Checked the two MYPGP agar plates from the batch created yesterday that had been incubated overnight at 37C and there was no visible contamination at all this time! It would seem that pouring the plates in the fume hood prevented the contamination that I have been seeing in my previous two batches of MYPGP agar.
Mysterious blob contamination.
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| Blood agar (gamma hemolysis) |
Both the indole test and the blood agar test today implicate Klebsiella spp. as being the contamination culprit.
Moving forward, I performed the BAD germination assay again as well as re-plated the samples from 6-10-14 that had since been stored at 4C.
Repeated procedure from 6-10-14 using Spore Stock A (3.05x10^6 CFU/mL P. larvae spores). Used the MYPGP agar from the most previous batch that had not been found to be contaminated. Drop plates were incubated at 37C overnight not inverted.
Three treatment groups: H2O, BHI+Thiamine (1ug/mg), B.A.D.
Briefly, the Spore Stock A was diluted ten fold down to 10^-4. Each of the ten fold dilutions (10^-1 to 10^-4) were diluted 1:5 in each of the treatment groups listed above in eppendorf tubes. Tubes were incubated at 35C shaking at 150rpm for 2 hours. Samples were blot plated to calculate CFU/mL and also diluted ten fold down to 10^-2 for each dilution. The incubated samples were then heated in a water bath at 65C for 15 minutes and the plating/diluting method was repeated.
//EWW
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