118

EXERCISES

FOR

THE

M,CROIlIOLOGY

LA80RATORY

COAGULASE

TESTS

Photographic Atlas Reference Coagulase Test Page 52

MATERIALS NEEDED FOR THIS EXERCISE

Per Student Group Three sterile rabbit plasma tubes (0.5 mL in 12 mm 75 mm test tubes) Sterile 1 mL pipettes Sterile saline Microscope slides Fresh slant cultures of: Staphylococcus aureus StaphyloCfoccus epidermidis
PROCEDURE
X

2. Place a drop of sterile water on side A and a drop of coagulase plasma on side B of each slide. ~""" 3. Transfer a loopful of S. aureus to each half of one slid{ making sure to completely emulsify the bacteria in the solutions. Observe for agglutination within 2 minutes. Clumping after 2 minutes is not a positive result. 4. Repeat step 3 using the other slide and S. epidermidis. 5. Record your results in the table provided. Confirm any negative results by comparing with the completed tube test in 24 hours. Lab Two 1. Remove all tubes from the incubator no later than 24 hours after inoculation. Examine for clotting of the plasma. 2. Record your results in the table provided. Refer to Table 5-27 when making your interpretations.
REFERENCES Collins, C. H., Patricia M. Lyne, J. M. Grange. 1995. Page 111 in Collins and Liyne's Microbiological M.ethods, 7th Ed. Butterworth-Heinemann, UK. Delost, Maria Dannessa. 1997. Pages 98-99 in Introduction to Diagnostic Microbiology. Mosby, Inc., St. Louis, MO. DIFCO Laboratories. 1984. Page 232 in DIFCO Manual, 10th Ed. DIFCO Laboratories, Detroit, MI. Forbes, Betty A., Daniel F. Sahm, Alice S. Weissfeld. 2002. Pages 266-267 in Bailey & Scott's Diagnostic Microbiology, 11th E .---;. Mosby, Inc., St. Louis, MO. Holt, John G. (Editor). 1994. Bergey's Manual of Determinative Bacteriology, 9th Ed. Williams and Wilkins, Baltimore, MD. Lanyi, B. 1987. Page 62 in Methods in Microbiology, Vol. 19, edited by R. R. Colwell and R. Grigorova, Academic Press Inc., New York. MacFaddin, Jean F. 2000. Page 105 in Biochemical Tests for Identification of Medical Bacteria, 3rd Ed. Lippincott Williams & Wilkins, Philadelphia, PA.

Lab One Tube Test 1. Obtain three coagulase tubes. Label two tubes with the organisms' names, your name, and the date. Label the third tube "control." ~2. Inoculate two tubes with the test organisms. Mix the contents by gently rolling the tube between your hands. Do not inoculate the control. 3. Incubate all tubes at 35C for up to 24 hours, checking for coagulation every 30 minutes for the first 2 to 4 hours. Slide Test 1. Obtain two microscope slides and divide them into two sides with a marking pen. Label the sides A and B.

TABLE

5-2 7
RESULT

COAGULASE

TEST TUBE

RESULTS TEST
INTERPRETATION

AND

INTERPRETATIONS
SYMBOL

Medium is solid Medium is liquid

Plasma has been coagulated Plasma has not been coagulated

S LID
RESULT

E T EST
INTERPRETATION SYMBOL

Clumping of cells No clumping of cells

Plasma has been coagulated Plasma has not been coagulated

SECTION

FIVE:

DIFFERENTIAL

TESTS

119

OBSERVATIONS AND INTERPRETATIONS

nefer

to Table 5-27 when recording and interpreting your results in the tables below. (See Photographic Atlas Figure 6-22 and 6-23.)

OBSERVATIONS TUBE
ORGANISM SYMBOL

AND TEST

INTERPRETATIONS RESULTS
INTERPRETATION

Un inoculated

Control

-S,~ S6

-+r'

.e-r~~~
ORGANISM

OBSERVATIONS
S LID
SIDE

AND
E TEST
SYMBOL

INTERPRETATIONS RESULTS
INTERPRETATION

Figure 6-21. Citrate Test Results. Simmon's citrate agar inoculated with Citrobacter diversus (+) on the left, Bacillus cereus (-) in the center, and an uninoculated control on the right.

Both tests utilize rabbit plasma treated with anticoagulant to interrupt normal clotting mechanisms. The Tube Test is performed by adding the test organism to rabbit plasma in a test tube. Coagulation of the plasma (including any thickening or formation of fibrin threads) within 24 hours indicates a positive reaction (Figure 6-22). The plasma is typically examined for clotting (without \ ~ shaking) after about 4 hours because it is possible for coagulation to take place early and revert to liquid within 24 hours. In the slide test, bacteria are transferred to a slide containing a small amount of plasma. Agglutination of the cells on the slide within one to two minutes indicates the presence of bound coagulase (Figure 6-23). Equivocal or negative slide test results are typically given the tube test for confirmation.

Coagulase Tests
COCCI.

PuRPOSE The Coagulase Test is typically used to differentiate Staphylococcus aureus from other Gram-positive

PRINCIPLE Staphylococcus aureus is an opportunistic pathogen that can be highly resistant to both the normal immune response and antimicrobial agents. Its resistance is due, in part, to the production of a coagulase enzyme. Coagulase works in conjunction with normal serum components to form protective fibrin barriers around individual bacterial cells or groups of cells, thus shielding them from phagocytosis and other types of attack. Coagulase enzymes occur in two forms-bound coagulase and free coagulase. Bound coagulase, also called "clumping factor," is attached to the bacterial cell wall and reacts directly with fibrinogen in plasma. The fibrinogen then precipitates causing the cells to clump together in a visible mass. Free coagulase is an extracellular enzyme (released from the cell) that reacts with a plasma component called coagulase-reacting factor (CRF). The resulting reaction is similar to the conversion of prothrombin and fibrinogen in the normal clotting mechanism. Two forms of the Coagulase Test have been devised to detect the enzymes: the Tube Test and the Slide Test. The Tube Test detects the presence of either bound or free coagulase while the Slide Test detects only bound coagulase.

Figure 6-22. Coagulase Tube Test. Coagulase-negative Staphylococcus epidermidis below and the more pathogenic coagulasepositive S. aureus above. Coagulase increases baqterial resistance
to phagocytosis and antibodies by surrounding infecting organisms with a clot.

Figure 6-23. Coagulase Slide Test. Emulsions of Staphylococcus aureus (+) on the right and S. epidermidis (-) on the left were prepared in sterile saline. Agglutination of the coagulase plasma is indicative of a positive result for bound coagulase.