Methods for isolation of pure culture

1. METHODS FOR ISOLATION OF
PURE CULTURE
DR. HARINATHA REDDY
Assistant professor

2. • Pure culture also known as axenic culture contain single
species.
• Pure culture technique develop by Robert Koch.

3. • A pure culture theoretically contains a single bacterial species.
• Simpler methods for isolation of a pure culture include:
1. Streak Plate Method:
2. Pour Plate Method:
3. Spread Plate Method:
4. Spread Plate Method:
• The purpose of spread plating and streak plating is to isolate
individual bacterial cells (colony-forming units) on a nutrient
medium.
• A pure culture may be isolated by the use of special media
with specific chemical or physical agents that allow the
enrichment or selection of one organism over another.

4. Streak Plate Method:
• This method is used most commonly to isolate pure cultures of bacteria.
• 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.
• The successive streaks “thin out” the inoculum and the micro-organisms
are separated from each other.
• These plates are incubated to allow the growth of colonies.
• The each separated bacteria form colony after inoculation.

5. Pour Plate Method:
• This method involves bacterial sample are mixed with melted agar
medium.
• Here, the mixed culture of bacteria is diluted directly in tubes
containing melted agar medium maintained in the liquid state at a
temperature of 42-45°C (agar solidifies below 42°C). The bacteria
and the melted medium are mixed well.
• After the agar has hardened, each cell is fixed in place and forms
an individual colony.
• When bacterial colonies develop, one finds that isolated colonies
develop both within the agar medium (subsurface colonies) and on
the medium (surface colonies).
• Plates containing between 30 and 300 colonies are counted. The
total number of colonies equals the number of viable
microorganisms in the diluted sample.

6. • Pour plate method has certain disadvantages as follows:
• (i) The picking up of subsurface colonies needs digging them out of the agar
medium thus interfering with other colonies.
• (ii) The microbes being isolated must be able to withstand temporary
exposure to the 42-45° temperature of the liquid agar medium; therefore this
technique proves unsuitable for the isolation of psychrophilic
microorganisms.

7. Spread Plate Method:
• In this method the mixed culture or microorganisms is not
diluted in the melted agar medium (unlike the pour plate
method); it is rather diluted in a series of tubes containing
sterile liquid, usually, water or physiological saline.
• A drop of so diluted liquid from each tube is placed on the
center of an agar plate and spread evenly over the surface by
means of a sterilized bent-glass-rod. The medium is now
incubated.
• The each separated bacteria forms separate colonies after
inoculation.

8. 4. Serial Dilution Method:
• Serial dilution technique developed by Joseph lister.
• It is one of isolation technique in which specific
microorganism isolated form mixed culture by serial dilution
technique.
• The first serial diluted bacteria is Lactobacillus lactis by
Joseph lister.

9. :
• 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.

10. USE OF SELECTIVEAND DIFFERENTIAL MEDIA:
• Many different types of media are used in the microbiology
laboratory.
• In general, these media can be divided into several
categories, including Natural, (Nutritive), Differential, and
Selective Media.
• Natural media: are defined as media types that support the
growth of a wide range of microorganisms.
• These media considered nonselective due to the fact that they
will grow most organisms.
• Examples of nutritive media include Tryptic soy agar, Nutrient
agar media.

11. Beef Extract……………………..…………..3.0 g
Peptone……..…………………….…………5.0 g
Agar………………………………………….15.0 g
Distilled Water………………………………….……1000 ml
Final pH 7.0 +/- 0.2.
Composition of Nutrient Broth: Nutrient broth contains
same ingredients except agar
Composition of Nutrient Agar:
Beef extract is an aqueous extract of beef tissues. It contains water-soluble
substances carbohydrates, organic nitrogen compounds, water soluble
vitamins, and salts.
Peptone is made by digesting proteinaceous materials e.g., meat, casein,
gelatin, using acids or enzymes.
Peptone is the principal source of organic nitrogen and may contain
carbohydrates or vitamins.

12. • Agar is a complex carbohydrate obtained from certain marine algae
(Gelidium sp).
• It is used as a solidifying agent for media and does not have any
nutritive value.
• Agarose is a linear polymer, made up of repeating units of agarobiose.
• Agar exhibits melting at 85 °C and solidifying from 32–40 °C.
• Solid medium contain 1.5 to 2 % solidifying agent.
• Semi solid medium contain 0.5% solidifying agent.
• Liquid medium also known as broth the solidifying agent is absent.

13. Enriched Medium:
• This nutritive medium may be enriched, i.e., by the addition of blood or
serum.
• Examples: sheep blood agar media and chocolate (heated blood) agar
media.
• Blood agar media is a type of growth medium (enriched with 5% sheep
blood) that encourages the growth of bacteria, such as streptococci,
that otherwise wouldn’t grow.
• Blood contains inhibitors for certain bacteria such as Neisseria and
Haemophilus genera and the blood agar must be heated to inactivate
these inhibitors and to release essential growth factors.
• Heating of blood agar converts it into chocolate agar (heated blood turns
a chocolate color) and supports the growth of these bacteria.

14. 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.
• Selective medium types include Eosin Methylene Blue agar
(EMB) and Mannitol Salt agar media.

15. Eosin Methylene Blue (EMB) agar:
• Eosin Methylene Blue Agar is a both selective and differential
culture medium. It is selective culture medium for gram-negative
bacteria (selects against gram positive bacteria) and is commonly
used for the isolation and differentiation of coliforms.
• 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. Rapid fermentation of
lactose & production of strong acids, thus rapid reduction in the
pH of the EMB agar the critical factor in the formation of the green
metallic shine observed with E.coli
• Other bacteria form dark purple.

16. 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 ).
• It is also a differential medium for mannitol-fermenting
staphylococci, containing carbohydrate mannitol and the indicator
phenol red, a pH indicator for detecting acid produced by mannitol-
fermenting staphylococci.
• Staphylococcus aureus produces yellow colonies with yellow
zones, whereas other staphylococci produce small pink or red
colonies with no colour change to the medium.

17. Differential medium:
• Organisms with differing growth characteristics typically show
visible differences in growth when placed on differential media.
Examples: MacConkey agar.
• MacConkey agar is a selective and differential media used for
the isolation and differentiation of gram-negative rods,
particularly members of the family Enterobacteriaceae.

18. • Peptone provide the essential nutrients, vitamins and
nitrogenous factors required for growth of
microorganisms.
• Lactose monohydrate is the fermentable source of
carbohydrate.
• The selective action of this medium is by crystal
violet and bile salts, which are inhibitory to most species
of gram-positive bacteria.
• Sodium chloride maintains the osmotic balance in the
medium.
• Neutral red is a pH indicator that turns red at a pH below
6.8 and is colorless at any pH greater than 6.8. .

19.  Lactose fermenting strains grow as red or pink.
 The red colour is due to production of acid from lactose,
and colour change of the dye when the pH of medium
falls below 6.8.
• Lactose non-fermenting strains, such as Shigella and
Salmonella are colourless and transparent and typically
do not alter appearance of the medium.

20. Thank you