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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
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
Solid Culture
Media
Petri dish
Slant
Stab (Deep)
Fig 4
Slant
Solid
Stab
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
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 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