TITLE: Counting Microalgae Culture Density
AUTHORS: Jakub Nedbal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Counting culture cell density offers a reliable way of consistend seeding density for freshly inoculated cultures and allows tracking the growth of the cultures. Cell densiry counting, as described in this protocol, is performed manually under the microscope in a Neubauer hemocytometer.</div><div class = "text-block">.</div></div>

[STEPS]
?. [Loading Microalgae into the  Hemocytometer]
Wipe down the sterile laminar flow cabinet with 70 % denatured ethanol solution.Prepare the hemocytometer, P20 pipette and pipette tips in the sterile cabinet.
?. [Loading Microalgae into the  Hemocytometer]
Mark down the temperature on the shaker.
?. [Loading Microalgae into the  Hemocytometer]
Dissociate any cell clumps by thorough vortexing.Do not vortex too vigorously to avoid the cotton wool plug getting splashed on.Vortex D. quadricauda for at least 5 seconds.Vortex C. vulgaris for at least 30 seconds.
?. [Loading Microalgae into the  Hemocytometer]
When C. vulgaris culture is left growing for a long time, the cells start clumping, forming a green rim of cell on the sides of the flasks near the surface of the medium. The cell clumps are difficult to dissociate and make assessing the cell culture density difficult if not impossible. In which case, the cells need to be dissociated by trituration in the sterile flow cabinet. This is done using a 20 ml syringe with a 21G syringe. The culture is aseptically drawn through the needle out of the culture flask into syringe. It is then expelled back into the flask, with the expelled jet aimed at the coagulated cells stuck to the walls of the flask. Repeating the sucking and expelling the culture suspension through the needle about 10 times dissociated the clumps and allows the cells to be counted in the hemocytometer.
?. [Loading Microalgae into the  Hemocytometer]
Bring a clean hemocytometer with a coverslip on into the laminar flow hood.Transfer the culture flask into the laminar flow hood.Remove the aluminium foil from the culture flask.Spray the tweezers with 70 % denatured ethanol. Let dry.Lift the cotton with pair of sterilized tweezers. Do not lay the wool on any surface. Keep it held in the tweezers until it is plugged back into the flask opening.
?. [Loading Microalgae into the  Hemocytometer]
Pipette out  sample of the algal culture.Load it into one chamber of the hemocytometer.
10 µl
?. [Loading Microalgae into the  Hemocytometer]
Shake the flask to resuspend any cells that started settling.Pipette out another  sample of the microalgae culture using a new tip.Load the second chamber of the hemocytometer, following the steps described above.
10 µl
?. [Loading Microalgae into the  Hemocytometer]
Reseal the culture flask with the cotton wool plug and aluminium foil cap.Return flask onto the orbital shaker.
?. [Counting Cell Culture Density]
Bring the hemocytometer to the microscope with a 10× phase contrast objective and sufficient working distance.
?. [Counting Cell Culture Density]
Desmodesmus quadricauda - counting coenobiaD. quadricauda cells form coenobia. Count the number of coenobia, not individual cells.Repeat the count three times - one for coenobia with two cells, one for coenobia with 4 cells, and one for coenobia with 8 cells.The counting in the hemocytometer app allows counting two cell types at the same time. This makes it possible to count coenobia with 4 and 2 cells at the same time, for instance.Count cells in both chambers and the 8 outer squares in each chamber.Sometimes, coenobia contain non-canonical number of cells, such as singlets, triplets, etc., as in the image below. To account for them, use a system that counts the approximate number of cells: For example, count two singlets count as a single doublet, two triplets count as one quadruple and one double, or a triplet and a singlet counts as one quadruple.Use the Hemocytometer Sidekick App to count the coenobia in each segment of the hemocytometer. The snapshot below shows the count of 8 squares, withe coenobiea with 4 cells in red and coenobia with 2 cells in blue.  ABCD1SquareDoubletsQuadruplesOctuples2LT13CT14RT15LM16RM17RB18CB19RB110LT211CT212RT213LM214RM215LB216CB217RB2
ABCD1SquareDoubletsQuadruplesOctuples2LT13CT14RT15LM16RM17RB18CB19RB110LT211CT212RT213LM214RM215LB216CB217RB2
?. [Counting Cell Culture Density]
Desmodesmus quadricauda - calculating cell culture density.Multiply the number of coenobia with two cells in each square by 2. Multiply the number of coenobia with four cells by 4. Multiply the number of coenobia with eight cells by 8. The sum of the three resultant numbers gives the total number of cells (not coenobia) in each square of the hemocytometer.The average of the number of cells in each of the 20 squares multiplied by 10000 ml-1 gives the cell culture density in cells per milliliter.
?. [Counting Cell Culture Density]
Clean the hemocytometer and the coverslit with 70 % denatured alcohol using Kimwipes or equivalent tissue paper.Load the C. vulgaris in the same way described above.
?. [Counting Cell Culture Density]
Chlorella vulgaris - counting cellsC. vulgaris cells form dense cultures. This means too many cells are in each square of the hemocytometer to count. On the other hand, the high number of cells means that the relative spread of counts in different squares of the chamber is relatively small.Count cells in both chambers of the hemocytometer, but only in the four squares forming a plus sign to simplify the process, as in the image below.When the cell density is very high (>5×106 ml-1), do not count cells in all 20 sub-rectangles of the hemocytometer. Instead count only a subset of the sub-rectangles in each square and scale the obtained number appropriately, to obtain the total in the entire square.  ABC1SquareCountSub-rectangles2CT10203LM10204RM10205CB10206CT20207LM20208RM20209CB2020
ABC1SquareCountSub-rectangles2CT10203LM10204RM10205CB10206CT20207LM20208RM20209CB2020
?. [Counting Cell Culture Density]
Chlorella vulgaris - calculating cell culture densityAs described in the above step. Counting cells in all sub-rectangles of each square for very dense cultures would be long and tedious, without adding much value. When only four (out of the total of 20) sub-rectangles were counted, multiply the obtained number by five to get the total of cells in each square.The multiplication can be done manually, or directly in the Hemocytometer Sidekick app by setting a dilution factor of 8 after the count is finished, as in the screenshot below.The average of the number of cells in each of the 8 squares multiplied by 10000 ml-1 gives the cell culture density in cells per milliliter.
?. [Counting Cell Culture Density]
Clean the hemocytometer and the coverslit with 70 % denatured alcohol using Kimwipes or equivalent tissue paper.Clean the sterile flow hood and tidy the spaces used.Turn off the lamp on the microscope.
?. [Counting Cell Culture Density]
ReferencesHemocytometer Sidekick appManual Cell Counting protocolgrowthCurves: Matlab file to analyse and graphically represent cell counting results.