Brucella Laked Blood Agar with Kanamycin and Vancomycin (LKV) is an enriched selective medium for the isolation and partial identification of obligately anaerobic gram-negative bacilli.  Brucella Laked Blood Agar with Kanamycin and Vancomycin contains vancomycin to inhibit the growth of gram-positive microorganisms and kanamycin to inhibit the growth of gram-negative, facultatively-anaerobic bacilli.  The sheep blood in the medium is laked and vitamin K₁ and Hemin are added to facilitate the recovery and pigment production of Prevotella melaninogenica.  This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.
 

Formula

Pancreatic Digest of Casein, 10.0 g

Soy Peptone, 3.0 g

Animal Tissue, 10.0 g

Dextrose, 1.0 g

Yeast Extract, 2.0 g

Sodium Chloride, 5.0 g

Sodium Bisulfite, 0.1 g

Hemin (0.1% soln), 5.0 ml

Vitamin K1 (1% soln), 1.0 ml

L-Cysteine, 0.4 g

Agar, 15.0 g

Kanamycin, 100.0 mg

Vancomycin, 7.5 mg

Sheep Blood (Laked), 50.0 ml

Distilled Water, 1000.0 ml

 

Final pH 7.1 +/- 0.4 at 25 degrees C.

Final weight 8.0 g +/- 0.8 for Biplates.

Final weight 16.0 g +/- 1.6 for Mono plates. 

Precautions

For IN VITRO DIAGNOSTIC USE only.  Approved biohazard precautions and aseptic techniques should be observed when using this product.  This product is for use only by properly-trained and qualified personnel.  Sterilize all biohazard waste prior to disposal. 

Storage and Shelf Life

Storage:  Upon receipt, store at room temperature (13°C - 27°C) in original container until used.  Avoid overheating or freezing.  Do not use medium if there are signs of deterioration (shrinking, cracking or discoloration due to oxidation of media) or contamination.  The expiration date applies to the product in its original packaging and stored as directed.  Do not use product past the expiration date shown on the container.

Shelf Life:  90 days from date of manufacture.

Procedure

Specimen Collection:  Specimens for anaerobic culture should be protected from air (oxygen) during collection, transport and processing.  Consult appropriate references for detailed instructions concerning collection and transport of anaerobes.

Methods for Use:  Brucella Laked Blood Agar with Kanamycin and Vancomycin should be inoculated directly with clinical material or a broth that has been previously inoculated from clinical material.  Inoculated plates should be streaked to obtain isolated colonies, immediately placed in an anaerobic atmosphere and incubated at 35-37^oC for 18-48 hours.  Additional periods of incubation may be necessary to recover some anaerobes.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references listed.

Materials Required But Not Provided

Standard microbiological supplies and equipment such as loops, saline blanks, slides, staining supplies, microscope, incinerator or autoclave, incubators, anaerobic chamber or anaerobic jars, disinfectant, other culture media and serological and biochemical reagents.

Limitations

Brucella Laked Blood Agar with Kanamycin and Vancomycin will not provide complete information for identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  Some organisms that would normally grow on the Brucella Laked Blood Agar with Kanamycin and Vancomycin medium may be inhibited.  It is recommended that a non-selective medium, such as Anaerobic Brucella Blood Agar, also be inoculated from clinical specimens to assure growth of all species present.  Some strains of facultative organisms, which should be inhibited, may grow on Brucella Laked Blood Agar with Kanamycin and Vancomycin. A test for aerotolerance should be used to confirm that each colony type is an obligate anaerobe.  Consult reference materials for additional information (1, 2, 3, 4, 5, 6).

Quality Control

Brucella Laked Blood Agar with Kanamycin and Vancomycin, if used properly, should support good growth of some obligately anaerobic gram-negative bacteria (e.g. Bacteroides fragilis and Fusobacterium mortiferum) and inhibit the growth of facultatively-anaerobic bacteria (e.g. Escherichia coli, Proteus mirabilis, Staphylococcus aureus) and gram-positive obligate anaerobes (e.g. Clostridium perfringens and Peptostreptococcus anaerobius). 

The following organisms are routinely used for quality control testing (nutritive / inhibitory capacity) at Anaerobe Systems.

Organisms Tested for LKV	ATCC #	Results	Time (Hours)	Special Reaction
Bacteroides fragilis	25285	Growth	24 h
Prevotella melaninogenica	25845	Growth	24 h	Pigmentt
Fusobacterium necrophorum	25286	Growth	24 h
Fusobacterium nucleatum	25586	No Growth
Clostridium perfringens	13124	No Growth
Peptostreptococcus anaerobius	27337	No Growth
Staphylococcus aureus   or    Enterococcus faecalis	25923     29212	No Growth
Escherichia coli	25922	Inhibited to No Growth
Proteus mirabilis	12453	Inhibited to No Growth
Propionibacterium acnes   or  Clostridium difficile	6919      9686	No Growth

  ^t  Pigment production may require more than 48 hours incubation

User Quality Control: The final determination to the extent and quantity of user laboratory quality control must be determined by the end user.

If sterility testing is to be performed on this product, the appropriate percentage of the original shipment amount should be incubated anaerobically and aerobically for 48 – 96 hours.

If the nutritive/inhibitory capacity of this media is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth/inhibition.

 

Organism	Expected Growth	Special Reactions
B. fragilis	24 hrs
C. perfringens	Inhibited
F. necrophorum	24 hrs
P. melaninogenica	48 hrs	Pigment
E. coli	Inhibited

 

Physical Appearance:  Brucella Laked Blood Agar with Kanamycin and Vancomycin should appear semi-opaque red in color. 

References

1.        Dowell, V. R., Jr. and T. M. Hawkins. 1974. Laboratory Methods in Anaerobic Bacteriology.  CDC Laboratory Manual. USDHEW C. D. C. Atlanta, GA 30333.

 

2.        Dowell, V. R., Jr. and G. L. Lombard. 1977.  Presumptive Identification of Anaerobic Non-sporeforming Gram-negative Bacilli. USDHEW, CDC.  Atlanta, GA 30333.

 

3.        Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson and A. Y. Armfiedl. 1977. Media for the Isolation, Characterization, and Identification of Obligately Anaerobic Bacteria. USDHEW, CDC, Atlanta, GA 30333.

 

4.        Holdeman, L. V., F. P. Cato and W. E. C. Moore. 1977. Anaerobe Laboratory Manual.  Virginia Polytechnic Institute and State University.  Blacksburg, VA 24061.

 

5.        Somer-Jousimies, H. R., Summanen, P., Citron, D. M., Baron, E. J., Wexler, H. M. and S. M. Finegold. 2002. Wadsworth – KTL Anaerobic Bacteriology Manual. Star Publishing Co., Belmont, CA 94002.

 

6.        NCCLS. Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard- Third Edition. (2004). NCCLS document M22-A3. NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898.