TECHNIQUES FOR COLLECTION,
ISOLATION AND PRESERVATION OF
MICROORGANISMS
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Sampling of microorganisms
from air
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Air can be sampled for the presence of microorganisms by
their collection on culture medium using open Petri dishes
containing a suitable medium. Now-a-days more accurate
sampling can be done by use of a slit-sampler device that
impringes air on agar surface.
Requirements
Air sampler
Nutrient agar plates (for bacteria)
Czapek Dox agar plates (for fungi)
Colony counter
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Procedure
Prepare plates of nutrient agar and Czapek Dox agar media.
Keep the plates in the chamber of air sampler.
Expose the plates for 05-10 minutes to allow air to expose
on the surface of medium.
Take out the plates from the chamber and incubate at 370 C
for 24 hours.(fungal culture-room temperature).
Result
Observe the growth of bacterial and fungal colonies. Count
the colony forming units (CFUs) of bacteria and fungi
which indicate the viable number of bacteria and fungi
present in air
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Hand-held Air sampler
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Sampling of microorganisms
from water
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Direct microscopic examination of water sample generally
reveals few microorganisms. The filtration technique is
used to sample the microorganisms for their estimation per
unit volume, as well as to study them morphologically.
The membrane filters provide a rapid and useful means of
sampling from water. Such filters are also used for viable
counting by laying them on a suitable agar plate and
allowing to form colonies. The filtration procedure for
viable counting is given below.
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Requirements
water sample
membrane filter assembly
Millipore filter (0.45micron meter)
side arm flask
suction pump
nutrient agar plates (pH 6.8)
Procedure
Place a Millipore filter in the membrane filter assembly and connect it
with flask.
Connect the other side of the flask with suction pump.
Allow the water sample to filter through millipore filter paper.
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Take out the filter paper and transfer on the surface of nutrient agar
medium in Petri plates.
Incubate the plates at 370C for 24 hours and observe the growth of
microbial colonies.
Result
The number of colonies can be calculated by counting in terms of
CFUs/mL of sample.
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Sampling of microorganisms
from soil
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Subsurface unsaturated zone
• Hand-auger
simple, cheap
0 – 20 ft
Rod inside hollow
stem for removing
plug
Hydraulic cylinder
press
Flight
• Hollow stem auger
Removable split spoon sampling
sampling
barrel push-tube sampling
Hinged teeth
(paring device)
20 – 100 ft
Bit or sampling
barrel
Aseptic soil 12
core
Sample Processing and Storage for Soil
• Store samples at 40C
• Process samples as quickly as possible
Surface soils
- air dry and sieve through a 2 mm mesh
- microbial communities remain essentially intact for 3 weeks
Subsurface samples
- perform analyses immediately under sterile conditions (if
not possible place samples in dry ice and ship overnight to
lab for analysis next day)
Analysis for microorganisms
1. bacteria
- cultural assay (choose culture medium carefully)
- direct counts
- antibodies
- extraction and analysis of nucleic acids
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There are two methods for direct visualization of soil
bacteria.
Now-a-days special glass capillaries of rectangular cross
section i.e. micro-slides having one wall quite thin (0.17
mm), are available. The tubes are made in a group of five
together lumped after filling the tubes with sterile distilled
water.
Second method is by placing the soil sample in the bottom
of a watch glass. After adding water below the sample
until the water surface is formed above the sample. With
the help of floating cover slip on the surface, sample is
drawn for further examinations.
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ISOLATION TECHNIQUE
In nature microbial cultures are mixed
Identification relies upon isolating individual colonies
Testing requires pure cultures
As a result isolation technique provides an essential
microbiological tool
In microbiology, the term isolation refers to the separation
of a strain from a natural, mixed population of living
microbes, as present in the environment, for example in
water or soil flora, or from living beings with skin flora,
oral flora or gut flora, in order to identify the microbes of
interest.
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Method of factional dilutions (Pasteur’s technique) is
based on mechanical disconnection of microorganisms by
serial dilution in liquid nutrient media.
Pour plate technique (Dilution in solid nutrient media
by R. Koch’s technique) is based on dilution of microbes
and pouring the tested material with gelatin. After cooling
the gelatin isolated colonies of microorganisms are formed
and they easily can be transferred on a fresh nutrient
medium by a platinum loop for obtaining a microbial pure
culture.
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Serial Dilution Technique
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Spread plate technique (Superficial dispersions by
Drigalsky’s technique)
It is more perfect method which is widely used in everyday
microbiological practice. This is quantitative technique that
allows the determination of the number of bacteria in a
sample.
Stages:
Pipette the required amount of bacteria (from your dilution)
on the surface of the Petri plate.
Spread the inoculum over the surface of the agar medium
using a hockey stick (spatula).
Repeat this action on 3-4 Petri plates without sterilization of
the hockey stick.
Incubate the plate inverted at 37 oC. 19
Streak plate isolation technique technique
In microbiology, streaking is a technique used to isolate a pure
strain from a single species of microorganism, often bacteria.
Samples can then be taken from the resulting colonies and a
microbiological culture can be grown on a new plate so that the
organism can be identified, studied, or tested.
Streak Plate Isolation Principle
An original inoculum containing a mixture of bacteria is spread
into 4 quadrants on solid media.
The goal is to reduce the number of bacteria in each subsequent
quadrant.
Colonies are masses of offspring from an individual cell therefore
streaking attempts to separate individual cells
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Discrete colonies form as the individual cells are
separated and then multiply to form isolated colonies in
the later quadrants.
The Goal is to isolate Colonies to Start Pure Cultures
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Streak plate technique
Advantages:
Spread millions of cells over the surface;
Individual cells deposited at a distance from all others;
Divide forming distinct colonies;
Distinct colonies do not touch any other colonies;
Clone of a single bacteria pure culture
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Stages:
· Using a sterile loop take a loopful of your bacteria
from the broth
· Streak a vertical line
· Then streak gently across section 1 in a
zig-zag pattern until a 1/3 of the plate is covered
· Do not dig into the agar
· Sterilize the loop let it cool
· Rotate the plate about 90 degrees and spread the
bacteria from the first streak into a second area
·
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Do only one streak (or very few) in the first area and once
you are in the second area do not go back to the first
· Do a zig-zag pattern until the 2nd area is covered
· Sterilize again do the same for 3rd area
· Make sure that your red hot loop is cool enough
prior to touch the bacteria
· Incubate the plate inverted at 37 oC.
In a day it is necessary to examine the colonies for
future investigation.
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Isolation Requires Aseptic Technique
Aseptic technique is the process of:
• Preventing contamination of a culture with environmental microbes
• Preventing contamination of yourself or the environment with the
organism in the culture
Remember everything is contaminated with a variety of environmental
microbes.
Remember microbes are invisible, you must “see with your minds eye”
during these procedures.
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PRESERVATION TECHNIQUES
OF
MICROORGANISMS
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WHY IS PRESERVATION IS IMPORTANT ?
• Until two decades ago the genetic resources were getting
depleted owing to the continuous depredation by man.
• It was imperative therefore that many of the elite, economically
important and endangered species are preserved to make them
available when needed.
• The conventional methods of storage failed to preent losses
caused due to various reasons.
• A new methodology had to be devised for long term preservation of
material
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Types of preservation techniques
•—
Vegetative
•—
Silica gel impregnation with skim milk
suspension
•—
Glycerol stocks
•—
Storage under liquid nitrogen
•—
Lyophilisation – freeze drying
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Vegetative stocks
•—
Very useful for frequent usage
•—
Easy to prepare
•—
Fast to transfer
•—
Easy to store
•—
However, the duration of survival is rather
short – days to months only, and —
Possibility
of mutating (changing form)
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Silica gel impregnation with skim milk suspension
•—
Technique that involves the storing of cultures in the
form of 10% skim milk suspension absorbed by the
granules of non-dyed silica gel granules
•—
Granules of silica gel are transferred to small test
tubes, cotton plugged and dry-sterilised
(180degrees Celcius for 2 hours)
•—
The tubes are immersed in ice cubes in a container
to avoid heat build-up when the suspension is placed.
— 30
•—
2 mls of 10% sterilised skim milk (sterilised by
wet sterilization – 121 deg. C for 20 minutes) is
transferred to a slant of actively growing culture
and the cells suspended.
•—
Eight to ten drops of the suspended cells are dropped,
one drop at a time (to avoid over-heat build up), onto
the ice-immersed tubes of silica granules.
•—
The granules are vortexed to break up the granules
from aggulitinating and placed back in the ice for a
short while
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• The tubes are then stored in a dessicator for a week.
• At the end of the week, the granules are again
vortexed, covered with parafilm and stored at
4deg. C until use.
• When required, asceptically one or two of the
crystals are placed onto a growth medium
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Glycerol stock-cultures
• Cultures are ensured to be actively growing.
• 20% glycerol in 2 ml. Bijou bottles or 1ml. Glycerol
in 1.5ml. Microfuge tubes are wet-sterilised
(121 deg. C for 20 minutes).
• The sterile glycerol is poured onto a slant of
culture and the cells suspended in the glycerol
asceptically.
•The suspended cells are then re-transfered to the
Bijou bottle / Microfuge tube, labeled and stored
at 20deg. C or lower.
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•When need to use, the culture is quickly thawed
under running water, a few drops transferred
asceptically to the growth medium, and the
balance kept back.
Storage under liquid nitrogen
• The culture to be stored is prepared as for the
glycerol stock, but transferred to a microfuge
tube, carefully labelled and transferred to a
vertical microfuge holder.
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• The whole holder is immersed into a tank of liquid
nitrogen. Liquid nitrogen is replanished regularly in
the tank.
•—When need to use, one of the tubes is carefully
removed from the holder, thawed rapidly by placing
under running water and part of the contents placed
on a growth medium.
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Lyophilisation – freeze drying
•—
This is the most difficult and the most expensive of the
storage techniques but is known to be the most preferred
of the storage techniques in view of the longitivity of the
survival of the micro-organism.
•—
It is the most expensive technique due to the requirement of a
special equipment called a freeze-dryer.
•—
The tubes too are special in nature.
• It is nearly always,with regards to long term storage of cell
suspensions, that contain greater than 10 to the power 8 cells
ml–1 (Miyamoto-Shinohara et al., 2000; Costa et al., 2000).
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• The reason for preserving high cell concentrations is on the
premise that the majority of cells die during long term
storage, but a sufficient number survive to enable the
continuation of the strain. Bozoglu et al. (1987) suggest
that survival of 0.1% of the original cell population is a
“sufficient number” of survivors of freeze drying to allow
continuation.
Cotton plug
Constriction
Absorbent filter paper
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•The tube used is as shown aside. A piece of absorbent filter
paper is placed inside the tube. The neck is constricted by
using flame to avoid the cotton wool to be place after the
constriction from dropping into the culture portion – at the
base. The cotton wool prevent cross contamination from
other cultures, whilst drying as well as when the culture is
reconstituted for growth.
• The diagram aside shows the tube after it is wet sterilised.
An aluminium foil is wrapped over the mouth of the tube
to prevent the cotton from getting wet.
• A suspension of the culture to be lyophalised is made, as
for the silica stock.
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• About 2 mls. of the suspension is placed onto the
filter paper asceptically , using a pasteur pippette.
•—The tube then is kept at below -40 deg. C to freeze
the sample.
•—The frozen cultures are then loaded onto a freeze
dryer, which is pre-set at -40deg. C.
•—The drying process commences, where the liquid is
directly converted into the gaseous state, by-passing
the liquid phase.
•—The freeze-dried samples are then paced in an holder
of the lyophilised tube and sealed under vacuum.
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•The tubes are then stored at 4deg. C until further use.
• It must however be noted that the tube can be used
only once. As such, several tubes of cultures are
prepared at a time.
Summary points
• Among the five methods, the most preferred is the
lyophilised form of storage, where 10%skim milk is the
medium of storage
•—The results from bacterial strain recovery efforts
following hurricanes Katrina and Rita are reported.
Over 90% of strains frozen in 10% skim milk were
recovered whereas various recovery rates were observed
for glycerol-stored stocks (56% and 94% of Escherichia
coli, depending upon the laboratory). 41
References
•—
Cody L.W, 2008, Skim milk enhances the preservation of
thawed − 80 °C bacterial stocks
•—
Lech, K., Brent, R.,1993. Growth on Solid Media. In:
Ausubel, F.M., Brent, R., Kingston, R.E.,Moore, D., Seidman,
J.G., Smith, J.A., Struhl, K. (Eds.), Current Protocols in
Molecular Biology. John Wiley and Sons, New York,
NY. 1.3.5.
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