On the Microbe Trail: Bacteria and Aseptic Technique Part of an Access Excellence Lesson http://www.accessexcellence.org/AE/AEC/AEF/1996/ziegenhirt_microbe.html Laura Ziegenhirt Modifications/Edits by Rachel Hughes Time: 2 class periods Preparation 1 hour Time: Materials Prepared NaOH agar plates & phenolphthalein solution, Inoculating loops, flame source if using metal inoculating loops, tape, storage box, low watt lamp, markers Prepared agar mixes available from Carolina Biological either Pre-measured Dehydrated Media or Luria Broth Agar Base - Abstract As a result of this lesson students learn how to pour, label, streak, seal and store plates in an incubator. Students will also be introduced to some of the microbes that live in their environment. 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. Objectives Students will be able to: 1. Pour, label, streak, seal and store plates in an incubator 2. Identify areas in their environment that provide a rich fauna of microbes once they have been swabbed. 3. Identify areas of contamination through a laboratory report. National Science Education Standard Content Standard A – Science as Inquiry Understandings of Scientific Inquiry Results of scientific inquiry--new knowledge and methods--emerge from different types of investigations and public communication among scientists. In communicating and defending the results of scientific inquiry, arguments must be logical and demonstrate connections among natural phenomena, investigations, and the historical body of scientific knowledge. In addition, the methods and procedures that scientists used to obtain evidence must be clearly reported to enhance opportunities for further investigation. Content Standard C – Life Science Interdependence of organisms Living organisms have the capacity to produce populations of infinite size, but environments and resources are finite. This fundamental tension has profound effects on the interactions between organisms. Content Standard F: - Science in Personal and Social Perspectives Personal and Community Health Teacher Background Resources http://www.accessexcellence.org/AE/AEC/AEF/1996/ziegenhirt_microbe.html http://www.fayette.k12.il.us/isbe/science/pdf/sci_13.pdf Activity Before Class: Prepare a 3% agar solution. One liter of agar solution (3g of Agar agar per l00 mL) of water will be sufficient for between 30- 40 plates. Place the agar and water in a flask and cover. Using a stir hot plate dissolve the agar into solution. Start the stir bar on low and increase slowly to avoid vigorous stirring that might produce bubbles. If you want to use a microwave avoid vigorous stirring because bubbles may form. The agar solution should be thick to prevent the phenolphthalein solution (used to simulate bacteria) from absorbing in too quickly. Add 25ml of NaOH to the melted agar. Pour the agar into the Petri dishes and let it cool. Prepare a phenolphthalein solution by adding 0.05 grams in 50 mL ethanol plus 50 mL water. Adding a small amount of glycerol to the phenolphthalein solution helps to prevent quick absorption of the solution into the agar. Separate into test tubes (enough for each working group). In addition, place some “bacto-broth” from the previous lesson at each group’s work station. You will check their clean up procedure at the end of the activity. The plates can be reused. Simply melt down the microwave, swirl until clear, and let cool. 1. Start by asking the students why we might want to grow bacteria (to isolate and identify a nuisance bacterium, or to grow useful bacteria). 2. Explain that 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 a gentle touch. Don’t rip the agar. 3. Tell students: “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.” 4. Let them know that one of the important outcomes of streaking plates is to end up with individual colonies at the end of their spread (see diagram). This will take some practice. 5. Instruct students to: i. 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. ii. Take the loop out of the wrap and dip it in the bacteria just to the top of the loop. (Hold the loop downward. Why?) iii. Carefully, (still holding the loop downward), lift the lid of your Petri dish at approximately a 30 degree angle and gently touch the loop of the agar. You don’t want to deposit all the bacteria in one place. iv. Drag the bacteria solution across the surface of the agar in a zigzag pattern from the top of and then to the bottom of the plate. (See the diagram) v. Bring your plate to the teacher for inspection. Day 2: Microbe Scavenger Hunt Before class: Prepare solutions of nutrient agar or luria broth. The easiest and most time efficent way to do this is to purchase a premixed solution or powder. Carolina Biological have these available as Pre-measured Dehydrated Media or Luria Broth Agar Base. Once it is heated let it cool enough so that students can pour their own plates. 1. Inform students that today they will be taking part in a Microbe Scavenger Hunt. As they do this they will learn how to pour, seal, label and store plates. 2. Go over the following instructions with students before they begin: i. Obtain four sterile Petri dishes. (Why do they have to be sterile?) KEEP THE LIDS ON. ii. Label the dishes with your name and class period on the outside edge of the Petri dish. iii. Bring your Petri dishes to the front where the agar is cooling and use the transfer pipettes to put 15ml of agar into the Petri dish. Do this by lifting the lid about 30 degrees. Make sure that you do not touch the pipette to the outside of the Petri dish (why?) iv. Close the lid and swirl very gently to distribute the agar. Leave the Petri dishes at your lab station while you prepare your data table. v. In the data section of your lab write-up, draw a representation of one Petri dish. Label the diagram to correspond to the labels on your Petri dish. vi. The agar should now be hardened. You are now ready to “hunt” bacteria! Remember your goal will be to spread bacteria on the surface of the agar to isolate a colony of bacteria. vii. Use a different cotton swab and Petri dish for each object (Why is this important?) in order to transfer bacterial cells by rubbing the fixture or object and then by gently streaking the surface of one of your Petri dishes in the same manner you used the inoculating loop. viii. For each specimen collected, record the following information: a. Location: _______________________(be as specific as possible) b. Conditions such as moisture, lighting, temperature c. Degree of human contact d. Other details or information ix. Once you have collected all the specimens use the tape that your teacher supplies to seal the Petri dish shut. IT CANNOT BE OPENED FROM NOW ON! This is a very important safety measure. x. Place the Petri dishes upside down in the storage box at the front of the class. (In your notebooks note why you think putting the Petri dishes upside down is important.) xi. You will need to come back periodically to check on your dish. You will check tomorrow and on the following day. Once all students have placed their dishes upside down in the storage box the teacher will close the box and place the low watt lamp onto the storage box. Ideally the temperature inside the box should be 98.6 degrees Fahrenheit. Why is it considered ‘ideal’ that the temperature be 98.6 degrees Fahrenheit? Following day xii. Today you will check for any growth on the Petri dishes. xiii. Remember it is very important not to open the Petri dishes. xiv. Note any difference in growth rates or types of growth among the quadrants. xv. Use a microscope or magnifying loop to look at the Petri dish. Describe what you see in terms of color, texture and shape. xvi. Finally write up what you did, pay particular attention to how the procedures reduced contamination and improved safety. Use diagrams to show what you saw on the plates. xvii. Place your Petri dishes in a location assigned by the teacher. It is important that they be disposed of correctly. Once all the Petri dishes have been collected, dip them into a bleach solution before placing them in appropriate bags for biological disposal. Now clean your lab station area again. Ask your teacher to inspect your area. Students should ask you to check their area for completeness of clean up. Using the “bactowand” described in the previous lesson, check their desks. Embedded Assessment 1. Questions throughout the exercise allow for assessment of students’ understanding of aseptic techniques and protocol. 2. The written laboratory report for an assessment of understanding. 3. Using the student-developed rubric to assess their own implication of protocols, especially the clean-up of their area.