MOSEEL, D. A. A. et at. (1970). J. appl. Bad. 33, 454-467.

Oxytetracycline-Glucose-Yeast Extract Agar for Selective Enumeration of Moulds and Yeasts in Foods and Clinical Material

D. A. A. MOSSEL, A. M. C . KLEYNEN-SEMMELINQ AND H. M. VINCENTIE

Central Institute for Nutrition and Food Research TNO, Zeist, The Netherlands

AND

H. BEERENS AND M. CATSARAS

Institut Pasteur, Lille, France

(Received 22 October 1969)

SUMMARY. The oxytetracycline-glucose-yeast extract agar of Mossel, Visser & Mengerink (1962) for the selective enumeration of moulds and yeasts in foods has been extensively tested on 4000 samples of various materials in two food microbiology and one clinical laboratory. Its performance was excellent in that it suppressed entirely the growth of Bacillaceae. Only when examining stools from patients under tetracycline therapy did the medium insufficiently inhibit Enterobacteriaceae. Substitution of oxytetracycline by 60 pg/ml of gentamicin ww useful in such instances.

AGAR MEDIA a t low pH values, as used for isolating moulds and yeasts from soil (Waksman, 1922)) are not entirely satisfactory for enumerating these organisms in foods and other biological materials, because it is virtually impossible to adjust such media to a pH value that suppresses adequately the acid-tolerant bacteria without a t the same time impairing the quantitative development of the moulds and yeasts (Mossel, Visser & Mengerink, 1962). Earlier investigations (Mossel, Visser & Mengerink, 1962) showed an oxytetracycline-glucose-yeast extract agar (OGY), as first suggested by Beech & Carr (1955), to be useful for this purpose, and Buttiaux & Catsaras (1965) and Sainclivier Q Roblot (1966) confirmed this. However, Put, van Reeven-Bouwhuis & Kruiswijk (1968) encountered difficulties when using OGY for enumerating spores of Byssochlamys fulva in soil and fruits; they observed particularly that spores of Bacillaceae developed copiously on the medium which rendered the isolation of moulds difficult, if not impossible. Because of this we undertook another extensive evaluation of the performance of OGY under various practical conditions and this paper describes the results obtained.

Materials and Methods

Media

The OGY (Mossel, Visser & Mengerink, 1962) was prepared by adding 10 ml of a freshly prepared, filter sterilized solution of a 0 1 % oxytetracycline (Terramycin, Pfher) solution to 100 ml of a 1% glucose+0.5% yeast extract agar, previously melted and cooled to 49 k 1". Pure cultures were streaked on plates of this medium

L4.541

Oxytetracycline agar for moulds and yeasts 455

and poured plates were used for examining foods, soil and clinical material. Incuba- tion was for up to 5 days a t 22+2".

Gentamicin agar was prepared by adding 1 ml of a filtered solution of 5 mg/ml of gentamicin (Garamycin, Schering) to 100 ml of the basal glucose-yeast extract agar, thus giving a final concentration of gentamicin of 50 pg/ml.

Spore counts in foods and soil were made in Tryptone-dextrose-peptonized milk agar, after heating suitable dilutions for 1 min a t 80" (Mossel 6 Krugers Dagneaux, 1959 ; Mossel, 1967), plates being incubated for up to 6 days at 30 + 2".

The Bacillus strains used in this study were maintained on Nutrient Agar (Oxoid) which often promotes sporulation (Galesloot 6 Labots, 1959).

Enterobacteriaceae in stools were enumerated by making poured plate counts in violet red bile salts-glucose agar (Mossel, Mengerink & Scholts, 1962). After solidification plates were covered with c. 20 ml of the medium and incubated for 18-24 h at 37".

Cultures In all, 65 strains of Bacillus spp. were studied, including B. cereus (25 strains), B. circulans (6), B. coagulans (l), B. jirmus ( l ) , B. licheniformis (2), B. mesentericw (l), B. polymyxa (2), B. pumilus (2), B. stearothermophilus (3), B. subtilis (4) and 18 unidentified strains, The majority of strains was obtained from the culture collection of the Laboratory for Microbiology, Technical University, Delft, The Netherlands. Roughly a third of the strains had been isolated from the foods listed in Table 1, using the method described in the previous section. The rest were isolated from clinical materials, whcre they had acted as contaminants.

Soil A total of 100 samples of garden soil was studied. Approximately $ of the material stemmed from strawberry and raspberry beds in the area of Zeist, the remainder being from similar sources in Lille, Prance. This type of soil was chosen because Put et nl. (1968) had encountered difficulties with such material. Samples were taken throughout a 12 months' period to eliminate seasonal influences.

TABLE 1 Food materials examined for moulds and Bacillus spores

No. of samples examined Food

cocoa Dried egg products Dried soups Fish flour Margarine Milk powder Mixed feeds Soya flour Sugars

250 100 180 200

2000 560 20 100 200

456 D. A. A. Mossel et al.

Foods About 3500 samples of foods, rich in Bacillus spores and containing low but significant numbers of moulds were examined. The types of food used are listed in Table 1. Roughly 90% were drawn from The Netherlands' market, the remainder being from French producers.

Clinical material One hundred samples of contaminated blood and pus were chosen a t random from the daily deliveries to the Clinical Laboratory of the Pasteur Institute, Lille, France.

Fifty stool samples from that Laboratory and 15 from the Department of Paedi- atrics, University of Utrecht, Utrecht, The Netherlands, were also selected. All were from patients under tetracycline therapy.

Results and Discussion The cultures of the Bacillus spp. tested grew prolifically overnight on nutrient agar a t 28-30" and none showed any visible growth after incubation for 5 days on OGY a t this temperature. The Bacillus strains isolated from clinical material had previously appeared to be entirely suppressed by oxytetracycline at 100 pg/ml, the level used in OGY.

Amongst the food samples c. 30% contained 102-103 Bacillus sporeslg, c. 50% contained los-104/g and c. 20% lo4-107/g. Even in OGY plates inoculated with the 10-1 dilution of these foods, no Bacillus colony ever grew.

The Bacillus spore counts of the soil samples examined varied from 0.4 x lo5 to 0.9 x 107/g, with an average count of c. 10s/g. In a few instances only Bacillus colonies were observed when 10-1 dilutions were platcd in OGY.

To challenge the medium further, small quantities of the mixed feeds (Table 1) containing oxytetracycline, or an antibiotic with a similar spectrum of inhibition, were spread over OGY plates to which 100 pg/ml of pimaricin had been added (Mossel & Sand, 1968). The purpose of this addition was to inhibit any moulds present and thus clear the way entirely for oxytetracycline resistant bacilli. Incubation was continued until one or more bacterial colonies had become visible. Slow growing Bacillus strains were indeed isolated from feeds in this way, similarly infrequently as from garden soil, after being heated a t 80" for 1 min. These strains were alike in that they did not survive well; their development on plates containing oxytetra- cycline at various concentrations wa.s highly irreproducible and they were all lost, eventually, after subculturing on agar media with, as well as without, oxytetracycline. It must be assumed, therefore, that oxytetracycline resistant types of Bacillaceae do not readily survive in natural habitats. This, in all probability, is the reason why, although Put et al. (1968) encountered such variants in their ecological studies on soil, we failed to isolate them at any level of significance from foods, and correspond- ingly never found them to interfere with the enumeration of moulds and yeasts in foods, when using oxytetracycline agar. This assumption finds further support in

Oxytetracycline agar for moulds and yeasts 457

observations of Connamacher (1969) who were not successful in increasing the oxytetracycline resistance in bacilli to levels > 5 pg/ml.

When examining patients’ stools, containing 106-109 Enterobacteriaceaelg, sparse bacterial growth was observed in OGY used in this instance for the enumeration of yeasts. The isolates appeared to be Enterobacteriaceae rather than Bacillus spp., an occurrence to be expected in patients under tetracycline therapy. Most of the Enterohacteriaceae could be suppressed by the use of 50 pg/ml of gentamicin, in agreement with reports made by Jao & Jackson (1964), Holt & Newman (1968), Santlrr & Blasius (1968) and Modde (1969).

Thus there are no reasons to replace OGY by another medium in the examination of foods for moulds and yeasts. For the past 8 years we have been using this medium succcssfully in our laboratories and the experiments reported in this paper confirm its sclective value.

We are indebted to Professor T. 0. Wiken of the Technical University, Delft, for making strains available for study.

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