identification and isolation of pure culture of microorganism and its preservation

1. MODERN COLLEGE OF PHARMACY
MOSHI, PUNE
Savitribai Phule Pune University
B.PHARM
Semester- III
By
Mrs. Sneha K. Patil
Assistant Professor
Dept. of Pharmaceutics
Isolation and
preservation methods

2. UNIT OUTCOMES
1
• To understand different techniques for isolation of
cultures
• To know the importance of isolation as well as
preservation
• To know different methods of preservation

3. CONTENT
2
1. Introduction
2. Isolation of pure culture techniques
3. Preservation of cultures
4. References

4. 1. Introduction
 Culture - act of cultivating microorganisms or the microorganisms
that are cultivated
 Pure culture – It consists of only one species of microorganisms
 Mixed culture – A culture contains more than one species of
microorganism
 Culture collection centres
e.g. 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
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5. 2. 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
(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
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6. (i) Streak plate method
 Sterilize the inoculating needle by flame to make red hot and
allow it to cool for 15 seconds.
 Streaking small amount of mixed culture over surface of the solid
medium in a petri plate with nichrome wire loop
 Then plates are incubated at specific temeperature and time and
observed the colonies on streak marks.
 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
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7. Streak plate technique continue….
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Fig. 1 streaking pattern

8. 2. Pour plate method
a. Loop dilution technique
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Mixed culture is diluted directly in tubes of liquid (cooled) agar medium
Medium is maintained at temp. of 45 C to allow thorough distribution of
inoculum
Inoculated medium is transferred into petri plate plates, allowed to solidify
and incubated
A series of agar plates shows decrease in number of colonies

9. 8
Fig. 2 Pour plate method – loop dilution

10. Pour plate method continue……
b. Serial dilution technique – Original inoculum may be diluted by
using sterile water or a sterile solution
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Mix 1 ml dilute sample + 20 ml liquid nutrient agar medium at 45 C
Shake liquid nutrient agar medium and pour in a sterile petriplate,
solidify and incubate it
Plates gets well isolated colonies
Count total number of colonies and multiply by dilution fator

11. Pour plate method continue……
 Disadvantages
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
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12. 3. Spread plate method
In this method, mixed culture is not diluted in the culture medium
but it is diluted in a series of tubes containing sterile water or a
saline solution
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Fig. 3 spread plate method

13. Spread plate method continue……
 Advantages
1. It is simple method
2. Only surface colonies are formed
3. This method is also used for counting the microbes present in
the inoculum
4. Microbes are not exposed to higher temperature
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14. 4. Micromanipulator method
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 It is devices that can pick a single microbial
cell from a colony of mixed culture
 They are used in conjunction with microscope to
pick single 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
 It reasonably sure of the pure culture coming from a single cell
 This device used with skill and precision

15. 5. Roll tube method
 It is used for isolation of stringent anaerobes
 A stoppered anaerobic culture tube
used for isolation and coated with
prereduced agar medium containing
oxygen free nitrogen
 When stopper is removed the tube is
kept anaerobic by continuously
flushing it with oxygen free Co2 from
gas canula
 Tube is rotated by motor and
inoculation starts from bottom to
upward with loop
 Tube is restoppered, incubate
anaerobically to get well isolated
colonies Fig. 4 Roll tube method 14

16. 3. Preservation of cultures
 Stock culture collection- cultures are maintained in viable condition
 Preservation – To maintain an isolated pure culture for extended
periods in a viable condition, without any genetic change
Objectives of preservation
1. Academic use
2. Fermentation industry
3. Biotechnology field
4. Research purpose
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17.  Preservation methods
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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. Lyophilisation/ freeze drying

18. 17
1. Periodic transfer to fresh Media
 Microorganism are preserved on agar slants and incubated for 24
hours or more and then stored in refrigerators
 These cultures are periodically transferred to fresh media
 Time interval at which the transfers are made varies with the
microbes and growth condition
 Advantages - it is a simple method and any special apparatus are
not required
 Disadvantage- failing to prevent changes in the characteristics of a
strain due to development of variants and mutants and risk of
contamination is also more in this process

19. 2. Storage at low temperature
 Culture medium can be successfully stored in refrigerators or cold
rooms, when the temperature is maintained at 4˚C
 At this temperature range the metabolic activities of microbes
slows down greatly and only small quantity of nutrients will be
utilized
 Useful for short time preservation and subculturing is necessary, if
the period exceeds four weeks
 Liquid nitrogen has provides long term preservation of cultures
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20. Liquid nitrogen method
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Microbes are prepared in dense suspension
medium containing protective agents (glycerol,
DMSO)
Cell suspension sealed into small ampoules/vials
and frozen at controlled rate to - 150 C
Ampoules/ vials are then stored in a liquid
nitrogen (- 196 C)
 Cells are remain viable for 10 to 30 years without
changing characteristics

21. 3. Storage in sterile soil
 It is mainly applied for the preservation of sporulating
microorganisms Fusarium, Penicillium, Alternaria, Rhizopus etc.
proved successful for store in sterile soil
 Soil storage involves inoculation of 1ml of spore suspension into
soil (autoclaved twice) and incubating at room temperature for 5-
10 days
 The initial growth period allows the fungus to use the available
moisture and gradually to become dormant
 The bottles are then stored at refrigerator
 Viability of organisms found around 70-80 years
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22. 4. Preservation by overlaying cultures with mineral oil
 In this method sterile liquid paraffin is poured over the slant
culture of microbes and stored upright at room temperature
 Where as cultures can also be maintained by covering agar slants
by sterile mineral oil which is stored at room temperature or
preferably at 0-5°C
 Remove some of the growth under oil with a transfer needle and
inoculate it in a fresh medium by preserving original culture
 It limit the oxygen access that reduces the microorganism’s
metabolism and growth, as well as to cell drying during
preservation
 The preservation period for bacteria from the genera Azotobacter
and Mycobacterium is from 7-10 years, for Bacillus it is 8-12 years
 It is simple method and mainly use for anaerobic microorganisms
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23. 5. Lyophilisation/ freeze drying
 Freeze drying can preserve different types of microorganism that
would be killed by ordinary drying
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A dense cell suspension is placed in small vials and frozen at
- 60 to - 78C and connected to high vacuum line
Ice presents in the frozen suspension evaporates (sublimes) under vaccum
Vials sealed of under a vaccum and stored in refrigerator

24. 23
Fig. 5 Freeze drying of cultures

25. Lyophilization continue….
 Advantages
1. Viability of culture for more than 30 years
2. Sub culturing is not required and culture can be maintained
without contamination
3. Lyophilised strain remains genetically stable
4. Minimal storage space is required
5. Easy for transport because of small vials
6. Cultures are easily revived by opening of vials and transferring of
rehydrated culture to a suitable medium
7. This method is employed for preservation of sera, toxins, enzymes
and other biologicals
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26. 5. References
 Kokare C. Pharmaceutical microbiology. 2nd ed. Pune: Nirali
prakashan; 2019. Pg. no. 4.1 -4.8
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27. Thank You