CRITERION™ AMPICILLIN DEXTRIN AGAR BASE

Cat. no. C7580 CRITERION™ Ampicillin Dextrin Agar Base 90gm
Cat. no. C7581 CRITERION™ Ampicillin Dextrin Agar Base 500gm
Cat. no. C7582 CRITERION™ Ampicillin Dextrin Agar Base 2kg
Cat. no. C7583 CRITERION™ Ampicillin Dextrin Agar Base 10kg
Cat. no. C7584 CRITERION™ Ampicillin Dextrin Agar Base 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ Ampicillin Dextrin Agar Base is recommended for the selective isolation, differentiation, and quantitative enumeration of Aeromonas species from water, food and environmental samples by the membrane filter method.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

Aeromonas spp. are gram-negative, oxidase-positive, non-spore-forming, rod-shaped, motile, facultatively anaerobic bacteria that are autochthonous to fresh and marine water settings and to some aquatic fauna. (1) In addition, they are readily isolated from both nutrient-rich and nutrient-poor environments and their numbers vary depending upon the temperature and trophic state of the territory.(2,5) Rising pollution levels and nutrient loading due to runoff may yield substantially greater and greater populations of these microbes, and may also affect the distribution of microorganisms in a given region. However, aeromonads have been isolated from municipal drinking-water systems, sometimes at high levels; consequently, there is great potential for them to enter distribution systems if water treatment is ineffective.(1,2,5)

Recent medical reports have increasingly associated Aeromonas spp. with human infections.(2) Mesophilic aeromonads have been isolated from patients with gastroenteritis, though their etiology in disease is unclear. They are associated with sepsis and wounds, and with eye, respiratory tract, and other systemic infections, particularly in young children and immunocompromised patients. Moreover, many systemic infections are the result of contamination of lacerations and fractures from Aeromonas-rich waters, and the severity of disease appears to correlate strongly with enterotoxin or toxigenic factor-producing species.(1)

Due to the increasing medical significance of these microorganisms, Havelaar et. al. designed a culture media based on a modification of the positive aspects of mA-Agar, described by Rippey and Cabelli, and Dextrin-Fuchsin-Sulphite (DFS) Agar to create Ampicillin Dextrin Agar.(3,4) Hardy Diagnostics' CRITERION™ Ampicillin Dextrin Agar Base, when combined with antimicrobial agents such as Ampicillin (Cat. no. SR136E), and vancomycin or the vibriostatic agent O129, is a selective medium that partially inhibits the growth of non-target bacterial species while allowing most Aeromonas spp. to grow.(5) Consequently, Aeromonas growth on this medium can be presumptively identified by the fermentation of dextrin and the presence of yellow colonies. Yellow colonies can then be counted and confirmed by testing for the presence of cytochrome c (oxidase test), the ability to ferment trehalose, and the production of indole.(1,2,5)

FORMULA*

Gram weight per liter: 36.7gm/L
Agar 15.0gm
Dextrin 10.0gm
Tryptose 5.0gm
Sodium Chloride 3.0gm
Yeast Extract 2.0gm
Potassium Chloride 2.0gm
Magnesium Sulphate Heptahydrate 0.2gm
Ferric Chloride 0.1gm
Sodium Deoxycholate 0.1gm
Bromothymol Blue 0.08gm

Final pH 8.0 +/- 0.2 at 25ºC.

Note: The addition of 10mg of sterile Ampicillin (Cat. no. SR136E) is needed to complete the ADA formula. An additional 2mg of sterile vancomycin may also be added to make Ampicillin Dextrin Agar with Vancomycin (ADA-V). If testing for Aeromonas in sea water, 50mg of the vibriostatic agent O129 may be added to inhibit the growth of Vibrio spp.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original beige.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 36.7gm of the dehydrated culture media in 1 liter of distilled or deionized water. Stir to mix thoroughly.

2. Heat to boiling to dissolve completely.

3. Sterilize in the autoclave at 121ºC. for 15 minutes.

4. Cool to 45-50ºC.

5. Add 10mg of sterile Ampicillin (Cat. no. SR136E) to inhibit Enterococcus faecalis .

Note: Alternatively, an additional 2mg of sterile vancomycin may be added to create Ampicillin Dextrin Agar with Vancomycin (ADA-V) to enumerate Aeromonas in finished waters. If detecting Aeromonas spp. in sea water, 50mg of the vibriostatic agent O129 can be added to the ADA medium.

6. Aseptically pour appropriate volume into desired sterile containers.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. G180.

LIMITATIONS

In some instances, Enterococcus spp. are reported to produce pinpoint-sized yellow colonies on Ampicillin Dextrose Agar. (5) Confirmation of presumptive Aeromonas colonies is necessary to rule out false-positives.

When examining food and environmental samples, where competing flora may be dominated by Pseudomonas spp., incubation at 37ºC. for 24 hours yields the best results due to the inhibition of most Pseudomonas spp. as proposed by Rippey and Cabelli.(2) At lower temperatures, the growth of P. aeruginosa may overwhelm the sample, making quantitative recovery extremely difficult.

Incubation under anaerobic conditions results in a lower rate of dextrin fermentation and faintly yellow to yellowish-green Aeromonas colonies that may fade to green upon removal of cultures from the incubator.(4)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, Ampicillin (Cat. no. SR136E), Vancomycin, O129, membrane filters, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Aeromonas hydrophila
ATCC ® 35654
A 24-48hr 15-30°C Aerobic Growth; yellow colonies
Escherichia coli
ATCC ® 25922
A 24-48hr 15-30°C Aerobic Variable, if present yellow colonies
Enterococcus faecalis
ATCC ® 29212
B 24-48hr 15-30°C Aerobic Partial to complete inhibition

User Quality Control

Physical Appearance

CRITERION™ Ampicillin Dextrin Agar Base powder should appear homogeneous, free-flowing, and beige in color. The prepared media should appear slightly opalescent, and blue in color.

REFERENCES

1. Sartory et. al. 2006. Guidelines for Drinking-Water Quality. 3rd ed. WHO, Switzerland; 1-17.

2. Rippey and Cabelli. 1979. Membrane Filter Procedure for Enumeration of Aeromonas hydrophila in Fresh Waters. Appl. and Environ. Micro.; 38:108-113.

3. Corry, Curtis and Baird. 1995. Culture Media for Food Microbiology. Elsevier. New York, NY.

4. Havelaar, A.H., M.During and J.F.M.Versteegh. 1987. Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration. Society for Applied Bacteriology.

5. Feige, M.A. 2001. Method 1605: Aeromonas in Finished Water by Membrane Filtration using Ampicillin-Dextrin Agar with Vancomycin (ADA-V). EPA. Washington, D.C.

6. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

7. Association of Official Analytical Chemists. Official Methods of Analysissm, AOAC, Washington, D.C.

8. Committee of Revision for The United States Pharmacopeia. 2004. The United States Pharmacopeia, 27th rev. United States Pharmacopeial Convention, Rockville, MD.

9. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

10. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

11. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm.


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050916gr