Investigating the Effect of on Bacterial Growth Developed by Ana Caldeira and Amanda Tsoi; HHMI Summer Workshop 2013

Transcription

1 Name Date Investigating the Effect of on Bacterial Growth Developed by Ana Caldeira and Amanda Tsoi; HHMI Summer Workshop 2013 Introduction You can find bacteria everywhere, even on your desk and on your skin. Although they are much too small to be seen individually without the help of a powerful microscope, you can see evidence of their presence by growing colonies in this lab. There are many variables that can affect the growth of bacteria. In this lab, you will be investigating the effect of a natural product on B. Subtilis bacterial growth. B. subtilis is rod-shaped bacteria found in soil and in the human gut. When it is in extreme environmental conditions, it has the ability to form a tough, protective endospore, allowing it to hibernate safely. When favorable environmental conditions return, the endospore can convert to a growing bacterial cell. Within 24 hours, one bacterial cell will divide by binary fission and produce over a million. This population of cells is called a colony. Because we only want to investigate B. Subtilis bacterial growth, we do not want to contaminate our experiment with other species of bacteria. In this lab, we will be using sterile technique, which means you will need to: o wear gloves, goggles, and lab coats o spray lab station with 70% ethanol and wipe clean before and after lab work o do not touch the inside of the Petri dish (including the inside of the Petri dish lid) o only remove the Petri dish cover when absolutely necessary o whenever the lid is removed, it should be held over the plate as a shield to prevent dust and bacteria from falling into the Petri dish In order to form your hypothesis and title your lab, you will need to decide what variable you would like to test. It needs to be a naturally occurring substance. Some examples are spices (i.e. cinnamon, turmeric), food plants (i.e. garlic, ginger), outdoor plants (i.e. pine needles, bark), etc. Some of these are provided in the lab; if you would like to test something else, you may bring it to class. Hypothesis If bacteria are exposed to (experimental variable), then the population of bacteria will (increase or decrease). Materials o 70% ethanol spray bottles o 2 petri dishes (plates) o LB agar (algae-based gelatin with nutrients for the bacteria to grow on) o B. Subtilis bacterial spores o parafilm (to seal plates) o 150mL beaker o hot hands o stirring rod o pipette o *your experimental variable*

2 Procedure Day 1 1. Put on goggles, gloves and lab coat. 2. Spray down work area with 70% ethanol and wipe clean. 3. Obtain two Petri dishes and on the bottom label one Control and one Experimental Variable. Put your initials and date on both. Refer to the diagram on the right. 4. Using the hot hands, obtain a 150mL beaker with 30mL hot LB agar. 5. Using the hot hands, carefully pour half of the LB agar into the Control Petri dish. Cover the plate immediately after. 6. Add a small, grape-sized amount of your experimental variable to the remaining LB agar in the beaker. Ensure that your experimental variable is in a homogenous (uniform), mushy or liquid form, without chunks. Stir this into your LB agar. 7. Using the hot hands, carefully pour the rest of the LB agar-mixture into the Experimental Variable Petri dish. Cover the plate immediately after. 8. Bring your plates to the storage area. Make sure they are properly labeled. Procedure Day 2 1. Put on goggles, gloves and lab coat. 2. Spray down work area with 70% ethanol and wipe clean. 3. Bring your plates from last class to your teacher. He/she will pipette 1mL of B. Subtilis bacterial spores onto your plate for you. 4. With the plate covered, gently tilt the plate from side to side to make sure the entire surface of agar is covered with the liquid containing the spores. 5. Seal both plates with parafilm and bring them back to the storage area. Procedure Day 3 1. Put on goggles, gloves and lab coat. 2. Spray down work area with 70% ethanol and wipe clean. 3. Bring your plates from last class to your station. 4. Observe your bacterial colonies. Use the key below to write your observations in your qualitative data chart. Criteria Used to Describe Bacterial Colonies Form - What is the basic shape of the colony? For example, circular, filamentous, etc. Elevation - What is the cross sectional shape of the colony? Turn the Petri dish on end. Margin - What is the magnified shape of the edge of the colony? Surface - How does the surface of the colony appear? For example, smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled), etc. Opacity - For example, transparent (clear), opaque, translucent (almost clear, but distorted vision, like looking through frosted glass), iridescent (changing colors in reflected light), etc. Pigmentation - For example, white, buff, red, purple, etc. From: projects/project_ideas/microbio_interpreting_plates.shtml

3 5. Tape your plate to graph paper. 6. Count the colonies of bacteria from top right to bottom left, as shown in the diagram below. If you have more than ~200 colonies, divide your plate into 4 quadrants, only count one quadrant, and multiply this number by Record your data in the quantitative data chart. 8. Clean up (keep gloves, goggles and lab apron on): a. With gloves on, submerge one plate at a time in the bleach-water container. While under water, remove the cover to expose the bacteria to the bleach. This will kill the bacteria. Take the plate out of the water and put it in the trash. b. Return all materials to their appropriate storage spaces. c. Spray down work area with 70% ethanol and wipe clean. d. Throw away gloves, return goggles and lab apron.

4 Data Qualitative Data Chart Observations Control Plate Experimental Plate Form Elevation Margin Surface Opacity Pigementation Smell Other Quantitative Data Chart Plate Control Number of Colonies of Bacteria Experimental Variable

5 Analysis Complete the bar chart below based on your quantitative data (for your lab report, you will need to insert a bar chart designed in Microsoft Excel). Title your graph and label the axes. The x-axis should be labeled with your independent variable (this rate/value remains the same regardless of the other variable). The y-axis should be labeled with your dependent variable (the value depends on the independent variable). Write three factual statements about your data Conclusion Answer the following question in paragraph form: A. Use your quantitative data to explain whether or not your experimental variable impacted bacterial growth. B. How does this compare to your hypothesis? C. Provide an idea for why you obtained the results that you did. Use at least one website or other source. D. Explain why you are unable to make a definitive conclusion about the impact of your experimental variable on bacterial growth based on this experiment alone. E. Did you make any mistakes? Explain. F. At what points during the lab could you have been more accurate (measuring, counting, following procedures, etc..)? G. If you performed this lab again, what would you do differently to obtain more accurate results?

6 Bibliography For all websites used, include the title, author, date and URL. For all other resources, include the title, author and date.