Gram Positive Bacilli

Laboratory diagnosis of : Clostridium

Laboratory diagnosis: Sample Direct smear Culture Identification of culture Colonies Film BR Animal pathogenicity special

Clostridium

THE GENUS CLOSTRIDIUM Morphology Cl. tetani Cl. perfringens Cl. botulinum

THE GENUS CLOSTRIDIUM Culture Cl. tetani Cl. perfringens Cl. botulinum Culture ch’: O2  obligate anaerobe CO2 0.03% Temp. 37°C PH 7.4 __________________________________________________________________________________ Media Ordinary media: grow Blood agar: haemolysis Robrtson cooked meat medium

THE GENUS CLOSTRIDIUM Biochemical Reactions Cl. tetani Cl. perfringens Cl. botulinum Sugar: -ve GMMLS GML indole +ve LM: acidified+clotted

Methods of anaerobiosis 2)Media containing reducing compounds 1)Deep agar 3)Absorption of O2 by Na-pyrogallate (Buchner`s tube or McLeod’s plate)

4) Replacement of Oxygen with hydrogen Gas-bag jar

Diagnosis of Tetanus Patient treated on a clinical basis without waiting for laboratory data. Samples: Tissues from wounds Direct smear: Gram positive bacilli terminal spherical spores (Drum stick appearance), motile with peritrichate flagella, non-capsulated.

Diagnosis of Tetanus Culture BR: Culture ch: Media On blood agar: O2: Obligate anaerobic, CO2 Temp. 37°C. Media On blood agar: complete haemolysis swarming, BR: Indole: +ve Suger fermantation: -ve.

Diagnosis of Tetanus Isolation of C.tetani must be confirmed by production of toxin and its neutralization by specific antitoxin.

Diagnosis of gas gangerene Patient should be treated without waiting for laboratory confirmation. Specimens: Material from wounds, Pus, Tissue, Exudates.

Diagnosis of gas gangerene Direct smear stained with Gram stain: Gram-positive, Sporulated: subterminal, not bulging non motile capsulated bacilli.

Diagnosis of gas gangerene Culture: Culture ch.: O2: Obligate anaerobe. CO2: … Temp.: … PH: …. Media: On blood agar complete haemolysis. double-zone haemolysis,(When examined with transmitted light), (inner clear zone &outer hazy zone) egg yolk-glucose agar or serum agar, colonies are surrounded by opaque white precipitate due to production of lecithinase (Nagler ‘s reaction).

Diagnosis of gas gangerene Nagler’s reaction: Use: Identification of Clostridium perfringens. Principle: Clostridium perfringens produces opalescence in human serum or egg yolk media due to the production of lecithinase C (phospholipase).

Diagnosis of gas gangerene Biochemical reactions Suger fermentation: C perfringens ferments GMLS  large amounts of gases & acids litmus milk stormy clot. Litmus milk: Use: differentiate bacteria based on various reactions they produce in milk supplemented with a litmus pH indicator Principle: Milk is a complex nutritional source contains proteins (mainly casein), lactose and minerals.

Diagnosis of gas gangerene Reactions on litmus milk Reaction Color of litmus milk Interpretation Acid production Indicator pink Glucose and lactose utilised Acid clot Indicator pink, medium clotted. Big amount of gas may be produced which splits the clotted milk into pieces (stormy clot) Casein precipitated by acid. This type of clot will dissolve if treated with NaOH Indicator blue Ammonia liberated

Diagnosis of botulism Confirmation of initial clinical diagnosis rests on demonstrating toxin in the patient’s feces, serum, or vomitus. Fecal samples are the best specimens for detecting toxin in food poisoning or infant botulism. Toxin is usually detected by lethal effect in mice neutralization by specific antisera.

Diagnosis of C difficile disease Diarrhea associated with antibiotic therapy in the preceding 4 to 6 weeks. The isolation of C difficile and the presence of toxin A and/or toxin B in the stool. Toxins can be detected by: Cellular cytotoxicity test: Latex agglutination test ELISA

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