Culture counter

Culture Counter, K. Cayemitte, May 14, 2025

Training by William Biggs

  1. Prep samples by dilution
    A. Always UV hood and sterilize gloves and the bench with ethanol first before moving cultures.
    B. Thick Cultures: 100 μL in 14.9 mL of filtered sea water (0.4 μm) C. Medium Cultures: 500 μL in 14.5 mL of filtered sea water (0.4 μm) D. Light Cultures: 1000 μL in 14 mL of filtered seawater (0.4 μm)
  2. Use the same filtered seawater to blank the culture counter (allows you to subtract particles in the filtered seawater from your sample).
  3. Rinse the cuvette with Milli-Q.
  4. Waste bucket is the one with the white funnel, just has bleach, sample, and Milli-Q, so you can pour down the drain and then refill with bleach.
  5. Sign in to use the culture counter should be a notebook on top that shows “Name”, “Date”, “Time”.
  6. Make sure that the culture counter has filtered seawater in it, and it is clean - this is the container left of the machine.
  7. Turn on the Coulter Counter using the on-off switch on the right side.
  8. Turn on the Computer, the box with “Multisizer 3” will come up, click “Ok”.
  9. Check that the lamp is on.
  10. Start with filtered seawater in a cuvette for a blank - sigma should be below 2000.
  11. System “Flush aperture tube” - Cleans the pipes in the equipment.
  12. Click “Start”.
  13. Red Value Integration under the curve - population size.
  14. Samples ½ of a ml.
  15. For filtered seawater (0.4 μm), it should be below 150 particles.
  16. Save under “Fiorella” -> “Kayla C”.
  17. Name the file “Seawater blank 5-14-25,” then click save.
  18. Setting -> “Load background run” -> “Load the saved seawater blank”.
  19. Load the sample by pouring your sample into the cuvette, pull down the sample tray by pushing the button under the tray, put the cuvette on the sample tray, push the button, and move the sample into the probe - it should go all the way up.
  20. Hit start, the box will now say “Backround subtraction”.
  21. Gate the cell amount by clicking and dragging where the curve starts and stops.

*NOTE- THIS DRAG OVER THE CELLS SIZE- look up cell size if you dont know *

  1. The red text that pops up is the number of cells per 0.5 ml.
  2. Write this number down in your Excel sheet, then calculate the number of cells per mil by multiplying by 2.
  3. Then, calculate how many cells you have in your sample by multiplying by the dilution factor: Concentration = (Total volume/ volume of cells added)* Number of cells/ ml.
  4. Rinse the culture counter probe with Milli-Q in between samples.
  5. When finished with all samples, wipe and rinse the probe with Milli-Q, then add the cuvette with Milli-Q back to the culture counter.
  6. Turn off the culture counter and exit from the program.
Written on March 2, 2026