Guide

Biology Laboratory Practice

2nd Part: Microbiology

Degree in Chemistry
Practice 1: Cell Counting
PRINCIPLES

Quantification of cells in microbial cultures

Microbial growth is measured by the increase in population, either by measuring the

increase in cell number or the increase in overall mass

There are several methods for cell counting. We will use two different direct counting
methods during this laboratory practice.

Direct methods

The total number of cells in a culture or natural sample can be determined by simply
counting the cells present in it. Therefore, the concentration will be the number of cells
present in a known volume of culture.

1. Direct microscopic counts using a counting chamber

Direct microscopic counts are possible using special slides known as counting
chambers (a microscope slide that is especially designed to enable cell counting in a
fixed volume).

There are different types of counting chamber but the most commonly used are the
Thoma cell counting chamber and the Neubauer cell counting chamber.

In these chambers the volume of culture medium used to count the cells is known,
therefore we can assess the cell concentration in the original undiluted culture solution.

Neubauer cell counting chamber

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Counting chambers consist of a grid with squares of known area etched on the surface
of a glass slide.

2. Viability counting

A viable cell is defined as a cell able to divide and increase cell numbers. The normal
way to perform a viable count is to determine the number of cells in the sample which
are capable of forming colonies on a suitable medium. Here it is assumed that each
viable cell will form only one colony. Therefore, viable count is often called plate count,
colony count or colonies forming unit (CFU) count. Colonies can then be counted and
based on the known volume of culture that was spread on the plate, the cell
concentration can be calculated (CFU/mL). Typically, a series of ten-fold dilutions are
made and plated. Acceptable values for counting range between 30-300 colonies per
plate.

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Practice 1 protocol

100 µl 100 µl 100 µl 100 µl

900 µl saline solution

in each tube
ml A? B
A B = 106 cell/ml 101 102 103 104
Dilution
Factor
Counting at Neubauer chamber 100 µl 100 µl

104 105

DAY 1

1. Count the total number of cells of an unknown concentration culture of the yeast
Saccharomyces cerevisiae (tube A) using the Neubauer cell counting chamber at light
microscope: dilute the tube A culture 10 times in saline solution and use this dilution to
calculate the number of cells/ml present in the original culture (tube A).

Count using the microscope 5 of the 0.2mm squares, evenly distributed throughout
the central square as indicated:

Use the microscope carefully. First use the 10x objective to focus the squares and
after the 40x objective to count.

Use this mathematical formula to obtain the number of cells per milliliter:

Depth = 0.1 mm
Width = 0.2 mm

Square volume (V) = 0.20 x 0.20 x 0.1 x 10-3 = 4 10-6 ml

Nº total cells counted x dilution = Nº cels/ml

Nº squares x Square volume

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2. Based on the previous result, calculate what volume has to be inoculate from tube
A to tube B, considering that the final of tube B is volume 40 ml, to obtain a final cell
concentration of 106 cells/ml in tube B. Once the teacher has reviewed the calculations,
proceed with the inoculation under sterility conditions.

3. The next objective of the practice is to determine the viable population (CFU/ml) in
tube B after inoculation. For this, make serial dilutions (of one order of magnitude in
each step) in saline solution (NaCl 0.9%) as indicated in the scheme. The dilutions are
necessary because the number of colonies on the plate has to be between 30 and 300
to avoid excessive error during the counting. As indicated in the scheme, use 100μl
from the dilutions in tube 103 and tube 104 to inoculate two plates of agar-YEPD
medium. The plates are inoculated by spreading the 100uL of diluted culture over the
surface of the agar with a Digralsky handle.

We will incubate the plates on the stove at 30ºC overnight, also tube B. The next day
we will count the colonies that have grown on each plate and observe the changes in
turbidity of tube B.

DAY 2

To count the plates discard the plate’s values when the number of colonies is outside
the range of 30 to 300 colonies. If the two dilutions of the sample are within the
recommended range, we will calculate the average value of the counting of the two
plates. If neither of the two plate counts is within the range, we will use the plate with
the closest value.

Based on the obtained results, calculate the viable population in culture B expressed
in CFU/ml (CFU= Colony Forming Units).

Notice that on each plate, we have inoculated 1/10 of the initial volume of the dilution
tube, therefore, the number of colonies that we count are 1/10 of the total cells present
in the dilution tube. For this reason, we multiply the dilution factor in the tube by 10 to
calculate the CFU/ml.

Example:

If we count 150 colonies on the plate obtained from the dilution tube 103, the dilution
factor to be used for calculations will be: 103 (tube) x 10 (plate) = 104

Therefore:

150 x 104 =1.5x106 CFU/mL

Follow the professor instructions to discard the culture wastes.

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Practice 2: Gram Staining
Gram staining is the most common technique used to differentiate two large groups of
bacteria based on their different cell wall constituents: gram-positive and gram-
negative bacteria. The procedure is based on the differential ability of microorganisms
to retain the color of the stains used during the gram stain reaction. Gram-negative
bacteria are decolorized by alcohol, losing the primary stain purple color. Gram-
positive bacteria are not decolorized by alcohol and will remain purple. After the
decoloration step, gram-negative bacteria are counterstained pink with safranin.

Practice 2 protocol

A culture of Lactiplantibacillus plantarum and a culture of Escherichia coli will be

provided by the teacher and you will have to proceed with the staining and the
subsequent observation of the slide under the light microscope.

The staining protocol is as follows:

1. Put a drop of distilled water on the slide. Sterilize Kolle's handle by placing the
part of the wire directly on the flame until it glows, then cool it. Aseptically
transfer a tiny amount of a colony from the Petri dish and gently stirring into the
water drop. Note that only a very small amount of culture is needed.

2. Fix the sample. Quickly pass the slide two to three times through a Bunsen
burner flame. Do not overheat, or the samples may become distorted.

3. Add a drop of crystal violet over the fixed culture. Let stand for 1 minute.
Bacteria will stain purple

Remove excess dye. Do not wash

4. Add a drop of iodin (this will trap the purple crystal violet color in the cell,
wherever it has stained). Let stand for 1 minute.

5. Add a few drops of decolorizer (70% ethanol) until no more violet color is visible
in the draining runoff. This step decolorizes gram-negative bacteria.

6. Counterstain with safranin for 1 minute.

7. Wash off the solution with water.

8. Remove the excess water with paper.

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 9. Place the slide under the light microscope and examine the slide using the 40x
objective.

Differences between Gram-positive and Gram-negative bacteria

Now, determine which bacteria culture is Gram positive and Gram negative from those
provided: L. plantarum and E. coli.

Follow the professor instructions to discard the culture wastes.