The document discusses various methods for isolating pure cultures of microbes and preserving microbial cultures. It describes four common isolation methods: streak plate technique, micromanipulator method, enrichment culture method, and serial dilution method. It also discusses four preservation methods: refrigeration, the paraffin method, cryopreservation, and lyophilization. The key isolation methods allow separating one type of microbe from a mixture to obtain a pure culture, while the preservation methods are used to maintain the viability and purity of isolated microbial cultures.

1. Culture Management:
 By
 Harinatha Reddy
 Department of Biotechnology
 biohari14@gmail.com

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3. Isolation of microbes:
 The term isolation refers to the separation of a strain from a
natural, mixed population of living microbes, as present in the
environment.

4. COMMON METHODS OF ISOLATION OF PURE
CULTURE
I. ISOLATIONBY
STREAKPLATE
II. MICRO
MANIPULATOR
III.ENRICHMENT
CULTURE
METHOD
IV .SERIALDILUTION
METHOD
TECHNIQUE ISOLATION
METHOD
METHODS
The process of screening a pure culture by separating one type of
microbes from a mixture is called Isolation.
Some common isolation methods are:

5. I. ISOLATION BY STREAKING OR STREAK PLATE
TECHNIQUE:
 This method is used most commonly to isolate pure cultures
of bacteria.
 In such cases,the colony from solid medium is streaked on the
surface of nutrient agar medium in a sterile petridish.

6. II-MICROMANIPULATOR METHOD:
• Micromanipulators have been built, which 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 bacterial cell) from a hanging drop
preparation.

7. ADVANTAGES OF MICROMANIPULATOR METHOD:
• The advantages of this method are that one can be reasonably sure
that the cultures come from a single cell and one can obtain strains
with in the species.
DISADVANTAGES:
• The disadvantages are that the equipment is expensive.
• It requires a skilled operator.

8. 3. Enrichment culture method:
 Enriched media are prepared by addition of specific growth
substances such as blood, serum and egg to a basal medium.
 Important examples of enriched media are:
 Blood agar for isolation of Streptococcus,
 Chocolate agar for isolation of Neisseria and Haemophilus

9. Chocolate agar media:
 Blood contains inhibitors for certain bacteria such as
Neisseria and Haemophilus genera and the blood agar must
be heated to inactivate these inhibitors.

10. Selective media:
 Selective medium types are formulated to support the growth
of one group of organisms, but inhibit the growth of another.
 These media contain antimicrobials, dyes, or alcohol to
inhibit the growth of the unwanted microorganisms.

11. Eosin Methylene Blue (EMB) agar:
 The combination of the two dyes eosin and methylene blue inhibits
most Gram positive bacteria but allows many Gram negative
organisms to grow.
 In EMB agar, most of the strains of E.coli colonies have a
characteristic green metalic shine.
 Other bacteria form dark purple.

12. Mannitol Salt agar media:
 It is selective media for Staphylococcus aureus bacteria.
 It contains a high concentration (about 7.5%-10%) of salt
(NaCl), making it selective for Gram-positive bacteria
(Staphylococcus ).

13. 4. Serial Dilution Method:
 This method is commonly used to obtain pure cultures of
microorganisms.
 Best method isolation of microorganisms form soil and water samples.
 The inoculum is subjected to serial dilution in a sterile liquid
medium.

14. Preservation of microbes.:
 Once a microorganism has been isolated and grown in pure
culture, it becomes necessary to maintain the viability and purity
of the microorganism.
 Normally in laboratories, the pure cultures are transferred
periodically into a fresh medium (subculturing).

15. Preservation methods:
These methods include:
1. Refrigeration,
2. Paraffin Method
3. Cryopreservation,
4. Lyophilization (freeze drying).

16. 1. REFRIGERATION:
• Pure cultures can be successfully stored at 0-4°C either in
refrigerators or in cold-rooms.
• This method is applied for short duration (2-3 weeks for bacteria and
3-4 months for fungi) because the metabolic activities of the
microbes are greatly slowed down but not stopped.

17. 2. Paraffin Method:
 This is a simple method of maintaining pure cultures of
bacteria and fungi.
 In this method, sterile liquid paraffin in poured over the slant
(slope) of culture and stored at room temperature.
 This condition helps microorganisms preserved for several
years.

18. Cryopreservation:
• In this method, the microorganisms of culture are rapidly frozen
in liquid nitrogen at -196°C in the presence of stabilizing agents
such as glycerol.
• Glycerol prevent the formation of ice crystals and promote cell
survival.

19. Lyophilization (Freeze-Drying):
• In this method, the culture is rapidly frozen at a very low
temperature (-70°C) and then dehydrated by vacuum.
• Under these conditions, the microbial cells are dehydrated and their
metabolic activities are stopped;
• as a result, the microbes go into dormant state and retain viability
for years.

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