Unknown bacteria

[Yersinia]

Moonwatcher,

You wrote to the microbiology mailing list:

<I recently inoculated two nutrient agar slants with an unknown bacteria. 
One will be kept at 25degrees and the other at 37degrees.  These will be 
kept for more than 24 hours. What is the best way to obtain the 
information needed in order to identify the bacteria.  Where would you 
start? >

I would start by taking a loopful of bacteria from each slant and 
streaking it for isolation onto TSA plates.  I'd incubate the plates for 
24h at 35C. The next day, I would check the plates and see how many 
different kinds of colonies were there (do you know if you have a pure 
culture, i.e., *only one* species of unknown bacteria? If you don't know, 
this is how you find out). On each different colony type, I would do a 
gram stain and find out if they are gram positive or gram negative, rods 
or cocci.  For gram negative rods, I would perform an oxidase test. For 
oxidase positive colonies, I'd streak them out on Cetrimide and/or PIA 
agar (Pseudomonas?). For oxidase negative, I would streak out on 
MacConkey or EMB (coliforms?), Bismuth Sulfite, Brilliant Green and XLD 
or Hektoen Enteric agar (Salmonellae?) Gram positive rods, I would go 
straight to the paragraph at the end of this email. For gram positive 
cocci, I would perform a catalase test and if positive, streak out on 
Vogel-Johnson annd/or Mannitol Salt Agar (pathogenic staphylococci, for 
instance, S. aureus?) or TSA with blood (hemolytic streptocci, example S. 
pyogenes?) I have no experience with gram negative cocci, so I don't know 
what to tell you about those offhand (anyone who'd like to help me out 
here, please do!). These plates would be incubated for 24 hours at 35C, 
then I'd look at the results:

Cetrimide and PIA: Turquoise or blue or bright green growth would 
indicate P. aeruginosa (or, on PIA, other pyocyanin-producing 
pseudomonads. Fluorescence would give further evidence of P. aeruginosa, 
P. putida or P. fluorescens. I'd streak those on additional TSA plates 
(or put them in a tube of TSB) along with a known P. aeruginosa control 
and see if they grow at 42C. Growth at 42C of a fluorescent pseudomonad 
would ID the organism as P. aeruginosa. Anything else that grew on those 
plates, but which were not blue-greenish nor flourescent, I would 
subculture onto TSA once more if you want to identify it further.  Same 
with the other fluorescent pseudomonads (see very end of this email).

MacConkey: If you see pinkish white colonies with a precipitate, you have 
a coliform of some sort. You could also put a suspected coliform into a 
tube of lactose broth containing an inverted Durham tube to look for gas 
production. 
EMB: Purplish/black colonies with a metallic green "sheen" indicate 
coliforms on this medium.  To ID to species level, at this point I would 
subculture to TSA (and go to the end of this email).

Bismuth Sulfite: If you get  brownish black colonies on this medium, it 
could possibly be S. typhi or S. typhimurium. Subculture to TSA and see 
the end of this email.

Brilliant Green: If the colonies are colorless but the agar turns red, 
could be a Salmonella species. Subculture to TSA....and see the end of 
this email.
XLD: Salmonella species form clear red colonies with black centers. If 
you see this, subculture to TSA.....etc.

Vogel-Johnson agar: Black colonies with yellow zones indicate S. aureus. 
You could perform a staphloslide or coagulase test at this point 
(positives strongly point to S. aureus as compared to other tellurite 
reducing organisms. Subculture to TSA...etc.

Mannitol Salt agar with golden colonies/yellow zones also indicates S. 
aureus. Subculture......

TSA with blood: Clear spots in which you can see through indicate beta 
hemolysis of the blood in this medium. S.  aureus and S. pyogenes will 
both produce this reaction, but I suggested only streaking streptococci 
here because the VJ  and MSA will indicate staph.  Subculture......etc.

Now we reach the end of this email. If you want to attempt species-level 
identifications, put your subcultured TSA plates in the incubator for 24 
h at 35C. and then run them through an identification system such as 
Vitek (my preference) or API.  When you get your results, compare them to 
your observations of the selective plates, the colony morphology on TSA 
and consider the original source of the bacteria, i.e., water sample? air 
sample? food? soil? your surface of a table? bathtub?  And so forth. 
Particular kinds of bacteria are more commonly found in particular 
environments. Look up the organism Vitek or API tells you in Bergey's 
too, while you're at it.

Happy hunting!

Infectionately,
Yersinia.

Mycelium is Yourcelium.