LAB 3 – STAPHYLOCOCCI AND MRSA

Question 1

You have a plate showing 3+WOP. The Gram is G+C, clps and the catalase is positive, the slide coagulase is positive, and the tube coagulase is negative.
a. What is the discrepancy in the results?
b. What could cause this discrepancy?

Answer 1

a. Slide coag (+) while tube coag (-), both should be (-)
b. Slide coag was observed after >10 seconds = false (+)

Question 2

You have a plate showing 3+WOP. The Gram is G+C, clps and the catalase is positive, the slide coagulase is negative, and the tube coagulase is positive.
a. What is the discrepancy in the results?
b. What could cause this discrepancy?

Answer 2

a. Slide coag (-) while tube coag (+), both should be (-)
b. false (+) can be due to non-sterile plasma (extracellular contamination)

Question 3

Why are tube coagulase tests supposed to be checked at 4 hours?

Answer 3

Some strains of Staphylococcus aureus produce fibrinolysin which may lyse clots formed earlier

Question 4

What is an important consideration of the catalase test when picking off of a BAP?

Answer 4

• RBCs can produce a wk+ result
• sampling should be done from the top of colonies

Question 5

Explain the two types of coagulase tests. How are they different and what do they test for?

Answer 5

Slide coag: detects bound coagulase (clumping factor) on cell wall of S. aureus
- bound coagulase directly binds fibrinogen = agglutination

Tube coag: Coagulase-reacting-factor (CRF) detects extracellular coagulase = thrombin-like complex that converts fibrinogen to fibrin (agglutination)

Question 6

How does the DNAse test work?

Answer 6

• Methyl green (indicator) forms a green complex with DNA at pH 7.5
• S. aureus DNAse cleaves DNA = colorless around colonies

Indicator: Methyl green or toluidine blue O

Question 7

You have an organism that is catalase positive and slide coagulase positive. What can you
report? Think about giving the most information possible to the doctor while not harming the
patient.

Answer 7

• Presumptive Staphylococcus sp.

Question 8

What does MRSA stand for?

Answer 8

Methicillin-Resistant Staphylococcus aureus

Question 9

What antibiotic is used to screen for MRSA?

Answer 9

Methicillin

Question 10

What component in MRSA CHROMagar is used to differentiate MRSA from other S.
aureus?

Answer 10

Cephalosporins differentiates between MRSA and methicllin-susceptible strains

Question 11

What component in MRSA CHROMagar is used to differentiate S. aureus from other
gram positive organisms?

Answer 11

S. aureus hydrolyzes a chromogenic substrate = mauve colonies

Question 12

What is the difference between DFA and IFA?

Answer 12

Direct Immunofluorescence Antibody: detects bacterial antigen in 1 step using fluorescein labelled antibodies
- more specific/ true negative

Indirect Immunofluorescence Antibody: detects either antigen/ antibody in 2 steps; “sandwiched” in a capture antigen and labelled AHG
- more sensitive/ true positive

AHG - Antihuman Globulin

Question 13

What immunodiagnostic test detects patient antibodies using a known antigen, and a labeled
antibody (anti-human globulin)? How many steps are there?

Answer 13

• Indirect Immunofluorescence Antibody (IFA)
• 2 steps; unlabelled antibody is added and binds known antigen
• excess antibodies are washed away
• labelled AHG binds to initial antigen-antibody complex = fluorescence

Question 14

Colonial and microscopic morphology of Rothia mucilaginosa

Question 15

Colonial and microscopic morphology of Micrococcus

Question 16

Discuss the epidemiology and clinical significance of: S. aureus, CNS, S. epidermidis, S. saprophyticus

Question 17

List steps to Kirby Bauer disk diffusion

Question 18

Sources of Error in disk diffusion and corrective action