Santiago de Cali University

DEPARTMENT OF BASIC SCIENCES

ACADEMIC PROGRAM IN MICROBIOLOGY
INDUSTRIAL MICROBIOLOGY LABORATORY

WORK GUIDE N°2: COUNTING IN NEUBAUER CHAMBER

GENERAL OBJECTIVE
Perform and interpret the technique for counting yeast and fungal spores in the chamber of
Neubauer.

THEORETICAL FUNDAMENTALS

The Neubauer chamber is a counting chamber used to determine the number of particles.
(yeasts, bacteria, red blood cells, etc.) per unit volume of a liquid through the microscope. It is
consisting of a thick block of glass marked by a grid (Fig. 1), which contains two zones
slightly depressed with known dimensions. The central depression of the coverslip is sunken
0.1 mm from the surface, so that when covered with a coverslip it is away from the surface
marked 0.1 millimeter, and the volume between the surface and the coverslip is 0.1 millimeter
cubic, that is 0.1 microliter.
It will usually be necessary to determine both the cell density in the suspension and the percentage.
of these that are viable. Different methods are used to determine cell viability. The most common is
the staining with methylene blue. Methylene blue is a dye that is introduced into the interior of the
cells that show damage to the membrane. Thus, the cells that appear blue are considered
non-viable.

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Figure 1. Top and side view of the Neubauer chamber

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METHODS

MATERIALS AND REAGENTS

MATERIALS REAGENTS
1 Micropipette 100-1000 ml with tips Yeast sample
1 calibrated balloon of 10 ml Mushroom cultivation (spore producing)
1 Dropper Distilled water
1 Neubauer Chamber Methylene blue 0.01% (water)
Cover glass Antiseptic alcohol
1 micropipette of 0.5-10 ul with tips
1 Microscope

PROTOCOLS

1- Yeast count

1.1- Sample Preparation.

Take 1 ml of the liquid culture (yeast sample) and dilute it to 10 ml with distilled water in a flask.
diluted. Add a drop of methylene blue. Cover and gently shake.
In case the culture is in solid medium, take a colony and resuspend it in 5 ml of
diluent; take 1 ml and make a 10-fold dilution-1.

1.2- Filling the chamber and cell counting

Clean the camera and the cover sheet with alcohol.

Place the slide over the chamber, it should be centered.
Fill the chamber with a small micropipette (0.5-10 ul). The filling must be continuous in a single attempt. Do not
Dry portions must be observed in the corners of the filling box, and it should not be flooded.
Take the camera to the microscope and focus the counting frame with the 4X eyepiece. You will observe an image.
similar to figure 2.

Figure 2. Neubauer Chamber 4X.

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If the cells you are going to count are small like yeasts, bacteria, etc. Position yourself in the central grid.
and when switching to the 10X eyepiece, it will be visualized as follows:

Figure 3. Neubauer chamber 10X.

In this grid, 25 squares are displayed, of which the cells present in 5 fields are counted.
Usually, the squares of the corners and the center (marked by arrows in Fig. 3) are counted, which
guarantees a random count. To perform the count, the 40X eyepiece is used, where each one is visualized
The fields and with the help of the microscope's cart, the grid is moved until all squares are counted.
The counting in these fields of the camera is known as AP (high power). With the 40X lens, each frame is
see as follows:

Figure 4. Neubauer chamber at 40X

The cells are counted box by box and a total is done. It is recommended to carry out the counting following the
arrows in Fig. 5 to prevent cells from being counted twice or not counted at all. Around each
In the grid, it can be seen that there are three lines that delimit the square, which are fundamental at the moment of
count since they define which cells are countable or which are out of the counting range. The cells that
They do not touch the second line, they are accountants; if they touch it or are on top of it, they are not included.

Figure 5. Guidelines for conducting the count

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The cells that have an X are the ones that should not be counted (Fig. 6)

Figure 6. Cell count in tertiary squares

After counting the cells, the number of cells per unit volume is calculated. For this,
You must take into account the area of each square and the space between the camera grid and the slide.
covers objects. The camera has a depth of 0.1 mm and the following dimensions:

Figure 7. Dimensions of the Neubauer chamber grid

Cell suspensions must be diluted enough so that the cells or other particles do not
they should overlap and be evenly distributed. For small cells, it is recommended to count four
corners and the square in the center.
Each square has an area of 0.2 * 0.2 = 0.04 mm2and 0.1 mm deep. Therefore, the total volume of
each square is 0.04 * 0.1 = 0.004 mm3if 5 squares were counted, the volume is 0.02 [Link] yes

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187 cells were counted in a volume of 0.02 mm3equivalent to 9350 cells/mm3There are 1000 mm3in 1 cm3,
giving a total of 9,350,000 cells/ml.
For practical purposes, the following formula can be applied:

Cells/ml = total counted cells * 50,000

Cells/ml = 187 * 50,000 = 9,350,000

But, in order to determine the number of cells in the original sample, this value must be multiplied by the
dilution, which in this case was 10.

Cells/ml = (total counted cells * 50,000) * dilution

Cells/ml = (187 * 50,000) * 10 = 93,500,000

1.3- Determination of cell viability

The blue methyl dye is able to penetrate the wall of dead cells, completely staining them, from
in such a way that living cells are observed without staining. To determine the cell viability of the culture is
it is necessary to apply the following formula:

Viability % = (total cells - dead cells) / total cells x 100

It is important to perform the counting as soon as possible after staining, because some
cells can die from oxidative processes, altering the count.

2- Fungal Spore Count

Take a loop handle part of the aerial mycelium of the fungus and dilute in 10 ml of distilled water. If applicable
necessary, filter through a mesh or gauze to eliminate the agar or remnants of mycelium. Load the chamber as
in the previous procedure.

In case the fungus has large spores, count the squares A, B, C, and D (Fig. 8) and apply the
next formula:

Spores/ml = (total spores counted * 10,000) / (number of squares * dilution factor)

Example: if a total count of 66 spores was obtained:

Spores/ml = (66 * 10,000) / (4 * 0.1)

Spores/ml = 1,650,000

If the spores are small, the counting can be done as for yeasts.

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 Figure 8. Grid for counting fungal spores.

BIBLIOGRAPHY

Autonomous University of Guadalajara, Manual of Chemical-Biological Sciences, Biotechnology: Appendix 1

quantification of yeasts, Invalid input for translation. Please provide text for translation.

Libkind, D, Tognetti, C & M, Moliné 'Theoretical-Practical Course on Microscopy and Yeast Counting for
beer producers [online], Applied Microbiology and Biotechnology Laboratory, Institute of
investigations en biodiversity y environment Bariloche- Argentina
The provided text is a URL and does not contain translatable content.
No text content was provided for translation.

INTERNAL LEGAL STATUS OF THE COURSE

He/She elaborated Valeria Pérez Luna E-mail valeriaperez@[Link]
Reviewed Sandra P. Rivera Msc. Department Basic Sciences
Approve x Area Microbiology

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