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close this bookGuidelines for Cholera Control (WHO; 1993; 68 pages)
View the documentPreface
View the documentAcknowledgements
View the document1. Introduction
View the document2. About cholera
open this folder and view contents3. Preventing cholera
open this folder and view contents4. Being prepared for a cholera epidemic
open this folder and view contents5. Early responses to the threat of an outbreak
open this folder and view contents6. Management of the patient with cholera
open this folder and view contents7. Preventing the spread of an outbreak
View the document8. Epidemiology: investigating an outbreak
open this folder and view contents9. The role of the laboratory
View the document10. After an outbreak
View the documentAdditional information on cholera control
close this folderAnnexes
View the documentAnnex 1. Building a ventilated improved pit latrine1
View the documentAnnex 2. management of the patient with cholera
View the documentAnnex 3. Sample health education messages
View the documentAnnex 4. Rules for safe food preparation to prevent cholera1
View the documentAnnex 5. Isolation of Vibrio cholerae O1 in a peripheral laboratory
View the documentSelected WHO publications of related interest
 

Annex 5. Isolation of Vibrio cholerae O1 in a peripheral laboratory

Vibrio cholerae O1, the causative agent of cholera, can be isolated and identified in any laboratory that has the basic equipment and supplies for bacteriological investigations. The vibrios are present in large numbers in the stools of patients with cholera before antibiotic therapy. They grow easily and rapidly on a variety of selective and non-selective alkaline media.

The following guidelines describe a simple and rapid method for the isolation and identification of Vibrio cholerae O1 in diarrhoeal stools.1

 

1For instructions on how to isolate Vibrio cholerae O1 from asymptomatic carriers, and water, sewage, or food samples, see the Manual for laboratory investigations of acute enteric infections (Geneva, World Health Organization, 1987: unpublished WHO document CDD/83.3 Rev 1, available on request from the Programme for Control of Diarrhoeal Diseases, World Health Organization, 1211 Geneva 27, Switzerland). This manual also describes additional tests to characterize Vibrio cholerae O1, identify atypical isolates, and distinguish Vibrio cholerae O1 from other vibrios and vibrio-like organisms.

A5.1 Collection and transport of faecal samples

Collect the stool sample before the patient is given an antibiotic. Use a clean cotton-tipped swab, and introduce it well into the rectum. When this is done properly, the swab will become moist and may be faecally stained.

Alternatively, collect freshly passed liquid stool in a bottle or on a cotton-tipped swab.

If it is possible to be certain that the sample will reach the laboratory within 2 hours, put the rectal swab or liquid stool into a sterile screw-cap bottle; seal the bottle tightly for transport.

If, however, the specimen will not reach the laboratory within 2 hours, put it into a tube containing Cary-Blair transport medium.

Alkaline peptone water (APW) may also be used if the transport time will not exceed 24 hours. At the laboratory, the specimen should be transferred to a fresh tube of APW for enrichment before inoculating solid media (see section A5.2).

When a transport medium is not available, soak strips of blotting paper with liquid stool. Send them to the laboratory in carefully sealed plastic bags to prevent drying.

Transport specimens in refrigerated boxes, if possible, or at ambient temperature.

A5.2 Culture and initial identification of Vibrio cholerae O1

Select and inoculate solid media

Laboratory technicians who are not experienced in identifying vibrios should use a selective medium and, if possible, a non-selective one. As experience is gained in recognizing typical colonies, the process may be simplified by using only the non-selective medium.

Satisfactory non-selective media include:

 

• meat extract agar (MEA), pH 8.5

• gelatin agar (GA), pH 8.2 to 8.5. Satisfactory selective media include:

• thiosulfate citrate bile salts agar (TCBS agar), pH 8.6

Note: TCBS plates should be used within 3 days of being prepared.

• taurocholate tellurite gelatin agar (TTGA), pH 8.5.

 

Instructions on preparing these media are given in section A5.3.

If possible, both the following procedures for inoculating the medium should be followed:

 

(1) Streak the specimen directly onto a non-selective or selective medium, or onto both. Incubate the plates overnight (12-18 hours) at 35-37 °C.

(2) Enrich the culture of Vibrio cholerae O1 by inoculating the swab, or by putting 2-3 loopfuls of stool into a tube of APW. Incubate the specimen for 6-8 hours at 35-37°C.

 

Then, inoculate plates from the APW tube and incubate as in (1) above.

If the incubation of APW exceeds 8 hours, inoculate a second tube of APW from the first, and repeat the incubation for 6-8 hours before inoculating solid media.

If only one procedure is followed, enrichment in APW followed by inoculation of solid media is recommended.


Identification of Vibrio cholerae O1

Identify suspicious colonies

Colonies of Vibrio cholerae O1 and Vibrio cholerae non-O1 have the same appearance:

 

• On MEA, they are colourless, translucent, flat, and 2-3 mm in diameter.

• On GA, they appear the same as on MEA, but have a halo.

• On TCBS agar, they are yellow, shiny, convex, and 2-3 mm in diameter. (Some strains of Aeromonas have a similar appearance.)

• On TTGA, they are translucent, flat, and 1-2 mm in diameter. At 24 hours they have dark pinpoint centres; later the colonies become gun-metal grey.

Perform tests to make a presumptive identification of Vibrio cholerae O1

Slide agglutination with specific antisera

Suspicious colonies should be tested for slide agglutination in polyvalent (group) Vibrio cholerae O1 antiserum.

Colonies may be tested directly from MEA, GA, or TTGA media. However, colonies from TCBS agar should not be tested directly because they are difficult to emulsify. Yellow colonies from TCBS agar should first be subcultured on MEA or GA for serological testing.

If possible, positive reactions should be confirmed with monovalent Ogawa and Inaba typing sera. Vibrio cholerae O1 will react with the O1 group antiserum and either Ogawa or Inaba typing serum.

A rapid presumptive diagnosis of Vibrio cholerae O1 can be attempted by streaking stool heavily on a pre-dried MEA or GA plate. This specimen should be incubated for 4-6 hours at 35 - 37 °C, and the confluent growth from the plate used to test for slide agglutination.

Other useful tests

Performing the above test with specific antisera is sufficient to diagnose a case of cholera when clinical and/or epidemiological patterns also suggest the disease.

However, if the required antisera are not available, the following tests may be used to support the identification of Vibrio cholerae O1, but will not differentiate serogroup O1 from other serogroups.

 

String test. Suspend an 18- to 24-hour growth from an MEA, GA, or TTGA plate, or a Kligler's iron agar (KIA) slant in a drop of 0.5% aqueous solution of sodium desoxycholate on a slide.

When positive, the suspension immediately loses turbidity and becomes mucoid (as with all vibrios). A mucoid "string" forms when the loop is drawn slowly away from the suspension. A few strains of Aeromonas show a weak string after about 60 seconds.

Oxidase test. Use fresh growth from an MEA, GA, or TTGA plate, or the KIA slant (but not a TCBS agar plate). All Vibrio cholerae (both 01 and non-01) are oxidase-positive, as are a number of other Gram-negative bacteria. However, Enterobac-teriaceae are oxidase-negative. (See section A5.3 for further instructions.)

Kligler's iron agar (KIA) reaction. Inoculate suspicious colonies into a tube of KIA. Vibrio cholerae (both 01 and non-01) produce an alkaline (red) slant, acid (yellow) butt, and no hydrogen sulfide or other gas. Some other Gram-negative bacteria also produce this reaction.

Send specimen to a reference laboratory for confirmation

Laboratory diagnosis of Vibrio cholerae O1, as described above, can be completed within 24-48 hours in a peripheral laboratory, and is sufficient for most purposes. However, when additional studies are desired to confirm cholera or to identify atypical isolates, these can be done at a reference laboratory. Such studies may include serotyping, biotyping, antibiotic sensitivity testing, and biochemical characterization of suspected Vibrio cholerae O1. They may also involve identification of atypical strains or related species.

A nutrient agar or trypticase soy agar (TSA) stab should be used to transport the specimen to a reference laboratory.

Note: In most cases, the specimen would be sent to a national reference laboratory. Special tests and training for laboratory staff, however, can be arranged with some international reference laboratories (see section 9.2).

A5.3 Preparation of media for transporting and isolating Vibrio cholerae O1

Media such as Cary-Blair, TCBS agar, and KIA are best provided to laboratories as premixed dry ingredients. However, other media can be prepared at the peripheral laboratory, according to the following instructions. (For more information on the composition of various media, see Manual for laboratory investigations of acute enteric infections.)

Alkaline peptone water (APW)

Peptone

10 g

Sodium chloride

10 g

Distilled water

1000 ml

Preparation. Add ingredients to the water and adjust pH to 8.5 with a concentrated solution of sodium hydroxide. Dispense in 5-10 ml amounts into screw-capped bottles. Autoclave at 121 °C for 15 minutes. (Store alkaline media in bottles with tightly screwed caps to prevent a drop in pH.)

Meat extract agar (MEA)

Peptone

10 g

Sodium chloride

10 g

Meat extract (concentrate)

3g

Agar

20 g

Distilled water

1000ml

Preparation. Add ingredients to the water and heat to boiling while stirring to dissolve the agar. Adjust the pH to 8.5 with a concentrated solution of sodium hydroxide. Autoclave at 121 °C for 15 minutes. Pour plates aseptically (20 ml per plate). Allow plates to cool slowly and store them in an inverted position at 4 °C. The plates should be used within 3-5 days.

Note: On this medium, colonies of Vibrio cholerae O1 are translucent, whereas those of Enterobacteriaceae are opaque.

Gelatin agar (GA)

Peptone

4 g

Yeast extract

1 g

Gelatin

15 g

Sodium chloride

10 g

Agar

15 g

Distilled water

1000 ml

Preparation. Add ingredients to the water and heat to boiling while stirring to dissolve the agar. Adjust pH to 8.5 with a concentrated solution of sodium hydroxide. Dispense into screw-capped bottles. Autoclave at 121 °C for 15 minutes.

Taurocholate tellurite gelatin agar (TTGA)

Trypticase

10 g

Sodium chloride

10 g

Sodium taurocholate

5 g

Sodium carbonate

1 g

Gelatin

30 g

Agar

15 g

Distilled water

1000ml

Preparation. Add ingredients to the water and heat to boiling while stirring to dissolve the agar. Adjust the pH to 8.5 with a concentrated solution of sodium hydroxide. Dispense into screw-capped bottles. Autoclave at 121 °C for 15 minutes.

Before use, add 0.5-1.0 ml of a filter-sterilized 0.1% aqueous solution of potassium tellurite to each 100 ml of the melted TTGA medium at 55 °C. Mix well. Pour plates aseptically (20 ml per plate).

A5.4 Oxidase reagent and test

Oxidase reagent is a 1% solution of tetramethyl-p-phenylenediamine dihydrochloride in distilled water. (1% dimethyl-p-phenylenediamine may also be used in the paper strip test.)

The reagent should be colourless and should be stored in a glass-stoppered, dark brown bottle, protected from the light, in a refrigerator. If only a clear glass bottle is available, it should be wrapped in aluminium foil or dark paper.

The oxidase test is performed as follows:

Use fresh growth from an MEA, GA, or TTGA plate, or the KIA slant (but not a TCBS agar plate). Place 2-3 drops of the oxidase reagent on a piece of filter paper in a Petri dish. Smear the culture across the wet paper with a platinum (not nichrome) loop or a clean, fine wooden toothpick.

A positive reaction is indicated by the appearance of a dark purple colour on the paper within 10 seconds. Among the Gram-negative rods. Vibrio, Campylobacter, Aeromonas, Plesiomonas, Pseudomonas, and Alcaligenes are oxidase-positive. All Enterobacteriaceae are negative.

Test a positive control using a species of Pseudomonas and a negative control using a strain of Escherichia coli at the same time.

 

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