Agar, 1.5% (Water Agar)

Cat. no. Q01 Agar, 1.5% (Water Agar), 16x125mm Tube, 9ml Deep 20 tubes/box

INTENDED USE

Hardy Diagnostics Agar, 1.5% (Water Agar) is used in the Kirby-Bauer alternative agar-overlay method of inoculation for antimicrobial disk diffusion susceptibility testing and for microbiological procedures that require an additive-free solid medium, such as when performing colony counts, testing antimicrobial disk potency, or in the modified fungal slide culture method.

SUMMARY

The Kirby-Bauer disk diffusion method for antimicrobial susceptibility testing has been widely accepted for the analysis of antimicrobial agents.(3-6,8,10) Historically, the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) has described an acceptable alternative method for standardizing the inoculum procedure using the agar-overlay method, though current literature no longer includes this method.

Barry et al. introduced the agar-overlay procedure in 1970 as an alternative to the method described by Bauer et al.(3,5) The modification incorporates placing antimicrobial disks onto a double pour plate consisting of a Mueller Hinton Agar base overlaid with 1.5% agar. Research by Barry and Badal concluded that broad quality control data established by CLSI for daily QC of the Kirby-Bauer method could be replicated using the agar-overlay procedure; this method is known to produce sharper zone edges, is easier to perform, and yields greater precision.(4) Similarly, research by Knowles and Gilmore found that laboratories using the agar-overlay technique reported 90 to 95% concordance with quality control ranges supplied by CLSI.(8) Barry et al. further modified the agar-overlay procedure for use as a rapid method for routinely determining antimicrobial disk potency prior to performing susceptibility testing.(2)

Many species of microfungi with delicate reproductive structures require complex and specific slide mounting preparations. Consequently, a variety of specialized procedures have been optimized. The modified fungal slide culture method uses an adaptation of the Koneman and Roberts methods, and was developed by Harris to facilitate the efficient visualization of sporulating cultures through semipermanent slide mounts.(7) This method employs prepared Agar, 1.5% plates used as a base for periodic low-power objective inspection of intact slide cultures.

Agar, 1.5% (Water Agar) contains agar as the solidifying agent and purified deionized water. Because Agar, 1.5% is additive-free, it is not recommended as a growth medium for most microorganisms, but may prove valuable for a variety of other uses.

FORMULA

Ingredients per liter of deionized water:*

Agar 15.0gm

Final pH 6.3 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: For plated media, upon receipt store at 2-8º C. away from direct light. For tubed media, upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Consult appropriate references or those listed for more information on performing specific procedures.(2-10)

Method of Use for Tubed Media:

Tubed media is traditionally intended as a suspending matrix for bacteria added to an appropriate plated medium and used in the agar-overlay procedure to provide an even inoculum.(2-6,8,9) Tubed media can also be melted and used to make pour-off plates for use in the modified fungal slide culture method listed below.(7)

Modified Fungal Slide Culture Method:

1. Prepare a pour-off plate or make plates using 15gm of agar per liter of deionized water.

2. Place a sterile 22mm2 cover glass (Cat. no. 2222HD) over the center of the pour-off agar plate.

3. Using a sterile, stainless steel spatula, cut a Nutrient Agar plate (Cat. no. W51) into blocks of approximately 5 to 8mm2.

4. Aseptically remove a block of Nutrient Agar and place it on top of the cover glass.

5. Inoculate the Nutrient Agar block on one or more sides with fungal hyphae or conidia and place a second sterile cover glass on the exposed top surface.

6. Replace the lid of the plate and incubate the culture at the appropriate temperature until adequate growth and conidiogenesis is observed. Note: periodic observation of fungal growth may be made by removing the lid of the plate and lowering a low- or high-power objective lens over the top cover glass. Alternatively, the lid of the plate may be left in place and a dissecting microscope used to observe the Nutrient Agar block for hyphal growth.

INTERPRETATION OF RESULTS

Consult appropriate references or those listed for information on the interpretation of results.(2-10)

LIMITATIONS

Use of the agar-overlay method is not applicable for use with fastidious or slow growing microorganisms and this method is not outlined in more recent publications of the CLSI M2 Document.(6)

For studies of geotropism in fungi, the modified slide culture chamber may be taped on its edge against an incubator wall.(7)

The modified fungal slide culture method may be used as an alternative to the nutrient shift method to stimulate sporulation in mucormycetes (formally zygomycetes).(7) Additionally, smaller cover glass slips may be used in the modified fungal slide method to prepare more permanent fungal mounts.(7)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, petri dishes (Cat.no. 330120), applicator sticks, spatulas, other culture media, such as Mueller Hinton Agar (Cat. no. G45), cover glass slips (Cat. no. 2222HD), incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Agar, 1.5% (Water Agar) is tested for pH and sterility only.

USER QUALITY CONTROL

Physical Appearance

Agar, 1.5% (Water Agar) should appear clear, and colorless to slightly white in color.

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. Barry, A.L., G.D. Fay, and F.W. Atchison. 1972. Quality control of antimicrobial disc susceptibility testing with a rapid method compared to the standard method. Antimicro. Agents and Chemo.; 2(6):419-422.

3. Barry A.L., F. Garcia, and L.D. Thrupp. 1970. An improved single.disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Amer. J. Clin. Pathol.; 53:149-158.

4. Barry, A.L. and R.E. Badal. 1982. Quality control limits for the agar overlay disk diffusion antimicrobial susceptibility test. J. Clin. Microbio.; 16(6):1145-1147.

5. Bauer, A.W., W.M.M. Kirby, J.C. Sherris, and M. Turck. 1966. Antibiotic sensitivity testing by a standardized single disk method. Amer. J. Clin. Pathol.; 45:493-496.

6. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS). 2009. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard - Tenth Edition. M02-A10. CLSI. Wayne, PA.

7. Harris, J.L. 1986. Modified method for fungal slide culture. J. Clin. Microbio.; 24(3):460-461.

8. Knowles, R.C. and B. F. Gilmore. 1981. Quality control of agar diffusion susceptibility tests: data from the Quality Assurance Service (QAS) Microbiology Program of the College of American Pathologists. Am. J. Clin. Pathol.; 76(Suppl): 590-596.

9. Larone, D.H. Medically Important Fungi: A Guide to Identification. American Society for Microbiology. Washington, D.C.

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

11. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


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