Guide for counting algae/spores with a counting chamber

• Quick reference
• Different counting chambers of the Daphnia lab and their technical specifications
• How to load the tricky chambers
• How to count properly
• The hard task: Calculating the algae concentration from the counted numbers

Quick reference for counting

Quick Formula: Algae cells per smallest square (total sum of counted cells = X divided by total smallest squares counted in = Y) multiplied by specific factor (X/Y) * specific factor.

Click here for a javascript program that helps you calculate the right concentration.

Of course parasite spores or other cells are counted in the same way!

Different counting chambers and their technical data

Code	Grid type	Depth	Length of smallest square	Chamber	Grid (smallest square marked red)
A	Thoma	0.02 mm	0.05 mm
B	Semen counting chamber	0.01 mm	0.1 mm
C	Neubauer improved	0.1 mm	0.05 mm
D	Bürker	0.1 mm	0.05 mm

How to load the tricky chambers

The chambers are constructed such that the outpart and the inner section are ground smooth and polished. The surface of the central part is deeper than that of the external supports. The counting nets are engraved in the central part (chamber base). If a cover glass is placed on the external support, a capillary gap is produced between the underside of this cover glass and the central part of the counting chamber.

To slide on the cover glass, get the slide ready on a level place, with the cover slip beside it ready to be grabbed.

1. Lick your fingers (purists may use distilled water for this) and wet the external support sections of the counting chamber (both to the left and to the right of the chamber base). Then push the cover glass slowly (but not too slow) forwards onto the counting chamber from the front (push perpendicular to the longest axis of the chamber). The formation of interference lines (Newton rings) between the external support and the cover glass shows that the cover glass is correctly positioned. Push the cover glass until a small section of the chamber base is still uncovered.
2. Put a drop of the solution you want to count on the chamber base (between the cover glass and the chamber base). As a result of the capillary effect the gap between the cover glass and the chamber base fills up. Before the thinned solution can overflow at the edges of the chamber section the tip of the pipette must be removed (if you use a specified volume this is not necessary, as all the solution will be sucked in without overflow). (See below for drop volume recommendations). If air bubbles are formed start again. Likewise, if the liquid overflowed over the edges and into the grooves, start all over.
3. Take the loaded counting chamber, put it under the microscope and check briefly with smallest magnification, if the distribution of algae cells is homogenous.
4. Count the cells (see next section).

Recommended volumes of liquid:

• Thoma (depth: 0.02mm): 2.2ul
• Semen counting chamber (depth: 0.01mm): 1.8ul
• Neubauer improved (depth: 0.1mm): 12ul
• Bürker (depth: 0.1mm): 12ul

How to count properly

Counting: green algae are included, red algae not

Counting Procedure: Pick two sides of the squares in the grid (here we'll pick TOP and LEFT). All algae cells which lie inside the smallest square unit (see diagram) without touching any grid lines, are counted. Additionally, all algae cells touching the TOP and/or the LEFT grid line of the square unit are counted as well, even if most of the algae cell lies outside the square. NO algae cell is counted, if it touches the BOTTOM and/or RIGHT grid line, even if most of the algae cell lies inside the square. Problematic cells: algae cells touching either the TOP and RIGHT grid lines, or the BOTTOM and LEFT grid lines. Decide before counting, whether to include either the TOP-RIGHT or the BOTTOM-LEFT algae cells to your counting. (Problematic cells are marked blue in the diagram)

Further, count the amount of square units you counted algae cells in. Best is to count algae in several square units lying apart from each other. For a better estimation, load the chamber again and make a second count.

The hard task: Calculating the algae concentration from the counted numbers

The exact formula: # algae cells / ml = ( X · 1000 mm3 ) / ( Y · w² · d )

Specific factor from the short cut: Factor = ( 1000 mm3 ) / ( w² · d )

X = # algae cells counted
Y = # smallest squares counted
d = depth of counting chamber
w = width of 1 square unit

w² · d is the volume of liquid over one (smallest) square

This page was initially composed by Patrick Mucklow