1. Science Projects using Bacteria Aseptic Lab Technique The following guidelines are meant to assist you in following proper sterile technique during bacteria experiments. By maintaining a sterile environment you avoid contaminating you, your bacteria and thus your results.

2. Pouring Plates In order to grow bacteria, you must provide a nutrient rich environment for them. This step will show how to create that environment by using Petri dishes with agar added to them. 45 cp/ 60 honors-my plates are divided into 4=4 trials

3. Prepare agar according to the directions on the bottle. Ex. add in a certain # of grams of agar to 1L of distilled water and boil…. (Nutrient agar uses 23grams to a liter) Allow agar to cool enough so that you can pick it up beaker with gloved hand. #1-day one

4. Make sure your Petri dish is up right (this means the lid is the larger piece and overlaps. While agar is heating, take your Petri dish and label the BOTTOM with the date, your initials, and a way to identify your bacteria or variables. Mrs. K dishes have 4 sections which are labeled 1,2,3,4 #2

5. Lift the lid off the Petri dish at an angle, just enough to pour in the agar into each of the 4 sections. Pour until the bottom surface is covered. #3

6. #4 Replace the lid at an angle as shown below, allowing steam to escape. Plates should take 30-45 min to set, Can be stored in a fridge for several days.

7. #1 Plating bacteria-day 2 Clear lab bench of all materials. Put on GLOVES Wipe down the surface with 70% ethanol. Gather materials: Prepared agar plates Test tube of Bacteria and rack 2 Small beakers Pipette with tips to dispense bacteria masking tape Alcohol (or you may need paper disks and tweezer or q-tips) Your variables –ex. soaps

8. #2A ONLY if using bacteria that I have bought Pour your bacteria into a clean beaker Set pipette to 50microliters. Withdraw 50 microliters into pipette

9. #3 A ONLY if using bacteria that I have bought Lift the lid of your plate just enough to insert to the pipette and add 50 microliters of bacteria to one section of plate-plate has 4 sections=4 trials Do this on all sections in each plate. Rub a q-tip around SURFACE of the agar plate in a z shape Dispose of q-tip in hazardous waster container DO NOT STAB THE AGAR. You want the bacteria to stay on the surface!

10. #3B Other ways to inoculate plate Use q-tip 1. Insert q-tip into sterile water 2. Rub q-tip on surface you want to obtain bacteria from- (ex. door handle) 3. Gently rub q-tip (z-shape rub) on agar surface of one section of your petri dish 4. Dispose of q-tip in container for autoclaving 5. Repeat with other q-tips in other sections of plate 6. When all plates are done, they MUST be taped closed 7. Put in warm incubator 24-48 hours Can be done for “where do you find the most bacteria” or “which cleaners kill the most bacteria on surfaces.”

11. #4 Your variable depends on your method 1. Dip a disk into your soap and insert it into center of one section of plate. 2. One variable in one dish One disk in each section

12. 1 way to Use Cleaners 1.Mark off a section of the surface you want to test (ex-desk top) 2. Use a clean, damp paper towel to rub test area (all the desk top) 3. Section off this area for each of your cleaners (ex. below) 4. Label areas 5. Use q-tip on each area as control=no detergent- put in one section of labeled petri dish 6. Use detergent on each area as directed on label 7. Then use new q-tip on each cleaned area and apply to petri dish Data will compare control to cleaned area

13. #5 Replace the lid on your petri dish. Tape it closed with masking tape Incubate your petri dish according to your procedure…usually 37 degrees C Petri dishes are turned upside down in incubator Clean up area with alcohol. Return all materials to teacher. Take gloves off. Wash hands.

14. Bacteria may look like this, the whitish colonies, or like this, diff. kinds. Not the white disks. Bacteria around the school.

15. How will you measure? You can measure the % of the plate covered by bacteria. It’s really an estimate. I’ll show you personally. You can measure the zone of inhibition You can count the number of colonies.

16. You may “count” bacteria using a grid like below. This is better explained in person. Choose 4 squares randomly and estimate (%) coverage in those 4 squares. Average the 4.

17. Can measure bacteria using a zone of inhibition = clear area where no bacteria grows. Bacteria is applied to plate in z shape as previously shown. Soak a small disk in your detergent and add to plate, see picture. Tape, incubate. Measure the clear area from the disk to where no bacteria grew.

18. All dishes are taped closed and never opened again They will be “pressured cooked” to kill anything later on. This is called an autoclave.