Vibrio cholerae (comma)—causes cholera
Escherichia coli—normally a nonpathogenic organism in the intestine, but if it gets into the abdominal cavity it can cause peritonitis. E. coli has also been known to cause urinary tract infections and diarrhea.
BACTERIOLOGIC METHODS
SMEARS
A smear can be made of almost all body discharges, lesions, or sediments obtained by centrifugation of spinal fluid.
Preparation of Smear
1. Smear specimen on a glass slide previously cleaned with alcohol or acetone and polished with lens paper. A thin and evenly spread smear is preferred for easier reading of the smear and identification of various organisms. Emulsify specimen with saline if thick.
2. Label the smear and circle material to be stained with a diamond point pen for easier identification and location of the material after staining.
3. Let the smear air dry. Forced heat drying will distort bacterial cells and other materials.
4. Fix the smear by passing it through a flame (smear side up) 3 to 4 times. DO NOT BURN SMEAR.
5. Let slide cool and then stain.
GRAM’S STAIN
The most common and useful staining procedure used in bacteriologic work is that of Gram. It is most likely to yield valuable information and should be done in all cases when staining is indicated. It is also used for the examination of cultures to determine purity and for purposes of identification.
Hucker’s Modification of Gram’s Stain Solution
1. Crystal violet/ammonium oxzlate solution (primary stain):
Yeast contamination is common and the stain must be filtered before use. Use only certified crystal violet. Gentian violet and methyl violet are not recommended because they contain impurities.
Solution A:
Crystal violet (certified) 2 gm
Ethyl alcohol (95 percent) 20 ml
Solution B:
Ammonium oxzlate 0.8 gmDistilled water 80 ml
Mix solutions A and B, store for 24 hours, filter, and store at room temperature, in a dark bottle, in a dark place, away from direct sunlight.
2. Iodine solution (mordant):
Iodine crystals (USP) 1 gmPotassium iodide 2 gm
Distilled water 300 ml
Grind iodine and potassium iodide in mortar. Dissolve potassium iodide in a flask in as small amount of water as possible. Add iodine crystals to potassium iodide solution. When dissolution is completed, add remainder of distilled water. Mix and let stand at room temperature for 24 hours. Filter and store in a dark bottle, away from direct sunlight.
3. Decolorizer
Acetone 1 volumeEthyl alcohol (95 percent) 1 volume
Mix 1 volume of acetone with 1 volume of ethyl alcohol and store in a tightly sealed bottle.
4. Safranine O counterstain:
Safranine O 0.25 gEthyl alcohol (95 percent) 10 ml
Distilled water 90 ml
Dissolve dye in ethyl alcohol, then add distilled water to dye solution and let stand at room temperature for 24 hours. Filter and store away from direct sunlight.
