Exercise 1: Ubiquity

Introduction

Microorganisms are organisms that are too small to see without the aid of a microscope. These organisms have existed for billions of years on Earth and have adapted to a wide range of habitats. Microorganisms are often described as being “ubiquitous in nature”. This means that they can be isolated from air, soil, water, plants, and animals (including humans). Microbes are even found in seemingly uninhabitable sites such as acidic hot springs, solid rock and deep sea thermal vents.

Many microorganisms are free-living and are not known to cause disease; they are non-pathogenic. Frequently they are saprophytes involved in decomposing organic matter – an important ecological role. Other microorganisms reside on or in another host species and benefit from the symbiotic association with their host(s) – they have a protected environment in which to reproduce successfully. If they damage their host causing disease, they are known as pathogens. In many cases the microbe may actually benefit their host, a relationship known as mutualism. Finally, some microbes are commensal organisms, where they benefit but have no significant effect on their host. However, commensal or mutualistic strains inhabiting our bodies are capable of becoming opportunistic pathogens if introduced into a suitable part of the body. Any area where a microbe resides and serves as a potential source of infection is called a reservoir.

Course Intended Outcomes

Demonstrate and apply basic aseptic techniques, standard lab safety procedures, and maintain aseptic environment in a microbiology laboratory.

Describe different types of media according to their physical state, chemical composition, and their function; list some of their applications; interpret microbial growth patterns.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Nutrient Agar (NA) plates
• Sterile swabs
• Additional materials will be assigned by your instructor

Procedure, Day 1

Students will inoculate several different specimen types onto plate media to demonstrate the presence of microorganisms. A single bacterial cell isolated on the media surface will grow into a visible colony. Most specimens will contain several different species of bacteria that will result in a variety of colony morphologies on plate media. Students will learn to recognize and describe the form, elevation and margin of colonies on plate media.

Technique:

1. Remove one sterile swab and moisten swab with dI deionized water.
2. Use the prepared swab to collect sample from desired surface.
3. Roll swab onto small area of media surface toward one edge.

• • • Agar medium like nutrient agar (NA) is semi-solid with a consistency like gelatin.
    • Use a low angle and light pressure while inoculating or you will damage the medium.
    • Keep the plates covered as much as possible.

1. Use swab to drag inoculum across medium in a zig-zag motion, see figure 1-1.
2. Repeat procedure with 2^nd swab for a different sample.

Sampling/Inoculation:

1. Your instructor will provide specific instructions for sample collection and inoculation.
2. Label plates 1 – 3 according to the instructions provided by your instructor.

• • • Warm the plates in your hands with lids closed before labeling.
    • Label plates with the group name, sample location and section day/time.
    • Always label plates on the bottom side containing the medium. Do NOT label the lids. Lids can become separated during storage and handling.

1. Inoculate plates as directed.
2. Collect all three plates and place lid side down in the rack for incubation at 35 ˚C.
3. One plate for the section will have the lid removed for the duration of the lab period
4. One plate for the section will remain unopened to serve as an uninoculated negative control.

Procedure, Day 2

1. Examine plates from previous week including the air exposure plate and the uninoculated control.
2. Determine which plates have microbial growth (G) or no growth (NG) after incubation.
3. Look for colonies of bacteria and/or fungi on the surface of the medium.
4. Use the table in figure 1-2 to determine the form, elevation and margin of one or two representative colonies from each plate.
5. Record your results in your lab notebook.

Colony Morphology Long Description

Lab Clean-up

Plates are disposable and should be thrown away in the biohazard container.

Tubes of sterile deionized water are placed in the tube rack for sterilization and cleaning.

Used swabs should be treated as contaminated and disposed in the biohazard container.

Paper wrappers are NOT biohazardous material.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

In your lab notebook, illustrate one or two colonies observed from each plate.

Use the following table to describe the characteristics of the colonies observed.

Critical Thinking Questions

How many bacteria are required to create a visible colony on plate medium?

What is the function and expected result for the uninoculated control plate?

Which sites that were tested had no growth? Do you think that these locations are sterile? Why or why not?

What was the most common colony morphology that your group identified on the plates?