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SAMPLING, PREPARATION AND SELECTION OF DILUTION, SPC, COLIFORM AND YEAST AND MOLD COUNT
S. S. SANNABHADTI e-mail address: sssannabhadti@yahoo.com Why Microbiological Analysis? To ensure safety, to indicate shelf life, to validate of process, to know employees carefulness and indicate efficiency to know whether it meets legal requirement the analysis is required. How tests are selected? It depends on its relevance to the product and the information required to be gathered. How to conduct the tests? Tests are conducted on samples Microbiological testing is destructive and microbes are dynamic in nature. What is a sample? Sample should be representative of the lot. Can it is to be achieved in all dairy products? No. So we have have standard methods and random sampling for a large lot of the product. What are the requirements of microbiological sampling? Peculiarities and nature of microbiological testing Contamination possibilities while sampling, growth of microbes during storage and/or transportation of sample, injured microbes in sample and difficulties in their recovery, reliability and relevance of test results with the information needed. How to prepare dilution and how dilution required is diluted? Dilution is done in a buffer like phosphate buffer or ringers solution or phosphate water or citrate buffer. It is based on the nature of the product. The flasks or tubes selected should have correct volume and pH. Temperature is maintained around 45C. The dilutions are based on the counts? In ice cream or paneer where suggested counts are not to exceed for plate count 2,50,000/g and 5,00,000/g, the dilutions chosen are 2 and 3 and 3 and 4, respectively. And plates are made in duplicate. In case of coliform count plates are prepared with first dilution 1 ml in duplicate or three plates of 3, 3 and 4 ml quantities of first dilution can be taken for counting in ice cream and paneer as the standards suggested are not more than 100 per gram and 90 per gram, respectively. Usually plates with 30 to 300 counts for total count and 10 to 150 for coliform count are chosen for counting.
Why total bacterial count, coliform count and yeast and mold counts are chosen in some products and in some only total bacterial and coliforms and in fermented products like dahi, chakka and shrikhand it is only coliform and yeast and mold count? It depends on the nature of the product, its environmental conditions and the relevance of the count as it has to give information on safety, shelf life, hygienic quality and has to satisfy legal requirements. It is necessary to keep control plate with each lot of plating. Asepsis during preparation of sample for dilution, preparation of dilution and plating is necessary. Each lot of medium should be compared with the previous lot for repeatability and accuracy. Manufacturers instructions in preparation and use of media is to be followed meticulously. When you are using incubators accurate of temperature maintenance and humidity maintenance is required. The plates should not be stacked too high and should be kept inverted during incubation. Check the accuracy of the thermometer in the incubator. If you are formulating and preparing the medium in the laboratory the ingredients should be weighed properly and also should be stored properly. The medium which is not being used immediately should be stored in refrigerator for not more than one to two weeks. In case it is stored for long either at room temperature or at refrigerated condition it may dry if it is not in air tight containers. If it is exposed to light and oxygen it may affect growth factors and dyes in the medium. This should adversely affect the medium performance and sometimes its selectivity. What is the information gathered by total bacterial count, coliform count and yeast and mold count? Total bacterial count indicates the influence of heat processing and survivors of processing, recontamination and likely influence on shelf life. The keenness of the observer, his ability to differentiate the colonies can be helpful in giving interpretation of the test results. Coliform are intestinal bacteria and gives indication on hygienic conditions of manufacture and storage. They do not survive pasteurization. It has some relevance with safety aspects as it has intestinal origin. It also indicates about adequacy of pasteurization treatment. Most of the yeast and molds occurring in milk do not survive pasteurization. So in products usually it gains entry from contaminated surfaces or through air. In long life products, acidic products and low water activity products, it has relevance with shelf life. It also indicates on air quality. I acknowledge the help given by the staff in Dairy Microbiology Department, especially Dr. J. B. Prajapati and Dr. R.K Shah for providing these material and also staff of Vidya Dairy for making it available to you. I thank Vidya Dairy authorities for giving me the opportunity to interact with you
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Coliform Count Standard Plate Count Coliform Count Coliform Count Yeast and Mold Count
IS:3509-1966 IS:13690-1992
4 5 6 7
Coliform Plate Count Coliform Count Standard Plate Count Coliform Count Total Bacterial Count Coliform Count
8 9 10
Yeast and Mold Count Coliform Count Yeast and Mold Count Total Bacterial Count Yeast and Mold Count Total Bacterial Count Yeast and Mold Count
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12
Paneer
Coliform Count Total Bacterial Count Yeast and Mold Count Coliform Count
Microbiological (Continued)
Sr. No. 13 14 15 16 17 Dairy Product Rasogolla Syrup Dahi Chakka Shrikhand Extra Powder Grade
Standards
Tests Recommended Plate Count Coliform Count Coliform Count Yeast and Mold Count Coliform Count Yeast and Mold Count Coliform Count Yeast and Mold Count Total Bacterial Count Coliform Count
for
Selected
BIS Standards 500/g Nil/g Not more than 10/g Not more than 100/g Not more than 10/g Not more than 20/g Not more than 10/g
Dairy
Products
BIS Bulletin IS:4079-1967 IS:9617 -1980 IS:13689- 1992 IS:2802- 1964 IS:1165 - 1992
Not more than 50/g Not more than 40,000/g Negative in 0.1g Absent in 0.1g Absent in 25g Not more than 50,000/g Not more than 90/g 75 million to 200 million/g
Coagulase Positive Staphylococcus aureus 18 Milk Powder, Dried Ice Cream Mix, Partially Skimmed Sour Milk Powder, Sweet Cream Butter Milk Powder Infant Foods and Processed Cereal Weaning Foods Salmonella and Shigella Total Count Coliform Count DMC (Dried milk)
Milk powder Second revision IS:1165 1975 Dried Ice Cream Mix IS:7839 1975 Sweet Cream Butter Milk Poder IS: 5163 - 1969 Infant milk foods IS:1547 1968 (First Revision) Malted Milk Foods IS:1806 1975 (First Revision) Processed Cereal Weaning Foods IS:1656 - 1969
19
50,000/g 10/g
Microbiological (Continued)
Sr. No. 20 Dairy Product
Standards
for
Selected
Standards
Dairy
Products
Whole Milk Powder, Skim Milk Powder, Infant Milk Powder, Malted Skimmed Milk Food, Malted Milk Food and Malted Milk Food Containing Cocoa Powder
P F A Standards