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THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER
THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER
183
THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER Important factors influencing the microflora of milk powder are the heat treatment given the milk prior to the drying process and the method of drying the milk. Where comparatively severe exposure to heat occurs—in roller-drying, for example—the resultant powder shows a more restricted flora than one in which the temperatures involved are less extreme (for example, powder produced by the spray-drying process). Further factors influencing the microflora of the powder are the extent of contamination from the milk-plant and the extent to which microbial multiplication can occur prior to the drying process. It is particularly important at this stage that numbers of coagulasepositive Staphylococcus aureus do not reach levels at which enterotoxin production creates a health hazard in the subsequent milk powder. The packaging process may also allow the introduction of contaminants, particularly atmospheric contaminants such as yeasts and moulds. On storage, numbers of micro-organisms gradually decline, and in year-old powders, spore-formers may comprise the dominant flora. When powdered milks are reconstituted, any surviving micro-organisms are capable of growth, and milks should therefore not be kept in this condition for a prolonged period.
A. Sampling A standard procedure for the sampling of milk powder, recommended in the British Standard (1963), requires that samples should be taken with a dry sterile metal spatula or spoon after mixing the top 150 mm (6 inches) of the contents and then transferring not less than 115 g (4 oz) to a sterile sample jar of sufficient size to allow mixing by shaking. A similar procedure, described by the International Dairy Federation (1958) suggests separate sampling of surface and subsurface contents of the packaged powder.
B. Microscopic Examination Prepare a 10 -1 dilution of the milk powder as described below and set up a Breed's smear preparation. Stain the smear with Charlett's improved Newman's stain and examine microscopically, using the oil-immersion objective. Determine the average number of micro-organisms per field and, using the microscope factor, calculate the total numbers present per gram of milk powder. This gives an indication of the extent to which micro-organisms may have proliferated in the milk prior to the actual drying process.
184
LABORATORY METHODS IN MICROBIOLOGY
C. Cultural Examination 1. Preparation of dilutions Use the method of Higginbottom (1945). Weigh out aseptically 10 g of milk powder and transfer to 90 ml of quarter-strength Ringer's solution at 50°C in a wide-mouthed bottle. Shake the bottle 25 times in 12 seconds with an excursion of 1 foot. Place the reconstituted milk in a water bath at 50°C for 15 minutes, then invert the jar several times and examine immediately. Prepare further dilutions in 9 ml amounts of quarter-strength Ringer's solution as required or as indicated by examination of the Breed's smear preparation. 2. Inoculation of plates and media Plate out 1 ml of each dilution on to suitable media for particular groups of organisms as follows. (a) General mesophilic flora {i.e. a general viable count). Use yeastrel milk agar or plate count agar and incubate at 30°C for 5 days. If required, a comparison can be made with numbers obtained after incubation for 3 days at 37°C, but higher numbers are generally obtained at 30°C. (b) Thermophilic micro-organisms. Use yeastrel milk agar or plate count agar and incubate at 55°C for 2 days. (c) Coli-aerogenes organisms. Use violet red bile agar and incubate at 30°C for 24 hours. Alternatively, inoculate 10-ml quantities of the 10_1 dilution into double strength MacConkey's broth and 1 ml quantities of each dilution into single strength MacConkey's broth. Incubate at 30°C for 48 hours. Presumptive positive results should be confirmed by subculture into fresh tubes of MacConkey's broth or streaking on to MacConkey's agar. (d) Yeasts and moulds. Use malt extract agar (pH 3-5) or Davis's yeast salt agar (pH 3-5) and incubate at 22° or 25°C for 3-5 days. (e) Pathogenic staphylococci. Use Baird-Parker's medium, spreading 0· 1 ml of each dilution on to the surface of well-dried plates. Incubate at 37°C for 24-48 hours. Confirm presumptive positive colonies by the coagulase test. (/) Aerobic spore-formers. Pour about 5 ml of the reconstituted milk (10 _1 dilution) into a sterile test-tube and heat at 80°C for 10 minutes after this temperature has been reached in a control tube. Cool under the tap and plate out 1-ml and 0 1 -ml quantities on to yeastrel milk agar or starch milk agar. Incubate at 30°C for 48 hours. (g) Anaerobic spore-formers. Pour 10 ml of the reconstituted milk (10 _1 dilution) into a sterile test-tube and heat at 80°C for 10 minutes after this temperature has been reached, in a control tube. Cool under the tap and seal the surface with sterile vaspar or add 2 ml of molten agar medium. Incubate at 37°C for 3 days. Stormy fermentation indicates a positive result.
THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER
185
D. Results and Suggested Standards Report results per gram of milk powder for each group of organisms examined. Davis (1963) suggests that freshly manufactured spray-dried milk powder should have a direct microscopic count of less than 10 million per gram, a general viable count of less than 20,000 per gram, and counts of coliaerogenes, yeasts, moulds and Staphylococcus aureus each of less than 10 per gram. For freshly manufactured roller-dried powder he suggests that the general viable count should be less than 5000 per gram and counts of coliaerogenes, yeasts and moulds should each be less than 10 per gram.
183
THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER Important factors influencing the microflora of milk powder are the heat treatment given the milk prior to the drying process and the method of drying the milk. Where comparatively severe exposure to heat occurs—in roller-drying, for example—the resultant powder shows a more restricted flora than one in which the temperatures involved are less extreme (for example, powder produced by the spray-drying process). Further factors influencing the microflora of the powder are the extent of contamination from the milk-plant and the extent to which microbial multiplication can occur prior to the drying process. It is particularly important at this stage that numbers of coagulasepositive Staphylococcus aureus do not reach levels at which enterotoxin production creates a health hazard in the subsequent milk powder. The packaging process may also allow the introduction of contaminants, particularly atmospheric contaminants such as yeasts and moulds. On storage, numbers of micro-organisms gradually decline, and in year-old powders, spore-formers may comprise the dominant flora. When powdered milks are reconstituted, any surviving micro-organisms are capable of growth, and milks should therefore not be kept in this condition for a prolonged period.
A. Sampling A standard procedure for the sampling of milk powder, recommended in the British Standard (1963), requires that samples should be taken with a dry sterile metal spatula or spoon after mixing the top 150 mm (6 inches) of the contents and then transferring not less than 115 g (4 oz) to a sterile sample jar of sufficient size to allow mixing by shaking. A similar procedure, described by the International Dairy Federation (1958) suggests separate sampling of surface and subsurface contents of the packaged powder.
B. Microscopic Examination Prepare a 10 -1 dilution of the milk powder as described below and set up a Breed's smear preparation. Stain the smear with Charlett's improved Newman's stain and examine microscopically, using the oil-immersion objective. Determine the average number of micro-organisms per field and, using the microscope factor, calculate the total numbers present per gram of milk powder. This gives an indication of the extent to which micro-organisms may have proliferated in the milk prior to the actual drying process.
184
LABORATORY METHODS IN MICROBIOLOGY
C. Cultural Examination 1. Preparation of dilutions Use the method of Higginbottom (1945). Weigh out aseptically 10 g of milk powder and transfer to 90 ml of quarter-strength Ringer's solution at 50°C in a wide-mouthed bottle. Shake the bottle 25 times in 12 seconds with an excursion of 1 foot. Place the reconstituted milk in a water bath at 50°C for 15 minutes, then invert the jar several times and examine immediately. Prepare further dilutions in 9 ml amounts of quarter-strength Ringer's solution as required or as indicated by examination of the Breed's smear preparation. 2. Inoculation of plates and media Plate out 1 ml of each dilution on to suitable media for particular groups of organisms as follows. (a) General mesophilic flora {i.e. a general viable count). Use yeastrel milk agar or plate count agar and incubate at 30°C for 5 days. If required, a comparison can be made with numbers obtained after incubation for 3 days at 37°C, but higher numbers are generally obtained at 30°C. (b) Thermophilic micro-organisms. Use yeastrel milk agar or plate count agar and incubate at 55°C for 2 days. (c) Coli-aerogenes organisms. Use violet red bile agar and incubate at 30°C for 24 hours. Alternatively, inoculate 10-ml quantities of the 10_1 dilution into double strength MacConkey's broth and 1 ml quantities of each dilution into single strength MacConkey's broth. Incubate at 30°C for 48 hours. Presumptive positive results should be confirmed by subculture into fresh tubes of MacConkey's broth or streaking on to MacConkey's agar. (d) Yeasts and moulds. Use malt extract agar (pH 3-5) or Davis's yeast salt agar (pH 3-5) and incubate at 22° or 25°C for 3-5 days. (e) Pathogenic staphylococci. Use Baird-Parker's medium, spreading 0· 1 ml of each dilution on to the surface of well-dried plates. Incubate at 37°C for 24-48 hours. Confirm presumptive positive colonies by the coagulase test. (/) Aerobic spore-formers. Pour about 5 ml of the reconstituted milk (10 _1 dilution) into a sterile test-tube and heat at 80°C for 10 minutes after this temperature has been reached in a control tube. Cool under the tap and plate out 1-ml and 0 1 -ml quantities on to yeastrel milk agar or starch milk agar. Incubate at 30°C for 48 hours. (g) Anaerobic spore-formers. Pour 10 ml of the reconstituted milk (10 _1 dilution) into a sterile test-tube and heat at 80°C for 10 minutes after this temperature has been reached, in a control tube. Cool under the tap and seal the surface with sterile vaspar or add 2 ml of molten agar medium. Incubate at 37°C for 3 days. Stormy fermentation indicates a positive result.
THE MICROBIOLOGICAL EXAMINATION OF MILK POWDER
185
D. Results and Suggested Standards Report results per gram of milk powder for each group of organisms examined. Davis (1963) suggests that freshly manufactured spray-dried milk powder should have a direct microscopic count of less than 10 million per gram, a general viable count of less than 20,000 per gram, and counts of coliaerogenes, yeasts, moulds and Staphylococcus aureus each of less than 10 per gram. For freshly manufactured roller-dried powder he suggests that the general viable count should be less than 5000 per gram and counts of coliaerogenes, yeasts and moulds should each be less than 10 per gram.