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please help me...

Dilworth dilworth at megsinet.net
Sun May 28 19:55:40 EST 2000

So, Lenny, first lesson is to redo the gram stain. Pay particular attention to
how long you decolorize.  My guess is that you didn't decolorize enough (if
they're truly gram negative - see end of this paragraph for gram positive
information).  You only need a little bit of one colony in a drop of saline to
make a gram stain.  Don't make them too thick!   H. influenzae is a gram
NEGATIVE tiny pleomorphic gram negative rod.  N. meningitidis is a gram NEGATIVE
diplococci, usually in pairs, and look like kidney beans together.  Are they
expecting you to name the bacteria totally from a gram stain?  You really need
some other identification here.  Have you performed oxidase testing,
subculturing to TM, using X&V factors?  Sugar fermentations?  This is a basic ID
in medical labs and should be able to be performed with overnight incubations
(remember, these are CSF's and you can't mess around with long protracted
ID's).  Look in your basic textbooks and see what you can do.  You can perform
more than one test on one day.  Also, make sure you're working from fresh growth
when you set up your identifications.  Old colonies won't work.  Sub them and
freshen them up first.  Also, make sure you streak for isolation; use a whole
plate for each isolate, and make sure they're not mixed!  That will really mess
you up!  Colony morphology between the two is totally different.  H. influenzae
is creamier, and smaller; N. meningitidis, is sticky.  Former won't grow on
blood agar, latter will grow on blood agar.   If these isolates, however, are
truly gram positive, then you're in the wrong ball park.  Gram positives should
grow on blood agar, and you can tell much more about their hemolysis and
morphology from that medium than from chocolate.

As to your urine specimen, E. coli is the most common urine isolate.  BASIC test
for E. coli is indole testing.  If isolate is Lactose positive on MacConkey,
indole positive, and citrate negative, it is E. coli.  You MUST perform indole
on blood agar, though, or other medium suitable for indole testing, as you need
tryptophan content of medium to produce indole.  Again, E. coli is an overnight
ID - actually the lab I work in now does a commercial test called PGUA that can
ID E. coli in a half an hour!  You heavily inoculate the agar in the tube and
incubate in a heat block at least a half an hour.  Then, if the medium turns
yellow, and you drop in Kovacs reagent and the indole is positive, it's E.
coli.  The isolate MUST be lactose positive on MacConkey with a typical
morphology, however, to use this screening test.

Crack open your textbook Lenny, and you'll find LOTS of information and charts
in there.

Judy Dilworth, M.T. (ASCP)
Microbiology, 25 years

Len wrote:

> hi my name is lenny and am studying some cases in microbiology:
> organism A provided on chocholate agar, isolated from cerebrospinal fluid of
> a 4yr old boy with clinical symptoms and lab results indicating bacterial
> meningitis....i suspect either Neisseria meningitidis, or Haemophilus
> influenzae..if this is right how can i be sure, which tests to do besides
> gram stain ...as they are similar under the scope, gram + cocci, versus g+
> coccobacilli...
> organism C provided on blood agar medium isolated in pure culture fom the
> clean catch urine of a 21 yr old woman with significant bacteriuria (>1 000
> 000 CFU/mL) is suspect E.coli
> any help with these case would be great
> Thanks in advance!!!

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