“Microbiological Challenge Test” for milk

Microorganisms

Are you mostly interested in a challenge test for milk? I saw in Chapter 6, Microbiological Challenge Testing, that the pathogens that may be considered for use in challenge studies for milk are Salmonellae, S. aureus, enterohemorrhagic E. coli (E. coli 0157:H7), and L. monocytogenes. We carry all these organisms. We do not sell one of the organisms recommended. It is Clostridium botulinum. Chapter 6 says that Clostridium sporogenes can be used as a surrogate for C. botulinum for certain applications. We also sell some of the other organisms listed in Chapter 6. They are Bacillus cereus, Clostridium perfringens, Listeria innocua, Shigella species, and Vibrio species. In addition we sell Campylobacter and Yersinia enterocolitica but Chapter 6 recommends using surrogates because these organisms are difficult to grow. Customers may also be interested in Chronobacter muytjensii because it can contaminate infant formula. We also sell organisms that may be considered normal flora in some foods. These organisms can indicate spoilage if found in high numbers. Lactobacillus fermentum and rhamnosus are examples. I made a few microorganism suggestions at the end of this letter. The suggestions are based on the list of pathogens at the end of Chapter 6.

Products

Several different types of products which can be used for challenge testing.

1. KWIK-STIK (P and S), LYFO DISK (V), KWIK-STIK Plus, Lab Elite: For a challenge test, the customer would grow the organism on an agar plate, make a suspension of the organism, calibrate it using a spectrophotometer of turbidometer, make serial dilutions as necessary, and then inoculate his test with the number of organisms which his test calls for. For example, it the customer wanted to inoculate his test with 100-990 microorganisms, he would use a turbidometer to make a suspension of organisms equivalent to a 0.5 McFarland (which is approximately 108 microorganisms). Then he would serially dilute the suspension until he had the concentration he needed in order to inoculate his test with 100-990 microorganisms. It would be much easier to perform the test with an enumerated product but some organisms may not be available in an enumerated form. Also, some customers may prefer to use fresh instead of lyophilized microorganisms.

The article from the Journal of Food Protection says, “When reviving strains from the frozen or lyophilized state, there should be one to two successive transfers in a non-selective growth medium.” Directions for maintaining organisms can be found on the Microbiologics website. If the customer wants a non-enumerated product, it might be best to use KWIK-STIK Plus (X) or Lab Elite (CRM) microorganisms because the journal says, “ The number of times a culture is transferred to product new working stock cultures should be minimized to avoid genetic changes…APAC International guidelines for laboratories indicate there should be not more than five passages from primary reference material. In order to keep passage numbers down, the customer can use KWIK-STIK Plus (2 passages) or Lab Elite (PFGE shows that it is equivalent to the original ATCC strain.) The article you sent says , “Biochemical characteristics, serology, genetic profile, virulence, or toxicity should be periodically reconfirmed as appropriate.” Genetic information is included with the Lab Elite product. The Lab Elite product has a unique genetic profile so it can be distinguished from other strains of the same species. http://www.microbiologics.com/Products/Lab-Elite If a laboratory is ISO:17025 accredited, they should use certified reference material (Lab Elite) when available. Sometimes you may prefer lyophilized organisms. The Journal article says, “A dry inoculum may be required for studies in low-moisture foods or when added moisture needs to be avoided. Inoculum can be prepared by freeze drying.”

2. EZ-FPC: EZ-FPC products are available at concentrations of 100-990 cfu per pellet (qualitative FPC) and at 1000-9900 (quantitative FPC) cfu per pellet. They are sold 10 pellets to a vial. They do not come with hydration fluid. They come with a Certificate of Assay. The following is a link to the product list. http://www.microbiologics.com/Products/EZ-FPC Just click on the orange box at the bottom of the page to see the list of microorganisms.

Qualitative EZ-FPC – 100-990 cfu per pellet
In the article you sent me from the Journal of Food Protection (Parameters for Determining Inoculated Pack?Challenge Study Protocols) it says under 5.1, Growth Studies, “When conducting studies to determine whether a pathogen grows in a product, ideally the number of organisms used should reflect the numbers normally expected in the product. Typically, an inoulum level of between 2 and 3 log CFU/g is use, even when this exceeds expected numbers because this level allows enumeration by direct plating. ” Our qualitative FPC product would meet this requirement because it ranges from 100 to 990 cfu per pellet.

If a Very Low Number of Organisms is needed: <100 cfu per inoculums.
If the customer wants to use a very low population of cells (<100 cfu per sampling unit), he can either dilute the EZ-FPC product further or use a product such as EZ-Accu Shot (A), EZ-CFU One Step (Z), or EZ-CFU (C). These 3 products deliver less than 100 cfu per inoculums when used as directed. Customers should always test the organisms on nonselective agar as well as selective agar because the organisms grow best on nonselective agar. Non selective agar is the control and is used to determine the number of cfu in the inoculum.

Quantitative EZ-FPC – 1000-9900 cfu per pellet
These products are used when testing the accuracy of one’s counting methods. A laboratory may perform counts when testing the shelf life of a food product.

When a laboratory performs a challenge test by inoculating a food product with a microorganism suspension, they should concurrently test the suspension on non-selective agar in order to determine the number of cfu per ml of inoculums.

The article that you sent me from the Journal of Food Protection, says in 4.3 that challenge studies should generally be conducted using three to five bacterial strains either individually or in combination (a cocktail). Your customers will probably want to purchase more than one strain for a study.

3. Epower: Epower products are sold as a vial of 10 pellets at a specific concentration for example, 104, 106, 108). They come with a Certificate of Assay. The following is a link to the product list. http://www.microbiologics.com/Products/Epower Just click on the orange box at the bottom of the page to see the list of microorganisms.

Epower products can be used in inactivation studies because (according to the journal article) “high numbers of organisms are typically used, e.g. 6 to 7 log CFU/g. the target level of reduction, which influences the inoculums level used, may depend on the regulations for specific food types, e.g., a 5-log reduction of the appropriate pathogen in juice (21CFR 120.24)”

We sell some strains of organisms at high concentrations. If we do not have the microorganism which the customer needs at a high Epower concentration, you can fill out a Lyophilized Microorganism Request Form. When the demand for a certain product is high we try to add it to our catalog. An alternative is to ask the technical support department what the approximate concentration of an organism is. Another solution is to purchase the KWIK-STIK or LYFO DISK and make an inoculom from freshly grown organisms.

4. Lab Elite: The article you sent says , “Biochemical characteristics, serology, genetic profile, virulence, or toxicity should be periodically reconfirmed as appropriate.” Genetic information is included with the Lab Elite product. The Lab Elite product has a unique profile so it can be distinguished from other strains of the same species. http://www.microbiologics.com/Products/Lab-Elite If a laboratory is ISO:17025 accredited, they should use certified reference material (Lab Elite) when available.

Microorganism	Reference culture #	Catalog #
Bacillus cereus	ATCC 10876	0998 P, S, V, X, FPC (103), E3, C, Z, CRM
Bacillus cereus	NCIMB 7464	0330 P, S, V, FPC (103), E3
Clostridium perfringens	ATCC 13124	0318 P, S, V, E3, Z, CRM
E. coli O157:H7 (requires special paperwork to send outside the USA)	ATCC 35150	0617 P, S, V, X, FPC (102)
Listeria monocytogenes	ATCC 19115	0687 P, S, V, X, FPC (102), E2,E3, C, Z
Listeria monocytogenes	SLR2249	0254 P, S, V, FPC (102)
Listeria innocua	ATCC 33090	0814 P, S, V, FPC (102)
Salmonella bongori	ATCC 43975	0595 P, S, V, FPC (102)
Salmonella enterica subsp. enterica serovar Abaetetuba	ATCC 35640	0817 P, V, S, FPC (102), E2
Salmonella enterica subsp. enterica serovar Abaetetuba	SLR156	0826 P, V, S, FPC (102)
Staphylococcus aureus	ATCC 25923	0360 P, S, V, X, FPC (103), E3, E4, C, Z, CRM
Staphylococcus aureus	ATCC 6538	0485 P, S, V, X, FPC (103), E3, E7, C, Z, A, CRM, EZ-PEC (includes hydrating fluid, pellets contain 2.0 – 9.9 E+07 cfu per pellet)
Vibrio parahaemolyticus	ATCC 17802	0818 P, S, V

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