REGAN-LOWE AGAR (Charcoal Blood Agar)

Cat. no. A65 Regan-Lowe Agar, 15x100mm Plate, 22ml 10 plates/bag
Cat. no. Q32 Regan-Lowe Semi-Solid, 13x100mm Tube, 4ml Deep 20 or 100 tubes/box
Cat. no. A63 Regan-Lowe Agar without Cephalexin, 15x100mm Plate,
24ml
10 plates/bag

INTENDED USE

Hardy Diagnostics Regan-Lowe Agar is a selective medium recommended for the isolation and cultivation of Bordetella spp. Regan-Lowe Agar without Cephalexin does not contain selective agents and is recommended for the cultivation of Bordetella.

SUMMARY

Several types of media have been developed over the years for the isolation of Bordetella pertussis. Regan and Lowe, in developing a medium for use in the transport of whooping cough specimens, discovered an enrichment medium useful for the selective isolation of B. pertussis and B. parapertussis.(6)

The basal medium of Regan-Lowe Agar consists of charcoal agar supplemented with defibrinated horse blood. Charcoal, along with starch, neutralizes fatty acids and peroxides, which are toxic to Bordetella. Horse blood is an added enrichment which supports the growth of Bordetella spp. Cephalexin inhibits the growth of normal flora of the nasopharynx. Yeasts and fungi are inhibited by the inclusion of amphotericin B (Cat. no. Q32). Beef extract and enzymatic digest are incorporated in the medium to supply amino acids and other nitrogenous substances that are necessary for bacterial growth. Osmotic equilibrium is maintained by the addition of sodium chloride. Niacin (nicotinic acid) is a vitamin which is added for growth promotion.

Katzko, et al., recently reported enhanced recovery of Bordetella spp. from nasopharyngeal swabs by extending the incubation of plated primary cultures beyond the usual seven days to a total of twelve days.(10)

FORMULA

Ingredients per liter of deionized water:*

Regan-Lowe Agar:
Charcoal Agar 66.5gm
Cephalexin 0.04gm
Horse Blood 100.0ml

In addition, Regan-Lowe Agar without Cephalexin (Cat. no. A63) is the same formulation as above, without cephalexin.

Regan-Lowe Deep (Cat. no. Q32) is a semi-solid medium. It contains half the amount of basal medium as the isolation medium (Cat. no. A65) and, in addition, contains amphotericin B. The half-strength formula allows Bordetella to be exposed to air and moisture, two parameters necessary for survival. (7,8)

Final pH 7.4 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If possible, it is recommended to inoculate the plate at the patient's bedside. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport medium and refrigerated until inoculation. Consult listed references for information on specimen collection. (1-5)

Refer to the keywords "Specimen Collection and Transport" on the Hardy Diagnostics Technical Document website for more information.

Method of Use for Regan-Lowe Deep (Cat. no. Q32): The medium should be brought to room temperature prior to inoculation. Providing the physical nature of the specimen is suitable for a stab inoculation, submerge the specimen into the medium. If using a swab, the tip must be submerged well into the medium. Break or cut any portion of the swab that is protruding from the tube. Tighten the cap and deliver immediately to the laboratory. Once received by the lab, the specimen should be immediately plated onto Regan-Lowe Agar and a non-selective medium. Return the swab to the Regan-Lowe Deep medium and incubate with CO 2 at 35ºC. for 48 hours, at which time a duplicate set of plates should be inoculated and incubated.

Method of Use for Regan-Lowe Agar (Cat. no. A65) and Regan-Lowe Agar without Cephalexin (Cat. no. A63): The medium should be brought to room temperature and the agar surface should be dry, prior to inoculation. Using aseptic techniques, inoculate the agar surface and streak for isolation. Incubate the plates in an aerobic, humidified atmosphere with CO 2 , at 35ºC. for up to 12 days. (10) It is recommended that the plates be incubated in a zip-lock bag with a moist cotton ball. Plates should be examined daily for typical colonial growth and morphology.

INTERPRETATION OF RESULTS

Plates should be examined daily with and without a dissecting microscope. When colonies of Bordetella spp. are visible to the unaided eye, they should appear small, gray, shiny, convex, smooth, and raised with a pearl-like luster; somewhat like a mercury droplet.

As incubation is lengthened, the colonies become whiter, resembling a bisected pearl. (9)

LIMITATIONS

This medium is intended for the primary isolation of Bordetella spp.

Often, selective media will inhibit specific strains of organisms for which they are designed to isolate. At the same time, contaminating organisms may prove resistant to the added antimicrobics, and thus result in growth. It is recommended, therefore, that Regan-Lowe Agar and a non-selective medium be inoculated in parallel, in order to ensure recovery of potential pathogens. (9)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Regan-Lowe Agar without Cephalexin:
Bordetella pertussis
ATCC ® 9797
B 48-96hr 35°C CO 2 ** Growth; small, pearly-white colonies
Additionally, the following organisms are tested on Regan-Lowe Agar and Semi-Solid:
Staphylococcus aureus
ATCC ® 25923
B 24hr 35°C Aerobic Partial to complete inhibition
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

B. pertussis growing in Regan-Lowe Agar

Bordetella pertussis (ATCC ® 9797) growing in Regan-Lowe Agar (Cat. no. A65). Incubated in CO 2 for 72 hours at 35ºC.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

3. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Regan and Lowe. 1997. J. Clin. Microbiol.; 6:303.

7. Brumfitt, W. 1959. J. Pathol. Bacteriol.; 77:95-100.

8. Preston, N.W. 1970. Lab. Pract., 19:482-486.

9. Gilchrist, Mary J.R., Ph.D. 1970. Clin. Micro. Newsletter; Vol. 12, No. 7.

10. Katzko, Gary, et al. 1996. J. Clin. Microbiol.; 34:1563-1564.


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