GENERAL: Microbes have exerted tremendous influence over our world long before humans existed. The very oxygen so necessary for human life would not exist in the proper proportions if it were not for these invisible microbes. They influence the food we eat; the soil for plant life; the recycling of organic and inorganic materials; and many industrial processes that just plain make life better. Of course, some cause disease, and throughout history, great plagues have killed more people than all the wars combined. Yet, we cannot live without them.

In order to study microbes, certain techniques must be used to "quarantine" the microbes being studied from all other microbes that may surround them. This is done by using sterile equipment (free from any microbial life), thus allowing the selected microorganisms a non-competitive environment, and allowing us to focus on that particular microorganism. Learning such techniques is fundamental to working with and studying microbes.

OBJECTIVES: This laboratory contains three exercises. In these activities, you will practice aseptic techniques and safety procedures needed when working with bacteria. You will learn how to prepare your lab areas for microbiology work; how to collect specimens for microbiological study; how to swab and inoculate agar plates; and how to seal, label and store plates.

INTRODUCTION: When handling bacteria we need to be very careful and treat everything as a pathogen. Thus, when working with bacteria it is very important that you disinfect your entire work area before and after each day's activity. To demonstrate the effort required to be thorough and those nooks and crannies that you might miss in your diligence, we have developed a way to visualize bacteria. "Bacto-broth" was spread on your lab area prior to class. Your job is to disinfect your lab station and eliminate all traces of the "bacto-broth." Our special bacto broth detector light will be used to inspect your lab area after you have wiped it down. This special bacto illuminator will allow us to see any remaining traces of bacto-broth.

EQUIPMENT:

· Disinfectant in one spray bottle (Lysol solution) per every 4 students
· Paper towels
· "Bacto-light" (instructor)

PROCEDURE:

1. Obtain a spray bottle and paper towels from the center of your table.
2. Wipe down your lab station and surrounding water fixtures and handles thoroughly with the disinfectant.
3. When you are finished, request an inspection with the "bacto-light" from your teacher. Any missed areas will need in a recleaning!
4. Have your instructor initial here to indicate successful disinfecting.

INTRODUCTION: Bacteria are grown and isolated on a nutrient substance that we call agar. Agar is a protein that is used in cooking and can be purchased at a health food store (agar agar). It is used as a thickening agent and when solidified has the consistency of jello. In microbiology, agar is the medium used to grow bacteria. Many different nutrients can be added to the agar depending on the type of bacteria you want to grow. Although we say that the agar has "hardened" it can still tear if you use too much force when swabbing with bacteria. Remember to use the gentle touch. Don't rip the agar.

To practice the technique of spreading bacteria on a plate we will use an indicator solution. Because bacteria are so small, you usually cannot see them when you spread bacterial solution on the agar. By using the indicator solution we may observe the spreading pattern.

EQUIPMENT:

· One (1)prepared NaOH agar plate per student
· Tube of phenolthalein solution (one tube per team of 4 students)
· Disposable inoculating loops

PROCEDURE:
1. Obtain a pre-poured petri dish of NaOH agar, an inoculating loop, and a vial of the simulated bacteria culture from the center of the table.
2. The inoculating loop is sterile in its wrap and is disposable. Take the loop out of the wrap and dip it in the bacteria just to the top of the loop.
3. Carefully lift the lid of your petri dish (at approximately a 30 degree angle) and touch the loop to the agar.
4. Drag the bacteria solution across the surface of the agar in a zig zag pattern from the top to the bottom.
5. Have your instructor inspect your inoculation and initial his/her approval here.
6. Dispose of inoculating loops and inoculated plates in the biohazard bags.

INTRODUCTION: In this activity, you will be searching for bacteria and fungi in the environment. Microbes can are found everywhere: on floor surfaces; in the air; in soil; on chairs; behind fire extinguishers. Most microorganisms are non-pathogenic, i.e., they do not cause disease, but rather enhance the environment through their interactions with other substances (Microbes on clover roots help "fix" nitrogen in the soil, thus promoting good root formation; bacteria and fungi aid in the decomposition of organic materials; bacteria are necessary for proper digestion of food; bacteria and fungi are fundamental in the production of basic foods, as bread, cheese and chocolate!)

Your job in this exercise is to find out some of these "hiding" places of microbes. You will select four different areas in this building or outside and sample them and inoculate them onto nutrient agar plates. These plates will be incubated, and next lab, we will look at them to see what you have uncovered.

EQUIPMENT:

· One (1) nutrient agar plate per student
· Sterile cotton swabs (4 per student)
· Permanent markers
· Storage containers for inoculated plates
· Data Sheets

PROCEDURE:

1. Obtain a sterile petri dish of nutrient agar from the center of the table. KEEP THE LID ON.
2. Invert the plate. On the BOTTOM of the petri dish, with a permanent pen, divide and number the quadrants. Label the dish with your name and class period.
3. In the data section of your lab write up, use your circle template to draw a representation of your petri dish. Label it to correspond to the labels on your petri dish. You are now ready to "hunt" bacteria.
4. Use the cotton swabs to transfer bacterial cells by rubbing the fixture or object and then gently swabbing the surface of one quadrant of your petri dish. For each specimen collected, record the requested information on the Data Sheet.

Important things to remember:

1. Keep the petri dish covered as much as possible.
2. Use a new cotton swab for each sample.
3. Be sure to record the information AS YOU DO THE COLLECTING.
4. Put the petri dish, upside down, in the plate containers indicated for incubation. (The plates are stored upside down so any moisture will collect on the top of the plate and not flood the bottom part where your bacteria are growing.
5. The plates will be incubated for 48 hours at 28-37 degrees Celsius. Next lab period, we will look at them and see what has grown.

Sample # 1

a. Location: _________________________ (be as specific as possible)

b. Conditions such as moisture content. lighting, temperature

c. Degree of human contact ; any other details or information

d. Why did you select this site?

Sample # 2

a. Location: _________________________ (be as specific as possible)

b. Conditions such as moisture content. lighting, temperature

c. Degree of human contact; any other details or information

d. Why did you select this site?

Sample # 3

a. Location: _________________________ (be as specific as possible)

b. Conditions such as moisture content. lighting, temperature

c. Degree of human contact; any other details or information

d. Why did you select this site?

Sample # 4

a. Location: _________________________ (be as specific as possible)

b. Conditions such as moisture content. lighting, temperature

c. Degree of human contact; any other details or information

d. Why did you select this site?

(These exercises were adapted from "On the Microbe Trail: An Introduction to Bacteria and Aseptic Technique" by Laura Ziegenhirt, from the Access Advantage Activities Exchange.)

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