[Microbiology] Problems identifying GRAM NEGATIVE ROD (Enteric)

Josip A. Borovac via microbio%40net.bio.net (by borovac from biogenevo.com)
Sun Mar 7 23:01:34 EST 2010

Hello folks, hows it going?
I have certain problems in my microbiology lab in identifying unknown bacteria. I hope that you experienced guys in the field may help me to clear this out a little bit.
So here is the thing, I've identified gram negative enteric under the microscope.
It grew on MacConkey and is LACTOSE POSITIVE (+).
This is confirmed by CTA Sugar test that revealed following.
GLUCOSE (+) positive + Gas formation at the bottom
MALTOSE, SUCROSE , LACTOSE - all positive (+)
DNAse Test (run three times) - NEGATIVE (-), no clearing around the streak
RUN TSI Sugars and two times I got A/A+ gas and once A/A + H2S (this one confused me)
Simmon's CITRATE came NEGATIVE (-) for three times I made the test
UREASE - always came out NEGATIVE (-)
PAD( Phenylalanine deaminase) - NEGATIVE (-)
MOELLER LYSINE - Negative (-)
MOELLER BASE - Negative (-)
SIM - completley black and huge H2S production (+), huge turbidity, very dark and dense
MIO - bottom of the tube YELLOW and top is PURPLE - totaly confusing, added Kovac's Reagent - nothing happened, suggests INDOLE negative (-). Run this three times, once out of these 3 times it turned completely yellow once I added Kovac's, other times it stayed purple on top a bit
MOTILITY - positive (+)
DNAse - always negative (-)

This all confuses me very much, I pretty much narrowed these to two choices, it should either be E. coli or Citrobacter freundii, however not all of the tests for these match.
In case of E. coli it should produce indole and should not produce H2S and the SIM should not be so filled with H2S and so radically black. E. coli should also be Lysine positive and I didn't got that. The rest pretty much agrees with expected results.
In case of Citrobacter freundii, everything pretty much matches up expect it should be Urease positive (+) and Citrate positive (+) which is not. Also this bacteria is reported to be only 50% positive for LACTOSE.
On MacConkey I got pretty intense pink color so it should be stronger Lactose fermenter than explained in C. freundii case.

This really is driving me nuts. I did these battery tests three times, strictly following the procedure and being really careful.I've spent whole last week in the lab trying to figure this out. Personally I would bet more for E. coli but then again I cannot be exactly sure for this one. Maybe some of you guys with a lot of clinical experience could help this poor undergraduate a little bit. :-)

On my second unknown I'm pretty sure I got Listeria monocytogenes, it had intense beta hemolysis present, catalase (+) and medium to small colonies. Cornybacterium should not have any beta hemolysis right? Just making sure.

Thank you very much folks, I would appreciate any of your input, opinions or advices!

All the best!


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