anaeroGRO™

LKV (Laked Blood with Kanamycin and Vancomycin) Agar

Cat. no. AG601 LKV Agar*, Monoplate 1 plate/pouch
Cat. no. AG061 BBE/LKV* Biplate 1 plate/pouch
Cat. no. AG064 BBE/LKV* Biplate 4Pak 4 plates/pouch
Cat. no. AG302 DuoPak A, BRU with Hemin and Vitamin K*, Monoplate; BBE/LKV*, Biplate 2 plates/pouch
Cat. no. AG303 MultiPak A, BRU with Hemin and Vitamin K*, Monoplate; PEA*, Monoplate; BBE/LKV*, Biplate 3 plates/pouch
Cat. no. AG313 MultiPak B, BRU with Hemin and Vitamin K*, Monoplate; LKV*, Monoplate; PEA*, Monoplate 3 plates/pouch

* All AnaeroGRO™ plated media is provided in standard 15x100mm monoplates or biplates. Each plate or set of plates is packaged in an oxygen-free gas flushed foil pouch containing a desiccant and an oxygen scavenger sachet.

INTENDED USE

Hardy Diagnostics AnaeroGRO™ LKV (Laked Blood with Kanamycin and Vancomycin) Agar is recommended for use in the selective isolation and partial identification of obligately anaerobic gram-negative bacilli, such as Prevotella spp. and Bacteroides spp.

SUMMARY

LKV Agar is useful for the rapid isolation of Prevotella species. Yeast and other kanamycin-resistant organisms, such as Capocytophaga species, sometimes grow on LKV; therefore, one should perform a Gram stain and determine the aerotolerance of all isolates.

Brucella Agar is the basal medium for LKV (Laked Blood with Kanamycin and Vancomycin) Agar. Dextrose, peptones, yeast extract, hemin, vitamin K and laked sheep blood are among the nutrients included in this 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 laked sheep blood. Hemin and vitamin K are incorporated to enhance the growth of Bacteroides species and to facilitate recovery and earlier pigment production of Prevotella spp.(7) Vancomycin inhibits the growth of gram-positive microorganisms and Porphyromonas spp. Kanamycin inhibits most gram-negative facultatively anaerobic bacilli, aerobes, and anaerobic gram-negative rods except for Prevotella and Bacteroides spp.

AnaeroGRO™ LKV Agar is packaged in an oxygen-free, reduced state to prevent the formation of toxic oxidized by-products that may damage obligate anaerobes and inhibit the growth of more fastidious species. Culture media that is exposed to environmental oxygen leads to a build-up of reactive oxygen species (ROS) that initiate damaging free radical reactions, which inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to the AnaeroGRO™ media to neutralize the growth inhibiting effects of peroxide and other reactive oxygen species (ROS) that may develop during the medium's brief exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free environment.

FORMULA

Ingredients per liter of deionized water:*

Peptamin 20.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Reducing Agents/Peroxide Inhibitors 1.5gm
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 15-30ºC. 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

Specimen Collection: Consult listed references for information on specimen collection.(1-6,8) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat, cold, and oxygen exposure. 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 (Cat. no. S120D) 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.(1-6,8) Minimize specimen exposure to ambient oxygen levels in air. Open the AnaeroGRO™ pouch just prior to use and immediately apply a liquid specimen directly to the agar surface. An enrichment broth, such as AnaeroGRO™ Thioglycollate Broth with H and K (Cat. no. AG22H), should be inoculated concurrently with primary isolation plates.

A large amount of inoculum should be used; streak inoculum to obtain isolated colonies. Incubate plates anaerobically at 35-37ºC. for up to 48 hours. Regardless of atmospheric system used, it is important to confirm anaerobiosis by using an anaerobic indicator, such as resazurin (Cat. no. BR55).

Aerotolerance Testing: Confirmation of obligate anaerobic microorganisms should be performed. A Chocolate Agar plate (Cat. no. E14) incubated in 5-10% CO2 is required for aerotolerance testing to detect isolates that require CO2, especially slow-growing, fastidious, facultative or microaerophilic species that do not grow alone on media containing blood (such as Haemophilus and Actinobacillus spp.). Use of traditional blood agar media alone for CO2 incubation may yield false-negative results. An additional Blood Agar plate (Cat. no. A10) incubated in air will further detail the atmospheric requirements and hemolytic properties of facultatively anaerobic microorganisms.

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.(2-6,8,9,11)

Refer to the Wadsworth-KTL Anaerobic Bacteriology Manual or other texts for more information on identification of anaerobes.(5)

LIMITATIONS

The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into an anaerobic atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic bacteria.

Some organisms may be inhibited on LKV Agar. Therefore, it is recommended that a non-selective medium, such as AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301), be inoculated in parallel to ensure growth of all species present.

Some species of facultative organisms may grow on LKV medium, so a test for aerotolerance should be used to confirm colony type.

Failure to cultivate and/or isolate obligate anaerobes may be due to the following:

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, transport media (Cat. no. S120D), incubators, incinerators, anaerobic culture materials, such as gas generators (Cat. no. AN25US), compact systems (Cat. no. AN010C), sealing clips (Cat. no. AN005C), chambers, jars (Cat. no. 16000), and oxygen indicators (Cat. no. BR55), 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

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

AnaeroGRO™ LKV Agar should appear clear, and reddish-brown in color.

B. fragilis growing on AnaeroGRO™ LKV Agar

Bacteroides fragilis (ATCC® 25285) colonies growing on AnaeroGRO™ LKV Agar (Cat. no. AG601). Incubated anaerobically for 48 hours at 35ºC.

AnaeroGRO™ LKV Agar

Uninoculated plate of AnaeroGRO™ LKV Agar (Cat. no. AG601).

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. Dowell, V.R., Jr. and T.M. Hawkins. 1987. Laboratory Methods in Anaerobic Bacteriology. In: CDC Laboratory Manual. DHEW Publication No. (CDC) 87-8272. U.S. Department of Health, Education and Welfare, Public Health Service. Center for Disease Control, Atlanta, GA.

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. 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.

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

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

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

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

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

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

ATCC is a registered trademark of the American Type Culture Collection.

083016gr