Practical 2 Part 1 : ASEPTIC TECHNIQUE AND TRANSFER OF MICROORGANISMS INTRODUCTION In natural environments, microorganisms usually exist as mixed populations.

However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. A pure culture is one in which all microorganisms are descendent of the same organism. In working with microorganisms we must also have a sterile nutrient-containing-medium in which to grow the organisms. Anything in or on which we grow a microorganism is termed a medium. A sterile medium is one which is free from all life form. It is usually sterilized by heating it to a temperature at which all contaminating microorganisms are destroyed. Finally, in which with microorganisms, we must have a method of transferring growing organisms (called the inoculums) from a pure culture to a sterile medium without introducing any unwanted outside contaminants. This method of preventing unwanted microorganisms from gaining access is termed aseptic technique. A. Aseptic Technique The procedure for aseptically transferring microorganisms is as follow: 1. Sterilize the inoculating loop The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange of the heat. In this way, all contaminants on the wire are incinerated. Allow the loop to cool a few seconds to avoid killing the inoculums. 2. Remove the inoculums a. Removing inoculums from broth culture ( organisms growing in a liquid medium) b. Removing inoculums from a plate culture ( organisms growing on agar surface in a petri dish ) 3. Transferring the inoculums (2a/b) into a broth tube 4. Transferring the inoculums into a petri plate B. Forms of Culture Media 1. Broth tubes are tubes containing a liquid medium. After incubation, growth (development of many cells from a few cells) as one or a combination of three forms: pellicle, turbidity sediment. 2. Slant tubes are tubes containing a nutrient medium plus a solidifying agent, agar-agar. The medium has been allowed to solidify at an angle in order to get a flat inoculating surface.

3. Stab tubes (deeps) are tubes of hardened agar medium which are inoculated by stabbing the inoculums into the agar. 4. Agar plate is sterile petri plates that are aseptically filled with a melted sterile agar medium and allowed to solidify. Plates are much less confining than slants and stabs and are commonly used in the culturing, separating and counting of microorganisms. Single colonies of microorganisms on agar plates can be described using the terms found in. C. Oxygen Requirements for Microbial Growth Microorganisms show a great deal of variation in their requirements for gaseous oxygen. Most can be placed in one of the following groups: Obligates aerobes, Microaerophiles, obligate anaerobes, Aerotolerant anaerobes and Facultative anaerobes. D. Temperature Requirements Microorganisms are divided into groups on the basis of their preferred range of temperature: Psychrophiles, Mesophiles, Thermophiles and Hyperthermophiles.

PART 2: OBTAINING PURE CULTURES FROM A MIXED POPULATION INTRODUCTION Microorganisms in natural circumstances are almost always found as mixtures of many species. For most purposes, it is necessary to isolate the various organisms in pure culture before they can be identified and studied. OBJECTIVES 1. To isolate single and pure colonies from a mixture of bacterial suspensions 2. To perform a serial dilution of bacterial suspension