I.
Introduction
So, you want to count and size cells? The Coulter Counter has made this task considerably easier and more precise than other methods. This guide will assist the novice cell counter to make good cell counts and store them on the computer. As always, I urge the beginning user to read through the operator’s manual to become more familiar with this instruments operation.
II.
Starting
the Instrument
1. Turn on the instrument
a. There are two switches on the instrument that must be switched ON. The first switch is on the main processor unit; it is a small green switch on the lower left front of the instrument. The second switch turns on the power to the sample stand and vacuum pump, and it is located on the back of the stand near the power cord.
b. Do not rapidly turn off and on the instrument as this can cause damage to electronic circuits.
c. When the main power switches are turned on, you should see the CRT come “to life”. You should also check that the vacuum pump has indeed started by looking at the tubing and noting whether or not some of the liquid in the tubing is “moving”. If no movement, then first check that waste trap is not full (thereby lifting the vacuum relief plug). Next, remove the right side panel from the sample stand and check to see if the vacuum pump inertial wheel is turning. Sometimes it gets stuck and gently turning it will get it started again. Allow the instrument to “warm up” for at least 15 minutes prior to use.
2. If not already on, turn on the computer and monitor and wait for Windows 98 to boot up. Double click on the Multisizer icon to start the software.
3. Select “Change Directory” from the File menu and select your data directory.
- If you are a new user, then choose “Create Directory” first to create your own personal data directory.
- If you do not do this step, your data will, by default, be stored in the c:\multi\user directory. Doing so, however, makes it more difficult to organize and archive data at a later date.
III.
Preparations
for Counting
You need to have at least 500 ml of 0.45 micron filtered seawater, a 50 ml volumetric flask, and a calibrated pipetter
1. Filter Seawater
Usually, you cannot count your sample without dilution because the likelihood that more than one cell passes through the orifice at the same time (coincidence) increases with concentration. You must carefully dilute your sample with filtered seawater. This seawater, preferably, should be exactly the same salinity as that in which your cells are growing to prevent subtle changes in size from differences in osmotic pressure. Use a 0.45 micron Millepore filter to filter the seawater into a clean vacuum flask. Note that it is better to do this freshly rather than use “old” filtered seawater as the “old” water has a habit of “growing” particulate material when left standing.
2.
Select the appropriate orifice for your samples
You must choose an orifice from the several sizes available that will best count and size your cells. The effective counting range for each orifice is from 2% to 60% of the orifice’s diameter. We have the following sizes: 70, 100, 200, 280, 400 microns. For most of the cultures we grow in our lab, the 70-micron orifice is used (range 1.4 microns to 42 microns). If your sample contains larger particulate material as well, then you perhaps will need to choose one of the larger orifices to prevent “clogging”.
If you must change the orifice tube,
a. Handle the orifice tube carefully! The orifice wafer itself is only tens of micrometers thick and can be easily broken.
b. Turn off power to sample stand. Release the vacuum by lifting the plug on top of the waste container.
c. On sample stand, set RESET/COUNT to RESET, and set FILL/CLOSE to FILL.
d. Lower the sample holder platform and remove the sample holder (beaker or 50 ml falcon tube)
e. Remove the rubber bands (if any) that hold the orifice tube to the support piece.
f. Gently twist and pull down on the orifice tube to remove it from the sample stand.
For the replacement orifice tube,
a. Lubricate the ground glass joint with low vacuum grease (there should be some in the Coulter drawer below the instrument). Do not use silicon grease!
b. Fit the orifice tube onto the sample stand support piece with a twisting action to ensure a good seal. The orifice itself should face toward the optics tube so that the orifice can be seen on the viewer screen.
c. Return the rubber bands (if any) to the hooks that hold everything together.
d. Fill the sample beaker with freshly filtered seawater, place it on the sample platform, and raise the platform until the orifice and the electrode are fully immersed.
e. Set RESET/COUNT to COUNT and FILL/CLOSE to CLOSE, then turn on the power to the sample stand.
f. Set FILL/CLOSE to FILL, then slowly rotate the RESET/COUNT to RESET. The orifice tube should fill rapidly with seawater from the refill purge reservoir next to the waste trap.
g. When the orifice has been purged, then turn the controls to CLOSE and COUNT, respectively.
3. If necessary, empty waste trap and refill purge reservoir
- Don’t over tighten the lids!
- A small amount of low-vacuum grease can help seal the lids without putting on the lids too tight.
- If you can’t loosen the lid, then just remove the tubing from the lid and pour the water out through the hose connections. Be sure to replace the tubing in the order in which they were removed.
4. Initial Settings of Instrument
a. Change the date
b. Select orifice that is actually installed on the instrument
c. Check that instrument is calibrated and Kd factors in memory. (See CALIBRATION section below)
d. Check that instrument settings are the same as those used for the calibration (Typically, this should be automatic current and gain, positive polarity, 256 channels, with autoscaling and pulse edit set to ON).
- To check these settings, Press the SET UP key and use the cursor keys to select MANUAL on the setup line. Press SET UP again to display the first page of the analysis setup. Press SET UP again to view the next page. Finally, Press SET UP repeatedly until the Main Setup window is once again displayed. Use the cursor keys to return the Setup line to AUTOMATIC.
Calibration is of primary concern when you wish to know the size of your particles accurately. It is not required for just cell counting, and on a day-to-day basis, the calibration should not change much. However, if the orifice becomes dirty or you use a radically different electrolyte counting solution, then re-calibration maybe necessary. The manufacturer recommends that calibration be checked at least once per month.
1. Select RECALL to check that the calibration constant for the orifice tube and electrolyte solution you are currently using is held in memory.
2. Press the CAL key and the stored value will appear in the next line against “Kd”.
3. If the value of Kd is not stored in memory, but is known, then press Kd and enter a value for Kd using the keypad. Press CAL to enter the new Kd value.
4. If the calibration is unknown or new, then select NEW
- the calibration standard bead size that will be used should be in the range of 10-20% of the aperture diameter.
- Prepare the calibration material by adding a couple of drops of the standard to filtered seawater in the sample cup. Remember you want to keep the coincidence factor below 10% so don’t add too much.
5. Enter the size of the calibration standard
6. Select mm as the size units.
7. Place the sample cup on the sample platform and raise the cup so that the orifice and electrode are fully immersed. Close the door of the sample compartment.
- Turn the RESET/COUNT knob to the RESET position (always turn in the clockwise direction)
- Following a short time, the light within the sample compartment should come on. The display on the Multisizer may or may not indicate that the current and gain are being adjusted. When this indication disappears (or if not present to start with) then you should see numerous pulses on the oscilloscope display.
8. For the greatest accuracy, the manufacturer recommends that the instrument be calibrated in the Narrow or Window range mode. However, you should perform a measurement in the Full Range mode in order to properly determine the proper size range to use for the other modes.
9. Use the following settings: X-Axis = LOG DIA
Y-Axis = Number Diff
Blank Subtract = NO
- Turn the RESET/COUNT knob to the COUNT position. In a short time the display on the multisizer will show the accumulation of particle counts.
- If not, then it is possible that the orifice is clogged. Try lowering the sample cup such that orifice will suck air. If few or no bubbles, then use the small brush (blueish handle) and gently brush the orifice until bubbling occurs ( remember to turn knob back to RESET to get vacuum). If a large amount of air is trapped within the orifice tube, then use the FILL knob to purge the orifice of air. Raise the sample cup and check the oscilloscope for “good” pulses. If good, then go back to step 7
10. Move the left and right cursors such that they encompass the accumulated calibration particle peak. The minimum separation of the cursors is approx 10% of the full range.
11. Turn RESET/COUNT knob to the RESET position again….wait for light in sample compartment to come on.
12. Press the NARROW key
- Set X-axis to Lin Vol (mode of curve is more sharply defined)
- Set Y-axis to Number Diff
- Blank subtract = NO
13. Move the left and right cursors until they are just on either side of the mode (ie the peak) or bring both the cursors together onto the mode.
14. Press the CAL button.
15. To make sure that the Kd factor has been recorded:
- Press the SETUP key to go back to the setup menu
- Make sure that ORIFICE DIAMETER, ORIFICE LENGTH, and Kd are correct, then select RECORD on the CALIBRATION line.
- Press the CAL button. The Kd value is now stored.
16. That’s it!…
V.
Count
Filtered Water only for Background subtraction
1. In the Main Setup Menu, move the cursor to the ANALYSIS line and select “BLANK”
2. Ensure that the sample cup and the orifice have been rinsed clean with filtered seawater to remove any contaminating particles from a previous count.
3. Put sample cup containing filtered seawater in place on the sample platform and raise it such that the orifice and electrode are completely immersed. Close the sample compartment door.
4. Turn the RESET/COUNT knob to RESET and wait for the light to come on in the sample compartment.
5. Press the FULL key. You may have to wait for auto current and gain setting to occur, then determine if the left and right cursors are in the correct position to encompass the size range of your cells in the NARROW range mode.
6. Once the cursors are properly set, press the NARROW key
7. Once the current and gain have been automatically set by the machine, you should check that the coincidence bar graph is below 10% (in fact, in the NARROW range it should be near 0% for filtered seawater). If not, it may be an indication that the orifice is clogged.
8. If all appears to be O.K., then turn the RESET/COUNT knob to the COUNT position. Shortly, the particles in the blank should begin to accumulate. Your blank count should be less than 1000 and the time it took to finish should be nearly the same every time (e.g. for the 70 um orifice it is nearly always 25 seconds). If it takes longer than normal than there was some temporary clog of the orifice, and you should discard that count.
9. If satisfied with the blank, then press SETUP key to return to the Main Menu
10. For repeated counting, remember to turn the RESET/COUNT knob (on the sample stand) to RESET before using the RESET button on the main control panel of the instrument.
VI.
Count
your sample
1. Move the cursor to the ANALYSIS line and select “SAMPLE”
2. Prepare a dilution of your sample using a calibrated pipette and a volumetric flask. Typically, we do a 50x dilution for a culture density around 3 x 105 cells per milliliter.
3. Pour sample into cup and place into position in the sample compartment. Close the door.
- If your cells do not “swim”, then you should work quickly from this point to avoid artifact from cells sinking or use a larger sample cup and the stirrer incorporated into the sample platform.
4. Turn the RESET/COUNT knob to RESET and wait for the light to come on in the sample compartment.
5. Press the NARROW key (initially, the current and gain may require auto setting; wait for completion)
6. Move cursor to Blank Subtract line and select “YES”; Check that the coincidence bar graph shows less than 10%.
7. Turn the RESET/COUNT knob to the COUNT position. Shortly, the display should show particle accumulation. Note the accumulation time. It should be consistently the same. If not, check for a clogged orifice.
8. Move left and right cursors to edges of your cells size distribution; the total number of particles between the cursors is displayed.
9. Download the data to the computer (See part VII).
10. For repeated counting, remember to turn the RESET/COUNT knob (on the sample stand) to RESET before using the RESET button on the main control panel of the instrument.
11. Three replicate numbers will give you a statistically robust number.
VII.
Download
to Computer
1. Remember to store your data in your own personal directory
2. From the Acquire menu, choose “Sample info”
- The Group ID will determine the filename under which the data is stored. Each subsequent data download using the same Group Id has an extension #01, #02, #03, etc. The Group ID should be limited to 8 characters.
- The Sample ID is primarily for your own reference
- Please enter the Operator info…it helps to identify whose data it is.
- Please use the Comments section to further identify the sample and enter dilution information
- Any other information entered is also helpful….eg volume setting for manometer etc…
3. From the Acquire menu, choose “Acquire from multisizer”
4. then press the PRINT key on the multisizer. You should see “receiving” on the computer monitor followed shortly by a display of the data. If all is O.K, then answer “Yes” to save the data.
- Occasionally, the multisizer “hiccups” and the data will not transfer correctly. If this should happen, you must turn off the multisizer and the computer and start all over again…. ie store a new blank and recount your sample.. I have not yet figured out why this is happening, but I suspect it has something to do with the order in which some of the keys are pressed.
VIII.
Shutdown
the instrument
1. Remove the sample cup; Rinse and fill it with distilled water and replace it back on to the sample stand.
2. Turn off power switches
3. Shut down the computer program
1.
Turn
on the machine and start the computer software (change data directory)
2.
Filter
seawater and prepare for sample (glassware, pipette, change orifice tube if
necessary)
3.
Check
waste trap and purge reservoir.
4.
Check
initial instrument settings (date, orifice size, calibration number, etc)
5.
Perform
calibration (if necessary)
6.
Count
your BLANK
a. Rinse orifice and sample cup with filtered seawater
b. Place sample cup containing filtered seawater onto sample platform; Close sample platform door.
c. Select BLANK as the analysis type
d. Turn the RESET/COUNT knob to RESET and wait for the light to come on in the sample compartment.
e. Press the FULL key and set left and right cursors for range to be used in NARROW mode
f. Press the Narrow key. Following automatic current and gain setting, check the coincidence bar graph (should be near 0%)
g. Turn the RESET/COUNT know to COUNT.
h. After accumulation is finished, check to see if accumulation time is consistent with the usual “no clog” time..e.g 25 seconds for 70-micron orifice. Blank should be less than 1000 counts.
i. Press SETUP to return to main setup display.
a. Select SAMPLE as analysis type
b. Prepare proper dilution of sample in filtered seawater and place in clean sample cup
c. Place sample cup into position and close door
d. Turn the RESET/COUNT knob to RESET and wait for the light to come on in the sample compartment. If needed, Press RESET on the main panel to remove any previous count data.
e. Press the NARROW key (initially, the current and gain may require auto setting; wait for completion)
f. Move cursor to Blank Subtract line and select “YES”; Check that the coincidence bar graph shows less than 10%.
g. Turn the RESET/COUNT know to COUNT and watch accumulation until finished. Check accumulation time!
h. Move left and right cursors to edges of your cells size distribution; the total number of particles between the cursors is displayed.
i. Download data to the computer
j. Repeat from step d for replicate analysis.
2.
Shut
down the instrument
a. Remove the sample cup; Rinse and fill it with distilled water and replace it back on to the sample stand.
b. Turn off power switches
c. Shut down the computer program