Isolation of pure culture

Isolation of Pure Culture

What is Pure (Axenic) culture?

It is laboratory microbial culture which contains only one specific species of microorganism.

Why do we need it?

Microorganisms are omnipresent. They are always found as mixed culture where different species of microorganisms live together. By using different techniques, we can isolate the particular microorganism and culture separately obtaining pure culture.

What is the purpose of isolating pure culture?

In order to study, explore and utilize the microorganism it is necessary to obtain them in the form of pure culture. After obtaining the pure culture, it is tested, verified and characterized in order to confirm the origin of microbial species. Isolation of pure culture has application in various fields like Molecular Biology, Bioprocess Technology, Clinical studies, Pathological and Agriculture, etc.

Who developed the concept of pure culture?

In mid of 19th century, Rober Koch developed the concept of pure culture.

What are different techniques of obtaining pure culture?

Plating technique like Spread plate, Pour plate and Streak plate are commonly used for isolation of pure culture.

For plating technique, the basic need is nutrient media. Read our article to learn how to prepare Nutrient media for bacteria and fungi.

Streak Plate technique for Pure Culture –

  • Requirements –
  • Inoculating loop
  • Sterile media
  • burner
  • Alcohol
  • Petri plate
  • incubator

Steps –

  • Clean your laboratory desk and wipe it with alcohol.
  • Switch on the burner.
  • Incinerate the loop by flaming it in burner. Flame it until it become red. This step sterilises the loop. Allow the loop to become cool. If you use the hot red loop for inoculation, it will kill the microorganisms and the purpose may not be achieved.
  • For broth/liquid culture, dip or submerged the loop into the broth forming a film of the loop. For solid media, pick up the single colony on the loop.
  • Streak the loop in one stroke (without lifting the loop) gently on sterile media petri plate in any of the following pattern. The streaking technique dilutes the
  • The loop is again incinerated.
  • Incubate the streaked petri plate in an incubator in an optimum temperature.
  • After incubation of 24 hours, you will see isolated colonies. Each colony represent a pure culture because a colony is formed by the multiplication of single bacteria. You can see the individual colony in the form of dot. The isolated colonies are further characterized and studied.
Streaking Technique Isolation of pure culture
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2. Pour Plate Technique for Pure Culture –

Requirements-

  • Sterile media
  • Petri plate
  • Alcohol
  • Burner
  • Pipette
  • Incubator

Steps –

  • Clean your working bench and wipe it with alcohol.
  • Switch on the burner.
  • Take 1 ml of mixed culture with the help of micro-pipette.
  • Inoculate it in sterile molten media.
  • Pour the inoculated molten agar on the sterile petri plate.
  • OR You can first inoculate the mixed culture in petri plate and pour molten nutrient agar over the inoculated culture (As shown in video).
  • Move the plate horizontally on the working bench. This will mix the bacteria with nutrient media uniformly.
  • Incubate the plate at optimum temperature.
  • After incubation of 24 hrs, colonies will be visible on the surface and inside the agar media.
Pour Plate Isolation of pure culture
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3. Spread Plate for Pure Culture-

Requirements –

  • Alcohol
  • Nutrient media
  • Sterile petri plates
  • Spreader
  • Burner
  • Incubator

Steps –

  1. Clean your working bench and wipe it with alcohol.
  2. Switch on the burner.
  3. Take 0.1ml of mixed culture with the help of pipette.
  4. Inoculate it on the surface of the sterile nutrient media plate.
  5. Dip the spreader in alcohol and sterilize it in the flame.
  6. Allow it to cool.
  7. Spread the inoculated mixed culture gently on the surface of the nutrient media plate using the sterile spreader.
  8. Incubate the plate in an incubator at optimum temperature.
  9. After 24 hours, the isolated colonies will be visible on the surface of nutrient media.
Spread plate Isolation of pure culture
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Difference between Spread and Pour Plate technique –

Difference between spread and pour plate - Isolation of pure culture
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The isolated colony obtained from either Streak, Spread or Pour technique is further inoculated in fresh media obtaining pure culture of required microorganism.

The following video shows the practical performance of all the above mentioned techniques.

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Dr. Sangha Bijekar has 7 years of Teaching Experience at University level. She loves to get engage in teaching and learning process. She is into blogging from last two years. She intends to provide student friendly reading material. She is avid Dog Lover and animal rescuer. She is learned Bharatnatyam and Khatak Dancer. She is into biking and She also loves to cook.

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