Module 4. Microbiological methods of milk testing

Lesson 21

ENUMERATION OF YEAST AND MOULDS IN MILK

21.1 Introduction

Aerial contamination is one of the major sources of moulds that are usually colonized on walls and roofs of the dairy farms.  When the milk is exposed to open area, moulds will enter in milk chain. Growth of yeasts and moulds is a common cause of the spoilage of dairy products, especially fermented milks, because these microbes are able to grow at low pH. Yeast spoilage is manifested as fruity or yeasty odour and/or gas formation. For proper quality control, it is important for the producer to evaluate yeast and mould counts in the milk used for making milk products.

21.2 Estimation of Yeast and Mould

The most common yeasts present in dairy products arc Kluyvermnyce marxianus and Debaromyces hansenii and their asporogenous counterparts, Candida famata, Candid. Kefyr and other Candida spp. The other prevalent ones are Rhodotorula spp, and Torulospora spp. The most common moulds belong to Penicillium spp., Cladosporium spp., Fusarium spp., Asperigillus spp., Rhizopus spp., Trichoderma spp., Geotrichum spp. and Mucor spp. Many of these cause defects in dairy products. These also produce potent toxins like aflatoxins, acrotoxins etc. Yeasts and moulds that spoil dairy products can usually be isolated from air, equipments and general environment (i.e. floors, walls, ventilation ducts, etc.).

21.2.1 Materials

Sterile sample bottle, pipettes, test tubes, water bath, hot air oven, incubator.

21.2.2 Media

Potato dextrose agar or Malt extract agar, Tartaric acid (10%)

21.2.3 Procedure

· Potato Dextrose Agar is prepared following standard procedure of media preparation.

· After sterilization, cool the media to 45°C and acidify with 2 ml of sterile 10% tartaric acid per 100 ml of the media immediately before pouring the plates (pH 3.5-3.7). Acidify potato dextrose broth similarly with 1 ml of the sterile tartaric acid.

· Prepare dilutions and transfer one ml of appropriate dilution to sterile Petri dish.

· Pour acidified melted potato dextrose agar and allow it to solidify.

· Do not invert the plates and incubate at 30-32°C. Count the plates after 72 h (3 days), but if the colonies are too small, extend the incubation time to 96-120 h. (4-5 days).

· Take the average of counts of duplicate plates, multiply by the dilution factor and record as the number of yeast or moulds per gram.

When counting mould or yeasts, use the following criteria:

A. Use plates that have maximum number of colonies and whose surfaces are not completely covered with mould and/or where each individual surface and sub-surface mould colony is discernible.

B. Count on any plate containing not more than 200 colonies and preferably not less than 20 colonies. The yeast counts may be subject to interference from moulds, etc., if so, count the plates, where the yeast colonies are most discernible

Yeast colonies will show blue-green or off-white tinge in colour and will form small, slimy colonies. Mould colonies tend to be larger and more diffused and are usually blue in colour. The results are recorded as number of yeast and mould count per ml or gram.

21.3 Mould Tests

1. Microscopic method -- Mould mycelia count

2. Macroscopic method -- Methylene blue borax test

3. Visual mould test -- Modified Methylene blue borax test         

21.3.1 Microscopic method -- Mould mycelia count

Mould mycelia count is made just like direct microscopic count method as described in earlier lessons. This method developed by ‘B.J. Howard’ requires the use of special slide to enumerate the mould mycelia.

21.3.2 Macroscopic method -- Methylene blue borax test

Five ml of warm milk is rinsed into a test-tube with 15ml of hot methylene blue and borax solution (30 gm of sodium borate and 10 methylene blue tablets, in 1 litre of water). The mixture is shaken well and poured into a shallow tin. The pan is rocked until all the mould present has agglutinated into blue-stained masses. These are gathered, by means of a scalpel, into a circular disc, diameter of which is measured and its area in square mm is calculated.

21.3.3 Visual mould test, modified methylene blue borax test

A small amount of milk is taken and is mixed with a mixture containing methylene blue and alkaline salts. The mixture is stirred while being heated. This causes fragments of mould mycelium that may be present to gather into a mass. The mixture is filtered and the mould mycelium retained is measured visually. For evaluating the quality of milk, it is assumed that when conditions are favorable for growth of the moulds, it is likely that bacteria and yeasts would grown extensively causing undesirable changes.