If you do not find black colonies, or fewer colonies, on the agar plate by the analyses of Clostridium perfringens or Sulfite-reducing bacteria, it can have several causes.
Checking the below items in your laboratory to solve the issue.
1. Media and filter (for water analysis) must be fit for purpose
For food analyses, these performances and criteria are:
ISO 7937 Media performance test TSC (SC)
ISO 15213-1 Media performance test ISA (IS or ‘TS’)
For water analyses, these performances and criteria are:
ISO 14189 Media performance test TSC
ISO 6461-2 Media performance test Sulfite Iron – Tryptose Sulfite (TS)
Did you perform a membrane filter validation according to ISO 7704?
The combination of media with filter must give a good performance.
If the media and or membrane filters are not fit for purpose, try to use a different batch of media or filter. Or try another media of membrane filter supplier.
Media from supplier to supplier can give different results in appearance and productivity.
For example:
CRM-CP was analyzed simultaneously on TSC (SC) media of 3 different suppliers according to ISO 7937.
The appearance of the TSC (SC) agar was different. One media, the Clostridium perfringens, is more enthusiastic with the black coloring than the other.
See the examples below of 3 different media suppliers.
Colorless colonies on ISA – a practical example
The reference material CRM-CP on ISA medium incubated for 24h at 37°C anaerobic gave no black colonies by a specific media lotnumber. However, not all the bottles of ISA give colorless colonies.
Some bottles are given black, and others of the same lotnumber of ISA give colorless colonies.
Another laboratory also found this phenomenon using the same ISA with the same lot number.
Inquiries with the supplier about the cause of this phenomenon could not find an explanation.
2. Media overlay for better black coloring
Cultures not overlaid with agar will have no or poor black colonies despite incubation under anaerobic conditions.
Some examples you will find below.
TSC (SC) with and without an overlay
TSC with and without an overlay
Sulfite Iron-Trypose Sulfite (TS) with and without an overlay
3. Incubation must be under anaerobic conditions
The incubation must be performed under anaerobic conditions. Ensure incubation conditions are correct.
You can check the anaerobic jar with an anaerobic indicator strip.
4. Heat treatment for the anaerobic spore in food analyzes
CRM-CPE.00007MH contains viable spores that survive a heat treatment of the first 1:10 dilution.
It turns out that when a higher dilution is pasteurized, the spores have a lower yield.
Therefore, it is recommended to carry out the heat treatment with a dilution not higher than 10x.
5. Diluent used in the heat treatment is crucial for a good yield by spore in water analyzes
Dilutions of CRM-CPE.0007BL is often made in water. Research has shown that different types of water can give different yields.
By Biosisto, softened water gave a significantly lower yield of spores. It is recommended to check with your laboratory whether this is the case.
