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THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM
THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM
189
THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM There are, at present, no statutory standards for the microbiological content of ice-cream in the United Kingdom. The Public Health Laboratory Service (Report, 1947) has devised a méthylène blue dye reduction test which is suitable for routine grading purposes and which provides a simple method of detecting ice-cream of poor hygienic quality. More detailed information can be obtained by cultural examination, which should be carried out on a gravimetric basis, and numbers of particular groups of micro-organisms determined as required by the use of appropriate media. A. Collection of Samples The sampling procedures described are those recommended by the British Standard (1963). 1. Hardened ice-cream For packages and tubs, one or more unopened packages constitute the sample which should be delivered intact to the laboratory in a sterile container. For multi-layered ice-cream, the sample should contain the same proportions of each layer as in the original ice-cream. In the case of ice-cream in bulk containers, first remove the surface layer with a sterile spatula or spoon and with a second sterile spatula take a sample of not less than 60 g (2 oz) into a sterile jar. For information on bulk ice-cream as served to the consumer, the sample is taken from the surface layer with the retailer's own server. 2. Soft ice-cream This is freshly frozen ice-cream sold direct from the freezer. In this case, fill the sample jars directly from the freezer outlet—the sample should be a minimum of 60 g (2 oz). 3. Transport of samples The British Standard (1963) recommends that samples should be transported to the laboratory in a refrigerated container and should be maintained at a temperature not higher than — 15°C until examined in the laboratory. A more convenient procedure, described in Report (1947), is to transport samples to the laboratory in an insulated container for delivery within 2 hours of time of sampling, but where this time limit is not practicable, samples are packed in ice and should arrive at the laboratory within 6 hours of sampling. o*
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LABORATORY METHODS IN MICROBIOLOGY
B. Treatment of Samples 1. If the sample is in the original retail package, transfer aseptically the whole, or a representative portion, to a sterile container of not less than 60 ml (2 fluid oz) capacity. 2. In the case of frozen samples, stand at room temperature for a maximum period of one hour until melted. Alternatively, the sample may be liquified by holding the sample jar in a water bath at 42-45°C for no more than 15 minutes as recommended in Memorandum (1948). 3. If the sample is unfrozen, examine immediately. C. Cultural Examination The analysis should be carried out on a gravimetric basis since, as shown by Patton (1950), the weight of 10 ml of ice-cream may range from 4-5 to 10-5 g. 1. Preparation of initial 10"1 dilution (a) Invert the sample bottle three times to mix the sample. (b) Using a sterile 10 ml pipette, weigh out 10 g of melted ice-cream into a sterile container. (c) Add 90 ml of sterile quarter-strength Ringer's solution and invert three times. This constitutes the 10 _1 dilution. 2. Inoculation of plates and media Prepare further dilutions in 9 ml of quarter-strength Ringer's solution up to 10~4 or as required and plate out 1 ml of each dilution on to suitable media for particular groups of organisms as follows. (a) General mesophilic flora {general viable count). Use yeastrel milk agar or plate count agar and incubate at 30°C for 3 days. (b) Coli-aerogenes organisms. Use violet red bile agar and incubate at 30°C for 24 hours, or, alternatively, inoculate 1 ml into MacConkey's broth and incubate at 30°C for 48 hours. To detect coli-aerogenes organisms in 1 g of ice-cream, inoculate 10 ml of 10 _1 dilution into 10 ml of double-strength MacConkey's broth. Presumptive positive tubes of MacConkey's broth should be confirmed by subculturing into fresh tubes of MacConkey's broth at 30°C, and at 44°C for Escherichia coli type 1. (c) Yeasts and moulds. Use malt extract agar (pH 3-5) or Davis's yeast salt agar (pH 3-5). Incubate at 22 or 25°C for 3-5 days. (d) Pathogenic staphylococci. Use Baird-Parker's medium, and streak 0· 1 ml of each dilution over the surface of well-dried plates. Incubate at 37°C for 24-48 hours. Confirm presumptive positive colonies by the coagulase test.
THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM
191
(e) Psychrophiles. Use yeastrel milk agar or plate count agar and incubate at 5°C for 7-10 days. (/) Thermoduric bacteria. Transfer 10 ml of the 10 _1 dilution into a sterile 6-inch x |-inch test-tube, close with a sterile rubber bung and heat in a water bath at 63-5 ± 0-5°C for 35 minutes. Remove the tube from the bath and cool under the tap. Then invert the tube three times and prepare further dilutions to 10 -3 in 9 ml amounts of quarter-strength Ringer's solution. Plate out 1 ml of each dilution on to yeastrel milk agar or plate count agar and incubate at 30°C for 4 days.
D. Méthylène Blue Dye Reduction Test The test should be carried out according to the report of the Public Health Laboratory Service (Report, 1947). The test should be set up at 5 p.m. on the day on which the sample is taken. (Any other convenient time may be chosen provided that the test is set up on the day the sample is taken and a continuous incubation period of 17 hours is maintained.) Procedure (a) Deliver 7 ml of quarter-strength Ringer's solution into a sterile tube with 10 ml mark. (b) Add 1 ml of standard méthylène blue solution (as used for dye-reduction tests on milk). (c) Using a wide-bore pipette, deliver ice-cream up to the 10 ml mark, thus adding 2 ml. (d) Insert a sterile rubber bung and invert the tube once. If the ice-cream contains much air the meniscus will fall as the air is freed and if this occurs, fill up with further ice-cream to the 10 ml mark. (e) Set up controls as follows. 1. Ice-cream colour: with a 10-ml graduated pipette, deliver 8 ml of quarterstrength Ringer's solution into a sterile 10-ml mark tube. With a wide bore pipette, add ice-cream to the 10-ml mark as in the test. Insert a sterile rubber bung, invert the tube and fill up with ice-cream to the 10-ml mark if required. 2. Méthylène blue : a méthylène blue control using heated ice-cream should be included in each test. About 5 ml of ice-cream should be heated in a boiling water bath for 15 minutes, cooled and used as in the test proper. 3. For coloured ice-cream, a separate colour control and méthylène blue control should be set up for each sample.
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LABORATORY METHODS IN MICROBIOLOGY
(/) Incubate the tubes in a water bath at 20 ± 0-5°C, until 10 a.m. on the following morning, i.e. a 17-hour incubation period. (g) Transfer the tubes to a water bath at 37 ± 0-5°C, and invert once every half hour until decolorization is complete as compared with the control tubes. Recording results Record the time taken for decolorization of the méthylène blue. If the méthylène blue is decolorized at the time of removal from the 20°C water bath, the time is recorded as 0 hours. £ . Results and Recommended Standards Report colony count per gram of ice-cream for each group of organisms examined. Davis (1963) suggests that ice-cream should have a general viable count of not more than 10,000 per gram, a count of coagulase-positive staphylococci of not more than 10 per gram, and a coli-aerogenes count of not more than 1 per gram. Suggested grades for ice-cream (Reports, 1947, 1950) Time taken to reduce méthylène blue
Provisional grade
Fails to reduce in 4 hours 2J-4 hours | - 2 hours 0
1 2 3 4
189
THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM There are, at present, no statutory standards for the microbiological content of ice-cream in the United Kingdom. The Public Health Laboratory Service (Report, 1947) has devised a méthylène blue dye reduction test which is suitable for routine grading purposes and which provides a simple method of detecting ice-cream of poor hygienic quality. More detailed information can be obtained by cultural examination, which should be carried out on a gravimetric basis, and numbers of particular groups of micro-organisms determined as required by the use of appropriate media. A. Collection of Samples The sampling procedures described are those recommended by the British Standard (1963). 1. Hardened ice-cream For packages and tubs, one or more unopened packages constitute the sample which should be delivered intact to the laboratory in a sterile container. For multi-layered ice-cream, the sample should contain the same proportions of each layer as in the original ice-cream. In the case of ice-cream in bulk containers, first remove the surface layer with a sterile spatula or spoon and with a second sterile spatula take a sample of not less than 60 g (2 oz) into a sterile jar. For information on bulk ice-cream as served to the consumer, the sample is taken from the surface layer with the retailer's own server. 2. Soft ice-cream This is freshly frozen ice-cream sold direct from the freezer. In this case, fill the sample jars directly from the freezer outlet—the sample should be a minimum of 60 g (2 oz). 3. Transport of samples The British Standard (1963) recommends that samples should be transported to the laboratory in a refrigerated container and should be maintained at a temperature not higher than — 15°C until examined in the laboratory. A more convenient procedure, described in Report (1947), is to transport samples to the laboratory in an insulated container for delivery within 2 hours of time of sampling, but where this time limit is not practicable, samples are packed in ice and should arrive at the laboratory within 6 hours of sampling. o*
190
LABORATORY METHODS IN MICROBIOLOGY
B. Treatment of Samples 1. If the sample is in the original retail package, transfer aseptically the whole, or a representative portion, to a sterile container of not less than 60 ml (2 fluid oz) capacity. 2. In the case of frozen samples, stand at room temperature for a maximum period of one hour until melted. Alternatively, the sample may be liquified by holding the sample jar in a water bath at 42-45°C for no more than 15 minutes as recommended in Memorandum (1948). 3. If the sample is unfrozen, examine immediately. C. Cultural Examination The analysis should be carried out on a gravimetric basis since, as shown by Patton (1950), the weight of 10 ml of ice-cream may range from 4-5 to 10-5 g. 1. Preparation of initial 10"1 dilution (a) Invert the sample bottle three times to mix the sample. (b) Using a sterile 10 ml pipette, weigh out 10 g of melted ice-cream into a sterile container. (c) Add 90 ml of sterile quarter-strength Ringer's solution and invert three times. This constitutes the 10 _1 dilution. 2. Inoculation of plates and media Prepare further dilutions in 9 ml of quarter-strength Ringer's solution up to 10~4 or as required and plate out 1 ml of each dilution on to suitable media for particular groups of organisms as follows. (a) General mesophilic flora {general viable count). Use yeastrel milk agar or plate count agar and incubate at 30°C for 3 days. (b) Coli-aerogenes organisms. Use violet red bile agar and incubate at 30°C for 24 hours, or, alternatively, inoculate 1 ml into MacConkey's broth and incubate at 30°C for 48 hours. To detect coli-aerogenes organisms in 1 g of ice-cream, inoculate 10 ml of 10 _1 dilution into 10 ml of double-strength MacConkey's broth. Presumptive positive tubes of MacConkey's broth should be confirmed by subculturing into fresh tubes of MacConkey's broth at 30°C, and at 44°C for Escherichia coli type 1. (c) Yeasts and moulds. Use malt extract agar (pH 3-5) or Davis's yeast salt agar (pH 3-5). Incubate at 22 or 25°C for 3-5 days. (d) Pathogenic staphylococci. Use Baird-Parker's medium, and streak 0· 1 ml of each dilution over the surface of well-dried plates. Incubate at 37°C for 24-48 hours. Confirm presumptive positive colonies by the coagulase test.
THE MICROBIOLOGICAL EXAMINATION OF ICE-CREAM
191
(e) Psychrophiles. Use yeastrel milk agar or plate count agar and incubate at 5°C for 7-10 days. (/) Thermoduric bacteria. Transfer 10 ml of the 10 _1 dilution into a sterile 6-inch x |-inch test-tube, close with a sterile rubber bung and heat in a water bath at 63-5 ± 0-5°C for 35 minutes. Remove the tube from the bath and cool under the tap. Then invert the tube three times and prepare further dilutions to 10 -3 in 9 ml amounts of quarter-strength Ringer's solution. Plate out 1 ml of each dilution on to yeastrel milk agar or plate count agar and incubate at 30°C for 4 days.
D. Méthylène Blue Dye Reduction Test The test should be carried out according to the report of the Public Health Laboratory Service (Report, 1947). The test should be set up at 5 p.m. on the day on which the sample is taken. (Any other convenient time may be chosen provided that the test is set up on the day the sample is taken and a continuous incubation period of 17 hours is maintained.) Procedure (a) Deliver 7 ml of quarter-strength Ringer's solution into a sterile tube with 10 ml mark. (b) Add 1 ml of standard méthylène blue solution (as used for dye-reduction tests on milk). (c) Using a wide-bore pipette, deliver ice-cream up to the 10 ml mark, thus adding 2 ml. (d) Insert a sterile rubber bung and invert the tube once. If the ice-cream contains much air the meniscus will fall as the air is freed and if this occurs, fill up with further ice-cream to the 10 ml mark. (e) Set up controls as follows. 1. Ice-cream colour: with a 10-ml graduated pipette, deliver 8 ml of quarterstrength Ringer's solution into a sterile 10-ml mark tube. With a wide bore pipette, add ice-cream to the 10-ml mark as in the test. Insert a sterile rubber bung, invert the tube and fill up with ice-cream to the 10-ml mark if required. 2. Méthylène blue : a méthylène blue control using heated ice-cream should be included in each test. About 5 ml of ice-cream should be heated in a boiling water bath for 15 minutes, cooled and used as in the test proper. 3. For coloured ice-cream, a separate colour control and méthylène blue control should be set up for each sample.
192
LABORATORY METHODS IN MICROBIOLOGY
(/) Incubate the tubes in a water bath at 20 ± 0-5°C, until 10 a.m. on the following morning, i.e. a 17-hour incubation period. (g) Transfer the tubes to a water bath at 37 ± 0-5°C, and invert once every half hour until decolorization is complete as compared with the control tubes. Recording results Record the time taken for decolorization of the méthylène blue. If the méthylène blue is decolorized at the time of removal from the 20°C water bath, the time is recorded as 0 hours. £ . Results and Recommended Standards Report colony count per gram of ice-cream for each group of organisms examined. Davis (1963) suggests that ice-cream should have a general viable count of not more than 10,000 per gram, a count of coagulase-positive staphylococci of not more than 10 per gram, and a coli-aerogenes count of not more than 1 per gram. Suggested grades for ice-cream (Reports, 1947, 1950) Time taken to reduce méthylène blue
Provisional grade
Fails to reduce in 4 hours 2J-4 hours | - 2 hours 0
1 2 3 4