Special Operations Forces Medical Handbook

8-45

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.

5. Examine the smears under oil immersion for:
   a. Gram stain reaction.
   b. Cell shape.
   c. Arrangements.


6. 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:

1. 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.


2. 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.


3. 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.


4. Shake the antigen well before using and place a drop of antigen on each drop of serum.


5. Mix the serum and antigen with an applicator stick.


6. Hold the glass plate in both hands.


7. Rotate the plate 15-20 times in an 8-inch circular motion.


8. Observe agglutination within 1 minute.


9. 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.


10. Report the results.
    a. Positive reaction and the highest dilution at which it occurred.
    b. Negative reaction occurring at a 1:20 dilution.