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 withC. perfringens antitoxin. Allow to dry

5- Inoculate also a non-toxin producing control organismthat 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 centreline (inoculum passes from the antitoxin-freehalf of the plate to the antitoxin-covered half.

A heavy inoculum of the test organism is incubated for up to4 hours in a tube containing litmus milk. Reduction of thelitmus milk is indicated by a change in colour of the mediumfrom 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 saccharolyticand 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 producesa fine film of growth. Use a hand lens to examine the plate.

On fresh blood agarC. 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 produceslarge 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