Clostridium

Lab Twenty One:- ________________ Medical Microbiology
Prepared by: Luma J. Witwit


Clostridium

Members of genus Clostridium are Gram positive spore forming rods that are anaerobic. These motile bacteria are ubiquitous in nature and are especially found on soil , under the microscope they appear as long drum sticks with a bulge located at their terminal ends.

A Gram-stain is a good method for identifying Clostridium because the cell incorporates the dye while the spore remains unstained. Clostridium shows optimum growth when plated on blood agar at human body temperatures. When the environment become stressed, however, the bacteria produce spores that tolerate the extreme conditions that the active bacteria cannot, in their active form, these bacteria secrete powerful exotoxins that are responsible for such disease as tetanus, botulism, and gas gangrene. The four clinically important species of Clostridium: C. tetani, C.perfringens, , C.botulinum and C. difficile.


Clostridium perfringens

Morphology

• Large rectangular bacilli (rod) staining gram positive.
• Spores rarely seen in vitro or in clinical specimens (ovoid, sub terminal).
• Non motile, but rapid spreading growth on blood agar mimics growth of motile organisms.
• Aerotolerant, especially on media supplemented with blood.
• Grow at temperature of 20-50 Co (optimum 45 Co) and pH of 5.5-8.0
• Four major lethal toxins (alpha (a), beta (b), epsilon (e), and iota (i) toxins, and an enterotoxin.
• C. perfringens sub divided into five types (A-E) on basis of production of major lethal toxins.
• C. perfringens reduces nitrates to nitrites a violet color which develops with in (5 min ) indicates presence of nitrites.


Lab Identification:

• Direct smear and Gram stain, capsules upon direct examination of wound smears.
• Cultures takes advantage of rapid growth in chopped meat media at 45 Co and then isolate on blood agar streak plate after four to six hours.
• Gas from glucose fermentation.
• Double zone of hemolysis on blood agar beta-hemolytic (theta toxin), alpha -hemolytic (alpha toxin).
• "Stormy fermentation" (coagulation) of milk due to large amounts of acid and gas from lactose (Iron-milk medium)


Clostridium tetani …. (A gent of tetanus)

Morphology

- Long thin gram-positive organism that stains gram negative in old cultures, round terminal spore gives drum stick appearance, motile by peritrichous flagella.

- Grow on blood agar or cooked meat medium with swarming. Beta-hemolysis exhibited by isolated colonies. spores resist boiling for 20 minutes.

- Binds gangliosides in synaptic membranes (synapses of neuronal cells) and blocks release of inhibitory neurotransmitters; continuous stimulation by excitatory transmitters.


Lab Identification

Use characteristics of resistance to heat, motility, and toxin production to help identify.

C. botulinum
a gent of botulism, a rare, but sever (lethal) neuroparalytic disease.

Morphology
- Heterogeneous group of fastidious, strictly anaerobic bacilli motile by peritrichous flagella heat-resistant spores (ovoid, sub terminal)
- proteolytic and non-proteolytic.
- species divided into four groups (I-IV) based on type of toxin produce and proteolytic activity.

Lab Identification
- Microscopic detection or (culture) are often unsuccessful (few organisms and slow growing )
- Toxin detected and typed in lab via toxicity and antitoxin neutralization tests in mice or by ELISA.

C. difficile
Motile bacterium (part natural intestinal flora ), Infection –acquired person to person in hospital.
- Antibiotics-associated diarrhea.
- Antibiotic – associated pseudo membranous colitis.
No terminal spores
Non-aerotolerant