2. Introduction
• Bacteria exist is nature in mixed forms
• To study every species independently, it is
necessary to obtain their pure culture
• The two major steps of obtaining a pure culture
are as follows :
Firstly, the culture has to be diluted until the
various individual microorganisms are separated
far apart on agar surface that after incubation
they form visible colonies isolated from the
colonies of other microorganisms. This plate is
called an isolation plate
Secondly, an isolated colony has to be aseptically
picked off the isolation plate
3. • Points to be taken into consideration during
the inoculation are :
The inoculation loop has to be sterilized
before every inoculation of colonies from the
agar plate
Every time the loop is sterilized by heat it
must be cooled before inoculating the next
colony
• There are several methods of isolating the
pure culture of bacteria, like, streak plate
method, pour plate method, spread plate
method and serial dilution
4. StreakPlateMethod
• The most common technique of isolation of
bacterial cells on the surface of agar plate is
the streaking method
• It is a simple and a rapid way of isolating
cells of bacteria on the agar plate surface
through mechanical means
• As the loop is streaked on the agar surface
the bacterial cells are rubbed off until
individual separate organisms are deposited
on the agar
5. • After incubation the area at the beginning
of the streaking will show confluent growth
of bacterial cells, whereas, the area at the
end of the streak will show discrete colonies
of bacterial cells
6. Pourplatemethod
• This method is used to count the number of
colony making bacteria in liquid specimen
• In this method a fixed amount of inoculum is
taken from a broth or say sample is placed in
the centre of a sterile Petri plate with the
help of a sterile pipette
• Molten cooled agar is then poured into the
Petri plate containing inoculum and mixed
well
• After the solidification of the Agar the
Petri plate is inverted and incubated at
37degree C for 24-48 hours
7. • Bacteria will grow both on the surface and within
the media.
• Colonies which grow within the medium are very
small in size and may be confluent
• Few bacteria which grow on the agar plate surface
are of same size, and appear as those on a streak
plate
8. Spread plate method
• This technique is used to readily quantify the
amount of bacteria present in a solution
• In this technique, the sample is diluted and then a
little amount of it is added to the agar plate
• Then the sample is spread over the agar surface
evenly with the help of a spreader
• After the colonies grow, the number of colonies is
counted and the original number of bacteria in the
sample is counted
• The end point of our analysis is the number of
colony forming units per milliliters
9.
10. Serial dilution
• It is a method of stepwise dilution of a substance
• The dilution factor is kept constant, resulting in the
geometric progression of concentration in a logarithmic
fashion
• Each dilution reduces the concentration of bacteria by a
specific amount
• By calculating the total dilution over the entire series, it is
possible to know how many bacteria were present when the
process was started
