262

A n t o n i e van L e e u w e n h o e k 37 (1971 )

Fig. I. Survival percentages in mice vaccinated with Bordetella pertussis whether or not in combination with Listeria L-form culture, and challenged I-7 days later with 300-700 LD.~o of Listeria monotTtogenes. 9 9 B. pertussis vaccine; o o B. pertussis vaccine i Listeria L-form culture.

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The question whether the second pbase of increased resistance after a B. pertussis injection reflects a developing celJ-mediated immunity cannot be answered with certainty. However, recent experiments have indicated that spleen cell suspensions from B. pertuss'is-treated donor mice, when transferred to recipients which are also given a small non-protective dose of B. pertussis vaccine, can produce a state of adoptive resistance against Listeria infection. MACKANESS, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo.--J. Exp. Med. 129: 973-992.

Comparison of selective media for the enumeration of Lactobacillus species F. C. GONZ~.LEZ 1'3, W . A. SCHEFFERS 1 a n d D. A. A. MOSSEL 2 t Laboratory o f Microbiology, University o f Technology, Delft, 2 Department c2[Bacteriology. Central Institute for Nutrition and Food Research TNO, Zeist

Lactobacilli are important agents in the spoilage of foods and beverages. They also play an essential role in the manufacture of certain fermented foods. For the enumeration of lactobacilli in food products and also in medical and dental specimens many selective media, based on various selective principles, have been described (see review by Sharpe. 1960; Sharpe and Fryer, 1965). However, not all of these media have been evaluated with respect to their suitability for the enumeration of the fastidious, heterofermentative lactobacilli, such as Lactobacillus brevis, L. buchneri, L. fi, rmenti, L. fi'uctivorans and L. pastorianus. We therefore decided to investigate the development of these bacteria in two of the most commonly used types of selective media, viz. liver infusion - sorbic acid agar (Emard and 3) Present address: Department of Microbiology, Faculty of Agriculture and Veterinary Sciences, University of Buenos Aires, Buenos Aires, Argentina.

A n t o n i e van L e e u w e n h o e k 37 (1971 )

263

Vaughn, 1952; Gasser, 1962; Vaughn, 1970) and acetate agar (Rogosa, Mitchell and Wiseman, 1951). As reference strains we used two homofermentative lactobacilli from the Delft culture collection: Lactobacillus casei and L. p]antarttm, Strains of the heterofermentative lactobacilli mentioned were obtained from Dr. M. E. Sharpe, National Institute for Research in Dairying, Reading, England. Cultures of all strains, to be subjected to comparative enumeration, were prepared by inoculation into M.R.S. broth and incubation for 24-72 hrat 30 C.We verified the identity of these cultures by determining their physiological key characteristics following Sharpe, Fryer and Smith (1966); no decisive discrepancies in properties were observed. All dilutions of primary cultures were carried out with 0.85 % saline containing 0.I % peptone (Straka and Stokes, 1957). Liver infusion agar with 0.12 % sorbic acid, pH 5.3, was prepared strictly according to Emard and Vaughn ( 1952); the use of phosphate in these media was avoided as recommended. Acetate agar according to Rogosa et al. ( 1951 ) was also prepared exactly as recommended by the authors; the final pH was hence adjusted to 5.4. As non-selective reference agars we used : a) de Man, Rogosa and Sharpe's (1960) agar (M.R.S. agar), prepared following the original description; b) tryptone-glucose-yeast extract-peptonized milk agar (T.D.Y.M. agar), that had previously been found quite suitable for the enumeration of lactobacilli, the fastidious types included (Mossel and Krugers Dagneaux, 1959; Mossel, 1964). All media were used in poured plates in glass Petri dishes of I I cm diameter. These plates contained I ml inoculum and 20 ml agar, tempered for l hr at 47 C prior to mixing with the inoculum. Subsequent to solidification the agar layers were covered with 20 ml identical sterile agar, tempered at 47 C. Four plates of each medium were used per dilution. Incubation was continued up to 6 days at 30 C. Only plates containing between 30 and 300 colonies were counted. The results of the counts of these ten strains in the two non-selective and the two selective agar media are summarized in Table I. With regard to the non-inhibitory media, we found identical productivity of M.R.S. agar and T.D.Y.M. agar, for the fastidious as well as for the normal strains. In general, colonies on T.D.Y.M. agar were smaller than on M.R.S. agar, but could readily be counted. The results confirm the earlier conclusion (Mossel, 1964) that T.D.Y.M. agar can be used for making total counts including fastidious lactobacilli. The liver infusion-sorbic acid agar of Emard and Vaughn gave excellent recovery of the homofermentative lactobacilli tested. The productivity of this medium for the heterofermentative, fastidious lactobacilli was less satisfactory, since: a) the colonies were small; as appears from the table, a number of strains even did not produce colonies which were visible with routine colony counters, although in these cases microcolonies frequently were found to be present after 6 days incubation; b) the recovery in repeated tests was rather variable (see also Scheffers, Gonz~ilez and Mossel, 1970). The productivity of the agar of Rogosa et aL was virtually identical to the productivity of the non-selective basal agars, and the colonies were of the same size as in M.R.S. agar. The general conclusion of this investigation with pure cultures is that the acetate agar of Rogosa, Mitchell and Wiseman at pH 5.4 incubated at 30 C secures complete recovery of lactobacilli, the fastidious strains included, whereas the liver infusion-sorbic acid agar of Emard and Vaughn at pH 5.3 gives unsatisfactory results with a number of fastidious strains. In this respect we consider the agar of Rogosa et al. as the more suitable medium for the enumeration of lactobacilli. Since in both types of selective media some other organisms than lactobacilli are known to develop, a further study will be performed on possible interference of these organisms (pediococci, leuconostocs; see also Scheffers et al., 1970) with the growth of lactobacilli in the acetate agar.

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264

Table I. C o u n t s in non-selective a n d selective agar media Non-selective agar media Species

L. casei L. plantarum L. brevis

Selective agar m e d i a

Strain

M.R.S. agar

T.D.Y.M. agar

Emard-Vaughn agar, pH 5.3

E.VIII.6.1.8 E.VII1.6.2.9

0.69 7:10 ~ 0.98 • 109

0.69 :.: 109 0.10 , 10 tO

0.58 "~ 109 0.10 ,: 10 t~

0.65 • 109 0.95 .( 109

0.77 >: 109 0.15•176 0.26: -~ 10 t~ 0.21 . 1 0 t~ 0.35;, 109 0.46 "-, 109 0.53 .~ 109 0.24~.:101~

0.76 :. 109 0.14 ~ 10 I~ 0.27-', 10 ~~ 0 . 1 9 ~ 1 0 l~ 0.34:,109 0.48 >, I 09 0.55 9 109 0.25 ~10 ~~

0.56 >: 109 0.77:~109 0 0.20:,',10 ~~ 0 0 0.27 :. 109 0.09>:109

0.74 >~ 109 0.15> 10 t~ 0.24 ~: 10 I~ 0.19>;10 I~ 0.35"<10 '~ 0.49 ~< 109 0.53 ~< 109 0.27• l~

X X L. buchneri X L, fermenti F F L. fi'uctivorans W L. pastoriamts T T

6 16 29 15 19 1 I 4

Rogosa agar, pH 5.4

0: no colonies suitable for counting. EMARD, L. O. a n d VAUGH~, R. H, 1952. Selectivity o f sorbic acid media for the catalase negative lactic acid bacteria a n d clostridia. - J. Bacteriol. 63: 4 8 7 4 9 4 . GASSER, F. 1962. Milieu d'isolement p o u r les Lactobacillus f6caux. - A n n . Inst. Pasteur 102: 239-243. DE MAN, J. C., ROGOSA, M. a n d SHARPE, M. E. 1960. A m e d i u m for the cultivation of lactobacilli. - - J . Applied Bacteriol. 23: 130-135. MOSSEL, D. A. A. 1964. Essentials of the a s s e s s m e n t o f the hygienic condition o f food factories a n d their products. - - J. Science F o o d a n d Agriculture 15: 349-362. MOSSEL, D. A. A. and KRUGERSDAGNEAUX,E. L. 1959. Bacteriological requirements for a n d bacteriological analysis o f precooked ( " i n s t a n t " ) cereals a n d similar foods. - - A n t o n i e v a n L e e u w e n b o e k 25: 230-236. ROGOSA, M., MITCHELL, J. A. a n d W1SEMAN,R. F. 1951. A selective m e d i u m for the isolation a n d e n u m e r a t i o n of oral a n d fecal laetobacilli. - - J . Bacteriol. 62: 132-133. SCHEEFERS,W. A., GONZ~LEZ, F. C. a n d MOSSEL, D. A. A. 1970. T h e e n u m e r a t i o n of lactobacilli with particular reference to fastidious species, In S y m p o s i u m on the Microbiology of Semi-preserved Foods, Liblice near Prague, Czechoslovakia, October 5-7, 1970. In press. SHARPE, M. E. 1960. Selective media for the isolation a n d e n u m e r a t i o n of lactobacilli. - - Lab. Practice 9: 223-227, SHARPE, M. E. a n d FRYER,T. F. 1965. Media for lactic acid bacteria. - - Lab. Practice 14: 697-701. SHARPE~ M. E., FRYER, T. F. a n d SMITH, D. G. 1966. Identification of the lactic acid bacteria, p. 65-79. h~ B. M. G i b b s a n d F. A. Skinner, [eds.], Identification M e t h o d s for Microbiologists. Part A. - - A c a d e m i c Press, L o n d o n and New York. STRAKA, R. P. a n d STOKES, J. L. 1957. Rapid destruction o f bacteria in c o m m o n l y used diluents a n d its elimination. - - Applied Microbiol. 5: 21-25. VAUCHN, R. H. 1970. Incidence o f various groups of bacteria in dehydrated onions a n d garlic. - - F o o d TechnO1.24: 189-191.