ENVR 421 Laboratory #1: Basic Bacteriology Techniques

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1 ENVR 421 Laboratory #1: Basic Bacteriology Techniques Introduction The purpose of this laboratory exercise is to familiarize you with two fundamental bacteriology techniques: the streak plate and the spread plate. The streak plate is a technique to produce isolated colonies of bacteria on a plate, which can be used to separate out different types of bacteria that are mixed together in a sample or a culture. The spread plate technique involves spreading a thin layer of liquid containing bacteria onto the surface of an agar plate. Bacteria in the liquid will spread out and form colonies on the surface of the plate, allowing us to count the number of bacteria in a sample. We will make a dilution series, which ensures that the bacteria spread onto a plate are few enough to be counted easily. The streak plate Purpose In this lab, you will streak tryptic soy agar and MacConkey agar with a liquid culture of Escherichia coli. The purpose of the streak plate is to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate. Isolated colonies represent a clone of cells, being derived from a single precursor cell. When culture media is inoculated using a single isolated colony, the resulting culture grows from that single clone. Historically, most microbiology research and microbial characterization has been done with pure cultures. Principle One bacterial cell will create a colony as it multiplies. The streak process is intended to create a region where the bacteria are so dilute that when each bacterium touches the surface of the agar, it is far enough away from other cells so that an isolated colony can develop. In this manner, spreading an inoculum with multiple organisms will result in isolation of the different organisms. Materials: Liquid culture of E. coli (one tube per student) Streaking stick four petri dishes with trypticase soy agar four petri dishes with MacConkey agar

2 Protocol 1. Label the petri dishes on the bottom (not the lid) with your name and the date 2. Take out one sterile wooden stick and hold it in one hand without touching it to anything else. 3. Holding the culture tube in your other hand, open the cap 4. Insert the stick into the culture tube until the end touches the liquid 5. Replace the cap of the test tube and put it back into the test tube rack. 6. Remove the lid and hold the plate in your hand 7. Touch the stick to the agar 8. Spread the bacteria in the first sector of the petri dish by moving the loop in a back and forth manner across about one-third of the dish, a zig-zag motion. Make the movements close together and cover the entire first region. The stick should glide over the surface of the agar; take care not to dig into the agar. 9. Discard the stick 10. Obtain a new stick for the second sector 11. Touch the stick to the first sector. Streak the second sector by passing the stick over the first sector 2-3 times, then streaking another third of the plate. 12. Discard the stick. 13. With a new stick, repeat for the last third of the plate D. Sue Katz Biology Department Rogers State University Claremore, OK

3 The dilution series Purpose We will count the bacteria in a sample using spread plating. Usually, you need less than 300 colonies on a standard petri dish for them to be easily countable. More than that, and the colonies grow together, making them difficult to distinguish. When measuring the number of bacteria in a sample, diluting the sample ensures that the colonies spread plated on the petri dishes are in a countable range. See the section at the end for an explanation of the dilution series. Principle The cultures we are working with, and many environmental samples, will contain high numbers of bacteria. If you spread these samples on a plate, there are so many bacteria that they will all grow together in one big continuous smear covering the entire surface of the plate, and you will not be able to count anything. To obtain a countable number of bacteria on a plate, we make a dilution series of the culture. Usually dilutions are done in a 10-fold series. This makes it simple to calculate the number of bacteria in the original sample based on the number of dilutions and the number of colonies on a plate containing that dilution. Materials Tubes of PBS Micropipette Pipette tips Vortexer Protocol 1. Set out 10 dilution tubes in a rack 2. Label the tubes 3. Set the micropipette to 100 µl 4. Put a tip on the end of the pipette 5. Vortex the culture tube 6. Open the tube and draw up 100µL with the pipette 7. Open the first tube and place the 100µL of culture inside 8. discard the tip 9. Close tube tightly and vortex

4 10. Open this same tube and draw up 100µL with the pipette 11. Open the second tube and place the 100µL inside 12. discard the tip 13. Close tube tightly and vortex 14. Repeat until you reach the last tube The spread plate Purpose One method of distributing bacteria evenly over the surface of an agar plate medium is commonly referred to as the spread plate method. Classically a small volume of a bacterial suspension is spread evenly over the agar surface using a sterile bent glass rod as the spreading device. The goal in evenly distributing the bacterial suspension is typically to permit the growth of colonies that can subsequently be enumerated and/or sampled following incubation. Each plate is spread with a single inoculum of the bacterial suspension. Principle Using the spread method a small volume of a bacterial suspension is distributed evenly over the surface of an agar plate using a smooth sterilized spreader Materials MacConkey plates Glass hockey sticks Beaker of ethanol Bunsen burner Micropipette tips Dilutions of bacterial culture Turntable Protocol 1. Label two MacConkey plates to correspond with each of your dilution tubes 2. Put three empty petri dishes on the benchtop 3. Dip three glass hockey sticks in the jar of ethanol 4. Pass them through the Bunsen burner CAREFULLY to flame sterilize

5 5. Place them in the empty petri dishes and let them cool 6. Start with the lowest dilution (the one with the least bacteria) 7. Put an open plate on the turntable 8. Use the micropipette to take 100µL of liquid from the lowest dilution tube 9. Pipette the liquid onto the surface of the plate 10. Take a glass hockey stick and move it lightly back and forth over the surface of the plate while spinning the turntable 11. Dip the glass hockey stick in the jar of ethanol 12. Pass it through the Bunsen burner 13. Place it back in the empty petri dish 14. Replace the lid and turn the petri dish upside down 15. Repeat with the remaining plates, alternating glass hockey sticks to let them cool

6 Original sample: 10 6 bacteria per milliliter 100µL=10 5 bacteria 10 5 bacteria (0.1 ml of original 100µL=10 5 bacteria 100µL=10 4 bacteria first dilution: 10 5 per ml 100µL=10 4 bacteria 10 4 bacteria (0.01 ml of original 100µL=10 3 bacteria second dilution: 10 4 per ml 100µL=10 3 bacteria 10 3 bacteria (0.001 ml of original

7 100µL=10 2 bacteria third dilution: 10 3 per ml 100µL=10 2 bacteria 10 2 bacteria ( ml of original 100µL=10 2 bacteria fourth dilution: 10 2 per ml 10 bacteria ( ml of original Dilution calculation: The last dilution has 10 colonies on the plate. After all the dilutions, there is a total of ml of the original sample on the last plate. 10 colonies / ml= 10 6 bacteria per ml