Editing Mycoplasma Assay Protocol (section)

====Running the Assay====

# Boot up the '''CLARIOstar program'''.
# Select '''Manage Protocols''' and then click on '''Myco Test'''.
# In the window, click on the '''Layout tab''' and '''Empty''' out all cell wells. You can do this by selecting the entire plate and clicking on the '''Empty''' button.
# Click '''Sample''' and select the wells on the plate that contain your samples (including the control). Click '''Start Measurement'''.
# In the second window that pops up, uncheck '''Gain Adjustment'''.
# Once '''your 5 minutes are up''', push the small black button above the myco tray insert on the machine to open it. '''Place your microtiter plate into the tray'''.
# On the new window in ''CLARIOstar'', double check that '''Gain Adjustment''' is unchecked. Then, hit '''Start Measurement''' when you’re ready.
# Once the program is finished, '''write down the values that pop up for each sample'''. These values are your '''first measurement (A)'''.
# Remove the microtiter plate from the machine.
# Next, '''add 100 µL of thawed MycoAlert Substrate''' (labeled “S” on aliquots) to each sample. '''Wait 10 minutes'''.
# Repeat steps 2-5. Ensure that '''Gain Adjustment''' is unchecked, and then run again.
# Once the program is finished, '''write down the values that pop up for each sample'''. These values are your '''second measurement (B)'''.
# Close the application and log off the computer (if in the ''Nathanson Lab'', leave it on). Return the microtiter plate to where you found it, if in the ''Nathanson Lab'' (no need to aspirate the samples), otherwise dispose of the media as if it were biohazardous. Sterilize your biohazardous secondary container with 70% isopropyl alcohol.

Next, calculate the following ratio for each of your samples, where '''B''' is your reading after adding MycoAlert Substrate and '''A''' is your reading after adding MycoAlert Reagent. Use the table below to determine the outcome of the assay. 

{| class="wikitable"
|+MycoAlert Result: Ratio = B/A
|-
! scope="col"| Ratio Value
! scope="col"| Result from the MycoAlert Assay
|-
|Less than 1.0
| Negative for mycoplasma.
|-
|1.0-1.2
|Inconclusive. Quarantine the cells for 24-48 hours and retest.
|-
|Greater than 1.2
|Positive for mycoplasma.
|}