LKV (LAKED BLOOD WITH KANAMYCIN AND VANCOMYCIN)AGAR

Cat. no. A60 LKV Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J87 Brucella with H & K / LKV, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J102 BBE / LKV, 15x100mm Biplate, 10ml/10ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Laked Blood with Kanamycin and Vancomycin Agar is recommended for use in the isolation and partial identification of anaerobic microorganisms.

SUMMARY

Brucella Agar is the basal medium for Laked Blood with Kanamycin and Vancomycin Agar. Dextrose, peptones, yeast extract, hemin, vitamin K and laked sheep blood are among the nutrients included in the medium. Dextrose serves as an energy source; peptones provide nitrogenous compounds, and yeast extract supplies B vitamins. Sodium chloride is incorporated to provide essential electrolytes. Sodium bisulfite, a reducing substance, is added to help maintain reduced conditions and a low pH.

Growth factors required by some anaerobic bacteria are provided by the added laked sheep blood, which also allows the demonstration of hemolytic reactions and enhances earlier pigmentation of Prevotella spp. Hemin and vitamin K are incorporated into the medium to enhance the growth of gram-positive spore-formers and Bacteroides species. (6) Vancomycin is employed to inhibit the growth of gram-positive organisms and kanamycin to inhibit gram-negative facultatively anaerobic bacilli.

FORMULA

Ingredients per liter of deionized water:*

Peptamin 20.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Dextrose 1.0gm
Sodium Bisulfite 0.1gm
Hemin 5.0mg
Vitamin K 1.0mg
Vancomycin 40.0ml
Kanamycin 40.0ml
Laked Sheep Blood 50.0ml
Agar 17.0gm

Final pH 7.3 +/- 0.2 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), 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: Consult listed references for information on specimen collection.(1-5) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Immediate and proper transport to the laboratory is essential for successful recovery of significant anaerobic pathogens. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate anaerobic transport medium and refrigerated until inoculation.

Recovery of anaerobes from clinical specimens requires reduced oxygen tension, low oxidation-reduction potential and the use of both selective and non-selective media.

Method of Use: Consult listed references for the correct inoculation procedure. (1-5,9) Prior to inoculation, the medium should be brought to room temperature. A large amount of inoculum should be used. This will minimize the damaging effects of toxic oxygen growth-limiting factors. Streak inoculum to obtain isolated colonies. Incubate plates anaerobically at 35-37ºC. for up 48 hours. Confirmation of anaerobic organisms should be performed by subculturing to an aerobic Blood Agar plate (Cat. no. A10). Examine anaerobic colonies for hemolytic reaction, colony morphology, gram stain, and further biochemical testing.

INTERPRETATION OF RESULTS

Examine for characteristic colonial growth and morphology. Use aerotolerance testing, biochemical testing, and/or gas-liquid chromatography for complete identification of anaerobes. Consult listed references for the interpretation of growth of anaerobic species. (2-5,9)

LIMITATIONS

It is suggested that LKV plates be reduced, prior to use, by placing them in an anaerobe jar at room temperature for a period of 6-24 hours.

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
Bacteroides fragilis
ATCC ® 25285
A 24-48hr 35°C Anaerobic Growth
Prevotella melaninogenica
ATCC ® 25845
A 24-48hr 35°C Anaerobic Growth
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Inhibited
Staphylococcus epidermidis
ATCC ® 12228
B 24hr 35°C Aerobic Inhibited

User Quality Control

PHYSICAL APPEARANCE

LKV Agar should appear clear, and brown in color.

B. fragilis growing on LKV Agar

Bacteroides fragilis (ATCC ® 25285) colonies growing on LKV Agar (Cat. no. A60). Incubated anaerobically for 48 hours at 35ºC.

LKV Agar

Uninoculated plate of LKV Agar (Cat. no. A60).

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. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

3. Tille, P.M., 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. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Onderdonk, A.B., et al. 1974. Infect. Immun; 10:1256.

8. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22-A. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

9. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual , 6th ed. Star Publishing Company, New York, N.Y.

10. Weinstein, W.M., et al. 1974. Infect. Immun.; 10:1250.


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031016gr