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HELP with microorganism identification/colony isolation!!?
In class I was assigned a TSB containing a mix of two organisms. I streaked this mix onto a TSA plate, a CNA plate, and a MAC plate. After 48 hours I observed growth on both the TSA and MAC plates, but not on the CNA plate.
Although the colonies on the TSA plate looked very similar, I thought I had identified two that were distinct and so I made a Gram stain of each.
The first stain went fine. The organism was clearly a pure sample of gram negative rods. HOWEVER, the stain of the other colony showed both gram negative rods and gram positive cocci! I thought I had somehow made a mistake so I repeated the stain using another colony resembling this one and arrived at the same result!
I am totally lost as to why there is a mix of organisms in this colony. Shouldn't a colony be purely one type of bacteria? I am also confused as to why there was no growth on the CNA plate when there appears to have been some Gram positive bacteria in my TSB mix of unknowns.
Any thoughts???? I would be really impressed if someone had a theory as to what's going on here!!


The best thing to do is pick one colony of each type and streak to another blood plate. Did you have two morphologies on the MacConkey also? Gram positives won't grow on Mac.

One thing beginners do is trail their loops instead of merely touching a colony. If you did not streak for isolation really really well, you may have gram positives underneath the gram negatives, although I have no explanation why these didn't come up on CNA.

Just out of curiosity, did you incubate in 5% CO2? Some gram positives, particularly Strep. pneumoniae, require CO2 for growth, which may be why your CNA was NG. Not sure how esoteric your unknowns are.

Did you have two morphologies on the MacConkey? Did you gram stain the TSB broth to see if you have gram positives in there?

You can pick your "mixed" colony and subculture it to TSA, Mac, and CNA. Be sure to flame your loop after the first turn. Streaking plates is somewhat of an art form. All of the techs I work with have different ways of streaking to accomplish isolation. I know that I streak differently depending if I'm picking a colony or streaking initial plates for isolation. Just remember: the heavier the concentration of bacteria, the more carefully you must streak. The best streaking, in my opinion, is a four quadrant streak.

You should be picking your colonies from the last two quadrants, preferably the last quadrant. Just touch the colony. Don't drag your loop.

Good luck.

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