# 3.1.1. Total or microscopic count

A haemocytometer (Fig. 10) is used to determine the
number of particles found within a demarcated region of a slide haemocytometer
containing a known volume. The number of cells counted in this volume is used
to extrapolate the number of cells in the total sample. There are several kinds
of haemocytometers*,* but they all
consist of a microscope slide with a grid etched into the bottom of a cavity
(the counting chamber, Fig. 11). The size of the counting chambers can vary
with model and manufacturer (e.g. Helber Z30000, Fuchs-Rosenthal, Neubauer,
Neubauer improved, Thoma, Thoma new). A typical chamber depth is 0.1 mm, but to
be able to count smaller particles (bacteria) a smaller depth (0.02 mm, e.g.
Petroff-Hausser) is required. The grid is divided in squares of different sizes
that allow for the counting of particles of different sizes. The number of
squares also depends on the model (Neubauer: 3x3; Neubauer improved 5x5; Thoma
4x4) as is the number of lines separating the squares (Figs. 12 and 13). A
cover glass closes up the top of the cavity, determining a specific chamber
volume. It is possible to obtain disposable counting chambers (e.g. Fastread, UK),
which have the advantage of not requiring cleaning between measurements.

Procedure to follow when using a haemocytometer

- Carefully clean haemocytometer and cover glass with lens paper with sterilised distilled water to avoid contamination or counting errors.
- Dry with lens paper.
- Slightly moisten the edges of haemocytometer.
- Apply cover glass.

Make sure to use the provided cover glasses - these glasses are thicker than the standard cover glasses so that surface tension will not deform them. - Press firmly until the Newton rings appear where slide and cover
come into contact.

This is important for accuracy of the measurement since only a proper placement ensures a correct volume and therefore counting. - Prepare
your sample according to description in other papers of the
*BEEBOOK*(American foulbrood, De Graaf*et al.*, 2013; European foulbrood, Forsgren*et al.*, 2013; fungi, Jensen*et al.*, 2013; nosema, Fries*et al.*, 2013; queen rearing and selection, Büchler*et al.*, 2013; instrumental insemination, Cobey*et al.*, 2013).

The samples, especially if they include bees or bee parts, should be carefully ground or dissected and mixed with water. The solution should contain 5-50 particles per square. If stock solution has more particles, it can be diluted until values in this range are obtained. This determines the dilution factor.

To facilitate calculations of the dilution factor, it is recommended to use one ml of water per sample or bee that has to be counted or to use 10 times dilution series. In this case, mixing the samples by vortexing during the dilution process is necessary to ensure a homogeneous suspension of the particles. Vortexing is also necessary to homogenise the solution before each counting. - Mix sample properly to ensure uniform/homogenous suspension before introducing the suspension to the periphery of one of the v-shaped wells with pipette. The area under the cover slip fills by capillary action.
- Place haemocytometer under microscope, adjust to appropriate magnification.
- Use a weak magnification to facilitate localisation of the grid.
- Adjust to appropriate
magnification for counting (see the
*BEEBOOK*papers on nosema, European foulbrood and fungi for more details, Fries*et al*., 2013; Forsgren*et al*., 2013; Jensen*et al.*, 2013, respectively).

Do not crash the objective into the cover glass when focusing! Remember the haemocytometer is much thicker than regular slides. - Allow 2 min for the particles to settle in the chamber before counting.
- Count
the particles in the appropriate squares depending on the size of the particles
to be counted, making sure that different areas of the chamber are counted
(e.g. for Nosema spore sized particles, Fig. 13).

Count at least 300 particles in order to minimise errors.

Particles that are only partially inside a particular square must be dealt with in a systematic manner to prevent double counting when the neighbouring square is counted. Count only those particles which are entirely within a square and only those crossing over the top and left boundaries (or bottom and right, if you prefer). If squares are separated by several lines, chose one as a boundary. - Calculate
the number of particles per ml of the original sample from the known volume of the counting chamber.

Formula:

- To obtain the total number of particles in
the sample, multiply the concentration obtained by the initial sample volume.

Example:

total number of counted particles: 288

area of
small squares counted: 24 x 0.04= 0.96 mm^{2}

chamber depth: 0.1mm

dilution: 1:200 (dilution factor d=200)

Say
total sample volume was 0.5 ml, there are 0.5 x 600,000 = 300,000 particles in
the samples.** **

**Pros: **Haemocytometers are
inexpensive and commonly used. They are long-lasting and versatile and a very
effective way to count particles.

**Cons:** Using a haemocytometer
requires a phase contrast microscope. Statistical robustness is lacking when
counting low concentrations. In addition subjectivity may be a problem among
users and it is a tedious and time consuming method (Hefner* et al.,* 2010). It is a monotonous and time consuming task, only
reliable for clearly recognisable particles or in samples without structures
looking similar to the particles of interest. The viability of the particles
counted is unknown.

The automated cell counting method, including flow cytometry, Scepter cell counters and vision based counters, may be a more reliable alternative method to use for particle counting. Not only is it less time consuming, it eliminates subjectivity and it also provides counting algorithms. In future it may even become a necessity in laboratories.

** Fig. 10.** A haemocytometer Photo: V Dietemann.

** Fig. 11.** A drawing of a haemocytometer by V Dietemann.

Haemocytometer
grid: red square = 1 mm

Fig. 12.

^{2}, 100 nl, green square = 0.0625 mm

^{2}, 6.25 nl, yellow square = 0.04 mm

^{2}, 4 nl, blue square = 0.0025 mm

^{2}, 0.25 nl, at a depth of 0.1 mm. Source: Wikipedia In an improved Neubauer haemocytometer total number of cells can be determined by number of cells found in grid (red square) x 10

^{4}(10000)

** Fig. 13.** Suggested counting of 24 squares in a haemocytometer.