1. Culture media(1) Mrs. Dalia kamal Eldien Msc in Microbiology Lecture (1)

2. Objectives  Introduction to culture media  Common consistent of culture media  Different methods of Classification  Classification based on concentration of agar

3. 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

4.  The important reasons for culturing bacteria in vitro 1. Diagnosing infectious diseases. 2. Studying its morphology and its identification 3.Obtain antigens from developing serological assays or vaccines 4.Genetic studies

5. 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

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.  Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth (blood cultures).  The growth of the bacteria on the fluid media give either granular or uniform turbidity  Sometimes the initial turbidity may be followed by clearing due to autolysis, which is in penumococci.

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

12. Liquid media show the surface pellicle

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

14. Solid media in petri-dish

15. Semi-solid agar:  Reducing the amount of agar to 0.4-0.5% renders a medium semi-solid  useful in demonstrating bacterial motility  Common example include, transport media such as Stuart’s and Amies media  Hugh & Leifson’s used for oxidation fermentation test.

16. Motility test in semi-solid media

17. Identify?

18. Biphasic media:  Sometimes, a culture system comprises of both liquid and solid medium in the same bottle. This is known as biphasic medium (e.g. 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.

19. Biphasic Medium

20.  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 Define inspissations???

21. Loffler's serum & LJ