Food Microbiology, 1984, 1, 223-227

Enumeration of yeasts and moulds-media trial

G. F. Thomson

Armed Forces Food Science Establishment, P.O. Box 147, Scottsdale, Tasmania 7254, Australia.

Received 29 May 1984

Media for the enumeration of yeasts and mot&s were compared. Dichloran-rose bengal chlortetracycline was found to enumerate at least equivalent numbers of fungi to the other media, and in some instances significantly P < 0.05) more. Media containing dichloran showed greater inhibition of mould colony size than the other media examined.

Introduction As a result of studies comparing anti- biotic based media with acidified media, for enumeration of yeasts and moulds, there has been a trend in recent years away from the use of acidified media. (Hup and Stadhouders 1972, Jarvis 1973, O’Toole and O’Neilll974, Mislivec and Bruce 1976, Beuchart 1978).

Oxytetracycline-glucose-yeast extract agar (with the optional use of gentamicin and rose-bengal) is currently specified in Australian Standard Methods for the Microbiological Examination of Food (A.S. 1766.2.2, Standards Association of Australia 1980) and in Microbiological Methods for the Dairy Industry (AS. 1095.3.3, Standards Association of Australia 1983). This medium replaced acidified potato dextrose agar in the former method and acidified malt extract agar in the latter. This media trial was undertaken wiith a view to future revi- sion of these standard methods.

Materials and methods Culture media Dichloran-rose-bengal chlortetracycline agar (DRBC) was prepared as described by King, Hocking and Pitt (1979). Rose-bengal chlor- 074@-0020/84/030223 + 05 $02.00/O

tetracycline (RBCT) was prepared as des- cribed by Jarvis (1973). Rose-bengal chloram- phenicol was supplied as a complete medium by Australian Hospital Services. Oxytetra- cycline-glucose-yeast extract agar (OGYE) and oxytetracycline-gentamicin-glucose yeast extract agar (OGGYE) were prepared as described by Mossell et al. (1962). Dichloran-glycerol medium (DGM) was pre- pared as described by Hocking and Pitt (19801. Potato dextrose agar (PDA) and malt extract agar (MEA) were prepared from Oxoid dehydrated media and acidified to pH 35 with 10% tartaric acid and 10% lactic acid respectively.

Testing procedure

Forty products were tested, consisting mainly of dehydrated products and a few fresh products. (Table 1). Ten independent samples of white pepper and kibbled onions were also t&S?d.

Ten gram samples of the products were homogenised in a Colworth Stomacher in 90 ml of 0.1% peptone solution. Further dilutions were prepared as necessary with the same diluent. Duplicate preparations were made of 1 ml of the homogenate with 15 ml of the chosen media in 90 mm petri dishes. Yeast and mould counts were determined at the appropriate dilution, after incubation at 25°C for 5 days, yeast colonies being con- firmed by microscopic examination. A measure of each medium’s ability to inhibit mould overgrowth was determined by record- ing the diameter of the largest colony at the counted dilution. When a mould colony over-

0 1984 Academic Press Inc. (London) Limited

224 G. F. Thomson

Table 1. List of products tested.

Almonds Garlic granules Barley Glucoden Beef booster Grapes Bread crumbs Ground ginger Capsicums-dried Honey Carrot rings--frozen Lentils Cheesecake Mixed spices Chocolate rich whip Muesli corn Mustard Cottage cheese--creamed Oats Cottage cheese-gherkin Onion-kibbled Creme of mushroom soup-powder Peas-frozen Currants Pepper-white Curry powder Potato and onion powder Dates-pitted Strawberries (sample a) Desiccated coconut Strawberries (sample b) Drinking chocolate Sultanas Edam cheese Sweet corn nibblets Espagnole sauce Wheat germ French onion dip Yoghurt-apricot

grew between half and the entire plate, it was subcultured onto the other media to confirm inhibition.

Total viable counts were also enumerated by the Pour Plate Method using appropriate dilutions and Standard Plate Count agar (Oxoid). Plates were counted after incubation at 30°C for 72 h.

Statistical analysis

Colony counts for each medium and product were evaluated using Duncan’s New Multiple Range Test of the log of the counts + 1, to correct for zero values. Mould colony diam- eters were also evaluated using Duncan’s New Multiple range Test (Beyer 1968). The statistical analysis was based only on those products for which results were available for all media.

Results Mould counts Table 2 summarizes the mean geometric mould counts obtained for kibbled onion, white pepper and various products. Counts ranged from <lo to 410 000 g-1.

Figure 1 depicts the results of Dun- can’s New Multiple Range Test at the 5%

level of significance. The media are listed in ascending level of enumeration. DRBC exhibited the highest mean geometric count in all situations but the interactions between media were not consistent within the three product groupings.

Yeast counts

No yeasts were detected in either kibbled onions or white pepper. When various products were tested, the medium from which the highest mean geometric counts were enumerated was DRBC (Table 2). Malt extract agar enumerated significantly less (P < 0.05) yeasts than DRBC or RBCT (Fig. 2). No other signifi- cant differences were detected.

Inhibition of mould colony size

The diameters of the largest mould col- ony isolated from the three product group- ings on each medium are summarized in Table 3. The diameter of these largest colonies was inhibited the most on

Yeast and mould media trial 225

Table 2. Means of logarithms (base 10) of mould counts on kibbled onions, white pepper and various products and for yeast counta on various products.

Medium

DRBC RBCT RBC OGYE OGGYE DGM PDA MEA

Kibbled onion*

2.53 2.39 2.43 2.24 2.32 2.02 2.02 2.12

Mould count

White pewera

449 4.86 4.87 4.74 4.71 4.70 4.89 4.74

Various product@

1.40 1.21 1.28 1.14 1.26 1.29 0.95 0.80

Yeast count

Various productsc

1.60 1.04 1.42 1.21 1.30 1.22 0.83 0.90

a Average of 9 samples for all media. b Average of 28 samples for all media. c Average of 10 samples for all media.

Kibbled onion PDA DGM MEA OGYE OGGYE RBCT RBC DRBC

white pepper DGM OGGYE MEA OGYE RBCT RBC PDA DRBC

Various products MEA PDA OGYE RBCT OGGYE RBC DGM DRBC

Fig. 1. Results of the treatment of geometric mould means by Duncan’s New Multiple Range Test. Media are listed in ascending order of enumeration. Means not connected by a line below are judged to be significantly different at the 5% level.

PDA MEA RBCT OGYE DGM OGGYE RBC DRBC

Fig. 2. Results of the treatment of geometric yeast means from various products by Duncan’s New Multiple Range Test. Media are listed in ascending order to enumeration. Means not connected by a line below are judged to be significantly different at the 5% level.

DRBC. Figure 3 shows the results of abilities of the media to isolate differing Duncan’s New Multiple Range Test on species of moulds. However when moulds these figures. This method of testing which overgrew more than half an entire does not take into account the different plate of a particular medium were sub-

226 G. F. Thomson

Table 3. Diameter (mm) of largest colony isolated from kibbled onion, white pepper and various products.

Medium Kibbled oniona White peppera Various product&

DRBC 19.5 18.3 16.0 RBCT 38.1 20.1 19.2 RBC 53.4 20.5 30.6 OGYE 48.9 26.5 31.5 OGGYE 54.5 27.1 31.6 DGM 31.2 23.8 16.8 PDA 28.4 26.7 18G3 MEA 26.8 26.4 21.0

* Average of 9 samples for all media. b Average of 6 samples for all media.

Kibbled onion OGGYE RBC OGYE RBCT DGM PDA MEA DRBC

White pepper OGGYE PDA OGYE MEA DGM RBC RBCT DRBC

Various products

OGGYE OGYE RBC MEA RBCT PDA DGM DRBC

Fig. 3. Results of the treatment of mould diameter means by Duncan’s New Multiple Range Test. Media are listed in descending order of diameter. Means not connected by a line below are judged to be significantly different at the 5% level.

Table 4. Average Standard Plate Counta for kibbled onion, white pepper and various products.

Kibbled onion White pepper Various products

130 000 1500 000 330 000

cultured onto the other media inhibition Discussion was confirmed. Based on the results of these trials the

Armed Forces Food Science Establish- Inhibition of bacteria ment now routinely uses DRBC. This The average Standard Plate Count for medium is preferred since at least equiv- the three product groupings is shown in alent number of fungi compared with the Table 4. No bacteria were isolated on the other media, and in some instances fungal enumeration media. significantly (P < 0.05) more, are

Yeast and mould media trial 227

enumerated. DRBC has the advantage over the non-rose-bengal based media, of the colonies being easier to count. This is because the dye is taken up by the colonies. DGM was found to be the hardest medium to count since the mould colonies tended to be ‘whispy’ and blend in with the medium.

Mould colonies were significantly smaller on the dichloran based media. Spreading colonies of Mucor and Rhizo- pus spp. were observed to overgrow plates on all media other than DRBC and DGM. The oxytetracyline media also failed to inhibit spreading colonies of Sporendonema sp.

Hocking and Pitt (1980) compared DRBC and DGM for enumerating fungal flora from dried commodities. They found that counts on DGM were signifi- cantly higher than on DRBC (P < 0.01). The results of this trial fail to substan- tiate these findings. The only significant

References Beuchat, L. R. (1979) Comparison of acidified and antibiotic supplemented potato dextrose

agar for three manufacturers for its capacity to cover fungi from foods. J. Food Protect. 42, 427428.

Beyer, W. H. (Ed.) (1968) Handbook of Tables of Probability. CRC pp. 371-373. Hocking, A. D. and Pitt, J. I (1980) Dichloran-glycerol medium for enumeration of xerophilic

fungi from low-moisture foods. Appl. Environ. Microbial. 39, 446492. Hup, G. and Stadhouders, J. (1972) Comparison media for the enumeration of yeasts and

moulds in dairy products. Neth. Milk Dairy J. 26, 131-140. Jarvis, B. (1973) Comparison of an improved rose bengal-chlortetracycline agar with other

media for the selective isolation and enumeration of moulds and yeasts in foods. J. Appl. Bacterial. 36, 723-727.

King, A. D., Hocking, A. D. and Pitt, J. I. (1979). Dichloran-rose bengal medium for enumeration and isolation of molds from foods. Appl. Environ. Microbial. 37, 959-964.

Mislivec, P. B. and Bruce, V. R. (1976) Comparison of antibiotic-amended potato dextrose agar and acidified potato dextrose agar as growth substrates for fungi. J. Assoc. Off. Anal. Chem. 59,720-721.

Mossel, D. A. A., Viser, M. and Mengerink, W. H. J. (1962) A comparison of media for the enumeration of moulds and yeasts in foods and beverages. Lab. PFUCt. 11, 109-112.

G’Toole, D. K. and O’Neil, G. H. (1974) Comparison of an antibiotic medium with acidified malt extract medium for the enumeration of fungi in butter. Aust. J. of Dairy Tech. Sept., 115-116.

Standards Association of Australia (1980) Colony counts of yeasts and moulds. In Methods for the microbiological examination of food. A.S. 1766.2.2.

Standards Association of Australia (1983) Yeasts and moulds. In Microbiological methods for the dair.y industry. A.S. 1095.3.3.

difference found between DGM and DRBC was in the enumeration of mould from kibbled onion where counts on DRBC were significantly higher than on DGM (P < 0.05).

The Standards Association of Aus- tralia gives the option of using rose- bengal (35 pg ml-l) if very large colonies or those with extensive aerial mycelia render colony counting difficult. Trials incorporating rose-bengal in the oxy- tetracycline media were not carried out but the results suggest that the best inhibition is achieved using a combina- tion of rose-bengal and dichloran.

Acknowledgements The author wishes to thank MS M. Geary and MS J. Brown for their technical assistance and Mr K. James and Dr G. Driver for their assistance with the statistical analyses.