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d. Rinse the smear with tap water.
e. Rinse the slide with decolorizer for 2-5 seconds.
f. Rinse the smear with tap water.
g. Flood the slide with safranin for 30-60 seconds.
h. Rinse the smear with tap water.
i. Blot the smear dry by placing it between paper towels or allow the smear to air-dry.
NOTE: Test each new batch of reagents with known gram-positive and gram-negative organisms.
Prepare quality control (QC) slides by adding a drop of a 24-hour culture to a glass slide and allowing
the broth to dry. Fix each slide and store them in a box until ready for QC testing. A slide containing
separate drops of gram-positive and gram-negative organisms should be stained each day to test the
quality of the gram stain reagent. Alternatively, a mixture containing both gram-negative and gram-positive
organisms can be used to prepare the control slides.
- Examine the smears under oil immersion for:
a. Gram stain reaction.
b. Cell shape.
c. Arrangements. - Record the results in the logbook and on the laboratory request form.
What Not To Do: Do not overheat the slide. Do not dislodge the smear when rinsing or drying the slide.
Lab Procedure: Brucellosis Test
18D Skills and Training Manual
When: To test a serum specimen for brucella microorganisms.
What You Need: A properly prepared serum specimen, a glass plate, a ruler, a wax pencil, Rose
Bengal Serum Agglutination brucellosis antigen (or other serum agglutination antigen as available), a 0.2ml
serological pipette, an applicator stick, a lab request form, and a logbook.
What To Do:
- Prepare the glass plate.
a. Make a series of 1 1/2-inch squares with a ruler and wax pencil.
b. Use 5 squares to test 1 antigen against the sera diluted.
c. Clean and dry the glass plate after each use. - Prepare the serum dilutions.
NOTE: 0.08 = 1:20 dilution, 0.04 = 1:40 dilution, 0.02 = 1:80 dilution, 0.001 = 1:160 dilution, and
0.005 = 1:320 dilution. - Pipet the specimen.
a. Use a 0.2 ml serological pipette.
b. Pipet the serum onto a row of squares on the plate.
c. Pipet the serum in this order, starting with 0.08, 0.04, 0.02, 0.001, and 0.005 ml. - Shake the antigen well before using and place a drop of antigen on each drop of serum.
- Mix the serum and antigen with an applicator stick.
- Hold the glass plate in both hands.
- Rotate the plate 15-20 times in an 8-inch circular motion.
- Observe agglutination within 1 minute.
- Obtain the correct results for the specimen given.
a. Agglutination occurs when clumping of the antigen occurs with a clearing of the background fluid.
b. Negative reactions are characterized by a homogenous suspension with no clumping or clearing. - Report the results.
a. Positive reaction and the highest dilution at which it occurred.
b. Negative reaction occurring at a 1:20 dilution.