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MITIS SALIVARIUS AGAR

INTENDED USE Remel Mitis Salivarius Agar is a solid medium recommended for use in qualitative procedures for selective isolation of Streptococcus mitis, Streptococcus salivarius, and enterococci from specimens of mixed bacterial flora and for differentiation of the viridans strains. SUMMARY AND EXPLANATION Mitis Salivarius Agar was developed by Chapman for the isolation of streptococci from specimens containing mixed microbial flora.1,2 Gold et al. used Mitis Salivarius Agar to differentiate streptococci isolated from the oral cavity.3 Other investigators used this medium in microbial studies of specimens collected from dental plaque and carious lesions.4,5 Because of its selective and differential qualities it is especially useful for the isolation of streptococci and enterococci from sources containing commensal microbial flora. PRINCIPLE Proteose peptone and tryptose supply amino acids and other nitrogenous compounds necessary for the growth of bacteria. Crystal violet, potassium tellurite, and trypan blue are selective agents that inhibit most gram-negative bacilli and gram-positive bacteria except streptococci and enterococci. Potassium tellurite is also a differential agent that is reduced by Enterococcus spp. to form black colonies. Sucrose and dextrose are carbon energy sources. S. salivarius metabolizes sucrose and develops luxuriant colonies with a “gum-drop” appearance. S. mitis and enterococci do not metabolize sucrose and, as a result, form smaller colonies on this medium. Trypan Blue is absorbed by colonies of streptococci producing a blue color. Dipotassium phosphate is a buffer and agar is a solidifying agent. REAGENTS (CLASSICAL FORMULA)* Sucrose ......................................................................... 50.0 g Trypan Blue ................................................................. 75.0 mg Proteose Peptone .......................................................... 10.0 g Crystal Violet ................................................................. 0.8 mg Tryptose ........................................................................ 10.0 g Potassium Tellurite 1% .................................................. 1.0 ml Dipotassium Phosphate ................................................... 4.0 g Agar ............................................................................ 15.0 g Dextrose .......................................................................... 1.0 g Demineralized Water ............................................... 1000.0 ml

pH 7.0 ± 0.2 @ 25°C

*Adjusted as required to meet performance standards. PROCEDURE 1. Inoculate and streak the specimen as soon as possible after it is received in the laboratory. 2. Incubate the plates in 5% CO2 at 33-37°C for 24 to 48 hours. 3. Include a nonselective agar plate (e.g., blood agar) to increase the chance of recovering organisms present in low numbers and to

provide an indication of other organisms present in the specimen. INTERPRETATION Typical colony morphology on Mitis Salivarius Agar:

Streptococcus salivarius - Large, pale-blue, mucoid colonies that are glistening (i.e., “gum-drop”) in appearance. Streptococcus mitis - Small, flat, hard colonies, blue in color with a domed center Streptococcus mutans - Raised, convex, undulate, opaque, pale-blue colonies that are granular (i.e., “frosted glass”) in appearance.

Colonies may exhibit a glistening bubble on the surface due to excessive synthesis of glucan from sucrose. Streptococcus sanguis - Raised, smooth, hard colonies embedded in agar Enterococci - Blue-black, shiny, and slightly raised colonies Coliforms - Colonies (inhibited) are brown in color QUALITY CONTROL All lot numbers of Mitis Salivarius Agar have been tested using the following quality control organisms and have been found to be acceptable. Testing of control organisms should be performed in accordance with established laboratory quality control procedures. If aberrant quality control results are noted, patient results should not be reported. CONTROL INCUBATION RESULTS Streptococcus salivarius ATCC® 13419 CO2, 18-24 h @ 33-37°C Large, pale-blue, mucoid colonies Enterococcus faecalis ATCC® 29212 CO2, 18-24 h @ 33-37°C Blue-black, shiny colonies Escherichia coli ATCC® 25922 CO2, 18-24 h @ 33-37°C Marked inhibition LIMITATIONS 1. Molds may grow on Mitis Salivarius Agar after 48 hours incubation.6 BIBLIOGRAPHY 1. Chapman, G.H. 1944. J. Bacteriol. 48:113. 2. Chapman, G.H. 1946. Am. J. Dig. Dis. 13:105. 3. Gold, O.G., H.V. Jordan, J. van Houte. 1973. Arch. Oral. Biol. 1973. 18:1357-1364. 4. Isenberg, H.D., D. Goldberg and J. Sampson. 1970. Appl. Microbiol. 3:433-436. 5. Hamada, S., N. Masuda and S. Kotani. 1980. J. Clin. Microbiol. 4:314-318. 6. MacFaddin, J.F. 1985. Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria. Vol. 1. Williams and Wilkins, Baltimore, MD. Refer to the front of Remel Technical Manual of Microbiological Media for General Information regarding precautions, product storage and deterioration, specimen collection, storage and transportation, materials required, quality control, and limitations. ATCC® is a registered trademark of American Type Culture Collection.

IFU 1615, Revised June 9, 2014 Printed in U.S.A.