Vibrio cholera

احمد معن سعدهللا •

بالل مؤيد جاسم •

بالل محمد حازم •

فراس الطائي: بأشراف الدكتور •

Genus : Vibrio

the main species:

.choleraCauses epidemic and pandemic : Vibrio cholerae .1

2. Non-cholera Vibrios: Cause ear, wound, soft tissue, and extra-intestinal infections.

3. Vibrio parahaemolyticus: Causes gastroenteritis, and

possibly extra-intestinal infections.

Vibrionacae

• Catalase +

• Oxidase +

• They ferment glucose, maltose, mannite, and sucrose with acid production only

• The TSI shows A/A profile (yellow colour)

Pseudomonas

• Catalase +

• Oxidase +

• Only glucose fermented without gas

• The TSI shows K/A , pink /yellow

Enterobacteae

• Catalase +

• Oxidase –

• LF : E.coli , klebseilla , citrobacter , enterobacter .

• NLF : shigella , salmonella , proteus .

Pseudomonas Vibrionacae Enterobacteae

Catalase +ve Catalase +ve Catalase +ve

Oxidase +ve Oxidase +ve Oxidase –ve

NLF Ferment

maltose,lactose,manitol

Ferment glucose

motile motile Motile / shigella ,

klebsiella ,yersinia

• Gram negative

• Short curved rods like commas with marked pleomorphism size

• These m.o. are : highly aerobic

• very actively motile (described as shooting stars or darting motility)

• The best way to detect motility is by dark-field or contrast microscopy

• These m.o. can tolerate temprature of range 18-37.

Biochemical characteristics of V. cholerae:

•They ferment glucose, maltose, mannite, and sucrose with acid production only.

• These m.o. are:

respiratory (oxidase positive)

fermentative (catalase positive).

• They give positive cholera-red reaction on nitrate peptone medium by reducing nitrate to nitrite, and produce indol (+ve) on tryptophan medium.

•The TSI shows A/A profile (yellow colour).

• The optimum pH for the growth of these m.o. is 7.0 , but they can tolerate up to 8.5 - 9.0.

• They are very sensitive to acidic pH (less than 6), therefore, they are quite susceptible to gastric juice (a major barrier against V. cholerae).

بالل محمد حازم

Culture of V. cholerae

This m.o. can be cultivated on different media:

Alkaline Peptone Water (APW;

Sea Water): This medium is of pH 8.5 – 9.0 and used for the

primary (first) isolation from clinical specimens. The

m.o. grow forming a surface pellicle within 8 hours.

Then, from this medium the m.o. can be sub-

cultured on solid media

Many m.o. can not tolerate such pH, but

Pseudomonas does.

2. Thiosulfate Citrate Bile Salt Sucrose Agar (TCBS):

The non-inoculated medium is olive green in colour, and contains an indicator called bromo-thymol blue.

V.Cholerae is sucrose- fermenter, and due to acid production, the pH drops and colour of the medium and colonies become yellow. Aeromonas can produce similar chages in this medium.

TCBS

3. Tellurite Taurocholate Gelatin Agar (TTGA)

On this medium the colonies show

iridescence and are surrounded by cloudy

zone of gelatin hydrolysis.

4. Meat Extract Agar: On this medium the colonies appear

translucent green to red bronzy in colour and

in old cultures the colonies become opaque

and rough

5. Taurocholate Pepton Broth:

This medium can be used primary

cultivation of the m.o., then sub-

cultured on other media

6. MacConkey’s Agar:

V.Cholerae colonies are of non-lactose

fermenters

7. Blood Agar:

El-Tor growth shows beta-haemolysis ( versus the

classical).

In general, the colonies of V. cholerae are convex,

smooth, granular.

احمد معن سعدهللا

Practical procedure of cultivation of V. cholera :

1. Direct stool examination:

a. Motility: Dark field or contrast microscopy.

b. Microscopical exam.: mucous,epith.cells,and large

number of m.o. , but no pus cells are seen.

c. Fluorescent antibody staining.

2. From the APW, TCBS and

another APW are inoculated and

left either for 5 hours or

overnight.

3.The colonies are subjected to

further identification.

Further Identification of V. cholerae:

1. String test: A drop of 0.5% Na-deoxycholate is mixed with a colony ;

after 60 seconds, with a loop there will a thread of

tenacious nature. If positive, it means V. cholerae.

2. Polyvalent anti-sera (anti-O1,anti- O139): To confirm that the V. cholerae belongs to O-1 or

O-139, agglutination test with specific polyvalent sera and

emulsified colonies is carried out.

3. Biochemical tests including cholera

red reaction.

4. Differentiation between classical & El-

Tor Biotypes.

5. Serotyping: by specific antisera

against A, B and A antigenic factors to

determine Ogawa, Inaba & Hikojima.