Lab

Corynebacteria

1. Pathogenic spp. ( C. diphtheria (Klebs- Loeffler Bacilli: KLB)
2. Nonpathogenic spp. (Diphtheriod). Normal flora of Upper Respiratory Tract (URT) ( C. ulcerans and C. urealyticum (can cause infections in immunocompromized patients).

Microscopical Characteristics:-

1. G + ve bacilli, non spore forming, non motile, club shaped (swallow at one end ) due to the presence of metachromatic granules (called volutin). Readily lose Grams stain; therefore use other specific stain like (Neisser stain, Albert stain).
2. Arrange in parallel or in acute angle, so called Chinese letters (dx. feature) according to its appearance. See below figure.



(Albert stain)

Diseases and Virulence factors:-
1. Local infections:
a.) Respiratory diphtheria due to Production of Potent exotoxin that Inhibits protein synthesis which leads to inflammatory response, necrosis and formation of gray pseudomembrane on tonsils, pharynx, and larynx leading to suffocation and death, mainly in young children.
b.) Cutaneous diphtheria where bacteria enter an injured skin forming gray pseudomembrane on that skin.


2. Systemic effects:
Spread of exotoxin via blood stream results in necrosis in different organs (heart, kidneys & liver) and nerve damage. This bacteria never invade blood only their toxin.

Laboratory Diagnosis:-

1. Specimens (Respiratory (Throat swab (from the pseudomembrane)
Requested as (KLB)
Cutaneous ( Skin swab.

2. Staining:

a) Grams stain ( G + ve bacilli, Chinese letter, colorless volutin granules.

b) Albert's stain ( Metachromatic granules (dark), bacilli (green), Chinese letter arrangement.

3. Culture:
Can grow on chocolate but there are more specific media:
a. Loefflers agar. (Enriched media only) contains serum or egg to enhance growth and metachromatic granules production.
b. K+ or Cystintellurite blood agar (selective & enrichment media). Colonies appears gray black due to tellurite reduction to telluride.
On culture differentiates between C. diphtheriae strains:
C. diphtheria gravis ( Large, non haemolytic, gray.
C.diphtheria mitis ( Small, haemolytic, black.
C. diphtheria intermidius ( medium, non haemolytic.


4. Gel-diffusion test (ELICK test): On a special plate agar, apply filter paper impregnated in antitoxin. Streak unknown toxin producer microorganisms in acute angle to the paper. After 24 hrs. notice precipitin line in 45 angles to the streaking.





4. Animal pathogenesity test (Virulence test):

a. Guinea pigs lethality (Antitoxin ( Not dead.
Without ( Dead.

5. Polymerase Chain Reaction (PCR). Detect gene of toxin.

6. Enzyme Linked Immuno Sorbent Assay (ELISA).
7. Immunoblot (immunochromogenic method).

Treatment:-

1. Antitoxin ------------ Neutralize toxin.
2. Antibiotics ---------- Erythromycin to eliminate bacteria.

Protection:-

1. Vaccines (DPT), booster dose DT after 10 years.


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Antitoxin

Precipitation line

Control (-)

Control (+)

Unknown tox -

Unknown tox +

BACTERIOLOGY

Lab.13

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