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Clostridium
- Microscopic appearance of different species.
- Differentiation between species according
to
biochemical reactions.
Clostridium perfringens
Clostridium perfringens
On Blood Agar C. perfringens produces large beta-haemolytic
colonies are produced. Some strains produce a double zone of
haemolysis.
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
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.
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
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.
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.
On lactose egg yolk medium, C. perfringens:
● Produces lecithinase C (alpha toxin)
● Ferments lactose
C.perfringens
produce proteolytic
enzyme (gelatinase)
that liquefy gelatin.
Gelatin Hydrolysis
On Robertson’s cooked meat medium C. perfringens is
saccharolytic
and slightly proteolytic.
Clostridium botulinum
Clostridium botulinum
Clostridium botulinum
On Blood Agar C. botulinum produces large semi-transparent
colonies with a wavy outline. Most strains are beta-
haemolytic
On Robertson’s cooked
meat medium C. botulinum
is proteolytic.
Clostridium tetani
Clostridium tetani
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).
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
Clostridium difficle
On Blood Agar
C. difficile produces
large non-haemolytic
colonies.
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