PLEASE HELP ME WITH MY MICROBIOLOGY UNKNOWN BACTERIA PROJECT

[xohailey]

I really appreciate any help I can get - experienced answers only please!

 

OK here is the deal - my lab professor mixed two bacteria in a broth tube, one is gram negative, one is gram positive. Before running tests on the two separate bacteria, I need to isolate them into an "Uknown A" and a "Unknown B" - after they are isolated we are then supposed to take the isolated bacteria and put each in a agar slant tube.

 

The first day of this project I put my sterilized loop in the broth - and then did a streak plate on each of the following agar dishes: mannitol salt, EMB, and TSA. I then put them in the incubator - on Wednesday I returned to lab, the results were:

 

TSA Plate - growth, white colonies, also 2 yellow dots (is this contamination?)

Mannitol Salt Plate - NO GROWTH

EMB Plate - growth

 

*** EMB plates only grow gram negative, I did a gram stain to ensure that this bacteria culture was pure and it seems to be pure (is this correct?)

*** Then I made a agar slant tube of my gram negative bacteria, assuming this is pure.

 

How do I isolate my gram positive? Do I just get this off the TSA plate and double check its gram positive?

PLEASE HELP!

Thank You

 

 

[CharonY]

Well, as you have indicated, your experiment basically failed as you were not able to enrich Gram-positive bacteria. As you pointed out correctly, EMB is slightly selective for Gram-negative. Now for what are the other two plate selective.

 

 

As a sidenote I want to point out that normally these plates are not 100% selective as there are always bacteria that can withstand the selectivity. But of course, for the course appropriate bacteria will have been selected.

 

Now back to topic. What you could do is think whether one of your plates may have Gram positive bacteria and then streak them out on the selective plates again. Of course, if you got clean colonies you can make dilution streaks and then characterize a clean colony from there.

[jorge1907]

It's not clear that enrichment was perfomed here at all and you shouldn't confuse these plated media with that effort.

 

Though some Gram postive microorganisms can gorw on the stuff, I'd say EMB is more than slightly selective for Gram negative bacteria. In any case, you should streak for isolation on nonseletive medium in preparing your pure culture slant as viable cells of the other bug may be present om EMB but not growing.

 

Why are you dismissing the "yellow dots" as contamination? There are Gram positive bacteria that produce yellow colonies. You could culture again on PEA (phenylethyl alcohol) agar if you can fnd some - this stuff inhibits Gram negative bacteria - esp. enterics.

Edited April 27, 2012 by jorge1907