Alkaline peptone water (APW) is recommended as an enrichment broth for isolating Vibrio cholerae from clinical samples and non clinical samples (suspected food & water samples). A number of other broth media have been described for enrichment of V.cholerae including Monsur’s enrichment medium and modification of APW with added potassium tellurite but they may not offer selective advantage over APW if used with a short incubation time (6 to 8 hours).
Enrichment media are liquid culture media which contain substances that inhibit the growth of unwanted organisms.
Enrichment broth should always be used with
- convalescent patients,
- suspected asymptomatic infections,
- environmental specimens, and
- whenever high numbers of competing organisms are likely to be present in the specimen.
but when the patient is in very early stages of illness and is passing liquid stool, it may not be necessary to enrich stool specimens.
Vibrio spp grow very rapidly in APW, and at 6 to 8 hours will be present in greater numbers than non-Vibrio organisms. Enrichment in APW enhances the isolation of V. cholerae O1 when few organisms are present, as in specimens from convalescent patients and asymptomatic carriers.
Preparation of Alkaline Peptone Water
- To make about 50 bottles (i.e. 500 ml), you will need 5 g of peptone and 5 g of Sodium chloride.
- Dissolve the peptone and sodium chloride in the water.
- Adjust the reaction of the medium to pH 8.6 to 9.0 using 1 mol/L sodium hydroxide.
- Dispense the medium in 10 ml amounts in screw-cap bottles.
- Sterilize by autoclaving (with caps loosened) at 121°C for 15 minutes. Tighten the bottle caps after the medium has cooled.
- Date the medium and give it a batch number. Label the bottles and record on each the expiry date of the medium (2 years from preparation).
- Store in a cool dark place with the bottle caps screwed tightly to prevent a change in pH .
pH of the medium (pH 8.6 ± 0.2 @ 25°C)
Shelf-life: Up to 2 years providing there is no change in the volume or appearance of the medium suggesting contamination.
Inoculation of APW from fecal specimens
APW can be inoculated with liquid stool, fecal suspension, or a rectal swab.
A. For use as enrichment medium:
- Inoculate the APW bottle with specimen (The stool inoculums should not exceed 10% of the volume of the broth)
- Incubate the tube with the cap loosened at 35°C to 37°C for 6 to 8 hours.
- After 6 to 8 hours’ incubation, subcultures to TCBS should be made with one to two loopfuls of APW from the surface and topmost portion of the broth, since vibrios preferentially grow in this area.
- Do not shake or mix the tube before subculturing.
- If the broth cannot be subcultured after 6 to 8 hours of incubation, subculture at 18 hours to a fresh tube of APW.
- This second tube should be subcultured to a solid medium after 6 to 8 hours incubation.
B. For use as a transport medium
- When cholera is suspected, transfer about 1 ml of specimen into 10 ml of sterile alkaline peptone water.
- The inoculated medium should reach laboratory within 8 hours of collection (Proteus species will grow eventually in alkaline peptone water.)
Growth and further processing
Vibrio cholerae grows rapidly on APW producing turbidity on and just below the surface of the medium within 4-6 hours. To confirm that the organisms are vibrios, examine a wet preparation/ perform hanging drop motility test, prepare gram stain of the smear (which may show gram negative curved bacilli) and/or culture in the TCBS medium.
If test for the detection of Vibrio cholerae lipopolysaccharide antigen in the stool sample is done after enrichment in APW, its sensitivity as well as specificity increases.
References and further readings:
- Cheesbrough M., District Laboratory Practice in Tropical Countries: Part 2:.Cambridge University Press .
- Laboratory Methods for the Diagnosis of Vibrio cholerae; Centers for Disease Control and Prevention