Clostridium - Microscopic appearance of different species. - Differentiation between species...
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Transcript of Clostridium - Microscopic appearance of different species. - Differentiation between species...
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Clostridium
- Microscopic appearance of different species.
- Differentiation between species according
to
biochemical reactions.
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Clostridium perfringens
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Clostridium perfringens
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On Blood Agar C. perfringens produces large beta-haemolytic
colonies are produced. Some strains produce a double zone of
haemolysis.
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1- Prepare a plate
of lactose egg yolk
milk agar
2- Turn the plate
over, and using a
wax pencil, draw
a line across the
centre of the
plate
3- Using a sterile swab, cover one half of the medium with
C. perfringens antitoxin. Allow to dry
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5- Inoculate also a
non-toxin
producing control
organism
that will grow
anaerobically.
6- Incubate the plate anaerobically at 35–37 ºC
overnight.
7- Look for an opacity around the inoculum in the half of the plate containing no antitoxin and no opacity in the half containing the antitoxin.
4- Inoculate the test
organism at right
angles to the centre
line (inoculum passes
from the antitoxin-free
half of the plate to
the antitoxin-covered
half.
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A heavy inoculum of the test organism is incubated for up to
4 hours in a tube containing litmus milk. Reduction of the
litmus milk is indicated by a change in colour of the medium
from mauve to white or pale yellow
Litmus Milk Reduction Test
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Nitrate Reduction Test
C.perfringens can reduce nitrate to nitrite which detected by
addition of sulfanilic acid which react with nitrite to form
diazonium salt which react with added alpha-naphthylamine to
form red colour.
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Lecithinase C activity: Seen as an opacity in the medium dueto the breakdown of lecithin in the egg yolk.
Lipase hydrolysis: Seen as (fatty) layer coveringcolonies and sometimes extending into the medium.
Lactose fermentation: There is a reddening in the medium.The colonies become red on exposure to air.
Proteinase activity (proteolysis): Shown by an area of clearingaround the colonies due to the breakdown of casein in themilk by the enzyme proteinase.
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On lactose egg yolk medium, C. perfringens:
● Produces lecithinase C (alpha toxin)
● Ferments lactose
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C.perfringens
produce proteolytic
enzyme (gelatinase)
that liquefy gelatin.
Gelatin Hydrolysis
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On Robertson’s cooked meat medium C. perfringens is
saccharolytic
and slightly proteolytic.
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Clostridium botulinum
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Clostridium botulinum
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Clostridium botulinum
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On Blood Agar C. botulinum produces large semi-transparent
colonies with a wavy outline. Most strains are beta-
haemolytic
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On Robertson’s cooked
meat medium C. botulinum
is proteolytic.
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Clostridium tetani
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Clostridium tetani
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On Blood Agar
C. tetani produces
a fine film of growth.
Use a hand lens to
examine the plate.
On fresh blood agar
C. tetani is
haemolytic
(alpha first followed
by beta haemolysis).
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When C.tetani is cultured in a medium which contains
tryptophan. Indole production is detected by Kovac’s reagent
which contains 4 (p)-dimethylaminobenzaldehyde which reacts
with the indole to produce a red coloured compound.
Indole Production
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Clostridium difficle
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On Blood Agar
C. difficile produces
large non-haemolytic
colonies.
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C. difficile can grow in bile esculin agar and turns the indicator
ferric ammonium citrate to a dark brown color results from
combination of esculetin end product of esculin hydrolysis
with ferric ions to form a phenolic iron complex.
Esculin
Hydrolysis