Culture Media PART 1- Solid media, Liquid Media & Use of Agar

    

1. Culture Media

·     In nature as well as in clinical samples, microorganisms exist in mixed populations. In order to study particular organisms or to know their clinical importance, their pure cultures have to be isolated from source by cultivating in suitable culture media. A suitable culture media makes the cultivation of microorganisms possible in laboratories. A culture media can be either solid or liquid which is used to grow, transport and store microorganisms. A culture media should contain all the necessary nutrients required for the growth and multiplication of the organisms. In general, all microorganisms require source of energy, micro and macro nutrients for growth.  However, the precise composition of the media will depend upon the types of organisms being cultivated as microbes vary considerably in their nutritional requirements. Knowledge of normal habitat of a particular organism is useful in the selection of media as their natural habitat reflects their nutritional requirements. In order to support the growth of microorganisms in culture media, physical factors like optimum temperature, pH etc. also have to be maintained properly.

·      Culture media can be classified on the basis of several parameters (Table 1.1). Based on physical nature or consistency media can be classified into Liquid, Solid and Semi-solid media.  Based on chemical constituents, media are classified into Simple, Complex, Defined or Synthetic media, Semi defined media or Semi Synthetic media. Based on their application or function media can be divided into Supportive media (General purpose media) and special media.  Special media includes a number of preparations like Enriched media, Enrichment media, Selective media, Differential media, Indicator media, Sugar media and Transport media. However, sometimes a single media may fulfil more than one functions. For example; Blood agar can act as both Enriched and Differential media.           

           Eosin Methylene Blue (EMB) agar can act as both Selective and Differential media

           MaConkey (MAC) agar can act as Selective, Indicator and Differential media

           Mannitol salt agar can act as both Selective and Differential media.

Based on oxygen requirements, media are classified as Aerobic media and Anaerobic media.

Table-1.1. Types of media
	Basis for Classification	Types
1	Physical nature or consistency media	Liquid media Solid    media                                                Semisolid media
2	Chemical constituents	Simple media Complex media Defined or Synthetic media  Semi defined media.
3	Application or function	Supportive media (General purpose media) Special media ·       Enriched media ·       Enrichment media ·       Selective media ·       Differential media ·       Indicator media ·       Sugar media ·       Transport media
4	Oxygen requirements	Aerobic media Anaerobic media

1.1.       Media based on physical nature

1.1.1.                  Liquid Media

Liquid media is liquid in consistency. Louis Pasteur used liquid media for first time to cultivate microbes. He used original urine and meat broth as liquid media. When inoculated into suitable media, microbes will produce clone (cells originating from single parental cell) of cells. On solid media they can be seen as discrete colonies with specific colony morphology which will be helpful in the identification of microorganisms. But in liquid media, microbes grow diffusely. Hence microbes cannot be easily identified from liquid culture. Also it is difficult to isolate pure culture from mixed populations of microbes when inoculated in liquid media. Inoculated liquid media are first incubated at 37⁰ C for 24 hrs, and then sub cultured to solid media to get pure, isolated colonies. However, liquid media are useful when large volume samples like blood and water are to be tested. They are also widely used for the bulk culture preparation of microorganisms for antigen and vaccine production.  Most widely used liquid media now a days is Nutrient broth. In liquid media microbes show growth patterns like uniform turbidity, pellicle formation at the top region, sedimentation at the bottom or floccules throughout the medium (Figure-1.1). 

1.1.1.                  Solid Media

Solid media is solid in consistency. Solid media contains the solidifying agent agar in it. Earliest solid media was cooked cut potatoes used by Robert Koch. Later he used gelatine to solidify the liquid media. But it was not satisfactory as gelatine liquefied at 24⁰ C and also digested by proteolytic bacteria. Use of agar as solidifying agent was suggested to him by Frau Hesse, wife of one of the investigators in Koch’s lab, who had seen her mother using agar as solidifying agent during preparation of jellies.

Solid media are widely used than liquid media. On solid media, microbes produce discrete colonies with particular colony morphology. Also organisms exhibit other characteristics like pigmentation or haemolysis.  For example, Serratia sp. and Pseudomonas sp. colonies   produce red and green pigmentation respectively on solid media.  Streptococci sp. produce α or β hemolysis on blood agar. Hence isolation of pure culture and identification of organisms are easier when cultivated on solid media compared to liquid media. Most widely used solid media is Nutrient agar. Its composition is same as nutrient broth except the use of additional ingredient, agar.  

Composition of Nutrient agar and Nutrient broth

Nutrient agar	Nutrient broth	Amount
Agar	--	20 g
Peptone	Peptone	5 g
Yeast extract / Beef extract	Yeast extract	3 g
NaCl (Sodium chloride)	NaCl (Sodium chloride)	3 g
Distilled water	Distilled water	1 Litre
pH	pH	7±0.2

1.1.1.1.              Agar

Agar, also called agar-agar is the universally used solidifying agent in the preparation of solid media. Agar is extracted from seaweeds or marine algae of species Gelidium, mainly Gelidium corneum. Agar is long chain polysaccharides with varying amount of inorganic salts and small quantities of protein like substances.   It is a sulphated polymer composed mainly of D-galactose, 3,6 anhydro- L-galactose and D-glucuronic acid. Commercially available agar is sulphuric acid esters of linear galactan, insoluble in cold water and soluble in hot water. Agar is well suited as a solidifying agent for many reasons. Agar is an excellent solidifying agent with no nutritive value and most microbes do not degrade it. Means, agar is not effected by the growth of the microorganisms. Most unique property of agar is that it melts at 90⁰ C and sets at 45⁰ C. Hence after being melted, it can be cooled to a temperature that is tolerable to human hands as well as to microbes. Also solid media with agar can be incubated at a wide range of temperatures depending on the type of organisms cultivated. Usually 2% agar is used for the preparation of solid media. Based on concentration of agar jellifying capacity also varies. Agar is commercially available as dehydrated powder or long shreds.

1.1.1.1.              Peptone

Peptone is another universal agent used in solid media. It is a complex mixture of partially digested proteins resulting from digestion of protenaceous materials like meat, casein and gelatine. Digestion of these materials can be done either by acid or with enzymes. As hydrolysis proceeds, large colloidal protein molecules are broken up into a series of smaller fragments like proteoses, peptones, polypeptides and amino acids respectively. Constituents of peptone usually include proteoses, polypeptides, amino acids, variety of inorganic salts like Phosphate, Potassium, Magnesium and accessory growth factors like riboflavin. It has nutritive value and main source of organic nitrogen and contain vitamins and carbohydrate based on the material digested. Commercially different brands of peptone are now available. Different brands differ in composition and growth promoting properties. Other common ingredients of solid media are meat/beef extract and yeast extract. Peptone also act as buffering agent.

1.1.1.1.              Beef Extract

Aqueous extract of lean beef tissue concentrated to paste like form is called beef extract. It has nutritive value and consists of water soluble substances of animal tissue which include carbohydrate, organic nitrogen compounds, water soluble vitamins and salts.

1.1.1.2.         Yeast Extract

It is aqueous extract of yeast cells available commercially in powder form. It is nutritious with rich source of B Vitamins, also contains organic nitrogen and carbon compounds.

1.1.1.3.         Sodium chloride (NaCl)

It is not necessary for bacterial growth and also does not act as buffer. The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. If the salt concentration is not similar, osmosis takes place transporting excess water into or out from the cell. Sometimes, either to cultivate some microorganisms or to recognize properties like haemolysis, blood agar which is an enriched media is usually used.  In this media blood is added. Red Blood Cells get haemolysed when added to water or to media having low osmotic pressure. This may be prevented by addition of NaCl.

1.1.1.4.         Distilled water

Water is necessary for the existence of living organisms. Usually distilled water is used for preparation of culture media since it is of definite composition. Calcium and Magnesium present in tap water react with phosphates present in peptones, beef extract and other ingredients of culture media to give insoluble phosphates. Insoluble phosphates may not form in cold, but during sterilisation such media throw down considerable precipitate.

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