MFO-10 Microbiological Examination of Frog Legs
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E95195BC802C445C863641BA6BF5B7C4 |
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0.03 |
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页数： |
7 |
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日期： |
2012-3-2 |
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Published by: POLYSCIENCE PUBLICATIONS, P.O. Box 1606, Station St-Martin, Laval, Quebec,Canada H7V 3P9. TEL.: 1-800-840-5870. FAX: (450) 688-1930.,Government of Canada Gouvernement du Canada,Official Method MFO-10,November 30, 1981,HEALTH PROTECTION BRANCH,OTTAWA,MICROBIOLOGICAL EXAMINATION OF FROGLEGS,1. APPLICATION,This method shall be used for the determination of the bacteria of the genus Salmonella in froglegs in,accordance with Section B.21.031 of the Food and Drug Regulations.,2. SAMPLING,2.1 Definition of Terms,2.1.1 Lot: A batch or production unit which may be identified by a code. When there,is no code identification, see Table II,2.1.2 Sample: The sample units (subsamples) taken per lot for analysis.,2.1.3 Sample Unit: A minimum of 25 g of whole froglegs. A sample unit is often referred,to as a subsample.,2.1.4 Analytical Unit: That amount of product withdrawn from the sample unit for analysis.,Here, it may be equal to the sample unit.,2.2 Collection of Samples,2.2.1 A sample, consisting of five sample units drawn at random from a lot, shall be taken.,(Table I),2.2.2 Each sample unit shall consist of at least 25 g of whole froglegs.,2.2.3 Employ aseptic techniques in collecting the sample units.,2.2.4 Place each collected sample unit into a separate sterile container.,2.2.5 Keep sample units frozen during transport.,MFO-10,November 30, 1981,- 2 -,3. PROCEDURE,The five sample units shall be analyzed individually or as one or more composite(s) for determining the,presence of bacteria of the genus Salmonella.,The test shall be carried out in accordance with the following instructions:,3.1 Handling of Sample Units,3.1.1 Keep sample units frozen in the laboratory prior to analyzing them.,3.1.2 Analyze sample units as soon as possible after receipt at the laboratory.,3.2 Preparation of Media,The following media, to be prepared and sterilized according to the manufacturer's,instructions, shall be used:,(1) Nutrient Broth (NB),(2) Selenite Cystine (SC) broth,(3) Tetrathionate Brilliant Green (TBG) broth,(4) Bismuth Sulfite (BS) agar,(5) Brilliant Green Sulfa (BGS) agar,(6) MacConkey Agar (MA),(7) Triple Sugar Iron (TSI) agar,(8) Lysine Iron (LI) agar,(9) Christensen's Urea (CU) agar,(10) Nutrient Agar (NA),3.3 Non-Selective Enrichment (Pre-enrichment),3.3.1 Thaw frozen sample units. Do not allow the temperature of any sample unit to exceed,45oC.,3.3.2 Place a minimum of 25g of whole froglegs (analytical unit) from each of the sample,units into a separate sterile container.,3.3.3 Alternatively, composite the analytical units and place each composite into a separate,sterile container.,Since compositing presents difficulties in preparation and disposal of the material,exercise care in handling bulk preparations.,3.3.4 Add sufficient NB to cover the froglegs.,3.3.5 Shake the container(s) to unite the contents.,3.3.6 Check the pH of each suspended analytical or composite unit. If the pH is outside the,range of 6.0 - 7.0, adjust to 7.0 with either sterile NaOH or HCl.,3.3.7 Inoculate NB with a known culture of Salmonella and subsequently make transfers to,all other media used in the analysis. This is the positive media control. Set up a,negative control by incubating appropriate uninoculated media during each step of the,analysis.,3.3.8 Incubate the inoculated pre-enrichment broth(s) and the controls at 35oC + 0.5o for,18-24 hr. In no circumstances shall the incubation be prolonged for more than 24 hr.,MFO-10,November 30, 1981,- 3 -,3.4 Selective Enrichment,3.4.1 Transfer 1 ml of the incubated pre-enrichment broth into each of 9 ml of SC and TBG,broths using a sterile pipette.,3.4.2 Incubate the SC broth and TBG broth for 24 + 2 hr at 35oC + 0.5o and at 43oC + 0.5o,respectively.,3.5 Selective Plating,3.5.1 After the incubation period, streak a loopful of each of the selective enrichment broths,onto BS agar and BGS agar plates to obtain well-isolated colonies. The broths may,also be streaked onto a third commercially available plating medium.,3.5.1.1 It has been observed that BS agar is inhibitory for Salmonella serotypes,other than S. typhi unless it is refrigerated at 4oC for at least 24 hr before,streaking. The possibility of an inhibitory effect of this medium should be,taken into consideration.,3.5.2 Incubate the plates at 35oC + 0.5o for 24 + 2 hr. It may be necessary to incubate the,BS agar plates for 48 + 2 hr.,3.5.3 Examine the plates after the incubation period for colonies indicative of Salmonella.,On BGS agar, such colonies are pink to fuchsia surrounded by red medium. On BS,agar, they are usually black, with or without a metallic sheen; with increasing time of,incu……
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