Bacillus and Clostridium species are capable of producing spores. A spore analysis can sometimes be a difficult challenge.
In most analyses, a heat treatment is necessary. The heat treatment is different per method and will kill the vegetative cells in the sample.
In the case of the thermoduric analysis, it kills the not heat resistant cells.
The heat treatment is crucial, and different aspects of the analysis are essential. In this article, we explain the impact of the different elements.
1. Method
- Choose the correct method.
There are different methods available. In the table below, you will find some examples:
| Spore analyse | Standard | Matrix | Heat treatment – °C | Heat treatment – Min. | Suitable CRM |
| Clostridia | ISO 6461-2 | Water | 75 ± 5 | 15 | CRM-CPE.00007BL |
| Clostridium perfringens | ISO 14189 | Water | 60 ± 2 | 15 ± 1 | CRM-CPE.00007BL |
| Clostridium spp. | ISO 15213-1 | Food | 80 | 10 ± 1 | CRM-CPE.00007MH |
| Geobacillus stearothermophilus | NEN 6809 | Milk and Milk products | 100 | 30 ± 1 | CRM-GSTEA |
| Mesophilic aerobic spores | NEN 6813 | Milk and Milk products | 80 ± 1 | 10 | CRM-BCE.MH, CRM-BSUBT |
| Thermoduric micro-organisms | NEN 6807 | Milk and Milk products | 63,5 ± 0,2 | 30 | CRM-BSUBT |
| Thermoduric streptococci | NEN 6808 | Milk and Milk products | 63,5 ± 0,2 | 30 | CRM-STHER |
2. Dilution fluid
- Be aware that the dilution fluid can influence the spore analysis. If you find too few spores, use another dilution fluid to notice the difference.
- Perform the heat step immediately after making a dilution. If you wait too long, there will be a decrease in spores.
- Note for Clostridia spores analyses in food:
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, carrying out the heat treatment with a dilution not higher than 10x is recommended.
3. Heat treatment
Perform the heat treatment correctly. Pay attention to the following aspects:
- Be aware that the temperature of the water is the right temperature. Use a calibrated thermometer.
- Ensure enough water is in the bath, so the sample you want to heat is sufficiently submerged.
- Run a control tube with the same amount of liquid and temperature during the analysis. Insert a thermometer here and start the heat treatment when this tube has reached the correct temperature.
- Do not put too many samples together in the water. This can cool the water too far down.
- Use a timer to set the heating time, and replace the samples in a cooling bath immediately after heating so that the heating does not continue too long.
- Read the standard for specific details in the heating treatment.
4. Culture Media and incubation
- Use media that works correctly and is tested according to ISO 11133.
- Use the correct supplement if necessary.
- Incubate under the right conditions and atmosphere
- At high incubation temperatures, ensure the culture plates do not dry out.
5. Certified Reference Material
Use a suitable Certified Reference Material as a positive control to ensure the method works correctly.
