APPLICATIONS OF THE PLASTIC FILM TECHNIQUE IN THEISOLATION AND STUDY OF ANAEROBIC BACTERIA

J. L. SHANK

Research Laboratories, Swift & Company, Chicago, Illinois

Received for publication 9 March 1963

ABSTRA CT

SHANK, J. L. (Swift & Co., Chicago, Ill.).Applications of the plastic film technique in theisolation and study of anaerobic bacteria. J.Bacteriol. 86:95-100. 1963.-The use of plasticfilms as oxygen barriers on the surface of agar

pour plates, in conjunction with thioglycolateand other selective and differential agents, allowsthe primary isolation and enumeration of clos-tridia and other anaerobes. Quantitative studiesreveal little if any inhibition of the test organismsunder these conditions, and toxin l)roduction,where it occurs, is shown to be virtually unim-paired.

In cultivating clostridia, an essential require-ment is the exclusion of atmospheric oxygen

from the medium. This requirement may be metin many ways. Freshly prepared infusion brothsare usually sufficiently reduced to allow thegrowth of these anaerobes. The use of gels or

agar in deep tubes or the incorporation of certainreducing chemicals in the media aids in main-taining a low redox potential. WVith fluid media,the bacteria may be counted statistically bydecimal dilution using the most probable numbermethod (Demeter, Sauer, and Mliller, 1933). Insemisolid or solid agar deeps, e.g., in oval tubes(Miller-Pickett), the colonies may be counteddirectly. These techniques, although suitable forpure culture work, cannot be relied upon forprimary isolation, since facultative anaerobes,especially streptococci, are not suppressed.Obviously, this will confuse total count deter-minations. Even the addition of chemical inhibi-tors or antibiotics to the media has yet to insurethe selection of clostridia (Mossel, 1959; Angelottiet al., 1962). The use of sodium azide for primaryisolation of anaerobic bacteria is tantamountto constructing a selective medium for strepto-cocei (Forget and Fredette, 1961). Moreover,azide has been shown to be inhibitory to certain

strains of Clostridium perfringens (Mossel et al.,1956). Apart from this, the growth of suchfacultative bacteria in clostridial cultures, partic-ularly in liquid cultures, often exerts an antag-onistic effect on the clostridia (Mossel andIngram, 1955).When clostridia are grown on the surface of

solid media or in conventional pour plates toobtain isolated colonies, the exclusion of oxygenbecomes a greater problem. The most directsolution is to inoculate the plates and incubatethem in anaerobic jars. There are many pro-cedures available to secure an anaerobic environ-ment: simple evacuation or physical displacementof oxygen with inert gases; evacuation andcombustion (Fildes and McIntosh, 1921; Evans,Carlquist, and Brewer, 1948); chemical con-sumption (Spray, 1930); biological respiration(McClung, KIlcCoy, and Fred, 1935). In general,however, these procedures require special equip-ment, and remain, as is well known, cumbersomeand time consuming. The use of anaerobicpetri dish lids in conjunction with thioglycolateagar (Brewer, 1942) allows the direct plating ofanaerobic bacteria. Although this method elimi-nates the anaerobic jar, the Brewer (1942) lidsare still relatively expensive. In use, moreover,a water film often develops on the agar surface,causing the colonies to coalesce.The use of plastic films as oxygen barriers on

the surface of agar pour plates inoculated withclostridia was first described by Kneteman(1957). An aerobic micrococcus was used toachieve anaerobiosis. This technique has severaldisadvantages. (i) It is necessary to preparefresh transfers of the micrococcus each time thetest is lerformed. (ii) The me(lium must be ableto support the growth of the micrococcus inaddition to the growth of anaerobic organisms.(iii) The incubation temperatures and pH mustbe limited to the optimum for the micrococcus.(iv) Certain selective agents to assist in theisolation of specific anaerobes (e.g., antibiotics)cannot be used.

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We have found, however, that the plasticfilm technique is not necessarily subject to suchdisadvantages. The use of thioglycolate (insteadof microorganisms) to preserve a reduced environ-ment, does not appear to exert any inhibitoryeffect. And the use of antibiotics in the mediumfurther extends the applicability of the plasticfilm technique by allowing its use for the primaryisolation and enumeration of specific anaerobesfrom foods and clinical materials.

MATERIALS AND METHODS

Medium. The basal medium was prepared asfollows: Tryptone (Difco), 15 g; yeast extract(Difco), 10 g; Sodium Thioglycollate (Difco),0.6 g; L-cystine (Matheson Co., Inc., Joliet, Ill.),0.1 g; agar, 15 g; and tap water, 965 ml. Themedium was autoclaved for 20 min (15 psi, 121 C)and cooled to 45 C. It had been reported that theincorporation of sulfadiazine to sulfite-polymyxinagar resulted in a medium that was selective forclostridia (Angelotti et al., 1962). Accordingly,these materials, along with ferrous sulfate, wereSeitz-filtered and added to the cooled basalmedium as follows: 5.0 ml of a 10% aqueoussolution of sodium sulfite (Na2SO3 .7H20); 10.0ml of a 0.06% aqueous solution of polymyxin Bsulfate (Nutritional Biochemical Corp., Cleve-land, Ohio); 10.0 ml of a 1.2% aqueous solutionof sodium sulfadiazine, USP (American CyanamidCo., New York, N.Y.); 10.0 ml of a 5% aqueoussolution of ferrous sulfate (FeSO4 .7H20) Thefinal pH was 7.1 i 0.1.

Anaerobic film. The film used in the presentreport was prepared from commercial rolledSaran (Dow Chemical Co., Midland, Mich.).The film was 1 mm thick with an oxygen trans-mission of 0.74 cc per 100 in.2 per 24 hr, as de-termined with a Dow Gas Cell using the SargeTechnique (Brown and Stauber, 1959). Circles(3-in. diameter), convenient to lay over thesurface of agar plates, were cut out and packedin petri dishes, separated from each other bypaper toweling. They were then sterilized bytreatment at atmospheric pressure with cylinderethylene oxide (Matheson Co., Joliet, Ill.) ina previously evacuated desiccator, for 24 hr atroom temperature.

Bacteria. C. novyi, C. sporogenes (Hall), C.tetani (Mueller, nontoxigenic), C. bifermentans,C. parabotulinum, C. difficile, and C. septicumwere obtained from L. S. McClung, Indiana

University, Bloomington. C. perfringens typesBPCK, Bi, B12, and B15 were originally securedfrom R. Angelotti, Robert A. Taft Sanitary En-gineering Center, Cincinnati, Ohio. Other clos-tridia used in this study were as follows: C. tetani47-S-3, from the American Type Culture Collec-tion, Washington, D.C.; C. sporogenes 3679,from C. F. Schmidt, Continental Can Co.,Chicago, Ill.; and C. botulinum A 33, from N.Grecz of the Quartermaster Food and ContainerInstitute, Chicago, Ill. The clostridia weremaintained in Brain Heart Infusion (BHI)-ground beef broth, prepared as follows. Freshground beef (0.2 g) was packed in the bottom ofscrew-cap tubes (14 X 1.5 cm). BHI (14 ml;Difco) was then added to each tube. The brothwas sterilized by autoclaving for 20 min at 121 C.Other bacteria used in this report are maintainedas stock cultures in our laboratory and wereoriginally secured from the American TypeCulture Collection. These are transferred monthlyon stock culture agar (Difco).

Toxin-antitoxin diffusion experiments. Sterileporcelain discs (4 cm in diameter) were sealedto the center of sterile petri dish bottoms. Theseal was achieved by using a sterile silicone greasefilm on the bottoms of the discs. Using the com-plete medium lacking the ferrous salt, suspensionsof C. tetani 47-S-3 and C. botulinum A 33 wereindividually poured into each plate. This resultedin four plates per organism. After solidificationof the agar, the discs were removed and uninocu-lated medium was poured into the empty wells.While the agar was still liquid, 1.2-cm antibioticdiscs, saturated with botulinum antitoxin A, B,and AB (Fort Dodge Laboratories, Inc., FortDodge, Iowa), or with tetanus antitoxin (ob-tained from Herald Cox, Lederle Laboratories,Pearl River, N.Y.), were aseptically inserted.By weighing the discs before and after saturation,it was determined that each disc adsorbed ap-proximately 125 mg of antitoxin. When the agarin the wells had solidified, anaerobic films werelaid over the surface of all plates. The plates wereincubated for 2 days at 37 C, and then held at21 C.

RESULTS

Clostridia from BHI meat deeps, incubatedfour days at 37 C, were serially diluted in steriledistilled water. Conventional pour plates wereprepared, using the complete medium described

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TABLE 1. Growth of Clostridium spp. under plastic filnz*

Anaerobic agar + film Peptone colloid + 0.1% FeSO4

Organism Growtht Degree of

Bacteria per ml Degree of blackeningt blacken-3 5 7 8 ingt

Clostridium novyi 1.95 X 107 ++ + + 0 0 +C. sporogenes Hall 84 1.9 X 109 ++++ + + + 0 +C. sporogenes 3679 9.6 X 108 ++++ + + + 0 4

C. tetani Mueller 2.8 X 108 ++++ + + + 0 +C. bifermentans 3.8 X 108 ++++ + + + 0C. parabotulinum 1.3 X 109 ++++ + + + 0 +C. difficile 3.9 X 109 + + + + 0C. septicum 6.5 X 105 + 0 0 0 -

C. perfringens BP6K 2.0 X 107 ++++ + + 0 0 iC. perfringens B6 1.3 X 109 ++++ + + + 0 +C. perfringens B12 6.8 X 108 ++++ + + + 0 +C. perfringens B15 2.8 X 108 ++++ + + 0 0 +C. botulinum A33 4.2 X 108 ++++ + + + 0 +

* Growth was for 48 hr at 37 C.t Degree of blackening (visual) was from light gray (+) to black (++++); 4 indicates a question-

able degree of blackening.t Growth was at dilutions shown (reciprocal of dilution); + = growth; 0 = no growth.

above. After the agar had solidified, an anaerobicfilm was laid over the surface of each plate andpressed out to remove air pockets. The plateswere then incubated for 48 hr at 37 C. At thesame time, Peptone Colloid tubes (Difco) wereinoculated and incubated along with the plates.All clostridia tested grew and produced easilycountable colonies within 24 to 48 hr (Table 1).All test organisms, with the exception of C.septicum, reduced sulfite to develop black colonies,owing to the formation of iron sulfide. It wasalso observed that all clostridial growth was con-fined to the center portion of the agar under thefilm. Very little growth of clostridia occurred atthe periphery of the film, and no growth at alltook place around the edges of the plates wherethere was no film covering. This pattern, alongwith H2S production (Fig. 1), would appear tobe an important feature in the general growthcharacteristics, especially of the putrefactiveclostridia, under these conditions.The addition of sulfadiazine to Mossel's (1959)

sulfite-polymyxin agar, as shown by Angelottiet al. (1962), produced a medium that inhibitsmost gram-negative bacteria. Strains of lacto-bacilli and staphylococci were also shown to beinhibited. This medium, however, was unable torestrict the growth of many facultative strepto-

cocci. These organisms, along with certain mem-bers of the genus Bacillus, when they did grow,generally did not form black colonies. Moreover,and significantly, their growth was not confined tothe most anaerobic region of the plate (beneath*i 7_ .- .....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....

FIG. 1. Growth of Clostridium sporogenes underplastic film.

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TABLE 2. Effect of plastic film on facultative and anaerobic bacteria

Organism

Bacillus cereus (graveolens)B. subtilisB. cereus

Streptococcus lactis C2S. lactis C10S. lactis ES. duransClostridium sporogenes 3679C. perfringens B15C. botulinum A33

Anaerobic agar + film

Total count(24 hr)

1.1 X 107<103<103<103<103<103

1.56 X 1088.0 X 106

1071.1 X 107

H2S

neg

negneg

4±

Distri-bution*

E

EC

C

C

* E = growth over entire plate including area withoutplate beneath film.

Total count(48 hr)

3.2 X 107<103<103<103<103

1.2 X 1081.56 X 1088.0 X 106

1071.1 X 107

H2S

neg

negneg

Distri-bution*

E

EE

C

C

C

Thioglycolate agar

Total count(48 hr)

3.0 X 1073.3 X 1078.0 X 1011.4 X 1011.36 X 1081.5 X 1081.6 X 108

<103<103<103

film; C = growth restricted only to center of

the center of the film), as were the clostridia. Onthe contrary, these facultative anaerobes, suchas B. cereus var. graveolens, Streptococcus dutrans,or S. lactis, were uniformly distributed throughoutthe agar, completely independent of the presenceor absence of the film (Table 2). The tentativecriteria for the genus Clostridium become, there-fore, (i) localized central growth beneath thefilm and (ii) development of black colonies(usually within 48 hr at 37 C). It is apparent thatthe 3-in. plastic film produces an oxidation-

FIG. 2. Toxin-antitoxin neuitralization bands un-

der film. Plate seeded with Clostridium botulinumA 33. Center disc satuirated with botulinum antitoxintype A.

TABLE 3. Toxin-antitoxin diffusion andneutralization under film*

Botulinum antitoxinOrganism Tetanusantitoxin

A B A-B

Clostridium 0 0 0 ++++tetani 47-S-3

C. botulinum ++++ ++++ ++++ 0A 33

C. sporogenes 0 ++ + 03679

C. sporogenes 0 0 0 0Hall

* Degree of visual distinctness of band is shownby + to ++++; 0 indicates no reaction.

reduction gradient. The uncovered region be-comes aerobic owing to the diffusion of air. Theperiphery of the film becomes microaerophilic.The central area of the film remains anaerobic.If the pour plate is prepared carefully, that is,to achieve maximal randomization of the cellsthroughout the agar, then the central areabecomes representative of the entire plate.With the toxin-antitoxin diffusion experi-

ments, after 4 days of incubation definite thinwhite lines of flocculation were observed in theuninoculated zone of some plates. These wereinterpreted as toxin-antitoxin neutralizationbands. Usually more than one band developed(Fig. 2). This experiment showed (Table 3)that both the A and B botulinum antitoxins re-acted with the type A organism. It also showed

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FIG. 3. Reactions of crude botulinum antitoxin type A against diffusion products of different clos tridia(a) Clostridirnt botuilinuinii A 33, (b) C. parabotulinum,, (c) C. sporogenes 3679, (d) C. sporogenes Hall,(e) C. tetani 47-S-3.

that antitoxin p)repared against type Albotulinumtoxin was able to cross-react with certain exog-enous materials produced by C. sporogenes. Onthe other hand, the tetanus antitoxin appearedto be quite specific. In other experiments, it wasdemonstrated that this type A botulinum anti-toxin reacted with diffusion products of C.parabotulinumn as well. Figure 3 shows theappearance of type A botulinum diffusion platesafter 10 davs of incubation at 21 C, where thediffusion area of the antitoxin had been de-natured by flooding the plate with 0.5% a-pro-piolactone for 10 min before photographing.No reaction occurred with C. tetani against typeA botulinum antitoxin, although multi)le bandswere observed with the other clostridia.

DIscussION

The anaerobic film functions as an effectiveoxygen barrier, thereby permitting the growthand the l)roduction of toxin by clostridia. Modi-fications of the medium, e.g., the exclusion ofantibiotics, makes this technique readily- adapta-ble to the cultivation of anaerobic bacteria in

general. The use of blood or ascitic fluid in themedium makes possible the isolation of manyslecies of Bacteroides. Since other plastic filmsare also available, with oxygen transmissionranges, for example, from 700 dowN-n to 0.7 ccper 100 in.2 per 24 hr (Shank and Lundquist,in press), this technique could be used to studymicroaerolhilic bacteria, under defined condi-tions, as well. The manufacturing of film dis-pensors for bacterial culturing remains a possi-bilitv. The ease and convenience of the filmtechnique makes it especially suitable for fieldsurveys, mobile laboratories, etc., for bothindustrial and clinical use.

In a clinical laboratory, the anaerobic filmtechnique was recently coml)ared with identicalplates incubated for an equal l)eriod of time in ananaerobic jar. (Anaerobiosis was achieved byusing acid pyrogallic and sodium carbonate.)A total of 155 individual specimens were ex-amined. In all but one case, recoverv from theanaerobic film l)lates was qualitatively andquantitatively identical with the flora recoveredfrom the l)lates incubated in the anaerobic jar.

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Included in this survey was a C. tetani that was form of McIntosh and Fildes anaerobic jar.isolated using the film but which did not grow Brit. J. Exptl. Pathol. 2:153-155.in the anaerobic jar (personal com?nunication J.' FORGET, A., AND V. FREDETTE. 1961. Sodium azideH. Silliker, St. James Hospital, Chicago Heights, selective medium for the primary isolation ofIll.), anaerobic bacteria. J. Bacteriol. 83:1217-1223.

KNETEMAN, A. 1957. A method for the cultivationof anaerobic spore forming bacteria. J. Appl.Bacteriol. 20:101-107.

ANGELOTTI, R., H. E. HALL, M. J. FOSTER, AND MCCLTJNG, L. S., E. McCoY, AND F. B. FRED. 1935.K. H. LEWIs. 1962. Quantitation of Clostrid- Studies on anaerobic bacteria. II. Furtherium perfringens in foods. Appl. Microbiol. extensive uses of the vegetable tissue anaero-10:193-199. bic system. Zentr. Bakteriol. Parasitenk. Abt.

BREWER, J. H. 1942. A new petri dish cover and II 91:225-227.technique for use in the cultivation of anaer- MOSSEL, D. A. A. 1959. Enumeration of sulfite-obes and microaerophils. Science 95:587. reducing clostridia occurring in foods. J. Sci.

BROWN, W. E., AND W. J. STAUBER. 1959. Gas Food Agr. 19:662-669.transmission by plastic films. Mod. Plastics MOSSEL, D. A. A., A. S. DE BRUIN, H. M. J.36:107, 110, 112-114, 116, 190. VAN DIEPEN, C. M. A. VENDRIG, AND G.

DEMETER, K. J., F. SAUER, AND M. MILLER. 1933. ZOUTEWELLE. 1956. The enumeration ofVergleichende Untersuchungen uber verschil- anaerobic bacteria, and of Clostridium speciesdene Methoden zur Coli-Aerogenes-Titer- in particular, in foods. J. Appl. Bacteriol. 19:bestimmung in Milch. Milchwirtsch. Forsch. 142-154.15:265-280. MOSSEL, D. A. A., AND M. INGRAM. 1955. The

EVANS, J. M., P. R. CARLQUIST, AND J. H. physiology of the microbiol spoilage of foods.BREWER. 1948. A modification of the Brewer J. Appl. Bacteriol. 18:232.anaerobic jar. Am. J. Clin. Pathol. 18:745-747. SPRAY, R. S. 1930. An improved anaerobic culture

FILDES, P., AND J. MCINTOSH. 1921. An improved dish. J. Lab. Clin. Med. 16:203-206.

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