We make our own as well.  I ask our Micro department to innoculate a broth with E. Coli, and Staph or Strep, and when it's ready, throw in some fresh cubed placenta.  I'll let it sit over night at RT and then fix and process it.  Works beautifully, and is very inexpensive.

Kathy Johnston
Tech II Special Stains
Anatomic Pathology
Calgary Laboratory Services
#9 - 3535 Research Road NW
Calgary, AB.
403-770-3572


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu]On Behalf Of Monfils,
Paul
Sent: April 26, 2006 12:06 PM
To: 'histonet@lists.utsouthwestern.edu'
Subject: RE: [Histonet] GRAM Stain Control Slides


Excellent gram controls are pretty easy to make. I order cultures of gram
positive and gram negative organisms from a biological supply company.  I
get them in liquid culture (broth), not on agar plates. They cost less than
$10.00 per culture.  I spin down the cultures to concentrate the organisms,
remove most of the supernatant, resuspend the organisms by vortexing, then
inject the concentrated culture into a small block of fresh lung.  I push
the needle almost all the way through, then withdraw the needle as I inject,
in order to distribute the organisms through the tissue. Lung works best
because there are a lot of small spaces into which the culture can flow.
Gently knead the tissue a bit with a fingertip (gloved of course) to further
distribute the organisms. Drop the lung into fixative, let it fix, then
process and embed as usual.  I inject gram positive organisms into one piece
of tissue, gram negatives into another, then after processing, cut the
tissue into smaller square pieces, and embed a gram positive piece and a
gram negative piece side by side in one paraffin block.

An easier way is to purchase cultures on agar plates, fix them as they are,
then process and embed small pieces of the agar with the organisms on it.
But even though it is a little more involved, I like the more "natural" look
of the organisms in tissue.  Also, in using normal lung you don't have to
deal with all the cellular infiltrate, necrosis, exudates, etc. that would
be present in an actual massive bacterial lung infection.

For gram positive I favor Bacillus subtilis and Streptococcus epidermidis.
For gram negative I use Escherichia coli or Enterobacter aerogenes, and
Neisseria subflava.  I like to have both a bacillus (rod) and a coccus
(sphere) in both the gram positive and the gram negative tissue.

I get my cultures from:  http://www.ctvalleybio.com



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