BACTERIOLOGYLABORATORY REPORTS / TECHNIQUESASEPTIC TECHNIQUE:
In handling bacteria specimens, do everything possible to assure that extraneous bacteria
do notcontaminate the specimen. This is
sterile technique.Preparation and Staining:
After air drying a slide, applying gentle heat helps the bacteria to adhere to the slide.
Simple Stain (Methylene Blue):
Stains all cells and can illuminate morphology.
stains both Gram+ and Gram- and makes all bacterial cells appear purple.
Both Gram+ and Gram- will stain with this dye.
CHELATING AGENT: Add
which will adhere only to cell walls that have a lot of peptidoglycan inthem (i.e. Gram+)
very briefly to wash away the Crystal Violet dye.
Gram-: The purple dye will be washed away.
Gram+: The purple dye will remain adherent.
COUNTERSTAIN: Then add
which will stain cells pink.
) -- Safranin doesn't stain it.
, as it takes up the
Dead Gram+ cells will appear as though they are Gram-
. In antibiotic-treatedspecimens, a certain portion of such cells is expected.
Stains Mycobacteria such as
LIPID cell wall gives them the property of
weak initial staining
strong retention of initial dye
(like Gram+)once stained.
dye is used initially, with heat, for 20 to 30 minutes (long time)
is then used as a decolorizing agent.
If the organism resist the decolorization, it is considered acid-fast.
It has the advantage that you can scan at low power until you find something that fluoresces, then you can hone inon it.
Certain organisms can agglutinate red blood cells. If you have antibodies to impedethat agglutination, then you have a positive test result (antibody is present).
Direct Fluorescent Antibody:
Fluoro tagged antibody checks for presence of antigen directly.
Indirect Fluorescent Antibody:
First allow antigen-antibody reaction, then use a second Fluoro tagged antibody(anti-antibody) to identify presence of the first antibody.
Usually avoided; expensive.
ACUTE and CONVALESCENT TITERS:
Take an antibody titer at initial infection (acute), and again four to fiveweeks later. You would expect a
four-fold increase in titer
if the suspected organism is indeed the infectingorganism.
There is usually a baseline level of antibody present with or without infection, because of cross-reactivitywith other antigens. Thus you use acute:convalescent titers as a means of comparison.
For some organisms, there are also absolute antibody levels that are diagnostic.
Radio-Immunoassay (RISA, ELISA):
Take the patient's direct specimen and try to detect residual evidence of theorganism's presence. Used when recovery of bacteria itself is not possible.
Polymerase Chain-Reaction (PCR):
Can also be used for direct detection but it's expensive.CULTURE:
EXCEPTIONS to the fact that organisms can be cultured in 24 hours.
takes up to eight weeks to culture.
organisms have never been cultured.
Streaking for Isolation:
Process of inoculating a plate such that you get single bacterial colonies once you culture the plate.