Zone of Inhibition Assay

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The night/ day before  create liquid cultures of the two bacteria that wish to work with

In the hood:

1. Heat bock set to 45 degrees

2. Vortexer

3. 10ul, 200ul, and 1000ul pipettor

4. Auto pipettor

5. 10ul, 200ul and 100ul pipet tips

6. 24 well plate

Create a beaker of verkon

1. Fill a beaker with 500ml of water

2. Add two ‘john dear’ spoon full of Verkon

Zone of Inhibition

1. Set the heat block to 45 degrees

2. Add 8 empty 13mm tubes with caps to the block

3. Warm up the BHI agar in the science microwave

a. Start at power level 6

b. Watch  if starts to boil lower the power level

c. Should take about 10 min

4. When all melted lave to cool for a little so it is not boiling

a. To cool a little faster put in a cool water bath

5. Add 1ml of agar to each well of a 24 well plate (carefully to not form bubbles)

Note: multiple plate can be made in advance, but don’t leave for longer then a week

6. Label plate

7. If the heat block says it has reached 45 degrees, feel the tubes, if they feel warm then proceed, if no wait a little longer

8. Warm the top agar

a. Power level 4

b. Should take about 5 min

9. Add 3ml to each tube

10. Wait until the heating of the block is fairly constant (the light is flicking on and off)

11. Read the optical densities of the two bacteria, to make sure they are at about .3

12. Take the catalase out of the 4 degree freezer

13. Take the TTC and optochin out of the -20 degree freezer

14. Let TTC and optochin thaw

15. Vortex the tube of Moraxella

16. Add 30ul of Streptococcus to one of the tubes with top agar

17. Vortex

18. Add 500ul to the top row of wells (skipping column 1)

19. Vortex the TTC

20. Add 3ul to one of the tubes with top agar

21. Vortex the Streptococcus and add 30ul to one tube that you just added TTC to

22. Vortex

23. Add 500ul to the wells in the second row (skipping column 1)

24. Repeat steps 15-23 with Moraxella, except using row 3 instead of 1 and row 4 instead of 2

25. Make sure top agar has solidified before moving on to the next step

26. Add 300ul of Moraxella to a tube containing top agar

27. Vortex

28. Add one drop (10ul) to wells A1, A3, B1, B3

29. Add 30ul of catalase to the tube

30. Vortex

31. Add one drop (10ul) to wells A4 and B4

32. Add 3ul of TTC to the tube

33. Vortex

34. Add one drop (10ul) to wells A5, B5

35. Add 3ul of optochin to the tube

36. Vortex

37. Add one drop (10ul) to wells A6 and B6

38. Repeat steps 24-35 with Staphylococcus and only using 4ul for a dot

39. Wait until top agar dries

40. Place in the 34 degree incubator

Clean up

1. Dump out all remain solution form tubes in verkon

2. Spray all tubes that contained pathogens with bleach

3. Vortex

4. Leave in verkon (for at least 1 hour)

5. Dup out the verkon in sink

6. Throw out all tubes in glass waist

7. Throw out all pipet tips in biohazard bins

8. Rinse tube caps

9. Rinse beaker with tap water and then miliQ water