Practical Musculoskeletal System

By

DR. MOHAMMED ANSAR QURESHI

Lecturer of Medical Microbiology and Immunology

 Septic arthritis & Osteomyelitis
 Septic arthritis: Infection of the joints.
 Osteomyelitis: Infection of the bones.
 Out of the causes of septic arthritis and
osteomyelitis, we will talk in this practical
course about:
o Staphylococci
o Clostridium tetani causing tetanus
o Histotoxic clostridia causing gas gangrene
 Staphylococci
 Microscopy: Gram positive, cocci arranged
in grape like clusters, non capsulated, non
motile and non spore forming.
 Culture:
 Facultative anaerobe
 Grows on nutrient agar on which it produces endopigment:
o Golden yellow: Staphylococcus aureus
o White: Staphylococcus epidermidis
o Lemon yellow: Staphylococcus saprophyticus
 Grows also on blood agar:
o Complete hemolysis: Staph. aureus
o No hemolysis: Staph. epidermidis and Staph. saprophyticus
 Mannitol salt agar:
o Selective medium for isolating Staph. aureus from stool in
case of food poisoning. Staph. aureus give yellow color
because of mannitol fermentation.
 Nutrient agar Blood agar

Golden yellow endopigment Complete hemolysis

on Nutrient Agar on Blood Agar
 Biochemical reactions:
 Catalase test:
o All staphylococci are catalase test positive.
 Coagulase test:
o Staph. aureus: coagulase positive.
o Staph. epidermidis and Staph. saprophyticus: negative.
 DNAase test:
o Staph. aureus: DNAase positive.
 Phage typing:
o To trace the source of infection in epidemiological
studies.
 Catalase test
o Catalase test helps in differentiate between staphylococci and streptococci
o Principle:
- 2H2O2 catalase 2H2O + O2
o Procedure:
- Pick up the test colony on a platinum loop and immerse it in few drops of 3 %
H2O2 (hydrogen peroxide).
o Interpretation:
- Rapid effervescence indicates oxygen production and a positive test. Eg-
staphylococci
- No effervescence Negative test-
- Eg- Streptococci
 Coagulase test
Slide method
o Detects the clumping factor bound to the organism.
o Homogenous suspension of the test organism is made in a drop
of saline on a slide then mixed with a drop of undiluted human
or rabbit plasma.
o Staphylococcus aureus clumps within 15 sec. because the
clumping factor precipitates the fibrin in the plasma on the cell
surface.
o Positve – Staph. Aureus
o Negative- Coagulase negative staph.
 Tube method
o Detects the free coagulase enzyme.
o It is done by adding 5 drops of an overnight broth culture of the
test organism to 0.5 ml of human or rabbit plasma diluted 1/10
in sterile saline.
o The tubes are incubated for 6-12 hours at 37 degree and
inspected hourly for coagulation.
o Positve – Staph. Aureus
o Negative- Coagulase negative staph.

Positive

Negative
 Laboratory diagnosis of staph. infections:
 Specimen:
o Pus, joint aspirate, blood, sputum, stool.
 Direct Gram smear:
o Clusters of Gram positive cocci + pus cells.

 Culture:
o Nutrient agar & blood agar at 37 degree for 24 hours.
o Mannitol salt agar is used for isolating Staph. aureus from
fecal specimens.
 Identification is done by:
 Colonial morphology:
o Color of the endopigment on nutrient agar.
o Presence of hemolysis on blood agar.
 Gram film:
o Gram positive cocci arranged in clusters.
 Biochemical reactions:
o Catalase test: to differeniate between staph. & strept.
o Coagulase test: Staph. aureus is positive.
o DNAase test: Staph. aureus is positive.
 Serological identification:
o By latex agglutination test.
  Molecular identification:
o PCR or DNA probes.
 Phage typing:
o To determine the source of infection.
 Antibiotic sensitivity testing:
 By disc diffusion method.

Staph. epidermidis: novobiocin sensitive

Staph. saprophyticus: novobiocin resistant

MRSA?
VRSA?
 Clostridia
 Anaerobic, spore forming Gram positive bacilli.
 Clostridia causing septic joint and bone
infections are:
 Clostridium tetani: causing tetanus
 Histotoxic clostridia: causing gas gangrene
 Clostridium tetani

 Microscopy:
 Gram positive bacilli
 Drum stick appearance due to terminal spherical
projecting spores
 Motile
 Non capsulated
Terminal spore
 Culture:
 Strict anaerobe
 Grows on nutrient agar
 Better growth on blood agar:
o Complete hemolysis due to production of tetanolysin toxin.
 Robertson cooked meat medium
 Biochemical reactions:
 No sugars are fermented.
Robertson cooked meat medium
 Animal inoculation:
 Laboratory mouse is injected IM with culture of the organism
(test animal).
 Within few hours, the tail becomes stiff.
 The injected limb shows spastic paralysis which spreads to
the rest of the body.
 The injection of the antitoxin few hours before injecting
culture protects the animal (control animal).
 Laboratory diagnosis of tetanus:
On clinical suspicion, antitoxin treatment should start
without delay. LAB diagnosis is done for confirmation
Specimen:
o Wound exudates from deep sites of the wound.
 Direct Gram smear:
o Gram positive bacilli with drum stick appearance.
 Culture:
o On blood agar which is incubated anaerobically.
o Robertson cooked meat medium.
Anaerobic jar
 Identification is done by:
 Colonial morphology:
o Presence of hemolysis on blood agar.
 Animal inoculation:
o Test and control animals.
o Test animal dies & control animal survives.
 Antitoxin test:
o The bacteria is subcultured on blood agar antitoxin
plate.
o Half of this plate is covered with the antitoxin.
o The plate is incubated anaerobically.
o The hemolysis is inhibited in the half containing the
antitoxin.
 Histotoxic clostridia
 Saccharolytic group:
o Clostridium perfringens (welchii)
o Clostridium novyi
o Clostridium septicum
 Proteolytic group:
o Clostridium histolyticum
o Clostridium sporogenes
 These clostridia cause gas gangrene.
 Clostridium perfringens is the commonest member.
 Clostridium perfringens

 Microscopy:
 Large Gram positive bacilli
 Spores are oval, subterminal and non projecting
 Non motile
 Capsulated in the tissues

Subterminal spore
 Culture:
 Strict anaerobe
 Grows on blood agar:
o Most strains produce complete hemolysis.
 Neomycin blood agar is a selective medium.
 Robertson cooked meat medium
 Biochemical reactions:
 Ferment glucose, lactose, maltose, sucrose with production
of large amount of acid and gas.

 Nagler reaction
Nagler reaction:
o Clostridium
perfringens produce
opalescence in 5% egg
yolk agar due to
production of lecithinase
enzyme.

o Such opalescence is
inhibited by specific
antitoxin.
 Stormy clot reaction:
o Clostridium perfringens ferments lactose in the litmus milk
producing large amount of acid and gas.
o Acid causes the milk to clot.
o The gas produced will split the clot.

 Animal inoculation:
 Laboratory mouse or guinea pig is injected IM with culture
of the organism (test animal).
 The injected limb will show edema and crepitations.
 Then, the animal will die.
 The injection of the antitoxin before injecting culture
protects the animal (control animal).
 Laboratory diagnosis of gas gangrene:
On clinical suspicion, treatment should start without
delay. LAB diagnosis is done for confirmation
Specimen:
o Wound exudates from deep sites of the wound.
 Direct Gram smear:
o Large Gram positive bacilli.
o Spores are rarely found.
 Culture:
o Robertson cooked meat medium.
o Neomycin blood agar.
o Blood agar which is incubated anaerobically.
 Identification is done by:
 Colonial morphology:
o Presence of hemolysis on blood agar.
 Animal inoculation:
o Test and control animals.
o Test animal dies & control animal survives.
 Biochemical reactions:
o Sugar fermentation.
o Nagler reaction.
o Stormy clot reaction.
GOOD LUCK