 In this tutorial, students will learn different isolation methods that are used for culturing of microbes. In coming series of videos, we will discuss study of fermented products on specified media. There are basically three different isolation methods that are used for culturing of microbes, straight-plate method, pore-plate method, and spread-plate method. First, we will discuss straight-plate method. For straight-plate method, falling procedure is used. Label the factory plates with isolation method and date. This will also help to identify the microorganisms and although it may take some time, but it may lead to help to avoid the big mistake later on. Secondly, check agar plate. If any moisture is present then it is preferable to place them in an incubator for overnight. Now, pour the melted agar on sterile petri plate. Sterile the mouth of glass with the help of separate lamp. Pour melted agar on sterile petri plate carefully. Place the petri plate on side. Now, check agar plates if any moisture is present then it is preferable to place them at room temperature for overnight. Step is addition of inoculum. Inoculum from another plate then it is preferable to use single colony from inoculated plates. For inoculation, first, flame this inoculating loop to red hot. Let it cool for one to two minutes. Single colony from plate, heat in a slightly rapid movement to avoid the gorging of agar plates. After inoculation, again red hot dilute the inoculated plate in an incubator in inverted position at 37 degrees centigrade for overnight. After incubation, observe the plate at a safe distance from space. Isolation method part two. Pour plate method. Pour plate method is also a pure culture technique which is used for isolation of microbes. It is very economical way for food labs to test, to perform tests on a sample that focused on specific number of bacteria. For pour plate method, following procedure is used. First, set the micro pipette on a specified volume which is 100 micro litre. Now, for second step, we use inoculum to inoculate the empty petri plate. For inoculum addition, we take culture, measure 100 micro litre of culture and spread it evenly on petri plate. Discard the micro pipette. Now pour melted agar on inoculated plate. For inoculation, sterilize the mouth of flowers and pour melted agar uniformly on plate. According to recommendation, it is preferable to fill the plate to 12 to 15 ml. Now, place the plate in laminar to dry. After this, place plate in incubator at 37 degrees centigrade for overnight. At desired temperature, at a quite 37 degree centigrade, actual temperature is 30 and it will set at 37 after few minutes. It will automatically adjust to the set point temperature. For night incubation, we will observe the plates and the colonies were calculated with the help of colony counter. After the calculation of colonies, we will also find out colony forming unit with the help of formula. This is the basic two isolation methods. Pour plate and straight plate. Facilitation method part three, bread plate method. Bread plate method is a technique which is used to place the liquid sample containing bacteria so that bacteria easily count and isolate. Successful spread plate method will have countable number of colonies that are evenly isolated on agar surface. For the bread plate method, calling procedure is used. Place the agar and allow it to cool less than 50 degrees centigrade. Now, take sterile petri plate and pour melted agar on plate. To pouring, place the plate for some time to dry. Pour plate and use spreader for spreading off inoculum. If you use glass spreader, then it is preferable to dip the spreader in alcohol and then flame it on burner. This is disposable spreader. Dip it in 1000 microlitre and take 1 ml culture. Inoculum on the surface of agar. Inoculum evenly on the surface of agar plate. Continue spreading until it fully absorbs on the surface of agar. It is recommended during the spreading, place the plate in front of flame to avoid the risk of contamination. As agar is fully absorbed on the agar surface, close the lid of plate and place in an incubator at 37 degree centigrade. For incubation, place the plates in an incubator at 37 degree centigrade for overnight. After overnight incubation, again observe the plates and at a safe distance from the face. Count the number of colonies with the help of colony counter and also calculate the colony forming unit from isolated colony.