Dishant Sharma

TYPES OF MEDIA FOR MYCOBACTERIA. MYCOBACTERIA.

Culture Media
Culture media are required to grow

the microorganism. It consist of water, electrolytes, peptone, meat extract, blood or serum, agar etc. Medium must contain all the nutrients the microorganism requires for growth.

Macronutrients
Analysis of microbial cell composition

shows that over 95% of cell dry weight is made up of a few major elements: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium , and iron. The first six (C, O, H, N, S, and P) are components of carbohydrates, lipids, proteins, and nucleic acids.

Macronutrients
The remaining four macro-elements exist in the cell as cations and play a variety of roles: 1- potassium (K) is required for activity by a number of enzymes, including some of those involved in protein synthesis. 2-Calcium (Ca2), among other functions, contributes to the heat resistance of bacterial endospores . 3-Magnesium (Mg2) serves as a cofactor for many

enzymes, complexes with ATP, and stabilizes ribosomes and cell membranes. 4-Iron (Fe2 and Fe3) is a part of cytochromes and a cofactor for enzymes and electron-carrying proteins.

Micronutrients
Trace elementsmanganese, zinc, cobalt,

molybdenum, nickel, and copperare needed by most cells.

Culture Media
isolation)

(Ideal Medium for

Support rapid and luxuriant growth of small

numbers of mycobacteria. Permit preliminary differentiation of isolates on the basis of pigment production and colony morphology. Inhibit the growth of contaminants. Economical and simple to prepare from readily available ingredients. Enable drug susceptibility tests.

Types of Media
Physical States- Liquid, Semisolid, Solid . Molecular Oxygen-Aerobic, Anaerobic.

Nutritional Factors 1-Simple Nutrient Broth, Nutrient agar. 2-Complex. 3-Synthetic-Dubos Medium with tween 80. 4-Special.

4-Special Media
1-Enriched-Blood agar; Loefflers Serum Slop-

Corynebacterium diptheriae. 2-Enrichment-Tetrathionate Broth, Selenite F broth-typoid & para thypoid . 3-Selective-Deoxycholate Citrate Agar; Bile Salt Agar-Salmonella(Enteric bacilli). 4-Differential-Mac Conkeys Medium-E.coli(LF-Pink). 5-Indicator-Wilson & Blair Medium-Salmonella typhi(black colour colonies). 6-Transport-Stuarts, Buffered Glycerol Saline (Enteric bacteria) 7-Sugar-Hisss Serum Sugars-Pneumococcus.

Liquid Media For Mycobacteria

Liquid media Glycerol Alanine Salts Middlebrook 7H9 Broth Dubos Broth Proskauer and Beck Appropriate techniques DNA preparation General General Surface-pellicle growth

Sautons
Lemco Broth Tryptic Soy Broth M9 Minimal Medium

General defined medium

General Phage infection Protein preparation

Solid Media For Mycobacteria

Solid media Middlebrook 7H11 agar Middlebrook 7H10agar Lemco agar Lowenstein Jensen (LJ) slopes M9 Minimal Agar General General General Strain maintenance Protein purification/ auxotrophy screening Appropriate techniques

Tryptic Soy Agar

Top agar BCG top BCG agar

Phage infection
Phage overlays Phage overlays for BCG Phage infection for BCG

Compositions Of LJ
Potassium dihydrogen phosphate Magnesium sulphate

Magnesium citrate
Asparagine Glycerol Malachite green (2%) Eggs

Ditilled Water

Composition of Middlebrook
7H9(Liquid) Ammonium Sulphate Monopotassium phosphate Disodium phosphate Sodium citrate Y Y Y Y 7H10(Solid) Y Y Y Y 7H11(Solid) Y Y Y Y

Magnessium sulphate
Calcium chloride

Y
Y

Y
Y

Y
NO Pancreatic D of Casein

Zinc sulphate
Copper sulphate

Y
Y

Y
Y

NO
NO

L-Glutamic acid
Ferric ammonium citrate

Y
Y

Y
Y

Y
Y

Pyridoxine hydrochloride
Biotin Malachite green Agar pH=6.6

Y
Y NO NO Y

Y
Y Y Y Y

Y
Y Y Y Y

Composition of Sautons Media

KH2PO4 MgSo4

Ammonium Ferric Citrate

Citric Acid Asparagine Glycerol pH=7.2

Egg based media-e.g.-LJ.

Advantages: Can stored for several months in refrigerator. Less likely to become contaminated by evaporation. Support growth of most mycobacteria. Disadvantages: Contamination covers total surface. DST difficult, as the concentration of drug loss by heating & interaction of the egg phospholipid with drug. Opaque

Agar based media-eg.-7H11.

Advantages: Less likely to support contamination.

Clear mediumobserve colony morphology.

DST more precise concentration of drug. Disadvantages: Dry out on prolonged storage. Daylight exposureFormaldehyde gas-Kill

MTb.

References

Textbook Of Microbiology- Dr.CP Baveja-

Culture Media. Mycobacteria Protocols- Tanya Parish & Neil G. Stoker.

Photos