Title 8. DIFFERENT METHODS USED IN ISOLATION OF BACTERIA.pdf ✅

PHARMD GURU Page 1 INTRODUCTION:  Microorganisms in nature are never found in pure populations.  Different types of mixed microorganisms are normally present in soil, water, food, air and various parts of the human or animal body.  To study the role played by a specific microorganisms in these environments, it is necessary to isolate the microorganisms in pure culture.  It is extremely important to maintain isolated pure cultures for extended periods in a viable condition.  Most microbiological laboratories usually maintain a large collection of pure cultures as well as subcultures of authentic species purchased from various culture collection centres. Ex: American Type Culture Collection (ATCC), USA National Collection of Industrial Bacteria (NCIB), Scotland National Collection of Yeast Cultures (NCYC), England National Collection of Type Cultures (NCTC), England National Chemical Laboratory (NCL), India etc PURE CULTURE TECHNIQUES: A pure culture consists of a population of only one species of microorganisms. The isolation of one kind of microorganism from a mixture of many different kinds is called the pure culture technique. The methods widely used for isolation of microorganisms are as follows: 1) Streak plate method 2) Pour plate method a) Loop dilution technique b) Serial dilution technique 3) Spread plate method 4) Micromanipulator method 5) Roll tube method. DIFFERENT METHODS USED IN ISOLATION OF BACTERIA
PHARMD GURU Page 2 1)STREAK PLATE METHOD: Streak plate method is the most widely used method for isolation of cultures. Streak plates are prepared by streaking a small amount of mixed culture over the surface of the solid medium in a Petri plate with a platinum or nichrome wire loop. The sample is streaked in such a way as to provide successive dilution (Fig. 4.1). The purpose of streaking is to thin out the innoculum (starter culture) successively so that microbes get separated. A second plate may also be streaked from the same loop/needle without reinnoculation. In the beginning of the streak, microbes are crowded and colonies develop closely but as the streaking proceeds cells gets separated as the needle contains less cells. Hence at the last streak, few and clearly separated colonies are developed. Transfer the well isolated colonies from streak plate to another new plate for isolation and purification (sub culturing). 2)POUR PLATE METHOD: In this method, the mixed culture is diluted directly in tubes of liquid (cooled) agar medium (Fig. 4.2). The medium is maintained in a liquid state at a temperature of 45°C to allow thorough distribution of the inoculum. The inoculated medium is transferred into Petri plates, allowed to solidify and incubated. A series of agar plates showing decreasing number of colonies resulting from the loop dilution technique is shown in Figure 4.2. In the serial dilution technique, the original innoculum may be diluted by using sterile water or a saline solution (by using pipette) so that the concentration of the microbes gradually becomes less. Mix 1 ml dilute sample in a 20 ml liquid nutrient