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[Microbiology] Re: TB Microscopy

JEDilworth via microbio%40net.bio.net (by bactitech from nospamhortonsbay.com)
Sat Aug 4 23:17:15 EST 2007

"Jennifer Keene" <Jennifer.Keene from students.olin.edu> wrote in message 
news:mailman.526.1186252737.11350.microbio from net.bio.net...
I see in the archives that people have asked about TB microscopy in the 
past,  but it looks like most of the replies were off list - would 
people send me any information about TB microscopy that they have??

I'm not sure what you need to know. There are basically two methods to 
see acid fast organisms under a microscope, of which M. tuberculosis is 
one: light microscopy and fluorescent microscopy.

Light microscopy involves either using the classic Ziehl-Nielsen 


(which involves heating the slide to make the dye pass through the 
coating of the bacteria in order for them to stain red) or the cold 
method, which uses carbol fuchsin dye in phenol. The gold standard is 
the ZN stain. There is a blue counterstain, and the acid fast bacteria 
stain red. They are also sometimes beaded in appearance. You need an oil 
immersion lens to read these smears.

The other methodology uses a fluorescent microscope and a different 
staining technique. I don't know the settings, wave lengths, or filters 
involved in setting up the scope (sorry). Perhaps someone else can fill 
in this information. One stains the slides with auramine/rhodamine.

http://www.polysciences.com/shop/product.asp?pf_id=24666&dept_id=82 - 
information on the stain itself

The acid fast organisms will fluoresce a golden yellow under the scope. 
Oil immersion is NOT used with this, although you can go down to oil to 
check on suspicious organisms.

Most laboratories use the fluorescent methodology to screen concentrated 
sputum specimens and other clinical specimens for acid fast bacilli, as 
it is much easier to see the rods fluoresce than see the red rods on a 
blue background, and there is less fatigue on the technologist reading 
the smears. Once the organism grows, it is then stained with ZN to prove 
it is acid fast.

Sputum specimens, ideally a first morning specimen and at least 5 ml in 
volume, are put through a concentration procedure.

http://ajrccm.atsjournals.org/cgi/content/full/161/5/1559 - scroll down 
under "methods" - this entire article is extremely interesting although 
quite technical. Our laboratory will not process sputum specimens for 
AFB with less than 5 ml - this article must be the benchmark on this 

Other specimens such as tissues are stained with auramine rhodamine 
without concentration. Sometimes histology will give the lab tissue 
sections to stain for acid fast bacilli.  Fluids are usually spun down 
at high speed and the sediment is examined for AFB by auramine 

Concentration procedures should only be performed by trained personnel 
in a biosafety cabinet, preferably in a room that has negative air flow 
to vent everything directly out of doors. All work on acid fast cultures 
are performed under a BSC. Slides are heat fixed at high temperature for 
two hours or chemically fixed with a phenol solution before staining to 
render the bugs non-infectious. Acid fast organisms have a tough outer 
waxy coat which renders them impervious to a lot of things - even the 
low concentration of sodium hydroxide used in the concentration 
procedure that kills off other bacteria in specimens with mixtures of 
bacteria such as sputum. It is this cell wall characteristic that makes 
AFB so difficult to treat, and why therapy must go on with 2-3 drugs at 
a time for months and months.

CAP, an accreditation agency for many laboratories, requires a maximum 
time frame of 24 hours for smear results from the time of specimen 
receipt.  That means that the specimen must be processed and smear read 
and reported within that 24 hour time frame. This is important due to 
the extremely infectious nature of tuberculosis. Many small laboratories 
must send out their specimens for AFB to a larger reference facility due 
to the lack of volume of specimens, personnel trained to work with AFB, 
lack of a BSC and/or fluorescent microscope. This unfortunately adds to 
specimen processing time, although with prompt reference lab couriers, 
this TAT can be accomplished.

Not sure if this is what you're looking for by the way of information. 
Hope this helps you out.

Judy Dilworth, M.T. (ASCP)

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