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