Corynebacterium Question And Answers

Corynebacterium

 

Question 1. Describe morphology, culture characteristics, and pathogenicity of Corynebacterium diphtheriae.
Answer:

It is gram-positive, non-acid-fast bacilli.

Corynebacterium Diphtheriae Morphology

• It is a thin, slender gram-positive bacillus.
• It is 3 to 6 µ in length and 0.6 to 0.8 µ in width.
• It is non-capsulated, non-motile, non-sporing.
• It shows pleomorphism, show granules or uneven staining.
• They contain metachromatic granules. Granules are composed of polymetaphosphate. Granules appear bluish-purple when stained with methylene blue, so they are also known as B abes Ernst granules or volutin granules. Granules represent energy storage.
• Special stains used for granules are Albert’s stain, Neisser’s stain and Ponder’s stain.
• They show clubbing at one or both ends due to the presence of metachromatic granules at their ends.
• Granules are always situated at the poles of bacilli and are also known as polar bodies.
• Bacilli are seen in pairs or groups. Bacilli forms various angles with each other resembling like letters V or L, this is called Chinese letter arrangement. A uniform pattern is also seen because of the incomplete separation of daughter cells after binary fission

Corynebacterium Diphtheriae Cultural Characteristics C. diphtheriae are grown best on media enriched with blood, serum, or egg. Growth is scanty on ordinary media. They are aerobic and facultatively anaerobic. The optimum temperature for growth is 37°C and the optimum pH is 7.2. Following are the usual media employed for the cultivation of diphtheria bacillus:

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• Hiss Serum Water: This is a liquid medium containing serum growth is seen as turbidity and pellicle formation.
• Loeffl’s Serum Slope: Diphtheria bacilli grow on this medium very rapidly. Colonies appear after 6–8 hours of incubation, long before other bacteria grow. The colonies are small, circular, white, or creamy and glistening.
• Tellurite Blood Agar Medium: It contains potassium tellurite (0.04%) which inhibits most other bacteria and thus acts as a selective agent. The organisms grow slowly on this medium and form grey or black colonies due to the reduction of potassium tellurite to tellurium.
  • The colonies may take 2 days to appear on this medium. Based on colony morphology on tellurite medium and other properties, three main biotypes of C. diphtheriae—gravis, intermedius, and mitis are distinguished.

Corynebacterium Diphtheriae Pathogenecity Diphtheria releases toxins and produces local and systemic effects.

Corynebacterium Diphtheriae Local Effects

• The bacilli remain confined to the site of entry where they multiply and start producing toxins.
• The toxin causes local necrotic changes along with superficial inflammatory reactions.
• The necrosed epithelium together with fibrinous exudate, leukocytes, erythrocytes, and bacteria, constitute the pseudomembrane, which is a characteristic feature of diphtheritic infection.
• The mechanical complications of diphtheria are due to pseudomembrane, whereas the systemic effects are due to toxins.

Corynebacterium Diphtheriae Systemic Effects

• Diphtheria toxin diffuses into the bloodstream and causes toxemia.
• The toxin has got affinity for cardiac muscle, adrenals, and nerve endings.
• It acts systemically on the cells of these tissues.
• The bacilli themselves do not play any part in systemic effects because they neither penetrate into the tissues nor pass into the bloodstream producing bacteremia.

Question 2. Write the morphology and staining reactions.
Answer:

Staining Reactions: The staining reaction of Corynebacterium diphtheriae is uneven

• With gram stain Corynebacterium diphtheriae appear as gram-positive bacilli arranged in a Chinese letter pattern with metachromatic granules.
• With Loeffl’s methylene blue stain, granules take up bluish purple color and are called metachromatic granules.
• In order to accentuate the granules and simple identification special staining methods are used. Special stains such as Albert stain, Neisser stain, and Ponder stain are used for staining the bacilli.
• Bacilli appear as green beaded slender rods in Chinese letter pattern and metachromatic granules appear bluish-black in color when Albert stain is used.
• By Neisser’s method, metachromatic granules are stained blue-black, and cytoplasm is brown in color.

Question 3. Write a short note on laboratory diagnosis of diphtheria.
Or
Write briefly on laboratory diagnosis of Corynebacterium diphtheriae.
Or
Describe laboratory diagnosis of diphtheria.
Answer:

Laboratory Diagnosis of Diphtheria

Collection of specimen: Two swabs from the lesions (throat, nose, larynx, ear, conjunctiva, vagina, or skin) are collected. One swab is used for smear examination and the other for culture.

Bacteriological Studies or Isolation of Bacteria

1. Direct smear examination:
   • Smears are stained with both Gram and Albert stains. Diphtheria bacilli show beaded slender green rods in typical Chinese letter patterns on
   • Albert’s staining. In gram-staining they resemble gram-positive organisms arranged in Chinese letter pattern with metachromatic granules.
2. Culture: The swabs are inoculated on the following culture media:
   • Loeffl’s serum slope: Growth appears within 6-8 hours on this medium. Subculture from Loeffl’s serum slope is made on tellurite blood agar and slate is incubated at 37°C for 48 hours.
   • Tellurite blood agar: These plates have to be incubated at 37°C for at least 48 hours before declaring these as negative, as growth may sometimes be delayed.
   • Blood agar: It is useful for differentiating streptococcal
     or staphylococcal pharyngitis which may simulate diphtheria.
3. Colony morphology and staining: On Loeffl’s serum slope, the colonies are small, circular, white, or creamy.
   • Diphtheria bacilli grow as black or gray-colored colonies on tellurite blood agar. Smears are prepared from suspected growth from various media.
   • These smears are stained with Albert- and Gram-stain to confirm the morphology of C. diphtheriae. Albertstaining shows green bacilli with bluish-black metachromatic granules. Gram staining reveals gram-positive organisms arranged in a Chinese letter pattern with metachromatic granules.
4. Biochemical reactions:
   • Fermentation of sugar in Hiss’s serum media with the production of acid-only, pathogenic diphtheria ferment sucrose and mannitol.
   • Starch and glycogen fermentation: Gravis ferments both, intermedius and mitis do not ferment any of them.
   • Nitrate reduction is positive
   • Hydrogen sulfide is positive.

Demonstration of Its Toxicity by Virulence Tests

In vivo tests:

• Subcutaneous test
• Intracutaneous test

In vitro tests:

• Elek’s gel precipitation test
• Tissue culture test.

In vivo tests:

• Subcutaneous Test:
  • Growth from an overnight culture on the Loeffl’s serum slope should be emulsified in 2 to 4 mL broth and 0.8 mL of emulsion is injected subcutaneously in two guinea pigs.
  • One guinea pig is protected with 500 units of diphtheria antitoxin 18–24 hours before the test. If the strain is virulent unprotected animals die in under 4 days.
• Intracutaneous Test:
  • Broth emulsion should be injected intracutaneously in two guinea pigs. Out of these two, one guinea pig acts as a control, i.e. it is protected with 500 units of diphtheria antitoxin.
  • Another guinea pig should be injected with 50 units of antitoxin intraperitoneally 4 hours after the test for prevention of death.
  • Toxigenecity should be indicated by inflammatory reaction at the site of injection, progressing to necrosis in 48–72 hours in test animals.

In vitro tests:

• Elek’s Gel Precipitation Test:
  • This is an immunodiffusion test. A rectangular strip of filter paper soaked in diphtheria antitoxin (1000 units per mL) is placed on the surface of a 20% horse serum agar plate, while the medium is still fluid.
  • When the agar solidifies, the test strain is streaked at a right angle to the filter paper strip.
  • The positive and negative controls are also put up. The plate is incubated at 37°C for 24 to 48 hours.
  • The toxin produced by the bacterial growth diffuses in the agar and produces a line of precipitation where it meets the antitoxin at optimum concentration.
  • There is no formation of precipitate in the case of nontoxigenic strains.
• Tissue Culture Test: In this toxigenicity of diphtheria bacilli is demonstrated by incorporating the strain in an agar overlay of cell culture monolayers. The toxin which is produced diffuses and kills the cells below.

Question 3. Name a few nonpathogenic strains of Corynebacterium and their differences to Corynebacterium diphtheria.
Answer:

An organism that is distinguished morphologically from Corynebacterium diphtheriae is called diphtheroid and is nonpathogenic. It is found on the conjunctiva, mucous membrane of the nasopharynx, oral cavity, and genitalia.

• C. hofmannii is found in the human throat.
• C. xerosis has been isolated from xerosis. Its relation with disease is uncertain. It may be found on skin as normal flares.
• Propionibacterium acnes is found in acne pustules and is anaerobic. It produces lipases that split free fatty acids from skin lipids.
• These fatty acids can produce inflammation and contribute to acne.
• Tetracycline can inhibit lipolytic action.
  • Morphology: It is short stumpy 1.5 to 2 µ in length with parallel sides and rounded ends. it does not exhibit pleomorphism and has no metachromatic granules.
  • lt occurs in palisades or bundles with uniform shapes and staining. Actual differentiation can be made by biochemical reaction and virulence tests.

 

Question 4. Write organisms causing a sore throat.
Answer:

The following are the organisms causing sore throat:

• Bacteria
  • S. pyogenes
  • Streptococcus groups C and G
  • Corynebacterium diphtheriae
  • Hemophilus influenza
  • Bordetella pertussis
  • Treponema vincentii
  • Leptotrichia buccalis.
• Fungus
  • Candida albicans.
• Virus
  • Epstein-B arr virus
  • Adenovirus
  • Coxsackievirus A6.