Bacterial Culture Isolation and Clostridium Tetani

Posted on Mar 3, 2025 in Microbiology

Variant 11

1. Pure Culture Isolation of Aerobic and Anaerobic Bacteria

Principles and Methods of Isolated Colonies’ Obtaining

Methods of Pure Anaerobes’ Culture Isolation

Isolation of Anaerobic Microorganisms

1st Day:

  1. Obtain specimen from patient.
  2. Examine specimen under light microscope using Gram’s technique.
  3. Inoculate specimen on Robertson cooked medium (glu + liver + layer of oil).
  4. Incubation, 24 hrs. 37°C.

2nd Day:

  1. Examine pureness under light microscope by Gram staining.
  2. Dilution of microorganisms in melted agar (MPA).
  3. Incubation of 3 pasteur tubes. 24 hrs. 37°C.

3rd Day:

  1. Describe colony in pasteur tube (size, shape, colour, surface, edges, consistency, opacity).
  2. Examine pureness under microscope by Gram staining by choosing 1 isolated colony.
  3. Inoculation on new Robertson cooked medium.
  4. Incubation, 24 hrs. 37°C.

4th Day:

  1. Take specimen from Robertson cooked medium.
  2. Examine under light microscope by Gram staining.
  3. Identification of microorganisms (study biochemical property & antigenic structures).

Isolation of Pure Culture of Aerobic Microorganisms

1st Day:

  1. Obtain specimen from patient.
  2. Examine specimen under light microscope using Gram’s technique for checking microflora type and amount of colony.
  3. Inoculate specimen on solid medium to obtain pure culture colony using spread or streak method.
  4. Incubation, 24 hrs. 37°C

2nd Day:

  1. Describe colony (size, shape, colour, surface, edges, consistency, opacity)…
  2. Examine pureness under microscope by Gram staining by choosing 1 isolated colony.
  3. Inoculation on meat peptone agar.
  4. Incubation, 24 hrs. 37°C.

3rd Day:

  1. Take specimen from meat peptone agar.
  2. Examine under light microscope by Gram staining.
  3. Identification of microorganisms (study biochemical property & antigenic structures).

2.

3. Clostridium tetani

Classification

  • Class: Clostridia
  • Order: Clostridiales
  • Family: Clostridiaceae
  • Genus: Clostridium
  • Species: C. tetani

Morphology

  • Gram “+” bacillus with terminal spore.
  • Due to this morphological shape, it is compared to that of a drumstick.
  • Motile by peritrichous flagella, non-capsulated.

Cultural Properties

  • Cl. tetani are obligate anaerobes.
  • They can grow in ordinary media such as nutrient agar or blood agar in anaerobic conditions.
  • Robertson cooked medium is also widely used for cultivation.
  • In blood agar, Cl. tetani form small hemolytic colonies. Hemolytic activity is a result of hemolysin action (tetanolysin).

Biochemical Properties

  • Cl. tetani does not ferment any sugar.
  • It does not produce catalase and oxidase. Cl. tetani is slightly proteolytic. It forms indole but no H₂S.

Antigenic Structure

  • O somatic antigen – common
  • H flagella antigen – type specific

According to the structure of flagella antigen, Cl. tetani is differentiated into 10 types.

  • The type of Cl. tetani is detected by agglutination test.
  • All types of Cl. tetani produce the same toxin which is pharmacologically and antigenically identical.
  • Tetanospasmin is a neurotoxin, oxygen-stable, and heat-labile. It is protein and rapidly destroyed by proteolytic enzymes. It is very toxic for humans and animals and causes spasm. Tetanospasmin is a good antigen and neutralized by antitoxin antibodies.

Pathogenesis

  • Tetanospasmin of Cl. tetani is the essential pathogenic product.
  • The toxin has a direct effect on the central nervous system.
  • When bacteria (vegetative forms) germinate from spores, toxin is produced and is absorbed by motor nerve endings which travels along the motor neurons of peripheral nerve to the anterior horn cells of the prevalent part.

Laboratory Diagnosis

  • It is not done due to bright clinical manifestations.
  • Clinical manifestations include: risus sardonicus and opistotonus

But in post-mortal examinations, diagnosis can be done by bacterioscopical, bacteriological, or biological method.

  • Biological method:
  • Animal inoculation is done for demonstration of toxigenicity which only establishes the pathogenicity of the isolated microorganisms.
  • 2 mice are taken, 0.2 ml culture filtrate is injected into the right side of the base of the tail of one mouse (test animal), the same amount of toxin filtrate is injected into the other animal (control) which has received tetanus antitoxic serum one hour before the test.
  • In a positive case, the test animal develops stiffness and spasm of the tail.
  • Death occurs in 1-2 days.
  • The control animal shows no change due to neutralization of toxin by antitoxin.

Treatment

  1. Administration of antitetanus antitoxic serum
  2. Surgical approach
  3. Antibiotic therapy
  4. Administration of tetanus toxoid

Prevention

Administration of tetanus toxoid.