1. Gram Positive Bacilli

2. Laboratory diagnosis of :
Clostridium

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

4. Clostridium

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

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

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

8. 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)

9. 4) Replacement of Oxygen with hydrogen
Gas-bag jar

11. 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.

12. 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.

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

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

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

16. 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).

17. 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).

18. 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.

19. 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

20. 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.

21. 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

22. Thank You
Niveen