Isolation of

microorganisms
Some basic terms
 WHAT IS A CULTURE?
Population of microorganisms grown under well defined
conditions.
 Nutrients
 Temperature
 pH and Humidity
 Time ( Incubation period )
 WHAT IS MIXED CULTURE?
When a particular species of microbe is present in a very
small number in comparison to the total number of
microorganisms, such culture is called as mixed culture.
 WHAT IS PURE CULTURE?
A culture containing only one species of microbe is called
pure culture.
 Storage at low temperature
• Culture medium can be successfully stored in
refrigerators or cold rooms, when the
temperature is maintained at 4˚C.
• Another: liquid nitrogen can provide long term
preservation of culture. In this method, dense
suspension of microbes is prepared in a medium
containing a protective agent(Glycerol or
dimethyl sulfoxide) which prevent cell damage
due to ice crystal formation. Suspension is sealed
into small ampoules or vials and then frozen at -
150°c
• At this temperature range the metabolic
activities of microbes slows down greatly and
only small quantity of nutrients will be utilized.
• This method cannot be used for a very long time
because toxic products get accumulated which
can kill the microbes.
• 10-30 Years without changing the characteristics.
Cont.…
 Incubation:-
• It refers to the maintenance of microbial cultures at specific
temperature for a given time period.
• An incubator is a device used to grow and maintain microbial
cultures or cell cultures.
 PROCEDURE:-
• Allow the cultured plate in the incubator at 37°C for 24 hours
for growth.

• After 24 hours observe the growth of bacteria on cultured plate.

INCUBATOR
 Introduction

• Microorganisms are generally found in nature (air, soil and water) as

mixed populations.
• To study the specific role played by a specific microorganism in its
environment, one must isolate the same in pure culture.
 The two major steps of obtaining a pure culture are as follows :
• Firstly, the culture has to be diluted until the various individual
microorganisms are separated far apart on agar surface that after
incubation they form visible colonies isolated from the colonies of
other microorganisms.
• Secondly, an isolated colony has to be aseptically picked off the
isolation plate.
YEAST
Unicellular Ciliated Protozoan under the Microscope.
VIRUS STRUCTURE
VIRUS MICROSCOPIC STRUCTURE
 COMMON METHODS OF
ISOLATION OF MICROORGANISMS
Isolation is defined as separating completely &
obtaining in pure form of particular type of
microorganisms, separating it from its habitat.
Some common isolation methods are;
I. Streak plate method
II. Pour plate method
III. Serial Dilution technique
IV. Spread plate method
V. Micromanipulator method
 I. STREAKING OR
STREAK PLATE TECHNIQUE
• In this method, the tip of a fine
structure wire loop called
Inoculation needle consist of a
wooden or glass handle with a
Nichrome wire the end of which is
bend to form a loop is used to
transfer microbes from culture.

• The straight wires are similar to wire

loop except they do not have loop.
These are used to transfer culture in
colony formed on solid culture
medium.
 Points to be taken into consideration during the
inoculation are :
• The inoculation loop has to be sterilized before every inoculation
of colonies from the agar plate
• Every time the loop is sterilized by heat it must be cooled before
inoculating the next colony.
This technique consist of the following steps-
• A. Hold the broth culture containing tube in left hand and shake it.
• B. Sterilize the wire loop of the inoculation needle on burner flame .
• C. Remove the cotton plug of the broth culture tube by little finger of right hand.
• D. Flame the mouth of the test tube immediately.
• E. Insert the wire loop to form a thin film and replace the cotton plug.
• F. The thin film in the loop is streaked in either a zig-zag manner by removing the
loop backwards and forwards firmly. Care should be taken that loop should not
be firmly pressed against the agar surface.
• G. Incubate the petri dish in incubator at a required temperature.
• H. Growth of the bacteria will be visible (after an overnight incubation)on the
streaked marks.
 II) Pour plate method

• The bacterial culture and liquid agar medium

are mixed together.
• After mixing the medium, the medium
containing the culture poured into sterilized
petridishes (petriplates), allowed solidifying and
then incubated.
• After incubation colonies appear on the surface.
 Disadvantages of Pour plate method
1. The microorganisms are trapped beneath the
surface of medium when it solidifies. Hence,
as well as subsurface colonies are
surface
developed and it is very difficult to isolate and
count the subsurface colonies.
2. This method is tedious, time consuming and
requires skill.
3. The microorganisms are subjected to hot shock
because liquid medium is maintained at 45°C
temperature.
4. This method is unsuitable for isolation of
psychrophile bacteria.
PROCEDURE FOR SPREAD AND POUR
PALTE METHOD
 a) SERIAL DILUTION

 This method is commonly used to obtain pure cultures of those

microorganisms that have not yet been successfully cultivated on solid media
and grow only in liquid media.
 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.
• If we take out 1 ml of this medium and mix it with 9 ml of fresh
sterile liquid medium, we would then have 100 microorganisms in 10
ml or 10 microorganisms/ ml.
• If we add 1 ml of this suspension to another 9 ml. of fresh sterile
liquid medium, each ml would now contain a single microorganism.
• If this tube shows any microbial growth, there is a very high
probability that this growth has resulted from the introduction of a
single microorganism in the medium and represents the pure culture
of that microorganism.
III) Spread plate method
Advantages of spread plate method
1. It is a simple method.
2. In this method only surface colonies are
formed.
3. Micro-organisms are notexposed to
higher temperature.
 IV-MICROMANIPULATOR METHOD
Micromanipulators
• 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.

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
• Disadvantages are that the equipment is expensive,
• Its manipulation is very tedious, and it requires a skilled operator.
 STAINING TECHNIQUES
• Staining is a technique used to enhance contrast in
samples, generally at the microscopic level.
• If contrast is not sufficient in the sample or the
microscopic method used, staining can provide the
necessary contrast.
 Purpose of staining:
 For greater visualization of cells.
 For study of their structures
 To differentiate the cells
Cont….

• Stains : stains are the organic dyes used for staining the
microorganisms.
e.g. crystal violet, methylene blue etc.
• Stains used for viewing bacteria via light microscopy are
typically positively charged chromophores (basic dyes)
• Chromophore = “color-bearing” ion of a salt
• Bacteria have a net negative charge (i.e., bind positive
ions).
 TYPES:

 ACIDIC: Negatively charged acid radicals imparts color in

eosin,
acid fuchsine, malachite green, nigrosin, Indian
ink.

 BASIC: Positively charged basic radicals combines

with
negatively charged particles in cytoplasm and gives color.
Ex: Haematoxillin, methylene blue, crystal violet, gention violet.

 NEUTRAL: Both positively and negatively charged imparts

different colors to different components.
Ex: Geimsa’s stain, Leishman’s stain, Wright’s stain.
 STAINING METHODS:

POSITIVE STAINING: - where the actual cells are themselves colored and appear
in a clear background.

 (a) Simple staining: A stain which provides color contrast but gives same color
to all bacteria and cells.
Ex: Loeffler’s methylene blue, Polychrome methylene blue, Diluted carbol
fuchsin.

 (b) Differential Staining: A stain which imparts different colors to different

bacteria is called differential stain(which contains more than one stain).
Ex: Gram’s stain, Acid fast staining, Special stains.
 NEGATIVE STAINING:
where the cells remain clear (uncolored) and the background is colored to
create a contrast to aid in the better visualization of the image.

 (a) Indian ink

 (b) Nigrosin .
BACTERIAL SMEAR PREPARATION:

Smear - is a distribution of bacterial cells on a slide for the purpose of

viewing them under the microscope.

Method:
-Aseptically a small sample of the culture is spread over a slide surface.

-This is then allowed to air dry.

-The next step is heat fixation to help the cells adhere to the slide surface.

-The smear is now ready for staining.

 SMEAR FIXATION:

 1) Heat fixation
 a) Pass air-dried smears through a flame two or three times. Do not
overheat.

 b) Allow slide to cool before staining.

2) Methanol fixation
 a) Place air-dried smears in a coplin jar with methanol for one minute.
Alternatively, flood smear with methanol for 1 minute.

 b) Drain slides and allow to dry before staining.

Simple Staining procedure:
• Make a thin smear on a slide.
• Heat fixes the smear by passing the slide 2-3 times gently over the
Bunsen flame with the smear side up.
• Pour Loeffler’s Methylene blue over the smear and allow it to stand
for 3 minutes.
• Wash the stained smear with water and air dry it.
• Observe the smear first under low power (10X) objective, and then
under oil immersion (100X) objective.
• Observe the presence of organisms and also the cellular content of
sample.
SIMPLE STAINING: LOEFFLERS
METHYLENE BLUE
GRAM STAINING
 Gram staining is most widely used differential staining in Microbiology.

 Gram staining differentiates the bacteria into 2 groups:

 Gram positive.

 Gram negative.
 Gram Staining Procedure

Christian gram was firstly discovered the differential

staining to differentiate types of
techniques, bacteria.
 Gram-positive staining:
i. First take the bacteria & spread on a clean slide.
ii. This smear is dried by passing it over blue flame.
iii. Add crystal violet solu. & allow for 30-60 sec.
iv. Remove excess stain with water.
v. Then add iodine solu. & allow for 60 sec.
vi. Wash with water.
vii. Finally wash the slide with alcohol.
viii. Wash with water & observe under microscope.
Cont..
• Observation:
If stain appears a deep violet or purple black,
then they are called as gram-positive bacteria.
e.g. staphylococci, pneumococci etc.

 Gram-negative staining:
i. Staining procedure is similar to gram-positive.
ii. If bacteria do not retain crystal violet stain then it
is counter stained by safranin for 10 sec.
iii. Washed with water & observe the smear under
microscope.
• Observation : if pink or red colour produced then
those are gram-negative bacteria. e.g. E.coli,
S.typhi etc.
Ziehl Neelsons acid fast staining method
• Certain organisms are not easily stained by the usual
dyes probably due to the presence of a water
repellant outer layer or high lipid content , but when
stained with acid fast stain they may retain the
colour even washed with acid.

• Mycobacterium tuberculosis & Mycobacterium

leprae.

• Ziehl & Neelsons discovered this method for

separation of “Mycobacterium Group”.
Procedure…
• A smear is prepared.
• Then added Ziehl-Neelson carol fuchin stain
,for 10mint.
• Then heated & wash with tap water.
• Then add 20% H2SO4 for one mint & wash
with water.
• Then add methylene blue for 30 sec.
• Wash the slide & dry & observe under oil immersion
lens.

• OBSERVATION: cells appears pinkish red are acid

fast cells/bacteria & those appears blue/green are
nonacid fast bacteria.
 CAPSULE STAINING

• The capsules serves as protective material by slowing

down or preventing penetration of chemicals and
body juices.
Procedure:
 For positive staining of smears:
• Make a smear from colony of S.pneumoniae on a clean grease
free glass slide, and allow it to air dry.
• Flood the smear with Crystal Violet and allow it to stain for 5-7
minutes.
• Wash the smear with 20% copper sulphate solution and dry it.
• Observe the smear first under low power(10X) objective, and
then under oil immersion (100X) objective.
• In the culture smear, the capsule is seen as a light blue in contrast
to the deep purple color of the cell.
 For negative staining of smears:
• Take a clean grease free glass slide.
• Put a large loopful of undiluted Indian ink on the slide.
• Then add a small loopful of liquid bacterial culture to the
Indian ink and emulsify.
• Take a clean, grease free cover slip and place on the ink
drop and press it down, so that the flim becomes very thin
and thus pale in color.
• Observe the wet flim under high power (40X)
objective.
• The capsule in negative staining method is seen as clear
refractile around the organism against a black
 ENDOSPORE STAINING

• Spores are highly resistant inactive forms.

• The morphology of bacterial endospores is best
observed in unstained wet flims under the phase
contrast microscope, where they appear as large ,
refractile, oval or spherical bodies within the bacterial
mother cells or else free from the bacteria.
• Different staining are available for staining of spores.
PROCEDURE
• Flims are dried and fixed with minimal flaming.
• Place the slide over a beaker of boiling water, resting it on the
rim with the bacterial flim uppermost.
• When, within several seconds, large droplets have condensed
on the under side of slide, flood it with 5% aqueous solution
of Malachite green and leave it for 1 minute while the water
continues to boil.
• Wash in cold water.
• Treat with 0.5% safranin and 0.05% basic fuchsin for 30
seconds.
• Wash and dry.
• This method colors the spores green and vegetative
bacilli red.
 Diseases Produced by Bacteria
NAME OF BACTERIA DISEASES PRODUCED
Staphylococci Localised suppurative
lesions

Streptococci Rheumatic fever

E-coli Urinary tract infection,

Gastroenteritis

Clostridium tetani Tetanus

Bacillus anthracis Anthrax

Pneumoccoci
Pneumonia

Salmonella Typhoid

Vibriocholera
Cholera

Mycoplsma pneumonae Pneumonia Mycobacterium

leprae Leprosy