Isolation and Preservation Pure Culture Method

Isolation and preservation are processes used in microbiology to separate and maintain pure cultures of microorganisms.

1. ISOLATION AND
PRESERVATION
METHODS
By
Ms. Fand S. B.
Assistant Professor
Arihantcollege of Pharmacy, Ahmednagar
PHARMACEUTICAL MICROBIOLOGY
B
Pharm
SEM III

2. Contents
▪Introduction
▪Pure Culture Techniques
▪Preservation of Culture

3. Unit Outcomes
▪To understand different techniques for isolation of cultures
▪To know the importance of isolation as well as preservation.
▪To know different methods of preservation

4. Introduction
▪Microorganisms are generally found in nature (air, soil and water) as mixed populations.
▪Even the diseased parts of plants and animals contain a great number of microorganisms, which differ markedly from the
microorganisms of other environments.
▪To study the specific role played by a specific microorganism in its environment, one must isolate the same in pure culture.
▪Culture collection centres:
1.ATCC - American type culture collection, USA
2.NCIB - National collection of Industrial bacteria, Scotland
3.NCYC - National collection of Yeast cultures, England
4.NCTC - National collection of type cultures, England
5.NCL - National chemical laboratory, India

5. Introduction

6. Isolation of Pure Culture Techniques
•The isolation of one kind of microorganism from a mixture of many different kinds is called pure culture techniques
•Methods used for isolation of microorganism are as follows:

7. 1. STREAK PLATE METHOD
•Streak plate method is the most widely used method for isolation of culture.
•Streak plates are prepared by streaking a small amount of mixed culture over the surface
of the solid medium in a petri plate with platinum or nichrome wire loop.
•Purpose - steaking is to thin out the innoculum (starter culture) successively, so that
microbes get separated.
•Subculturing - transfer the well isolated colonies from streak plate to another new plate
for isolation and purification.

8. 2. POUR PLATE METHOD
•In this method, the mixed culture is diluted directly in tubes of liquid (cooled) agar
medium.
•The medium is maintained in a liquid state at a temperature of 45ºC to allow through
distribution of the inoculum.
•The inoculated medium is transferred into petri plate, allowed to solidify and incubated.
•A series of agar plates showing decreasing number of colonies resulting from the loop
dilution techniques.

9. Mix 1 ml dilute sample + 20ml liquid nutrient agar
medium at 45ºC.
Shake liquid nutrient agar medium and pour in sterile
petri plate, solidify & incubate it.
Pates gets well isolated colonies
Count total no of colonies and multiply by dilution
factor
Serial dilution technique:
2. Pour plate method

10. Advantages:
1.Difficult to count surface and subsurface colonies.
2.This method is tedious, time consuming and requires skill.
3.Microorganism are subjected to heat shock because liquid medium is maintained at 45 °C temperature.
4.This method is unsuitable for isolating psychrophile bacteria.
2. Pour plate method

11. 3. SPREAD PLATE METHOD
•In spread plate technique, the mixed culture is not diluted in the culture medium.
•It is diluted in a series of tube containing sterile water and saline solution.
•A sample is removed from each dilution tube (0.01ml) 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 plate are incubated and the isolated colonies are observed after 24 hour and counted.

12. ▪Advantages:
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 nt exposed to higher temperature.
3. Spread plate method

13. 4. MICROMANIPULATOR METHOD
•Micromanipulators have been built. Which permit one to pick out a single cell from a mixed culture.
•This instrument is used in conjugation with a microscope to pick a single cell (particularly bacterial cell) from
a hanging drop preparation.
•The single microbial cell is gently sucked into the micropipette and transferred on to a large drop of sterile
medium on another coverslip.

14. Advantages
▪The advantages of this method are that one
can be reasonably sure that the cultures
comes from a single cell and one can obtain
strains with in the species.
Disadvantages
▪This equipment is expensive.
▪Its manipulation is very tedious and it
requires a skilled operator.
4. Micromanipulator method

15. 5. 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
preproduced 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.
•Inoculations is done with a transfer loop held against the agar surface as the tube is being
rotated by a motor.

16. Preservation of Cultures
▪Once a microorganism has been isolated and grown in pure culture, it becomes necessary to
maintain the viability and purity of the microorganism by keeping the pure cultures free
from contamination.
▪Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh
medium (subculturing) to allow continuous growth and viability of microorganisms.
▪The transfer is always subject to aseptic conditions to avoid contamination.

17. Objectives of Preservation
▪To maintain isolated pure cultures for extended periods (future use) in a visible conditions.
▪To avoid the contamination.
▪To restrict genetic change (Mutation)

18. Applications of Preservation
▪Academic use
▪Research Purpose
▪Fermentation Industry
▪Biotechnological field
1.Once a microorganism has been isolated and grown in pure culture, it becomes necessary to
maintain the viability and purity of the microorganism by keeping the pure culture free from
contamination.
2.Normally in laboratories, the pure cultures are transferred periodically onto or into a fresh
medium (sub-culturing) to allow continuous growth and viability of microorganisms.

19. Applications of Preservation
3.The transfer is always subject to aseptic conditions to avoid contamination. Since repeated
sub-culturing is time consuming,
4.It becomes difficult to maintain a large number of pure cultures successfully for a long time.
5.In addition, there is a risk of genetic changes as well as contamination.
6.Therefore, it is now being replaced by some modern methods that do not need frequent sub-
culturing.
7.These methods include refrigeration, paraffin method, cryopreservation, and lyophilization
(freeze drying)

20. Methods of Preservation
1.Periodic transfer to fresh media (Sub culturing)
2.Storage in sterile soil
3.Storage at low temperature
4.Preservation by overlaying culture with mineral oil
5.Freeze drying/ Lyophilization

21. 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.

22. 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.
•This method is only used for short Preservation of cultures and subculturing is necessary if the period exceeds
four week.
•In another very low temperature method, liquid nitrogen has provided long-term preservation of cultures.

23. 2. STORAGE AT LOW TEMPERATURE
•Microorganisms are prepared as a deitse suspension in a medium containing a protective agent (glycerol or
dimethyl sulfoxide), which prevents cell damage microorganism due to ice crystal formation.
•Then liquid nitrogen method has been successfully used for many species and cells remain viable for 10 10 30
years without changing their characteristics

24. 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.

25. 4. PRESERVATION BY OVERLYING 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 inoculate 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.

26. 5. LYOPHILIZATION / FREEZE DRYING
•Freeze-drying is a process where water and other solvents are removed from a frozen product via sublimation.
•Sublimation occurs when a frozen liquid goes directly to a gaseous state without entering a liquid phase.
•It is recommended using slow rates of cooling, as this will result in the formation of vertical ice crystal
structures, thus allowing for more efficient water sublimation for the frozen product.

27. ▪Procedure:
✓In this process, a dense cell suspension is placed in small vials and frozen at-60 to 70°C.
✓The vial are immediately connected to a high vacuum line.
✓The ice present in the frozen suspension evaporates (sublime) under the vacuum.
✓This results in dehydration of bacterial cell and their metabolic activities are stopped; as a result, the
microbes go into dormant state and retain viability for years.
✓The vials are then sealed off under a vacuum and stored in the dark at 4°C in refrigerators.
✓Only minimal storage space is required; hundreds of lyophilized cultures can be stored in a small area.
5. Lyophilization / freeze drying

28. ▪Advantages:
✓Remained viable for more than 30 years.
✓Frequent sub-culturing is not required.
✓Maintained without contamination Lyophilized strains remains genetically stable.
✓Small vials can be sent conveniently through the mail to other microbiology laboratories when
packaged in a special sealed mailing containers.
✓This method is employed for the preservation of sera, toxin, enzymes and other biologicals.
5. Lyophilization / freeze drying