AGAR PREPARATION:

 In a beaker, 28 grams of the dehydrated powder or lab-prepared

media is added to 1000 milliliters of distilled or deionized water.
 The suspension is then heated to boiling to dissolve the medium
completely.
 The dissolved medium is then autoclaved at 15 lbs pressure (121°C)
for 15 minutes.
 Once the autoclaving process is complete, the beaker is taken out
and cooled to a temperature of about 40-45°C.
 If enrichment is desired, the addition of blood or biological fluids can
be done after the autoclaving process.
 The media is then poured into sterile Petri plates under sterile
conditions.
 Once the media solidifies, the plates can be placed in the hot air oven
at a lower heat setting for a few minutes to remove any moisture
present on the plates before use.

STORAGE OF NUTRIENT AGAR:

The media in the powder form should be stored between 10 to 30°C in a

tightly closed container, and the prepared medium should be stored at 20-
30°C.
 After opening, the product should be appropriately stored when dry,
after tightly capping the bottle in order to prevent lump formation as
the medium is hygroscopic in nature and thus, absorbs moisture
relatively quickly.
 The container should be stored in a dry ventilated area protected
from extremes of temperature and sources of ignition.
 The product should be used before the expiry date on the label.
USES OF AGAR:
 The container should be stored in a dry ventilated area protected
from extremes of temperature and sources of ignition.
 The product should be used before the expiry date on the label.
 Nutrient agar is often used for the demonstration and teaching
purposes as it doesn‟t contain harmful substances and can be used
for the isolation of multiple microorganisms.
 The use of Nutrient agar is recommended by standard methods as it
has a simple composition which can even be prepared within a
laboratory.
 It is one of the most common media used for the enumeration of
bacteria from environmental samples.
 The addition of biological fluids like horse or sheep blood, serum, egg
yolk, etc. to the nutrient agar makes the medium more selective for
certain fastidious organisms.

STREAK PLATE METHOD:

In microbiology, streaking is a technique used to isolate a pure strain from
a single species of microorganism, often bacteria.
 The dilution or isolation by streaking method was first developed by
Loeffler and Gaffky in Koch‟s laboratory, which involves the dilution of
bacteria by systematically streaking them over the exterior of the agar
in a Petri dish to obtain isolated colonies which will then grow into the
number of cells or isolated colonies.
 Streaking is rapid and ideally a simple process of isolation dilution.
 The technique is done by diluting a comparatively large concentration
of bacteria to a smaller concentration.
 The decrease of bacteria should show that colonies are sufficiently
spread apart to effect the separation of the different types
of microbes.
 Streaking is done using a sterile tool, such as a cotton swab or
commonly an inoculation loop.
  Aseptic techniques are used to maintain microbiological cultures and
to prevent contamination of the growth medium.

PRINCIPLES OF STREAK PLATE:

 The streak plate method is a rapid qualitative isolation method.

 The techniques commonly used for isolation of discrete colonies
initially require that the number of organisms in the inoculums be
reduced.
 It is essentially a dilution technique that involves spreading a loopful
of culture over the surface of an agar plate.
 The resulting diminution of the population size ensures that, following
inoculation, individual cells will be sufficiently far apart on the surface
of the agar medium to effect a separation of the different species
present.
 In the streaking procedure, a sterile loop or swab is used to obtain an
uncontaminated microbial culture. The process is called “picking
colonies” when it is done from an agar plate with isolated colonies
and is transferred to a new agar or gelatin plate using a sterile loop or
needle.
 The inoculating loop or needle is then streaked over an agar surface.
 On the initial region of the streak, many microorganisms are
deposited resulting in confluent growth or the growth of culture over
the entire surface of the streaked area.
 The loop is sterilized by heating the loop in the blue flame of the
Bunsen burner, between streaking different sections, or zones and
thus lesser microorganisms are deposited as the streaking
progresses.
 The streaking process will dilute out the sample that was placed in
the initial region of the agar surface.
T STREAK:

 The three-phase streaking pattern is known as the T-Streak.

 The streaking is done using a sterile tool, such as a cotton swab or
commonly an inoculation loop.
 The inoculation loop is first sterilized by passing it through a flame.
 When the loop is cool, it is dipped into an inoculum such as a broth or
patient specimen containing many species of bacteria.
 The inoculation loop is then dragged across the surface of
the agar back and forth in a zigzag motion until approximately 30% of
the plate has been covered.
 The loop then is re-sterilized and the plate is turned 90 degrees.
 Starting in the previously streaked section, the loop is dragged
through it two to three times continuing the zigzag pattern.
 The procedure is then repeated once more being cautious to not
touch the previously streaked sectors.
 Each time the loop gathers fewer and fewer bacteria until it gathers
just single bacterial cells that can grow into a colony.
 The plate should show the heaviest growth in the first section.
 The second section will have less growth and a few isolated colonies,
while the final section will have the least amount of growth and many
isolated colonies.
 QUADRANT METHOD:
 In the quadrant method, four equally sized sections are streaked. The
continuous streaking method typically involves inoculating the top
half of the plate, rotating it 180 degrees, and inoculating the other
half of the plate without sterilizing the loop or dragging bacteria from
the previous section.

DISCONTINUOUS STREAKING METHOD:

1. Sterilize the inoculating loop in the bunsen burner by putting the loop
into the flame until it is red hot. Allow it to cool.
2. Pick an isolated colony from the agar plate culture and spread it over
the first quadrant (approximately 1/4 of the plate) using close parallel
streaks or insert your loop into the tube/culture bottle and remove
some inoculum. You don‟t need a huge chunk.
3. Immediately streak the inoculating loop very gently over a quarter of
the plate using a back and forth motion.
4. Flame the loop again and allow it to cool. Going back to the edge of
area 1 that you just streaked, extend the streaks into the second
quarter of the plate.

1. Flame the loop again and allow it to cool. Going back to the area that
you just streaked, extend the streaks into the third quarter of the
plate.
 2. Flame the loop again and allow it to cool. Going back to the area that
you just streaked, extend the streaks into the center fourth of the
plate.
3. Flame your loop once more.

SIGNIFICANCE OF STREAK PLATE METHOD:

 The streak plate technique is the most popular method for isolating
specific bacteria from a sample containing a mixture of
microorganisms.
 Streak plate technique is used to grow bacteria on a growth media
surface so that individual bacterial colonies are isolated and
sampled.
 Samples can then be taken from the resulting isolated colonies and
a microbiological culture can be grown on a new plate so that the
organism can be identified, studied, or tested.
 When the bacteria are streaked and isolated, the causative agent of a
bacterial disease can be identified.

LIMITATIONS IF STREAK PLATE:

Streak plating is a microbiology laboratory method that has two major
disadvantages.
 Firstly, users will not be able to grow obligate anaerobes using this
method.
  Secondly, only organisms that were viable in the original sample are
able to be grown.


ASEPTIC TECHNIQUE:
 Aseptic technique is a set of routine measures that are taken to
prevent cultures, sterile media stocks, and other solutions from being
contaminated by unwanted microorganisms (i.e., sepsis).
 While such actions are sometimes called “sterile technique,” that
terminology is appropriate only in reference to preventing the
introduction of any organisms to the laboratory or medical equipment
and reagents (e.g., during surgery).
 Since the goal of a biologist is to grow microorganisms or eukaryotic
cells without the introduction of extraneous organisms, aseptic
techniques are crucial for accurate and meaningful experimentation.
 One should always remember that a completely sterile working
environment does not exist.
 However, there are a number of simple, common sense procedures
that will reduce the risk of culture contaminations.
 The aseptic techniques control the opportunities for contamination of
cultures by microorganisms from the environment, or contamination
of the environment by the microorganisms being handled.

GENERAL RULES:
 Close windows and doors to reduce draughts and prevent sudden
movements which might disturb the air.
 Make transfers over a disinfected surface. Ethanol disinfection is
recommended because of its rapid action. If the bench surface is
difficult to clean, cover the bench with a sheet of tough material
which is more easily disinfected.
 Start the operations only when all apparatus and materials are within
immediate reach.
 Complete all operations as quickly as possible, but without any hurry.
 Vessels must be open for the minimum amount of time possible.
 While vessels are open, all work must be done close to a Bunsen
burner flame where air currents are drawn upwards.
 On opening a test tube or bottle, the neck must be immediately
warmed by flaming (see below) with the vessel held as near to
horizontal as possible and so that any movement of air is outwards
from the vessel.
 During manipulations involving a Petri dish, limit exposure of the
sterile inner surfaces to contamination from the air.
 The parts of sterile pipettes which will be put into cultures or sterile
vessels must not be touched or allowed to come into contact with
other non-sterile surfaces, such as clothing, the surface of the
working area, or the outside of bottles/ test tubes.
 All items which come into contact with microorganisms must be
sterilized before and after each such exposure. This could be either
by the technical team preparing for and clearing up after a piece of
practical work (for example, in the case of glassware to be used), or
by the worker during the course of the practical (for example, in
flaming a wire loop).

 STERILE HANDLING:

Always wipe your hands and work area with 70% ethanol.
 It is recommended to wear gloves. This will prevent any foreign
contaminants from coming in contact with the customers and sample
during testing. If gloves are not used, it is necessary to wash hands
before and after testing.
 Wipe the outside of the containers, flasks, plates, and dishes with
70% ethanol before placing them in the cell culture hood.
 Avoid pouring media and reagents directly from bottles or flasks.
 Use sterile glass or disposable plastic pipettes and a pipettor to work
with liquids, and use each pipette only once to avoid cross
 contamination. Do not unwrap sterile pipettes until they are to be
used. Keep pipettes at the work area.
 Always cap the bottles and flasks after use and seal multi-well plates
with tape or place them in resalable bags to prevent microorganisms
and airborne contaminants from gaining entry.
 Never uncover a sterile flask, bottle, Petri dish, etc. until the instant
you are ready to use it and never leave it open to the
environment. Return the cover as soon as you are finished.
 If you remove a cap or cover, and have to put it down on the work
surface, place the cap with opening facing down.
 Use only sterile glassware and other equipment.
 Be careful not to talk, sing, or whistle while performing sterile
procedures.
 Perform experiments as rapidly as possible to minimize
contamination.

INOCULATING AGAR PLATES, SLOPES AND

CULTURES:

Carry out the transfer of cultures as quickly as possible, with tubes
and plates open to the air for the minimum length of time.
 Normal practice is to open agar plates away from the body and
without removing the lid completely from the base.
 In instances when the lid of the Petri dish may be removed for longer
periods than normal, work very close to the Bunsen burner flame to
reduce the chances of contamination.
 If you experience frequent contamination of plates with fungal spores,
reduce the chance of draughts further, and consider inoculating
plates from below with the agar surface facing downwards. In this
way there is perhaps less chance of spores settling onto the plate
from the air.

USING A WIRE LOOP:
 While using a wire loop, hold the handle of the wire loop close to the
top, as you would hold a pen, at an angle that is almost vertical. This
leaves the little finger free to take hold of the screw cap/ cotton wool
plug of the bottle/ test tube. It also ensures that any liquid culture on
the loop will run down into the flame.
 Sterilize a wire loop by heating to red hot in a roaring blue Bunsen
burner flame before and after use. This ensures that contaminating
bacterial spores are destroyed.
 The flaming procedure should heat the tip of the loop gradually. This
is because after use it will contain culture, which may splutter on
rapid heating and possibly release small particles of culture, forming
an aerosol.
 Position the handle end of the wire in the light blue cone of the flame.
This is the coolest area of the flame.
 Draw the rest of the wire upwards slowly into the hottest region of the
flame – immediately above the blue cone.
 Hold there until it is red hot.
 Ensure the full length of the wire receives adequate heating.
 Allow to cool for a few seconds in the air, then use immediately.
 Do not put the loop down, or wave it around.
 Re-sterilize the loop immediately after use.

USING A PIPETTE:
 Sterile graduated or dropping (Pasteur) pipettes are used to transfer
cultures, sterile media and sterile solutions.
 Remove the pipette from its container/ wrapper by the end containing
a cotton wool plug, taking care to touch as little of the pipette as you
need to take a firm hold.
  Fit the teat. It is sometimes helpful to dip the teat first in sterile liquid
to lubricate it.
 Hold the pipette barrel as you would a pen, but do not grasp the teat.
This leaves your little finger free to take hold of the cap/ cotton wool
plug of a bottle/ test tube and your thumb free to control the teat.
 Depress the teat cautiously and take up an amount of fluid which is
adequate for the amount required, but does not reach and wet the
cotton wool plug.
 Squeezing the teat with the pipette tip beneath the liquid surface
introduces air bubbles which may cause „spitting‟ and, consequently,
aerosol formation. Avoid this by squeezing the teat before placing the
tip into the liquid.
 Then gently release the pressure until the required amount of liquid is
drawn up, and lift the pipette tip out of the liquid.
 Return any excess gently.
 Immediately after use put the contaminated pipette into a nearby
discard pot of disinfectant.
 Remove the teat only once the pipette is within the discard pot
otherwise drops of culture will contaminate the working surface.


FLAMING THE NECK OF BOTTLES AND TEST TUBES:

 This ensures that no microorganisms enter the mouth of the vessel to

contaminate the culture or the medium. Passing the mouth of the
bottle through a flame produces a convection current away from the
opening, and helps to prevent contamination. The hot part of the
flame is above the inner bright blue „cone‟ and the vessel needs to be
moved through the flame, not held in place.
 Loosen the cap of the bottle so that it can be removed easily.
 Lift the bottle/ test tube with your left hand.
 Remove the cap/ cotton wool plug of the bottle/ test tube with the little
finger curled towards the palm of your right hand. (Turn the bottle, not
the cap.)
  Do not put down the cap/ cotton wool plug.
 Flame the neck of the bottle/ test tube by passing the neck forwards
and back through a hot Bunsen burner flame.
 After carrying out the procedure required, for example, withdrawing
culture, replace the cap/ cotton wool plug on the bottle/ test tube
using your little finger. Take care! The bottle will be hot. (Turn the
bottle, not the cap.)
 If cotton wool plugs have partly lost their shape, they can be more
easily guided back into the neck of the vessel by slowly twisting the
mouth of the vessel as the plug is pushed down.

DISINFECTING SURFACES:
 For technicians, ethanol disinfection is recommended because of its
rapid action (around 5 minutes). Technicians will be more
experienced and able to deal with the associated fire hazards of
working with ethanol.