ALKALINE PEPTONE WATER

Code: CM1028

A broth medium for the enrichment of Vibrio species from food, water and clinical samples.
Formula                       gm/litre
Peptone                          10.0
Sodium Chloride             20.0
pH 8.6 ± 0.2
Directions
Add 30 g to one litre of distilled water. Mix well, distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes.
Description
Alkaline Peptone Water (CM1028) is for the enrichment of Vibrio cholera and Vibrio species from food, water and clinical samples. This broth can also be used for direct microscopic examination of samples using the hanging drop method.
Alkaline Peptone Water was first formulated by Shread, Donovan and Lee¹ to be used as a non-selective enrichment broth for the cultivation of Aeromonas species. Cruickshank reported that when the pH is raised, the medium can be used to effectively cultivate Vibrio species².

The 2% (w/v) sodium chloride incorporated in this medium promotes the growth of Vibrio cholerae, while the alkalinity of this medium inhibits most of the unwanted background flora.
Technique
There are various methods available for the isolation of Vibrio species from environmental, food and clinical samples. These generally involve a pre-enrichment step followed by plating onto a solid medium and morphological, biochemical and serological identification.
 Many different enrichment media have been described but of these only Alkaline Peptone Water has achieved wide acceptance. Refer to the appropriate guidlelines or standards for formulations and exact methodology.
Clinical Samples
Inoculate swab specimens directly into Alkaline Peptone Water. Material not being cultured directly from a swab may be transferred into the medium using a sterile microbiological loop. For faecal specimens, aseptically transfer approximately 1 g of the sample to the medium and mix well. The inoculated broths are generally incubated at 35-37°C for 5-6 hours or 18-20 hours at 18-20°C³.
Food and Water Samples
Refer to the appropriate standard such as APHA^4,5 FDA-BAM⁶ and ISO⁷.
For all methods plating media should be incubated overnight and then inspected for typical colonies:
Sodium Dodecyl Sulphate Polymixin B Sucrose Medium (SPS Medium): Sucrose positive vibrios such as Vibrio cholerae and Vibrio alginolyticus are yellow in colour. Sucrose negative species such as Vibrio parahaemolyticus and Vibrio vulnificus produce blue-green colonies. Organisms producing sulphatase e.g. Vibrio vulnificus are also usually surrounded by a halo of precipitation.
Cholera Medium TCBS (CM0333): Sucrose positive vibrios such as Vibrio cholerae and Vibrio alginolyticus are yellow in colour on TCBS. Sucrose negative species such as Vibrio parahaemolyticus and Vibrio vulnificus produce blue-green colonies.
MacConkey Agar (CM0007): Lactose negative Vibrio species produce colourless colonies.
Biochemical Confirmation : Vibrio spp. are oxidase positive and ferment glucose with the production of acid only. As oxidase testing may lead to false negative results on media containing carbohydrates (such as TCBS) subculture to nutrient or blood agar before testing.
Storage conditions and Shelf life
The dehydrated medium should be stored at 10-30°C and used before the expiry date on the label.
Store the prepared medium may be stored for up to 1 month at room temperature
Appearance
Dehydrated Medium: Straw coloured, free-flowing powder.
Prepared medium: Clear, straw coloured liquid
Quality Control
Positive control:                                               Expected results
Vibrio parahaemolyticus ATCC®17802 *       Turbid growth
Vibrio vulnificus ATCC®27562             *       Turbid growth
Vibrio furnissii ATCC®11218               *        Turbid growth
Negative control :
Uninoculated medium                                       No change
References
1. Shread, P., Donovan T.J., and Lee J.V. (1991) Soc. Gen. Microbiol. Q. 8:184.
2. Cruickshank, R. (1968) Medical Microbiology. 11th ed. Livingstone Ltd, London, UK.
3. Janda J.M., et al. (1988) Current Perspectives on the Epidemiology and Pathogenesis of Clinically Significant Vibrio spp. Clinical Microbiology Reviews July 3:245-267.
4. Standard Methods for the Examination of Water and Waste Water 20th Edition 1998 APHA.
5. Compendium of Methods for the Microbiological Examination of Foods, Fourth Edition 2001, APHA.
6. FDA BAM on line 2001 http://www.cfsan.fda.gov/~ebam/bam-9.html.
7. Methods for Microbiological examination of food and animal feeding stuffs Part 14 Detection of Vibrio parahaemolyticus. BS5763: Part 14 : 1991 ISO 89.