to each of a series of homolo-1gous sebacates (Stahl and Pessen, 1953). The sameauthors (1954) reported that internally plasticizedpolymers resist degradation by fungi.

Visual rating of growth of microflora and changes iiflexibility and breaking strength have been used in theselaboratories as indices of deterioration of plastic filmsand coated fabrics. These observations were madefollowing exposure to soil burial or by single-specie andmixed-spore suspension culture tests. These tests forevaluation of the deterioration of plastic films andcoated fabrics on the basis of weight of coating and enduse of the fabric have been found to be unreliable.Therefore, laboratory tests are being designed which

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will determine the probable effects of several types ofexposure on the treatment, rather than the effects ofthe specific type of exposure under which the fabricmight be used.Methods for exposing coated fabrics are variable

with respect to duration of burial in soil and composi-tion of mixed-spore suspensions.

In studies to establish appropriate methods2, thereappeared to be a comparative correlation betweenresults obtained from a 21-day mixed-spore cultureexposure and a 14-day soil-burial exposure. The cri-teria used for deterioration were tensile strength, flexi-bility, and visual rating of fungus growth. However, nostatistical test of correlation was carried out.The studies reported herein were initiated in 1953

and are the result of a cooperative effort among fourindustrial laboratories and the Chemicals & PlasticsDivision, Quartermaster Research & DevelopmentCenter.The objectives of these investigations were to deter-

mine: (a) adequacy of previously used test proceduresand criteria for evaluation of mildew resistance ofcoated fabrics; (b) whether a base fabric subject to thecellulolytic activity of fungi should be treated with anappropriate fungicide even when coated with a nonsus-ceptible plasticizer (for example, DOP); and (c)whether partial substitution of DOP by a plasticizersubject to microbial deterioration (for example, P4)would require the addition of a toxicant to the basefabric, the plasticizer, or both.

EXPERIMENTAL METHODS

Materials

The coated fabrics tested were as shown in table 1.3The fabrics were coated with vinyl organosols formu-lated as shown in table 2.The cotton sheeting was coated on one side only with

three coats of each of the above formulations. The firsttwo coats were knife-coated with oven heat at 270 F.Oven heat was then raised to 350 F to flux the vinyl.The cotton duck was coated with three coats of the

above formulations. The first two coats were applied toone side of the fabric at an oven temperature of 270 F.The fabric was then reversed, and the other side wasgiven one coat with an oven temperature of 350 F toflux the vinyl.The cotton sateen was coated with five coats of each

of the above formulations on one side only. The firstfour coats were applied with the oven heat at 270 F anda fifth coat was applied with the oven temperature at350 F to flux the vinyl coating.

3Coated fabrics were prepared by Films & FilamentsSection, Chemical & Plastics Division, Quartermaster Re-search & Development Center.

TABLE 1

Untreated Cu-8-TreatedCotton Base Cotton Base Plasticizers & Toxicants

Fabric Fabric

Sheeting a) DOPSheeting b) DOP-P4Sheeting c) DOP-P4 + CU-8, PMA*

Sheeting a) DOPSheeting b) DOP-P4Sheeting c) DOP-P4 + CU-8, PMA

Duck a) DOPDuck b) DOP-P4Duck c) DOP-P4 + CU-8, PMA

Duck a) DOPDuck b) DOP-P4Duck c) DOP-P4 + CU-8, PMA

Sateen a) DOPSateen b) DOP-P4Sateen c) DOP-P4 + Cu-8, PMA

Sateen a) DOPSateen b) DOP-P4Sateen c) DOP-P4 + Cu-8, PMA

*Cu-8 (copper-8-quinolinolate) and PMA (phenyl mercuricacetate). 1.0 per cent and 0.5 per cent, respectively, based onweight of plasticizer.

TABLE 2

Material

Vinyl resin...........................DOP ................................Barium ricinoleate stabilizer.........PbSiO2 stabilizer.....................Solvent thinner......................P4..................................DOP + Cu-8 (1% Solubilized Cu-

8-quinolinolate based on weight ofDOP)..............................

Pigment .............................PMA (0.5% PMA based on weight of

plasticizer) .........................

Fornulation (Parts)

A B C

100 100 10070 35 301.5 1.5 1.51.5 1.5 1.5As required

35 35

7.0As required

0.35

Methods1. Mixed-spore culture test, 21 days: The mixed

spore suspension was comprised of spores of Penicilliumpiscarium (BPI 15.1), Penicillium lutteum (BPI 66),Aspergillus flavus (QM 4m), Aspergillus niger (USDATC-215-4247), and Trichoderma viride (BPI T-1). Thecoated fabrics, prepared as cut-strips (1 in x 6 in) withlong dimension parallel to the warp, were placed onmineral salts agar4 and inoculated with the mixed-sporesuspension. After incubation for 21 days the strips wereremoved, washed, conditioned at 73 i 2 F and 50 ± 4

4NH4NO3, 3.0 g; KH2PO4, 1.0 g; MgSO4.7H20, 0.50 g;KCl, 0.25 g; agar, 15.0 g; distilled water, 1,000 ml.

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per cent relative humidity for 24 hr and broken on theScott Tester5 or tested for stiffness in a Clark FlexibilityTester.6

2. Soil-burial, 14 days: 114 in x 10 in strips were sub-jected to soil burial in a tropical chamber for 14 days.The tropical chamber was held at 86 i 2 F and 85 i 2per cent relative humidity. Soil burial was followed bydetermination of breaking strength retained (ScottTester using the cut-strip method and conditioning thefabric strips at 68 dt 1.8 F and 50 i 2 per cent relativehumidity for 24 hr), and measure of stiffness of thefabric on the Clark Flexibility Tester.

RESULTS

The data are summarized in the scatter diagramsshown in figures 1 through 4. The figures show theaverage data reported by each laboratory for breakingstrength and stiffness of these coated fabrics. The agree-ment of the data relative to the suggested tolerancesfor these physical tests may be deduced from thesegraphs. An increase in stiffness not exceeding 30 percent, and a loss in breaking strength not in excess of15 per cent are proposed as tolerances for each of thesephysical tests. In the graphed data, these tolerancesare represented by horizontal lines drawn at 30 and85 per cent, as required. These allowances are not to beconsidered final but, rather, are subject to changeshould other tolerances be considered more character-istic and/or realistic.The results for each of the five participating labora-

tories are presented in all cases except, as in figures 3and 4, where four laboratories reported.

Soil burial and breaking strength retained (figure 1).Unprotected duck and sateen coated with vinyl plasti-cized with DOP are variable in resisting microbiologicaldeterioration. This is evidenced by the wide scatterobtained, showing that some laboratories would passwhile others would fail the same fabric relative to thetolerance established. Although all laboratories reportmore than tolerable loss in breaking strength for asimilar coating applied to sheeting, the range of averagebreaking strength is rather wide.No substantial difference is indicated for DOP + P4

in vinyl applied to the base fabrics. When the plasticizerprotected with Cu-8, and PMA is applied in vinyl coat-ing to unprotected base fabric, or when protected basefabric is coated with DOP; DOP + P4; and DOP +P4 + Cu-8, and PMA, a somewhat larger measure ofresistance to mildew is evidenced by this method of test.In these instances, the reproducibility between labora-tories, though poor, is enhanced as indicated by theslightly reduced scatter. This method, soil burial, fol-lowed by breaking-strength determination, appears to

5Scott Testers Inc. Providence, R. I.6Thwing-Albert Instrument Co. Philadelphia 44, Pa.

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FIG. 1. Scatter-diagram of per cent breaking strength re-tained after 14-day soil burial for each of 3 coated fabrics re-ported by 5 laboratories designated as 0, a,P, El, and M.

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F1G. 2. Scatter-diagram of per cent breaking strength re-tained for 3 coated fabrics after 21-day mixed spore culture byeach of 5 laboratories designated as 0, a, I, El, and *.

be poorly reproducible and hence unfeasible as a meas-ure of deterioration for these coated fabrics.

Mixed-spore culture and breaking strength retained(figure 2). Unprotected sheeting and duck coated withvinyl plasticized with DOP only were found by alllaboratories to deteriorate in excess of tolerance. Thiswas found also to be the case for sateen by all but onelaboratory. When P4 supplemented DOP as plasticizer,all laboratories reported greater than tolerable loss inbreaking strengths. The breaking strength of unpro-

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120

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FIG. 4. Scatter diagram of per cent stiffness increase after14-day soil burial of 3 coated fabrics by each of 5 laboratoriesdesignated as 0, (1, 0, El, and *.

tected sheeting coated with vinyl containing DOP +P4 + Cu-8, and PMA was reported by one laboratoryto be slightly in excess of tolerance. Similar results forboth duck and sateen were reported by one otherlaboratory.

All coatings applied to protected sheeting resisteddeterioration. One laboratory reported more than toler-able loss in breaking strength for protected duck andsateen plasticized with DOP + P4, containing Cu-8 andPMA.

Mixed-spore culture followed by breaking strengthretained as the criterion for deterioration is poorly re-producible, as evidenced by this wide scatter. There-fore, this combination of test and criterion is unaccept-able as a procedure for test.Mixed spore culture and stiffness increase (figure 3).

Data obtained from 21-day mixed-spore culture fol-lowed by flexibility measure afforded better reproduci-bility than the procedures reviewed previously. It isevident that when Cu-8 and PMA are added to plasti-cizer of vinyl applied to unprotected base fabrics, orwhen plasticizer with or without toxicants is applied incoats to Cu-8-treated base fabrics, deterioration doesnot occur. This is indicated by loss in flexibility notexceeding the tolerance. Further, the reduced scatterof the average data indicates a more consistent per-formance of these coated fabrics, as evaluated by thisprocedure. A wide scatter ranging from a tolerable to agreater-than-tolerable change in stiffness is notablewhen the mixed plasticizer (1:1 of DOP and P4) wasused for vinyl coated on unprotected sheeting, duckor sateen. Sheeting, too, coated with vinyl plasticizedwith DOP alone afforded a wider-than-desirable scatter.

Reproducibility in performance of coated fabricssubjected to mixed-spore culture' followed by deter-mination of stiffness, is better than when soil-burial ormixed-spore culture was followed by breaking-strengthdetermination.

Soil burial and stiffness increase (figure 4). Soil burialfollowed by flexibility measure afforded the best repro-ducibility for evaluation of deterioration of the coatedfabrics. Although scatter of the data for some of thecoated fabrics is inclined to be wide, the data reportedare completely consistent and reproducible within theestablished tolerance.

Therefore, it would be reasonable to assume thatthese coated fabrics (sheeting, duck, and sateen) andthe coating as applied may best be evaluated for micro-biological deterioration by using soil burial followed bystiffness measure.

DISCUSSION

Examination of the data indicates that the least vari-able results, predicated on range of scatter, were ob-tained with Clark flexibility measure following soilburial of these coated fabrics. Greater variability isevident with flexibility measure after mixed-spore cul-ture and with breaking-strength determinations aftereither soil-burial or mixed-spore culture.The particular biological conditions attending fabric

deterioration in soil, further complicated by unmeasuredintegrated physico-chemical processes, have never beenfully investigated and, therefore, are not clearly under-stood. Because of this, spore-culture techniques havebeen developed to measure deterioration with thethought that such techniques can be closely controlled

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and replicated. This implies a complete understandingof biological conditions pertaining to degradation offabric in such test procedures. An understanding of thedeteriorative processes in culture, in fact, still remainsto be achieved. Mixed-spore cultures have been recog-nized by some to be more efficacious than single culturesof fungi and these, in turn, more desirable than soilburial. The mixed-spore population was regarded to bemore representative of conditions in nature. If "repre-sentativeness" of biological material is desirable, thenthe use of a mixed-spore culture in preference to soil israther inconsistent since the soil affords the more com-plete biological spectrum for deterioration evaluation.

Further, the mixed-spore suspension was developedto test treated fabrics against a wider microfloral spec-trum. This was believed to afford a more complete eval-uation of deterioration than would be possible withsingle cultures. The culture of even a single fungus on acellulosic substrate is accompanied by an exceedinglycomplex and incompletely understood physiologic ac-tivity. The influence of several species of fungi eachupon the other in competing for this cellulosic substrateis even more complex and not necessarily additive.

In deterioration testing, variation of biological mate-rial (substrate and test organism) is axiomatic. Theuniversality of this variation permits no more than anempiric procedure and evaluation at this time. Culturetests, and particularly the mixed-spore suspension,therefore, are no less empiric than soil burial.The soil-burial procedure was found to be more re-

producible than mixed-spore culture and, when coupledwith flexibility measure, more diagnostic of deteriora-tion than when coupled with breaking-strength deter-minations. The dual nature of coated fabrics (basefabric and plastic coating) could possibly influence thecriterion to be used for evaluation. The quantitativepredominance of either the base fabric or the plasticcoating might be the determining factor for the use ofeither breaking strength or flexibility measure.

Breaking-strength determinations were more vari-able than flexibility measures following mixed-sporeculture or soil burial. It is entirely possible that smalldeviations (less than 5 per cent) from tolerance forbreaking strength or stiffness may not be significant.There still remain, however, a considerable number ofbreaking-strength determinations in rather serious dis-agreement. As all fabrics were prepared by one agency,the wide variability as evidenced by the scatter may beascribed to: (a) those aspects of soil-burial and mixed-spore culture previously discussed; (b) variations inconditions between laboratories for test and procedure;and (c) personal variations in the reading of results.Most laboratories submitted data in a form which

precluded statistical evaluation as it would apply toreproducibility. Although consideration of data in termsof scatter affords an excellent means of appreciating

TABLE 3

Coated Fabric Method of Test

Type Wt. of Thicknessbase of Procedure Criterion for evaluationfabric coating

I Light Light Soil burial FlexibilityII Light Heavy Soil burial Flexibility

III Heavy Light Soil burial Breaking strengthIV Heavy Heavy Soil burial Flexibility

trends for reproducibility, this can be regarded only asa substitute for detailed data.

Breaking strength as a measure of degradation offabrics is a basic determination, and its usefulnessshould not be discounted for coated fabrics. Comparedto flexibility as here tested, however, breaking strengthis not quite as reproducible.

Relatively light coatings on light-base fabrics, as hereinvestigated, and heavy coatings on relatively light-base fabrics may be best measured for deterioration bythe Clark flexibility measure. However, breaking-strength determination would possibly be more desir-able in evaluating degradation of light-coated heavy-base fabric combinations. If relative weights of coatingand base fabric are factors in selection of an appropriatemeasure of deterioration, the test procedures shown intable 3 might be feasible. Further investigation is indi-cated to determine whether this relationship affords anappropriate means for selection of test and procedureto determine mildew resistance of each type of coatedfabric.

CONCLUSIONS

1. Testing for mildew resistance of these coated fab-rics prepared with vinyl organosols fluxed on to the basefabric should be predicated on stiffness by the Clarkflexibility measure following soil burial for 14 days-incubation at 86 i 2 F and 85 i 2 per cent relativehumidity. More consistent results were obtained withthis procedure and measure than with any other methodor criterion investigated.

2. Coated sheeting, duck, and sateen may be pro-tected adequately against microbiological deteriorationby addition of a fungicide to the base fabric or to theplastic coating. No evidence was obtained to indicatethat both the base fabric and the coating should beprotected.

3. In view of the fact that adequate protectionagainst physical deterioration was obtained withcopper-8-quinolinolate, which is fungicidal but notbactericidal, it appears that fungi are primary degradersof plastic-coated fabrics and bacteria are of secondaryimportance. However, the methods used are total innature and would not characterize deterioration by onekind of microflora or the other.

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SUMMARY

A cooperative study was made among four industriallaboratories and these laboratories. The efficacy ofmixed-spore culture and soil burial, followed by break-ing-strength and flexibility measure, were evaluated asprocedures and criteria to determine resistance tomicrobiological deterioration of variously plasticizedvinyl-coated fabrics.Data presented indicate, for the coated fabrics

studied, that soil burial for 14 days, followed by theClark flexibility measure, afforded best reproducibilityfor measure of degradation of these materials. Sheeting,duck, and sateen base fabrics may be protected againstdeterioration when solubilized copper-8-quinolinolateand phenylmercuric acetate at 1.0 per cent and 0.5 percent, respectively (based on the weight of the plasti-cizer) are incorporated into the vinyl coat. Coatingsbased on unprotected duck and sateen evidenced nodeterioration when dioctylphthalate, alone, was usedas the plasticizer; the same coating based on sheeting,however, did deteriorate. No explanation for this ap-parent anomaly is offered.

Resistance to biological deterioration was impairedwhen methyl acetylricinoleate was mixed with di-

octylphthalate as the plasticizer. All coated fabrics con-sidered, a larger measure of resistance to the degrada-tive effects of fungi and bacteria is obtained with afungicide added to the base fabric or to the plasticizer,compared to the response of unprotected base fabriccoated with vinyl plasticized with a resistant plasti-cizer. There is no evidence that addition of fungicide tothe base fabric and the addition of bactericide andfungicide to the plasticizer is more efficacious thantreatment of either the base fabric or the plastic coating.The feasibility of testing coated fabrics with regard torelative weight of fabric and thickness of coating issuggested by these data, but must be confirmed byintensive study.

REFERENCES

ABRAMS, E. 1948 Microbiological deterioration of organicmaterials: its prevention and method of test. Nat. Bur.Standards (U. S.) Misc. Publ., 188.

STAHL, W. H. AND PESSEN. H. 1953 The microbiologicaldegradation of plasticizers. I. Growth on esters and alco-hols. Appl. Microbiol., 1, 30-35.

STAHL, W. H. AND PESSEN, H. 1954 Funginertness of inter-nally plasticized polymers. Modern Plastics., 31, 111-112.

The Coexistence of Pathogens and Pseudomonads inSoluble Oil Emulsions1

R. SAMUEL-MAHARAJAH, HILLIARD PIVNICK, W. E. ENGELHARD, AND SHEILA TEMPLETON

Department of Bacteriology, University of Nebraska, Lincoln, Nebraska

Received for publication June 1, 1956

This investigation is concerned primarily with thegrowth and virulence of pathogenic bacteria introducedinto soluble oil emulsions. The soluble oils are petroleumor fatty oils emulsified with soaps of petroleum sulfonicacids, naphthenic acids, fatty acids, rosin, or tall oil.When mixed with water, the soluble oils form stableoil-in-water emulsions and, as such, are used in machineshops as coolants and lubricants in the cutting andgrinding of metals. Unfortunately, they serve as excel-lent substrates for the growth of bacteria.The predominating microflora indigenous to soluble

oil emulsions used industrially belongs to the genusPseudomonas (Lee and Chandler, 1941; Duffett et al.,1943; Pivnick, 1952, 1955; Pivnick et al., 1956; Sabina,1956). However, members of the genera Achromobacter,

' This investigation was supported by a research grant(RG-4266) from the National Institutes of Health, U. S.Public Health Service.

Bacillus, and Vibrio, as well as yeasts and molds havealso been found (Duffett et al., 1943; Pivnick, 1952;Davis and Updegraff, 1954). In addition, coliformbacteria persist in these emulsions. For example, Pageand Bushnell (1921), Duffett et al., (1943), and Bennett(1956) isolated Escherichia coli and Aerobacter aerogenes,while Pivnick and Fabian (1954) found similar bacteriain about one-half of the emulsion samples obtainedfrom industrial sources. Pivnick and Fabian (1954) alsofound that the bacteria in fecal matter undergo rapidcell division in soluble oil emulsions.With regard to the pathogenic bacteria, those of the

genera Salmonella and Klebsiella grow readily in theseemulsions and members of the genus Shigella survivefor several days (Pivnick et al., 1954). Bennett andWheeler (1954) investigated survival but not growth ofpathogens in soluble oils which differed from those usedby Pivnick et al. They found viable Shigella paradysen-

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