Description
I need help with writing me a formal lab report for one of my biology courses that talks about drug tolerance. I did the complete experiment and have all the data required to complete this lab report. I need your help in summarizing and organizing the data in a formal lab report format, plus making two bar-charts for the results section using PowerPoints or any other graphing program. The first graph compares the drug response for weeks one and two of the experiment where the second graph shows the results for week 3 of the experiment. I have a photo that shows a similar graph that our instructor draw for us so we can make our graph such.Please check the attachments.
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I need help with writing me a lab report for one of my biology courses that talks about drug tolerance. I
did the complete experiment and have all the data required to complete this lab report. I need your help
in summarizing and organizing the data in a formal lab report format, plus making two bar-charts for the
results section using PowerPoints or any other graphing program. The first graph compares the drug
response for week one and week two of the experiment where the second graph shows the results for
week 3 of the experiment. I have a photo that shows a similar graph that our instructor draw for us so
we can make our graph such.
drug of addiction and abuse formal lab report notes: the mice lab (drug tolerance)
first week, we tried different doses of the same drug on different mouses to see the animal tolerance to
the drug. This is specifically called ‘dose response’
second week, came in and the mice was injected with TCPOBOP drug the night prior and that made
what they call as ‘cross tolerance’. The second week of the experiment we had reduced sedition times
because the mice received injection of TCPOBOP one day before the day of the second week
experiment.
In the third week we measured the metabolic tolerance using aminopyrine. We compared the rate of
metabolism of aminopyrine in liver that had come from control mice with that in liver taken from mice
that have had the level of the enzymes induced by treatment with TCPOBOP with a single treatment.
TCPOBOP induces the same liver enzymes which metabolise pentobarbitone.
focus on the result section and have a descriptive results section and not discussion. straight results and
in details. (text description of your results) pull the data out of this excel files and make a graph that
describe the data then describe them in detail but do not go into discussing the data.
Think about the following when looking at the data to write your results section:
how long does it take to loos the righting reflex? make a graph that compare the time ‘ loss of righing
reflex in minutes’ against the doess ‘ low dose, medium dose and high dose’ for both week one and two.
make only one graph that compare your findings for both week one and two, so one graph that has two
columns each for one week. check the picture attached and make something similar.
use journal article that did similar experiment.
This lab involved the use of a life mice and we had to measure some aspects of the mice. VIVO is the use
of animals in lab.
The mice was given a drug called Pentobarbital which would make them relaxed.
Righting reflex is when an animal flip back after it is putting on its back.
Highest dose = the highest sedation
We monitored duration of loss of righting reflex, breathing rate, strength of pedal reflex and eye blink
reflex in minutes.
Day 1 – 1st part – Mice received injection of pentobarbital – 3 doses
Day 7 – Mice received injection of TCPOBOP – Agonist of the constitutive androstane receptor (CAR) (EC50
= 20 nM). Induction of enzymic activity. (week 2 of the experiment )
Day 8 – 2nd part – Mice received injection of pentobarbital – 3 doses
Background info:
When drugs of abuse are taken repeatedly, the response they produce is reduced. This is a
phenomenon known as tolerance.
Tolerance response depends on the microsomal enzymes within individuals’ bodies that are
responsible for metabolising those drugs.
The barbiturate group of drugs develop tolerance very rapidly and very intensely. This is a result of
the induction of the microsomal enzymes that are responsible for metabolising them. In this case
the tolerance is referred to as metabolic or drug disposition tolerance.
The barbiturates are CNS depressants and their effect can be measured by monitoring a number of
different activities. This can include the level of arousal or activity of an animal. When a normal
mouse is placed on its back it will immediately “right” itself. Barbiturates and other CNS depressants
can cause loss of this “righting reflex”. The duration of loss of the “righting reflex” can be used as a
measure of CNS depression and dose response curves can be drawn up and potencies of different
CNS depressants compared.
What happened in the labs of this experiment:
The experiment was carried over 3 weeks.
Over the first two laboratory sessions we monitored the response of mice to pentobarbitone sodium.
In the first session we were provided with normal (control) mice. In the second session, we were
provided with the same mice except they had had been pre-treated with TCPOBOP (3 mg/kg) on the
previous day. The aim of the experiment was to relate the response to pentobarbitone to the dose
given and to determine how much tolerance had developed by exposure to TCPOBOP. Remember
these groups of experiments are about pentobarbitone tolerance and not TCPOBOP per se. Do you
know which member of the P450 enzyme family is induced by pentobarbitone?
In the third week we measured the metabolic tolerance using aminopyrine. We compared the rate
of metabolism of aminopyrine in liver that had come from control mice with that in liver taken from
mice that have had the level of the enzymes induced by treatment with TCPOBOP with a single
treatment. TCPOBOP induces the same liver enzymes which metabolise pentobarbitone.
Method
Week 1
CNS depressants inhibit the thermoregulatory centre. The body temperature of the mouse will
drop if nothing is done. You must ensure that the mice are kept warm by using the heated mat to
minimise loss of body heat.
•
The mice will have been randomly divided into groups of 5 mice with two groups at each of
the doses of pentobarbitone sodium.
•
Each mouse is weighed and labelled. Each mouse will receive a dose volume of
Pentobarbitone solution (10ml/kg or 0.1 ml per 10 grams body weight).
•
The mice will be injected by the licensee and you will be told the dose which you record.
Note the time at which each individual mouse loses its “righting reflex”. The depth of CNS
depression can be monitored by doing a number of simple tests.
After the loss of righting reflex and at 10 minute intervals:
1. lightly touch the corner of the eye with a piece of moistened tissue and record the strength
of the blink as strong, medium or weak
2. estimate the breathing rate in breaths per minute (this is done by counting the number of
breaths in 15 seconds and multiplying the value by 4)
3. squeeze the toes on one of the hind paws and record the withdrawal reflex as strong,
medium or weak. Record these observations in the Record sheet provided.
Record the time at which the mouse regains its “righting reflex”. The duration of loss of “righting
reflex” is the difference between the loss time and the regain time.
Results and Analysis
You will have recorded the results for only one dose of pentobarbitone sodium in 5 mice. You must
collect all the results for the other doses from the other groups in the class to obtain results for all 10
mice at each dose.
•
For each group of 10 mice calculate the mean (and SEM) time and median time for loss of
“righting reflex”.
•
For each group of mice calculate the mean breathing rate and predominant blink and
withdrawal strength at each time for each dose.
•
Plot dose response curves for the duration of loss of “righting reflex” for the class results
using the mean results, showing error bars.
•
Plot the time course for the average breathing rate at each dose.
•
Plot the time course for the predominant blink and withdrawal at each dose.
Week 2
The same measurements are taken in week two as in week 1 except that the mice in week 2 will have
been pre-treated with TCPOBOP. Plot the same results and compare the results of week one with
those of week 2.
Does pre-treatment with TCPOBOP affect the responses? If so are the differences statistically
significant?
Week 3
Part A will have already been done for you. You do part B.
Part A
Livers from mice exposed to TCPOBOP or control mice (no treatment) were
harvested and stored at -20°C. A 25% homogenate of liver was prepared in
KCl/buffer (1.15% KC1 in 0.1M NaH2P04/Na2HPO4 buffer (pH 7.4))
The enzymes responsible for metabolising pentabarbitone were isolated by
centrifugation at 4 K rpm for 15mins, 4°C.
Part B
The chemicals and tissues used in this assay produce a colour as well so this needs to be both
subtracted from both control and TCPOBOP samples. These are referred to as “Normal liver control”
and (TCPOBOP treated control” and they have no aminopyrine added to their reaction tubes
Set up the following mixtures in test tubes:
Table 1
Tube
numb
er
Liver
homo
genat
e
(ml)
Cofactor
(ml)
Semicarbazide
(ml)
KCLbuffer
(ml)
Aminopyri
ne
(ml)
1
0.5
0.25
0.2
1.05
–
2
0.5
0.25
0.2
0.55
0.5
TCPOBOP treated
control
3
0.5
0.25
0.2
1.05
–
TCPOBOP treated
test
4
0.5
0.25
0.2
0.55
0.5
Normal liver control
Normal liver Test
Co-factor:- Glucose-6-PO4 (3×10-2M),NADP (5×10-4), Mg Cl2 (1×10-2M), Nicotinamine (6
x 10-2M)
Semicarbazide:- Semicarbazide – HCl (7.5 x 10-2M)
Aminopyrine:- Aminopyrine (5x 10-2M)
The tubes are covered with parafilm and incubated in a shaking water bath at 37°C for 45 minutes.
During this incubation aminopyrine is converted to formaldehyde.
Standard Curve
While the samples are being incubated prepare a series of dilutions of formaldehyde for a calibration
curve using the following concentrations:
2 ×10-5M,
5 ×10-5M,
1 × l0-4M,
2 ×10-4M
1×10-2M
1+9 ml
ml
1×10-3M
1+9
2 + 8 ml
1×10-4M
2×10-4M
2 + 8 ml
5 + 5 ml
2×10-5M
5×10-5M
5+5
2+8
-5
1×10 M
The dilutions are made up using the KCl-buffer. There is no requirement to incubate the
formaldehyde standards.
Centrifugation Step
During the incubation period label 4 centrifuge tubes 1-4, 5 test tubes labelled 5-9 (table 2), and a
further 9 test tubes labelled 1-9 (table 3). To the 4 centrifuge tubes and the 5 test tubes labelled 5-9
add 1.5ml of 12.5% trichloracetic acid (TCA) to each one (table 2). TCA cause any proteins in the
reaction to precipitate, including the enzymes responsible for converting aminopyrine to
formaldehyde i.e. it will terminate this reaction. If this step is omitted then (i) formaldehyde will still
be produced and (ii) proteins can interfere with the spectrophotometric step.
At the end of the 45 minute incubation period:
To each tube (centrifuge and test) add 1 ml of tissue sample derived from table 1 or formaldehyde
standard. The following table summarises the tubes contents (table 2).
Tube 9 is used to blank the spectrophotometer and contains 1 ml of the KCI-buffer and 1.5 ml TCA.
Table 2
Centrifuge
Tube 1
Centrifuge
Tube 2
Centrifuge
Tube 3
Centrifuge
Tube 4
Test Tube 5
Test Tube 6
Test Tube 7
Add
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
Add
1ml – Normal liver control from 37°C
incubation
1ml – Normal Liver test from 37°C
incubation
1 ml -TCPOBOP treated control from
37°C incubation
1ml – TCPOBOP treated test from 37°C
incubation
1ml – 2 x10-5 M formaldehyde in KCl
1ml – 5 x10-5 M formaldehyde in KCl
1ml – 1 x10-4 M formaldehyde in KCl
Action
Centrifuge
Centrifuge
Centrifuge
Centrifuge
Test Tube 8
Test Tube 9
1.5ml of 12.5%
trichloracetic acid
1.5ml of 12.5%
trichloracetic acid
1ml – 2 x10-4 M formaldehyde in KCl
1ml KCl buffer (blank)
Tubes 1 to 4 (containing the liver homogenate) are centrifuged in a refrigerated centrifuge for
15mins at 4000 rpm (table 2).
Tubes 5 to 9 do not need centrifuging.
Second incubation/heating and absorbance reading
2ml of clear supernatant liquid from each of tubes 1 to 4 are transferred to respective test tubes
(table 3). 2ml of the solutions in tubes 5 to 9 are transferred to another 5 test-tubes (table 3). To
each test tube add 1 ml of Nash* reagent (table 3). Nash reagent contains the chromogen that turns
yellow in the presence of formaldehyde.
(*Nash reagent: 112.5g ammonium acetate; 1.5 ml acetylacetone and 2.25 ml acetic acid are
dissolved in distilled water to a final volume of 250ml)
Table 3
Test Tube 1
Origin
Normal liver control after centrifuge – 2ml
Test Tube 2
Normal Liver test after centrifuge – 2ml
Test Tube 3
Test Tube 5
TCPOBOP treated control after centrifuge –
2ml
TCPOBOP treated test after centrifuge –
2ml
2ml from previous test tube 5
Test Tube 6
2ml from previous test tube 6
Test Tube 7
2ml from previous test tube 7
Test Tube 8
2ml from previous test tube 8
Test Tube 9
2ml from previous test tube 9
Test Tube 4
Add
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
1ml Nash
reagent
Action
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Mix and incubate @ 60°C x
10mins
Wait and heat tubes 1-9 at 60°C (table 3) all at the same time: see step below.
Cover the test tubes with parafilm and mix the contents of each test tube thoroughly. Heat for
10mins in a 60°C water bath.
Remove from the bath and once they have cooled to room temperature measure the absorbance at
412 nm using the tube 9 as the “blank”.
To control for liver tissue colouration use the tubes without substrates added. With the absorbances
from tubes 1-4, take the ‘control’ values from the ‘test’ values to obtain corrected measures for
‘normal’ and ‘TCPOBOP’ treated liver.
Normal liver absorbance = tube 2 abs – tube 1 abs
TCPOBOP liver absorbance = tube 4 abs – tube 3 abs.
Then apply these values to the standard curve to provide the two liver concentrations.
Task-Specific Assessment Criteria Sheet for Induction of Drug Tolerance in Mice
Learning Outcomes
This practical should give you an insight into the development of drug tolerance in vivo. You
should also understand the contribution played by drug metabolism as well as cross-tolerance.
Marking Criteria
Introduction (15%)
Methods (0%)
what is the point of your investigation based on what is known
in the literature and what results do you expect and why
(physiological & pharmacological). Word limit of 200 words
refer to schedule.
Results (60%)
tables, graphs, figures etc should include a caption and your
results should be described in prose. There should be no
discussion of your results in this section.
Discussion (15%)
were your results as expected, if not why, if yes why (scientific
explanations!). Preventable sources of error are unacceptable
as these should have been rectified in the laboratory session.
This section should contain references to other studies/papers
that back up your argument. Word limit of 200 words
Conclusion (10%)
What do you conclude from your results? There should be no
discussion in this section. Word limit of 50 words
References (0%)
Although no marks are awarded for this section failure to cite
sources properly will be taken into account when the marks for
the introduction & discussion are awarded. Only references
from peer reviewed articles should be included. References
derived from web sites (e.g. ‘www.askthedoctor.com’) are
unacceptable.
Learning Outcomes for the Drug Tolerance laboratory and Associated ASPA Lecture
1) a strong understanding of the physiological and pharmacological mechanisms involved
in the development of drug tolerance;
2) a strong understanding of the ethical and legal aspects of the use of animals;
3) a strong understanding of the principles of experimental design, experimental
observation and care in the collection of samples in investigations involving animals;
4) a strong appreciation of the importance of animal care for the integrity of results;
5) skill in the interpretation and critical analysis of the results of research;
6) an appreciation and understanding of the principles and practice of the 3Rs;
7) an appreciation of whether in vivo studies of biological phenomena in protected animals is
essential to obtain knowledge and understanding of physiological and pharmacological
principles;
8) an understanding of how to handle animals;
9) a demonstration of best practise in the use of animals for scientific procedures and a culture
of care.
II. Assessment Criteria
Marking
Criteria
Introduction
(15%)
FIRST
Exceptional High 1st Mid 1st
Low 1st
1st
Clear, relevant & contextualized aims /
objectives
Introduction is accurate and refers to
relevant information that aligns with the
topics under investigation.
Exceptional knowledge and understanding of
the area of study that considers /relates all
relevant information.
Work is of an exceptional standard and draws
on material beyond the prescribed range.
Results
Analysis &
Presentation
(60%)
Exceptional demonstration of laboratory
skills producing high quality data
Exceptional data analysis handling
Accurate / defined calculations with
examples
Excellent and appropriate presentation of
data (e.g. tables figures with comprehensive
titles and legends).
Results are described concisely and
appropriately and reflect the data presented.
Work is of an excellent standard with strong
scientific writing evident.
Discussion (15%) Logical and detailed interpretation of the data
supported by critical evaluation/ synthesis/
analysis and evidenced with appropriate
references.
Work is of an excellent standard with strong
scientific writing evident and drawing on
material beyond the prescribed range
UPPER SECOND
High 2.1
Mid 2.1
Low 2.1
Clear &relevant aims / objectives
Introduction is accurate and refers to
relevant information that aligns with
the topics under investigation.
Very good knowledge and
understanding of the area of study
supported by relevant information but
with a few omissions.
Introduction is of a very good standard
that draws mainly on taught /prescribed
material.
Very good demonstration of
laboratory skills producing good
quality data
Very good data analysis handling
Accurate / defined calculations with
examples
Appropriate presentation of data (e.g.
tables figures with sound titles and
legends).
Results are described appropriately
and reflect the data presented.
Work is of a very good standard with
very good scientific writing evident.
Some omissions evident.
LOWER SECOND
High 2.2
Mid 2.2
Low 2.2
THIRD
High 3rd
Mid 3rd
Relevant but descriptive aims /
objectives
Simple/descriptive aim
Introduction is limited
detail, sufficient only to
Introduction is mostly sound but lacks
depth and may include irreverent detail. terminology and basic
Good knowledge and understanding of Work is of a sufficient s
mostly superficial, low
the area of study but contains
several omissions. Very
inaccuracies and omissions.
references.
Introduction is of a good standard that
relies heavily on a few set sources.
Good demonstration of laboratory
skills producing competent data
Good data analysis handling
Calculations include some errors.
Presentation of data is good but
unbalanced (e.g. poor use of
titles/legends).
Results are described but with some
inaccuracies, inconsistencies and in
limited context.
Work is of a good standard but
scientific writing could be improved.
Inaccuracies and omissions evident.
Sufficient demonstratio
skills producing adequa
variable data
Sufficient data analysis
Calculations may be inc
inaccurate.
Presentation of data is
sufficient (e.g. poor use
tables/figures with irre
Minimal description of
several inconsistencies
contextualisation.
Work is of a sufficient s
contains several inaccu
omissions.
Interpretation of the da
relate to relevant infor
make meaningful conn
Logical interpretation of the data,
supported by some critical
evaluation/analysis.
Interpretation of the data is mostly
sound but low in
evaluation/synthesis/analysis.
The work typically relates
facts/concepts together applying
known/taught concepts
The work is balanced towards the
descriptive rather than analytical.
Shallow or unbalanced in parts with
some inaccuracies /irrelevant
material.
Good
Verbose and contains some irrelevant
material
The work is descriptive
limited reference to tau
References are limited and generally
reliant on set sources.
Presentation and performance are
good, but unbalanced in some areas.
References are irreleva
Presentation and perfo
limited, unbalanced an
Conclusion
(10%)
Excellent
Succinct and accurate reflection of results
obtained
Very good
Succinct with a few omissions
Resources/
References &
Presentation
References are relevant and current and are
cited appropriately. Presentation is of a
professional standard.
Performance in all areas deemed beyond
expectation of the level and the prescribed
range.
References are relevant and current
and are cited appropriately.
Presentation is of a professional
standard. Sound performance in the
majority of areas.
Advice and Tips for writing your Drugs of Addiction and Abuse Drug Tolerance formal report
1. Whichever group you are from you can analyse Group B’s class data, Group A’s class data or
you can pool the data from both groups.
2. Plotting the loss of righting reflex data – this can be done with a bar chart showing the low,
medium and higher doses for weeks 1 and 2
3. Plot the loss of righting reflex data as a mean with error bars for Standard error of the mean
(SEM = SD/sqrt(n))
4. You can statistically compare the means by use of ANOVA and student’s t-test
5. The breathing rate at each time-point can be calculated by taking the average, don’t include
values which are missing (from the animals which have recovered from the sedation).
6. The reflex responses can be plotted by taking the following approach – assign a number to 0,
W, M and S – eg 0, 1, 2, 3. Now it is possible to plot the pedal and blink reflexes as means
over time.
7. You need text explanation as well as comprehensive figure legends in your results section.
Adequate
But mostly ‘discussion’
Statistics and comparison of means
Comparison of week 1 vs week 2 means
Comparison of dose groups for week 1 and week 2
Introduction: Background pharmacology and aims
Results: Explanation of your results and statistics
Discussion: Analysis of results with respect to the literature – pharmacological mechanisms of action
Conclusion: What are the findings from your experiment
Aminopyrine data
Standard curve – tubes 5-8
Control liver – tube 2 – tube 1
TCPOBOP treated liver – tube 4 – tube 3
Use standard curve to read off formaldehyde concentrations for liver samples.
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