Microbiological Examination of Foods - NCBI

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THE processing and use of frozen food products has increased to large proportions and, with the perfection and availability of freezing anid storage facilities ...
April, 1946

Microbiological Examination of Foods* Tentative Methods for the Microbiological Examination of Frozen Foods Food and Nutrition Section 0

THE processing and use of frozen food products has increased to large proportions and, with the perfection and availability of freezing anid storage facilities, should continue to increase. The following tentative drafts represent methods which have proved workable and, while by no means complete, will form the basis for the perfecting of standard methods for frozen foods. Modifications and improvements will be effected as more experience in this field is gained. In testing frozen vegetables for microbiological content, consideration must be given to the fact that freezing storage at temperatures of approximately -10° F. (-23° C.) brings about a reduction in viable micro6rganisms. This may amount to roughly 50 per cent in 6 months or 75 per cent in 1 year. Thus, while a high bacterial plate count on frozen vegetables may be taken as proof of poor sanitary history, a low or moderate count does not by itself prove the opposite. It is recommended that in the preparation of samples of frozen fruits and vegetables for microbiological analysis a mechanical " blender " be employed. This has been arrived at after considerable experience with various methods of preparation on a wide variety of frozen products. In comparison with or hand grinding the mechaniwashinZ cal disintegration will, as a rule, yield higher bacterial counts. However, more uniform results will be obtained, and the method is less laborious. Simi-

larly the choice of tryptone glucose extract agar rests on the demonstrated superiority of this medium over nutrient agar or glucose agar, although glucose agar as a medium for frozen peas appears to be only slightly inferior.

Examination of Frozen VegetablesSelection, transportation, and storage of samples Select from the lot to be examined a suitable number of packages, say 3 or 4, from each code lot of the pack. Transport in dry ice to the laboratory for analysis and place the samples in a refrigerated (O° F.) storage chest until they. are to be analyzed. The temperature preferably should not rise above 00 F. (-18° C.) in the handling of the material.

Preparation of Sample: A. Peas, Lima Beans, Cut Corn, Whole or Regular Cut Green Beans, etc. 1. Macroscopic examinationOpen the package and note condition of the product, especially if ice crystals are present on the inner walls of the package and the vegetables appear to be somewhat shriveled. This condition is indicative of thawing and subsequent refreezing. Record observations and any abnormalities, such as unnatural color or odor, or pink colonies of torulae which are indicative of improper handling practices. 2. Sampling for plate countsThe sample, if not loose-frozen, should

* Report of the Committee on Standard Methods for the Microbiological Examination of Foods. COMMITTEE ON MICROBIOLOGTCAL EXAMINATION OF FOODS. Organized 1932. Published reports: Year Books, 1935-1936, 1937-1938, 1941-1942, 4.J.P.H., June, 1943; August, 1945.

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be broken up into small units. This can be done by tapping the unopened package sharply against the table edge or by sharply striking the package with a dull instrument, being careful not to break open the package. After opening the package remove sample with sterile spoon, taking the sample from various parts of the broken out package-e.g., from the center and corners. A 50 gm. sample is aseptically weighed into a sterile borosilicate glass mechanical blender jar; 450 ml. of sterile water is added to the jar and the contents of the jar blended for 2 minutes. If a Variac transformer is available it is advisable to increase the speed of the motor gradually and then run the blender for the required time at the 110 V. setting of the transformer. Allow the sample to stand for 2 to 3 minutes to permit foam to subside. Pipette 1 ml. of the mixture into a 99 ml. sterile water blank. Replace the cap on the dilution bottle and shake the bottle briskly 25 times through a 1 ft. arc. Pipette 1 ml. aliquots of this mixture into each of two Petri dishes (1:1,000 dilution) and also 0.1 ml. aliquots into each of two more Petri dishes (1:10,000 dilution). A 1:100 dilution may be obtained by pipetting 0.1 ml. aliquots of the original mixture into each of two Petri dishes. Pour melted tryptone glucose extract agar (pH 7.0) cooled to 450 C. into the Petri dishes immediately, and thoroughly mix the dilution water with the agar by gently rotating the plates in a figure 8 motion with slight tilting of the Petri dish. Cool to harden, invert and incubate at 320 C. for 4 days. Dilutions of 1:100, 1:1,000, and 1:10,000 will usually suffice for commercially packed frozen vegetables, although further dilutions should be made if the history or the appearance of the samples warrant. It is of prime importance that the

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agar be poured immediately after the inoculum is introduced; otherwise many bacteria will adhere to the glass and an inaccurate count will result. After the incubation period is complete the plates are counted, using a suitable colony counter. Results are recorded as " Plate count of microorganisms per gram." 3. Direct microscopic c o u n t method-The direct microscopic method has an advantage over the plate count method in that it is quicker and requires less equipment and glassware. Also it detects dead microorganisms, and indicates sanitary history, irrespective of the viable count. Weigh 50 gm. of the vegetable (peas, lima beans, and cut corn) into a 250 ml. iodine flask. Add 100 ml. water, stopper flask; and shake briskly 50 times through a 1 ft. arc. Using a Breed pipette, transfer 0.01 ml. of the washings to a microscope slide and, using a needle, spread the drop evenly over a 1 sq. cm. area of the slide. Dry and fix with heat or with methyl alcohol. Stain with Gray's double dye stain,' or with North aniline oil methylene blue stain,2 rinse, dry, and examine under the microscope, using oil immersion. Use an ocular micrometer, such as a Whipple disc or Howard disc, with the microscope tube so adjusted that the side of the graduations is equal to 0.1 mm. (area of field 0.01 sq. mm.). Count the cells in 100 fields, and multiply the number by 20,000 to bring to the gram sample basis. Express results as "Direct microscopic estimate, microorganisms per gram." In the direct method the following assumptions are made. (A) That all the cells are removed from the surface of the vegetable by the washing, (B) that the suspension of bacterial cells is uniform, and (C) that the drop of liquid is evenly spread over 1 sq. cm. Interpretation of results - Plate

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counts of more than 400,000 per gram of peas or direct microscopic counts of over 1,000,000 per gram may be considered indicative of poor sanitary conditions in the plant, or poor handling practice in transit or in warehousing. In regard to other frozen vegetables it would appear that counts tend to run higher. In vegetable freezing plants plate counts of over 500,000 per gram of newly frozen produce are not encountered unless there is some degree of carelessness in the plant, such as faulty clean-up practice or prolonged holding of the material after blanching. B. Frozen Spinach For proper comminution of spinach in a " blender " it is necessary to allow the package of spinach partially to defrost by standing at room temperature for 1 2 to 2 hours. Open the package and weigh 50 gm. of the contents into a sterile borosilicate mechanical blender jar. Assemble the sample from various portions of the package, taking care to select petiole and blade portions in about the same ratio as in the sample as a whole. Add 450 ml. sterile water. Blend for 2 minutes, and proceed as in analysis of A, peas, lima beans, etc. C. Frozen Broccoli and Cauliflower Allow partially to defrost at room temperature. Using a sterile scalpel, cut portions from the curd and stem of several representative pieces of the vegetable. Aseptically transfer 50 gm. of these portions into the sterile borosilicate glass mechanical blender jars, add 450 ml. sterile water, and proceed as directed for A, frozen peas, etc. D. Frozen Asparagus iPartially defrost at room temperature. Cut spears into short lengths, using sterilized scalpel. Transfer aseptically 50 gm. of the segments into a sterile borosilicate glass mechanical blender jar, add 450 ml. sterile water, and proceed as in analysis of A, frozen peas, etc. When weighing out sample portions, a number of spears should be

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selected and the proportion of butts and tips maintained. The " cuts and tips " type of asparagus pack can be weighed directly inta the blender cup.

Examination of Frozen Fruits Preparation of Sample Hold the package of frozen fruit at r6om temperature for 1 to 2 hours before opening in order partially to defrost the contents. While the fruit is still partially frozen, cut portions from various parts of the contents of the package, using a sterilized scalpel. The proportion of fruit to syrup should approximate that of the whole package. Weigh 50 gm. of fruit and syrup into a sterile borosilicate glass mechanical blender jar. Add 450 ml. sterile water, and blend for 2 minutes. Make a 1:1,00G dilution by adding 1 ml. of the blended mixture to 99 ml. sterile water and further dilutions in the usual manner. Plate 1 ml. portions from the various dilutions on tryptone glucose extract agar. Incubate for 3 days at 320 C. Colonies are counted under a suitable colony counter. Record results as " Plate count of microoirganisms per gram." Direct microscopic counts for molds and yeasts are made according to the methods given in Official Methods, A.O.A.C.3 for microscopic analysis of tomato products. 'Care must be taken to distinguish between mold hyphae and fruit setae. REFERENCES 1. Gray, P. H. H. Two Stain Method for Direct Bacteria Count. J. Milk Tech., 6, 2:76, 1943. 2. North, W. R. Aniline Oil-Methylene Blue

Stain for the Direct Microscopic Count of Bacteria in Dried Milk and Dried Eggs. J. Assoc. Off. Agri. Chem., 28, 2:425-427, 1945. 3. Official and Tentative Methods of Analysis of the Association of Official Agricultural Chemists, 5th Ed., 1940.

HARRY E. GORESLINE, PH.D.,

Chairman, U. S. Department of Agriculture, Washington, D. C.

Vol. 36

EXAMINATION OF FOODS

M. THOMAS BARTRAM, PH.D. JAMES A. BERRY EDWIN J. CAMERON, PH.D. JOHN L. ETCHELLS, PH.D. MATTHEW E. HIGHLANDS ALBERT C. HUNTER, PH.D.

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LLOYD B. JENSEN, PH.D. CARL S. PEDERSON, PH.D. PAUL S. PRICKETT, PH.D. JOHN M. SHARF, PH.D. OSCAR B. WILLIAMS, PH.D. FREDERICK WV. FABIAN, PH.D., Consultant

LETTER TO THE EDITOR TO THE EDITOR: I have been told that a set of Journals representing the numbers of the American Journal of Public Health published during the war years, which were lost when our Medical Library was burned during the Battle of Manila, are to be given by the American Public Health Association to the Institute of Hygiene at Manila for the use of public health workers in the Philippines. May I express our gratitude to the Association and to its members for

this demonstration of thoughtfulness which is characteristic of the bighearted way your people act. While I am in this country I want to say on behalf of my colleagues in the Philippines that the donation is very gratefully appreciated.

Sincerely, HILARIo LARA, M.D., DR.P.H. Member Philippine Public Health Association and Philippine Medical Association; Director, Institute of Hygiene, Manila, P. I. (The Association continues to receive copies of the Journal made available by members for other libraries overseas like those in the Philippines. The Association is constantly being reminded of the enormous value which such Journals have in devasted countries.)