Cell Counting

Objective: To become familiar with the measurement of viable and non-viable cell counts.

Tools of the Trade:

  • Olympus Flourescence Microbiology Microscope System with Digital Documentation
  • Improved Neubauer Hemacytometer
  • 4mM Calcein AM anhydrous DMSO
  • 2mM Ethidium Homodimer in DMSO/H20
  • Nalgene Cryogenic Vials

Introduction:

  • We will be using a stain that needs to be prepared just before use. There is an incubation time involved during that time it would be wise to get the microscope and hemacytometer ready.
  • If this is new to you it will be wise to practice first in getting the hemacytometer set-up with the grid clearly focused.
  • Also please refer to the SOP for the microscope system. A clear understanding will help you to be wise!

Prepare working stain:

  1. Add 3 ul of 2 mM ethidium homodimer to a 1.2 ml cryogenic vial.
  2. Add 997 ul of Ham's F12 medium to the vial.
  3. Freeze until needed. (May have been already made for you.)
  4. Thaw just before use. Will quickly thaw in your hand.

A Word about your cell sample:

  • Cells should be kept on ice in the time lag between the actual sampling and the mixing with the stain.
  • It is important to get an equal cell distribution at sampling time. This is important to all measurements regarding cells.  Swirl cell cultures just prior to mixing with the stain and again just before loading the hemacytometer. Your goal is to achieve an accurate cell distribution with cell clumping kept to a bare minimum.

Stain Cells:

  1. Combine 20 ul of cell suspension with 20 ul of working stain (1:1) in a clean 1.5 ml Eppendorf tube. Mix gently.
  2. Incubate at room temp. for 30-45 minutes.

During incubation:

Prepare microscope system. Refer to SOP, familiarize yourself with the UV system.

Prepare hemacytometer:

  1. Obtain the hemacytometer from under the counter, it"s in a red vinyl case.
  2. Refer to the attached diagrams.
  3. Clean hemacytometer with Di H20 and then with 70% ethanol and dry off.
  4. Place hemacytometer on microscope stage and clearly focus using white light.
  5. At 100X you will clearly see the counting chamber and get a feel for the process.
  6. To count the cells you need to see the grid and have the UV light on. This is best accomplished by having the white light just barely on, this enables you to see the grid in relationship to the cells. The UV light causes the stained cells to fluoresce.

Load hemacytometer:

  1. Place hemacytometer on prefocused microscope stage.
  2. At 100X and with white light only (close UV shutter) you will clearly see the cells and the grid. Open the UV shutter and the cells will flouresce.
  3. RED IS DEAD
    GREEN IS ALIVE
  4. Change to 200X. Refocus if necessary. It will be easier to see the counting areas properly under white light, then diminish the white light and re-open the UV shutter.
  5. Refer to the attached diagrams. Count all cells in the 
  6. 4 areas marked ³W² By common convention count cells touching the top and left borders but not the bottom and right borders. Cells outside the counting areas are NOT to be counted.

 

Perform documentation:

Refer to microscope SOP for specific info on digital documentation.

Formula to determine cell counts:

C= ( N/V ) X "D"

C= cell count in cells per milliliter 
N= number of cells counted in counting area 
V= volume of counting area. The volume of each "W" is .0001 ml 
D= dilution factor. For this protocol the dilution factor is 2.

Typically:

C= ( N/ .0004 ) X 2 in cells per ml.

Clean up:

  1. Soak slides in 10% Clorox solution before disposing.
  2. Clean counter.
  3. Clean microscope.
  4. Turn off UV burner and microscope system.
  5.  

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