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Isolation of pure cultures and
preservation of cultures
Agricultural Microbiology (ABB 156)
College of Horticulture and Forestry
Rani Lakshmi Bai Central Agricultural University,
Jhansi-284003
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
What is Pure cultures ???????
Cultures that contain only one type of cell, ideally with
the culture derived from an initial single cell.
A pure culture is a population of cells or multicellular
organisms growing in the absence of other species or
types.
OR
Pure culture
Mixed culture
Isolation of Pure cultures
1. POUR PLATETECHNIQUE
2. SERIAL DILUTION TECHNIQUE
3. SPREAD PLATETECHNIQUE
4. ENRICHMENT METHOD
5. STREAK PLATETECHNIQUE
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
1. POUR PLATETECHNIQUE
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
Solid samples e.g. Soil,
Food material, root crush
etc.
Liquid samples e.g. water,
milk, blood, urine, leaf
sap etc.
Serial Dilution method
1 ml
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
➢ In nature, microbial populations do not segregate
themselves by species but exist with a mixture of many
other cell types.
➢ The pour-plate technique requires a serial dilution of the
mixed culture by means of a loop or pipette.
➢ Molten agar cooled to 45°C, is poured into a Petri dish
containing a specified amount of the diluted sample.
➢ Following the addition of the molten-agar media, the plates
gently rotated in a circular motion to achieve uniform
distribution of microorganisms.
➢ Incubated overnight to grow and multiply the microbial cell.
➢ Count the colonies
Purpose of pour plate method
➢ To check the microbial load in a samples (water, soil, and
plant sample).
The most common method for determining the total
viable count is the pour-plate method.
The pour plate technique can be used to determine the
number of microbes/ mL in a specimen.
It has the advantage of not requiring previously
prepared plates and is often used to assay bacterial
contamination of foodstuffs.
The use of relatively hot agar carries the risk of killing
some sensitive contaminants, so giving a low result.
Small colonies may be overlooked.
Advantages of pour plate technique
Disadvantages of pour plate technique
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
2. SERIAL DILUTION TECHNIQUE
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
✓ A serial dilution is the stepwise dilution of a
substance in solution.
✓ Usually the dilution factor at each step is constant,
resulting in a geometric progression of the
concentration in a logarithmic fashion.
✓ The heavy population of microbes is diluted for
further isolation purposes.
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
➢ Prepare a series of at least 6 test tubes containing
9 ml of sterile distilled water.
➢ Using a sterile pipette, add 1ml of sample in the
first tube of the set. Label it as 10-1.
➢ Mix the contents well by swirling the tube upside
down few times.
➢ From the first tube, take 1ml of the sample and
transfer to second tube. Label it as 10-2.
➢ Repeat the procedure with all the remaining tubes
labelling them until 10-6.
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
Advantages
1. It helps to reduce a dense culture of cells to a more
usable concentration.
2. A specific amount of bacteria are reduced with
every dilution.
3. The number of colonies cultured from serial
dilutions of the sample are counted to estimate the
concentration of an unknown sample.
Disadvantages
1. It does not separate bacteria like a streak plate.
3. SPREAD PLATE TECHNIQUE
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
✓ Spread plate technique is a method employed to
plate a liquid sample for the purpose of isolating or
counting the bacteria present in that sample.
✓ A perfect spread plate technique will results visible
and isolated colonies of bacteria that are evenly
distributed in the plate and are countable.
✓ The technique is most commonly applied for
microbial testing of foods, plant samples or to
isolate and identify variety of microbial flora
present in the environmental samples e.g. soil.
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
➢ Pipette out 0.1 ml from the appropriate desired dilution
series onto the center of the surface of an agar plate.
➢ Dip the L-shaped glass spreader (hockey stick) into
alcohol.
➢ Flame the glass spreader over a Bunsen burner.
➢ Spread the sample evenly over the surface of agar using
the sterile glass spreader, carefully rotating the Petri
dish underneath at an angle of 45o at the same time.
➢ Incubate the plate at 30-37°C for 24 hours.
➢ Calculate the conlony forming units (CFU) value of the
sample.
Method of Spread Plate
To make accurate dilutions using pipettes (master
serial dilution technique).
To apply a balanced spread technique using a glass
spreader to spread the inoculum evenly on the agar
surface.
To respect the necessary “short” time interval between
agar inoculation and spreading.
➢ Once you count the colonies, multiply by the appropriate
dilution factor to determine the number of CFU/mL in the
original sample.
Rani
Lakshmi
Bai
Central
Agricultural
University,
Jhansi
Check Points
For example: Suppose the plate of the 10-6 dilution
yielded a count of 130 colonies. Then, the number of
bacteria in 1 ml of the original sample can be
calculated as follows:
CFU (Bacteria)/ml = 130/0.1ml x 106
= 130 × 107
=1.3 x 109 or 1300,000,000 Cells/ml
Total No. of colonies
Volume of culture Spreaded on plate
x Dilution factor
CFU/ml =
Calculation of result:
4. ENRICHMENT METHOD
➢ Enrichment culture is basically an isolation technique designed
to make conditions of growth very favorable for an organism of
interest while having an unfavorable environment for any
competition.
Or
“Enrichment culture is the technique that is used to enhance the
population density of a particular group of microorganisms within
the total microbial population of a sample”.
➢ Enrichment is generally done by introducing nutrients into the
certain growth media to favour the growth of a particular
microorganism over others, enriching a sample for the
microorganism of interest.
➢ Enrichment culture techniques are used to increase a small
number of the desired organisms to detectable levels. This allows
for the detection and identification of microorganisms with a
variety of nutritionalneeds.
For example: 1. Methanogenic enrichments are usually
performed in media that has a nutrient composition,
environmental pH value, temperature and oxygen-free
conditions, similar to those of natural methanogenic
environments.
2. For the isolation of phosphate solubilizing microbes
generally calcium triphosphate is supplemented in the
media, So that targeted microbes can be isolated.
3. Similarly for the isolation of salt tolerant microbes, the
media is supplemented with NaCl with different
concentration.
Observable Characteristics for the isolation of
Desired Bacteria
1. Odor Production: Some bacteria produce a unique aroma (gas) as
a result of their metabolic processes. Microbiologist will always
make note of the specific aroma of the organism under study.
Examples of aroma are ammonia (NH3), rotten egg (H2S), alcoholic (-
OH), fecal, etc.
2. Pigment Production: Pigments produced by
bacteria are either cell bound (limited to cells
and the colony), extracellular (pigment is
released into the medium) or both. The color of
the pigment should be noted, as this
characteristic could be very helpful in the
identification of the organism.
3. Colonial Morphology: This category includes the colony density,
texture, elevation, cohesiveness, shape and margin.
a. Colony density: Hold the plate in front of a light source to
determine if the colonies are clear, opaque (no light passes through
the colony) or translucent.
b. Colony texture: This
is best observed when
direct light is reflected
off the colonies. The
texture is usually
either smooth (even
surface) or rough
(irregular, non-smooth
surface).
5. STREAK PLATE TECHNIQUE
✓ It is used for isolating individual colonies of bacteria. During
streaking, you use a sterile loop to pick a single colony from the
culture plate.
✓ This involves just lightly touching the loop to the colony of
interest.
✓ Then, you smear, or “streak” the loop with the bacteria onto a
fresh plate to isolatethe different bacteria colonies.
✓ The modern streak plate method has progressed from the efforts
of Robert Koch.
Quadrant streaking
Zig-Zag streaking
Single Line streaking
✓ To produce isolated colonies of an organism (mostly
bacteria) on an agar plate.
✓ This is useful when we need to separate organisms in
a mixed culture (to purify/isolate particular strain
from contaminants) or when we need to study the
colony morphology of an organism.
✓ To identify the organism: biochemical tests to identify
bacteria are only valid when performed on pure
cultures.
Purpose of streaking
Preservation of Cultures
✓ The primary aim of culture preservation is to maintain the
organism alive, uncontaminated and without variation or
mutation.
➢ Refrigerator or cold room storage is of use only for short time
preservation of cultures.
✓ Live cultures on a culture medium can be successfully stored in
refrigerators or cold rooms, when the temperature is
maintained at 4ºC.
✓ At this temperature range the metabolic activities of microbes
slows down greatly but do not altogetherstop.
✓ As a result, bacterial metabolism will be very slow and only
less 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.
1. Storage at Refrigerator Or Cold Room Storage
Cultures are storage at Refrigerator
➢ Freezing is a common process for storage of bacteria. Thus, thick
bacterial suspensions can be frozen at a temperatureof - 30ºC.
➢ Metabolicrates are reduced by lowering the temperature.
➢ Freezing and thawing is a well known technique for actually
disrupting cells.
➢ Cultures can be preserved very effectively if frozen in the
presence of a cryoprotectant (Glycerol or dimethyl sulphoxide
(DMSO), which reduces damage from ice crystals.
➢ The simplest way to preserve a culture is to add 15%(v/v) glycerol
to the culture and then to store it at -20ºC or -80ºC in a freezer.
2. Storage By Freezing
➢ Cultures can be preserved for a number of years in glycerol, at a
temperatureof -40ºC in a freezer.
Freeze-drying (lyophilization) is one of the most economical and
effective methods for long-term preservation of bacteria and other
microorganisms.
➢ In this method, about 2 ml of glycerol solution is added on to
the agar slant culture. Shaking can emulsify the culture.
Emulsion is then transferred to ampoules, with each ampoule
having 5 ml of the culture .
In this method bacterial cultures or virus suspensions are dried
and kept in the dry stateunder suitableconditions.
3. Freeze-drying (lyophilization)
Freeze- drying is a multistageprocess:
➢ It begins with freezing below 0ºC (e.g. -20ºC), a temporary stop
to metabolicactivity.
➢ Then continues with the removal of water without thawing
(sublimation).
➢Ends with a dried product. The dried product is
sealed either under vacuum or under an inert gas,
can be stored at room temperature with no further
metabolic activity until water and nutrients are
restored.
Ampoule containing lyophilized
microorganisms
4. Storage In Silica Gel
➢ Both bacteria and yeast can be stored in silica gel
powder at low temperature for a period of 1-2 years.
➢ In this method, finely powdered, heat sterilized and
cooled silica powder is mixed with a thick
suspension of cells and stored at low temperature.
➢ The basic principle in this technique is quick
desiccation at low temperature, which allows the cell
to remain viable for a long period.
5. Preservation using Paraffin and mineral oil
➢ Various fungi such as Fusarium, Penicillium, Alternaria,
Rhizopus, Aspergillus etc. proved successful for storage in
sterile soil.
➢ Soil storage involves inoculation of 1 ml of spore suspension
into soil (that has been autoclaved twice) and incubating at
room temperaturefor 5-10days.
➢ This initial growth period allows the fungus to use the available
moisture and gradually to become dormant. The bottles are
then stored at refrigerator.
➢ Spraying few soil particles on a suitable medium retrieves
culture.
6. Storage in Sterile Soil
8.Isolation of pure cultures and preservation of cultures.pdf

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8.Isolation of pure cultures and preservation of cultures.pdf

  • 1. Isolation of pure cultures and preservation of cultures Agricultural Microbiology (ABB 156) College of Horticulture and Forestry Rani Lakshmi Bai Central Agricultural University, Jhansi-284003
  • 2. Rani Lakshmi Bai Central Agricultural University, Jhansi What is Pure cultures ??????? Cultures that contain only one type of cell, ideally with the culture derived from an initial single cell. A pure culture is a population of cells or multicellular organisms growing in the absence of other species or types. OR Pure culture Mixed culture
  • 3. Isolation of Pure cultures 1. POUR PLATETECHNIQUE 2. SERIAL DILUTION TECHNIQUE 3. SPREAD PLATETECHNIQUE 4. ENRICHMENT METHOD 5. STREAK PLATETECHNIQUE Rani Lakshmi Bai Central Agricultural University, Jhansi
  • 4. 1. POUR PLATETECHNIQUE Rani Lakshmi Bai Central Agricultural University, Jhansi Solid samples e.g. Soil, Food material, root crush etc. Liquid samples e.g. water, milk, blood, urine, leaf sap etc. Serial Dilution method 1 ml
  • 5. Rani Lakshmi Bai Central Agricultural University, Jhansi ➢ In nature, microbial populations do not segregate themselves by species but exist with a mixture of many other cell types. ➢ The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. ➢ Molten agar cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample. ➢ Following the addition of the molten-agar media, the plates gently rotated in a circular motion to achieve uniform distribution of microorganisms. ➢ Incubated overnight to grow and multiply the microbial cell. ➢ Count the colonies Purpose of pour plate method ➢ To check the microbial load in a samples (water, soil, and plant sample).
  • 6. The most common method for determining the total viable count is the pour-plate method. The pour plate technique can be used to determine the number of microbes/ mL in a specimen. It has the advantage of not requiring previously prepared plates and is often used to assay bacterial contamination of foodstuffs. The use of relatively hot agar carries the risk of killing some sensitive contaminants, so giving a low result. Small colonies may be overlooked. Advantages of pour plate technique Disadvantages of pour plate technique Rani Lakshmi Bai Central Agricultural University, Jhansi
  • 7. 2. SERIAL DILUTION TECHNIQUE Rani Lakshmi Bai Central Agricultural University, Jhansi ✓ A serial dilution is the stepwise dilution of a substance in solution. ✓ Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. ✓ The heavy population of microbes is diluted for further isolation purposes.
  • 8. Rani Lakshmi Bai Central Agricultural University, Jhansi ➢ Prepare a series of at least 6 test tubes containing 9 ml of sterile distilled water. ➢ Using a sterile pipette, add 1ml of sample in the first tube of the set. Label it as 10-1. ➢ Mix the contents well by swirling the tube upside down few times. ➢ From the first tube, take 1ml of the sample and transfer to second tube. Label it as 10-2. ➢ Repeat the procedure with all the remaining tubes labelling them until 10-6.
  • 9. Rani Lakshmi Bai Central Agricultural University, Jhansi Advantages 1. It helps to reduce a dense culture of cells to a more usable concentration. 2. A specific amount of bacteria are reduced with every dilution. 3. The number of colonies cultured from serial dilutions of the sample are counted to estimate the concentration of an unknown sample. Disadvantages 1. It does not separate bacteria like a streak plate.
  • 10. 3. SPREAD PLATE TECHNIQUE Rani Lakshmi Bai Central Agricultural University, Jhansi ✓ Spread plate technique is a method employed to plate a liquid sample for the purpose of isolating or counting the bacteria present in that sample. ✓ A perfect spread plate technique will results visible and isolated colonies of bacteria that are evenly distributed in the plate and are countable. ✓ The technique is most commonly applied for microbial testing of foods, plant samples or to isolate and identify variety of microbial flora present in the environmental samples e.g. soil.
  • 12. Rani Lakshmi Bai Central Agricultural University, Jhansi ➢ Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. ➢ Dip the L-shaped glass spreader (hockey stick) into alcohol. ➢ Flame the glass spreader over a Bunsen burner. ➢ Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri dish underneath at an angle of 45o at the same time. ➢ Incubate the plate at 30-37°C for 24 hours. ➢ Calculate the conlony forming units (CFU) value of the sample. Method of Spread Plate
  • 13. To make accurate dilutions using pipettes (master serial dilution technique). To apply a balanced spread technique using a glass spreader to spread the inoculum evenly on the agar surface. To respect the necessary “short” time interval between agar inoculation and spreading. ➢ Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample. Rani Lakshmi Bai Central Agricultural University, Jhansi Check Points
  • 14. For example: Suppose the plate of the 10-6 dilution yielded a count of 130 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows: CFU (Bacteria)/ml = 130/0.1ml x 106 = 130 × 107 =1.3 x 109 or 1300,000,000 Cells/ml Total No. of colonies Volume of culture Spreaded on plate x Dilution factor CFU/ml = Calculation of result:
  • 15. 4. ENRICHMENT METHOD ➢ Enrichment culture is basically an isolation technique designed to make conditions of growth very favorable for an organism of interest while having an unfavorable environment for any competition. Or “Enrichment culture is the technique that is used to enhance the population density of a particular group of microorganisms within the total microbial population of a sample”. ➢ Enrichment is generally done by introducing nutrients into the certain growth media to favour the growth of a particular microorganism over others, enriching a sample for the microorganism of interest. ➢ Enrichment culture techniques are used to increase a small number of the desired organisms to detectable levels. This allows for the detection and identification of microorganisms with a variety of nutritionalneeds.
  • 16. For example: 1. Methanogenic enrichments are usually performed in media that has a nutrient composition, environmental pH value, temperature and oxygen-free conditions, similar to those of natural methanogenic environments. 2. For the isolation of phosphate solubilizing microbes generally calcium triphosphate is supplemented in the media, So that targeted microbes can be isolated. 3. Similarly for the isolation of salt tolerant microbes, the media is supplemented with NaCl with different concentration.
  • 17. Observable Characteristics for the isolation of Desired Bacteria 1. Odor Production: Some bacteria produce a unique aroma (gas) as a result of their metabolic processes. Microbiologist will always make note of the specific aroma of the organism under study. Examples of aroma are ammonia (NH3), rotten egg (H2S), alcoholic (- OH), fecal, etc. 2. Pigment Production: Pigments produced by bacteria are either cell bound (limited to cells and the colony), extracellular (pigment is released into the medium) or both. The color of the pigment should be noted, as this characteristic could be very helpful in the identification of the organism.
  • 18. 3. Colonial Morphology: This category includes the colony density, texture, elevation, cohesiveness, shape and margin. a. Colony density: Hold the plate in front of a light source to determine if the colonies are clear, opaque (no light passes through the colony) or translucent. b. Colony texture: This is best observed when direct light is reflected off the colonies. The texture is usually either smooth (even surface) or rough (irregular, non-smooth surface).
  • 19. 5. STREAK PLATE TECHNIQUE ✓ It is used for isolating individual colonies of bacteria. During streaking, you use a sterile loop to pick a single colony from the culture plate. ✓ This involves just lightly touching the loop to the colony of interest. ✓ Then, you smear, or “streak” the loop with the bacteria onto a fresh plate to isolatethe different bacteria colonies. ✓ The modern streak plate method has progressed from the efforts of Robert Koch.
  • 21. ✓ To produce isolated colonies of an organism (mostly bacteria) on an agar plate. ✓ This is useful when we need to separate organisms in a mixed culture (to purify/isolate particular strain from contaminants) or when we need to study the colony morphology of an organism. ✓ To identify the organism: biochemical tests to identify bacteria are only valid when performed on pure cultures. Purpose of streaking
  • 22. Preservation of Cultures ✓ The primary aim of culture preservation is to maintain the organism alive, uncontaminated and without variation or mutation.
  • 23. ➢ Refrigerator or cold room storage is of use only for short time preservation of cultures. ✓ Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms, when the temperature is maintained at 4ºC. ✓ At this temperature range the metabolic activities of microbes slows down greatly but do not altogetherstop. ✓ As a result, bacterial metabolism will be very slow and only less 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. 1. Storage at Refrigerator Or Cold Room Storage
  • 24. Cultures are storage at Refrigerator
  • 25. ➢ Freezing is a common process for storage of bacteria. Thus, thick bacterial suspensions can be frozen at a temperatureof - 30ºC. ➢ Metabolicrates are reduced by lowering the temperature. ➢ Freezing and thawing is a well known technique for actually disrupting cells. ➢ Cultures can be preserved very effectively if frozen in the presence of a cryoprotectant (Glycerol or dimethyl sulphoxide (DMSO), which reduces damage from ice crystals. ➢ The simplest way to preserve a culture is to add 15%(v/v) glycerol to the culture and then to store it at -20ºC or -80ºC in a freezer. 2. Storage By Freezing ➢ Cultures can be preserved for a number of years in glycerol, at a temperatureof -40ºC in a freezer.
  • 26. Freeze-drying (lyophilization) is one of the most economical and effective methods for long-term preservation of bacteria and other microorganisms. ➢ In this method, about 2 ml of glycerol solution is added on to the agar slant culture. Shaking can emulsify the culture. Emulsion is then transferred to ampoules, with each ampoule having 5 ml of the culture . In this method bacterial cultures or virus suspensions are dried and kept in the dry stateunder suitableconditions. 3. Freeze-drying (lyophilization) Freeze- drying is a multistageprocess: ➢ It begins with freezing below 0ºC (e.g. -20ºC), a temporary stop to metabolicactivity. ➢ Then continues with the removal of water without thawing (sublimation).
  • 27. ➢Ends with a dried product. The dried product is sealed either under vacuum or under an inert gas, can be stored at room temperature with no further metabolic activity until water and nutrients are restored. Ampoule containing lyophilized microorganisms
  • 28. 4. Storage In Silica Gel ➢ Both bacteria and yeast can be stored in silica gel powder at low temperature for a period of 1-2 years. ➢ In this method, finely powdered, heat sterilized and cooled silica powder is mixed with a thick suspension of cells and stored at low temperature. ➢ The basic principle in this technique is quick desiccation at low temperature, which allows the cell to remain viable for a long period.
  • 29. 5. Preservation using Paraffin and mineral oil
  • 30. ➢ Various fungi such as Fusarium, Penicillium, Alternaria, Rhizopus, Aspergillus etc. proved successful for storage in sterile soil. ➢ Soil storage involves inoculation of 1 ml of spore suspension into soil (that has been autoclaved twice) and incubating at room temperaturefor 5-10days. ➢ This initial growth period allows the fungus to use the available moisture and gradually to become dormant. The bottles are then stored at refrigerator. ➢ Spraying few soil particles on a suitable medium retrieves culture. 6. Storage in Sterile Soil