Isolation And Preservation Methods 5 Best

Isolation And Preservation Methods

INTRODUCTION:-

Microorganisms in nature are never found in pure populations. Different types of
mixed microorganisms are normally present in soil, water, food, air and various parts of the human or
animal body. To study the role played by a specific microorganisms in these environments, it is
necessary to isolate the microorganisms in pure culture.

Isolation And Preservation Methods

It is extremely important to maintain isolated
pure cultures for extended periods in a viable condition.

Most microbiological laboratories usually maintain a large collection of pure cultures as well as subcultures of authentic species purchased from
various culture collection centres.

Isolation And Preservation Methods

Isolation And Preservation Methods

E.g. American Type Culture Collection (ATCC), U.S.A; National
Collection of Industrial Bacteria (NCIB), Scotland; National Collection of Yeast Cultures (NCYC), England;
National Collection of Type Cultures (NCTC), England; National Chemical Laboratory (NCL), India etc.

PURE CULTURE TECHNIQUES:-
A pure culture consists of a population of only one species of microorganisms. The isolation of one kind
of microorganism from a mixture of many different kinds is called the pure culture technique. The
methods widely used for isolation of microorganisms are as follows:

Isolation And Preservation Methods

(i)Streak plate method
(ii)Pour plate method
(a) Loop dilution technique
(b) Serial dilution technique
(iii) Spread plate method

(iv)Micromanipulator method
(V) Roll tube method.

(i) Streak plate method: Streak plate method is the most widely used method for isolation of cultures.
Streak plates are prepared by streaking a small amount of mixed culture over the surface of the solid
medium in a Petri plate with a platinum or nichrome wire loop (Refer Fig. 16 of Appendix I). The sample
is streaked in such a way as to provide successive dilution (diagram).

The purpose of streaking is to thin out the innoculum (starter culture) successively so that microbes get
separated. A second plate may also be streaked from the same loop/needle without reinnoculation. In
the beginning of the streak, microbes are crowded and colonies develop closely but as the streaking
proceeds cells gets separated as the needle contains less cells.

Hence at the last streak, few and clearly
separated colonies are developed. Transfer the well isolated colonies from streak plate to another new
plate for isolation and purification (sub culturing).

(ii) Pour plate method: In this method, the mixed culture is diluted directly in tubes of liquid (cooled)
agar medium (Diagram )). The medium is maintained in a liquid state at a temperature of 45°C to allow
thorough distribution of the inoculum. The inoculated medium is transferred into Petri plates, allowed
to solidify and incubated.

A series of agar plates showing decreasing number of colonies resulting from
the loop dilution technique is shown in Diagram .

In the serial dilution technique, the original innoculum may be diluted by using sterile water or a saline
solution (by using pipette) so that the concentration of the microbes gradually becomes less. Mix 1 ml
dilute sample in a 20 ml liquid nutrient agar medium at 45°C. Shake the liquid nutrient agar medium and
pour in a sterile Petri plate, solidify and incubate it.

The plate gets well isolated colonies and then counts
the total number of colonies in the sample, multiply the number of colonies by dilution factor.

Disadvantages of pour plate techniques:

(i) The microorganisms are trapped beneath the surface of the medium when it solidifies.
Hence, surface as well as subsurface colonies are developed and it is very Difficult to isolate
and count the subsurface colonies.
(ii) This method is tedious, time consuming and requires skill.
(iii)The microorganisms are subjected to heat shock because liquid medium is maintained at 45°C
temperature.
(iv)This method is unsuitable for isolating psychrophile bacteria.

(iii)Spread plate method: In spread plate technique, the mixed culture is not diluted in the culture
medium. It is diluted in a series of tubes containing sterile water or a saline solution. A sample is
removed from each dilution tube (0.1 ml) and placed onto the surface of an agar plate. The culture is
spread by using a glass spreader on the surface of the agar plate. The plates are incubated and the
isolated colonies are observed after 24 hours and counted (diagram).

Advantages of spread plate method:
1. It is a simple method.
2. In this method, only surface colonies are formed.
3. This method is also used for counting the microorganisms present in the inoculum.
4.Microorganisms are not exposed to higher temperature.

(iv) Micromanipulator method:
Micromanipulators are devices that can pick up a single microbial cell from a colony of mixed culture.
Micromanipulators are used in conjunction with microscopes to pick up a single bacterial cell from
hanging drop preparations. The single microbial cell is gently sucked into the micropipette and
transferred on to a large drop of sterile medium on another coverslip.

Micromanipulator method makes
one reasonably sure of the pure culture coming from a single cell. The device however has to be used
with skill and precision.

(v) Roll tube method:
Roll tube method is used for the isolation of stringent anaerobes. A stoppered anaerobic culture tube is
used for isolation which has been coated with a prereduced agar medium containing oxygen free
nitrogen. When the stopper is removed the tube is kept anaerobic by continuously flushing it with
oxygen free CO₂ from a gas cannula. Inoculation is done with a transfer loop held against the agar
surface as the tube is being rotated by a motor. Inoculation starts from the bottom and draws the loop
gradually upward (DIAGRAM). After inoculation the tube is restoppered and incubated anaerobically to get
well isolated colonies.

PRESERVATION OF CULTURES
Microbial species are isolated and characterized by microbiologists and deposited in culture collection
centres. The cultures are maintained in viable condition and are referred to as the stock-culture
collection. To maintain an isolated pure culture for extended periods in a viable condition, without any
genetic change is referred to as preservation. Different types of cultures are preserved in laboratories by
avoiding contamination as well as genetic changes (mutations). The various objectives of preservation
are as follows:

Academic use: In microbiology subject, microbes are studied along with their practical applications.
Microorganisms are needed for laboratory classes, to study morphology, basic staining techniques,
isolation, counting, biochemical reactions, reference strains for taxonomic studies etc. For the
fulfillment of such academic requirements, microorganisms are preserved in laboratories.

Fermentation industry: The fermentation industry is engaged in production of various compounds,
produced by several microorganisms e.g. antibiotics, enzymes, vitamins, amino acids, vaccines, antisera
etc. For the process of preparation of the above compounds, an innoculum (starter culture) is required;
hence the fermentation industry must maintain microbial cultures.

Biotechnology field: Novel microorganisms are produced with the help of biotechnology e.g. insulin
producing Escherichia coli. Such types of genetically modified microorganisms are not found in the
environment. Hence, it is preserved for their biotechnological applications.

Research purpose: Many microorganisms are used for the study of byproducts produced from them.
Some microorganisms produce infections (pathogenic) in human beings and are used for vaccine
production. Some microorganisms are genetically modified and studied for different pharmaceutical
applications. Many microbes have the potential to degrade pollutants.

These and several related fields
of research need stock cultures of microorganisms, which are preserved for their important uses.
A number of methods are used for maintaining microorganisms in a viable condition over long periods:

1. Periodic transfer to fresh media.
2. Storage at low temperature.
3. Storage in sterile soil.
4. Preservation by overlaying cultures with mineral oil.
5. Lyophilization or freeze drying.

1. Periodic transfer to fresh media:
In all microbiology laboratories, microbes are preserved on agar slants. The slants are incubated for 24
hours or more and then stored in refrigerators. These cultures are periodically transferred to fresh
media. The time interval at which the transfers are made varies with the microorganisms and the
conditions of growth. It is a simple method and any special apparatus is not required. But risk of
contamination is more which may change the genetic and biochemical characteristics of the cultures.

Isolation And Preservation Methods

2. Storage at low temperature:
Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms when the
temperature is maintained at 4°C. This method is only used for short time preservation of cultures and
subculturing is necessary if the period exceeds four weeks.

Isolation And Preservation Methods

In another very low temperature method, liquid nitrogen has provided long-term preservation of
cultures. Microorganisms are prepared as a dense suspension in a medium containing a protective agent
(glycerol or dimethyl sulfoxide), which prevents cell damage due to ice crystal formation. The cell
suspension is sealed into small ampoules or vials and then frozen at a controlled rate to -150°C. The
ampoules or vials are then stored in a liquid nitrogen refrigerator (- 196°C). The liquid nitrogen method
has been successfully used for many species and cells remain viable for 10 to 30 years without changing
their characteristics.

3.Storage in sterile soil:
This method is mainly applied for the preservation of sporulating microorganisms e.g. Bacillus,
Streptomyces, Aspergillus, Penicillium species etc. Pure culture (spores) of microorganisms are kept in a
sterile soil medium and preserved for a number of months under refrigeration.

4.Preservation by overlaying cultures with mineral oil:
Many bacterial species can be preserved by covering their growth on the agar slant with sterile mineral
oil or liquid paraffin. The oil must cover the slant completely. In this method, we can remove some of
the growth under the oil with a transfer needle and innoculate it in a fresh medium by preserving the
original culture. It is a simple method and is mainly used for anaerobic microorganisms. This is cost
effective method of preserving cultures of bacteria and fungi for 15 to 20 years but changes in
characteristics still occur.

5.Lyophilization or freeze drying:
Freeze drying can preserve different types of microorganisms that would be killed by ordinary drying. In
this process, a dense cell suspension is placed in small vials and frozen at -60 to -78°C. The vials are
immediately connected to a high vacuum line. The ice present in the frozen suspension evaporates
(sublimes) under the vacuum. This results in dehydration of bacteria with a minimum of damage to
delicate cell structures (Fig. 4.5). The vials are then sealed off under a vacuum and stored in a
refrigerator.

This method has many advantages:
(i)Cultures preserved by freeze drying method have remained viable for more than 30 years.
(ii)Sub-culturing is not required and the culture can be maintained without contamination.
(iii) The lyophilised strain remains genetically stable.
(iv)Minimal storage space is required. Many lyophilised cultures can be stored in a
Small area.
(v) The small vials can be easily sent to other microbiology laboratories through
The mail.
(vi) Lyophilized cultures are easily revived by opening of vials and transferring of
The rehyderated culture to a suitable growth medium.
(vii) This method is also employed for the preservation of sera, toxins, enzymes and other biologicals.

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

Isolation And Preservation Methods

 

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