Media formulation, media constituents

1. Media formulation:
Dr. Harinatha Reddy
Department of Biotechnology
Bangalore

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3.  Medium formulation is done by changing one of independent
variables such as nutrients, pH, temperature, etc. by fixing rest of
others at specific level.
Carbon + Nitrogen source + O2 + Other requirements ----> Biomass + Products + CO2 + H2O+ Heat

4.  In most cases, the medium is independent of the bioreactor
design and process parameters.
 Even small modifications in the medium could change cell line
stability, product quality, yield, operational parameters, and
downstream processing

5.  Media must satisfy all nutritional requirements of the organism.
 Generally must provide a carbon source (for energy and
for biosynthesis) nitrogen, phosphorous and sulfur.
 Sources of – Macro and Micro (trace) elements.
 Some require vitamins e.g. biotin and riboflavin.
Medium composition:

6. Ion
Examples of enzymes
containing this ion
Cupper Cytochrome oxidase
Ferrous
Catalase
Cytochrome
Magnesium
Glucose 6-phosphatase
Hexokinase
DNA polymerase
Manganese Arginase
Molybdenum
Nitrate reductase
Nitrogenase
Nickel Urease
Zinc
Alcohol dehydrogenase
Carbonic anhydrase
DNA polymerase

7. Essential characteristics of formulated media:
 Media must satisfy all nutritional requirements of the organism.
 It should produce the maximum yield of biomass and product.
 It should also have minimal problems during media designing
recovery of desired product.

8.  The role of trace elements (Iron, Zinc, Copper, Selenium,
Manganese & Molybdenum) in medium formulation might be quite
significant.
 The concentration of NaCl is needed to adjust the osmolarity of
media and pH is commonly maintained by CO2-bicarbonate system

9. Constituents of media:

10.  Many elements are needed for microorganism nutrition and their
physiological functions.
 Thus, these elements have to be supplied in the culture medium to
support adequate growth of microbial cultures.
Constituents of media:

11. The culture media usually contain the following constituents:
1. Inorganic nutrients
2. Carbon and energy sources
3. Organic supplements
4. Antibiotics
5. Solidifying agents
6. Salts

12. 1. Inorganic Nutrients:
 The inorganic nutrients consist of macronutrients and
micronutrients.
 C, H, O, N, S. P, K, and traces of Fe, Ca, Mn Zn, Co, Cu, and Mo
elements are found in the form of inorganic ions in water,

13. Macronutrient elements:
 The six elements namely nitrogen, phosphorus, potassium,
calcium, magnesium and sulfur are the essential macronutrients
for tissue culture.
Micronutrients:
 Although their requirement is in minute quantities,
micronutrients are essential for plant cells and tissues.
 These include iron, manganese, zinc, boron, copper and
molybdenum.

14. 2. Carbon and energy sources
 Majority of culture media contain glucose which serves as an
important source of energy.
 Glucose is degraded in glycolysis to form pyruvate/lactate.
 These compounds on their further metabolism enter citric acid
cycle and get oxidized to CO2 and H2O .
 .

15. 3. Organic supplements:
 Several additional organic compounds are usually added to the
media to support cultures.
 These include certain proteins, peptides, Vitamins, lipids,
nucleosides, serum and citric acid cycle intermediates.

16. Serum:
 Serum is a natural biological fluid, and is rich in various
components to support cell proliferation.
 The most commonly used sera are calf serum (CS), fetal
bovine serum (FBS), horse serum and human serum.
 While using serum, it must be screened for viral diseases
(hepatitis B, HIV).

17. Vitamins:
 The medium should be supplemented with vitamins to achieve
good growth of cells.
 The vitamins added to the media include thiamine, riboflavin,
niacin, pyridoxine, folic acid, pantothenic acid, biotin,
ascorbic acid and vitamin E.

18. Amino acids:
 The media supplemented with amino acids stimulate cell
growth and help in establishment of cells lines.
 Further, organic nitrogen (in the form of amino acids such as L-
glutamine, L-asparagine, L- arginine, L-cysteine) is more
readily taken up than inorganic nitrogen by the cells.

19. 4. Antibiotics
 Antibiotics were added to reduce contamination.
 The most commonly used antibiotics were ampicillin, penicillin,
gentamycin, erythromycin, kanamycin, neomycin and tetracycline.

20. 5. Solidifying agents:
 For the preparation of semisolid or solid tissue culture media,
solidifying or gelling agents are required.

21. Agar:
 Agar, a polysaccharide obtained from seaweeds (Red algae:
(Gelidium and Gracilaria) is most commonly used as a gelling
agent for the following reasons.
 It does not react with media constituents.
 It is not digested by microbial or plant enzymes and is stable at
culture temperature.
 Agar at a concentration of 0.5 to 1% in the medium can form a
gel.

22. Gelatin:
 It is used at a high concentration (10%) with a limited success.
 This is mainly because gelatin melts at low temperature (25-
35°C), and consequently the gelling property is lost.

23. 6. Salts:
 The salts contribute to cations (Na+, K+, Mg2+, Ca2+ etc.) and
anions (CI–, HCO–
3, SO2-
4, PO3-
4), and are mainly responsible for
the maintenance of solute concentration.
 Na+, K+ and CI– ions regulate membrane potential.
 PO3-
4 is required for ATP synthesis.

24. Advantage of Salts in Media:
 Supply essential inorganic ions.
 Provide requisite pH.
 Maintain the solute concentration in media.

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