ANAEROBIC DIFFERENTIATION DISKS

Cat. no. Z8411 Colistin 50 disks/cartridge
Cat. no. Z7191 Kanamycin 50 disks/cartridge
Cat. no. Z7501 Vancomycin 50 disks/cartridge

INTENDED USE

HardyDisk™ Anaerobic Differentiation Disks (also referred to as "special-potency disks") are used to verify gram stain reactions of anaerobic bacteria, and are especially helpful with certain clostridia that tend to stain gram-negative. They are useful in separating anaerobic, gram-negative bacilli into several groups, aiding in their presumptive identification. These disks can also differentiate Fusobacterium species from other true gram-negative bacilli.(2,3,6,7)

SUMMARY

Special-potency antimicrobial disks of colistin, kanamycin, and vancomycin can be used as an aid in clarifying gram stain reaction of anaerobes, and in preliminary grouping of anaerobic gram-negative bacilli. In general, gram-positive organisms are resistant to colistin and susceptible to vancomycin, while most gram-negative organisms are resistant to vancomycin.(3,7,10) Most anaerobes have a characteristic susceptibility pattern to these special-potency, high concentration disks. Therefore, most clinically significant anaerobic gram-negative rods can be separated into broad groups, based on relatively few tests. For example, the B. fragilis group and Porphyromonas species can be presumptively identified by their disk patterns and a few other simple tests (such as; indole, catalase, esculin, etc.).(2)

The use of these anitbiotic susceptibility patterns to aid in the identification of anaerobic bacteria was first reported in 1967 by Finegold, et al. In this study, high concentration disks were used to differentiate between the main groups of gram-negative anaerobic bacteria. It was determined that the susceptibility tests were rapid and easy to perform.(8,9)

Complete identification of anaerobes can be costly, and often requires various biochemical tests, GLC (gas liquid chromatography), etc. Many clinical labs do not perform complete identification of anaerobes, since presumptive identification can be just as useful in determining the appropriate therapy.(3) HardyDisk™ Anaerobic Differentiation Disks are helpful in the presumptive identification of these organisms. Due to the high concentrations of these antibiotics, these disks are not intended for use in determining antibiotic susceptibility for therapeutic purposes.

FORMULA

Each HardyDisk™ Anaerobic Differentiation Disk is prepared by impregnating the below specified concentration of the appropriate antibiotic onto a 6mm diameter high quality filter paper disk.

Colistin 10mcg
Kanamycin 1,000mcg
Vancomycin 5mcg

STORAGE AND SHELF LIFE

Storage: Upon receipt store at -20 to +8ºC. away from direct light. The disks should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect from light, excessive heat, and moisture.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for the primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

If testing is performed on the open bench, all plates should be promptly (within 20 minutes) incubated anaerobically. Some clinical isolates may die after relatively short oxygen exposure.(3,7)

1. Allow disks to equilibrate to room temperature.

2. Select one, well-isolated colony of an anaerobic organism to be tested from a primary or pure subculture.

3. Inoculate a non-selective anaerobic blood agar plate (Brucella with Hemin and Vitamin K (Cat. no. A30), by streaking the first quadrant back and forth several times, to ensure an even, heavy lawn of growth. Streak the other quadrants for isolation.

4. Using forceps, place the colistin, kanamycin, and vancomycin disk (one each) in the first quadrant well apart from each other (nitrate, SPS, and bile disks may be placed in the second quadrant of the plate at this time, refer to listed references for procedure information).(3,7)

5. Incubate the plates anaerobically at 35-37ºC. for 24-48 hours, or until sufficient growth is observed. Longer incubation time may be required for some organisms.

INTERPRETATION OF RESULTS

Sensitive (S) - Zone of inhibition is greater than or equal to 10mm.

Resistant (R) - Zone of inhibition is less than 10mm.

LIMITATIONS

The definitive identification of most species requires some additional biochemical testing.(2)

Rare strains of B. fragilis are susceptible to colistin.(3)

When using these special-potency disks for rapid identification, it is important to remember that the results of the test does not give information about the antimicrobial agents that can be used for therapy.(6,9)

Occasionally, some clostridia and lactobacilli can appear resistant to vancomycin (5ug).(7)

Porphyromonas species are gram-negative bacilli, but are susceptible to vancomycin.(7)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, disk dispensers, 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
Colistin Kana Vanco
Fusobacterium nucleatum

ATCC ® 25586
*
48-72hr
35°C
Anaerobic
S S R
Bacteroides fragilis

ATCC ® 25285
*
48-72hr
35°C
Anaerobic
R R R
Clostridium perfringens

ATCC ® 13124
*
48-72hr
35°C
Anaerobic
R S S

User Quality Control

PHYSICAL APPEARANCE

HardyDisk™ Anaerobic Differentiation Disks are 6mm (in diameter) filter paper disks and should appear white in color.

The disks can be identified by a letter code printed on both sides of the disk. The Colistin disk (Cat. no. Z8411) is identified by the letters CT, the Kanamycin disk (Cat. no. Z7191) has the letters KAN, and Vancomycin (Cat. no. Z7501) has Va5 printed on both sides of the disk.

B. fragilis Resistant to Vancomycin

Showing Vancomycin Resistance
Bacteroides fragilis (ATCC® 25285) growing on Brucella Agar with Hemin and Vitamin K (Cat. no. A30) with a Vancomycin disk (Cat. no. Z7501). Incubated anaerobically for 48 hours at 35ºC.

C. perfringens Sensitive to Vancomycin

Showing Vancomycin Sensitivity
Clostridium perfringens (ATCC® 13124) growing on Brucella Agar with Hemin and Vitamin K (Cat. no. A30) with a Vancomycin disk (Cat. no. Z7501). Incubated anaerobically for 24 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. 1998. Essential Procedures for Clinical Microbiology. 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. Engelkirk, P.G., Ph.D., J. Duben-Engelkirk, Ed.D., V.R. Dowell Jr., PhD. 1992. Principles and Practice of Clinical Anaerobic Bacteriology. Star Publishing Company, Belmont, CA.

7. Jousimies-Somer, H., Ph.D., P. Summanen MS, D.M. Citron, BS, E.J. Baron, Ph.D., H.M. Wexler, Ph.D., S.M. Finegold, MD. 1996-2005. Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, Belmont, CA.

8. Leigh, D.A. and K. Simmons. 1977. Identification of non-sporing anaerobic bacteria. J. Clin. Pathology; 30:991-992.

9. Sutter, V.L. and S.M. Finegold. 1971. Antibiotic disc susceptibility tests for rapid presumptive identification of gram-negative anaerobic bacilli. Applied Microbiology; 21:13-20.

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


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080216gr