TITLE: Determination of Auxenochlorella protothecoides Cell Density With a Hemocytometer
AUTHORS: Dimitrios Camacho, Sabeeha Merchant

[DESCRIPTION]
This protocol describes a reliable method for counting the cell densities of Auxenochlorella protothecoides cultures using a Neubauer hemocytometer. This method is accurate for cell densities > 106 cells / mL. 

[BEFORE_START]
Make the phosphate buffer solution.
 
1. Make a stock of 1 Molarity (M) NaH2PO4 and 1 Molarity (M) Na2HPO4
 
2. Dissolve 5.38 g of NaH2PO4 • H2O (monobasic monohydrate, M.W. = 138 g / mol) in 39 mL of Milli-Q H2O.
 
3. Dissolve 8.66 g of Na2HPO4 (dibasic anhydrous M.W. = 142 g / mol) in 61 mL of Milli-Q H2O.
 
4. Mix solutions together to make 100 mL 1 Molarity (M) sodium-phosphate buffer, pH 7. 
 
5. Add 10 mL of 1 Molarity (M) sodium-phosphate buffer to 990 mL of MilliQ-H2O to make a 10 millimolar (mM) phosphate buffer, pH 7.
 

[GUIDELINES]
The key to obtaining accurate and precise cell counts is to mix the samples well before every step. Cells will settle to the bottom of the tube or flask if left standing, even for a few minutes. 

[STEPS]
SECTION: Procedure
1. Move the culture flask to a clean sterile hood.
SECTION: Procedure
2. Swirl the culture several times to ensure thorough mixing.
SECTION: Procedure
11. Hold the stage clip open, set the hemocytometer on the microscope stage, and release the stage clip gently to secure the hemocytometer.
SECTION: Procedure
10. Remove the dust cover from the microscope. Rotate the nosepiece to set the objective lens to 20x. Rotate the turret condenser to the brightfield setting denoted as BF. Lower the microscope stage using the coarse focus knob. Switch the light source on. 


 
 

Adjust the inter-pupillary distance by pushing or pulling the eye tubes together or apart.
SECTION: Procedure
9. Ensure that the sample is well mixed and pipette 10 µL of sample into each of the two chambers. 

SECTION: Procedure
8. Use a KimwipeTM to wipe the hemocytometers dry.
SECTION: Procedure
7.1. 5 s If the H2O is not uniformly dispersed within 5 s, (fig. 2 A) you may have the wrong side of the cover slide facing down. Wipe the cover slide and hemocytometer with KimwipeTM, then flip the cover slide over. Retest the dispersion of H2O. 
 



SECTION: Procedure
7. Pipette 10 µL of water into both chambers of the hemocytometer. The cover slides have a coating on one side which alters the dispersion of the Milli-Q H2O and your sample on the hemocytometer.

SECTION: Procedure
6. Place the hemocytometer on a KimwipeTM. Place the cover slide over the hemocytometer
SECTION: Procedure
5. Remove the hemocytometer from its protective case and wipe both the hemocytometer and cover slide with a KimwipeTM.
SECTION: Procedure
4.1. Dilute samples with 10 millimolar (mM) sodium-phosphate pH 7 or with the growth medium in which the cells were cultured. Dilutions generally do not exceed 200-fold.

SECTION: Procedure
4. Dilute the sample until the suspension is almost clear (fig. 1). Record your dilution factors. 
 

SECTION: Procedure
3. Using sterile technique, pipette 100 µL of culture into a 1.5 mL tube.

SECTION: Procedure
11.1. Carefully raise the stage using the fine focus knob until the gridlines of the hemocytometer come into focus. 

Warning: The cover slide will collide with the objective lens if the stage is raised too high. This may result in damage to the cover slide, hemocytometer, and objective lens. 
SECTION: Procedure
12.6. Look into socket with your naked eye.
SECTION: Procedure
12.5. Remove one eyepiece from the tube socket.
SECTION: Procedure
12.4. Open the luminous field diaphragm until the edge of the diaphragm disappears from the field of vision.
SECTION: Procedure
12.3. Center the luminous field diaphragm using the two centering screws.
SECTION: Procedure
12.2. Close the luminous-field diaphragm until a ring is visible.
SECTION: Procedure
12.1. Raise the condenser using the substage height adjustment knob to max height.

Make sure that the condenser does not collide with the bottom of the hemocytometer. There is an adjustable stop screw that you may set so that the condenser never collides with the hemocytometer.
SECTION: Procedure
12. The contrast for transmitted light brightfield microscopy can be optimized according to the Köhler illumination technique, summarized below.
SECTION: Procedure
11.3. Slowly increase the light intensity until you are comfortable with the contrast of cells and the background.
SECTION: Procedure
11.2. Use the diopter adjustment to compensate for the difference of vision between your two eyes.

Close one eye and adjust the stage using the fine focus knob to focus on the gridlines of the hemocytometer.

Open the opposite eye and close the other eye. Adjust the focus using the diopter adjustment without touching the fine adjustment knob.
SECTION: Procedure
17. Remove the cover glass and wipe the cover slide and hemocytometer clean with KimwipeTM. Pipette 10 µL of Milli-Q H2O into each chamber. Remove the cover slide and wipe it dry with a KimwipeTM. Clean the hemocytometers with 70% ethanol or 10% bleach, rinse with Milli-Q H2O, and dry with a KimwipeTM. Store the hemocytometer and cover slide in their protective case.

SECTION: Procedure
16. After completing the count, carefully lower the microscope stage and remove the hemocytometer. Turn off the microscope’s light source and replace the dust cover.
SECTION: Procedure
15. Calculate the average number of cells counted across both chambers and multiply this number by your dilution factor and 104 cells. This number is the cell density (cells / mL). See calculation below.

volume = l × w × h = 1 mm × 1 mm × 0.1 mm = 0.1 mm3 = 0.1 µL
 
cell density = (cells counted / 0.1 µL) × 104 = cells / mL 
SECTION: Procedure
14.1. If more than 10 cells are present within the first 0.2 mm × 0.2 mm grid alone, re-dilute the sample and record the new dilution factor.
SECTION: Procedure
14. Count between 100 – 200 cells inside the triple grid lines that outline the border of the 1 mm x 1 mm grid (5 × 5 squares) (fig. 3). Cells that are partially through the triple grid line should be counted only if more than half of the cell is inside of the innermost line; if more than half of the cell is outside the innermost line, it should not be counted (panel B, fig. 3).
 
 

    
SECTION: Procedure
13. After pipetting the sample into the chambers, wait 1 min for the sample to disperse evenly in both chambers. Cells should settle in place and should stop moving. All cells should also come into the same plane of focus as the gridlines as they become evenly suspended in the Z-axis.

SECTION: Procedure
12.8. Replace the eyepiece. These are the optimal settings for most applications. Repeat steps 12 to 12.7 after switching between objective lenses.
SECTION: Procedure
12.7. Use the lever on the condenser to adjust the aperture so that the luminous field diaphragm is about 2/3 – 4/5 of the diameter of the exit pupil of the objective.
SECTION: Procedure
18. Autoclave or kill all samples and dilutions with 10 % (v/v) Bleach.