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Morphology
Cl. tetani
Cl. perfringens
Cl. botulinum
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
Biochemical Reactions
Cl. tetani
Cl. perfringens
Sugar: -ve
GMMLS
indole +ve
LM: acidified+clotted
Cl. botulinum
GML
Methods of anaerobiosis
1)Deep agar
2)Media containing reducing compounds
3)Absorption of O2 by Na-pyrogallate (Buchner`s tube or McLeod’s plate)
4) Replacement of Oxygen with hydrogen
Gas-bag jar
 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.
 Culture
• Culture ch:
 O2: Obligate anaerobic,
 CO2
 Temp. 37°C.
• Media
 On blood agar:
 complete haemolysis
 swarming,
 BR:
• Indole: +ve
• Suger fermantation: -ve.
of C.tetani must be confirmed by
production of toxin and its neutralization
by specific antitoxin.
 Isolation
 Patient
should be treated without waiting for
laboratory confirmation.
Specimens:
 Material from wounds,
• Pus,
• Tissue,
• Exudates.
 Direct
smear stained with Gram stain:
• Gram-positive,
• Sporulated: subterminal, not bulging
• non motile
• capsulated bacilli.
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).
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).
 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.
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
Reaction
Indicator blue
Ammonia liberated
 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.
 Diarrhea
associated with antibiotic therapy in
the preceding 4 to 6 weeks.
isolation of C difficile and the presence of
toxin A and/or toxin B in the stool.
 The
 Toxins can be detected
• Cellular cytotoxicity test:
• Latex agglutination test
• ELISA
by: