ISOLATION OF PURE CULTURE BY POUR PLATE METHODS | SERIAL DILLUTION | lOOP DILLUTION

S.Y B.PHARM SEMESTER III

PHARMACEUTICAL MICROBIOLOGY PRACTICAL

EXPERIMENT NO. 08

ISOLATION OF PURE CULTURE BY POUR PLATE METHOD

AIM: - To carry out the isolation of pure culture by using pour plate technique.

REFERENCE: -

    1.     Practical pharmaceutical microbiology book by Prof Rageeb, K.D Baviskar, N.G Patil, Published by S. Vikas And Company(Medical Publisher), Edition 2018, Page No 61-63

    2.     Experimental Microbiology book (As Per PCI Syllabus) by Savita Mandan, Umesh Laddha, Sanjay Surana, Published by Career Publication, First edition, Page No 71-72

    3.     Pharmaceutical Microbiology (As Per PCI Regulation) by Prof Chandrakant Kokare, Published by Nirali Prakashan, First Edition, Page No. 4.2 – 4.3

REQUIREMENT’S: -

CULTURE: - Soil isolated culture, Escherichia coli, and staphylococcus aureus.

APPARATUS: - Conical flask, test tubes, Petri plates, nichrome wire loop, beaker, test tube rack, Pipettes, Glass marking pencil, Busen burner.

CHEMICALS: - Beef extract,  Peptone,  NaCl, agar, alcohol.

EQUIPMENTS: - Autoclave, Colony Counter with magnifying glass, Hot air Oven, Incubator, Balance, pH meter, Water bath.

MEDIA: - Nutrient Agar Media

INTRODUCTION: - Pour plate method is usually the method of choice for counting the number of colony-forming bacteria present in a liquid specimen.  In this method, fixed amount of inoculum (generally 1 ml) from a broth/sample is placed in the center of sterile Petri dish using a sterile pipette. Molten cooled agar (approx. 15mL) is then poured into the Petri dish containing the inoculum and mixed well.  After the solidification of the agar, the plate is inverted and incubated at 37°C for 24-48 hours.

PRINCIPLE OF POUR PLATE TECHNIQUE: - In the pour plate dilution of mixed culture is carried out directly in test tubes of liquid agar medium. The medium is maintained in a liquid state by keeping media at high temperature to allow proper distributions of the inoculum. The medium is then prepared into Petri plates and allowed to solidify. Incubate all plates under suitable conditions for growth of microorganism. A series of agar plates showing decreasing numbers of colonies resulting from the dilution technique. 

PROCEDURE OF POUR PLATE TECHNIQUE: -

     1.     Dilute the bacterial sample in such a way that final concentration of bacteria will be in range of 30-300 cfu/0.1-1.0ml

    2.     Prepare nutrient agar media and transfer 15 to 20 ml medium in each test tube.

    3.     Sterilizeall glass wares in hot air oven and cool to 42 to 45°C temperature.

    4.     Take three sterilized test tubes and three Petri plates and mark by using glass marking pencil as A,B and C.

    5.     Transfer one loopful of culture in first test tube. Gently mix the culture by rolling the tube between the palms.

    6.     Transfer one loopful of culture from first tube A to second tube B and mix a first inoculated tube.

    7.     Transfer one loopful of culture from second B to third test tube C and mix as stated in step one.

    8.     All inoculated liquid medium is poured into separate sterile Petri plates. After the medium solidifies, place the Petri plates in an incubator at 30 to 35°C for 48 hour in an inverted position. 

OBSERVATION AND RESULT: -

Colonies appearing in Petri plates A are not distinct, small and overlap with one another. In Petri plates B colonies are large, discrete and generally do not mixed with other colonies. In Petri plates C larger and isolated colonies with distinct shape, color, texture, size and form are observed. From Plate A to C there is dilution of the inoculums and only less number of microorganism are allow to grow in Petri plates C . Colonies are developed on the surface and subsurface for the medium.

ADVANTAGES OF POUR PLATE METHOD: -

    1.     This method is used to count viable cells.

    2.     Distinct separate colonics are obtained on solid medium.

DISADVANTAGES OF POUR PLATE METHOD: -

    1.     Microorganisms are uniformly spread in liquid agar therefore microbes are also present below surface. Hence surface as well as sub surfaced colonies ate developed and It is very difficult to isolate and count the subsurface colonies.

    2.     Those microbes which are sensitive to higher temperature cannot be isolated by pour plate technique because the temperature of agar should be always high to maintain at liquid consistency. Hence pour plate method is unsuitable for isolations of psychrophilic microorganism.

    3.     This method is tedious, time consuming and requires skill.

    4.     Aerobic bacteria which get trapped inside the agar fail to survive due to lack of oxygen.

    5.     For each dilution you need separate tubes.

    6.     Need to control the specific temperature, otherwise too hot media kills the bacteria present in the sample.