MIDDLEBROOK AGAR
CAT Nº: 2043
For the isolation and cultivation of Micobacteria,
used with the ENRICHMENT OADC SUPPLEMENT
FORMULA IN g/l
Disodium Phosphate
1.50
Pyridoxine
1.0 mg
Monopotassium Phosphate
1.50
Zinc Sulfate
1.0 mg
Ammonium Sulfate
0.50
Cooper Sulfate
1.0 mg
L-Glutamic Acid
0.50
Biotin
0.5 mg
Sodium Citrate
0.40
Calcium Chloride
0.5 mg
Magnesium Sulfate
0.025
Malaquite Green
250 µg
Ferric Ammonium Citrate
0.04
Bacteriological Agar
15.00
Final pH 6.6± 0.2 at 25ºC
PREPARATION
Suspend 19 grams of the medium in 900 ml of distilled water. Add 5 ml of Glycerol. Mix well and dissolve by heating
with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121ºC for 10 minutes.
Cool to 45-50ºC and aseptically add one 100 ml flask of the Enrichment Supplement OADC (Cat. 6037). Homogenize
gently and dispense in Petri plates. The prepared medium should be stored at 8-15°C. The color is light amber, slightly
opalescence.
The dehydrated medium should be homogeneous, free-flowing and beige in color. If there are any physical changes,
discard the medium.
ENRICHMENT OADC SUPPLEMENT (Cat. 6037)
(Composition: each flask for one liter of medium)
Sodium Chloride……………………8.5 g
Bovine Albumin Fraction V…… 50 g
Dextrose………………………………20 g
Catalase……………………………….0.03 g
Oleic Acid……………………………..0.6 ml
USES
MIDDLEBROOK AGAR can be used when supplemented with glycerol, Bovine Albumin Fraction V, dextrose, catalase and
Oleic Acid to cultivate and isolate a wide variety of Mycobacteria, including M.tuberculosis, excepting M.bovis that is
inhibited by glycerol.
Inorganic salts provide substances essential for the growth of Mycobacteria. Sodium citrate, when converted to citric
acid, serves to hold certain inorganic cations in solution. Albumin neutralizes toxic products that form during the
development of the organisms. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen.
Dextrose is is the fermentable carbohydrate providing carbon and energy. Sodium chloride supplies essential electrolytes
for transport and osmotic balance. Supplementation with glycerol enhances the growth of mycobacteria.Bacteriological
Agar is the solidifying agent.
M. fortuitum, M. fortuitum and M. kansasii grow on this medium while most other mycobacteria strains are inhibited.
Inoculate and incubate at 35 ± 2°C under 10% of CO2, and observed after 21 days
1
LABORATORIOS CONDA, S.A.
www.condalab.com
MICROBIOLOGICAL TEST
The following results were obtained in the performance of the medium, with glycerol and the Enrichment OADC
Supplement, after incubation at a temperature of 35 ± 2°C under 10% of CO2, and observed after 21 days
Microorganisms
Mycobacterium tuberculosis H37RV
Mycobacterium fortuitum ATCC 6841
Mycobacterium kansasii ATCC 12478
Growth
Good
Good
Good
BIBLIOGRAPHY
Middlebrook, G., M. L. Cohn, W. B. Dye, W. B. Russell, Jr., and D. Levy. 1960. Microbiologic procedures of value in tuberculosis. Acta.
Tubercul. Scand., 38:66. Cohn, M. L., R. F. Waggoner, and J. K. McClatchy. 1968. The 7H11 Medium for the cultivation of mycobacteria.
Am. Rev.
Resp. Dis., 98:295. Tenover, F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter, C. R. Horsburgh, Jr., and R. C. Good. 1993. The
resurgence of tuberculosis: is your laboratory ready. J. Clin. Microbiol. 31:767-770. Isenberg, H. D. (ed.). 1994. Clinical microbiology
procedures handbook.
Chelikani P, Fita I, Loewen PC (January 2004). "Diversity of structures and properties among catalases". Cell. Mol. Life Sci. 61 (2): 192–
208. doi:10.1007/s00018-003-3206-5. PMID 14745498.
STORAGE
25ºC
Once opened keep powdered medium closed to avoid hydration.
2ºC
2
LABORATORIOS CONDA, S.A.
www.condalab.com