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On the Microbe Trail:
An Introduction to Bacteria and Aseptic Technique

Laura Ziegenhirt



(Written in collaboration with Jenny Cuccinello. Adapted from an activity developed by Fellows in the Howard Hughes Biology Institute.)

TYPE OF ENTRY:

  • lesson/activity

TYPE OF ACTIVITY:

  • hands on
  • introduction/reinforcement of lab skill
  • simulation

TARGET AUDIENCE:

  • Life Science
  • Biology
  • Advanced/AP Biology
  • Genetics
  • Biotechnology
  • ESL
  • LEP
  • NEP


ABSTRACT OF THE ACTIVITY:

In this series of exercises, the students will predict the conditions necessary for bacterial growth, test their predictions and at the same time practice the aseptic techniques and safety procedures needed when working with bacteria. In the first exercise, the students' prior knowledge of bacteria is determined including the conditions they believe necessary for bacterial growth. Armed with petri dish and cotton swabs they are dismissed to the campus to obtain samples from the places with these conditions. Students learn how to prepare their lab areas for microbiology work, pour and swab plates, and seal, label and store plates.


BACKGROUND INFORMATION:

This activity is a series of exercises that introduces students to bacteria and aseptic microbiology lab techniques. The content exercises and the lab skill exercises run side by side. The students will determine the conditions necessary for bacterial growth as well as the techniques used to culture and isolate bacteria. To complete the series of exercises takes one full 55 minute class period on the initial day and two half periods on the second and third days. However, there are many logical stopping points and you can tailor it to fit your time frame.

There is no prep time needed for the first exercise. The prior knowledge activity works well as a journal entry and, if done at the end of the period prior to beginning the activity allows you to read through their responses and get an idea of what the students know and don't know. I prefer to use the four square method but the questions could be answered in narrative form . I also allow the students to draw pictures if necessary.

In exercise two the students learn how to prepare their work area. We used spray bottles filled with a solution of 50% Lysol. A 10% bleach solution is very effective also. The simulated "bacto-broth" solution is made from fluorescent Elmer's glue (any color) diluted with water. One small bottle of glue should suffice 5 classes. Spread on the students' lab tops and water faucets before each class. The "bacto-light' is a hand held black light. Any missed spots will fluoresce nicely and show the students if they were thorough.

The students practice swabbing plates in exercise three. In order to assess student competency we devised a method so we could see their swabbing pattern. Prepare a 3% agar solution. You want it thick to prevent the phenothalein solution used to simulate bacteria from absorbing in too quickly. Add 25 mL of NaOH to the melted agar. Pour the agar into petri dishes and let cool. Prepare a phenothalein solution. Adding a small amount of glycerol to the phenothalein solution helps to prevent quick absorption of the solution into the agar. Separate into test tubes (enough for each working group). These plates can be reused. Simply melt down the microwave, swirl until clear, and let cool.

In the concluding exercise the students are sent on a microbe scavenger hunt. We used this activity to show the students how to pour their own plates as well as how to label them, seal them and store in the incubator. For our incubator we used two plastic drip trays from the garden nursery and a heating pad. Works great!


ACTIVITY DESCRIPTION

MATERIALS:

exercise two: disinfectant in spray bottles (50% Lysol solution), old rags, "bacto-broth" and "bacto-light" (see background notes)

exercise three: prepared NaOH agar plates and phenolthalein solution, inoculating loops, flame source if using metal inoculating loops

exercise four: nutrient agar, sterile petri dishes, cotton swabs, permanent markers

PROCEDURE:

Exercise one:
In your journals answer the following four questions.

1. Where I think bacteria grow

2. What I think bacteria look like

3. How bacteria can be bad/good

4. Questions I have about bacteria

Using the conditions you described in question one, make a list of places you have access to on campus that you would expect to find bacteria.

Exercise two:
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 disinfectant 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.

Obtain a spray bottle and cloth from the equipment table. Wipe down your lab station and surrounding water fixtures and handles thoroughly. When you are finished, request an inspection with the "bacto-light" and your teacher. Any missed areas will need in a recleaning! Make sure to get a stamp on your lab write up to indicate successful disinfecting.

Exercise three:
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 on. 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.

Obtain a pre-poured petri dish, an inoculating loop and a vial of the simulated bacteria culture from the equipment area. 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. Carefully lift the lid of your petri dish (at approximately a 30 degree angle) and touch the loop to the agar. Drag the bacteria solution across the surface of the agar in a zig zag pattern from the top to the bottom.

Bring your plate to your teacher for inspection.

Exercise four:
Your teacher has prepared a stock solution of nutrient agar. It has been cooling so that it can be poured. Obtain a sterile petri dish. KEEP THE LID ON. 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 according to the instructions from your teacher. Bring your petri dish to the front where the agar is cooling and use the transfer pipette to put 15 ml of agar in your dish. Do this by lifting the lid at about a 30 degree angle . Close the lid and swirl gently to evenly distribute the agar. Leave at your lab station while you prepare your data table.

We want to spread the bacteria on the surface of the agar to try to isolate a group of bacteria cells called a colony. Please be very careful when swabbing the surface so as not to tear the "hardened" agar.

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. 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 following information:



   a. Location: _________________________ (be as specific as possible)
   b. Conditions such as moisture content. lighting, temperature
   c. Degree of human contact
   d. Any other details or information

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, where your teacher directs for incubation. We store the plates upside down so any moisture will collect on the top of the plate and not flood the bottom part where your bacteria are growing.


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