I have tried numerous times to get plate counts of inoculated D/E Broth with B. subtilis.
Let me start with my procedure. I make a test suspension from a 24 hour culture of B. subtilis on a TSA slant. I make the test suspension using 8.5 % physical saline 10 ml. I then make a serial dilution and use 10^-7 dilution which I have found to be the right dilution. When I plate the suspension directly onto the Letheen agar I get perfect spread of colonies in the 50 cfu/ml range. When i Inoculate D/E Broth with the same suspension and plate it over night I get cloudy grouped colonies. I have attached pictures of the Test suspension plates and the broth + Innoculum.
Is anybody familiar with this problem I am having, And what can I do to remedy the issue? I am doing method suitability to test finished cosmetic products. I have not been able to move forward with this specific procedure.
Usually, 0.85% saline is used for resuspension (0.85g/100mL). If you are using 8.5% saline, maybe the high sodium content of D/E is pushing them over the salt tolerance limit.
I meant 0.85% saline. The D/E broth does seem to have a fair amount of sodium based compounds for neutralization. Could that still possibly be the issue? Should I try using something else for resuspension?
If you need to incubate in D/E to neutralize antimicrobial compounds, can you make the final plating dilutions in saline? I don't know what effect the plating the D/E on Letheen would be. Could it be creating a weird surfactant effect that permits more motility?
Kathryn Marjorie Jones I was just thinking when i autoclave my test tubes with the saline I lose about .6 ml of water. I wonder if that increases the salinity to high.My salt loving staph always gives me perfect colonies. The increased motility was what Ive been thinking could be the issue. Thank you for your assistant.
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