STAPHYLOCOCCUS
By DR Mohammed Elmustafa ismail
Main pathogenic species
S. aureus
S. epidermidis (albus)
S. saprophyticus.
General Characteristics:-
Gram +, cocci, 1 μm, grape-like clusters
Facultative anaerobe (grows in the presence of
O2 or without)
Catalase positive.
Non-spore forming
Some strains have notable capsules
Relatively heat resistant
Resistant to high concentrations of salt
Can survive long periods on dry inanimate
objects
STAPHYLOCOCCUS
AUREUS
Pathogenicity
Skin infection (abscesses, boils, impetigo)
Wound infection and burns infection.
Conjunctivitis
Hospital acquired infection
Pneumonia
Mastitis (inflammation in breast).
Antibiotic associated enteritis.
Food poising.
Scalded skin syndrome.
Toxic shock syndrome.
UTI
DAY 1
Specimens collection
Skin infection
Wound infection
Conjunctivitis
Ear swab
Toxic shock syndrome
Scalded skin syndrome
Pneumonia sputum
Antibiotic associated enteritis
Food poising. Stool or
vomiting
UTI urine
Culter in
Neutral agar
Blood agar
Chocolate agar
Monitol salt agar
Macconky agar
CLED agar
Method of stricken
Using sterile wire loop or swab of the specimens held
First make a well:
by streaking full loop on top surface of media.
Area A:
Sterilization loop and take four line from well to make area
(A).
Area B:
Sterilization loop by flame take four line from area (A) to
make area (B).
Area C:
take four line from area (B) to
make area (C)
Zigzag:
make zigzag form area (C )
incubation
Best incubation aerobic or anaerobic at 37c
to over night (about 18 hours).
DAY 2
Colony morphology
In neutral agar:
- Produce yellow-gold colony, circular, convex
Smooth, modrate in size, moist.
Blood agar
- most strain B hemolytic, white in color,
circular, convex, Smooth, modrate in size,
moist.
Chocolate agar
- white in color, circular, convex, Smooth,
moderate In size, moist.
Monitol salt agar
- Yellow colony (Monitol fermentable),
circular, convex, Smooth, modrate in size,
moist.
In macconky agar:
- most strain pink color (lactose fermentable),
circular, convex, Smooth, modrate in size,
moist.
CLED agar
- Yellow colony (lactose fermentable),
circular, convex, Smooth, modrate in size,
moist.
Indirect gram stain
Aim:
To differentiate b/w gram positive and gram
negative and to appearance morphology of
the cell.
Procedure:
Prepare smear and fixation
Crystal Violet was poured and kept for
about 30 seconds to 1 minutes and wash
with water.
Flood the gram’s iodine about 30 seconds
to 1 minutes and wash with water.
Then ,wash with 95% alcohol or acetone
for about 3 seconds and rinse with water.
Add sufranin for about 1 minute and wash
with water.
Air dry, and Observe under Microscope
100X.
Result:
gram positive cocci arrange in cluster
Biochemical test
Main biochemical test:
I. Catalase test
II. Coagulase test (tube method and slide
method)
III. DNAse test
Catalase test
Aim:
To differentiate b/w staphylococcus and
streptococcus group.
Principle:
Catalase acts as a catalyst in the
breakdown of hydrogen peroxide to oxygen
and water.
Required:
I request 2ml from 3% H2O2 and wooden
stick to do catalase test.
Method:
pour 2ml of the hydrogen peroxide in test
tube
By using a sterile wooden stick take colony
from test organism and immerse in
3%H2O2.
Look for immediate bubbling.
Result:
Appear bubbling positive test.
Absent bubbling negative test.
Coagulase test
Aim:
Test used to differentiate [Link] which produce
coagulase enzyme, from [Link] and
[Link] which do not produce coagulase
enzyme.
Two type of coagulase test:
Free coagulase which converts fibriogen to fibrin by
activation a coagulase reacting factor present in
plasma, detected by tube test
Bound coagulase (clumping factor) which converts
fibriogen directly to fibrin without requiring a
coagulase reacting factor, detected by slied
Required:
I request sterile undiluted human plasma to
do coagulase test.
Method for slide test (bound coagulase):
By sterile loop take loop full from normal
saline and place in two side from slide.
Take small amount from colony and mix
with saline in one side other side using as
control.
Add drop form undiluted human plasma in
two side from slide and mix.
Result:
Clumping within 10 scent [Link]
No Clumping within 10 scent other
coagulase positive staphulococcus
DeoxyriboNucleAse test
Test is used to differentiate [Link] which
produces the DNAse from other
staphylococci which do not produce DNAse.
Principle:
Deoxyribonuclease enzyme hydrolyzes DNA.
Required:
I request sterile DNAse media to do DNAse
test.
Method:
Sterilization wire loop by flame and allow to
cooling.
Take part from colony, and make line in
DNAse media.
incubation 18h at 37c in O2
DAY 3
Result of DNAse
Add 1%HCL in surface of media, to
demonstration of DNA.
Result:
Positive result appear clear zoon
around colony
Negative result no appear clear zoon.
List of enzymes &toxins Includes:
1. hemolysins,
2. coagulase,
3. lipases,
4. leukocidin,
5. hyaluronidase,
6. fibrinolysin, &
7. exfoliatin (scalded skin
syndrome)