1 Lab 1: Aseptic Technique
Lab Objectives
After completing this lab, the student should be able to:
- Use aseptic technique to inoculate a broth, deep, plate and slant.
- Explain how aseptic technique prevents contamination, maintains culture purity, and why this is a key concept of microbiology.
- Apply microbial terminology to describe growth patterns on each media type.
- Determine when each media type should be used to cultivate bacteria.
Introduction
Aseptic Technique was developed in the early to mid-1800s through the efforts of such scientists as Robert Koch, Louis Pasteur, Ignaz Semmelweis, and Joseph Lister. As with many standardized methods in microbiology, Aseptic Technique has been modified over time as more knowledge was gained within the field of microbiology and culturing methodologies advanced. However, throughout its development, Aseptic Technique has maintained three essential goals:
- Prevent contamination (unwanted microbial growth) of specimen or culture
- Prevent contamination of self
- Prevent contamination of work area (the “lab space”)
In this lab, you will learn the methods we use in the microbiology laboratory to maintain a clean environment and prevent contamination. These methods will include:
- Utilizing a good hand washing technique
- Disinfecting work surfaces before and after working with microbes
- Using a Bunsen burner or Bacti-cinerator to sterilize inoculating loops/ needles and test tubes or flasks
- Disposing of used or contaminated equipment and specimens properly
Media comes in four basic types – broths, deeps, plates, and slants. Plates (also called Petri plates), deeps and slants are all solid forms of media that rely on agar isolated from seaweed to provide a solid growth surface while broths are a liquid type of growth medium. It is important to note that bacterial cells will have different characteristics when grown on solid versus liquid media.
Colonies are formed only on solid media where cells can form visible masses on top of agar surfaces. Slants and plates differ in their surface area – slants will have a smaller surface area and a smaller exposure to the environment when the test tube is opened compared to the much larger surface area of plates. Because plates are the easiest form of media to contaminate, it is important to hold a plate in such as way as to minimize air exposure. Whereas Petri plates can be used to obtain isolated colonies, slants lack sufficient surface area to achieve isolation in most cases. Deeps, while a solid form of media, lack the ability to provide isolate colonies because growth only occurs along a narrow inoculation band termed the stab line or slightly away from the stab line in motile species. It should be noted that deeps will contain an oxygen gradient where oxygen is plentiful at the top at the deep but limited at the bottom of the deep due to poor oxygen diffusion across thick agars.
Growth in broths in indicated by a general cloudiness called turbidity. Cells