I need to inoculate spores into milk and then count on the plate. I never see a spore, so how i recognize it from the germ cell? does anyone know the procedure and if there are differences from a normal count in agar colonies?
If you need to inoculate milk with a known number of Bacillus subtilis spores then you will have to go follow a "sporulation" protocol - (going by your question I am not 100% certain whether this is what you want to do).
However, please find a sporulation protocol, developed by the Universities of Bonn and Freiburg, below:
- Inoculate your culture of Bacillus subtilis in 4 ml LB-medium
- Let them grow overnight at 37°C, 200 rpm
- Measure the OD600 of your overnight culture and dilute it in LB-medium to an OD600 off around 0.1-0.2/ml in 10 ml LB
- Let the cells grow to an OD600 of 0,8/ml (37°C, 200 rpm)
- Centrifuge 10 ml of the cells at 13,000 x g for 1 minute
- Wash the pellet with 1 x PBS
- Re-suspend the pellet in 5 ml DSM (Difco Sporulation Medium)
- Let the cells grow for 24 hours at 37°C (200 rpm)
Lysozyme treatment (additional for spore purification)
- Treat the samples with lysozyme (15 mg/ml) at a dilution of 1:6
- Incubate for 1 h at room temperature
- Wash 6 times with 1 x PBS
- count spores using a Neubauer improved counting chamber and make aliquots with a defined number of spores per aliquot (e.g. 100 Million spores per 500 μl)
(Note: always use fresh DSM since the FeSO4 is rusting when it is in dilution)
May I make special mention of the Bacillus subtilis Manual here which was put together by the iGEM team and has proved as an excellent resource. The protocol above was taken from it.
Once you have your spore counts you can then inoculate the milk to your liking. However, please be aware that milk, unless completely pasteurised prior to inoculation, is not a sterile product. This means that other organisms might grow on whatever agar you decide to plate out on.