Culturing of Bacteria Culturing of Bacteria

Major Contributions in MicrobiologyMajor Contributions in Microbiology

Media and CultureMedia 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: Culture Media: Used to grow bacteria and it can be used to:Used to grow bacteria and it can be used to:

Enrich the numbers of bacteria Enrich the numbers of bacteria

Select for certain bacteria and suppress othersSelect for certain bacteria and suppress others

Differentiate among different kinds of bacteriaDifferentiate among different kinds of bacteria

Culture and MediumCulture and Medium

CultureCulture is the term given to microorganisms that are cultivated in is the term given to microorganisms that are cultivated in the lab for the purpose of identifying and studying themthe lab for the purpose of identifying and studying them

Pure culturePure culture

Mixed cultureMixed culture

MediumMedium is the term given to the combination of ingredients that is the term given to the combination of ingredients that will support the growth and cultivation of microorganisms by will support the growth and cultivation of microorganisms by providing all the essential nutrients required for the growth that is providing all the essential nutrients required for the growth that is multiplication, in order to cultivate these microorganisms in large multiplication, in order to cultivate these microorganisms in large numbers to study themnumbers to study them

Classification of Culture MediaClassification of Culture Media

Media can be classified according to their properties:Media can be classified according to their properties:

1- according to 1- according to physical statephysical state into solid, semi-solid, broth into solid, semi-solid, broth

a)a) LiquidLiquid - Peptone water, Nutrient broth - Peptone water, Nutrient broth

b) b) SemisolidSemisolid - Nutrient agar, stabs- Nutrient agar, stabs

c) c) SolidSolid - Blood agar, Serum agar ( - Blood agar, Serum agar (Figs-1, 2, 3 and 4)Figs-1, 2, 3 and 4)

2- According to 2- According to chemical compositionchemical composition into into

a) a) Natural (Empirical) mediaNatural (Empirical) media• don’t have any addition of specific nutrients e.g. milk, don’t have any addition of specific nutrients e.g. milk,

vegetable juices, diluted blood. It is called empirical vegetable juices, diluted blood. It is called empirical because the exact chemical composition of the constituents because the exact chemical composition of the constituents is not exactly knownis not exactly known

.

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 1

Nutrient Broth Nutrient Agar

Fig 2

Solid Culture Media

Petri dish Slant Stab (Deep)

Fig 3

Solid

Slant

Stab

Fig 4

bb)) Semi-Synthetic MediaSemi-Synthetic Media

It is one in which the chemical composition of the medium isIt is one in which the chemical composition of the medium is

• partially known, in other words the medium has natural partially known, in other words the medium has natural component (unknown chemical composition) andcomponent (unknown chemical composition) and

• certain specific nutrients (known chemical composition) e.g. certain specific nutrients (known chemical composition) e.g. potato-dextrose agarpotato-dextrose agar

c) Synthetic Mediac) Synthetic Media (chemically defined media) (chemically defined media)

One in which all chemical ingredients of known composition One in which all chemical ingredients of known composition are mixed in definite proportionsare mixed in definite proportions

d) Living Mediad) Living Media

A living media consists of living cells or tissues which are A living media consists of living cells or tissues which are used for the culture of strictly parasitic organisms like viruses used for the culture of strictly parasitic organisms like viruses or or rickettsiaerickettsiae which can not be cultured on a non living which can not be cultured on a non living mediummedium

3- 3- According to According to functional type general purposefunctional type general purpose in to selective, in to selective, enriched, enrichmentenriched, enrichment

4- Based on Based on oxygen requirementoxygen requirement::

• Aerobic mediumAerobic medium

• Anaerobic medium Anaerobic medium

Basic requirements of culture mediaBasic requirements of culture media NutrientsNutrients

- Energy source - Energy source - Carbon source - Carbon source - Nitrogen source - Nitrogen source

Mineral salts – Sulphate, phosphates, chlorides and carbonates Mineral salts – Sulphate, phosphates, chlorides and carbonates of K, Mg and Caof K, Mg and Ca

A suitable pH about 7.2 – 7.4A suitable pH about 7.2 – 7.4

Accessory growth factorsAccessory growth factors - Tryptophan for - Tryptophan for Salmonella typhiSalmonella typhi

WaterWater

Many of the ingredients used in culture media are of natural origin, Many of the ingredients used in culture media are of natural origin, the most important are:the most important are:

Beef extractBeef extract

PeptonesPeptones

Yeast extractYeast extract

GelatinGelatin

AgarAgar

Agar – AgarAgar – Agar

Complex polysaccharide Complex polysaccharide

Used as solidifying agent for Used as solidifying agent for culture media in Petri plates, culture media in Petri plates, slants, and deepsslants, and deeps

Generally not metabolized by Generally not metabolized by microbesmicrobes

Liquefies at 98°CLiquefies at 98°C

Solidifies ~42°CSolidifies ~42°C

Added to culture media in a Added to culture media in a concentration of 1.2% to 2% to concentration of 1.2% to 2% to render it solidrender it solid

Peptone:Peptone: Water soluble protein (milk, meat, soya)spray Water soluble protein (milk, meat, soya)spray dried dried product product obtained from hydrolysis(acid, enzyme obtained from hydrolysis(acid, enzyme

e.g. papin, pepsin, trypsin)e.g. papin, pepsin, trypsin)

Meat extract: Meat extract: Concentrated aqueous infusion of fresh beefConcentrated aqueous infusion of fresh beef

Mineral salts:Mineral salts: Sulphate, phosphate, ironSulphate, phosphate, iron

CarbohydrateCarbohydrate provides the microorganisms with carbon which is provides the microorganisms with carbon which is the source of energythe source of energy

Water deionizedWater deionized

OROR

Distilled water Distilled water free fromfree from chemicals that can suppress the growth of chemicals that can suppress the growth of microorganisms 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 MethodsA 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