P. larvae germination in BAD medium assay

I performed a Gram stain of the mysterious blob and it revealed a large number of Gram negative (pink) rod shaped bacterium. This was what was believed, but still doesn't necessarily narrow down the possibilities. The reason I performed a Gram stain and am so interested in identifying what the contaminant is is because I was worried it was my P. larvae that was the culprit.

Gram Stain Procedure

60 seconds Crystal Violet
60 seconds Iodine
10 seconds Ethanol
30 seconds Safranin

There were a few areas of the Gram stain that appeared to have Gram positive (purple) rods, but due to their clumping and vicinity they were likely really Gram negative. Regardless, I created crude genomic lysates of three separate contamination zones and performed the AFB PCR amplification. The results (not pictured) did not implicate these mysterious contamination blobs to be P. larvae.

Discussed with Dr. Gibbs and Heather V. about the contamination issues with my MYPGP agar. Upon inspection of the contamination, the culprit was believed to be Klebsiella spp. due to the slimey nature and morphology.

I isolation streaked a loop full of a mysterious blob onto a MacConkey and Blood agar retrieved from the Gibbs lab. Plates incubated at 37C overnight. The results of this will further identify the bacteria.

Performed Indole and oxidase tests:

Oxidase Test: Top right = mystery blob, top left = Klebsiella aerogenes, bottom right = E. coli B/r

Indole Test: Mystery blob,  Klebsiella aerogenes,  E. coli B/r

Mystery blob was found to be Indole negative and Oxidase positive. However, I am not too confident about the oxidase test (I have never very good luck with performing or concluding that test). 

Indole : Positive = no color change

            Negative = color change to red

Oxidase: Positive = purple color change

              Negative = no color change

Klebsiella aerogenes is:

Catalase positive

Indole negative

Non-motile

Oxidase negative

Note: not all Klebsiella spp. display these tendencies.

I will check the MacConkey and Blood agar plates tomorrow and further conclude what this mystery bacterium is.

Made up a new batch of MYPGP agar using Fisher Lab reagents (0.5 Liter), this time the plates were poured in the fume hood after it had been thoroughly sterilized. Two of the plates were placed in the 37C incubator and will be checked tomorrow for contamination. The remaining plates were stored in the cold room. 

//EWW