Raw materials for culture Media
Raw materials play an important role in media preparation. The quality of media depends on the quality of raw materials. The most important raw materials are used for preparations of media are water, agar, peptone, casein hydrolysate, meat extract, yeast extract, and malt extract.
1. Water as solvent: Water plays a vital role in the preparation of media. Water should be free from copper ion because copper ion inhibits the growth of microorganisms. The conductivity of water should be less than 15 µS (microelements) and the pH of the water should be slightly acidic but should not be less than 5.5.
2. Petri dishes: Petri dishes are generally sterilized with ethylene oxide (EtO) or gamma-irradiated. If EtO is sterilized they should be then checked for residual EtO toxicity which affects the growth of the microorganisms. The maximum permissible limit for residual EtO is 1 µg/g. Only borosilicate glassware should be used because soda glass can leach alkali into the media.
3. Energy source: The most common substance added to culture media is glucose that acts as a source of energy and also increases the rate of growth of organisms. Other carbohydrates are also added to media at 5-10 g/lt as biochemical substrates to detect the production of specific enzymes in the identification of organisms.
4 Nutrients: The nutrients of culture media are selected to recover the required spectrum of organisms in the sample e.g. coliforms or anaerobes. Proper nutrients help for bacterial growth.
5. Essential Metals and Minerals: The inorganic essential components of culture media are divided on a semi-quantitative basis: Macro-components (gm/liter): Na, K, Cl, P, S, Ca, Mg, Fe. Micro-components (mgm-microgram/liter): Zn, Mn, Br, B, Cu, Co, Mo, V, Sr, etc.
6. Buffering Agents: The pH of a culture medium is poised around the optimum necessary for the growth of the desired micro-organisms. The use of buffer compounds at specific pK values is especially necessary when fermentable carbohydrates are added as energy sources. Such examples of buffering agents are phosphates, acetates, citrates, zwitterion compounds, and specific amino acids that are added to culture media.
7. Indicator Substances: The addition of colored indicator substances in the medium is very effective in detecting the fermentation of specific carbohydrates. They change color distinctly and rapidly at critical pH values. Examples: phenol red, Bromo-cresol purple, fuchsin, etc., are toxic and it is essential to use in low concentrations. Known sensitive strains of micro-organisms are also used in the screening tests.
8. Selective Agents: Chemicals or antimicrobials are added to culture media to make them selective for certain micro-organisms. They are added at specific concentrations to suppress the growth of unwanted organisms in a polymicrobial sample. Examples: bile salts, dye-stuffs, selenite, tetrathionate, tellurite, and azide. Thereafter antimicrobial agents are used in mixtures when suppressing polymicrobial contaminating flora. Antimicrobials are more specific in their selective action.
9. Gelling Agents: Gelatin is still used for a few specific media and carrageenans, alginates, silica gel, and polyacrylamides are sometimes used as gelling agents but the most important gel-forming substance used in culture media is agar. It is inert to microbial action and melting temperatures (38°C and 84°C respectively) the high gel strength which allows low concentrations of agar to be used. It has low toxicity and is also not hydrolyzed by agarase enzyme at high temperatures. Microbiological agar is specially processed to yield low toxicity, high clarity, low mineral, and high diffusion gel.
10. Other Components: Many other substances are added to culture media for specific purposes like growth factors for fastidious organisms, pH-reducing compounds for anaerobic organisms (thioglycollate and cysteine), whole blood to detect hemolytic enzymes, etc.
11. Meat extract: It is obtained by hot water extraction of lean beef and then concentrated by evaporation. It contains gelatin, albumoses, peptones, proteoses, amino acids, creatinine, purines, and accessory growth factors.
12. Yeast extract: It is prepared from washed cells of bakers’ yeast and contains a wide range of amino acids, growth factors, and inorganic salts.
13. Malt extract: It is prepared by extracting soluble materials from sprouted barley in water at 55o C and concentrated by evaporation. It contains maltose, starch, dextrin, glucose, and small amounts of protein and protein breakdown products and growth factors.
Nutritional Requirements for Culture Media
Most of the microorganisms are grown in specific culture media in laboratory conditions and for proper growth, nutrition plays an important role in the structural and functional properties of the cells. Nutrition is generally composed of:
- Major macronutrients: C, H, O, N, S, P, K, Mg, Fe, Co, and Mn.
- Major micronutrients: Zn, Co, Cu, and Mo.
- Carbon and energy sources.
- Growth factors.
Carbon: The main source is organic compounds and CO2. It is the main component of cellular materials.
Hydrogen: The main source is organic compounds. It is the main component of cell water.
Oxygen: The main source is organic compounds, CO2, and O2. It is the main constituent of cell material and cell water. It is an electron acceptor in aerobic respiration.
Nitrogen: The main source is organic compounds, NH3, NO3, N2, etc. It is the main constituent of amino acids, nucleic acids nucleotides, and coenzymes.
Sulfur: The main source is organic sulfur compounds. It is the main constituent of some amino acids like cysteine, methionine, glutathione, and several coenzymes.
Phosphorus: The main source is inorganic phosphates. It is the main component of nucleic acids, nucleotides, phospholipids, etc.
Potassium: The main source is potassium salt. It is the main component of cellular inorganic cation and cofactor for certain enzymes.
Magnesium: The main source is magnesium salt. It is the main component of inorganic cellular cation, a cofactor for certain enzymatic reactions.
Iron: The main source is iron salt. It is the main component of cytochromes and certain non-heme iron-proteins and a cofactor for some enzymatic reactions.
Calcium: The main source is calcium salt. It is the main component of inorganic cellular cation, a cofactor for certain enzymes, and a component of endospores.
Manganese: The main source is manganese salt. It is the main component of inorganic cellular cation, a cofactor for certain enzymes.
They are also known as trace elements because they require a very small amount. They usually act as cofactors for essential enzymatic reactions in the cell. They are cations required for bacterial nutrition. Examples: Mn, Co, Zn, Cu, and Mo.
Zn2+ is present at the active site of some enzymes but is also involved in the association of regulatory and catalytic subunits in E. coli aspartate carbamoyltransferase. Mn2+ aids many enzymes in catalyzing the transfer of phosphate groups. Mo2+ is required for nitrogen fixation and Co2+ is a component of Vitamin B12. Cu2+ is an important micronutrient required as a redox cofactor of multiple proteins.
Carbon and Energy Sources for Bacterial Growth:
A source of carbon as an energy source and other nutrients as growth parameters are required in a permissive range of physical conditions viz. O2 concentration, temperature, and pH. Sometimes bacteria are referred to as individuals or groups based on their patterns of growth under various chemical or physical conditions. For example, phototrophs are organisms that use light as an energy source; anaerobes are organisms that grow without oxygen; thermophiles are organisms that grow at high temperatures.
All living organisms require an energy source. Organisms that use light energy are called phototrophs, when organisms that use an organic form of carbon are called heterotrophs, and when organisms that oxidize inorganic compounds are called lithotrophs. The source of carbon is either organic carbon or CO2. Organisms that use organic carbon are heterotrophs and organisms that use CO2 as a sole source of carbon are known as autotrophs (Table).
Table: Prokaryotes as per nutritional requirement
|Nutritional Type||Energy Source||Carbon Source||Examples|
|Photoautotrophs||Light||CO2||Cyanobacteria, some Purple and Green Bacteria|
|Photoheterotrophs||Light||Organic compounds||Some Purple and Green Bacteria|
|Chemoautotrophs or Lithotrophs||Inorganic compounds, e.g. H2, NH3, NO2, H2S||CO2||A few Bacteria and many Archaea|
|Chemoheterotrophs or Heterotrophs||Organic compounds||Organic compounds||Most Bacteria, some Archaea|
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