Jacobs University Bremen gGmbH

General Introduction Manual

for the BCCB Teaching Laboratory
Academic Year 2022 / 2023

Authors: S. Diercks-Horn and S. Illenberger

General Introduction Manual BCCB Teaching Laboratory 2022/2023

Table of content
0 PREFACE .................................................................................................................................................. 2
1 LABORATORY SAFETY .............................................................................................................................. 2
1.1 BIOSAFETY LEVEL S1 ...................................................................................................................................... 2
1.1.1 S1 Safety Instructions ...................................................................................................................... 3
1.1.2 Safe Laboratory Practice ................................................................................................................. 3
1.1.3 Safety Rules ..................................................................................................................................... 3
1.2 HANDLING OF CHEMICALS ............................................................................................................................... 6
1.2.1 Hazard Labels according to the Globally Harmonized System (GHS) .............................................. 6
1.2.2 Introduction to H- and P-phrases from GHS .................................................................................... 7
1.2.3 Handling of chemical bottles in the lab ........................................................................................... 7
1.2.4 Storage of Chemicals and Solutions ................................................................................................ 7
1.2.5 Disposal of Chemicals ...................................................................................................................... 7
1.2.6 Cleaning Lab Ware .......................................................................................................................... 8
1.3 SAFETY DATA SHEETS (SDSS) ........................................................................................................................... 9
1.3.1 How to prepare SDSs ....................................................................................................................... 9
1.4 LAB COURTESY ............................................................................................................................................ 11
1.5 HANDLING DISASTERS................................................................................................................................... 11
1.5.1 Chemical Spills ............................................................................................................................... 11
1.5.2 Chemical Spills onto Skin or into Eyes............................................................................................ 11
1.5.3 Other Accidents ............................................................................................................................. 11
1.5.4 Fire................................................................................................................................................. 11
2 OPERATION OF SPECIFIC EQUIPMENT ................................................................................................... 12
2.1 MICROPIPETTES........................................................................................................................................... 12
2.2 BALANCES .................................................................................................................................................. 13
2.3 STIRRER AND HOT PLATE ............................................................................................................................... 13
2.4 PH METER .................................................................................................................................................. 14
2.5 HANNA PH TESTER CHECKER ®PLUS ................................................................................................................ 14
2.6 SPECTROPHOTOMETRY AND SPECTROPHOTOMETERS .......................................................................................... 15
2.6.1 Spectrophotometry ....................................................................................................................... 15
2.6.2 Spectrophotometers ...................................................................................................................... 17
2.6.3 Spectrophotometric measurements – general aspects ................................................................. 18
2.6.4 Single-wavelength absorbance measurements ............................................................................. 18
2.6.5 Wavelength Scans (Spectra) .......................................................................................................... 19
2.7 COOLED MICRO CENTRIFUGES......................................................................................................................... 19
2.8 LABORATORY CHEMICAL HOODS ..................................................................................................................... 19
2.9 BUNSEN BURNERS ........................................................................................................................................ 20
3 NOTES ON EXPERIMENTAL BIOCHEMISTRY ........................................................................................... 21
3.1 WATER QUALITY ......................................................................................................................................... 21
3.2 METRIC UNITS ............................................................................................................................................ 21
3.3 GIVING CONCENTRATIONS ............................................................................................................................. 22
3.4 BASIC CALCULATIONS .................................................................................................................................... 22
3.4.1 Dilutions and Ratios ...................................................................................................................... 23
4 REQUIRED INDIVIDUAL STUDENT PREPARATION .................................................................................. 24
4.1 READINGS AND CALCULATIONS ....................................................................................................................... 24
4.2 SAFETY DATA SHEETS (SDSS) ......................................................................................................................... 24
5 DOCUMENTATION OF EXPERIMENTAL RESULTS .................................................................................... 25
5.1 RESULT REPORTS AND LAB REPORTS ................................................................................................................ 25
5.2 REFERENCES (HARVARD SYSTEM).................................................................................................................... 25
6 REFERENCES .......................................................................................................................................... 26

1
General Introduction Manual BCCB Teaching Laboratory 2022/2023

0 Preface
Welcome to the BCCB Teaching Laboratory. Working in the laboratory is exciting and a lot of
fun, but only if everybody is well prepared, knows what to do and follows the common rules.
Therefore: Please read through this document as a preparation for each lab course. You also
need to bring this manual to the lab on every single lab day!

The BCCB Teaching Laboratory is a Safety Level S1 Laboratory, and special safety regulations
apply here. Before you are allowed to work in the BCCB Teaching Laboratory, you need proof
of participation in the Safety Instruction "User Regulations to the Laboratory Safety Level S1",
which has to be repeated each academic year.

When participating in a laboratory course, you are expected to know all safety guidelines for
work in the lab (Section 1) and how to use relevant equipment (Section 2). Of course, you will
get additional hands-on instructions for the relevant equipment during the laboratory courses.
Some helpful information on experimental biochemistry can be found in Section 3. Section 4
contains all information for successful participation in laboratory courses, which includes
thorough preparation. Section 5 contains information about Result and Lab reports.

1 Laboratory Safety
1.1 Biosafety Level S1
Biosafety laboratories are specialized laboratories in which scientists may work with
genetically modified organisms (GMOs) or infectious agents. Laboratories falling under
Biosafety regulations are marked with the Biohazard Pictogram (Fig. 1) and the Safety level is
indicated (e.g., in German "Sicherheitsstufe 1" for the BCCB Teaching Laboratory, see image
on front page).

Fig. 1: Biohazard pictogram

This pictogram indicates that the laboratory falls under special safety regulations.
The biosafety level is usually indicated (e.g., in German it is marked
"Sicherheitsstufe 1") on the doors to the BCCB Teaching laboratory.) Image taken
from: https://commons.wikimedia.org/wiki/File:Biohazard.svg

2
General Introduction Manual BCCB Teaching Laboratory 2022/2023

There are four biosafety safety levels (1-4) with Biosafety Level S1 representing the lowest
level. In a Biosafety level 1 laboratory scientists may work with agents that usually pose a
minimal potential threat to laboratory workers and the environment. Furthermore, these agents
do not consistently cause disease in healthy adults. Experiments with these agents can generally
performed on standard open laboratory benches without the use of special containment
equipment. However, special safety instructions and lab rules apply.

1.1.1 S1 Safety Instructions

Everybody, who enters the BCCB lab must have had an instruction to the “User Regulations to
the laboratory area safety level S1” and signed his or her attendance to these instructions.
During the first year, these safety instructions are given in combination with the respective
laboratory courses. For all other students and personnel, yearly safety instructions are given at
the beginning of the semester / academic year.

1.1.2 Safe Laboratory Practice

Works in laboratories that are assigned S1 require a strict compliance with regulations of safe
laboratory practice (see also section 1.1.3.). The most important rules are:

• Absolute prohibition of drinking, eating, chewing gum, smoking as well as of storage of

any food or tobacco in the S1 lab area.
• Keep doors and windows of the laboratories closed.
• Always use mechanical or electronic pipetting devices.
• Use sharps and cannulas (syringe needles) only if absolutely necessary. Dispose them
safely into designated containers; do not throw them into the waste bins!
• Avoid aerosols whenever possible. Do not shake bottles and close them after use.

1.1.3 Safety Rules

The following two pages are a short summary of the safety rules that apply in the BCCB
Teaching Laboratory. You will need to sign a declaration at the end of this Manual after the
MANDATORY Safety Instruction (Fall Semester 2022).

With your signature, you will certify that you understand and accept the general rules
about working in the S1 laboratory and dealing with hazardous chemicals.

3
General Introduction Manual BCCB Teaching Laboratory 2022/2023

General Safety Rules – BCCB Teaching Laboratory

The following rules serve to make the laboratory a safe place for yourself and your colleagues.
Their observance is mandatory, and offenders may be banned from the lab (and the course).

➢ Cotton lab coats (knee length) must be worn upon entering the lab and must not be removed
until exiting.

➢ Short pants or skirts that expose the legs are not permitted in the lab. Feet must be covered
with long socks and sturdy shoes or boots while in the lab. Open footwear is not permitted.

➢ Long hair must be tied back to avoid contact with chemicals or flames.

➢ Long (and artificial) fingernails are not permitted in the lab.

➢ Smoking, eating, drinking, chewing gum, and applying cosmetics are forbidden in the lab.

➢ All items not required for the performance of experiments must not be brought into the
laboratory. Such items are to be stored in a hallway locker.

➢ Never sit on bench tops, the heating or the floor.

➢ Never run in the lab.

➢ Never work alone in the lab. An instructor must be present at all times when students are in
the lab.

➢ Only authorized experiments may be performed. Any changes to the procedures given in a
particular manual may be made only with the specific authorization of the instructor.

➢ Never smell or taste chemicals.

➢ Never use mouth suction for filling pipettes. Use a pipetting device (e.g., a pipette bulb).

➢ No guests may be brought into the lab without the express permission of the instructor. For
guests, all safety rules apply.

➢ Broken glassware and equipment must be returned to the instructor. Broken items must not
simply be tossed in the trash.

➢ Small glass items (Pasteur pipettes, slides, cover slips) and sharp items (e.g., scalpels and
cannulas) have to be disposed in the yellow waste containers provided.

➢ Notify your instructor of any dangerous lab conditions that exist. This may include anything
from improperly functioning equipment to reckless behavior of someone else in the lab.

4
General Introduction Manual BCCB Teaching Laboratory 2022/2023

General Biological and Chemical Safety Rules

The following rules serve to make the laboratory a safe place for yourself and your colleagues
with respect to potential hazards arising from the handling of hazardous chemicals. All rules
are explained in more detail in this General Introduction Manual. Their observance is
mandatory.

➢ During all lab courses, you are going to work with biologically or chemically hazardous
materials. In addition to the instructions given before and during the lab course, it is your
responsibility to check the Manual and the Safety Data Sheets (SDSs) before the lab starts
to get familiar with the special substances used during the respective day.

➢ The quizzes will include questions concerning lab safety and SDSs.

➢ Use the appropriate waste containers for S1 (security level 1) waste whenever working with
GMOs (genetically modified organisms).

➢ Use the appropriate waste containers for all solutions used (stated in the Manuals).

➢ If a chemical spill occurs, first alert everyone in your immediate area, then consult your
demonstrator for instructions. Receive full instructions on spill cleanup and waste disposal
before touching spilled chemicals.

➢ Eye and skin contact with chemicals and burns on the skin:
Rinse the affected area thoroughly with large amounts of cool water. Remember that a heat
of reaction may occur when water comes into contact with some chemicals, so make sure
the rinsing is done thoroughly and for at least 15 minutes. Do not apply ointment. Have
someone notify the demonstrator.

➢ All accidents, regardless of how minor they may seem, must be reported immediately to the
instructor. No injuries may be ignored.

➢ In case of fire, notify the instructor at once. When an evacuation is necessary, turn off power
to any equipment and proceed to the nearest fire exit in an orderly manner.

5
General Introduction Manual BCCB Teaching Laboratory 2022/2023

1.2 Handling of Chemicals

All hazardous chemicals carry a standardized hazard label. It provides a first-glance idea of the
specific hazard from this substance. Since December 2010 these labels follow the "Globally
Harmonized System of Classification and Labeling of Chemicals (GHS)", a labeling system
aiming at a common classification of chemicals worldwide. For the preparation of the SDSs
you will have to use the GHS system (for more information see:
http://www.unece.org/trans/danger/publi/ghs/ghs_welcome_e.html).

1.2.1 Hazard Labels according to the Globally Harmonized System (GHS)

Fig. 2 shows the hazard labels for different groups of chemicals according to the new GHS
System, with the descriptions and pictograms according to GHS and the names that will be used
for these chemicals in laboratory courses held in the BCCB laboratory.

Description Pictogram Name Description Pictogram Name

Explosives Corrosives

Acute Toxicity
Flammables

Caution

Oxidizing
Health Hazard

Gases under Environmental

Pressure Hazard

Fig. 2: Hazard Labels according to the Globally Harmonized System (GHS)

(http://www.sigmaaldrich.com/sigma-aldrich/help/help-welcome/hazard-and-precautionary-statements.html)

6
General Introduction Manual BCCB Teaching Laboratory 2022/2023

1.2.2 Introduction to H- and P-phrases from GHS

Hazard statements (H-phrases) form a set of standardized phrases about the hazards of chemical
substances and mixtures that can be translated into different languages. For example, “H223:
Flammable material”. The current H-phrases can be found at: https://www.msds-europe.com/h-
statements/

Precautionary statements (P-phrases) are a set of standardized phrases giving advice about the
correct handling of chemical substances and mixtures. For example, “P223: Avoid release into
the environment”. The current P-phrases can be found at: https://www.msds-europe.com/p-
statements/
The P-phrases from the GHS have been grouped into general statements and those for
prevention and response. P-Phrases can be combined (as indicated with a "+") yielding the final
P-phrase, e.g., “P305+351+338 IF IN EYES: Rinse continuously with water for several
minutes. Remove contact lenses if present and easy to do – continue rinsing”.

EUH-Phrases
Some R-phrases from the previous European System do not have simple equivalents under the
new GHS. These phrases have been retained as European Union Specific Hazard Statements
(EUH-phrases) where the numbering mirrors the number of the previously used system in
Europe (R-phrase). For example, EUH 014 – Reacts violently with water“. The current EUH-
Phrases can be found at: https://www.msds-europe.com/h-statements/

1.2.3 Handling of chemical bottles in the lab

All containers with chemical substances, including your own samples, need to be properly
labeled. Read all labels very carefully. The use of a wrong solution may give undesired results.
Solutions may be available in different concentrations, and many chemicals have very similar
names. Be careful!

1.2.4 Storage of Chemicals and Solutions

Chemicals and solutions should always be kept at the temperature indicated in the manual.

1.2.5 Disposal of Chemicals

All waste chemicals must be properly disposed of in designated containers. As wastes often
pose a serious hazard, consult your demonstrator on how to dispose of them if you are unsure.

7
General Introduction Manual BCCB Teaching Laboratory 2022/2023

According to German law, good laboratory practice and common sense the following
substances must not be disposed of down the drain or in the trash (this list is not exhaustive):

• solid inorganic chemicals (powders) that have been removed from their stock container or
that have been obtained as the product of a chemical reaction
• solid organic chemicals (powders) that have been removed from their stock container or
that have been obtained as the product of a chemical reaction
• halogenated solvents (such as chloroform, dichloromethane)
• non-halogenated solvents (such as ethanol, methanol, acetone)
• inorganic acids (such as HCl, H2SO4)
• inorganic bases (such as NaOH)
• solutions containing heavy metals (Fe, Pb, Cu, Ag)
• filter paper contaminated with chemicals.

Dispose of these substances into the provided labeled containers. If you need assistance, please
contact the teaching assistants. Contamination of our waste water with any of the above
chemicals can cause very serious problems for Jacobs University Bremen.

1.2.6 Cleaning Lab Ware

Both, glass and plastic containers should be washed with water as soon as possible after use.
After washing, they should be rinsed with deionized water (WEK) and air-dried (for water
types see section 3.1.)

8
General Introduction Manual BCCB Teaching Laboratory 2022/2023

1.3 Safety Data Sheets (SDSs)

Working in the BCCB laboratory may involve handling of hazardous substances. By German
law, you need to familiarize yourselves with the proper procedures for handling of or working
with a particular substance. Hence, each participant has to prepare Safety Data Sheets
(SDSs) for each hazardous substance. SDSs include information such as physical data (melting
point, boiling point, flash point etc.) toxicity, health effects, first aid, reactivity, storage,
disposal, protective equipment, and spill/leak procedures. In case of getting substances into the
eyes or on your hands, the SDS provides all information about what to do. Should you have to
see a doctor after exposure to a hazardous substance, the SDS will help to find the proper
medical treatment. Further information is available at: http://www.ilpi.com/msds/.

1.3.1 How to prepare SDSs

In the laboratory courses, we usually use an adopted, condensed SDS format (see example on
Page 10). In each manual to individual experiments, you will find which SDSs you need to
bring to the lab day. The SDSs will be collected at the beginning of the course day and the
course assistants will check them. SDSs contribute to your course grade (10 % in the BCCB
First Year Laboratory Courses). Here is a little checklist for preparing SDSs:

• Each course participant has to prepare individual SDSs. Copies or print outs from
books, catalogs etc. are not accepted. Chemical structures have to be drawn by hand!
• Check on the manual which SDSs you need to bring.
• For each substance use the highest concentration and purity, e.g., 100 % concentration and
analytical grade (p.a.)
• Only use web pages from companies selling chemicals, because only those companies
update their databases regularly. Indicate your source on the SDS and on which date
you retrieved it. We recommend you use the following link to start with:
https://www.sigmaaldrich.com/DE/en

• Use the template provided in Campusnet and fill in all information required. Do not
forget to indicate the "role in the experiment".
• Print your name and sign the SDSs to confirm personal preparation.

9
General Introduction Manual BCCB Teaching Laboratory 2022/2023

SAFETY DATA SHEET (Sigma-Aldrich, 33209-M, 2021-07-29)

Name (chemical and trivial) Acetic acid
CAS-no. 64-19-7
Chemical formula CH3COOH; C2H4O2
Structural formula (hand drawn)

Molar mass/Molecular weight 60.05 g/mol

Density (liquids only) 1.049 g/cm3 at 25°C
General classification Organic acid
Functional groups (organic compounds only) Carboxyl
GHS Pictograms Flammables, Corrosives
H statements (write out) H226 Flammable liquid and vapour
H314 Causes severe skin burns and eye damage

P statements (write out)
P210 Keep away from heat, hot surfaces, sparks, open flames
and other ignition sources. No smoking.
P233 Keep container tightly closed.
P240 Ground and bond container and receiving equipment.
P280 Wear protective gloves/ protective clothing/ eye
protection/ face protection/ hearing protection.
P303 + P361 + P353 IF ON SKIN (or hair): Take off
immediately all contaminated clothing. Rinse skin with
water.
P305 + P351 + P338 IF IN EYES: Rinse cautiously with water
for several minutes. Remove contact lenses, if present and
easy to do. Continue rinsing.

Signal word Danger

Gloves Yes
Safety goggles Yes
Hood Yes

Role in the experiment incl. explanation

Internet Source: https://www.sigmaaldrich.com/DE/en, retrieved 20. July 2022

_________________________________
Printed Name and Signature

10
General Introduction Manual BCCB Teaching Laboratory 2022/2023

1.4 Lab Courtesy

• Do not leave your equipment drawer open for a classmate to collide with, possibly
causing injury or a spill.
• Never take a communal reagent to your own bench. It is frustrating for the others if they
cannot find what they need. Keep common areas clean!
• Do not take more of a chemical than you need, and do not take any of a communal
reagent before you know how much you will need.

Be helpful! If one of your colleagues is inexperienced in proper lab work, do not hesitate to
help him/her. On the other hand, if you have any questions concerning the experiment or the
lab work, ask your supervisors or the lab assistants. Questions help avoid accidents!

1.5 Handling Disasters

1.5.1 Chemical Spills
If a chemical spill occurs, first alert everyone in your immediate area. Then consult your
demonstrator for instructions. Receive full instructions on spill cleanup and waste disposal
before touching spilled chemicals.

1.5.2 Chemical Spills onto Skin or into Eyes

Rinse the affected area thoroughly with large amounts of cool water. Remember that a heat
reaction may occur when water comes into contact with some chemicals, so make sure the
rinsing is done thoroughly and for at least 15 minutes. Do not apply ointment. If you splash
chemicals in your eye, quickly flood the eye with a stream of water (eye showers at the sinks).
Do this for 15 minutes. In the meantime, have someone notify the instructor.

1.5.3 Other Accidents

All accidents, regardless of how minor they may seem, must be reported immediately to the
instructor. This includes minor cuts to your fingers etc. No injuries may be ignored, they need
to be documented.

1.5.4 Fire

In case of fire, notify the instructor at once. Call the porters at 911 (internal) or 0421 200-4815
(using a mobile phone). If an evacuation becomes necessary, turn off the power to any
equipment and proceed to the nearest fire exit in an orderly manner. Stay together as a group
and go to the meeting point on the campus green.

11
General Introduction Manual BCCB Teaching Laboratory 2022/2023

2 Operation of specific equipment

2.1 Micropipettes
These precision instruments (price!) are also called "Gilsons", "Finnpipettes", "Pipetmen", or
"Eppendorf pipettes" (all brand names). They come in different sizes; in the course, we will use
those of 1-20, 20-200, and 100-1000 µL range from Eppendorf (Fig. 3) which use tips of
different size.

plunger

tip ejector

volume adjustment

Fig. 3: Eppendorf Micropipettes. The volume range that can be pipetted accurately is shown on the pipette
opposite the tip ejector.

Here is a brief introduction to pipetting (Eppendorf, 2001, p. 24-26):

1. Find the right micropipette for the desired volume, and the right tips.
2. Set the micropipette to the correct volume.
3. Set the micropipette down onto a new tip in the box, and tighten the connection by
pushing down gently. If you put on the tip by hand, you should wear gloves.
4. Hold the micropipette vertically over the solution. Depress the plunger to the first positive
stop.
5. Put the tip into the solution to a depth of 2-4 mm.
6. Slowly let the plunger come up while the solution flows into the tip. Wait for
1-2 seconds to make sure no more liquid flows.
7. Move the tip to the recipient tube. Touch the tip to the side of the tube.
8. Depress the plunger slowly to the first, then to the second stop. Wait for 2-3 seconds to
allow the liquid to flow out.
9. Discard the tip using the tip ejector.

12
General Introduction Manual BCCB Teaching Laboratory 2022/2023

2.2 Balances
There are two types of balances in the BCCB Teaching Lab: The Lab Balances and the Mini-
Balances (Fig. 4).

Fig. 4: Lab Balance (on the left) and Mini-Balance (in the middle), Mini-Balance with beaker (on the right)

The balances do not need to be calibrated. Make sure for the Lab Balance that the level in the
back is in the right position (bubble in the middle of the ring). Place the vial you wish to weigh
into, or the weighing boat, onto the balance platform. The Mini-Balance can only be used with
a beaker for weighing (Fig. 4, on the right). Press TARE to set the balance to zero. Weigh in
your substance. Clean the balance after use. Ask an instructor.

2.3 Stirrer and Hot Plate

• The right hand–side switch and knob control the stirring speed, the left hand–side switch
and knob control the heater.
• Before putting a plastic beaker on the stirring plate check whether it is hot.
• Never drop a stirring bar into an empty glass beaker that is on the stirrer (the beaker may
break).
• When finished using the equipment, always set both controls (stirrer and heater) back to
zero (0).

13
General Introduction Manual BCCB Teaching Laboratory 2022/2023

2.4 pH meter
• Measuring the pH of a sample (WTW, 2002, p. 15-22): Withdraw the electrode from the
storage solution. Rinse with distilled water (using a squirt bottle) into the waste container.
Gently dry with a soft tissue. Immerse the electrode into the solution to be measured. If
necessary, press "M" until "pH" and the pH value appear on the display. Rinse the electrode
with distilled water and replace into storage solution.
• Make absolutely sure that the electrode cannot be damaged by a rotating stir bar or by other
equipment. The electrode tip is very sensitive.
• Never immerse the electrode into solutions that are not clear or that contain un-dissolved
material or protein.
• Our pH Meters are usually calibrated already.
• If you need to calibrate the pH meter yourself, get an instructor. You need to use the
automatic calibration (AutoCalTEC) function and standardized buffer solutions (pH 7 and
pH 10 if measuring basic solutions, and pH 4 and pH 7 if measuring acidic solutions).

2.5 Hanna pH tester Checker ®Plus

Fig. 5: Hanna pH tester Checker ®

plus (image taken from manual by
Hanna instruments,
http://hannainst.com/usa/index.cfm)

• Measuring the pH of a sample with a Hanna pH tester:

• Turn the pH tester (Fig. 5) on by sliding the switch on the top.
• Remove the protective cap and rinse pH electrode with distilled water (using a squirt bottle)
into a waste container. Do not be alarmed if white crystals appear around the cap, this is
normal with pH electrodes and they dissolve when rinsed with water.
• Immerse the tip of the electrode (bottom 4 cm/1½") in the sample to be tested (Fig. 6).

14
General Introduction Manual BCCB Teaching Laboratory 2022/2023

• Make sure that the electrode cannot be damaged by a rotating stir bar or by other equipment.
Stir gently and wait for stable reading.
• Never immerse the electrode over the maximum immersion level. The connector must be
always clean and dry.
• After use, rinse the electrode with water and store it with a few drops of storage solution
in the protective cap. Do not use distilled or deionized water for storage purposes.
• Always replace the protective cap after use.

Fig. 6: Proper immersion of Hanna pH tester (http://hannainst.com/usa/index.cfm)

2.6 Spectrophotometry and Spectrophotometers

2.6.1 Spectrophotometry
To characterize substances or biomolecules, one can use spectrophotometry (see Fig. 7). In
biochemistry, this technique is based on the measurement of the absorption of light by a solution
that contains a light-absorbing solute (dissolved chemical) (Nelson and Cox, 2005, p. 82). It is
intuitive that the color of the solution will be deeper when there is more solute in it. In fact, the
intensity of light decays exponentially when it travels through a cuvette containing a colored
solution.

Fig. 7: The principle of spectrophotometry using light of a defined wavelength and a cuvette of defined
width that defines the path length (d, usually 1 cm). The labels generally correspond to the Lambert-Beer-
Law (see text). Image modified from Nelson and Cox (2005).

15
General Introduction Manual BCCB Teaching Laboratory 2022/2023

This logarithmic relationship is described by the law of Lambert-Beer, which (by convention)
usually assumes a path length (d) of 1 cm (distance light needs to travel through the solution).
This is only given, if the cuvette is positioned in the instrument with the right orientation. Here
is the equation of the Lambert-Beer-Law:

I  I 
E = − log  = log 0  =    c  d with
 I0  I 

Eλ extinction (or: absorption) at wavelength λ

I0, I Intensity of incident (I0) and transmitted light (I)
d path length: by convention d = 1 cm
c the molar concentration of the solute
ελ the molar extinction coefficient at wavelength λ

Biomolecules are usually analyzed for their absorption in the UV / visible light ranges (Fig. 8).
The molar extinction coefficient ε is a substance-specific parameter that varies with the
wavelength. This also explains why colored substances or pigments preferentially absorb light
at certain wavelengths and not at others and thus appear colored (Voet and Voet, 2004, p. 282).
The wavelengths of visible light are shown at the bottom of Fig. 8 (the top shows the wider
electromagnetic spectrum including UV light and Infrared):

Fig 8: Subdivision of light wavelengths (http://www.scienceline.ucsb.edu/images/wavelength1)

Some spectrophotometers (machines that measure extinction) can record extinction values at
many wavelengths from the same sample and plot the absorption against the wavelength. Such
a graph is called a spectrum. For biochemists, spectra of substances serve as "fingerprints" for
their identification. To compare spectra that one has taken from a sample with spectra from the

16
General Introduction Manual BCCB Teaching Laboratory 2022/2023

literature, one often compares the position (wavelength) of the "peaks" and "valleys / troughs"
in the spectra because these are rather characteristic for a substance. The exact position of the
peaks depends on the solvent (e.g., buffer or organic solvent) so it is important to note what
your substance is dissolved in (Silberberg, 2003, p 267-268).

One technical point about spectrophotometry: the percent transmittance (= percentage of light
transmitted through a cuvette and thus reaching the detector) is defined as

I 100
%T = 100 = E .
I0 10

Therefore, when E = 1, only 10 % of the incident light is transmitted. This is where

spectrometers begin to haveproblems with accuracy. At E = 2, only 1 % of the incident light is
transmitted; with so little light, no accurate measurements are possible anymore. Therefore,
meaningful readings for E are always between 0 and 1 (both in single-wavelength
measurements and in spectra). If a solution of a colored substance is more concentrated, it needs
to be diluted for an accurate reading (Boyer, 2000, p. 156-157).

2.6.2 Spectrophotometers
In the laboratory, we have spectrophotometers from different companies but with a similar
general setup, and most spectrophotometers have integrated printers. Fig. 9A depicts the main
type (Foodalyt). Please familiarize yourself with the instruments BEFORE you measure your
samples. You may also ask the course instructors for help ☺.

A B C

Fig. 9: A representative spectrophotometer of those types that will be used in the BCCB Teaching Lab (A)
and a semi-micro cuvette (B). The correct positioning of the cuvette in the Foodalyt spectrophotometer is
shown in (C). The sample compartment is not covered (grey oval), and the cuvette is directly inserted from
the top Please note that the triangle on the cuvette needs to point towards the triangle on the left of the
sample compartment. (Image A was taken from: https://www.foodalyt.de/de/foodalyt-photometer-light-
photometer-light-extra.html, image B can be found at:
https://www.fishersci.co.uk/shop/products/polystyrene-semi-micro-cuvette/10151710, image C was
provided by S. Illenberger).

17
General Introduction Manual BCCB Teaching Laboratory 2022/2023

The sample compartment holds standard size cuvettes (Fig. 9B). In the laboratory course, we
mainly use so-called semi-micro cuvettes that need to be inserted into the instruments in the
correct orientation to ensure accurate measurements with a light path of 1 cm. (for further
details see also section 2.6.1). To help you find the right orientation, these cuvettes have a little
triangle at one top indicating the light path orientation (Fig. 9B). In the Foodalyt
spectrophotometer the triangle should face one of the arrows next to the sample compartment
(Fig. 9C).
Depending on the substances analyzed and the wavelength at which samples are measured,
cuvettes of different materials are used. Make sure that you use the right type of cuvette for the
job. We have the following cuvettes:
o Glass: for water and organic solvents, 360 to 1100 nm. Not for Bradford assays (the
dye binds to glass). Very expensive.
o Plastic UV Half-Micro and UV Micro: for aqueous solutions only. 220 to 1100 nm.
Expensive.
o Plastic Visible Half-Micro and Micro: for aqueous solutions. 360 to 1100 nm.

2.6.3 Spectrophotometric measurements – general aspects

For any spectrophotometric measurement, there are some general rules that need to be followed:
• Make sure you use the proper type of cuvette (check the laboratory manual!).
• Check for proper settings before you start (e.g., correct wavelength and mode).
• Never pipette any liquid into cuvettes while the latter are still in the sample compartment.
• Always remove the cuvettes from the instrument before you add or withdraw any liquid.
• Use a cuvette rack to transport and transfer cuvettes containing your blanks and samples.
• If you accidentally spill any liquid into the instrument, notify the instructor and clean up
immediately.
• Always measure / scan a blank/ baseline first before you measure your samples.
• KEEP ALL YOUR SAMPLES until all data have been checked by a supervisor.

2.6.4 Single-wavelength absorbance measurements

Single-wavelength absorbance measurements are usually performed at a wavelength where the
solute has an absorption maximum. At the desired wavelength, you first have to measure the
"Blank" to automatically correct for the absorbance by the cuvette and solvent. For single
wavelength measurements, you are expected to record the values manually in your laboratory
manuals.

18
General Introduction Manual BCCB Teaching Laboratory 2022/2023

2.6.5 Wavelength Scans (Spectra)

Different compounds have characteristic absorption maxima and minima. Measuring the
absorbance of a compound in a wavelength range can thus be used as a characteristic
"fingerprint" for that compound. The curve that is obtained by measuring the absorbance over
a continuous range of wavelengths is called an absorption spectrum of a compound. Again, you
will first have to measure the "Blank" sample to automatically correct for the absorbance by the
cuvette and solvent throughout the whole wavelength range. The result you obtain is a curve
that is referred to as the "baseline". Most of the procedure is self-explanatory, but you should
ask a supervisor before you use the instrument for the first time.

2.7 Cooled micro centrifuges

(Eppendorf; 2000, p. 16-22)

• The temperature should normally be left unchanged during the lab course day, and will
be either 4 ºC or 22 ºC. Centrifuges in use should be kept close (especially if the centrifuge
is set to 4 ºC, to avoid condensation). When switching of centrifuges, open the lid.
• Load the centrifuge rotor symmetrically (i.e., with tubes of equal fill height facing each
other; the lid joints must point outwards). Caution: Each tube must be counterbalanced
by another tube of the same weight. Attach the rotor lid if applicable and close the
centrifuge.
• Set the speed and run time. Following the run, press "Open" to open the lid.

2.8 Laboratory Chemical Hoods

Sometimes a scientist needs to work with hazardous chemicals that are volatile, generating
vapors or are highly flammable. To minimize the exposure to airborne contaminants, these
chemicals should be worked with in a laboratory chemical hood. A hood is a partially enclosed
workspace that is exhausted to the outside of the building. When used properly, hazardous gases
and vapors generated inside the hood are captured before they enter the breathing zone.

• Our Lab Manuals clearly state whether you need to perform parts of the experiment in the
hood. In addition, consult the SDSs to determine whether a chemical is likely to require a
fume hood for safe usage. If there is any doubt, a hood should be used.
• Pull down the "sliding front" (sash) as far down as possible without interfering with your
work, but for sure as far down as your breast. Never stick your head into the fume hood.
• If you have finished working, completely pull down the sash.

19
General Introduction Manual BCCB Teaching Laboratory 2022/2023

2.9 Bunsen burners

collar, adjustable air hole

needle valve, gas control

Fig 10: Common Bunsen burner (image from http://www.usbeck.eu/en/index.php?artikel&gruppe=1020 ).

• a Bunsen burner (Fig. 10) can produce 4 different types of flames

o "Pilot” flame for switching on and “pause”
o the “coolest” flame (300°C) is yellow/orange, it is also called the safety flame
o the medium flame, the blue flame or the invisible flame (500°C) is difficult to see in
a well lit room
o the hottest flame (>700°C) is called the roaring blue flame, it is characterized by a
light blue triangle in the middle and it makes a noise
• a Bunsen burner should be lid with the air hole and the needle valve closed
o make sure the air hole and the needle valve are closed
o get the lighter and try it once or twice
o open the gas supply valve at your bench (push and turn)
o keep your head out of the burner’s area
o light the Bunsen burner, a very small flame appears – the pilot flame
o open the needle valve to get the safety flame
o adjust the flame by moving the collar of the adjustable air hole down, letting more or
less air in
o when you do not use the burner at least close the air hole and the needle valve to only
have the pilot flame, which just keeps the Bunsen burner on
o do not leave your place unattended, when using a Bunsen burner
Be aware that the lighting with the pilot flame does not always work and you then have to open
the needle valve a little bit to light the Bunsen burner – directly a high flame, even if it is called
safety flame.

20
General Introduction Manual BCCB Teaching Laboratory 2022/2023

• for safely turning off the Bunsen burner

o close the adjustable air hole
o close the needle valve
o close the main gas valve

3 Notes on Experimental Biochemistry

3.1 Water Quality
The most common solvent in biochemistry and cell biology is water.
• Tap water (designation on the faucets in the lab: WBK) contains impurities and should only
be used in special cases.
• Deionized / demineralized water (designation on the faucets in the lab: WEK) is used for
rinsing glassware after cleaning. Do not waste this water as it is energy-consuming to
prepare it.
• ddH2O (double demineralized water, distilled water) comes from a special filter system in
the preparation room. This water (and only this water) should be in the water bottle at your
place and may be used for buffer preparations and dilutions.

3.2 Metric Units

In the natural sciences, metric units and their derivatives, such as meter, gram, liter etc. are
used. Common prefixes are:

• kilo (103)
• milli (10-3)
• micro (10-6)
• nano (10-9)
• pico (10-12)
• femto (10-15)

All calculations always need to include the correct units. Without unit identification,
calculations are meaningless as they do not provide any information on the actual scale. For
description of concentrations (c), different ways of defining the amount of a solute (n) in a given
volume (V) exist (see section 3.3).

Since many biochemical reactions, molecular interactions and cellular processes occur at low
concentrations (micromolar range and lower) scientist use the exponential depiction of
concentrations (usually given in x powers of 10: 10x).

21
General Introduction Manual BCCB Teaching Laboratory 2022/2023

3.3 Giving concentrations

In biochemistry and cell biology, the concentration of a solution can be given in different ways.
The most common are:

• Molarity (M): a one molar (1 M) solution has one mole of substance per liter solution. One
mole equals the molecular weight of the substance in grams. In biochemistry, one
frequently uses the following concentration ranges:
millimolar (mM, 10-3 M)
micromolar (µM, 10-6 M or 10-3 mM)
nanomolar (nM, 10-9 M or 10-6 mM or 10-3 µM).

• Weight per volume: one gram per liter (1 g/L) means one gram of solute (dissolved
material) in one liter final volume of solution.
• Percent weight per volume: one percent weight per volume (1 % wt/vol) means one gram
of solute in 100 mL final volume of solution.
• Percent volume per volume: one percent volume per volume (1 % vol/vol) means one
milliliter (mL) of solute in 100 mL final volume of solution.

Note: There has to be a space between the number and the unit.

3.4 Basic calculations

Most concentrations in Biochemistry and Cell Biology will be given as molarity (M). You will
often be asked to prepare an "x-molar solution" or calculate the number of moles (n) present in
a certain weight (m) of a salt or in a defined volume (V) of a solution with known concentration
(c). With the following equations you will be able to calculate number of moles, masses,
concentrations, molarities etc. through simple rearrangements:
𝒎
𝐧 = m refers to mass (the unit is gram)
𝑴

M refers to molecular mass (the unit is grams/mole)

Therefore, dividing gram by grams per mole leaves you the unit mole.

n = cV c refers to molar concentration (the unit is moles/liter)

V refers to volume (the unit is liters)

Therefore, multiplying (moles per liter) by liter leaves you the unit moles.

Another task to be performed is the preparation of dilutions, e.g., for creating a working solution
of lower concentration from a more highly concentrated stock solution, such as: "How do you

22
General Introduction Manual BCCB Teaching Laboratory 2022/2023

prepare 75 mL of a 50 mM solution of NaCl using water and a 1.5 M stock solution?" The
formula that may be used for such calculations is:

c1V1 = c2V2 c1 = concentration of stock (the unit is moles/liter)

V1 = volume of stock required (the unit is liters)
c2 = final concentration needed (the unit is moles/liter)
V2 = final volume needed (the unit is liters)

One way (everyone ends up doing it slightly differently) to calculate the above mentioned tasks
is:
𝒄
V1 = 𝒄𝟐 ∗ 𝑽𝟐
𝟏

(50∗ 10−3 𝑚𝑜𝑙𝑒𝑠/𝐿)

𝑉1 = ∗ (75 ∗ 10−3 𝐿) = 2500 ∗ 10−6 𝐿 = 2.5 ∗ 10−3 𝐿 = 2.5 𝑚𝐿 ☺
1.5 𝑚𝑜𝑙𝑒𝑠/𝐿

3.4.1 Dilutions and Ratios

Please be aware that there are two terms with different meaning commonly used in BCCB and
Biology: dilutions and ratios:

A dilution gives the volume of the (stock) solution to be diluted in comparison to the total
volume of the final solution (= stock + solvent). For example, a 1:10 dilution of a stock solution
X contains 1 part X in a total of 10 parts solution. It is generated by adding 1 part (e.g., 1 mL)
of X to 9 parts (9 mL) of solvent. The dilution factor is 10.

In contrast, ratios give the number of parts of the stock compared to the number of parts of the
solvent. A ratio of 2:3 ("two to three") means that the whole is made up of 2 parts of one thing
and 3 parts of another — thus, the whole contains five parts in all. Hence, the ratio of the stock
solution X and the solvent in the 1:10 dilution mentioned above is 1:9.

Based on these explanations you may notice that there is a general discrepancy between
chemical and biological terms!!!

23
General Introduction Manual BCCB Teaching Laboratory 2022/2023

4 Required Individual Student Preparation

For any laboratory course you need to come prepared: knowing what you are doing is the best
protection measure, and experimental work is a lot more fun, if you know what you are doing.
Furthermore, you are expected to complete the experiment within the given time frame, and
there is not enough time to start reading about the experiment during the lab course.

4.1 Readings and Calculations

To carry out the experiments in the course successfully, preparation is essential. All
information needed is provided in the Course Manual (the document that contains the
instructions for each experiment) and the General Introduction Manual (this document). You
need to have read both documents before each laboratory course, so that you already know
what you are going to do, what the scientific background of the experiment is, and why you
need to perform the experiment in a particular fashion and with certain safety measures. The
preparatory questions at the beginning of each manual provide some guidance as to how to
prepare.

You can also save some time, if you familiarize yourself with the calculations needed, e.g., for
the different lab days, you can already pre-calculate the dilution series and the amount of salts
needed for the buffer preparation.

4.2 Safety Data Sheets (SDSs)

Each participant has to prepare Safety Data Sheets (SDSs) for each hazardous substance
(substances are indicated in the respective course manuals). The SDSs will be collected at the
beginning of the lab day and will be checked by the course assistants. Without submission of
the correct SDSs, you will not be able to participate in the lab day. Furthermore, a grade penalty
will apply. General information about SDSs is available at: http://www.ilpi.com/msds/.
IMPORTANT: The personal SDS printouts have to be signed by the student to confirm
personal preparation of the data sheet and familiarity with its contents !!!

24
General Introduction Manual BCCB Teaching Laboratory 2022/2023

5 Documentation of experimental Results

5.1 Result Reports and Lab Reports
The point of the result and lab reports is to give you and your instructors an overview over what
you have done and to record your experimental approach and your results for future
reproduction. Furthermore, it will also contain the interpretation of your results and the
conclusions that can be drawn from them. Knowing how to write such reports is an essential
soft skill in scientific research and application in both, science and industry.

In general, the lab reports are group reports and both group members are responsible for
content and timely submission of the report. Hence, the grade will always count for both group
members. The only exemption is the individual Lab Report for the “Carbohydrates”
Experiment. Lab reports must be typed and handed in both, in printed and electronic version.
The latter may have to be submitted to the Turnitin Program (your respective course instructors
will inform you). Whereas result reports are hand-written mostly individually during the lab
day and handed in at the end of the same lab day. Here, preparation by reading the respective
experimental manual is essential.

For BCCB lab courses, lab reports follow the same style that scientific publications usually
have in the Natural Life Sciences. This will later help you write your Thesis and future
publications. For further information, please refer to the "General Formal Criteria" Document
and the general instructions provided in CampusNet.

5.2 References (Harvard System)

References indicate which sources support the claims and information provided in your lab
report. A reference list at the end of the paper contains detailed information about your sources.
In-text references in your paragraphs relate these sources to particular statements. In the BCCB
lab courses the reference style employed follows the "Harvard System" by the Council of
Science Editors (CSE) that lists the references by the author names and publication years. For
further information, you may refer to:
http://www.scientificstyleandformat.org/Tools/SSF-Citation-Quick-Guide.html.

Internet sources are usually given in a separate section below the reference list. Please stick
to the style given in the Lab Manuals (it may vary for different lab courses). In any case, you
have to list the whole link and the date of retrieval. Only peer-reviewed internet sources should

25
General Introduction Manual BCCB Teaching Laboratory 2022/2023

be cited. This means that Wikipedia is not a citable source. An exception may be images,
because they are often free of copyrights.

6 References
IMPORTANT: The citation style in this manual follows the same "Harvard System" style that
you need to use for your lab reports.

Boyer RF, (2000). Modern Experimental Biochemistry. 3rd ed., Benjamin Cummings, Redwood City.
Eppendorf, (2000). Operating Instructions for Centrifuge 5415 R. Eppendorf AG, Hamburg.
Eppendorf, (2001). Instruction Manual for Eppendorf Research Family fix, variable, multi. Eppendorf
AG, Hamburg.
Nelson DL and Cox MM, (2005). Lehninger Principles of Biochemistry. 5th ed., W.H. Freeman and
Company, New York.
Silberberg MS, (2003). Chemistry: The Molecular Nature of Matter and Change. 3rd ed., McGraw-Hill,
New York.
Thermo Spectronic, (2002). Operator’s Manual for GenesysTM 10 Series Spectrophotometers. Thermo
Spectronic, Cambridge.
Voet D and Voet JG, (2004). Biochemistry.3rd ed., Wiley & Sons, New York.
Wissenschaftlich-Technische Werkstätten GmbH & Co.KG (2002). Instruction Manual for WTW
inoLab pH Level 1. WTW, Weilheim.

Internet Sources and Links

http://hannainst.com/usa/index.cfmn (retrieved 2022-07-20)
http://www.ilpi.com/msds/ (retrieved 2017-08-16)
https://www.msds-europe.com/h-statements/ (retrieved 2022-07-20)
https://www.msds-europe.com/p-statements/ (retrieved 2022-07-20)
http://www.scienceline.ucsb.edu/images/wavelength1 (retrieved 2015-08-21)
http://www.scientificstyleandformat.org/Tools/SSF-Citation-Quick-Guide.html (retrieved 2017-08-16)
http://www.sigmaaldrich.com/sigma-aldrich/help/help-welcome/hazard-and-precautionary-
statements.html (retrieved 2012-03-08)
http://www.unece.org/trans/danger/publi/ghs/ghs_welcome_e.html (retrieved 2014-03-05)
http://www.usbeck.eu/en/index.php?artikel&gruppe=1020 (retrieved 2011-03-03)
https://commons.wikimedia.org/wiki/File:Biohazard.svg (retrieved 2022-07-20)
https://www.fishersci.co.uk/shop/products/polystyrene-semi-micro-cuvette/10151710 (retrieved 2018-
08-07)
https://www.foodalyt.de/de/foodalyt-photometer-light-photometer-light-extra.html (retrieved 2018-08-
07)

26
General Introduction Manual BCCB Teaching Laboratory 2022/2023

Note: All links provided in this manual were retrieved on the dates indicated and checked on
July 20, 2022. Please note that link for the foodalyt -photometer is no longer available.

27
General Introduction Manual BCCB Teaching Laboratory 2022/2023

Declaration of Acceptance of General Rules in the BCCB Teaching

Laboratory

Hereby, I state that I have read the complete General Introduction Manual to the BCCB
Teaching Laboratory. With my signature, I also accept all rules and regulations stated therein,
and I certify that I understand that any violation may be penalized. Furthermore, I am aware of
all risks that arise from experimental work in a laboratory.

Printed Name and Date Signature

28