Toxico gy
olog
Lab
b
Ma al
anua
Fall­
­2009
Pharm y and
macology
Toxico partment
ology dep t
Contents
Instructors
Safety guide lines
Tentative course schedule
Course evaluation
Experiment (1)­Ames test
Experiment (2)­DNA fragmentation
Experiment (3)­Comet Assay
Experiment (4)­Drugs of Abuse screening test
Toxicology Lab Manual­Fall 09
Toxicology Lab Team
Course Supervisor
Dr Ola Ahmed Heikal ola.heikal
Teaching Assistants
Amira Emad Abdel Aziz amira.abdel Aziz
Alyaa Mahmoud Abdel Haleem alyaa.abdelhaleem
Maha Ibrahim El Zeiry maha.elzeiry
Mina Naguib Rashad mina.naguib
Nagwan Nabil Shanan nagwan.shanan
Shady Saad Ibrahim shady.ibrahim
Toxicology Lab Manual­Fall 09
General Rules and Safety Guidelines
The safety of all students, staff, and laboratory personnel is of high priority and
the responsibility of everyone in the laboratory.
The proper handling of chemicals and adherence to lab regulations will be graded
for 20% of your final grade.
General
1. You are allowed to attend the lab ONLY according to your schedule. Only signed
excuse forms by the course supervisor will be accepted for compensation labs.
2. Lab doors will be closed 10 min later than the indicated lab time. In case of late
arrival (max 10 min.) 50% of the lab grade will be deducted.
3. The lab work will be conducted in groups of 3. Working places will be assigned
for each group.
4. For each experiment, you are required to hand in a  Group Report . You must
submit your report before the announced deadline as a hard copy. Rerorts
submitted after the deadline will not be graded.
5. In the lab, you MUST wear a lab coat. Don t take it off as long as you are in the
lab. If you don t have a lab coat, you will not be allowed to enter the lab.
6. Gloves MUST be worn while performing your experiments. Never handle any
chemicals or biological samples bare handed. Take off your gloves BEFORE
leaving the lab.
7. If you wear gloves, NEVER touch taps, door handles, equipment, keyboards,
computers, light switches...Etc.
8. It is strictly prohibited to eat or drink in the lab.
Safety
1. Special attention must be given to hazardous chemicals/reagents.
2. Wear appropriate protective equipment when handling chemicals (for example
gloves and protective clothing).
3. Minimize the inhalation of chemicals. Do not leave chemical containers open
4. Dispose chemical/biological waste in appropraite containers (see point Waste
Disposal).
5. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
instructor s procedures for clean up.
6. Wash your hands before you leave the lab
Cleaning and Waste disposal
1. Keep the lab tidy. Everyone has to clean his/her place BEFORE leaving the
lab.
2. Prepare separate container for disposal of contaminated tips and eppendorfs
at your working place.
3. Discard waste in the appropriate bags/container.
4. Cleaning of spilled EtBr:
In case of little spilling, clean area with tissues soaked in alcohol 70%.
Dispose the tissues in the special buckets for EtBr waste. In case of large
spilling, mark but avoid the contaminated area and call your instructor.
5. Cleaning of spilled chemicals (especially near the balances):
Always clean spilled chemicals immediately. In case of spilling hazardous
chemicals, mark but avoid the contaminated area and contact your
instructor.
Toxicology Lab Manual­Fall 09
6. Close all reagents containers and switch off all equipment you have used at
the end of the lab session.
Chemical wastes hazard
Some wastes produced during performing your experiments are potentially hazardous
and can cause injury, illness, or death. Special care should be taken during handling
these chemicals, for example:
Ethidium bromide causes eye, skin, and respiratory tract irritation and is a known
mutagen (i.e., it can change genetic material in a living cell and has the potential to cause
cancer).Wear appropriate protective clothing, and gloves whenever dealing with
ethidium bromide.
Biological samples hazard
Biological samples such as tissues have the potential to transmit infectious diseases.
Wear gloves whenever handling biological samples. Dispose your gloves, sample and
any tools that came in contact with any biological samples in appropriate bags
 Biohazard labelled bags . These bags will be autoclaved before disposal.
In Experiment (1) ­you will be dealing with Salmonella typhimurium: a genus of
harmful bacteria, which cause gastrointestinal diseases, through ingestion of
contaminated materials. Disposal of bacterial samples should be done in the  Biohazard
bags to be autoclaved before disposal.
In Experiment (2) ­you will be dealing with liver homogenates from rats and mice.
Disposal of the liver homogenate samples should be done in the  Biohazard bags to be
autoclaved before disposal.
Safety precautions in the handling of carcinogens / mutagens
Every effort is made in this laboratory to isolate the areas where carcinogens/mutagens
are used in order that the entire laboratory is not contaminated unknowingly by these
compounds.
Safety precautions in the handling of the Salmonella tester strains:
Salmonella typhimurium can cause diarrhea and food poisoning. The particular
S.typhimurium strain, LT2, the parent of all of our tester strains, is used by geneticists all
over the world and is not very virulent. The deep rough mutation (rfa) present in the
standard tester strains (see Table I) lowers virulence by orders of magnitude and these
strains should be relatively harmless. The pKM101 plasmid, with one antibiotic
resistance marker for ampicillin, and contained in the tester strains TA98 and TA100
should be a minimal hazard. Plasmids are extremely common in the enteric population
in nature and most of the RTF plasmids isolated from Salmonella in hospitals contain
many antibiotic resistance genes. Nevertheless, as a matter of routine with any
Salmonella strain we use plugged pipettes and autoclave any material containing
Salmonella before it is washed or disposed of.
Toxicology Lab Manual­Fall 09
Toxicology lab tentative schedule­Fall 09
Toxicology Lab Manual­Fall 09
Course Evaluation
" A Final lab exam is required for completion of this lab course.
" The final grade will be the total of the individual labs assessment.
Deadline for Report submission Weight
Report (1) Thursday,5th of Novemeber 17:00 10%
Report (2) Thursday, 19th of November 17:00 10%
Report (3) Thursday, 26th of November 17:00 10%
Report (4) Thursday, 3rd of December 17:00 10%
Final Lab Exam To be announced 40%
Evaluation   20%
Toxicology Lab Manual­Fall 09
Experiment (1) Ames Test
Background
Principle
Ames test optimized protocol
Experiment (1)­Lab report­ To be submitted to your corresponding
TA as a hard copy
Reagents and buffers
Toxicology Lab Manual­Fall 09
Background
A wide variety of chemicals exist in our environment, and many can have
mutagenic effects.
" Mutagens typically produce base pair substitutions or insertions or
deletions.
" Most cancer development results from accumulated mutations in a
number of genes (oncogenes, tumor suppressor)
First developed by the American microbiologist Bruce N.Ames in 1971,  Ames
test is a widely used technique for screening potential carcinogens by testing for
mutagenesis of bacteria. It relies on the observation that the most common
cause of cancer is somatic mutations brought about by DNA damage. Chemicals
that damage bacterial DNA, and induce mutations, are also likely to cause
mutations in mammalian cells. Performing the assay in bacteria gives much
faster and less expensive results than animal studies, with results usually being
available in 2 days.
Ames test can be done in two different ways:
I. Qualitative "spot test" that allows the rapid screening of multiple
compounds for possible mutagenicity.
II. Quantitative assay using sequential dilutions of the test chemical
added to the top agar overlay to find out what concentrations of the
test chemical are mutagenic and/or toxic.
Further improvements to the assay include the use of strains mutated for DNA
repair and cell wall synthesis (to increase sensitivity to mutagens). Enhanced
results have also been achieved by the treatment of potential mutagens with
liver cell extracts, to allow for the conversion of compounds that are not
mutagenic into carcinogenic compounds by liver cell enzymes.
Principle
In the original version of Ames test, auxotrophic histidine mutants of Salmonella
typhimurium, unable to synthesize their own histidine, were utilized. A gene for
the very long biosynthetic pathway of the amino acid histidine is defected by a
point mutation. These mutants, therefore, are unable to synthesize histidine and
depend on histidine supplements for normal growth (on a mineral salt medium).
Because the primary mutation is caused by a point mutation, the addition of a
test substance can cause a secondary mutation which corrects the point
mutation.
This process of  back mutation is termed  Reversion , i.e. synthesis of histidine,
and the bacteria in which this reversion occurs are called  Revertants . Thus the
back mutation  Reversion restores the wild type phenotype and cause the
auxotrophic test strain to be able to grow prototrophically, i.e. capable of growth
Toxicology Lab Manual­Fall 09
on a histidine free mineral medium.  Revertants can be readily recognized by
their ability to form colonies on a mineral medium. The test can be used to
compare between efficacies of different mutagens. The higher the concentration
of mutagen used the more  Revertant colonies will result.
Spontaneous reversion may, however, occur. The relation between the induced
back mutations to the spontaneous back mutations is a measure of the
mutagenicity of a substance. An abnormally high spontaneous  Reversion rate
may indicate contamination.
The test has been adapted for use in detecting chemicals which are potential
human carcinogens or mutagens by adding homogenates of rat (or human)
liver directly to the petri plates thus incorporating an important aspect of
mammalian metabolism into the in vitro test. In this way, a wide variety of
carcinogens requiring metabolic activation can be detected easily as mutagens.
Rat liver is the most convenient source of activating enzymes.
The economy of the bacterial/ mammlian microsomal assay suggests its
usefulness as a tool in rapidly obtained information about the potential
mutagenic/ carcinogenic activity of uncharacterized compounds in complex
mixtures.
Ames in his original publication, where he tested 300 carcinogenic chemical
compounds, concluded that a high correlation exists between the mutagenic
potential and the carcinogenicity of a chemical substance. From the 300
carcinogenic substances he tested, more than 90% showed a mutagenic effect;
similarly, 87% of the non carcinogenic substances showed no mutagenic effects.
Therefore, a positive Ames test is a very strong indication that the substance
under test is carcinogenic. However, the missing 10% correlation  from Ames
study indicates that in these cases, the findings must be further tested in
experimental animals. For those substances that  pass the test and are possibly
of interest for pharmaceutical, cosmetics, or food industries, testing using
experimental animals remains the final indispensable confirmatory test.
The sensitivity of the test can be enhanced by the following
modifications:
1. To increase the uptake of hydrophobic test substances (and thereby
manifests its mutagenic effect), mutants in the rfa gene  deep rough gene
are used. These mutants are defective in the biosynthetic pathway of
some hydrophilic polysaccharides that exist in the cell envelope, hence,
resulting in defective lipopolysaccharide barrier.
2. Using S. typhimurium mutants with a deleted uvrB gene, which is
involved in DNA repair, guarantees that the secondary mutations caused
by the test substance would not be corrected by the cellular repair
machinery.
Toxicology Lab Manual­Fall 09
3. Strains harbouring the plasmid pKM101, lead to further improve in the
sensitivity of the method. The plasmid carries gene that codes for
ampicillin resistance.
Bacterial strains utilized in Ames test
Strain Histidine Other genotype Type of
mutation features Histidine
mutation
TA 100 His G46 rfa­uvrB­PKM101 Point mutation
TA 2635 His G428 Rfa­PKM101 Frame shift
mutation
Safety precautions in the handling of the Salmonella tester strains
Salmonella typhimurium can cause diarrhea and food poisoning. The particular S.
typhimurium strain, LT2, the parent of all of our tester strains, is used by
geneticists all over the world and is not very virulent. The deep rough mutation
(rfa) present in the standard tester strains lowers virulence by orders of
magnitude and these strains should be relatively harmless. The pKMI0I plasmid,
with one antibiotic resistance marker for ampicillin, and contained in the tester
strain TA100 should be a minimal hazard. Nevertheless, as a matter of routine with
any Salmonella strain we use plugged pipettes and autoclave any material
containing Salmonella before it is washed or disposed of.
Toxicology Lab Manual­Fall 09
Ames test Optimized Protocol
Aim of experiment
To demonstrate the mutagenic effect of a benzo(a)pyrene. Ethidium bromide will
be used as the positive control.
Protocol
I. Pre­culture of the frozen bacterial strain (TA100)
1. In an Erlenmeyer flask, prepare 100 ml of LB agar.
2. In LB agar, add histidine* 87.5źM (100 źl from stock 87.5 mM) and
ampicillin 100źg/mL (100źl from stock 100 mg/ml) & then pour it onto
the plate and leave it to cool.
3. Inoculate the plate with a crust of the Salmonella tester strain
cryopreserved at  80 C TA100 or strain TA2635.
4. Incubate the pre culto
re overnight (15 h) at 37°C.
Toxicology Lab Manual­Fall 09
II. Second Pre­culture of the test organism
1. In a 50 ml Erlenmeyer flask, add 10ml LB NaCl medium, mixed with 100
µg/ml Ampicillin (10źl from stock 100 mg/ml) and 87.5µM histidine*.
2. Inoculate the mix prepared with a single colony of the Salmonella tester
strain TA100 or strain TA2635 from the first pre culture.
3. Incubate the pre culture overnight (15 h) at 37°C.
III. Working culture of the tester strain
1. Use 0.2ml of the pre culture to inoculate 10ml of LB NaCl medium
containing 100µg/ml ampicillin (10źl from stock 100 mg/ml) without
the hisitidine.
2. Shake the culture for 3 h at 37°C. This cell suspension is used directly in
subsequent steps; it should not be stored on ice to avoid heat shock!
#Metabolizing buffer (M  Buffer)
The M buffer is prepared as mentioned (refer to the appendix), the
substrate G 6 P with the cofactor NADP+ are freshly added to the sterile
M buffer just before being used in the experiment.
#Top agar
The Top agar is prepared as mentioned (refer to the appendix), the
hisidine* and biotin are freshly added to the sterile Top agar just before
being used in the experiment.
*The addition of histidine should trigger the bacterial cell division & reduce the
rate of spontaneous mutation. This small amount of histidine in the growth
medium allows the bacteria to grow for an initial time and have the opportunity to
mutate. When the histidine is depleted only bacteria that have mutated to gain the
ability to produce its own histidine will survive. We can say that: it supports few
rounds of cell division as many mutagens works only on replicating DNA.
III. Procedures:
1. Label three glucose minimal salt agar plates (AT agar): Positive control,
Negative control and Test. Please write your group number and date of
performing the experiment at the side of the AT agar plates as instructed
by your teaching assistant.
2. Label three reaction mixtures tubes (1.5ml eppendorf) exactly as you
labelled the AT agar plates in step (1)
3. Aseptically, pipette 100źl of the provided bacterial strain (TA100 or
TA2635) in each reaction tube.
4. Add 500źl of the M­buffer to each reaction tube.
5. Add 20źl of benzo(a)pyrene dissolved in DMSO and 10źl of rat liver
extract S9* (an activator) to the  Test reaction tube only.
Toxicology Lab Manual­Fall 09
* As requir ent S­9 frac wed (at roo ature) and kept
red, sufficie ction is thaw om tempera
on ice; the carded at th he day
e unused portion is disc he end of th
6. Add 20źl DMSO to each control rea tube (positive and negative
O c action e e a
contro
ols).
7. Add 10 Eth µg/ml solved
0­20µl hidium Bromide (2µ diss in DMSO) to the
positive con on tube onl
ntrol reactio ly.
8. Incuba all reaction tub at 37 °C for 1h in a ther shake at
ated bes h rmo er
600rpm
m.
9. Aseptically, pour ction mixt 3 gar medium
r each reac ture onto 3ml top ag m at
40­47°C. Mix* well then po esponding AT
w our each sample onto the corre
agar pl g histidine.
late lacking
*The contents ar mixed (b rotating the tube between t palms) and
c re by g the
poured on minim glucose agar plat Uniform distribut of the top
d mal e tes. m tion
agar on the surf of the plates is accomplish by gen tilting and
o face e hed ntly
rotatin overed plat n setting th own to har
ng the unco te and then he plates do rden.
The mixing, pour stribution should take d the
ring and dis s e less than 20 sec and
plates should be left to hard s. It is impo
l den for several minutes ortant to follow
Tox b Manual­Fa
xicology Lab all 09
these time limits. If the top agar starts to harden in mid­operation a
stippled plate surface will result which makes scoring of revertants difficult
10. Within an hour, the plates should be put in a dark, 37' incubator. Incubate
the AT agar plates at 37°C for 48­72h. After 2 days the colonies
(revertants to histidine prototrophy) in both test plates and controls are
counted, and the presence of the light background lawn of growth (due to
the trace amounts of histidine added) is confirmed.
You have to collect your plates from the microbiology lab after the
incubation period is over.
11. Score the number of colonies per plate and write it down in your lab
report.
The number of colonies on the test and control plates is
compared. The negative control plate may show a few
spontaneous histidine­synthesizing revertants. The
mutagenicity of a substance is proportional to the number of
colonies observed. If the test chemical is indeed a mutagen
and potential carcinogen, the higher the concentration of the
mutagen used, the more revertant colonies will be observed.
Additions/replicate Negative Test Positive
control control
M buffer(ml) 0.5 0.5 0.5
S9 extract(µl) 0 10 0
DMSO(µ) 20    
Test strain(µl) 0.1 0.1 0.1
Benz(a)pyrene (µl) 0 20 0
Ethidium Bromide 0 0 10 20µl
Number of
colonies/petri dish
Toxicology Lab Manual­Fall 09
Experiment (1)­ Lab Report
Name:
ID:
Group Number:
Instructors names:
Aim of the experiment:
Principle
Results
Interpretation
Conclusion
Toxicology Lab Manual­Fall 09
Reagents and Buffers
AT­solid medium
Salt ingredients Preparation
Autoclave the salt, agar­ and the
K HPO 7.5g
2 4
magnesium solutions, sterile filter the
NaNH HPO X 4 H O 2.63g
4 4 2
glucose solution and cool to about 50°C
Citric acid 1.5g
before mixing
monohydrate
SL6 75µl
H O q.s.300ml
2
pH 7.4
Agar­Agar components
Agar­Agar 11.25g
H O q.s.350ml
2
Magnesium solution
MgSO4 X 7 H O 0.15g
2
H O q.s.37.5ml
2
Glucose solution
Glucose 15g
H O q.s.75ml
2
Additional
Ampicillin
100źg/l
Top­Agar
NaCl­Agar Preparation
NaCl 0.45g Autoclave the NaCl­agar and let it cool down
Agar Agar 0.59g in a water bath set at 42°C. Then add 0.75
­
H O 900ml ml of sterile filtered histidine­biotin
2
solution.
Histidine­Biotin solution
Histidine 7mM
Biotin 5mM
Ampicillin
100źg/L
Toxicology Lab Manual­Fall 09
NB­NaCl­broth
Pepton 5g
Supplements
NaCl 5g
Ampicillin: working concentration = 75
Meat extract 3g
mg/l (i.e. 75µg/ml)
H O q.s.1000ml
2
Stock solution 100 mg/ml H O
2
Sterile filter and store at ­20°C
M­buffer (Metabolisation buffer)
Buffer constituents Preparation
KP­buffer pH 7.4 200ml First add 200 ml of KP­buffer (1M,
KCl 4.1g pH7.4), then add the he indicated
MgCl X 6H O 2.65g amounts of the salts and fill up to 1000
2 2
H O q.s.1000ml ml using distilled water. Subsequently
2
dissolve the substrate or the cofactor in
Substrate and co­factor
the indicated end volume of buffer.
concentration
Glucose­6­phosphate 8mM
NADP+ 6mM
KP­buffer (1M)
Basic components Preparation
K HPO 68g Prepare both solutions separately, mix
2 4
H O q.s.500ml amounts of the solutions to get the
2
required pH value. If required, dilute the
Acidic components
buffer with distilled water (possibly after
KH PO 87g
2 4
addition of other salts) to get the final
H O q.s.500ml
2
concentration
Toxicology Lab Manual­Fall 09