Bacteria We look not at the things which are seen, but at the things which are not seen. For the things which are seen are temporal; but the things which are not seen are eternal. 2 Cor 4:18 Introduction Bacteria are simple unicellular prokaryotes, having a nucleus but no organelles and inhabit virtually every environment on earth. In this experiment you will test several environments for the presence of bacteria. Although many bacteria are harmless, some bacteria cause disease. Microbiologists are scientists who study microorganisms such as bacteria. To study bacteria, they often use a technique called microbial culture. A microbial culture (or culture for short) is a method to multiply the microbes from a just a few cells to many thousands or millions. The cultures are grown on specific culture media under conditions that promote fast growth and reproduction of the microbes. In laboratories today, scientists grow bacterial cultures in a Petri dish filled with culture media called nutrient agar. Agar is a gelatinous-type substance derived from seaweed and enriched with beef serum on which many bacteria will thrive. In early laboratories, scientists used gelatin and beef nutrients which is what you will use today to make Petri dishes to grow cultures. Learning Objectives: Use an early method to build your own Petri dish Use the Petri dishes to test different surface environments for the presence of bacteria Materials Required: From Biology Kit Measuring Cups Measuring Spoons Pipette Student Supplied 3 shallow clear containers with lids (such as deli containers) 12 grams plain Knox Gelatin -or- 1-½ packages 1 beef bouillon cube -or- 1 tsp granules 2 tsp sugar Cotton swabs Antiseptic wash (such as for hands) Safety ALWAYS wear gloves when working with Petri dishes during this experiment. Although most bacteria are harmless, assume that the bacteria you are handling are hazardous. Do not breathe in or ingest growing bacteria Keep the Petri dishes away from children and animals. Never open the Petri dishes. Boil the gelatin carefully as it will tend to boil over quickly and may cause burns. DO NOT OPEN THE PETRI DISH once the bacteria have begun to grow. When finished with the experiment, flush with chlorine bleach and dispose in trash. Wash hands with soap and water after handling Petri dishes and samples Experiment Introduction Agar contains nutrients which will allow bacteria to grow and reproduce in colonies. Bacteria will be collected by moistening a cotton swab, running it over a surface you suspect holds 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 1
bacteria, and then rolling that cotton swab over a Petri dish. This form of transfer from a surface to a Petri dish by means of an inoculator (cotton swab) is called bacterial culturing. 1. Prepare Gelatin (Agar) Mixture (Directions for 3 Petri dishes): Mix together the following: 1 cup water 1 ½ packages plain gelatin (12 grams) 1 beef bouillon cube (or 1 tsp beef granules) 2 tsp sugar Bring mixture to boil on stove, stirring frequently Watch carefully until the gelatin has completely dissolved about 3 minutes Note: Do not let mixture boil over. The mixture should not have solid particles floating. Note: Gelatin requires the boiling time in order to become gel-like. Make sure that the mixture comes to a boil, without boiling over Remove the mixture from the heat and allow to cool about 15 minutes Take the lids (the larger piece) off the containers Carefully, pour the warm mixture so that the containers are between 1-3 1 full 2 Set the lid on the bottom portion so that it is slightly ajar following: This is to allow the escape of excess moisture during the cooling process Allow the agar dishes to cool completely and harden Replace the lid on the base Note: If the Petri dishes are not going to be used right away (for more than 1 hour), place the lid back on the base and turn the Petri dish upside down. This prevents moisture from ruining the agar. Store them in a cool place such as a refrigerator. Plates should be used within 2-3 days. 2. Divide your 3 Petri Dishes in the following manner: When working with the Petri dishes, keep lids on whenever possible to prevent contamination Turn the dish upside down, use a permanent marker to divide the base into fourths With 3 Petri dishes, you have 12 sections to use 3. Mark one Petri dish in the following manner: On one quarter section write Control No surfaces will be tested, but test your ability to keep the plates and swabs clean while performing the experiment Mark one quarter section as With Antiseptic ; and an adjacent one as Without Antiseptic Here you will streak samples from the nastiest thing you can think of on BOTH quarters (dog s teeth, bathroom bowl, grocery bathroom sink ) 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 2
But, on the quarter marked With Antiseptic, you will place 1-2 drops of handsanitizer in the middle The last quarter section can be marked with a sample of your choice 4. Make your Plan for Petri dishes: At this time, come up with a plan for obtaining 11 surface samples Try to think of places where bacteria often grow unchecked Avoid areas frequently cleaned with soaps or antibacterial detergents If the samples are not all from your home, you can transport the cotton swabs in separate baggies On the base, write the name of the surface that you will test Identify the 11 surfaces sampled in Column 1 of Table A 5. Streak the Plate: Use a clean cotton swab for each sample If the surface you are testing is not wet, dampen the cotton swab with water Roll the cotton swab on the selected surface be sure to cover all sides of the swab Remove the lid of the Petri dish and lightly draw a zig-zag line on the quarter designated for that surface Start at the top and work to the bottom (or center), working back and forth as shown, rolling the cotton swab as you draw the line (see figure) Repeat for all 11 surfaces For the Control: Do not touch the cotton swab on any environmental surface Dampen with water and streak it on the plate For the With Antiseptic: First, streak with the same source as streaked on the Without Antiseptic Second, take a pipette and place 2 drops of antiseptic in the middle of the streak Third, take a permanent marker and outline where you have dropped the antiseptic Later, you will measure how far the antiseptic influence reaches Make a hypothesis of the effect of the antibacterial agent 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 3
6. Incubate: Close the lids of the Petri dishes Secure the edges with plastic wrap Invert the dishes (agar side up) to prevent condensation from falling into the bacteria Place the Petri dishes in a warm dark place for about ~3 days, more or less Warm: around 98 o F (37 o C); such as near heater, laptop, cable box Dark: such as in a box, a closet, drawer 7. Data Analyses and Conclusions When the Petri dishes have growth, examine them for the different types of bacterial growth Place the kinds and numbers in Table A Take a ruler and measure the centimeters between the place where Antiseptic was place and the first sign of bacterial growth Answer Question 1 in Data Analysis Perform data analyses and make your conclusions 8. Disposal Please make sure you dispose the Petri dishes properly Flood the Petri dishes with chlorine bleach in a baggie for 20 minutes After emptying the bleach, seal the Petri dishes in a larger bag and dispose in trash 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 4
Lab Report for: Table A: Bacterial Summary of Results Three Petri Dishes Sample Surface Petri Dish 1 # different Types Color and Description of Control Note: All lines may not be needed Color, Shape and Description of Location: (surface, center of agar, both) Smell or other Observations Sample Surface Petri Dish 2 # different Types Color and Description of Color, Shape and Description of Location: (surface, center of agar, both) Smell or other Observations 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 5
Sample Surface Petri Dish 3 # different Types Color and Description of Color, Shape and Description of Location: (surface, center of agar, both) Smell or other Observations Data Analysis 1. Antibacterial: a. Describe what you streaked on the two quarters of the plate marked With Antiseptic and Without Antiseptic. b. What kinds of colonies grew on the portion that did not have antiseptic? c. Describe the effect of the antiseptic. d. How many centimeters were between the antiseptic and first sign of bacterial growth? Explain what you think happened. 2. Which surface grew the most bacterial species? Explain. Identify the number of colonies and their shapes 3. Was your Control plate free of bacteria? Why or why not? 4. Which result and growth was the most interesting and surprising? Explain in detail. 5. After performing this experiment, might there be a change in how you perform cleaning duties? Explain. 2016 Catholic Initiatives in Math and Science, LLC All Rights Reserved 6