Yeast

You can either use fresh (wet) yeast or dried yeast. In either case, take a small amount and dissolve in a little bit of water, until the liquid becomes turbid. Use this suspension for microscopy. (read The hemocytometer (counting chamber) to sell how yeast cells look like in a counting chamber).

Yogurt

One of the more difficult specimens. Yogurt contains many bacteria, these are a bit difficult to see with bright-field microscopy. You can stain them (read ). Take a small sample (knife-tip) and dissolve in water. Then apply a drop to the slide, apply a cover glass, and observe under the microscope.

Cheese

Here you have to take the right kind of cheese. The toast-cheese (the one where each one is wrapped separately in plastic foil) won’t work. They don’t have any fungus growing on them (Do not let it rot, you may be growing poisonous fungi). I’m a cheese lover and I consider Camembert, Brie, Gorgonzola blue cheese not only good for eating but also a valuable source for the fungi Penicilium.

Honey

Some of them contain pollen. If the honey is turbid (opaque) then this may be due to sugar crystals or due to pollen. Clear honey won’t work.

Counting chamber: This one is called the Neubauer improved. There are other standards with different grids available as well.

Yeast cells in the hemocytometer. The grid is clearly visible.

Yeast cell suspension applied to the chamber. Notice that some of the cell suspension has gone into the overflow area.

One counting chambers has grids of different sizes. Consult the manual to find out the size.

Do not count cells on the top and right lines. Here it's necessary to count the in the big square because there are too few cells in individual small squares.

Counting chamber seen from the side.

Grid layout of the Neubauer Improved hemocytometer.

Purpose of the hemocytometer

The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of sperm cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.

Preparing the sample

The fluid containing the cells must be appropriately prepared before applying it to the hemocytometer.

• Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed.
• Appropriate concentration: The concentration of the cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares (which takes time). Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000. A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts water (or better saline of correct concentration to prevent bursting of the cells). The dilution must later be considered when calculating the final concentration.

Counting the cells

• Counting cells that are on a line: Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted.
• Number of squares to count: The lower the concentration, the more squares should be counted. Otherwise one introduces statistical errors. How many squares? To find out one could calculate the cell concentration per ml based on the numbers obtained from 2 different squares. If the final result is very different, then this can be an indication of sampling error.

Calculating the cell density

Here it is necessary to do some simple math. The following numbers are needed: number of cells counted in a square, area of the square, height of the sample, dilution factor. The objective is to find the number of cells in 1ml of original solution.

• Step 1 – Averaging: If one did not count all of the cells in a large square (1mmx1mm) then it is necessary to average the results first before proceeding. For the purpose of this example, I use an average cell count of 123.456 cells.
• Step 2 – Computing the volume: It is necessary to determine the volume represented by the square. The width and height of the square (e.g. 0.25mm x 0.25mm) must be multiplied by the height of the sample (often printed on the hemocytometer, in this example it is 0.1mm): v = 0.25mm x 0.25mm x 0.1mm = 0.00625mm³ = 0.00625ul (where ul is microliters).
• Step 3 – Calculating the number of cells in 1 ml: if there are 123.456 cells in 0.00625ul, then how many cells are there in 1ml (=1000ul)? We do simple direct proportion:

  123.456cells/0.00625ul = X/1000ul
  (123.456cells*1000ul)/0.00625ul = X (the ul cancel out)
  X = 19 752 960 cells

• Step 4 – Correcting for dilution: If the sample was diluted before counting, then this must be taking into consideration as well. We assume that the sample was diluted 1:10. The final result is therefore 19 752 960 cells x 10 = 197 529 600 cells in 1 ml. That a lot of cells.

Things to watch out for

• Type of counting chambers: There are different types of counting chambers available, with different grid sizes. One counting chamber also has grids of different sizes. Take care that that you know the grid size and height (read the instruction manual) otherwise you’ll make calculation errors.
• Use the provided cover glasses: They are thicker than the standard 0.15mm cover glasses. They are therefore less flexible and the surface tension of the fluid will not deform them. This way the height of the fluid is standardized.
• Moving cells: Moving cells (such as sperm cells) are difficult to count. These cells must first be immobilized.
• Objective The hemocytometer is much thicker than a regular slide. Be careful that you do not crash the objective into the hemocytometer when focusing.

Why bacteria are difficult to see

Bacteria are difficult to see with a bright-field compound microscope for several reasons:

• They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
• Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
• They are transparent: Bacteria will show their color only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
• Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

Research organizations and advances amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artifacts (“fringes”) around the individual cells. One possibility is to stain the bacteria, but in this case there fixing and staining process may introduce artifacts.

What is a safe source of bacteria? For recreational or educational purposes, one should never use spoiled food or (heaven forbid!) use bacteria obtained from the human body and grown on agar plates. The risks involved are simply not worth it, especially when working with students. Other sources, such as soil or humus have other disadvantages. The impurities make it difficult to keep bacteria from other particles apart, especially if one uses bright-field optics. Rather I recommend the use of yogurt. It should be possible to see small circular cells (cocci), which may also occur in pairs. It is also possible to scratch some bacterial cells off from certain kinds of cheese. Brevibacterium can be found on Limburger cheese, for example. One has to be aware that some cheeses use a combination of bacteria and fungi, however, and that the larger fungal cells may outweigh the bacteria.

In summary, there are easier (and maybe also more interesting) specimens to observe than bacteria. I you want to see individual cells, then I do recommend that you start out with yeast suspensions. These eukaryotic cells are much larger and can be more easily identified.

For pictures of bacteria in phase contrast read the following post: Bacteria in phase contrast