Lab Ten:-
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Medical Microbiology
Prepared by: Luma J. Witwit
Corynebacterium
Corynebacteria belong to the family Mycobacteriaceae and are part
of the CMN group (Corynebacteria , Mycobacteria and Nocardia).
The most important member of the genus Corynebacterium is
C.diphtheriae. It was first observed and described by Klebs but was first
cultured by Loeffler. Therefore, it is known as the Klebs- Loeffler
bacillus.
Corynebacterium diphtheria infects the nosophyrnx or skin. Toxigenic
strains secrete a potent exotoxin which may cause diphtheria. The toxin is
distributed to distant organs by the circulatory system and may cause
paralysis and congestive heart failure.
Morphology:C. diphtheria is pleomorphic, club shaped, Gram positive bacilli
arranged in V forms or palisad, an aerobic organism characterized by
non-encapsulated, non-sporulated , immobile , non acid fast, straight,
curved rods with a length of 1-8μm and width of 0.3-0.8μm which form
ramified aggregation in culture looking like "Chinese letters".
Cultures in vitro, contain thin spots in their cell walls that allow
decolorization during the Gram stain and result in a Gram variable
reaction. Older culture often contain metachromatic granules
(polymetaphosphate ) which stain bluish – purple with methylene blue.
Most cell contain 2 or 3 of these granules, and they tend to be on the
poles. With Albert stain, the granules stain bluish- black and the
cytoplasm green. The presence of this granules in thin slender bacilli
helps to distinguish C.diphtheriae from short, thick, plumpy, nonpathogenic diphtheriods which lack them.
Specimens:Dacron swabs from the nose, throat, or throat suspected lesions must
be obtained before antimicrobial drug are administered. Swabs should be
collected from beneath any visible membrane. The swab should be placed
in semisolid transport media such as Amies.
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Lab Ten:-
________________
Medical Microbiology
Prepared by: Luma J. Witwit
Diagnosis
Smears:Smears stained by Albert stain or gram stain show beaded rods in
typical arrangement.
- primary isolation:They are aerobes and facultative anaerobes. Optimum temperature for
growth is 37C°. They can grow on ordinary nutrient agar, but their
growth is improved by the presence of animal protien.
A verity of media may be used as:* Loeffler’s serum slop: which is used to obtain early growth of this
bacteria, colonies can be seen in 6-8 hours, long before other bacteria
grow, for rich production of metachromatic granules and for conducting
toxigenicity test of this bacteria. The colonies are at first small, white,
opaque disc, but on continued incubation increase in size and may
aquired a yellow tint.
* Blood tellurite agar (BTA): is a selective media for corynebacteria
by inhibiting most other pathogenic and commensal bacteria. On this
media, colonies become gray to black because tellurite is able to diffuse
through the cell wall and membrane and are reduced to tellurium metal,
which is participated inside the cell.
On the basis of colonial morphology on BTA, diphtheria bacilli can be
divided in to three biotypes- mitis, intermedius and gravis and hemolysis
on sheep blood agar plates. Most strains the biotype mitis show βhaemolysis, gravis strains are often weakly haemolytic, but intermedius
strains are non- haemolytic.
Virulence tests of C.diphetheriae:There are two types:
1- In vitro.
* Elek’s gel precipitation test.
2- In vivo (rabbit).
* Subcutaneous.
* Intradermal.
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Lab Ten:-
________________
Medical Microbiology
Prepared by: Luma J. Witwit
In vivo testing is rarely done because the in vitro method is
reliable, lees expensive, and free from the need to use animal.
le s el per p
o es
The most common in vitro assay for toxigenicity of C. diphtheria
strains.
This test is based on the double diffusion of diphtheria toxin and
antitoxin in an agar medium.
A sterile filter paper impregnated with diphtheria antitoxin is
imbedded in agar culture medium. Isolates of C. diphtheria are then
streaked across the plate at an angle of 90 o to the antitoxin strip.
Toxigenic C. diphtheria is detected because secreted toxin diffuses
from the area of growth and reacts with antitoxin to form lines of
precipitin .
Figure 1 : Elek s immunodiffusion
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