Gear list
A gear list for counting cells might look like these, below. There's any number of options and there's no apparent ceiling on the price
you can pay for each item. However, broadly, the higher prices offer greater accuracy and high-use or specific-use functionality; most
of which is wasted on a cell counting exercise. If we're doing a handful of counts a day and more interested in precision than accuracy,
then we can happily settle on low- or mid-range options.
I've built this list with price foremost in mind, but also precision. In addition to the list, I've provided some colour on the purpose
of each component. This is only guidance and you must consider your specific needs and seek advice where necessary.
The following guidance ranges from the "cheap and cheerful" Option A to the high precision and ease of use of Option C. All three should provide acceptable results for brewing, distilling or wine making.
The minimum hardware requirements for CellMate:
- computer w/ Windows; one available USB port (faster processor ~= faster results)
- (Mac/OSX & Linux versions in future)
- microscope - compound, brightfield microscope w/ 40x objective - this is the most common microscope spec
- microscope camera - Cellmate is only tested with the camera specified, below.
- However, any 2+MegaPixel camera with a 0.5x reduction lense is very likely to be work well
- standard haemocytometer counting chamber (Neubauer, Bürker-Türk/Thoma)
|
Price (GBP, inc VAT) |
|
| Item |
Option A |
Option B |
Option C |
Shop |
| Camera |
138.00 |
138.00 |
138.00 |
Brunel Microscopes |
| Microscope (min. 400X) |
127.00 |
220.00 |
290.00 |
Apex Microscopes |
| Pump Pipette Filler (25mL) |
30.43 |
|
|
Camlab |
| 2x Serological Graduated Pipette (25mL) |
15.26 |
|
|
Camlab |
| Pump Pipette Filler (10mL) |
|
22.88 |
|
Camlab |
| 2x Serological Graduated Pipette (10mL) |
|
8.18 |
|
Camlab |
| Pump Pipette Filler (0.2mL) |
24.61 |
|
|
Camlab |
| 2x Serological Graduated Pipette (1.0mL) |
6.43 |
|
|
Camlab |
| Disposable Pastuer Pipettes |
4.79 |
|
|
Camlab |
| Adjustable-Volume Pipette (0.5-5mL) |
|
|
148.80 |
Cole-Parmer |
| Adjustable-Volume Pipette (20-200μL) |
|
176.40 |
176.40 |
Cole-Parmer |
| Cytometer & 2x Cover Glasses |
60.83 |
60.83 |
60.83 |
Camlab |
| 10x Spare Cover Glasses |
|
14.40 |
14.40 |
Camlab |
| Methylene Blue Dye (5g) |
6.78 |
6.78 |
6.78 |
Camlab |
| 500x 1.5ml Graduated Microtubes |
10.84 |
10.84 |
10.84 |
Camlab |
| TOTAL |
424.97 |
658.31 |
846.05 |
|
| TOTAL (ex. VAT) |
354.14 |
548.59 |
705.04 |
|
(Prices as at 5th November 2018)
Microscope
Cell counting isn't especially demanding on microscope hardware. A perfectly usable image can be achieved on inexpensive hobby
microscopes. The necessary features are: 10x & 40x objectives (20x is a useful, but optional); mechanical stage (not clip in);
and a "substage condensor" - it's a construction between the light source and the stage. There's a small lever which opens and closes an
iris to allow more or less light to pass. Closing the iris and limiting the light gives greater contrast and is generally better to our
cytometry exercise.
These suggested microscopes offer a bright, consistent image and good build quality. For counting cells, these are more than sufficient.
CellMate is compatible only with brightfield microscopy. Phase contrast, darkfield, etc. are not compatible.
Camera
There's an increasingly wide range of microscope cameras. Cellmate has not been comprehensively tested with different
devices, however a basic minimum specification:
- MegaPixels: 2.0+MP
- No material benefits at 5.0MP for this particular exercise
- 0.45x/0.5x reduction lense
- this is very important for the appropriate field of vision - how many squares we see in the image
- pixel size: less than 2.8 x 2.8µm*
Liquid Handling
There are two popular solutions for moving small, precisely measured volumes of liquid. At low cost, serological graduated pipettes and pumps provide a good level of potential accuracy, however this is
dependent on the operator, who controls exactly how much liquid is pumped in and out. At the higher end, micropipettes are similar in principle, but offer excellent accuracy & repeatability (see below)
for measures as small as 10μL (0.01mL) with less reliance on a careful operator.
Ultimately, we're trying to achieve consistent dilutions to bring slurries with billions of cells down to a level where we,
(or rather the computer,) can feasibly count a sample. Typically, a yeast slurry collected from a beer fermentation will need to
diluted upto 1000x; less so for the computer.
Micropipettors
Using the micropipettors suggested in Option C, above, a 500x dilution can very easily and quickly
be achieved by pipetting 20μL of the yeast slurry and 10mL of dilutant (ie. distilled water). Then, by pipetting equal measures
(ie. 200μL) of the first dilution and viability dye, we reach our 1000x dilution target.
A micropipette will come with a few tips, which are reusable, though prices for large quantities makes them potentially disposable, if you prefer.
More...
These micropipettes may account for a significant amount of our cell counting investment and warrant a closer look at the precision they offer.
| Micropipette |
Type |
Min (μL) |
Max (μL) |
Inaccuracy (%) |
Imprecision* (%) |
Price (inc VAT) |
Datasheet |
| Cole-Parmer (WZ-21600-20) |
Adjustable |
500 |
|
6.0 |
2.0 |
148.80 |
Cole-Parmer |
|
|
|
5000 |
0.6 |
0.2 |
|
|
| Socorex Acura® 825 |
Adjustable |
20 |
|
1.5 |
0.6 |
213.60 |
Socorex |
|
|
|
200 |
0.6 |
0.2 |
|
|
| Brand Transferpette® S Digital D-200 IVD |
Adjustable |
20 |
|
3 |
0.6 |
210.55 |
Brand |
|
|
|
200 |
0.6 |
0.2 |
|
|
| Cole-Parmer (GY-21600-68) |
Adjustable |
20 |
|
1.5 |
0.2 |
176.40 |
Cole-Parmer |
|
|
|
200 |
0.6 |
0.3 |
|
|
| Brand Transferpette® S |
Fixed |
20 |
20 |
0.8 |
0.4 |
123.01 |
Brand |
| Socorex® Acura® 815 |
Fixed |
20 |
20 |
0.7 |
0.5 |
131.64 |
Socorex |
| Cole-Parmer (WZ-21600-02) |
Fixed |
200 |
200 |
0.3 |
0.2 |
92.40 |
Cole-Parmer |
Prices as at Nov 2018. * Coefficient of Variation (CV, %)
The first thing to note is that adjustable volume models offer significantly less accuracy at their lower volume bounds. However, our principle aim in liquid handling for cell
counting is to ensure our dilutions are the same week in, week out. This repeatability is also known as "Imprecision" (and statistically as Coefficient of Variation (CV)).
Personally, I own the Cole-Parmer (CP) 0.5-5mL, CP 200μL & Socorex 20μL (CP don't currently produce a fixed volume 20μL). The build quality of the Socorex feels significantly better, but,
for now, I can only conclude that CP's precision and price make these superior, on balance, for cell counters.
Serological Pipettes
Serological pipettes are long, thin glass stems to which a pump is attached. By virtue of that long, thin shape the operator can clearly and accurately determine how much liquid has been
drawn (or dispensed from the drawn amount). Graduations on a 1ml pipette are at 10μL (0.01mL) and can be accurate to +/-0.007%. They're also useful for sucking samples out from specific points of
a sample or vessel.
The negative aspect of the long pipette shape is that it is slightly more fiddly to use, but, as with most things, that comes with practice. It is glass and it is delicate; hence
they are often sold in pairs. Finally, each pipette type (1/5/10/25mL) is paired with a pump ensuring the operator has a fair chance of making accurate adjustments. Although the
1mL pipette fits, for example, the 10mL pump, I can assure you that making small adjustments is challenging. Just get the right pump.
Using one, the other, or a combination of serological and micro-pipettes, we can achieve our dilutions.
Disposable Pastuer Pipettes
This type of pipette allows us to transfer small volumes from small or narrow-access vessels and place them carefully on, for example, a cytometer. They aren't much designed for accuracy
and precision. It sort of defeats the purpose of the exercise.
Haemocytometer & Cover Glasses
The haemocytometer (hemocytomter, if you prefer) gives us a fixed set of squares forming a grid, including fixed depth. By counting the cells in a good sample of these fixed-volume squares, we're able extrapolate to calculate how many
cells we expect in our undiluted sample or slurry. There is varying quality (accuracy of the grid), but agreeable accuracy for brewing is entirely achievable for ~GBP60, as above. Additionally,
there is a choice of "standard" and "metallised". This refers to the material with which the grid is printed. Metallised cytometers show white lines, which some manual counters prefer over
dark lines. Cellmate prefers standard cytometers, but can work with either.
A pair of cover glasses are often supplied with a cytometer, but in a busy environment, it's easy to lose or break these, particularly
during cleaning. If you can't ensure careful handling, have spares on hand. Also, be aware that cover glasses for cytometers are a
distinct type - being slightly heavier, they hold the sample in place. Non-cytometer specific cover glasses may affect counting accuracy.
Viability Dyes
There's a choice of a few dyes and minor variations on each. Methylene Blue (aka. Methylthioninium Chloride) is the most popular, but I understand Erythrosine B is more reliable and non-toxic.
The manufacturer or supplier should provide instruction to dilute, if necessary; it can be obtained in a pre-diluted format.
Microtubes
These very small vessels make it easier to shake our sample manually or by way of some invention. My multi-tool and a makeshift
attachment work well. Some form of vigorous shaking ensures that the cells are distributed evenly within our sample and that clumps
of cells are broken up enough to count.
; "Microscope & camera")
; "A micropipettor")
; "Pipette pumps & glass pipette")