ISOLATION OF BACTERIA | STREAK PLATE METHOD | MICROBIOLOGY PRACTICALS

S.Y B.PHARM SEMESTER III

PHARMACEUTICAL MICROBIOLOGY PRACTICAL

EXPERIMENT NO. 06

ISOLATION OF PURE CULTURE BY STREAK PLATE METHOD

AIM: - To carry out the isolation of pure culture using streak 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 56–59.

2. Experimental Microbiology book (As Per PCI Syllabus) by Savita Mandan, Umesh Laddha, Sanjay Surana, Published by Career Publication, First edition, Page. No 68-70.

3. Pharmaceutical Microbiology (As Per PCI Regulation) by Prof Chandrakant Kokare, Published by Nirali Prakashan, First Edition, Page No. AI25 – AI27

PURE CULTURE TECHNIQUE: -

INTRODUCTION: -

Microorganisms are isolated from various substrates or sources like soil, air, fresh water, sea water, food, body surface etc. Microbes in nature are found in mix populations. In the laboratory, these populations can be separated into pure cultures. It is possible to study different characters of microbes like morphological, cultural, bio chemical, serological and genetic with the help of pure cultures. In pure culture techniques, colonies are individual, isolated, visible masses and representing the multiplication of single organism.

MIX CULTURE: - A culture which contains more than one kind of microorganism is called mixed cultured.

PURE CULTURE: -A culture that contains only one kind of microorganism is called pure culture.

METHODS OF ISOLATION OF PURE CULTURE: -

METHODS OF ISOLATION OF BACTERIA

The techniques to obtained pure culture may be classified as follows.

1. Streak plate method.

2. Pour plate method.

3. Spread plate method.

4. Roll tube method.

5. Single cell isolations. (Micromanipulator)

STREAK PLATE METHOD: -

It is common method use to separate and isolate particular bacterial colony from mixture of bacteria.

REQUIREMENTS: -

CULTURE: - 24 hrs cultures of Escherichia coli, Bacillus subtitles or any other soil isolated culture.

CHEMICALS: - Beef extract, Peptone, NaCL, Agar-Agar, alcohol.

APPARATUS: - Sterile Petri dishes, inoculating nichrome wire loop, burner, marking pen.

EQUIPMENTS: - Autoclave, Colony Counter, Hot air Oven, Incubator, Balance, pH meter.

PRINCIPLE: -

In streak plate method microbial culture is inoculated with the help of nichrome wire loop and streaked over surface of solidified agar. Concentration of bacterial colony is more at the starting point and it goes on decrease toward last point of streak. It helps to isolate individual colony from mix colonies and each colony considered as pure colony. So that size, shape, colour, and other physical characteristics of isolated colonies can be easily studied.

PROCEDURE: -

1. Petri dish is labelled on the bottom. Labels usually include the organism name and date.

2. Prepare nutrients agar media as per experiment 2nd and sterilize by autoclave at 121°C, 15 Ibs pressure for 15 minutes and pour the medium into Petri plates carefully.

3. Rotate the petri plate to allow for uniform distributions of the medium and solidify.

4. All procedures should be carried out in aseptic conditions to avoid contaminations.

5. Take a small amount of bacterial inoculum by nichrome wire loop from agar slants or agar plates. Carefully lift the lid and streak the cultures on the agar medium.

6. Streak the culture as the first zone by using inoculums in zigzag manner and forms T-shaped streaking, and sterilize it after streaking. Sterilization of inoculating loop is carried out by holding it in flame of Bunsen burner. This technique of sterilization is known as incineration.

7. Sterilization of loop is carried out to destroy the culture presents on wire loop.

8. The streaking in second zone is carried out by taking the culture from first zone. Sterilized inoculating loop is touched to first streaking zone to receive the culture and streaking is carried out in second zone. Again sterilize the inoculating loop.

9. Third zone is prepared by using second zone while fourth zone is prepared with the help of third one finally sterilizes the wire loop.

10. Different techniques of streaking may be performed on different petri plates using the same inoculums.

11. Incubate the plate for 24 hours and you will see isolated colonies in the fourth sector. There will be less growth in third sector and heaviest growth in first and second sector.

OBSERVATIONS AND RESULTS:

The effect of different techniques of streaking on the growth and separation of colonies may be observed. It is observed that well developed and isolated colonies have developed at the fourth zone of streaks.

CLICK BELOW TOPIC TO READ

1. INTRODUCTION OF MICROBIOLOGY

2. BRANCHES OF MICROBIOLOGY

3. SCOPE OF MICROBIOLOGY

4. HISTORY OF MICROBIOLOGY {PART 1}

5. HISTORY OF MICROBIOLOGY {PART 2}

6. HISTORY OF MICROBIOLOGY {PART 3}

7. PROKARYOTES VS EUKARYOTES DIFFERENCES

8. MORPHOLOGY OF BACTERIA

9. ULTRASTRUCTURE OF BACTERIA

10. NUTRITIONAL REQUIREMENTS OF BACTERIA

11. RAW MATERIAL USED FOR CULTURE MEDIA