1. Culturing of Bacteria

2. Major Contributions in Microbiology

3. Media and Culture
Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats
Culture: The propagation of microorganisms using various media
Culture Media: Used to grow bacteria and it can be used to:
Enrich the numbers of bacteria
Select for certain bacteria and suppress others
Differentiate among different kinds of bacteria

4. Culture and Medium
Culture is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying them
Pure culture
Mixed culture
Medium is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth that is multiplication, in order to cultivate these microorganisms in large numbers to study them

5. Classification of Culture Media
Media can be classified according to their properties:
1- according to physical state into solid, semi-solid, broth
a) Liquid Peptone water, Nutrient broth b) Semisolid - Nutrient agar, stabs c) Solid Blood agar, Serum agar (Figs-1, 2, 3 and 4)
2- According to chemical composition into
a) Natural (Empirical) media
don’t have any addition of specific nutrients e.g. milk, vegetable juices, diluted blood. It is called empirical because the exact chemical composition of the constituents is not exactly known

6. Culture Media( physically)
Fig 1
Culture Media( physically)
Solid
Semi Solid adding small amounts of agar to fluid
And mainly used as transport media or in motility tests.
Liquid (broth) .

7. Fig 2
Nutrient Broth
Nutrient Agar

8. Fig 3

9. Fig 4
Slant
Solid
Stab

10. It is one in which the chemical composition of the medium is
b) Semi-Synthetic Media
It is one in which the chemical composition of the medium is
partially known, in other words the medium has natural component (unknown chemical composition) and
certain specific nutrients (known chemical composition) e.g. potato-dextrose agar
c) Synthetic Media (chemically defined media)
One in which all chemical ingredients of known composition are mixed in definite proportions
d) Living Media
A living media consists of living cells or tissues which are used for the culture of strictly parasitic organisms like viruses or rickettsiae which can not be cultured on a non living medium

11. 4- Based on oxygen requirement: Aerobic medium Anaerobic medium
3- According to functional type general purpose in to selective, enriched, enrichment
4- Based on oxygen requirement:
Aerobic medium
Anaerobic medium

12. Basic requirements of culture media
Nutrients Energy source Carbon source Nitrogen source
Mineral salts – Sulphate, phosphates, chlorides and carbonates of K, Mg and Ca
A suitable pH about 7.2 – 7.4
Accessory growth factors Tryptophan for Salmonella typhi
Water

13. Many of the ingredients used in culture media are of natural origin, the most important are:
Beef extract
Peptones
Yeast extract
Gelatin
Agar

14. Agar – Agar Complex polysaccharide
Used as solidifying agent for culture media in Petri plates, slants, and deeps
Generally not metabolized by microbes
Liquefies at 98°C
Solidifies ~42°C
Added to culture media in a concentration of 1.2% to 2% to render it solid

15. Peptone:. Water soluble protein (milk, meat, soya)spray dried. product
Peptone: Water soluble protein (milk, meat, soya)spray dried product obtained from hydrolysis(acid, enzyme e.g. papin, pepsin, trypsin)
Meat extract: Concentrated aqueous infusion of fresh beef
Mineral salts: Sulphate, phosphate, iron
Carbohydrate provides the microorganisms with carbon which is the source of energy
Water deionized OR
Distilled water free from chemicals that can suppress the growth of microorganisms

23. c. Streak plate method

24. Direct Measurement of Microbial Growth
A standard plate count reflects the number of viable microbes and assumes that each bacterium grows into a single colony. Because it is impossible to say that each colony actually arose from an individual cell (cells clump, fact of life) plate counts are reported as the number of colony-forming units (CFU) instead of the number of cells
If the concentration of bacteria is too great the colonies will grow into each other and the plate will be uncountable
To insure a countable plate a series of dilutions should be plated.  The serial dilutions should give at least one countable plate in the series ( or , depending on preference of the individual lab)
In a direct microscopic count, the microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide - Hemocytometer

29. Estimating Bacterial Numbers by Indirect Methods
A spectrophotometer is used to determine turbidity i.e. cloudiness by measuring the amount of light that passed through a suspension of cells - Fig
More cells = more turbidity; more turbidity = less light passing through the suspension
%T is percent transmission - fewer cells present (less turbidity) will allow more light to pass through, the %T is higher when the cell number is lower
Absorbance is the opposite of %T.  More light is absorbed when more cells are present - some people like this measure better because absorbance goes up as turbidity or cell number goes up
After counting the cells in all 4 corner-squares, divide by 4 to get the average number of cells per 1 mm2 cell area. Multiply this number by 1x104 to obtain the average number of cells per ml