12.2: Procedures
- Page ID
- 16028
A. ELISA
Per Table: 1 “strip” of test wells; viral antigen solution (EBV), positive and negative control solutions, anti- IgG peroxidase conjugate (second antibody solution), Phosphate buffered saline (PBS, for washing steps), substrate solution, 4 “unknown” donor serum samples.
Note
Follow all directions carefully. Be particularly careful to avoid cross-contamination of the solutions.
1. Label the first 6 wells of your strip 1-6.
2. Add 50 µl of EBV (viral antigens) to each of the 6 wells using the P200 pipettor.
3. Incubate for 5 minutes at room temperature.
4. Remove the liquid using a transfer pipet. Discard the liquid in the beaker labeled “liquid waste.”
5. Using a transfer pipet, add PBS to each of the 6 wells until almost full (do not allow them to spill over). Be careful not to touch the transfer pipet to the well.
6. Remove the liquid from the well as demonstrated by your instructor (Important: do NOT use the PBS transfer pipet to remove the liquid- this will result in contamination of the PBS with antigen.
7. Add 50 µl of the appropriate test reagent to each well as outlined in the table below. Use a new pipet tip for each solution!
| Well # | Test Reagent | Description |
| 1 | (-) PBS | Negative control |
| 2 | (+) | Positive control |
| 3 | DS 1 | Donor serum 1 |
| 4 | DS 2 | Donor serum 2 |
| 5 | DS 3 | Donor serum 3 |
| 6 | DS 4 | Donor serum 4 |
8. Incubate at 37˚C for 15 minutes.
9. Remove all the liquid from each well with a new pipet tip for each.
10. Wash each well once with PBS. (You can use the same tip for all of the wells provided you don’t contaminate the tip.)
11. Add 50 µl of anti-IgG peroxidase conjugate (secondary antibody) to each of the 6 wells (see above).
12. Incubate at 37˚C for 15 minutes.
13. Remove the liquid from each well.
14. Wash each well twice with PBS.
15. Add 50 µl of the substrate to each of the 6 wells.
16. Incubate at 37˚C for 5 minutes.
17. Remove the test strip from the incubator and examine each well. Development of a color reaction is an indication of a positive result.
Note
Wells may be incubated for a longer period of time if the color reaction does not develop after 5 minutes
B. Biolog
Before you begin: make note of the appearance of growth on your agar plate. Record appearance of growth as you have before. More information about the contents of the wells and how the test is set up can be found by watching the videos found at this link: www.biolog.com/videos.php
Per table: one Biolog microplate, 1 swab for picking up bacterial growth, 1 tube of inoculation fluid, 1 reservoir, 1 multichannel pipettor (octapet), pipet tips, and an empty pipet tip box
1. Unwrap your Biolog microplate and label it on the side of the plate with your table number. Place the plate over a white sheet of paper to make the wells more visible.
2. Use a swab to pick up a small amount of bacterial growth from your agar plate (your instructor will guide you as to how much to pick up).
Note
It’s very important not to add too many cells to the tube. It is easier to correct the mistake of not adding enough than it is to correct adding too much, so go slowly!
3. Transfer the cells you have picked up into a tube of inoculating fluid to make a suspension.
4. Mix thoroughly and break up any clumps (vortex gently if needed).
5. Use the turbidimeter (a machine that estimates the concentration of cells in a suspension based on its turbidity) to determine the % transmittance (how much light passes through) for your sample. Ideally this should be 90-98%. Your instructor will assist you with this part.
6. When your sample is at the proper % transmittance, add the contents of the entire tube into a clean reservoir.
7. Secure 8 pipet tips onto the octapet. Make sure all the tips are firmly attached.
8. Use the pipet tips to transfer 100 µl to each of the 98 wells (8 at a time)
Note
It is very important to do this step carefully.
a) Be sure you are pipetting the correct amount of fluid. (If you pipet too much you will run out of your suspension before inoculating all wells).
b) After drawing up the suspension, check the pipet tips—they should all have the same amount and should be free of bubbles. If they do not, release the suspension back into the reservoir and start again.
c) Always start on the left side of the plate (so A1, the negative control, gets inoculated first).
d) Make sure your tips (all 8) are directly over the wells before beginning to release the bacterial suspension from the pipet tip.
e) DO NOT let the tips touch the bottom of the wells. If you do this you will be transferring small amounts of substrate/tetrazolium dye, which could lead to false positive results.
f) After each 2-3 transfers, use the empty pipet tip holder to press the tips back into place (they may become loose after a few uses).
9. Replace the lid on the plate, and incubate your sample at 33˚C.
10. Your plate will be incubated until color development is complete (3-36 hours) and then refrigerated until you can read the results.
11. Discard all contaminated materials (inoculation fluid tube, inoculator, reservoir and pipet tips as instructed. DO NOT put any of these items in the regular garbage.