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 Help w/ unknown, please!  

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Ok, so I need some help determining what unknown bacteria I have. I cannot repeat any tests. I have two in mind that I'm between but I'm not sure if I should mention them here yet....I would like a fresh perspective with nothing but the results to influence your thinking...

Here's what I have:

Gram neg rods

Not aerobic

Positive for glucose fermentation: supported by PRG and TSIA slant results

Positive for lactose fermentation: supported by EMB, Litmus Milk, PRL, and TSIA slant results

SIM tube: positive for H2S (also positive with PIA and the TSIA slants), positive for indole (also positive with Tryptone broth), positive for motility

positive for urease

negative for gelatinase

negative for deaminase

negative for lipase

negative for starch hydrolysis/amylase

negative for oxidase

positive for catalase

Nitrates reduced to Nitrites

MR positive/VP negative

Citrate negative

All tests were incubated at 37 C for 1 week...I know many tests are supposed to be incubated at 30 C for 48 hrs. This is how this particular class/school always does it. I'm not sure how much this would skew results. Any help is much appreciated!


Check this out. Your reactions do not fit. Is your culture mixed?


First, thanks for your reply. Second, no it is supposed to be pure. This is my first microbiology lab, so it's possible I screwed up a test. I'm positive it's H2S positive because three tests supported that result...I'm also positive it is a lactose fermenter because two-three tests back that up.

I also forgot to add that it's positive for sucrose fermentation and gas.

I *think* I may have messed up the indole may be indole negative...

I'm still between those two bacteria I initially had in mind but I'm leaning more towards one now.

I would be so appreciative if you could add your thoughts. Even though some results may not fit, if u could give me ur best guess, I would really appreciate the input.


If your bug is indole negative, then it is probably Citrobacter freundii. An H2S positive E. coli is extremely rare. The VP negative throws it out of Klebsiella/Enterobacter, as this group is always VP positive. KE group is always citrate positive also. Citrobacter can be weakly positive for urea. Proteus/Providencia sp. would be non-lactose fermenting.

If you are using SIM for motility and H2S, all you have to do is put a drop of indole reagent on top of the SIM after you read your other reactions. Indole positive will be bright pink. Not sure how you can screw that up. If it's colorless, then it's negative. It has to be done the day after you set it up. I have no idea why they're telling you to incubate your stuff for one week. Everything would be overgrown.

I have never incubated ID media at 30 degrees for 48 hours, with the exception being the API NFT kit, which we use to identify non-fermenters when our automated media doesn't do it for us, or if there's a problem.

Are you using tubed media or a kit? If it's a kit, then you definitely do NOT want to incubate the kit for a week.

You must read your reactions the day after you set them up. You can't let the tubes sit in an incubator for days. The readings will not be accurate.shaking head

Hope this helps you.


Thank you for your help. I was between E. coli and Citrobacter freundii, leaning towards the latter. It helps to have an experienced person's reassurance.

We inoculate and incubate the media for one week. Then we add the reagents, if any are required, and read the results. We incubate them for one week because our laboratory period is only once per week for three hours. My professor realizes this may skew some results but I guess this is more of a matter of scheduling than anything else.

We used tryptone broth and SIM to test for indole. We were doing about 20 tests that day on a time crunch. I wrote in my lab book "no red +" I remember the test and I know they weren't red. I think in my haste I wrote the positive sign instead of the negative sign.

It was definitely positive for H2S...the SIM tube, TSIA slant, and peptone iron agar all tested H2S positive.

I made the comment about 30 degrees for 48 hrs bc that's what it said in my lab supplement for many of the tests. I will take your word on this since I'm sure you're more familiar with the practical application of these tests.

We're using tubed media.

I really appreciate you taking the time to help me. Thank you! grin


You're quite welcome.

Just curious - no way you can get back into the lab the day after you inoculate your media?

E. coli are urea negative. That was a big clue that you didn't have it.

Back in the olden days, when we used tubed media for ID's (1970's), we would set up SIM, Citrate, Lysine, Ornithine, and MRVP tube for lactose fermenters. Nowadays, we use automated ID's for 99% of our gram negative identification. Vitek2 uses 64 well cards and we ID organisms much more precisely now than 39 years ago.

We are able to produce ID/sensitivities in a bit over 8 hours. Our day shift sets up ID's and sensis on positive urine cultures in the morning, and we send out results in the evening. We set up on second shift and day shift signs out in the morning. The sensitivities are better because they are reported as MIC's (minimum inhibitory concentrations) instead of zone sizes (sensitive, intermediate, or resistant), which the infectious disease docs appreciate, especially if they're following complex infections. We still have to use plate sensitivities if we have to (Kirby Bauers, with discs) but also supplement with eTests, which can be used on plates but will give MIC results. Some organisms don't work in the Vitek2, and some organisms still require KB's as the only kind of sensi that can be performed.

Microbiology has come a long way since I started back in 1974. The organisms are, unfortunately becoming more resistant, and signing out sensitivities accurately has become much more of a challenge for the technologists, as we are dealing with ESBL's and carbapenemase producers nowadays. Those weren't around, even in the 90's, and are a product of overuse of antibiotics :-(.

I plan on working at least a couple of more years. The future of microbiology lies in molecular testing. Our lab has been performing it for about 10 years now, and more testing is coming out all of the time.

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