Procedure for Grams Staining
After the smear has been dried, heat-fixed, and cooled off, proceed as follows:
1. Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.
2. Wash briefly in tap water and shake off excess.
3. Cover specimen with iodine solution and let stand for 1 minute.
4. Wash with water and shake off excess.
5. Tilt slide at 45° angle and decolonize with the acetone-alcohol solution until the purple color stops running. Wash immediately with water and shake off excess.
6. Cover specimen with safranine and let stand for 30 seconds to 1 minute.
7. Wash with water, shake off excess, and gently blot dry. The smear is now ready to be read. (Use oil immersion lens.)
The crystal violet stain is the primary stain, which stains everything in 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, gramnegative organisms. The gram-negative organisms will stain a much deeper pink than the pus cells, and mucus will stain even lighter pink than the pus cells.
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 things: 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.
Example: Smear shows numerous gramnegative 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 gramnegative, 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.
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 surfaces.