9 Reading: Prokaryotes
Introduction
Prokaryotes include the domains Bacteria and Archaea. All of the organisms that we study in this lab will be in the domain Bacteria.
This exercise is designed to familiarize students with some basic equipment and techniques used in the study of microorganisms. In addition, students will learn some basic techniques used in identifying prokaryotes and make and view microscope slides of some common prokaryotes.
Microbiology Laboratory Equipment
Sterilization
It is important that all instruments and media discussed below be sterile, that is, free of any living organisms. The use of sterile equipment, media, and techniques prevents unwanted microorganisms from contaminating your cultures.
Media
Culture media containing the necessary nutrients are used to grow microorganisms in a laboratory. Four kinds of commonly used culture media are shown below.
Broth
Broth is a liquid that contains nutrients for bacteria to grow. It is kept in glass tubes and capped with a metal or plastic sleeve.
Agar
Agar is solid or semisolid. It liquefies at 100º C and solidifies at 40º C.
Agar plates are Petri dishes that contain agar for growing microorganisms. They have a large surface area and are useful for isolating and studying microorganisms. After they are inoculated, they are incubated in an inverted position. This prevents condensation from dripping from the cover onto the agar.
Agar slants are useful for maintaining cultures. Microorganisms grow on the surface of agar plates and slants.
Transfer Instruments
Subculturing refers to transferring microorganisms from one medium to another. For example, bacteria growing in broth may be transferred to an agar plate.
Wire loops are used to transfer microorganisms from liquid media to liquid or solid media.
Pipettes are used to transfer liquids. A mechanical device must be used with pipettes to create a vacuum.
Incubation
Bacterial cells on the agar or in the broth will reproduce rapidly if other environmental conditions such as temperature are favorable. A single cell on the agar will shortly produce a colony of cells that is easily visible to the naked eye.
An incubator is a chamber that maintains a constant temperature. After microorganisms are transferred to broth or agar, they are placed in an incubator (incubated) for a period of time while the cells reproduce.
Refrigerators are useful for maintaining stock cultures for long periods of time because microorganisms grow (reproduce) very slowly at low temperatures. They can also be used to store subcultures after they have been incubated.
Culture Transfer Techniques
The procedure discussed below can be used to transfer microorganisms from a tube of broth to another culture tube.
Microorganisms are often transferred from one medium to another with a wire loop. Before the loop is used to remove a sample of microorganisms, it must first be sterilized. A bacti-cinerator or Bunsen burner can be used to heat the loop. Figure 3 shows a bacti