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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?

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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:

Sporulation

  1. Overnight culture

    • Inoculate your culture of Bacillus subtilis in 4 ml LB-medium
    • Let them grow overnight at 37°C, 200 rpm
  2. Exponential growth

    • 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)
  3. Sporulation

    • 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)
  4. 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
  5. Additional:

    • 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.

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    $\begingroup$ Thank you, but i have already a culture of b.subtilis spores (10^8), i have only to put into milk. Then this milk (+spore) will be treated (temperature etc) to try to kill the spores. After treatment i have to.count how much we have reduced the number of spores. I put 1 ml for inclusion in petri plate with pca+skim milk. Now, how i count spores? As a common colonies? Or are different? If there are spore and germinative in the same plate how i recognize spores? $\endgroup$ – StarChips Oct 9 '17 at 18:20
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    $\begingroup$ Ah, OK. I'm with you now @StarChips. I don't think there's a way to detect spores as such on agar plates. They are easy to small. You would rather operate on the assumption that all the colonies that you can see on your PCA plates stem from spores. Given that you only inoculate with spores. So at the end you just count the colonies. $\endgroup$ – Johnny Oct 9 '17 at 18:41
  • $\begingroup$ Yes, but i don't know if the condition of treatment could cause sporulation. I want to be sure to count only spores $\endgroup$ – StarChips Oct 9 '17 at 18:49
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    $\begingroup$ Chances are that the treatment will cause sporulation given that some live, vegetative Bacillus subtilis cells are present in the sample. If you really want to be 100% certain that all you count are spores or colonies from spores then you have to go through far more effort. This can't be done on agar plates only. Unfortunately this article is behind a paywall but in its abstract it outlines a wee bit what is required to get an accurate answer on spore counts in your sample: sciencedirect.com/science/article/pii/… $\endgroup$ – Johnny Oct 10 '17 at 10:37

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