Haveman, Shelley 1-2

Automatic Acridine Orange Cell Counts on Nikon Microscope

(Room 406C)

  1. Turn on UV light (mercury), then 2 other boxes on shelf (right to left), waiting a few seconds between each one, then turn on computer. The monitor should be on already.
  2. Sign in with your start time and the initial reading on the UV light box.
  3. You should see the blue light on the microscope when the computer comes on.
  4. Microscope settings: shutter open (down), filter B-2A, thicker 100x lens, bino/photo lever pushed in (Bino).
  5. DO NOT use a Kimwipe to clean a microscope lense! Use only the lense paper.
  6. On the computer, open Simple PCI (desktop icon).
  7. From the File menu > Open > Workfile document
  8. C://Documents&Settings/Shelley/count cells_2
  9. Click on Start collecting (panel on left), type in a filename (if it’s the date, don’t use slashes or colons) and save in your folder. The keyboard is in the middle drawer.
  10. Click OK after making sure that every box in the Data components window is selected.
  11. Click Stop collecting (panel on left).
  12. Top left panel: click icon with 2 peaks, deselect Runtime menu box, click OK.
  13. Top left panel: click icon with beaker, deselect Runtime menu box, click OK.
  14. Click Start collecting (panel on left)
  15. Click Start > (panel on left)
  16. Choose Fluorescein, Gain 5, Exposure 0.1112.
  17. Click OK (bottom), you should see blue UV light from the microscope.
  18. Put a drop of immersion oil on the cover slip, focus on your slide while looking through the eyepiece, pull the Bino/photo lever out, and click OK.
  19. Push the Bino/photo lever back in and repeat the previous 2 steps for a total of 10 fields of view.
  20. To delete a bad field, click Stop collect (bottom of window), Delete previous (panel on left), Start collecting (panel on left).
  21. When you’re finished collecting all 10 fields, click Stop collect (bottom of window), then Stop collecting (panel left).
  22. Top left panel: click icon with 2 peaks, select Runtime menu box, click OK.
  23. Top left panel: click icon with beaker, select Runtime menu box, click OK.
  24. Close the Image display window.
  25. Select Original image 1 in Field 1 folder in Filename [Active] window.
  26. Muktak’s Note: the following steps can be skipped if you find it easier to count cells by eye on the screen and write down the number!
  27. Click the stoplight icon at the top of the Filename [Active] window, Replace, click OK in the Data components window, Start > (panel on left).
  28. Adjust levels in the Identify objects window (everything green is counted), OK.
  29. Adjust levels in the Qualify objects window (red too small, blue too big, neither is counted), OK.
  30. If you make a mistake, it’s hopeless. DO NOT stop; your field will be deleted!!
  31. Repeat for all 10 images. Settings don’t need to be adjusted every time if images are similar.
  32. When you’re finished, record the count for each field in the Field Data window and the total count for all fields in the Object Summary Statistics
  33. For the next slide, close Filename [Active] window and click Start collecting (left)
  34. When you’re all finished, turn off computer, then 3 boxes on shelf (left to right), clean oil off the lenses with lense paper (not Kimwipes!) and sign the book, recording the number of hours shown on the UV light box.
  1. The area of each field is 5826 um2. The filtration area (chimney cross-section) is 213.8247 mm2. Therefore, the number of cells on the whole slide is the average number per field multiplied by 36701.802. Multiply this by your dilution factor (e.g. 7 ml of oxalate extract, but only 1 ml used to make the slide; 1 ml of glutaraldehyde-fixed cells contains 0.9 ml of culture).