Determination of cell counts with microscope

To determine the number of suspended particles in a given volume (e.g. cells, spores of fungi), counting chambers are generally used (Fig. 11). These are microscopic slides into which cross-channels are grooved. The slide is thinner at these channels, so the height of the liquid column is known. As the size of the grid is also known, the volume between the slide and cover slip can also be precisely defined. These slides are usually thicker than the normal ones to avoid deformations. The overflowing liquid can freely exit.

The size and shape of grids can differ to fit the purpose of analysis. In the case of Thoma-chambers, the area of the big square is 1 mm², which comprises 16 smaller squares. The area of the smaller squares is 1/16 mm²and with more divisions, the area of the smallest squares is 1/400 mm². The height of the more widely used Bürker-chambers is 0.1 mm (Fig. 11). The size of the big square is 1/5 mm x 1/5 mm = 1/25 mm², and that of the rectangle is 1/5 mm x 1/20 mm = 1/100 mm². There are several other counting chambers available with different rulings (e.g. Neubauer-, Türk-, Jensen-, Fuchs-Rosenthal). To get the correct count, it is important to repeat counting many times, possibly on several subsamples.

Fig. 11. Bürker-chamber.(a) Parts of the chamber 1. cover glass 2. clamp 3. counting chamber (drop the spore suspension here) 4. facet with grid (b) Enlarged grid 5. big square of 1/25 mm²area 6. rectangle of 1/100 mm²

area. (c) Micrograph of spores fromAspergillus nigerin a Bürker chamber.

EXERCISE 13: DETERMINATION OFASPERGILLUS NIGERSPORE CONCENTRATION WITH BÜRKER-CHAMBER

Object of study, test organisms:

Aspergillus nigerculture in Petri dish Materials and equipment:

inoculating loop sterile water in test tube Bunsen burner

pipette, sterile pipette tips Bürker-chamber

alcohol

light microscope Practise:

1. Prepare a suspension fromAspergillus nigerspores in sterile distilled water using an inoculating loop.

2. Degrease the Bürker-chamber with alcohol over a Bunsen burner.

3. Fix the cover slip to the chamber.

4. Put one drop of the spore suspension beside the cover slip of the chamber. The chamber will be filled with the suspension due to the capillary action.

5. Wait for 1-2 minutes until flow of the suspension stops.

6. Put the chamber under the microscope and adjust focus. Check your sample with 16x or 40x objective.

7. Count the number of spores in 10 big squares (or rectangles).

8. Average these values and then calculate the concentration of spores as spore count/mL for the suspension.

The cells to be enumerated can be stained for better observation. Classical staining procedures are reviewed in chapter 7.4.1. Additionally, fluorescent dyes are also widely used to determine cell counts in various environmental samples. One of such dyes is DAPI, which binds to the DNA of virtually every microorganism, but this stain is not suitable to assess cell viability since it fails to differentiate between living and dead cells.

EXERCISE 14: ENUMERATION OF MICROBES WITH DAPI STAINING

DAPI molecules are able to penetrate cell membranes and bind to the double helix of the DNA. Cells can be easily counted, if a known volume of fixed water sample is filtered through a membrane and, after staining the filter, surface is investigated by an epifluorescence microscope (Fig. 12).

Fig. 12. Fluorescence microscopic image of DAPI stained bacterial cells.Bacterial cells from drinking water show blue fluorescence on the membrane filter.

Object of study, test organisms:

microbes of surface water samples Materials and equipment:

disposable gloves laboratory scales paraformaldehyde (PFA) beaker

magnetic stirrer

phosphate buffered saline (PBS) (see Appendix) cc. NaOH solution

Pasteur pipette

membrane filtration apparatus

polycarbonate and cellulose nitrate membrane filters (0.22 or 0.45 µm pore size) 50 mL Falcon tube

plastic Petri dishes scalpel

pipette with pipette tips DAPI solution (1µg/mL) 80% ethanol

double distilled water glass slide

cover slip

Vectashield Mounting Medium (H-1000, Vector Laboratories Ltd) immersion oil (non-fluorescent)

epifluorescence microscope with a mercury lamp digital camera

computer with adequate softwares Practise:

1. For the preparation of fixative solution, dissolve 1 g PFA in 50 mL PBS. (PFA causes irritation when inhaled, therefore the use of a fume hood is recommended.) Dissolution can be aided with heating (ca. 60°C), permanent stirring and adding some drops of cc. NaOH solution.

2. Adjust pH to 7.0.

3. Filter the solution through a 0.22 µm pore size membrane filter. (The prepared 2% PFA solution can be stored in the fridge for one week).

4. Filter the water sample (2-50 mL, depending on the type of sample) using polycarbonate membrane filter (slowly with occasional stirring). To help uniform cell distribution, place a 0.45 µm pore size cellulose nitrate membrane filter between the sieve of the filtration unit and the polycarbonate filter.

5. Fill the Falcon tube with fixative (PFA solution) and immerse the filter in it with sterile forceps (PFA solution must cover the entire membrane filter).

6. Incubate the filter overnight at 4°C.

7. Fill PBS into an empty Petri dish, then transfer the filter into the PBS solution for 1-2 minutes (liquid must cover the entire membrane filter).

8. Transfer the filter to another empty Petri dish and let it dry.

9. Cut a 0.5 by 0.5 cm piece from the filter with a scalpel or scissors, and pipette 30 µL DAPI solution onto its surface. From this step onwards, work in a dark place. The filter piece can be marked with a soft pencil.

10. After 2 minutes, transfer the filter into 80% ethanol for a few seconds.

11. Dip the filter paper into double distilled water for a few seconds.

12. Dry the filter.

13. Place the filter onto the surface of a glass slide, put a drop of Vectashield Mounting Medium onto the filter and then cover with a cover slip. Cover with paper towel and gently press the cover slip to remove any excess of mounting medium.

14. Examine the slide with epifluorescence microscope using a 100x objective and immersion oil under UV excit-ation (the absorption maximum of DAPI is at 358 nm, and emission maximum is at 461 nm).

15. Record images from at least 20 different microscopic fields with a digital camera.

16. Count the cells on each picture and determine the mean values.

17. Determine the cell count for one mL water sample based on the amount of filtered water and the size of the membrane filter field. Evaluate the variability of cell morphology.

(See also Supplementary Figure S21.)