3.iii. CELL CULTURE METHODS & PROTOCOLS

 

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USE OF THE HAEMOCYTOMETER

(for improved Neubauer Haemocytometer)
(Click here to download a text version of these instructions)

PLEASE NOTE:

DESCRIPTION.

The haemocytometer has a central area, slightly lower (0.1 mm) than the rest of surface of the slide. This depression forms the counting area/ chamber (Figure 1a; below).

Looking at the surface of the slide; finely ruled areas that form grids can be seen (Figure 1b; above). These define an area on the slide of a known size. The area measured from the grid and the height between the slide and the base of the depression of the counting area defines a known volume. By counting the cells or other objects in this volume the no. of cells etc. per unit volume can be calculated and this value used to give the number in the original volume.

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INSTRUCTIONS FOR USE

(1). Ensure both the haemocytometer and it's cover slip are clean. If they are dirty wash them in distilled water and then wipe them over with alcohol. When dry breath on the surface of the haemocytometer and quickly place the cover slip in position so that it is centred over the counting chamber.

(2). Gently, but firmly press down on the cover slip, using a thumb placed either side, holding the haemocytometer firmly between both hands, until Newton's rings (refraction rings of light of a rainbow/oil slick coloration) are seen. If no rings are seen try again. If problems are experienced obtaining rings, e.g. on a hot day, then cool the haemocytometer under running cold water for few minutes before drying and then trying to the mount the cover slip again.

(3). Take (suck) up a small amount of the cell suspension/solution to be counted in a fine tipped Pasteur pipette. Gently touch the pipette against the side of the cover slip where it touches the base of the depression (Figure 3, below). A drop of liquid should be drawn out of the pipette and into the chamber. If it is not then gently and slowly squeeze out one drop. This will fill one counting chamber. If both chambers are to be used then repeat for the other chamber.

(4). Place the haemocytometer under a microscope. Turn the microscope on and under low power (*5/*10) focus on the grid area of the counting chamber. PLEASE NOTE: do not take too long to count as the liquid will start to evaporate from the sides of the chamber, and the chamber dry out, with the heat of the lamp.
(5). Under the microscope the grids area can be seen to be formed of 9 large squares in a 3*3 grid (Figure 4; below). Each of these squares covers an area of 1 mm2 which, with the depth of the chamber being 0.1 mm, means that the volume of each large square is 0.1 mm3.

The central area of the grid is made up of triple ruled lines that in the centre form a 5*5 grid of 25 squares with a total volume of 0.1 mm3 (Figure 4, above, and Figure 5a, below ). Each of these 25 squares has an area of 0.04 mm2 and a volume of 0.004 mm3. Each one of these 25 squares is further divided up into 16 smaller ones in a 4*4 grid (Figure 5b, below) . Each of these 16 smaller squares has a surface area of 1/400 mm2 (0.0024 mm2) and a volume of 0.00025 mm3.

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COUNTING A HIGH-DENSITY CELL POPULATION.
When counting high densities of cells squares use the 40x objective and count the four corner and one centre 0.04 mm2 square (Figure 6a; below, left). When counting a 0.004 mm2 square scan from the top left of one of 16 1/400 mm2 squares and move to the bottom right as shown in Figure 6b, below right.

Cells falling on the bordering triple lines should only be counted if they are on either the top or on the left lines only. Exclude those touching the bottom and the right hand side lines. When performing cell counting, the most accurate count is achieved when 120-200 cells can be counted. If you have more than this in the five squares then you should dilute your sample and re-count. If you have less than this you should use either the low density cell population method as described below or centrifuge and resuspend your cells in a smaller volume.

Calculate the concentration of cells as below:
The centre triple ruled grid has a volume of 0.1 mm3. You have counted the cells in 5/25 of this grid, i.e. 5 out of the 25 squares. Thus to convert to the number of cells in 0.1 mm3 you have to divide by 5 and then multiply by 25. To convert this answer from cell/0.1 mm3 to cells/ ml you have to multiply by 10,000 ( 0.1 mm3 * 104 =1000 mm3= 1 mm3).

Let no. of cell counted in 5 squares = X.
no. of cells/ ml suspension = (X/5) x 25 x 10,000


NOTE: This does not take into account any dilutions made in preparation of the stain or any other dilutions prior to staining. You will have to calculate the effects of these for yourself.

COUNTING A LOW-DENSITY CELL POPULATION.
In this case use 10x objective lens and count the squares (0.1 mm3) in each corner of the 3*3 grid, i.e. outside the triple ruled area, ( labelled A, B, C and D in Figure 4 above). Divide the number of cells counted by 4 and multiply by 10,000 to get the number of cells per ml.


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PRACTICAL 1: INTRODUCTION TO MICROSCOPES & CELL COUNTING

STUDENTS SECTION

(Click here to download a text version of these instructions)

Before you start tissue culture it is important that you are familiar with the techniques and equipment that you will be using in handling and monitoring your cells.
This practical is designed to:

1). Instruct you in the use of some of the equipment used in tissue culture, namely

2). In addition to practising the basic lab skills you have gained during your time at Luton, you will be taught in the techniques of:

There is no overall protocol for this practical session as it is part of the exercise for you to get experience in organising your own work in the laboratory and using your lab notebooks to help you do this.

YOU must therefore plan your work, construct protocols and record the results as instructed in your lab note books!!
Don't forget to construct tables before you start in which you can enter the results, i.e. with: lines dividing the columns and rows up; appropriately labelled columns; headings, etc.

After being instructed in the use of the microscope and haemocytometer, you are asked to do the following:

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Cell counting

Take the suspension of cell you have been given and, using the cell counting protocol given to you, count the number of cells in the solution following the procedure below:

i). Take 25 to 100 ml of cell suspension in to a clean test tube or Eppendorf tube
ii). Add an equal volume of methylene blue stain,
iii). Mix gently and count using both upper and lower chambers, record the mean of the count as well as the raw counts in your laboratory note book. (NOTE: you will be given training in the use of the microscope and haemocytomters)
iv). Repeat the count a total of three times.
v). Calculate the mean and standard deviation of the three counts and check with the supervisor to ensure that your count is in the expected range, i.e. that your counting technique is working.
vi). Remember when calculating the number of cells to take into account the dilution caused by the addition of the stain!

If you do not get the correct count then discuss what might be going wrong with the supervisor /demonstrator and repeat the process until you get the expected counts.

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Additional notes:

i). Cells in suspension tend to "settle out" with time, i.e. fall to the bottom of the test tube. You will thus need to gently resuspend and mix them by hand to redistribute them evenly throughout the solution before you take a sample to count (NOTE: this mixing technique will be demonstrated to you).
ii). Make a note of the actual name, model number and manufacturer of any equipment that you are using in a procedures and quote this in your methods in the notebook.
iii). If special conditions are set on a piece of equipment that you are using then you need to note these down and to quote them in your methods. Things to note, for example might be: eyepiece magnification and focal objectives magnification on microscopes; speed, temperature and time on centrifuges, etc.
iv). If you do not complete the practical then do not worry, as you will be able to carry on in the session the following week.


*(NOTE: these techniques are also covered in the recommended text for this module)

References

1). Immunochemistry in Practise. 3rd Ed. A. Johnstone & R. Thorpe. Blackwell Science. 1996. [ISBN: 0 86542 633 3] see pages 105-110 (covers cell counting and some staining)

2). Practical Immunology. 3rd Ed. Huson LH & Hay FC. Blackwell Sientific. 1991 [ISBN 0 632 01491] see pages 92-7110 (covers cell counting and some staining)

3). Practical skill in biology 2nd Ed. A. Jones, R. Reed, J. Weyers. 1998. Addison Wesley Longman. [ISBN 0 582 29885] see pages 108, 127 (covers use of the microscope, cell counting and some staining).

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PRACTICAL 1: INSTRUCTORS SECTION

(Click here to download a text version of additional instructions for Instructors/ Demonstrators/ Tutors for Parctiacl 1: Introduction to Microscopes & Cell Counting)

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PRACTICAL 2: DILUTION & COUNTING OF CELLS

STUDENTS SECTION

(Click here to download a text version of these instructions)


This practical continues and expands on the previous one (Practical One) and provides an opportunity to develop and expand on your cell handling and counting abilities. Many of the teaching objective are thsu the same.
However, once again there is no practical schedule and YOU must therefore plan your work, construct protocols and record the results as instructed in your lab. Note books!! Don't forget, as before, construct tables before you start in which you can enter the results, i.e. with lines dividing the columns and rows up; appropriately labelled columns; headings, etc.

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PART ONE: Counting of cells at serial (doubling) dilutions.

i). Take the suspension of cell you have been given and prepare the following dilutions.

Tube number:
1
2
3
4
Dilution factor:
1
0.50 (1/2 )
0.25 (1/4)
0.0125 (1/8)
ml of cell suspension
200 ml
200 ml
200 ml from tube 2
200 ml from tube 3
ml of buffer
200 ml
200 ml
200 ml
200 ml

NOTE: above volumes are microlitres and not millilitres. The same is also true of the other volumes quoted below. Some browsers could show the volumes as the wrong units. If in any doubt consult the text version (down-loadable - see above.)

Prepare by first adding 200 ml buffer to each tube, as shown below:

 

Add 200 ml cell to tube one (neat) and tube 2 (diluted by half). Mix tube 2 gently and then transfer 200 ml of diluted cells from tube 2 to tube 3 (diluted to one quarter). Mix tube 3 gently and then transfer 200 ml of diluted cells from tube 3 to tube 4 (diluted to one eighth), finally mix all tubes gently before removing aliquots of cells for counting.

ii). Take out 50 ml aliquot of cells from each of the above tubes (1-4 above) and add to a fresh test tube. Then add 50 ml of methylene blue stain to the same tube

iii). Mix gently and count as before i.e. using both upper and lower chambers and record the mean of the count as well as the raw counts in your laboratory note book).

iv). Repeat once more (i.e. a total of twice)

v). Plot a graph of dilution versus cell number/tube, plotting the line through the mean of the two counts but mark the individual counts on the graph as well (it should look something like the following quick sketch). Don't forget to take into account any additional dilution stages, such as caused by adding the stain, etc.


Don't forget- all graphs need titles, the axis accurately and clearly labelled and a footnote explaining what the graphs is about and what it shows, including any findings, conclusions, etc. (look at a scientific paper in one of the journals for examples if you are not sure). Please note, the graph above is just a rough guide as to what the line and points should like when there are plotted. It should not be taken as an example if a final "finished" graph!!

PART TWO: Counting of cells at linear dilutions.

i). Take the suspension of cell you have been given and prepare the following dilutions.

Tube number:
1
2
3
4
Dilution factor:
1
0.75 (3/4 )
0.50 (1/2)
0.25 (1/4)
ml of cell suspension
200
150
100
50
ml of buffer
0.0
50
100
150

NOTE: above volumes are microlitres and not millilitres. The same is also true of the other volumes quoted below. Some browsers could show the volumes as the wrong units. If in any doubt consult the text version (down-loadable - see above.)

ii). Take out 50 ml aliquot of cells from each of the above tubes (1-4 above) and add to a fresh test tube. Then add 50 ml of methylene blue stain to the same tube

iii). Count, etc. as before. This time the results should look something like this:

iv). Calculate the mean of the three counts and also the standard deviation for all six (3 times upper + lower chamber) . Check with the supervisor to ensure that your count is in the expected range, i.e. that your counting techniques is working.

v). Remember that when calculating the number of cells, to take into account the dilution caused by the addition of the stain.

If you do not get the correct result then discuss what might be going wrong with the supervisor/demonstrator and repeat the process until you get the expected counts.

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ADDITIONAL NOTES

i). Cells in suspension tend to "settle out with time, i.e. fall to the bottom of the test tube. You will thus need to gently resuspend and mix them by hand to redistribute them, evenly throughout the solution before you take a sample to count (NOTE: this mixing technique will be demonstrated to you).

ii). Make a note of the actual name, model number and manufacturer of any specialised equipment that use in procedures and quote this in your methods in the notebook.

iii). If special conditions are set on a piece of equipment that you are using then you need note these down and to quote them in your methods. Things to note, for example might be eyepiece, focal objective magnification on microscopes; speed, temperature and time on centrifuges, etc.

iv). If you do not complete the practical then do not worry, as you will be able to carry on in the session the following week.

*( NOTE: see previous references list)

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PRACTICAL 2: INSTRUCTORS SECTION

(Click here to download a text version of additional instructions for Instructors/ Demonstrators/ Tutors for Practical 2: Dilution & Counting of Cells)


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