Download Department of Microbiology Bacteriology Lab

Lab.13
BACTERIOLOGY
Corynebacteria
1. Pathogenic spp.  C. diphtheria (Klebs- Loeffler Bacilli: KLB)
2. Non–pathogenic 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 Gram’s 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.
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Lab.13
BACTERIOLOGY
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) Gram’s 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. Loeffler’s agar. (Enriched media only) contains serum or egg to
enhance growth and metachromatic granules production.
b. K+ or Cystin–tellurite 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.
Precipitation line
Antitoxin
Control (-)
Control (+)
Unknown tox Unknown tox +
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Lab.13
BACTERIOLOGY
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.
END
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