A pure culture is usually derived from a mixed culture by transferring a small sample into a new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample several times before inoculating the new medium. Hence, pure culture is defined as a laboratory culture containing a single species of organism.
Importance of Pure Culture:
• Desired known species of microorganisms can grow once pure
culture is isolated.
• Pure culture can be correctly identified for accurate
study and testing of microorganisms in a clinical environment.
• Experimenting with pure culture ensures the same results
after repetition of the experiment many times.
• In pure culture, the spontaneous mutation rate is low.
Hence, pure culture is required to isolate which is carried
out by:
1. Streak plate method
2. Pour plate method
3. Spread plate method
4. Serial dilution method
5. Special methods: (a) Single Cell Isolation Methods (b) Enrichment Culture Method
Flowchart: Types of isolation of pure culture
1. Streak Plate Method: The principle of this method
is that, by streaking, a dilution gradient is established across the face of
the Petri plate where bacterial cells are deposited on the agar surface. This
method is used most commonly to isolate pure cultures of bacteria where a small
amount of mixed culture is placed on the tip of an inoculation loop and is
streaked across the surface of the agar medium by different fashions (Fig) and
the plates are incubated to allow the growth of colonies and each colony is the
progeny of a single microbial cell thus representing a clone of pure culture.
Finally, the micro-organisms are separated from each other. The isolated
colonies are picked up separately using a sterile inoculating loop and
re-streaked onto fresh media to ensure purity.
Fig: Different types of streak plates
2. Pour Plate Method: The main principle of this
method is the dilution of the inoculum in successive tubes containing liquefied
agar medium to permit a thorough distribution of bacterial cells within the
medium.
In this method, the mixed culture of bacteria is diluted
directly in the liquid agar medium tube (42-45°C) and mixed well. In this
method, a fixed amount of inoculum (generally 1 ml) from a broth/sample is
placed in the center of the sterile petri dish using a sterile pipette. The
contents of each tube are poured into separate petri plates and then allowed to
solidify. These plates are incubated to develop bacterial colonies both within
the agar medium (subsurface colonies) and on the medium (surface colonies).
Finally, isolated the colonies are picked up by an inoculation loop and
streaked onto another petri plate to ensure purity (Fig). This method is also
used for determining the number of viable bacterial cells present in a culture.
3. Spread Plate Method: The principle of this method
involves using a sterilized spreader with a smooth surface made of metal or
glass to apply a small amount of bacteria suspended in a solution over a plate
that needs to be dry and at room temperature so that the agar can absorb the
bacteria more readily. A successful spread plate will have a countable number
of isolated bacterial colonies evenly distributed on the plate.
The technique makes it easier to quantify bacteria in a
solution. A drop of diluted liquid from each tube is placed on the center of an
agar plate and spread evenly over the surface using a sterilized bent-glass rod.
The medium is incubated for the colonies development on the agar medium plates.
The isolated colonies are picked up and transferred onto fresh medium to ensure
purity (Fig).
Some useful differences between pour plate and spread plate
methods
|
Parameters |
Pour plate method |
Spread plate method |
|
Procedure |
Inoculum from a sample is placed in the center of a sterile petri
dish using a sterile pipette. Molten-cooled agar (approx. 15 ml) is then
poured into the petri dish containing the inoculum. |
Inoculum from a sample is pipette out and spread evenly on sterile
nutrient agar by a spreader. |
|
Amount |
The inoculum is 1 ml. |
The inoculum is 0.1 ml. |
|
Medium |
Colonies are grown in a solid culture medium. |
Colonies are grown in a solid culture medium. |
|
Sample Application |
More surface area is covered as the sample is spread throughout the
media. |
The sample is spread only to a limited area i.e. only on the surface
of agar. |
|
Uses |
It is used to determine CFU/ml or PFU/ml. |
It is used to isolate specific clonal colonies. |
|
Advantages |
1. It allows the growth of microaerophiles. 2. It helps in the identification of various bacteria, like aerobe,
anaerobe, or facultative aerobe bacteria. |
1. Picking surface colonies will not interrupt other colonies by
digging out of agar. 2. Get an even distribution of colonies. |
|
Disadvantages |
1. Picking subsurface colonies can interrupt other colonies by
digging out of agar. 2. Do not get an even distribution of colonies. |
1. It does not allow the growth of microaerophiles. 2. It does not allow the growth of obligate anaerobes. |
|
Benefit |
It is beneficial to isolate certain bacteria which are motile and do
not grow as a colony. |
It is beneficial for the isolation of bacteria from soil or water. |
|
Application |
This technique is used in Bacteriologic examination of milk, and bacterial
number of urine to signify urinary tract infection. |
This technique is used in enrichment, selection, and screening
experiments. |
4. Serial Dilution Method: A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.
This dilution aims to inoculate a series of tubes with a
microbial suspension so dilute that some tubes show the growth of only one
individual microbe. Example: A culture containing 10 ml of liquid medium,
containing 1,000 microorganisms i.e., 100 microorganisms/ml of the liquid
medium. 1 ml of this medium is taken out and mixed with 9 ml of fresh sterile
liquid medium, resulting in a medium have 100 microorganisms in 10 ml or 10
microorganisms/ml. Further, 1 ml of this suspension is added to another 9 ml of
fresh sterile liquid medium, each ml is now contained a single microorganism.
If this tube showed any microbial growth, there is a very high probability that
this growth has resulted from the introduction of a single microorganism in the
medium and represents the pure culture of that microorganism (Fig).
Fig: Serial dilution method
5. Special Methods
1. Single Cell Isolation methods: An individual cell is picked out by this method from the mixed culture and is allowed to grow to procure pure culture. There are two methods used for the isolation of single cells:
(i) Capillary pipette method: Several small drops of
a suitably diluted culture medium are placed on a sterile glass coverslip by a
sterile pipette. Examine then each drop under the microscope until find one
microorganism in one drop. This drop is then removed with a sterile capillary
pipette to a fresh medium for multiplication to yield a pure culture (Fig).
Fig: Capillary pipette method
(ii) Micromanipulator method: Micromanipulators
permit one to pick out a single cell from a mixed culture. This instrument is
used in conjunction with a microscope to pick a single cell (particularly a bacterial
cell) from a hanging drop preparation. The micromanipulator has micrometer
adjustments with a micropipette that can move in all directions. A series of
hanging drops of a diluted culture are placed on a special sterile coverslip by
a micropipette. Hanging drop is searched for a single microorganism cell. This
cell is drawn into the micropipette by gentle suction and then transferred to a
large drop of sterile medium on another sterile coverslip. When the number of
cells increases in that drop, the drop is transferred to a culture tube having a
suitable medium which yields a pure culture of the required microorganism.
Advantage: It can be reasonably sure that the cultures come from a single cell.
It can obtain strains within the species. Disadvantage: The equipment is
expensive and it requires a skilled operator.
2. Enrichment Culture Method: This method is used to isolate those microorganisms that are present in relatively small numbers or have slow growth rates in the mixed culture. The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and specific conditions of incubation favors the growth of desired microorganisms. This method is unsuitable for the growth of other types of microorganisms.