yersinia at GATE.CYBERNEX.NET
Sat Mar 29 00:05:51 EST 1997
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
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
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
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.
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.
Mycelium is Yourcelium.
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