Name: Date: __________________

Section: Score: __________________

Exercise No. 9
Staphylococci
Staphylococci are normally found on the skin and mucous membranes, a well as in our
environment. It is, therefore, necessary to determine whether given staphylococci isolate is
pathogenic or not. There are no absolute criteria, which distinguish pathogenic form non-
pathogenic strains, but pathogenic strains generally hemolyze red blood cells, produce catalase
and plasma coagulase and ferment mannitol.
Objectives:
1. The student must be able to know and perform the methods and techniques of isolation
2. and identification of bacteria.
3. The student must be able to cultivate Staphylococcus sp.
4. The students must be able to perform Gram staining and biochemical tests for
Staphylococcus.
5. The student must be able to differentiate the species of Staphylococcus based on their
colonies and reactions to different biochemical tests.
Materials:
Bacterial suspension of S. aureus & S. epidermidis
Blood Agar plate
Glass slide
Wire loop
Alcohol lamp
Compound microscope
Sterile applicator stick
Sterile test tubes
Mannitol Salt Agar
Human blood plasma
Cedarwood oil
Reagents:
3% H2O2
Gram stain reagents
Procedures:
A. Gram stain:
1. Prepare a bacterial smear from the bacterial suspension. (always practice aseptic
technique)
2. Stain with Gram stain.
3. Examine under LPO, HPO and OIO.
S. aureus under LPO S. aureus under HPO S. aureus under OIO

S. epidermidis under LPO S. epidermidis under HPO S. epidermidis under OIO

B. Hemolysis Test:
1. Divide with a glass market a blood agar plate into 2 sectors and streak on each sector
one of the two organisms above. Label. Incubate at 37oC for 18 to 24 hours.
2. Following incubation, read results. Hemolysis is indicated by a clear zone around the
colonies on BAP.
3. Study the hemolytic activity of the 2 organisms.
4. Make necessary drawings.
S. aureus Hemolysis Test S. epidermidis Hemolysis Test

C. Catalase Test
1. Emulsify a colony using sterile applicator stick in 1 drop of 3% H2O2 in a glass slide
2. Observe for the production of gas bubbles
3. Interpretation: Gas bubbles = positive; No gas bubbles = negative
4. Make necessary drawing.
S. aureus catalase test S. epidermidis catalase test

D. Coagulase Test
1. Place 0.5ml of fresh human plasma in a small sterile tube. Inoculate the tube with one
loopful of Staphylococcus aureus. Do the same with the Staphylococcus epidermidis.
2. Incubate at 37°C for 4 hours.
3. Observe for coagulum formation.
4. If no coagulum is formed, incubate further up to 18 hours.
5. Interpretation: Clot formation = positive; No clot formation = negative
 S. aureus coagulase test S. epidermidis coagulase test

A. Mannitol Fermentation Test

1. Divide with a glass marker a MSA into 2 sectors and streak organisms on each sector.
Label. Incubate.
2. After incubation, read results. A change of color of the corresponding compartment of
MSA from pink to yellow indicates fermentation of mannitol.
3. Examine which specie ferments mannitol and make necessary drawing.
Mannitol Fermentation test
Experiment worksheet on Staphylococci
I. Complete the table below.
a. Indicate the diseases that is caused by the different specie of Staphylococcus and its treatment
Staphylococcus specie Disease and Treatment
characteristics
S. aureus

S. epidermidis

S. saphrophyticus

Reference:
a. Indicate the use of the differential test for Staphylococcal specie
Differential test Use
Coagulase test

Mannitol Fermentation test

Tellurite Glycine test

Polymyxin Sensitivity test

Voges Proskauer test

Reference:

b. Identify the differential test applicable for the different Staphylococcal and the corresponding
result
Microorganism Differential test Result
S. aureus vs. S.
saphrophyticus

S. aureus vs. S.
epidermidis

S. saphrophyticus vs. S.
epidermidis