Procedure for Grams Staining
After the smear has been dried,
and cooled off, proceed as follows:
Place slide on staining rack
specimen with crystal violet. Let stand for
Wash briefly in tap water and shake off
Cover specimen with iodine solution and
let stand for 1 minute.
Wash with water and shake off excess.
Tilt slide at 45° angle and decolonize
with the acetone-alcohol solution until the
purple color stops running. Wash im-
mediately with water and shake off excess.
Cover specimen with safranine and let
stand for 30 seconds to 1 minute.
Wash with water, shake off excess, and
gently blot dry. The smear is now ready to
be read. (Use oil immersion lens.)
Example: Smear shows numerous gram-
negative bacilli. If two or more types of bacteria
are seen in a smear, the rule is to report them in
order of predominance, for example:
1. Numerous gram-positive cocci in clusters
2. Few gram-negative bacilli
Gram-positive organisms are easy to see
because they stain a deep blue or blue-black.
Gram-negative organisms stain a deep pink, but
since the background material is also pink, minute
and detailed inspection is necessary before
reporting the results.
In the presence of gonorrhea the smear will
reveal large numbers of pus cells with varying
numbers of intracellular and extracellular gram-
negative, bean-shaped cocci in pairs. Such a
finding can be considered diagnostic. It is
important to point out that only a few of the
thousands of pus cells on the slide may contain
bacteria and sometimes it requires considerable
search to find one.
Principle of Grams Stain
The crystal violet stain
which stains everything in
is the primary stain,
the smear blue. The
Grams iodine acts as a mordant that causes
the crystal violet to penetrate and adhere to the
gram-positive organisms. The acetone-alcohol
mixture acts as the decolorizer that washes the
stain away from everything in the smear except
the gram-positive organisms. The safranine is the
counter-stain that stains everything in the smear
that has been decolorized: pus cells, mucus, gram-
negative organisms. The gram-negative organisms
will stain a much deeper pink than the pus cells,
and mucus will stain even lighter pink than the
READING AND REPORTING SMEARS
Place a drop of oil in the slide and, using the
oil immersion objective of the microscope, read
the smear. All body discharges contain extraneous
materials, such as pus cells and mucus. Of interest,
however, are the types of bacteria that may be
present. The stained smear reveals only two
the morphology and the staining
characteristics of the bacteria present. Positive
identification requires cultures and further studies.
The hospital corpsman reports only what he
or she sees.
Serology consists of procedures by which
antigens and reacting serum globulin antibodies
may be measured qualitatively and quantitatively.
Serologic tests have been devised to detect either
antigens present or antibodies produced in a
number of conditions. Most are based on
agglutination reactions between an antigen and
a specific antibody.
Antigen is a substance that, when introduced
into an individual who does not already possess
that substance, may stimulate the individuals cells
to produce specific antibodies that react to
this substance in some detectable way. The four
basic characteristics of an antigen are it must be
foreign to the body, it must possess a high
molecular weight, it must gain entrance into
the body, and it must have a high specificity to
stimulate tissues to produce a defensive protein
substance called antibody.
Antibodies are the specific defensive proteins
produced when an antigen stimulates individual
cells. They are produced by the host in response
to the presence of an antigen and are capable of
reacting with antigens in some detectable way.
The antigen-antibody reaction takes place as
a result of a reaction between specific antibodies
in the plasma and antigen present on cell