Basic Laboratory Techniques

Learning Objectives
Know the lab equipments and culture media needed to develop pure cultures Concept of aseptic technique and procedures needed for subculturing of microorganisms Streak-plate inoculation of microorganisms for pure culture isolation

Introduction
Pure Culture a culture containing single species of microbes. Culture Media
a favorable growth environment with supply of nutrients. Liquid(broth medium), semisolid or solid (agar) Solid media can be agar slant (slanted position), agar deep (upright position), or agar plate (large surface area)

Introduction
Aseptic techniques
Techniques used to achieve sterility Sterilization is process of rendering material free of microbes. eg:autoclave

Equipments
Test tubes, petridishes, bunsen burner, inoculating loop, autoclave and refrigerator

Culture Transfer Techniques

Learning Objectives
Perform the technique for aseptic removal and transfer of microorganisms for subculturing Correctly sterilize inoculating instruments in the flame of the bunsen burner Correctly manipulate your fingers to remove and replace the test tube closure

Culture Transfer Techniques

Subculturing
A mechanism of transferring microorganisms from one medium to another Important in preparing and maintaining stock cultures as well as in microbiological test procedure Proper aseptic technique is important to prevent external contamination

Subculturing Procedure
Label the tube to be inoculated with the name of the organism and initial Sterilize an inoculating loop until the wire become red hot (cover max. portion) and allowed to cool for 10 20 secs. Uncap the tubes by lifting the closure upward and never put the closure on lab. bench Flame the necks of the tube by briefly passing them over the flame Transfer microorganisms by using d/t techniques depending on the culture medium ( slant to broth, broth to slant) Reflame the necks of the tubes and replace the caps Reflame the inoculating loop to destroy any remaining organisms
See Figure 1.1

Isolation of Pure Culture

Important for studying single organisms morphological and biochemical properties Uses different techniques designed to produce discrete colonies. Colonies are visible microbial growth on a solid media, each colony representing multiplication of a single organisms Discrete colonies can be used for subsequent pure culture isolation

Streak-plate Technique
A method used for isolating a discrete colonies from a mixed culture A rapid isolation method that involves spreading a loopful of culture over the surface of an agar plate Can be called four-way or quadrant technique

Streak-plate technique
Label the plate agar with name of the organism and your initial, and date it. Place a loopful of culture on the agar surface in Area 1. flame the loop and cool, then streak it rapidly across the surface of Area 1. Reflame and cool the loop, and turn the petri dish 90. Touch the corner of the culture in Area 1 and streak it in Area 2. Reflame and cool the loop and again turn the dish 90. Streak Area 3 in the same manner Reflame and cool the loop, turn the dish 90 and drag the culture across Area 4 in the same manner (use a wider streak) Donot let the loop touch any of the previously streaked areas except the corners Donot forget Flaming the loop in between. flaming is to dilute the culture (few organisms are streaked in each area)