Lab 1- Cell Counting and Calculations Flashcards
What was the objective of the experiment?
- To count cells via the trypan blue exclusion method using a neubauer haemocytometer
- Revise basic microscopy
What materials were used?
- 100 ul cell suspension of unknown concentration
- Naubauer haemocytometer
- Trypan blue
What does trypan blue do?
Trypan blue dye is described as being a vital stain allowing discrimination between viable cells and cells with damaged membranes that are usually considered to be dead cells. It turns dead cells blue.
Why does trypan blue turn dead cells blue?
Trypan blue is a diazo dye that has been widely used to color dead tissues or cells selectively. The mechanism of trypan blue staining is based on it being negatively charged and not interacting with cells unless the membrane is damaged.
Undamaged cells with intact membranes are very selective concerning the compounds that pass through their membrane, and thus should not take up trypan blue. Therefore, all the cells that exclude the dye are considered viable. By contrast, cells with damaged membranes are stained in a distinctive blue color readily observed under a microscope.
We mixed 20 ul of cells to the 80 ul of trypan blue. We took out 15 ul of the cell/trypan solution to each side of the haemocytometer and count the live and dead cells in the large squares (1mm x1 mm x0.1mm). What is our dilution factor and what do we need to multiply for our hemocytometer?
(20 ul to 80 ul) 2:8 > 1:4
1+4=5 = dilution factor
For example, a 1:5 dilution (verbalize as “1 to 5” dilution) entails combining 1 unit volume of solute (the material to be diluted) + 4 unit volumes of the solvent medium (hence, 1 + 4 = 5 = dilution factor)
Dilution factor: 5x10^4
Why multiply with 10^4 hemocytometer?
As each of the “large” squares is 1mm x 1mm, and the area between the slide and the coverslip is 0.1mm, then the volume above each “large” square is 0.1 mm3= 0.0001 ml
To give you cell count per ml, you need multiply to 10000.
10000 is equivalent to 10^4 to give you cell count per ml
What is our equation?
Live cells x5 x10^4 /4
Dead cells x 5 x10^4 /4
Viability: live cells / (live cells + dead cells) x 100
Why divide by 4?
Divide number of large squares counted (4)
If you need to transfer 0.18 ml, which pipette would you use and why?
P-200
Because 0.18ml =180 ul. The P-200 will measure this volume with greater accuracy and precision.
What are some general rules of pipetting?
- Never draw any fluid into the white barrel of the pipette itself
- Never turn the adjustment scale below or above the full range settings
- To maximise precision, always use the smallest volume pipette for a given total volume
- Only use reverse pipetting for viscous liquid e.g. serum, glycerol or when bubbles must be avoided.
What are serial dilution?
Serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.
What is the dilution factor?
A dimensionless number that describes the strength of the solution. It is equal to the volume of the stock used (V1), divided by the total volume of working solution produced (V2)
What is the diluent?
The fluid used for diluting concentration
What is difference between stock solution and working solution?
Stock solution is the concentration which is being diluted while working solution is already diluted, ready to use.
What is the dilution factor if you add 0.2 ml of a sample to 9.9 ml of diluent?
The final volume - the aliquot volume + the dilute volume;
0.1 ml +9.9 ml = 10 ml
The dilution factor = the final volume/the aliquot volume;
10 ml / 0.1 ml = 1:100 dilution (10^2)
What is the dilution factor if you add 40 ul of sample to 160 ul of diluent?
The final volume - the aliquot volume + the dilute volume;
40 ul + 160 ul = 200 ul
The dilution factor = the final volume/the aliquot volume;
200 ul / 40 ul = 1:5 dilution (5^1)
For our experiment, what was the goal for performing serial dilutions?
In this experiment, we performed serial dilutions of trypan blue viability dye in a microtiter plate. We performed these dilutions in duplicate and thus were able to visualise your pipetting accuracy. In addition, we compared our plate to our lab partner’s plate.
Are the end points for each serial dilutions as expected?
Results for 2 fold were much easier to see and results were as expected. All wells were visible. The 4 fold end point was at E and 10 fold dilutions were harder to see but were also as expected and accepted.
Are your replicates acceptable?
Yes, our samples had the same end results.
How might your serial diluting technique be improved?
Thorough mixing at each step to achieve a homogenous mixture with a constant concentration over the sample volume. This can dramatically affect the downstream concentrations. Manual pipetting will always produce some error (use a computer robot). Avoid bubbles. Pipetting was performed by two people which will have different pipetting techniques. Best to duplicate our results. Ideal to repeat testing.
