FORMULAS AND HELPFUL TIPS

(I) CONCENTRATION CALCULATIONS

(1)

Example:
1 L sample + 9 L water (diluent) = 10 L total volume Dilution factor = 10
1 L solution (dilution factor 10) + 1 L water (diluent) = 2 L total volume
Dilution Factor = 20 (2 x 10)
5 L water (diluent) + x L sample = 5 + x L total volume
Reduction of concentration should be = 6
6 = 1 part of sample + 5 parts of dilution (all parts are equal in volume)
5 equal parts of dilution = 5 L water (diluent) / 5 = 1 L
All 6 parts have the volume of 1 L
x L sample = 1 L
Conc. Reduction = initial volume of sample : total volume of the diluted solution
= parts of sample : all parts of the dilution
Example:
1 L sample + 9 L water (diluent) = 10 L total volume
Reduction of concentration = 1 : 10
1 L 1:10 solution + 1 L water (diluent) = 2 L total volume
Reduction of concentration = 1:20 (1:2 x 1:10)
5 L water (diluent) + x L sample = 5 + x L total volume
Reduction of concentration should be = 1:6
1:6 = 1 part of sample + 5 parts of diluent (all parts are equal in volume)
5 equal parts of dilution = 5 L water (diluent) / 5 = 1 L
All 6 parts have the volume of 1 L
x L sample = 1 L

(2)

(3)

C1 x V1 = C2 x V2
C2 = Wanted
C1 = Concentration
concentration of
of the solution
the end-solution

(4)

V1 = Volume of
V2 = Wanted
solution in the
Volume (volume
mixture
of all parts)
Example:
Concentration of the solution: C1 = 20 mg/mL
Wanted concentration: C2 = 10 mg/mL
Wanted volume: V2 = 5 mL
C1 x V1 = C2 x V2 => 20 mg/mL x V1 = 10 mg/mL x 5 mL
V1 = (10 mg/mL x 5 mL) / 20 mg/mL
V1 = 2,5 mL
2,5 mL solution (20 mg/mL) + 2,5 mL dilution = 5 mL solution (10 mg/mL)
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(II) PHOTOMETRIC MEASUREMENTS

DNA and RNA absorbs in the ultraviolet range, at a wavelength of about
260 nm because of the nitrogenous bases
Proteins absorb at 280 nm
Beer-Lambert Law:
A = cl
A = absorbance value (no units)
= extinction coefficient (constant for each substance)
=0.027 (g/mL)-1 cm-1 for ssDNA
=0.020 (g/mL)-1 cm-1 for dsDNA
=0.025 (g/mL)-1 cm-1 for ssRNA
c = concentration of substance (units for DNA/RNA = g/mL)
l = light path length
= 1 cm for standard cuvettes
For the light path length in 96 well plates use the formula
4


V = sample volume
d = mean diameter of the well
Does not account for the meniscus of the liquid
= 0.29 cm for a 96 well plate with a 100 L sample volume
= 0.56 cm for a 96 well plate with a 200 L sample volume
Example
10 L ssDNA + 90 L water mixed in the cuvette (Conc. Reduction = 1:10)
Spectrophotometric measured absorbance (OD) of ssDNA A = 0.9
Measured blank (10 L buffer of ssDNA + 990 L water) A = 0,225
ssDNA absorbance without the background of solutions: A ssDNA A blank
ssDNA A = 0.9 blank A = 0,225 A = 0.675
0.675 = 0.027
x c x 1 cm x (Concentration Reduction)
.

(5)

c=
.

c = 250 g/mL
The original 10 L ssDNA has a concentration of 250 g/mL

(6)

The ratio of absorbance at 260 to 280 nm (A260/A280) should be

In case of DNA: 1.8 (>1.75)
In case of RNA: 1.8 - 2.1

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(III) UNITS
Unit prefix - indicate multiples or fractions of the units
tera
T
= 1012
giga
G
= 109
mega
M
= 106
kilo
k
= 103
Unit
=0
Unit
Milli
Micro
Nano
Pico

n
p

103 = 1,000
10-3 = 0.001

=0
= 10-3
= 10-6
= 10-9
= 10-12

106 = 1,000,000
10-6 = 0.000001

109 = 1,000,000,000
10-9 = 0.000000001

1012 = 1,000,000,000,000
10-12 = 0.000000000001

Solids in liquid
1 g/L = 1 mg/mL = 1 g/L = 1 ng/nL = 1 pg/pL
1 g/L = 0.001 g/mL = 0.001 mg/L
1 mg/mL = 1000 g/mL = 1000 mg/L

(IV) GENERATION TIME

Generation time is per definition the time interval required for the cells
(or population) to divide:
t
G
n

(7)

G = generation time
t = time (in minutes or hours)
n = number of generations (number of times the cell population doubles during
the time interval)
Number of bacteria
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval

(8)

B x 2

n = 3.3 log(b/B)
This equation is an expression of growth by binary fission
t
G
b
3.3 log
B
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(7)
+
(8)
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(V) CELL NUMBER WITH HEMOCYTOMETER

1 mm3 = 1 L
Thoma Chamber

(9)

Size of the smallest square:

0.0025 mm x 0.1 mm Depth = 0,00025 mm = 0,00025 L
Size of the next bigger square:
Next bigger square = 16 x smallest square
16 x 0.0025 mm x 0.1 mm Depth = 0.004 mm = 0.004 L
1. Count the cells in a square of your choice
2. Calculate the cell number to the standard unit cells per mL
3. If the sample was diluted, multiply the calculated cell concentration by the
dilution factor
Example
12 cells are in the next bigger square (0.004 L)
12 cells per 0.004 L = 12 cells/0.004 L
Use the rule of three

3000 000 cells/mL

The sample was diluted (1 part + 4 parts dilution) with a dilution factor of 5
3000 000 cells/mL x 5 = 15000 000 cells/mL
The smaller the volume of interest- the smaller the number of cells
Example of the same concentration
2 points in the smaller dotted square (1/4 the size of the big one)
8 points in the big square (4 times more than in the small one)
Example of a dilution
2 points in the smaller dotted square (1 part)
Transfer into a big square filled with dilution solution (3 parts)
Concentration Reduction (2) = 1:4
Calculate the concentration of the dilution
Original concentration: 8 points in the big square, dilution 1:4
8 x = 8 4 = 2 points in the dilution

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(VI) TRANSFORMATION EFFICIENCY

Transformation efficiency
# of colonies
x 106pg x volume of transformants
pg plasmid DNA
1 g
X L plated

(10)

# of colonies = colonies counted in the petri dish

pg plasmid DNA = used amount of pUC19 DNA for the tansformation in pg
106 pg / 1 g = Calculation from the unit pg to the unit g
volume of transformants = total amount of the mixture (in L) before X L were
taken out to plate it
X L plated = amount of liquid, which was given on agar in the petri dish (in L)
Example
30 successfully transformed blue colonies are on the agar
300 pg Bluescript plasmid DNA were used
1053 L were mixed as a volume of transformants
50 L competent cells
3 L Bluescript DNA
1000 L LB medium
200 L were spread
30 colonies
x 1000000 pg x 1053 L transformants
300 pg Bluescript DNA
1 g
200 L plated
30 colonies
x 106 pg x 1053 L
300 pg Bluescript DNA
1 g
200 L

526500 colonies
g Bluescript DNA

In this example, 526,500 colonies were transformed per g Bluescript plasmid.

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UNIT 1. ACCURATE PIPETTING OF LIQUIDS, SERIAL DILUTIONS AND STERILE

TECHNIQUE
This unit aims to introduce you to a number of crucial skills to be able to succeed in
the laboratory. These skills include accurate pipetting of liquids, preparation of serial
dilutions, data analysis and sterile technique.
I. ACCURATE PIPETTING OF LIQUIDS (PART I)
Answer these following questions before you join the practical course.
1.1 Name the three main types of pipettes.
1.
2.
3.
1.2 Name the volume ranges of the threemicropipettes:
Micropipette
Range in L
P20
P200
P1000

1.3 In which situations do you have to use the forward technique and in which the
reverse technique?
Technique
Sort of liquid
Forward technique
Reverse technique

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INTRODUCTION
The first part of todays session is dedicated to give you experience in using various
types of pipettes usually found in a research lab. There are three main types of
pipettes used in the lab to move liquids from one reservoir to another: (i) Pasteur
pipettes, (ii) volumetric or serological pipettes, and (iii) micropipettes.

Figure 1. Three main types of tools used in the laboratory to move liquids. (i) Pasteur pipettes,
(ii) pipettes, and (iii) micropipettes.

(i) Pasteur pipette: small, tapered glass tube, not graduated, and used with a bulb. It
is used to dispense liquid when the volume is not critical.
(ii) Volumetric or serological pipette: glass or plastic, calibrated to deliver any amount
in the graduated scale from 1 to 25 mL, and used with a bulb or Pipette-Aid. A
Pipette-Aid is an electric pipettor that has two buttons which control the flow of liquid
in and out of the pipette. Pipettes are available that hold 1 mL, or 2 mL, or 5 mL, or
10 mL, or 20 mL. The proper pipette must be chosen for the task at hand. For
example, a 1 mL pipette will more accurately measure 1 mL than will a 5 mL pipette.
Thus, you should choose the smallest volume pipette that will do the job.
(iii) Micropipette: used with plastic pipette tips, calibrated to dispense smaller
volumes ranging from 0.5 L to 1000 L. There are different micropipettes from
different brands designed for a particular range of volume. The volume capacity is
indicated at the top near the plunger. The micropipettes available in the lab cover
three volume ranges: 2- 20 L (p20), 20- 200 L (p200), and 100- 1000 L (p1000).
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You must choose the correct pipette for the volume to be measured. The lowest end
of each volume range is subject to the greatest percentage error. For accuracy, you
must stay within the calibrated range of each micropipette: 2- 20 L (p20), 20- 200 L
(p200), and 100- 1000 L (p1000). Do not push the micropipette outside of the
indicated volume range. If you do so, this will lead to inaccurate measurements and
damage to the pipette. For each of these micropipettes, a different tip is used.
(iii a) Micropipette overview

Figure 2. Moving the button/piston displaces air which moves the liquid. 1. The piston moves to
the appropriate position when the volume is set. 2. When the operating button is pressed to the first
stop, the piston expels the same volume of air as indicated on the volume setting. 3. After immersing
the tip into the liquid, the operating button is released. This creates a partial vacuum and the specified
volume of liquid is aspirated into the tip. 4. When the operating button is pressed to the first stop again,
the air dispenses the liquid. To empty the tip completely the operating button is pressed to the second
stop (blow out).

(iii b) Pipetting Technique: Forward versus Reverse

The forward technique is the standard for pipetting aqueous liquids, whereas the
reverse technique is used for pipetting viscous solutions or liquids with a tendency to
foam.

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Figure 3. Forward versus reverse pipetting technique. Forward pipetting modus: 1. Press the
operating button to the first stop. 2. Dip the tip into the solution to a depth not more than 3-4 mm, and
slowly release the operating button. Wait 1-2 seconds and withdraw the tip from the liquid, touching it
against the edge of the reservoir to remove excess liquid. 3. Dispense the liquid into the receiving
vessel by gently pressing the operating button to the first stop and then press the operating button to
the second stop. This action will empty the tip. Remove the tip from the vessel, sliding it up the wall of
the vessel. 4. Release the operating button to the ready position. Reverse pipetting modus: 1. Press
the operating button to the second stop. 2. Dip the tip into the solution to a depth not more than 34 mm, and slowly release the operating button. This action will fill the tip with a volume that is larger
than the set volume. Wait 1-2 seconds and withdraw the tip from the liquid. 3. Dispense the liquid into
the receiving vessel by pressing the operating button gently and steadily to the first stop. This volume
is equal to the set volume. Hold the button in this position. Some liquid will remain in the tip, and this
should not be dispensed. 4. The liquid remaining in the tip can be pipetted back into the original
solution or disposed of together with the tip. 5. Release the operating button to the ready position.

(iii c) Preventing cross-contamination

Pipette-to-sample: A contaminated pipette, or its tips, can contaminate your samples.
You can avoid such contamination by wiping your pipette with 70 % ethanol, by using
sterilized tips, tips with filters, and by changing the tip after pipetting each sample.
Sample-to-pipette: Samples or aerosols can enter the cone of the pipette. You can
avoid such contamination by keeping the pipette vertical when pipetting in order to
prevent liquid from running into the pipette body, by releasing the push-button slowly,
by using filter tips, and by storing the pipette vertically.
Sample-to-sample: The remains of sample A can mix with sample B inside the tip
and may cause a false result. You can avoid such contamination by changing the tip
after each sample.
(iii d) Calibration of micropipettes (accuracy versus precision)
Calibration of micropipettes means determining the difference between the dispensed
volume and the selected volume (accuracy). Precision refers to the repeatability of
the pipetting. It is expressed as standard deviation (SD) or coefficient of variation
(CV). What is needed is both, accuracy and precision.

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Figure 4. Accuracy versus precision. When the set volume is for example 20 L, accurate but not
precise: The mean volume is the correct (set) volume but individual pipettings differ from the set
volume. Precise but not accurate: There is not variation between the separate pipettings, but the mean
volume differs from the set volume. Accurate and precise: The mean volume is the set volume and
there is no variation between different pipettings.

(iii e) Factors affecting micropipettes accuracy

Factors affecting micropipettes accuracy include:
Position: Pipettes should be held vertical during the aspiration of liquids; however,
some users often hold pipettes at many different angles during a pipetting interval.
Holding a pipette 30 off vertical can cause as much as 0.7 % more liquid to be
aspirated due to the impact of hydrostatic pressure.
Tips: To ensure accurate results, use tips specified by manufacturers. If using other
tips, the properties should be tested (proper hold, dispensing of the correct and
whole volume).
Release of Plunger: Releasing the plunger abruptly can cause liquid to accumulate
inside the instrument which in turn can be transferred to other samples causing
variability in sample volume and is a potential source for cross-contamination. It is
recommended that a smooth, consistent pipetting rhythm is employed since it helps
to increase both accuracy and precision.
Immersion Depth: The pipette tip should only be inserted into the vessel containing
the liquid to be transferred about 3-4 mm.
Environmental conditions: Temperature, air pressure, and humidity are the main error
sources from the environment. The greatest contributor to error is liquid temperature
(see Figure 5).
Density: Density is the mass/volume ratio of the liquid. The density varies with the
temperature and air pressure. The density of water is at room temperature (20-25 C)
0.996 g/mL.

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Figure 5. Impact of liquid temperature and density on pipetting accuracy.

MATERIALS
Deionized distilled H2O (ddH2O in 100 mL Flask)
Tween-20 (in 100 mL Flask)
Pasteur Pipette
5, 10 and 25 mL Pipette
Pipette-Aid
Set of micropipettes (P20, P200 and P1000)
Set of tips
100 mL beaker
Reaction tubes (1.5 mL microcentrifuge tube and 15 mL reaction tube)
Rack for microcentrifuge tubes and reaction tubes
Analytical balance
EXPERIMENTAL PROCEDURE
(i) Macropipetting with a Pasteur pipette:
When you need to take a liquid, but do not need a specific volume, you can use a
Pasteur pipette.
1.4 Using this pipette, fill in the 15 mL reaction tube with H2O from the 100 mL flask.
How many times did you need to pipette in order to fill in the 15 mL reaction
tube?
(ii) Macropipetting with a pipette and a Pipette-Aid:
You will practice dispensing different volumes of liquid using the 5, 10 and 25 mL
plastic disposable pipette.
Using this pipette, practice pipetting with the Pipette-Aid. Place the tip of the
pipette just deep enough into solution to obtain the volume of solution needed but
do not hit the bottom of the beaker (or any reservoir). Get used to the rate at

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which the fluid is drawn up and expelled. The rate varies depending on how hard
you push the buttons. Expel the ddH2O into an empty beaker.
Pay attention on the pipette calibration markings. For example, as the 10 mL
pipette is filled from the tip, the volume it contains when the bottom of the
meniscus is at the 7 mL mark is actually 3 mL. Thus, the markings indicate the
volume of solution that has been let out of the pipette.
Learn how to insert the pipet into a flask without touching the sides and hold it
there while withdrawing the liquid.

1.5 Draw up the following volumes: 1, 2.5, 3, 5, 10.5, and 20 mL. Which pipette did
you use to achieve the most accurate measurement of the volume needed?
Explain why.
(iii) Micropipetting using a micropipette:
A micropipette is used to dispense small volumes (< 1 mL) of liquid. In this
experiment you will practice working with three micropipettes of different volume
ranges.
Take the P20, P200 and P1000 micropipettes and look at the digital volume
indicator.
The P20 has 3 display spaces:
The top space reads tens of L
The middle space reads ones of L
The bottom space reads tenths (in red) of L
The P200 has 3 display spaces:
The top space reads hundreds of L
The middle space reads tens of L
The bottom space reads ones
The P1000 has 3 display spaces: The top space reads of thousands of L
The middle space reads hundreds of L
The bottom space reads tens of L
Once you have understood the volume display, take the P1000 micropipette to
measure 500 L ddH2O and deliver it into a microcentrifuge tip.
Set the volume and make sure that the volume you choose is within the range of
the micropipette.
Load the correct tip onto the micropipette by firmly pushing the barrel into the tip
which is still in the pipette-tips box. Tap it up and down to make sure it is seated.
Before you draw up the liquid from its reservoir, check that you are using the right
micropipette and doublecheck if the set volume is correct.
Draw up the liquid by using the forward pipetting technique mentioned above.
Expel the ddH2O into an empty microcentrifuge tube and deposit the tip directly
into the sharps container by pressing the ejector button.
Practice with P20 and P200 pipettes until you get used to them.
1.6 Draw up the following volumes of ddH2O: 12.5 L, 25 L, 200 L, 650 L,
666 L, 970 L, and 1300 L. Expel the ddH2O into an empty microcentrifuge
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1.7

1.8

1.9
1.10

tube. Which pipette did you use to achieve the most accurate measurement of
the volume needed? Explain why.
Take the P200 and the 1.5 mL reaction tube, which contains 30 L water and
pipette 20 L out of it. Use the P200 again together with the 1.5 mL reaction
tube, which contains 190 L water and pipette 180 L out of it. Is there any
difference between the positions of the first stops?
Try to find out the amount of water inside of the 1.5 mL reaction tube labeled
with X-number. Look at the liquid and the scale on the side of the tube, to get an
idea of the best fitting pipette. Take up the liquid into the tip. If there is any liquid
left in the tube, dispense the water carefully and increase the numbers on the
digital volume indicator. Try again to take up the liquid. If there are bubbles
inside of the tip, decrease the numbers on the digital volume indicator
(Attention! Please check the pipettes range before using it!). Take another
pipette with a different range, if necessary. Get closer to the actual amount of
water with every attempt and note your result.
You will now practice the reverse pipetting technique by drawing up 500 L
Tween-20. What did you observe?
Name the different steps for pipetting with the micropipette. Mind the correct
order:
Step
Legend
1.
2.
3.
4.
5.

6.

7.
8.

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9.
10.

Changed after: http://www.accessexcellence.org/AE/AEPC/geneconn/smallvol/part1.php,

http://www.clker.com/cliparts/o/Z/8/K/a/X/eppi-lysat-b-hi.png,
http://biology.hunter.cuny.edu/tech/pipetman-gif.gif,
http://www.starlab.ch/images/products/E1415-0200.gif

1.11 Name the donts during pipetting with the micropipette:

DONTS
Legend

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II. ACCURATE PIPETTING OF LIQUIDS (PART II)

Answer the following questions before you join the practical course.
1.12 Mention the density of water.
1.13 What does BSA means and what is it?
1.14 Explain the principle of a standard curve.
1.15 Calculate the concentration reduction (2) and the dilution concentration of each
tube in the serial dilution in Figure 7.
initial sample initial
Diluent Total
Concentration Dilution
concentration sample volume volume
reduction (2)
concentration
volume
500+500= 500 : 1000 =
x 2 mg/mL=
2 mg/mL
500 L 500 L
1000 L
1:2=
1 mg/mL
1 mg/mL

(iv) Micropipette calibration:

In this exercise you will calibrate your P1000 micropipettes which will tell you how
accurate your measurements are. You will pipette ddH2O into a number of
microcentrifuge tubes. The amount of water dispensed, and therefore the accuracy
with which you pipette, will be determined by weighing.
Take five sets of three microcentrifuge tubes, label them (A1-3 to E1-3) and weigh
each tube
Using a P1000 pipette, dispense volumes of 0.2 mL to each tube in set A,
0.4 mL to each tube in set B, 0.6 mL to each tube in set C, 0.8 mL to each tube
in set D, and 1 mL to each tube in set E
1.16 In your computer use a spreadsheet to make the following calculations:
- Substract the two weights for each test to determine the volume dispensed into
each (assume that the density of H2O is 1.0 g/mL)
- Determine the mean and standard deviation for each set of triplicates done
- Plot the volume dispensed (Y) versus the micropipette setting (X) on a line
graph
- Calculate the best fit for this data using linear regression and add the fit to the
line graph. See the following example:

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Figure 6. Example of visualized P1000 Micropipette calibration results. Volume dispensed (Y) is
plotted versus the micropipette setting (X).

DISCUSSION
1.17 What volume is being measured?
The charts below show the type of micropipette being used and its digital
volume indicator setting. What volume is being measured in each case?
Type
P1000

Settings
0

1.

4.

2.

5.

3.

6.

Type
P200

Settings

Unit 1

Volume measured (L)

1.

4.

2.

5.

3.

6.

Type
P20

Volume measured (L)

Settings

Volume measured (L)

1.

4.

2.

5.

3.

6.

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1.18 Calculate accuracy and precision of your P1000 micropipette when measuring
200 L ddH2O according to the following formulas:

III. SERIAL DILUTIONS

INTRODUCTION
A common procedure in molecular cell biology, microbiology and biochemistry is the
quantitative determination of some compound in a sample. In most cases a
colorimetric assay is used for such determinations. Colorimetric assays involve the
use of a spectrophotometer to determine the absorbance of light due to the presence
of a colored analyte. The quantitative aspects of these assays are based on BeerLamberts Law which states that the concentration of a colored compound in a
solution is directly proportional to the absorption of light by the solution.
A=lc
A = absorbance
= molar absorptivity (varies with the wavelength of light used in the measurement)
I = path length
c= analyte concentration
In this part you will use an assay (Bio-Rad protein assay, Bio-Rad Laboratories,
Munich, Germany) to measure protein concentration. The Bio-Rad Protein Assay is a
dye-binding assay in which a differential color change of a dye occurs in response to
various concentrations of protein (1). The absorbance maximum for an acidic solution
of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when binding
to protein. The Coomassie blue dye binds primarily to basic and aromatic amino acid
residues (2). Today you will prepare solutions containing various concentrations of a
protein (Bovine Serum Albumin, BSA) and read the absorbance of these solutions at
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595 nm with a spectrophotometer. This will allow you to prepare a standard curve.
Comparison of the absorbance of a sample to a standard curve will provide you a
relative measurement of a samples protein concentration.
LITERATURE
1. Bradford, M., Anal. Biochem., 72, 248 (1976).
2. Compton, S. J. and Jones, C. G., Anal. Biochem., 151, 369 (1985).

MATERIALS
BSA (2 mg/mL)
Phosphate Buffer Saline PBS
Coomassie Brilliant Blue G-250 dye reagent (contains dye, phosphoric acid and
methanol)
Sample (cell lysate)
Set of micropipettes (P20, P200 and P1000)
Set of tips
Reaction tubes (1.5 mL microcentrifuge tube)
Microtiter plates
Spectrophotometer
EXPERIMENTAL PROCEDURE
Using serial dilutions (1/2 serial dilutions) prepare BSA solutions in buffer (PBS)
with the following concentrations: 2, 1, 0.5, 0.25, 0.125 mg/mL. Prepare each of
these solutions. Find the pipetting scheme below (Figure 7).

Figure 7. Pipetting scheme of a serial dilution with a dilution factor of 2, which leads to a
1:2 concentration reduction of the starting solution. One part of the starting solution (very right
tube, concentration of 2 mg/mL) is diluted with one part diluent. It is mixed 1 Part : 1 Part. The
concentration of the tube in the black box is to be determined with the help of the standard curve.
Changed after: http://www.clker.com/cliparts/o/Z/8/K/a/X/eppi-lysat-b-hi.png,

1.19 Draw a pipetting scheme before you start pipetting.

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Add 97 L of diluted dye reagent to each well (A1 to A12, total = 12 wells) of a
microtiter plate.
Pipet 3 L of BSA solution 0.125 mg/mL to well A1 and A2;
3 L BSA solution 0.25 mg/mL to well A3 and A4;
3 L BSA solution 0.5 mg/mL to well A5 and A6;
3 L BSA solution 1 mg/mL to well A7 and A8;
3 L BSA solution 2 mg/mL to well A9;
3 L sample solution of unknown concentration to well A10 and A11 and
3 L PBS -as a blank- to well A12.
Remember to change the pipette tips while using a new BSA concentration (to
avoid cross-contamination).
Mix sample and reagent by repeated pipetting. Be careful not to produce air
bubbles while mixing.
Incubate at room temperature for at least 5 minutes and measure absorbance at
595 nm.

DISCUSSION
1.20 Why do we need to measure the absorbance of PBS? What is meant with PBS
as a blank?
1.21 Plot absorbance versus concentration to get a standard curve.
1.22 Use the standard curve to determine concentration of protein in the sample (cell
lysate).
1.23 Calculate the dilution factor according to formula (1) and complete the chart
below.
total volume of the dilution
initial volume of starter solution

Dilution factor

all parts of the dilution

parts of starter solution

50 mL starting solution in a final volume of 500 mL

(1)

Dilution Factor
10

200 mL starting solution in a final volume of 500 mL

250 mL in 500 mL
320 L in 80 mL
2 Parts starting solution in a total of 5 Parts solution
50 mL starting solution mixed with 500 mL dilution solution

11

200 mL starting solution mixed with 500 mL dilution solution

250 mL with 500 mL
320 L with 80 mL
2 Parts starting solution with 5 Parts dilution solution

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1.24 Calculate the concentration reduction of the dilution according to formula (2)
and complete the chart below.
Conc. Reduction initial volume of starter solution : total volume of the dilution
parts of starter solution : all parts of the dilution

(2)

Concentration Reduction
50:500 = 1:10

50 mL starting solution in a final volume of 500 mL

200 mL starting solution in a final volume of 500 mL
250 mL in 500 mL
320 L in 80 mL
2 Parts starting solution in a total of 5 Parts solution
50 mL starting solution mixed with 500 mL dilution
solution
200 mL starting solution mixed with 500 mL dilution
solution

50:550 = 1:11

250 mL with 500 mL

320 L with 80 mL
2 Parts starting solution with 5 Parts dilution solution
1.25 We use a BSA stock solution with a concentration of 2 mg/mL, which means,
that there are 2 mg BSA in 1 mL solution. The rule of three can be used to
calculate the amount of BSA in 1 L:
(3)

Calculate the amount of BSA or the amount of liquid

BSA stock 2 mg / 1 mL
BSA stock 2 mg / 1 mL
1 g
0.5 L
85 mg
42.5 mL
2 L

10 mg

20 g

270 L
1.5 mL

7 g

3.5 mg

1 mL
500 L

14.4 g

1g
Unit 1

13 L
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IV. STERILE TECHNIQUE

INTRODUCTION
By using sterile techniques you can minimize the exposure of your solutions and cell
cultures to contaminants (present in the air, hand and mouth while talking, etc.) that
would interfere with your results and would even kill your cell cultures. Thus, it is very
important to know how to work properly to keep your cells and solutions free of
contaminants. Today, and in next lab course units, you will practice how to work in a
cell culture hood.
(i) Basic rules
The working area should be as far away from traffic as possible.
Set the working area up to minimize the time that sterile cultures and media are
open. That means: Clear the working area beforehand and have everything you
will need ready at hand.
If you are using a cell culture hood, wipe it down with an antiseptic/cleaning agent
before use.
You can only touch sterile items with other sterile items
Hold caps from sterile bottles face side up in your hand when you uncap them.
Do not touch the sterile side of the cap.
You are the most likely source of bacteria and fungi that could contaminate your
cultures. Talking close to an open culture is the surest way to contaminate it.
Minimize the amount of open bottles and the time bottles are open and dont talk
while working with open bottles.
(ii) Common mistakes
Pipetting up too far in the pipette.
Touching the tip of the pipette against the outside of a bottle, the ground, bench
(anywhere not sterile).
Dropping an opened container or tube to the ground.
Talking into an open dish or bottle.
(iii) Rules when working with a cell culture hood
Although the danger of contamination is diminished in a cell culture hood, sterile
technique is still essential in keeping your cultures contaminant free. In a cell culture
hood, the major cause of contamination is movement of the arms in and out of the
cabinet, which breaks the air curtain and disturbs the air flow required to prevent
contamination.
Check that the cell culture hood is on and the air is circulating.
Do not block airflow by placing items on it.
Do not bring more items into the cell culture hood than necessary.
Wipe down the surface with 70 % EtOH before and after each use.
Minimize hand movement in and out of the cell culture hood.
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MATERIALS
70 % EtOH
Cell culture Media
Paper towels
Wrapped 10 mL pipettes
Pipette-Aid
T25 Cell culture flask
Gloves
EXPERIMENTAL PROCEDURE
Today you will learn how to dispense liquids from a bottle with cell culture media into
a cell culture flask. After preparing your culture, you will place your cell culture flask in
an incubator at 37 C and let it incubate for a week. In next lab course unit, you will
be able to check whether you worked properly by examining your cell culture for
contaminants.

Check that the cell culture hood is on and the air is circulating.
Bring needed items into the cell culture hood.
Wear gloves and wipe them with 70 % EtOH.
Wipe down the surface with 70 % EtOH before use.
Wipe down the cell culture media bottle surface with 70 % EtOH before use.
Take a 10 mL pipette. Pull the plastic sleeve down and away from the pipette to
snap it open. Only pull it about 1/5 of its length. Insert the pipette into the PipetteAid. Hold the Pipette-Aid in one hand while you remove the pipette from the
wrapping with your other hand (be careful not to touch the pipette to any surface
as you do this).
Open the cell culture media bottle and place the cap face side up. Do not touch
the sterile side of the cap.
With the pipette, draw up 8 mL of cell culture media without touching the outside
of the bottle.
Open the cell culture flask and place the cap face side up. Do not touch the sterile
side of the cap.
Expel the liquid into the cell culture flask without touching the outside of the bottle.
Close both the cell culture flask and cell culture media without touching the sterile
side of the cap.
Place the cell culture flask into the incubator.
Remove items from the cell culture hood.
Wipe down the surface with 70 % EtOH.
Switch off the cell culture hood.

LITERATURE
1. Barker, Kathy. At the bench: A Laboratory Navigator. Cold Spring Harbor
Laboratory Press, 2005.
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UNIT 2. BASIC MICROBIOLOGY LABORATORY TECHNIQUES (PART I)

The aim of the next two units is to introduce you to a number of basic techniques
used to study microorganisms. These techniques include Grams staining to identify
bacteria, bacterial culture methods, measurement of bacterial growth and growth
limitation. In this unit you will learn to distinguish between gram positive and gram
negative bacteria by following the Gram staining method. Gram staining is a valuable
diagnostic tool in clinical settings as both bacterial types react to antimicrobial agents
differently. Antimicrobial activity is measured by determining the smallest amount of
agent needed to inhibit the growth of a test organism, a value called the minimum
inhibitory concentration (MIC). In this section you will also learn how to determine the
MIC-value of an antimicrobial agent.
The correct handling of a light microscope
In this unit you will work with a microscope. Microscopes are expensive scientific
instruments. The Rhine-Waal University of Applied Sciences provides Zeiss
microscopes. Handle them properly and carefully and they will last for many years!
When moving your microscope, always carry it with both hands. Grasp the arm with
one hand and place the other hand under the base for support.

Figure 8. Parts of the Microscope.

Turn the revolving nosepiece so that the lowest power objective lens (4 x
magnification, labeled with the red ring) is "clicked" into position. Attention: Use the
100 x objective lens (white ring) only after consulting the lab instructors. There
is the risk of a damage in case of incorrect use. Place the microscope slide on the
stage and fasten it with the stage clips. You can push down the back end of the stage
clip to open it. Look at the objective lens and the stage from the side and turn the
focus knob so that the stage goes upward. Move it as far as it will go without touching
the slide! Use the fine adjustment knob only, when the distance of the stage to the
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objective goes under 0.5 cm. Now, look through the eyepiece and adjust the dimmer
switch and diaphragm for the greatest amount of light. Slowly turn the coarse
adjustment so that the stage goes down (away from the slide). Continue until the
image comes into focus. Use the fine adjustment, if necessary, for fine focusing.
Do not prepare your sample on the stage! The work with liquids on the stage is
forbidden.
Move the stage with the microscope slide around so that the image is in the center of
the field of view and readjust the dimmer switch or diaphragm for the clearest image.
When you are able to identify the image of your sample, change to the next objective
lenses with only minimal use of the focusing adjustment. Use the fine adjustment. If
you cannot focus on your specimen, repeat the previous steps with the higher power
objective lens in place. Do not allow the objective lens to touch the slide! Do not
touch the glass part of the lenses with your fingers. Use only special lens paper to
clean the lenses. When finished, lower the stage, click the low power lens into
position and remove the slide. Turn out the light before you unplug the microscope.
Always keep your microscope covered when not in use.
I. GRAMS STAINING
Answer these following questions before you join the practical course.
2.1 Name the bacteria which are examined.
1.
2.
2.2 Which microscopes objective lens is only to be used after consulting the lab
instructors?
2.3 Sequence the steps of the experiment. If things happen at the same time give
the same number.
Number
Steps of the experiment
Iodine is added
Decolorizer is added
1

Crystal violet solution is added to the bacteria

Insoluble crystal complex are formed
Safranin is added

Crystal violet solution enters the cells

Cells with peptidoglycan-poor cell walls get colorless
All bacteria are stained
Gram negative cells are stained
Gram positive cells are stained
Lipid layer from the gram negative cells are dissolved

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INTRODUCTION
The Gram staining method is one of the most important staining techniques in
microbiology. It is almost always the first test performed for the identification of
bacteria.
In order to understand how staining works, you should know about the physical and
chemical nature of stains. Stains are generally salts (a compound composed of a
positively charged ion and a negatively charged ion) in which one of the ions is
colored. For example, the dye methylene blue is actually the salt methylene blue
chloride which will dissociate in water into a positively charged methylene blue ion
which is blue in color and a negatively charged chloride ion which is colorless. Dyes
or stains are divided into two groups: basic and acidic. If the color portion of the dye
resides in the positive ion, as in the above case, it is called a basic dye (examples:
methylene blue, crystal violet, safranin). If the color portion is in the negatively
charged ion, it is called an acidic dye (examples: nigrosin, congo red). When using a
basic dye, the positively charged color portion of the stain combines with the
negatively charged bacterial cytoplasm and the organism becomes directly stained.
An acidic dye reacts differently. Since the color portion of the dye is on the negative
ion, it does not combine with the negatively charged bacterial cytoplasm. Instead, it
forms a deposit around the organism, leaving the organism itself colorless. This type
of staining is called indirect or negative as the organism is seen indirectly, and is
used to get an accurate view of bacterial size and shapes.
Today, we will make a direct stain of two bacterial strains by using the basic dye
crystal violet as a primary stain. The bacteria that retain the crystal violet-iodine
complex appear bluish purple under microscopic examination and are classified as
gram positive bacteria. Others that are not stained by crystal violet are referred to as
gram negative bacteria and appear pinkish red.
Principle and interpretation
Gram staining is based on the ability of bacterial cell wall to retain the crystal violet
dye during solvent treatment. Gram positive bacteria have very thick cell walls
consisting primarily of peptidoglycan whereas most of the cell wall in gram negative
bacteria is composed of an outer membrane containing lipids, proteins and
polysaccharides. Only 10 % of the gram negative total cell wall is made of
peptidoglycans (Figure 9). When bacteria are incubated with crystal violet, dye enters
into the cell. Iodine is subsequently added as a mordant to form an insoluble crystal
violet-iodine complex. This step is commonly referred to as fixing the dye.
Subsequent treatment with a decolorizer (a mixed solvent of ethanol and acetone)
dissolves the lipid layer from the gram negative cells which enhances the leaching of
crystal violet from the cells to the surroundings. In contrast, in gram positive cells, the
solvent dehydrates the thicker peptidoglycan cell wall thereby closing the pores as
the cell wall shrinks during dehydration. Thus, the diffusion of the violet-iodine
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complex is blocked and bacteria remain stained. Finally, a counterstain of safranin is

applied to the smear to give colorless-gram negative bacteria a pink color.

Figure 9. Schematic diagrams of gram-positive (a) and gram-negative (b) cell walls. The
Gram stain photo in the center shows cells of Staphylococcus aureus (purple, gram-positive) and
Escherichia coli (pink, gram-negative). Source: Figure 3.13 Brock Biology of Microorganisms 13/e.

MATERIALS
Deionized distilled H2O (ddH2O in 100 mL Flask)
Microscope slides
Grams crystal violet solution
Grams iodine solution
Grams decolorizer solution
Grams safranin solution
Micropipette (P1000) and tips
Bunsen burner
Gram positive bacteria: Bacillus subtilis
Gram negative bacteria: Escherichia coli
EXPERIMENTAL PROCEDURE
(i) Prepare a slide smear
Transfer a drop of the Bacillus subtilis suspended culture on a slide. It should only
be a very small amount of culture.
Spread the culture with a pipette tip to an even thin film over a circle of 1.5 cm in
diameter. Since it is possible to put two small smears on a slide, transfer and
spread a drop of Escherichia coli suspended culture into the same slide.
Allow to air-dry the slide and fix it over a gentle flame, while moving the slide in a
circular fashion (three times, during 1 s/time) to avoid localized overheating. The
applied heat helps the cell adhesion on the glass slide to make possible the
subsequent rinsing of the smear with water without a significant loss of the
culture.
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(ii) Gram staining

Flood the fixed smear with Grams crystal violet solution. Let stand for
60 seconds.
Pour off the stain and gently wash with water.
Flood with Grams iodine solution. Allow it to remain for 60 seconds.
Pour off the iodine solution and gently wash with water. Shake off the excess
water from the surface.
Decolorize with Grams decolorizer solution until the blue dye no longer flows
from the smear. Further delay will cause excess decolorization in the gram
positive cells, and the purpose of staining will be defeated.
Gently wash the smear with water.
Counterstain with Grams safranin solution for 60 seconds.
Wash off the red safranin solution with water. Allow the slide to air-dry.
Place 3 drops of Mounting Medium on the dried samples and cover it with a cover
slide
Examine the slide under a microscope. Attention: Use the 100 x objective lens
(white ring) only after consulting the lab instructors (oil immersion objective).
Attention! Wash off any spilled stain immediately with water to avoid leaving
permanent marks in the sink, lab bench, or glassware.
DISCUSSION
After performing the staining, answer following questions:
2.4 Why do we heat fix the bacteria to the slide before staining?
A. So the bacteria hold the stain better.
B. So the bacteria dont wash off the slide.
C. To kill them.
2.5 A basic dye has the color in the_____________ ion.
A. positive
B. negative
C. neutral
2.6 All bacteria have a slight negative charge; a basic dye has the color in the
positive ion. Opposite charges attract. This is the principle behind:
A. Indirect staining.
B. Why Gram-positive bacteria dont stain pink.
C. Direct staining.

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2.7 Is this a direct stain or an indirect stain?

A. Direct stain
B. Indirect stain
2.8 Is this a direct stain or an indirect stain?

A. Direct stain
B. Indirect stain
II. MINIMUM INHIBITORY CONCENTRATION
Answer these following questions before you join the practical course.
2.9 What does MIC stand for?
2.10 Calculate the concentration reduction (2) and the dilution concentration of each
tube in the serial dilution in Figure 11.
initial sample initial
Diluent
Total
Concentration
Dilution
concentration sample volume volume reduction (2)
concentration
(mg/mL)
volume
(mg/mL)

2.11 What is measured via a spectrophotometer?

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INTRODUCTION
In this section you will perform the antimicrobial agent susceptibility assay using
dilution methods. The assay defines the minimum inhibitory concentration (MIC)
which is the smallest amount of agent needed to inhibit the growth of a
microorganism. You will be able to apply the knowledge acquired in previous Unit 1
regarding preparation of serial dilutions. To determine the MIC, a series of culture
tubes is prepared and inoculated with the same number of microorganisms. Each
tube contains culture medium with an increasing concentration of the agent. After
incubation, the tubes are checked for visible growth (turbidity). The MIC is the lowest
concentration of agent that completely inhibits the growth of the test organism. This
procedure is also called the tube dilution technique (
Figure 10).

Figure 10. Antimicrobial agent susceptibility assay using the tube dilution technique to
determine the minimum inhibitory concentration (MIC). Growth, which is assessed as turbidity,
takes place in those tubes with antimicrobial agent concentrations below the MIC.

MATERIALS
15 mL culture tubes
LB culture medium
Antimicrobial agent Kanamycin
Escherichia coli
Set of micropipettes (P20, P200 and P1000)
Beaker
Set of tips
Gloves

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EXPERIMENTAL PROCEDURE
(i) Antimicrobial agent susceptibility assay
Perform following steps under a laminar flow hood:
By using serial dilutions, prepare 4 culture tubes containing each 2.7 mL of LB*1
culture media with the antimicrobial agent Kanamycin*2 at following
concentrations (in mg/mL): 5x10-1, 5x10-2, 5x10-3 and 5x10-4. Your stock solution
is 5 mg/mL. Do not forget to label each culture tube (e.g. [Kan] (5x10-1 mg/mL)).

Figure 11. Serial dilution of Kanamycin*2 with a diluting factor of 10 for the Antimicrobial agent
susceptibility assay. The stock solution (5 mg/mL) is placed in the very left tube and one part is
mixed with nine parts of LB*1 culture media in another tube, which leads to a 1/10 reduction of the
Kanamycin*2 concentration. Figure changed after: 1.5 mL tube, Test tube and Beaker:
http://www.clker.com/cliparts/o/Z/8/K/a/X/eppi-lysat-b-hi.png,
http://preview.turbosquid.com/Preview/2010/12/16__02_12_33/test%20tube_render.jpgff4d2eff-890e47c6-ab72-da933fe249ecLarge.jpg,
http://upload.wikimedia.org/wikipedia/commons/thumb/4/47/Beakers.svg/800px-Beakers.svg.png

Prepare an additional tube with 2.7 mL LB culture media without antimicrobial

agent. Do not forget to label the tube (e.g. [Kan] (0 mg/mL))
Add 10 L of Escherichia coli suspension into each tube. Be sure that you shake
the bacterial suspension before taking the 10 L aliquot. Take the 10 L aliquot
as fast as possible (before bacteria sediments) and inoculate it into the 2.7 mL
culture medium.
Let the bacteria grow by shaking overnight @ 37 C.

(ii) Measurement of bacterial growth by turbidity (spectrophotometry)

Increased turbidity in a culture is an index of bacterial growth. A spectrophotometer
does emit light and the amount of light transmitted through the sample is measured.
The amount of transmitted light decreases as the number of cells in the sample
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increases. Therefore the reading, called absorbance or optical density, indirectly

reflects the number of bacteria. This method cannot distinguish between dead and
living bacteria. An alternative method, called plate count method, reveals information
related only to live bacteria. For more information on this latter method, please read
Chapter 5, Brock Biology of Microorganisms, 13/e.

Visualize the culture tubes and determine MIC.

Determine MIC by measurement of bacterial growth via spectrophotometry as
follows:
- Take 1 mL of LB culture media without Kanamycin and measure the optical
density (O.D.) at 600 nm. Set this value as blank.
- Take 1 mL of LB culture media with 5 mg/mL Kanamycin and measure O.D. at
600 nm.
- Take 1 mL of LB culture media with 5x10-1 mg/mL Kanamycin and measure
O.D. at 600 nm.
- Take 1 mL of LB culture media with 5x10-2 mg/mL Kanamycin and measure
O.D. at 600 nm.
- Take 1 mL of LB culture media with 5x10-3 mg/mL Kanamycin and measure
O.D. at 600 nm.
- Take 1 mL of LB culture media with 5x10-4 mg/mL Kanamycin and measure
O.D. at 600 nm.

LB*1 culture media: Lysogeny Broth (LB) or Luria-Bertani broth is the most widely
used medium for the growth of bacteria. Formulation per one liter: 10 g Tryptone, 5 g
Yeast Extract and 10 g NaCl.
Antimicrobial agent Kanamycin*2: Kanamycin sulfate is a water-soluble antibiotic
originally purified from the bacterium Streptomyces kanamyceticus. Kanamycin acts
by binding to the 30S subunit of the bacterial ribosome and inhibiting protein
synthesis in susceptible bacteria.
DISCUSSION
2.12 Why do we discard 0.3 mL of the last tube of the Kanamycin dilution?
2.13 What do we measure in our sample with the help of the photometer, when we
set the LB culture media as blank?

LITERATURE
1. Gregersen, T., Rapid method for distinction of gram-negative from gram-positive
bacteria, Eur. J. Appl. Microbiol. Biotechnol., 5, 123, 1978.
2. Madigan, M.T., Martinko, J.M., Stahl, D.A., and Clark, D.P., Brock Biology of
Microorganisms, Chapters 3, 5 and 26, ed. Benjamin Cummings, San Francisco,
CA, 2012.
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