..fungal titers?

Dilworth bactitech at hortonsbay.com
Thu Feb 22 12:17:52 EST 2001

Thanks for the birthday wishes, NM!  Those wishes are very much
appreciated, even though I could have done without the birthday

"Scotch tape preps" are used in clinical mycology to preserve the
morphology of the fungus for microscopic identification.  One inoculates
a fungus on a nonselective medium, typically Sabs or Potato Dextrose,
and allows the fungus to grow.  Rapid growers only need 2-4 days, while
dermatophytes need 1-3 weeks as they grow more slowly.  Then, under a
safety hood, one opens the plate, gently presses clear Scotch tape onto
a representative part of the colony, lifts, and puts it face down on a
microscope slide that already has a couple of drops of Lactophenol
cotton blue stain.  This is available in bulk or individual dropper
ampules.  If you want, you can then apply another drop over the top and
cover slip the whole shebang with a large cover slip.  This tends to
flatten the tape more, especially if the slide you're using is one of
the big 2 x 3 inch or so glass slides.  Then, trot your prep over to the
nearest microscope and start looking.  Sometimes you have to look for
awhile to see textbook picture stuff, but it's faster than performing
slide cultures and 90% or so of clinical specimens can be ID'd like
this.  At my previous job we had lots of fungus from our podiatry
clients (yes, people, feet and toenails are full of fungus in some
people......) and we ID'd virtually all of the isolates in this manner. 
Some weeks we had 15-20 isolates to ID, so messing around with slide
cultures took a lot more time.

A slide culture consists of cutting a small hunk of agar aseptically
from a plate, placing it on a slide in some sort of moist chamber
(rigged up from sterile Petri plates with a moist towel in the bottom
and some applicator sticks to raise up the slide).  Then the agar chunk
is inoculated at the edges only and a coverslip placed over the top. 
It's best to do these in duplicate.  Then, in a few days when the hyphae
start to grow, you can gently peel off the coverslip, plop it on another
slide that already has a drop of LPCB stain, and start looking.  This
preserves the morphology of the isolate much better, but is more
tedious.  An easier way to do this is to cut the chunk from near the
edge of the plate, plop the chunk down on another part of the plate,
coverslip that, and incubate with the agar side down and the coverslip
up.  This automatically keeps the prep moist and you don't have to
gerry-rig the above setup.  You can do two preps to a plate depending on
the size chunk you cut and the size of coverslip you use.

Hope this helps explain what we clinical people do in our lab.  It's
mostly a lot of microscopic examination. We match growth rate, colony
morphology, type of specimen and pictures in good reference books.  We
also get unknowns twice a year to check out our expertise.  Usually the
CAP unknowns are a couple of yeasts, which aren't difficult, and three
fungi, one of which is easy, and two of which are pretty difficult. 
Sometimes they send formalinized preps of fungi you don't want to mess
with in real life, like Coccidioides immitis or Histoplasma species.

For the record, I've never used a Waring blender to do medical mycology
:-).  That is the stuff of research labs.  It is quite a mental picture,
I must say - whirring fungus!!  Makes sense what he said about breaking
up the mycelia, though.

Judy Dilworth, M.T. (ASCP)

nmaccallister at webtv.net wrote:
> Well thanks again for your kind comments and interesting points of view.
> I appreciate hearing these informations from you.  I like the usages of
> "Waring blenders" and "scotch tape preps".  I admire the kind of
> practical ingenuity these ideas represent.  I still love that story
> identifying the first production centrifuges as recycled washing
> machines...(Isn't that great!).....

> .....Judy, is the "scotch tape prep" a method of collecting spores for
> transfer?.. or freezing?
> Or do you let the spores germinate on the tape in order to view the
> mycelium? ..(Maybe all 3?)......
> Well, thanks again,..and "Happy Birthday, Judy!!!"
> Thanks to each of you!  NM

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