4.Precautions and common problems during testing:
1) Both pre-enrichment and secondary enrichment are essential processes.
The content of salmonella in food is generally very small and there are many kinds of bacteria at the same time. The pre-enrichment can help the injured bacteria to be repaired, while the enrichment can inhibit the growth of some miscellaneous bacteria.
2) In the separation culture, it should be noted that when there are no typical or suspected salmonella bacterial colonies, the following steps should be followed to test the atypical salmonella bacterial colonies.
HE and XLD: atypical salmonella present yellow colonies on HE and XLD AGAR, with or without black centers. Two or more atypical salmonella colonies were selected after HE and XLD were cultured for 24 + / -2 hours without typical salmonella colonies.
BS AGAR: some atypical strains produce green colonies with little or no dark surrounding medium. If there are no typical colonies after 24 + / -2 hours of BS plate culture, no colonies will be selected for the plate culture for 24 + -2 hours. Two or more atypical colonies were selected if no typical or suspicious colonies were found after 48 + / -2 hours of culture.
3)TSI should be prepared as a high column incline, which is conducive to the interpretation of the results. In the experiment, it should be underlined before puncturing.
If H2S are blackened, it is difficult to observe the acidification on the bottom layer, and if more gas is produced, the medium will be pushed out very high, or even the tube will be pushed open. The typical salmonella bacteria on TSI have a red inclined surface, a yellow bottom, gas production, 90% H2S formation, and black AGAR. But lactose positive salmonella also has a yellow inclined surface, so salmonella cannot be identified based on the results of iron trisaccharide culture alone.
4)At present, no single medium can accurately screen salmonella, so multiple selective media must be selected for simultaneous screening.
The chromogenic medium was only more selective, and the experimenter could not report the result only if the selective medium and TSI characteristics were consistent without completing the critical biochemical reaction and serological identification, which might lead to false positive results.
5) Serological identification is an important method for the identification of salmonella.
That includes the antibacterial antigens (O), the trichogenic antigen (H) and the Vi antigen identification. Pure bacteria within 24 hours should be used for serum detection, the results should be observed within a specified time, and self-coagulation experiments should be conducted. If the coagulation is not good at room temperature (do not put the refrigerator), it may be better if it is kept at room temperature for 1-2 days or if it is passed through the generations again. If laboratory conditions permit, it is recommended to purchase imported serum (preferably from Thailand and Denmark), and if domestic serum is used, try to use both manufacturer's products to verify each other.
6) The determination of salmonella results should be based on the biochemical test results and on this basis the serological judgment should be made.
It is absolutely not advisable to test directly with polyvalent serum without biochemical test. Since serology is based on antigen antibody binding, non-salmonella microbes may also carry salmonella antigens similar to salmonella, which may lead to false positive results. Therefore, when we make identification, we must first conduct biochemical test, and then conduct serological identification on the basis of biochemical test.
7)If we only need to determine whether A particular strain belongs to salmonella, we only need to make O polyvalent and H polyvalent serum (most foodborne salmonella agglutinate A-F group O polyvalent).
If you want to identify what is a strain of concrete salmonella, it needs to test with serum credits, from polyvalent serum do O antigen and single factor H antigen, and with reference to the 4789.4 GB - 2016 national food safety standards of food microbiology inspection salmonella "appendix B of table lookup corresponding salmonella.
8)In serological identification, the O polyvalent serum cannot be directly determined as negative without agglutination, because the effect of Vi antigen has to be considered.
Vi antigen belongs to K antigen group, which will block the binding of O antigen and corresponding antibody. Therefore, when Vi antigen exists, O polyvalent serum will not agglutinate. Therefore, the effect of Vi antigen must be removed. The specific method is to take the fungus moss and make a concentrated bacteria solution in 1ml physiological saline, boil it on the flame of alcohol lamp, and then check if it is still not agglutination, then it can be determined as negative.
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