Culture medialecture (1)

Mrs. Dalia kamal eldienMsc in microbiology

introduction

The study of microorganisms requires techniques for isolating from natural sources and growing them in the laboratory on synthetic media.

Thus, development of synthetic culture media and culture techniques have played important roles in the advance of this field.

culture medium is a liquid or gel, designed to support the growth of microorganisms 

Microbiologists use bacterial culture media for many purposes and applications

1. isolate and identify of bacteria

2. long-term storage of pure cultures

3. Antibiotic sensitivity test

The important reasons for culturing bacteria in vitro diagnosing infectious diseases. studying its morphology and its identification obtain antigens from developing serological assays or

vaccines genetic studies

Basic Composition of culture media

When culturing bacteria, it is very important to provide similar environmental and nutritional conditions

bacteria need the basic nutritional conditions to grow invitro, include:

Amino Acids Carbohydrates Vitamins Fatty acids inorganic salts Water

1.Amino acid: Are the building blocks of proteins. ‘Essential’ amino acids must be added to culture media as

cells are not able to synthesize these themselves. Example: cystine, leucine, lysine, methionine,

phenylalanine, tryptophan, and valine.

2. Carbohydrates The main source of energy is derived from sugars. The major sugars used are glucose and galactose, however,

some media contain maltose or fructose.

3.Vitamins Serum is an important source of vitamins in culture media. Many vitamins needed by bacteria especially group B

vitamins, are necessary for cell

4. Fatty Acids and Lipids Are important in serum free media since they are normally

present in serum e.g. cholesterol and steroids essential for specialized cells.

5.Inorganic Salts The inclusion of inorganic salts in media performs several

functions. Primarily they help to retain the osmotic balance of the cells

and help regulate membrane potential by provision of sodium, potassium and calcium ions.

6. Water

All bacteria require some amount of water. Buffering Systems• Most cells require pH conditions in the range 7.2-7.4 and

close control of pH is essential for optimum culture conditions.

Classification:

Bacterial culture media can be classified in at least three ways;

- Based on consistency- Based on nutritional component - Based on its functional use.

Classification based on consistency, Culture media are liquid semi-solid solid biphasic.

Liquid media: These are available for use in test-tubes, bottles or flasks,

sometimes referred as “broth” (e.g nutrient broth). In liquid medium, bacteria grow producing turbidity. Certain aerobic bacteria and those containing fimbriae

(Vibrio & Bacillus) are known to grow as a thin film called ‘surface pellicle’ on the surface of broth.

Sometimes the initial turbidity may be followed by clearing due to autolysis, which is in penumococci.

Long chains of Streptococci when grown in liquid media tend to entangle and settle to the bottom forming granular deposits

Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth. This is the practical approach in blood cultures.

Sterile Liquid media(right) turbid liquid media(left)

Liquid media show the surface pellicle

Solid media: Any liquid medium can be rendered by the addition of

certain solidifying agents. Agar agar (simply called agar) is the most commonly used solidifying agent.

Agar is a polysaccharide obtained from the cell membranes of some species of red algae

It melts at 90-95oC and solidifies at 32–39 C. doesn’t contribute any nutritive property However, it may be a source of calcium & organic ions. it is used at concentration of 1.5% w/v to make a solid agar

medium. Solid media are used mainly in Petri dishes as plate

cultures. Also in bottles or tubes as stab (deeps) or slope cultures.

Solid media in petri-dish

Semi-solid agar: Reducing the amount of agar to 0.4-0.5% renders a

medium semi-solid useful in demonstrating bacterial motility Certain transport media such as Stuart’s and Amies

media are semi-solid in consistency Hugh & Leifson’s oxidation fermentation test medium as

well as mannitol motility medium are also semi-solid.

Motility test in semi-solid media

Identify?

Biphasic media: Sometimes, a culture system comprises of both liquid

and solid medium in the same bottle. This is known as biphasic medium (Castaneda system for blood culture).

The inoculums is added to the liquid medium and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid medium.

Biphasic Medium

Besides agar, egg yolk and serum too can be used to solidify culture media. While serum and egg yolk are normally liquid, they can be rendered solid by coagulation using heat.

Serum containing medium such as Loffler's serum slope and egg containing media such as Lowenstein Jensen medium and Dorset egg medium are solidified as well as disinfected by a process of inspissations

Loffler's serum & LJ