Culturing of Bacteria

Major Contributions in Microbiology

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

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

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

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) .

Fig 2 Nutrient Broth Nutrient Agar

Fig 3

Fig 4 Slant Solid Stab

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

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

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

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

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

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

c. Streak plate method

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 (25-250 or 30-300, 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

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