Gram Positive Bacilli Laboratory diagnosis of Clostridium Laboratory
Gram Positive Bacilli
Laboratory diagnosis of : Ø Clostridium
Laboratory diagnosis: Ø Sample Ø Direct smear Ø Culture Ø Identification of culture Ø Colonies Ø Film Ø BR Ø Animal pathogenicity Ø special
Clostridium
THE GENUS CLOSTRIDIUM Morphology Cl. tetani Cl. perfringens Cl. botulinum
THE GENUS CLOSTRIDIUM Culture Cl. tetani Cl. perfringens Cl. botulinum Culture ch’: • O 2 � obligate anaerobe • CO 2 0. 03% • Temp. 37°C 37 • PH 7. 4 __________________________________ Media • Ordinary media: grow • Blood agar: haemolysis • Robrtson cooked meat medium
THE GENUS CLOSTRIDIUM Biochemical Reactions Cl. tetani Sugar: -ve Cl. perfringens GMMLS GML indole +ve LM: acidified+clotted Cl. botulinum
Methods of anaerobiosis 1)Deep agar 2)Media containing reducing compounds 3)Absorption of O 2 by Na-pyrogallate (Buchner`s tube or Mc. Leod’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 • Culture ch: �O 2: Obligate anaerobic, �CO 2 �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. : • O 2: Obligate anaerobe. CO 2: … 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 p. H 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 Casein precipitated by clotted. Big amount of gas may acid. This type of clot will be produced which splits the dissolve if treated with clotted milk into pieces (stormy Na. OH clot) Reaction 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 • Cellular cytotoxicity test: • Latex agglutination test • ELISA by:
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