MORPHOLOGY OF CLOSTRIDIUM BOTULINUM
Shape – Clostridium botulinum is a thin, rod shape (bacillus) bacterium.
Size – The size of Clostridium botulinum is about 5 µm × 1.0 µm (micrometer).
Arrangement Of Cells – Clostridium botulinum is arranged singly, in pairs or in short chains. They are pleomorphic organisms.
Motility – Clostridium botulinum is a motile bacterium.
Flagella – Clostridium botulinum is a flagellated bacterium with Peritrichate flagella arrangement.
Spores – The Oval, Sub-terminal & Bulging spores are present in C. botulinum.
Capsule – C. botulinum is a Non-capsulated bacterium.
Gram Staining Reaction – Clostridium botulinum is a Gram +ve (Positive) bacterium.
CULTURE REQUIREMENTS OF CLOSTRIDIUM BOTULINUM
⇒ Special requirements – Unlike C. perfringens, Clostridium botulinum has no complex nutritional requirements and readily grow in an ordinary medium like NAM. Also, it grows well in the media containing Blood or Serum, commonly Blood Agar medium & Robertson Cooked meat broth medium is used for the cultivation of Clostridium botulinum in Laboratory.
⇒ Optimum temperature – The optimum temperature for the growth of Clostridium botulinum is 35°C. Some strains of C. botulinum can grow at very low temperatures (1-5°C).
⇒ Optimum pH – 4.6 – 8.9
⇒ Oxygen requirements – Clostridium botulinum is an obligate anaerobic bacterium i.e. can only grow in the absence of oxygen (Strict anaerobe).
⇒ There are various culture media used for the cultivation of C. botulinum in the laboratory and most commonly the Sheep Blood Agar medium and CMB medium is used, the other media are as follows –
- Nutrient Agar medium
- Columbia Horse Blood Agar medium
- Alkaline Glucose gelatin medium
- Egg yolk agar medium
- Clostridium botulinum isolation (CBI) agar medium (Selective medium)
- The liquid medium (Nutrient Broth medium, TSB medium, etc.).
The Clostridium botulinum isolation (CBI) Agar medium, a selective medium for the cultivation of C. botulinum in the laboratory can easily be prepared by adding Antibiotics – Cycloserine, Sulfamethoxazole & Trimethoprim to the Egg yolk agar medium.
CULTURE CHARACTERISTICS OF CLOSTRIDIUM BOTULINUM
|Cultural Characteristics||Sheep Blood Agar Medium||Nutrient Agar medium (NAM)||Egg yolk agar medium|
|Shape||Large, Irregularly circular||Large, Irregularly circular||Large, Irregularly circular|
|Size||2-3 mm||2-3 mm||2-3 mm|
|Elevation||Slightly raised||Raised||Slightly raised|
|Color||Grey||Whitish grey||Greyish White|
|Hemolysis||β - hemolysis||-----||-----|
In Sheep’s Blood Agar medium, Clostridium botulinum grows well, and many strains of C. botulinum shows b-hemolysis (complete hemolysis characterized by a clear zone around the colonies) which is caused by the production of botulinolysin (a hemolysin toxin). However, there are many strains which do not produce this toxin hence shows no hemolytic activity in blood agar medium.
In Roberson’s cooked meat broth medium, C. botulinum grows well, produce turbidity and the meat is not digested but may turn Black with foul odor due to the proteolytic action of Clostridium botulinum. It also produces gas and turns pH of the medium Acidic.
In liquid culture media like Trypticase soy broth or Nutrient broth, the growth of the bacterium occurs as a turbidity in the broth medium, which is further analyzed for the morphology (under the microscope), gram reaction, biochemical tests, and Clostridium botulinum specific tests.
Moreover, in alkaline glucose gelatin medium, the spores of Clostridium botulinum are consistently produced when incubated at 20–25°C. In, CBI agar medium (Clostridium botulinum isolation agar), which is a selective medium for Clostridium botulinum the colonies are similar as that on Egg yolk agar but it prevents the growth of contaminant and only allows the growth of Clostridium species especially C. botulinum and commonly employed for its isolation from fecal matter where a large no. of contaminant bacteria are present
That’s all about the Morphology & Culture Characteristics of Clostridium botulinum