Corynebacterium

Lab Ten:- 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).

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 1-8?m and width of 0.3-0.8?m 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. The cell wall sugars include rbinose , galactose and mannose.

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.


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 protein .

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.

Virulence tests of C.diphetheriae:-

There are two types:

1- In vitro.
* Elek’s gel precipitation test.

2- In vivo(rabbit).
* Subcutaneous.
* Intradermal.
Elek test

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 from lines of precipitin.