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Table of Contents:
FHSB 1214 Biology I
FHSC 1214 Cell Biology
Experiment Description Page
Introduction Writing of Lab Reports 5
Practical 1 Cell Biology Studies I
Practical 1 Biological molecules I
Identification of Biomolecules 13
Practical 2 Cell Biology Studies II
Practical 2 Biological molecules II
Identification of Unknown Carbohydrate Solutions and
Investigation of Action of Saliva and HCl in Carbohydrate Solution
at Two Different Temperatures
20
Practical 3 Cell Biology Studies III
Practical 3 Enzyme studies I (Experiment 1) Optional: Practical
3 Enzyme studies I (Experiment 2)
Investigation of the Effects of Catalase Concentration on
Hydrogen Peroxide Decomposition Synthesis of Starch Using an Enzyme
Extracted from Potato Tuber
24
27
Practical 4 Cell Biology Studies IV
Practical 4 Enzyme studies II
Investigation of the Effects of Different Catalytic Conditions
on Hydrogen Peroxide Decomposition
29
Practical 8 Cell Biology Studies VIII
Practical 5 Cell studies I
Microscopy 32
- Practical 6 Cell studies II
Extraction of Cell Organelles by Cell Fractionation
47
Practical 5 Cell Biology Studies V
Practical 7 Cell studies III
Determination of Solute Potential of Potato Cell Sap
54
Practical 6 Cell Biology Studies VI
Practical 8 Cell studies IV
Effects of Different Treatments on Stained Potato Cells
64
Practical 7 Cell Biology Studies VII
Practical 9 Energetics I
Respiration of Germinating Beans
67
Practical 9 Cell Biology Studies IX
- Microscopic Examination of Cells at Various Stages of Plant
Mitosis and Meiosis
71
Practical 10 Cell Biology Studies X
- DNA, Mitosis and Meiosis Modelling 89
- Practical 10 Energetics II
Respiration of Yeast
93
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Important rules on tests and lab assignments Details:
If a student fails to submit an assignment or misses a test, the
lecturer will NOT remind you to submit a new assignment nor to sit
for a replacement test. The replacement test will be announced to
everyone in general and not to individual absentees. Those who are
supposed to attend must turn up and will not be reminded. It will
be conducted at the end of the semester on a different topic
(usually more difficult) when all students are so busy with tests
and assignments.
It is the responsibility of the student who misses a graded/full
report (with valid reasons) to submit a replacement report, which
is based on a different experiment than the one carried out for the
graded/full report.
If a submission is done online, a minimum of 7 days are given to
submit your assignment. As such, no excuses will be entertained if
theres a server/ IT failure or technical problems with your UTAR
account. Hence, you have an option to submit your assignment on day
1 to be safe, or on day 7 to be stupid. You may submit a
replacement report upon the approval of the practical lecturer.
However, it is NOT the responsibility of the lecturer to remind you
about it.
I acknowledge reading the above & agree to be bound by terms
therein. Your signature: ___________________ Name: Student ID:
Date:
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How YOU can do well in BIOLOGY
Follow the 4As and you can expect As. ttitude
Attend ALL lectures, tutorials and practicals on time without
fail.
Be attentive in class and revise your notes after class while
the topic is still fresh in your mind. Why waste time re-reading
2-3 months later?
Do your assignments faithfully as they carry marks for the
finals.
Come prepared for lessons (i.e. read up beforehand).
Read up beforehand before attending lectures so that you wont be
lost and wasted hours of your life week after week.
Why stress yourself out if you can avoid it? Do NOT count on
last minute revision for tests and examinations, as it will be too
late to catch up and seek help in areas where you may find
confusing or unclear of.
Why panic before exams because you cant find this or that? Keep
separate files for lecture, tutorial and practical. File up the
respective notes systematically so that you do not lose them along
the semester.
Do you expect the lecturer/ tutor to be available all the time
to answer your questions? It is YOUR responsibility to take the
initiative to clear your doubts or satisfy your curiosity to
understand certain scientific phenomena by reading up on the
relevant topics.
ttendance for lectures, tutorials and practicals
Lectures, tutorials and practicals carry marks that count
towards your finals.
You are expected to be present at ALL lectures, tutorials and
practicals.
Absence from any lesson must be accompanied by a photocopy of
your medical certificate presented to your lecturer/ tutor at your
next meeting.
If you know in advance that you will not be able to attend the
practical for a particular week, you are expected to inform your
tutor latest by the Friday before the affected week.
A
A
Based on a true story A professor at the National University of
Singapore recounts how on one occasion a student consulted him days
before the exam. Student: Prof, could you explain this page to me
please? Professor: What dont you understand about this page?
Student: EVERYTHING. Professor: But I already went through this
during lecture. Student: Oh, I didnt attend most of the lectures
actually. As for the next page, could you explain this page to me
please? ... and this page too and that too Prof: Im sorry, I cant
help you. Student: (Hmmmph, HES so selfish. Hey, I paid to study
here!) What do YOU think?
If the student failed, whose fault was it?
Was this student clever in skipping lectures?
Was it fair for the student to make demands on the lecturers
precious time to answer his questions?
How would the student have benefited himself if he looked up
books and other sources of information for himself first?
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ssignments
Use proper A4 foolscap for all handwritten assignments.
Write neatly and legibly in blue or black ink. Your tutor
reserves the absolute right to reject your assignment and ask you
to re-do the assignment should he/she consider it to be below the
expected quality.
Submit your assignment on time. Late submissions may entail mark
deduction or not be graded at all.
ssessments
ALL academic tests and examinations help prepare you better for
the finals.
As such, to sit for them all is not only compulsory, but
beneficial. After sitting for one, youll just want to sit for
another, and another, and another
Absence from tests and examinations MUST be covered by a medical
certificate, or will be considered to have failed the tests.
A
A
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Introduction Writing of Lab Reports
hy should I bother writing lab reports in the correct way? The
Foundation Programme is designed to prepare you for undergraduate
studies at UTAR which will require the writing of lab reports all
years generally. At the end of your third
year, you may have an opportunity to work on scientific projects
which will culminate in an official scientific report. Depending on
the quality of your report, the golden chance remains of publishing
your report in a scientific journal. Such recognition may open
doors of opportunity (e.g., strengthen application for scholarships
and further studies etc.). Science professors are evaluated in most
parts of the world by the papers they write. Format of a lab report
Your lab report should be preceded by a cover page which contains
the following:
Name
Partners name Group
Date
Program
Unit code
Unit description
Year and semester of study
Title of lab report
Lecturers name Example:
W
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Your lab report should contain the following sections:
Title
Objective
Apparatus
Materials
Procedure
Results/ observations
Discussion with citations
Conclusion
References
The following guidelines on report writing are those required by
the actual internationally-recognized scientific community. The
text in quotation marks in the following section is taken from
Warren D. Dolphin of Iowa State University. Credit has been given
to the author by citing the source. This is good practice as
opposed to plagiarism, in which copied material is claimed as the
possession of the copyist.
1 Apparatus, materials and procedure As the name implies, the
materials and procedure used in the experiments should be reported
in this section. The importance in writing this section is to
provide enough detail for the reader to understand the experiment
without overwhelming him or her. When procedures from a lab book or
another report are followed exactly, simply cite the work, noting
that details can be found in that particular source. However, it is
still necessary to describe special pieces of equipment and the
general theory of the assays used. This can usually be done in a
short paragraph, possibly along with a drawing of the experimental
apparatus. Generally, this section attempts to answer the following
questions:
1. What materials were used? 2. How were they used? 3. Where and
when was the work done? (This question is most important in field
studies.)
2 Results and observations Results The results section should
summarize the data from the experiments without discussing their
implications. The data should be organized into tables, figures,
graphs, photographs, and so on. But data included in a table should
not be duplicated in a figure or graph. All figures and tables
should have descriptive titles and should include a legend
explaining any symbols, abbreviations, or special methods used.
Figures and tables should be numbered separately and should be
referred to in the discussion by number, for example:
Figure 1 shows that the activity decreased after five
minutes.
The activity decreased after five minutes (fig. 1). Figures and
tables should be self-explanatory; that is, the reader should be
able to understand them without referring to the text. All columns
and rows in tables and axes in figures should be labelled. This
section of your report should concentrate on general trends and
differences and not on trivial details. Many authors organize and
write the results section before the rest of the report.
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2.1 Recording Qualitative Data Qualitative experiments include
those that require observations of non-quantifiable data such as
observations of colour, slides and whole specimens. Below are
guidelines on reporting a segment of qualitative experiments.
Liquid in container: Be careful to distinguish accurately among
solution, suspension and emulsion. It is your responsibility to
look up the definitions as studied in secondary school.
KI solution was added to the starch suspension
emulsion of lipid droplets in water Amount of light penetrating
solution Be careful to distinguish accurately among clear,
cloudy/murky and milky. It is your responsibility to look up the
definitions as studied in secondary school. Colour Some
descriptions of colour are unacceptable as they are ambiguous.
Light/pale brown, instead of beige
Murky/ cloudy white, instead of milky If theres a change in
colouration, you may choose to report as follows.
The initial blue colouration of the solution turns green, then
yellow and may finally appear brick red.
If the transition cannot be easily seen, at least state the
initial and final colours. If there is no change, one must state
the colour (e.g., it remained blue). It is incomplete to only
report there was no colour change without at least recording the
initial colour. Precipitate One should comment on the precipitate
colour and relative quantity. To do so, the mixture must be left to
settle.
Colour of precipitate - green, yellow, brick red precipitate
Amount of precipitate - a little, moderate amount, abundant
Example: When describing observations involving Benedicts test, one
should report that when one shakes the test tube containing
Benedicts solution and precipitate, the entire mixture will take
the colour of the precipitate. This colour upon shaking is recorded
and also the amount of light penetrating solution (transparent/
translucent/ opaque).
Moderate amount of brick red precipitate suspended in solution,
which bore a tinge of blue. Solution was milky.
Note: Particles cannot be regarded as precipitate. (e.g.
groundnut particles in water.)
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2.2 Recording Quantitative Data Quantitative experiments include
those that require observations of quantifiable data such as time,
quantity, weight, etc.
Tabulation and graphing
There are two categories of data normally used in reporting
quantitative results raw data and processed data. Raw data refers
to the readings obtained from measurements (e.g., length, weight,
height, quantity, etc.).
The table must be accompanied by the following features:
Informative table title
Gridlines
Columns/ rows with appropriate headings and units (units and
calculations should not be in the table body)
All processed data related to and required for plotting graph
must be shown in the table. E.g. Averages, rate of yeast
respiration in terms of no. of bubbles formed per minute.
Precision and decimal places: One must express data according to
the precision afforded by the instrument. E.g., if the instrument
can weigh an item as light as 0.1 g, then do not record it as 0.10
g, so as to correctly reflect the precision of the instrument. Note
that the decimal places in the table must be the same for the same
unit of measurement, and reflect the precision of the instrument.
If a measurement unit is converted to percentage or any other unit,
one is not bound by the precision of the instrument. However, the
recording should maintain a consistent and reasonable use of the
number of decimals (e.g., avoid too many decimals 88.8888888 %).
Note that the table and graph below feature such consistency of
decimal places.
Precision of processed data can be presented in the following
manner:
Averages calculated should follow the decimal places of the raw
data.
Processed data involving summation and/ or subtraction should
follow decimal places of the raw data.
Decimals arising from processed data involving multiplication
and/ or division should be reasonable (e.g., not unnecessarily
long).
Sample table: Title: Mass of precipitate of standards at various
concentrations of glucose solutions.
Precipitate mass (g)
Glucose concentration (%)
Reading 1 Reading 2 Reading 3 Ave.
4 0.1 18.6 18.4 18.7
2 8.2 9.3 9.0 8.8
1 5.2 4.5 4.8 4.8
0.5 2.3 1.8 2.1 2.1
0.1 0.4 0.3 0.4 0.4
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Graph
Plot a graph that will show the trend of the investigation.
Include the following in the plotting of graph:
Informative title
x-axis : labelled, including units (independent variables)
y-axis : labelled, including units (dependent variables)
appropriate scale used
points plotted
centroid point
Shape of graph can only be drawn using pencil, blue and black
ink pen
points plotted according to table of data
best fit line/ curve
Sample graph:
Note: The line of the plot does not go beyond the concentrations
used (no extrapolation of points).
Hence, one should not extrapolate otherwise it is a claim that a
certain y value is predicted for a certain concentration.
Avoid clashing headings with clashing units (e.g., headings with
two different units gram eggs vs. gram nutrients per gram plain
feed)
Mass of eggs laid in a week (g)
Amount of nutrients (g/ g plain feed)
0.30
0.25 0.20 0.15 0.10 0.00
Mean 78.0 74.0 69.3 62.7 59. 7 58.0
Average mass of precipitate of standards at various
concentrations of glucose solutions
0
2
4
6
8
10
12
14
16
18
20
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Concentration of glucose solution (%)
Ave. precipitate mass (g)
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2.3 What if I do not obtain desired results? For the purpose of
your UTAR lab report, if you dont obtain the desired results, just
record them as they are. By right, you should repeat it however,
you may be constrained by a limited amount of supplied solutions in
the UTAR lab and time. Hence, if your repeats involve consuming
more solutions, please ask your tutor first. You may put a footnote
concerning the expected results. In your discussion, be sure to
explain the possible reasons for the anomaly.
3 Discussion with citations This section should not just be a
restatement of the results but should emphasize interpretation of
the data, relating them to existing theory and knowledge.
Speculation is appropriate, if it is so identified.
Start your discussion with a brief summary of the experiment. Be
careful not to repeat the procedure here. This summary should
briefly introduce the readers about the experiment.
This brief summary should be then followed by a brief
introduction on the biological theory behind the experiment.
Explain how the independent variables affected the dependent
variables, you may use equations provided and show the
dependent/independent variables.
Do not include all the list of each and every number on the data
sheet. Suggestions for the improvement of techniques or
experimental design may also be included here. In writing this
section, you should explain the logic that allows you to accept or
reject your original hypotheses. You should also be able to suggest
future experiments that might clarify areas of doubt in your
results. When citing references in the text, do not use footnotes;
instead, refer to articles by the author's name and the date the
paper was published.
Fox in 1988 investigated the hormones on the nest-building
behavior of catbirds.
Hormones are known to influence the nest-building behavior of
catbirds (Fox, 1988).
When citing papers that have two authors, both names must be
listed. When three or more authors are involved, the Latin et al.
(et alia) meaning "and others" may be used. A paper by Smith,
Lynch, Merrill, and Beam published in 1989 would be cited in the
text as:
Smith et al. (1989) have shown that... This short form is for
text use only. In the References, all names would be listed,
usually last name preceding initials.
3.1 General Comments on Style 1. All scientific names (genus and
species) must be italicized. Underlining indicates italics in a
typed paper. 2. Use the metric system of measurements.
Abbreviations of units are used without a following
period.
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3. Be aware that the word data is plural while datum is
singular. This affects the choice of a
correct verb. The word species is used both as a singular and as
a plural. 4. Numbers should be written as numerals when they are
greater than ten or when they are
associated with measurements
6 mm or 2 g
two explanations of six factors.
When one list includes numbers over and under ten, all numbers
in the list may be expressed as numerals; for example,
17 sunfish, 13 bass, and 2 trout. Never start a sentence with
numerals. Spell all numbers beginning sentences.
5. Be sure to divide paragraphs correctly and to use starting
and ending sentences that indicate the purpose of the paragraph. A
report or a section of a report should not be one long
paragraph.
6. Every sentence must have a subject and a verb.
7. Avoid using the first person, I or we, in writing. Keep your
writing impersonal, in the third
person. Instead of saying, "We weighed the frogs and put them in
a glass jar," write, "The frogs were weighed and put in a glass
jar."
8. Avoid the use of slang and the overuse of contractions.
9. Be consistent in the use of tense throughout a paragraph--do
not switch between past and
present. It is best to use past tense. 10. Be sure that pronouns
refer to antecedents. For example, in the statement, "Sometimes
cecropia caterpillars are in cherry trees but they are hard to
find." Does "they" refer to caterpillars or trees?
After writing a report, read it over, watching especially for
lack of precision and for ambiguity. Each sentence should present a
clear message. The following examples illustrate lack of
precision:
"The sample was incubated in mixture A minus B plus C." Does the
mixture lack both B and C or lack B and contain C?
"Protection against Carcinogenesis by Antioxidants" The title
leaves the reader wondering whether antioxidants protect from or
cause cancer.
The only way to prevent such errors is to read and think about
what you write. Learn to reread and edit your work.
Identify trends/ patterns by in words the trend shown in the
graph. Remember to make reference to the values shown on the graph.
Explain all the observations or trend obtained during the
investigation.
As temperature increases from 25 oC to 50OC, rate of yeast
respiration/ mean number of bubbles formed per 3 mins. increases
proportionately/ linearly from 7 to 28.
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In summary, the discussion should be correctly applying the
theoretical concept involved in the experiment.
4 Conclusion State the general trend obtained through the
investigation and provides a concise conclusion about the
investigation. Conclusion should be an attempt to answer the
experimental objective.
5 References This section lists all articles or books cited in
your report. It is not the same as a bibliography, which simply
lists references regardless of whether they were cited in the
paper. The listing should be alphabetized by the last names of the
authors. Different journals require different formats for citing
literature. For articles: Fox, J.W. 1988. Nest-building behavior of
the catbird, Dumetella carolinensis. Journal of Ecology 47: 113-17.
For Books: Bird, W.Z. 1990. Ecological aspects of fox reproduction.
Berlin: Guttenberg Press. For chapters in books: Smith, C.J. 1989.
Basal cell carcinomas. In Histological aspects of cancer, ed. C.D.
Wilfred, pp. 278-91. Boston: Medical Press. For electronic
resources:
For web page with personal author
Irving, I. (2009, August 25). Crime, punishment and poverty in
the United States. Retrieved from
http://ideas.repec.org/p/dal/wparch/uspov.html/
For web page with corporate author
U.S. Food and Drug Administration. (2009). Smoking cessation
products to help you quit. Retrieved from
http://www.fda.gov/hearthealth/riskfactors.html/
For web page without author & without date
Perceptions of university student leadership and achievement
(n.d.). Retrieved from http:/www.cc.edu/user_surveys/1998-10/
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Practical 1 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)
Identification of Biomolecules
______________________________________________________________________
Objective: To identify the biomolecules in a solution using various
food tests and state the justifications. Introductory
instructions:
You may perform this experiment in groups of 2-3. Important
notice: Any heating that has to be done in the following tests
should be carried out in a water bath at 95oC. Direct heating of
test-tubes should not be taking place. Apparatus & Equipments:
Test tubes Test tube rack Boling tubes Wooden holder Water bath,
95oC Spatula Materials: Iodine 0.1 M hydrochloric acid Sudan III
Starch solution Corn oil Egg albumin 1% copper sulphate
solution
1% sucrose solution 0.1 M Sodium hydroxide 1%
glucose/fructose/lactose solution Absolute ethanol DCPIP
(dichlorophenolindophenol) solution Ascorbic acid
Introduction The nutrients in the food you eat supply your body
with energy for growth and repair. These principle substances
include carbohydrates, proteins, fats, minerals and vitamins. We
can test for the presence of these important compounds in food by
using chemical reagents that react in predictable ways in the
presence of these nutrients. Please refer to the notes given above
on:
How to record qualitative data. (Marks will be awarded based on
proper recording.)
What to do if you dont obtain the desired results. Flowchart
Students will be allowed to proceed with the experiment only if
they have come into the laboratory with a flowchart of the days
experiment.
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Procedures: Part 1: Identification of Carbohydrates (A) Test for
reducing sugars
The reducing sugars include all monosaccharide, such as glucose
and fructose, and some disaccharides, such as maltose and lactose,
using 0.1 1% sugar solutions. Common tests for reducing sugars
include Benedicts test (described below) and Fehlings test (not
described here). See basis of test below for explanation of the
following reaction:
Benedicts test for reducing sugars:
Procedure* Basis of test Observation
Reducing sugar test
Add 2 cm3 of any one solution of the reducing sugar(s) provided
into a boiling tube. Add an equal volume (2 cm3) of Benedicts
solution into the same boiling tube. Using a wooden holder, shake
and heat the mixture in 95C water bath for one minute, shaking
continuously to minimize spitting.
Benedicts solution contains copper sulphate. Reducing sugars
reduce soluble alkaline blue copper sulphate containing copper (II)
ions, Cu2+ to insoluble red-brown copper oxide containing copper
(I). The latter is seen as a precipitate.
[Note: report after shaking and after contents settle down; see
introduction pg. 7]
*: Please do NOT remove measuring cylinder or any other item
from the stations provided. Observe and report characteristics of
tube contents before and after precipitate settles to bottom of
tube, taking note of liquid, colour and precipitate. (B) Test for
non-reducing sugars
The most common non-reducing sugar is sucrose, a disaccharide.
If reducing sugars have been shown to be absent (negative result
for test (A)), a brick-red precipitate in the test below indicates
the presence of a non-reducing sugar. If reducing sugars have been
shown to be present, a heavier precipitate will be observed in the
following test than with the reducing test if non-reducing sugar is
also present. The proper procedure to test for the presence of an
unknown carbohydrate sample containing non-reducing sugars
involves:
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First test for reducing sugars: Benedicts test on the unknown
fresh sample Why is this step necessary? What results will one get
which will cause this step to be called a negative test?
Second test for reducing sugars: Benedicts test on the
acid-hydrolysed unknown sample What results will one get which will
cause this step to be called a positive test?
Procedure* Basis of test Observation
Non-reducing sugar test
Add 2 cm3 of fresh sucrose solution into a boiling tube. Add 1
cm3 of 0.1 M hydrochloric acid. Using a wooden holder, heat the
mixture at 95C for one minute. Carefully neutralize the mixture
with equal volume (1 cm3) of 1 M sodium hydroxide. Finally, add an
equal volume (4 cm3) of Benedicts solution to the acid-hydrolysed
sugar solution. Using a wooden holder, shake the mixture
continuously to minimize spitting while heating at 95C for one
minute.
A polysaccharide or disaccharide can be hydrolyzed to smaller
component constituents by boiling with 0.1 M hydrochloric acid.
Sucrose is hydrolyzed to glucose and fructose, both of which are
reducing sugars and give the reducing sugar result with the
Benedicts test.
[Note: report after shaking and after contents settle down; see
introduction pg. 7]
Additional Information The mixture is likely to bump violently
during heating and extra care should therefore be taken. The test
is semi-quantitative, that is, a rough estimation of the amount of
reducing sugar present will be possible. The final precipitate will
appear green to yellow to orange to red-brown with increasing
amounts to reducing sugar. The initial yellow colour blends with
the blue of the copper sulphate solution to give the green
colouration. Is the precipitate that of reducing sugar or copper
oxide?
*: Please do NOT remove measuring cylinder or any other item
from the stations provided. Observe and report characteristics of
tube contents before and after precipitate settles to bottom of
tube, taking note of liquid, colour and precipitate.
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(C) Test for starch Starch is only slightly soluble in water, in
which it forms a colloidal suspension. It can be tested as a mainly
solid in suspension.
Procedure* Basis of test Observation
Iodine test
***Note: The starch prepared for you is already cooked starch.
Add a few drops of 1% cooked starch solution on a white tile. Add a
few drops of I2/ KI solution (iodine). Be sure to mix them together
on the tile with a glass rod.
A polyiodide complex is formed with starch.
*: Please do NOT remove measuring cylinder or any other item
from the stations provided. Part 2: Identification of Lipids Lipids
include oils (such as corn oil and olive oil), fats and waxes.
Procedure* Basis of test Observation
Sudan III
Sudan lll is a red dye. Add 2 cm3 of oil to 2 cm3 of distilled
water in a test tube. Add a few drops of Sudan III and shake.
Fat globules are stained red and are less dense than water.
[***Note: report after shaking and after contents settle
down]
Emulsion test
Add 2 cm3 fat or oil to a test tube containing 2 cm3 of absolute
ethanol. Dissolve the lipid by shaking vigorously. Add 4 cm3 volume
of distilled water.
Lipids are immiscible with water. Adding water to a solution of
the lipid in alcohol results in an emulsion of tiny lipid droplets
in the water which reflect light and give a white, cloudy/milky
appearance.
[***Note: report after shaking and after contents settle
down]
*: Please do NOT remove measuring cylinder or any other item
from the stations provided.
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Part 3: Identification of Proteins A suitable protein for these
tests is egg albumin.
Procedure* Basis of test Observation
Millons Test
Add 2 cm3 of protein (albumin) solution into a boiling. Add 1
cm3 of
Millons reagent. Using a wooden holder, heat the mixture at 95C
for one minute. Millons reagent is poisonous: be extremely
careful!
Millons reagent contains mercury acidified with nitric acid,
giving mercury (II) nitrate and nitrite. The amino acid tyrosine
contains a phenol group which reacts to give a red mercury (II)
complex. This is a reaction given by all phenolics and is not
specific for proteins. Protein usually coagulates on boiling. Thus
appearing solid. The only common protein lacking tyrosine likely to
be used is gelatin.
Biuret Test
Add 2 cm3 (albumin) protein solution into a test tube. Add an
equal volume (2 cm3) of 5% sodium hydroxide solution and mix. Add 2
drops of 1% copper sulphate solution and mix. No heating is
required.
A test for peptide bonds. In presence of dilute copper sulphate
in alkaline solution, nitrogen atoms in the peptide chain form a
purple complex with copper (II) ions, Cu2+. Biuret is a compound
derived from urea which also contains the CONH group and gives a
positive result.
*: Please do NOT remove measuring cylinder or any other item
from the stations provided.
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Part 4: Identification of Vitamin C (ascorbic acid) ***Note: If
more than 5 drops of ascorbic acid are required to turn DCPIP to
colourless, please dilute the latter significantly. This test can
be conducted on a quantitative basis if required, in which case the
volumes given below must be measured accurately. A suitable source
of vitamin C is a 50/50 mix of fresh orange or lemon juice with
distilled water. Vitamin C tablets may also be purchased.
Procedure* Basis of test Observation
DCPIP test
Using 0.1% ascorbic acid solution as a standard. Add 1 cm3 of
DCPIP solution to a test-tube. ***Add the 0.1% ascorbic acid to the
DCPIP drop by drop until it becomes approximately colourless (or by
stirring gently if youre provided with a syringe needle/ glass
rod). Note the no. of drop(s) of ascorbic acid solution used.
DCPIP is a blue dye which is reduced to a colourless compound by
ascorbic acid, a strong reducing agent.
Additional Information Shaking the solution would result in
oxidation of the ascorbic acid by oxygen in the air. The effects of
shaking and of boiling could be investigated.
*: Please do NOT remove measuring cylinder or any other item
from the stations provided.
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REPORT GUIDELINES Results/ observation: Tabulation of
qualitative data 1. Tabulate your observations above for each
biochemical food test executed,
according to the guidelines provide in the introduction on
writing lab reports. Note: The table in the lab manual for this
task is not presented correctly.
2. Wrong results are alright for this experiment. 3. No need to
write procedure, basis of test, discussion or conclusion. 4. You
may choose to construct one or more tables. 5. For tests involving
carbohydrates, observe and report characteristics of tube
contents before and after precipitate settles to bottom of tube,
taking note of liquid, colour and precipitate as above.
o Liquid mixture, solution, suspension, emulsion? clear,
cloudy/murky, milky?
o Colour state initial and final colours?
o Precipitate (if any) colour of precipitate? amount of
precipitate?
Discussion: 1. How could you determine the concentration of
ascorbic acid in an unknown sample?
2. You are provided with three sugar solutions. First one
contains glucose, second one
is a mixture of glucose and sucrose, and lastly is sucrose
solution. (a) How could you identify each solution? (b) Supposing
that the apparatus were available, and time permitted, briefly
discuss
any further experiments you could perform to confirm your
results. 3. After carrying out Benedicts test, a student concludes
that the obtained positive results prove that glucose is present.
True or false? Provide a reason. 4. After carrying out Benedicts
test, a student identifies the coloured precipitate as reducing
sugar. True or false? Provide a reason. 5. A student pours
Benedicts solution into a tube containing a carbohydrate. No colour
change is obtained. The student concludes that the carbohydrate is
not a reducing sugar. True or false? Provide a reason. 6. A student
adds acid to a solution of sucrose followed by neutralization and,
finally, Benedicts test. A negative (instead of positive) result is
obtained. Explain why. 7. Why does sucrose yield positive results
after carrying out the non-reducing sugar test? What are the
components of sucrose?
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Practical 2 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)
Identification of Unknown Carbohydrate Solution and Investigation
of Action of Saliva and Hydrochloric Acid in Carbohydrate Solution
at Two Different Temperatures
______________________________________________________________________
Objective: Students are expected to state the objective of this
experiment. Introductory instructions:
You may perform this experiment in groups of 2-3. Apparatus
& Equipments: Boiling tubes Metal test tube racks Water bath,
~37-40oC Water bath, ~90-95oC Beaker Dropper Wooden holder
Materials: Carbohydrate solution A Carbohydrate solution B
Benedicts solution 3 M Hydrochloric acid 3 M Sodium hydroxide
Flowchart Students will be allowed to proceed with the experiment
only if they have come into the laboratory with a flowchart of the
days experiment. Procedures: This experiment is to be done in
pairs. To avoid congestion, each pair should collect the following
before beginning the experiment:
4 ml NaOH
18 ml Benedicts Solution 2 ml Solution A
10 ml Solution B
8 ml HCl
1 dropper
5 ml measuring cylinder (to be washed with distilled water each
time before reuse)
Metal test tube racks Overview Please see tables 1 & 2 on
the next page to get a rough idea of what is required in the
experiment. Can you identify in the instructions that follow, how
the tubes are to be placed under various temperatures and time
periods? Carry out your investigation as follows. 1. Prepare two
boiling tubes containing 1 ml solution A and 1 ml solution B
respectively.
Add 1 ml Benedicts solution into each test tube. Heat both tubes
together in the hotter (~90-95oC) water bath for two minutes.
Record the results in table 1.
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2. Add a few drops of fresh solution A and B separately spaced
on a white tile. On each solution, add 1-2 drops of I2/KI solution
(iodine). Be sure to mix them together on the tile with a glass
rod. Record your observations in the Table 1.
3. Pipette 2 ml solution B into each of four test-tubes and,
label the tubes 1, 2, 3 and 4 respectively with labelling paper (or
masking tape) near mouth of tube. Write the initials of your group
name or individuals.
4. Place tubes 1 and 2 in a water bath of ~37o (it doesnt matter
how long you put it in at this stage as no saliva or HCl have been
added yet).
5. Salivate into a measuring cylinder till it reaches 5 ml.
6. Ensure that the following two steps (6 and 7) adding of
saliva or HCl into the
respective tubes (mentioned in the next sentence and below) is
done approximately at the same time. (Why is this necessary?)
7. Pipette 2ml of saliva each into 1 and 4. Shake the contents
of the tubes well to ensure
thorough mixing. 8. Measure 4 ml HCl and pipette 2 ml each into
tubes 2 (already in water bath of ~37oC)
and 3. Place tubes 3 and 4 in a water bath set at 95 oC. Let
tubes 1, 2 (already in water bath of ~37oC), 3 & 4 (recently in
water bath of ~95oC) incubate at their respective temperatures (see
Table 2) for 35 minutes from this moment.
9. Label 4 more new tubes (either test tubes or boiling tubes)
as follows: 1, 2, 3 and
4. After 5 minutes of incubation of tubes labelled 1 to 4
prepared previously, pour out about half of the total volume of the
contents from all these tubes into the respective newly labelled
test tubes (e.g., 1 into 1, 2 into 2 etc.). Straightaway, place
back the original tubes (labelled 1-4) back into the respective
temperatures of incubation.
10. Neutralize the acid in each of tube labelled 2 and 3 with
1ml of sodium hydroxide (each). Shake each tube (2 and 3) to ensure
uniform mixing.
11. To carry out Benedicts test, add an equal volume of
Benedicts solution (2 ml) for
each tube. Using a wooden holder, shake and heat at 95C for one
minute, shaking continuously to minimize spitting. Record your
observations in Table 2.
12. After 35 minutes of incubating tubes 1 to 4, neutralize the
acid in each test tube
labelled 2 and 3 with 1ml of sodium hydroxide. (Why is
neutralization necessary?) Remove 2ml of solution from each tube 1
to 4 and carry out Benedicts test with an equal volume of Benedicts
solution (2 ml) for each tube. Remember to heat your sample (please
see previous steps). Record your observations in Table 2.
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REPORT GUIDELINES Results/ observation: Table 1: (title)
Observations Conclusions
Solution A
Benedicts test: Iodine test:
Absence/presence of what type of carbohydrate?
Solution B
Benedicts test: Iodine test:
Absence/presence of what type of carbohydrate?
Table 2: (title)
Tube Contents Temp (C)
Benedicts TestColour Observation
After 5th min (from tubes 1 4
into 1 4)
After 35th min (tubes 1 4)
1 2 ml solution B 2 ml saliva
37
2 2 ml solution B 2 ml 3 M HCl
37
3 2 ml solution B 2 ml 3 M HCl
95
4 2 ml solution B 2 ml saliva
95
For Benedicts test and Iodine tests, please follow lab manual
guidelines for students on writing lab report on the following:
o Liquid Solution, suspension, emulsion? Clear, cloudy/murky,
milky?
o Colour State initial and final colours?
o Precipitate Colour of precipitate? Amount of precipitate?
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Discussions: 1. Discussion should contain:
1) Name of the enzyme involved 2) Specific action(s) of enzyme
involved 3) Effect of HCl on Solution B 4) Effect of temperature on
saliva and/ or solution B 5) Product:
a. Identification (make suggestion(s)/ educated guesses) b.
Structure (e.g., chemical classification etc.)
6) Bases of chemical test(s) used 7) Which carbohydrate is more
complex, A or B? Give a reason.
Conclusion: How do saliva, HCl and temperature affect the
carbohydrate?
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Practical 3 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)
Investigation of the Effects of Catalase Concentration on Hydrogen
Peroxide Decomposition
Objective: To investigate the effects of different catalase
concentration on the decomposition of hydrogen peroxide.
Introductory instructions:
You may perform this experiment in groups of 2-3. Apparatus and
Materials: 5 test or boiling tubes Scalpel/ pen knife 1 beaker
(500cm3) White tile 1 beaker (250cm3) Mortar and Pestle 4 test
tubes Weighing boat 1 rubber bung with delivery tube Measuring
cylinder 1 Retort Stand (optional) 4 filter funnel and filter paper
(optional) 1% hydrogen peroxide solution ** Potato Water bath 37C
**Caution: Hydrogen peroxide is formed continuously as a by-product
of chemical reactions in living cells; it is a very toxic
(poisonous) substance. Note to lecturer: This experiment may be
done together with Experiment 2 if the lab session is 3 h long.
Introduction: Enzymes are proteinaceous molecules that speed up
chemical reactions within living systems. In this experiment, the
effect of catalase on hydrogen peroxide is investigated. Catalase
is an enzyme present in the cells of plants, animals and aerobic
(oxygen requiring) bacteria. It promotes the conversion of hydrogen
peroxide, a powerful and potentially harmful oxidizing agent, to
water and molecular oxygen.
2H2O2 + catalase 2H2O + O2
Warning: H2O2 is corrosive. For the person handling, please wear
gloves. Flowchart Students will be allowed to proceed with the
experiment only if they have come into the laboratory with a
flowchart of the days experiment. Procedures: 1. Depending on the
size of the rubber bung holding the delivery tube, select either
one
boiling or test tube and label it as tube A.
2. Cut the potato and weigh 5g of potato using a weighing
boat.
3. Cut the 5g potato samples into smaller pieces (the smaller
the easier for you to mash) and mash the potato sample using the
mortar and pestle. Note: dont spend too much time on this.
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4. Add 6 cm3 of distilled water to the potato samples during the
mashing process.
5. Separate the solid mashed potato from the liquid either (i)
pouring the liquid into a test tube; or (ii) by filtering the
mashed potato sample (with filter paper and funnel) and collect the
filtrate in a test tube. Note: using filter paper and funnel is
more time-consuming.
6. Fill an empty test tube with tap water (see Figure 1).
7. Add 5cm3 of hydrogen peroxide into tube A.
8. Seal the end of the delivery tube furthest away from the
rubber bung with a piece of
parafilm.
9. Add 1cm3 of the filtrate from the mashed potato samples into
tube A. 10. Immediately close the test tube with a rubber bung that
has been connected with a
delivery tube. 11. Set up the apparatus as shown in Figure 1.
(if retort stand is provided; if not just use
each others hands).
Figure 1 Experimental Setup
12. Remove the parafilm and immediately immerse the tube
containing the mixture in a
beaker containing 37C water.
13. Start your watch and count the number of gas bubbles
produced for 2 minutes and record it. After you finish, return the
water you took back to the electric water bath. [Note: water can
maintain the heat in it for quite some time.]
14. Get a 2nd measurement by disposing the contents of tube A to
repeat step 7 to 13. After you finish, return the water you took
back to the electric water bath.
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15. Repeat the experiment with 10g of potato, then 15g and
finally 20g (optional, depends on time available).
16. Record the data in Table 1. Calculate the averages in order
to plot graphs. REPORT GUIDELINES Results/ observation: 1)
Tabulation of quantitative data Table 1: (title)
(What heading should you write here?)
5g 10g 15g *20g
Number of Attempt
1st 2nd *3rd 1st 2nd *3rd 1st 2nd *3rd *1st *2nd *3rd
Number of gas bubbles produced
*Optional, depends on time available. 2) Graphing of
quantitative data Present your graph (pasted from Excel) of the
average number of bubbles produced against potato samples used. Use
a best fit curve. Discussion:
From the data you have collected in the practical, account fully
for the results which you have obtained. Discuss any anomalous
data/ results that you might have. Explain the trend or pattern of
the graph.
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Practical 3 Experiment 2 (FHSC 1214 Cell Biology ONLY) Synthesis
of Starch Using an Enzyme Extracted from a Potato Tuber
______________________________________________________________________
Objective: To investigate which compounds the enzyme in the potato
can act on to produce starch (investigate three possible
substrates: glucose, maltose and glucose-1-phosphate). Introductory
instructions:
You may perform this experiment in groups of 2-3. Apparatus and
Materials: Centrifuge and centrifuge tubes Glucose-1-phosphate (2%)
Test tube rack Glucose solution (2%) Pestle and mortar Maltose
solution (2%) Knife Iodine solution Labelling paper (or masking
tape) Potato tuber Test tubes Measuring cylinder White tile
Procedures: Introductory instructions:
Create a flowchart before you enter the lab in order to
understand the steps in this experiment. Show this to your tutor
before starting the experiment.
Perform this experiment in pairs.
Take 5 ml iodine only when ready to begin the experiment.
Groups may have to take turns to centrifuge, depending on the
number of groups and holders in the centrifuge.
NOTE: After carrying out steps 1 to 2, proceed to Experiment 2.
Return to Experiment 1 only during the waiting periods of
Experiment 2.
A. Extracting the enzyme from potato tissue 1. Peel a
medium-sized half potato. Cut half of it into small cubes on a
white tile (the
smaller the easier for you to grind). Grind a few pieces of
potato cubes in a pestle and mortar with 20cm3 of water.
2. Separate the aqueous part of the extract from the solid as
best as possible. You can
do this by pouring it out while restraining the solids with your
fingers or an appropriate instrument. Divide the aqueous part of
the extract into two equal portions and pour them into two
centrifuge tubes. As far as possible, avoid letting sand and solid
matter to get into the tubes.
3. Spin the extracts in a centrifuge for ten minutes at 5000 rpm
so that the starch, cell
walls and other solid matter will settle at the bottom of the
centrifuge tubes. The starch-free liquid above the deposit, or
supernatant, should contain the enzyme.
4. Using a teat pipette, carefully, without disturbing the
deposit beneath, withdraw as
much the clear enzyme solution as possible from the centrifuge
tube. 5. To check whether this enzyme solution is starch-free,
transfer a few drops of it into a
test tube and add 2 drops of iodine solution onto it. If a blue
colour appears, then the potato extract would need to be
centrifuged again.
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B. Attempting starch synthesis 6. Label three clean test tubes
G, M and G1P, respectively. Use a separate teat pipette
and measuring cylinder in each case to place 3 cm3 of glucose
solution in the G tube, 3 cm3 of maltose solution in the M tube,
and 3cm3 of glucose-1-phosphate solution in the G1P tube.
7. To synthesise starch, pour 2 cm3 of the enzyme solution (the
liquid or supernatant you
obtained after centrifuging above) into the substrate tube (G, M
and G1P), mix well and note the time.
8. For each substrate, place 15 discrete drops of iodine
solutions on clearly labelled piece
of white tile. 9. After one minute of the reaction use a teat
pipette to place one drop of enzyme-
substrate solution onto one existing drop of iodine solution on
the white tile. Stir with a suitable object (e.g. woodsplint or
tooth pick) and record the colour produced. Repeat at intervals of
1 minute over 15 minutes, all the three tubes simultaneously.
REPORT GUIDELINES Discussion Discuss the following questions: 1.
Draw the structural formula of the substrates. What features of the
starch-synthesizing
substrate molecule might have been recognized by the
starch-synthesizing enzyme? 2. The synthesis of polymers such as
starch requires metabolic energy. What was the
energy source in the successful reaction? 3. The enzyme isolated
from potatoes is known as starch phosphorylase. In the intact
potato tuber it is also used to break down starch. How did
conditions in the test tube favor starch synthesis? In what
circumstances does the enzyme bring about starch synthesis in a
potato?
4. In plant leaves, starch accumulates in chloroplasts. The
synthesis of starch requires
ATP. Where do you think this ATP comes from?
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Practical 4 (FHSB 1214 Biology I & FHSC 1214 Cell Biology)
Investigation of the Effects of Different Catalytic Conditions on
Hydrogen Peroxide Decomposition
Objective: To investigate the catalytic effect at different
conditions on the hydrogen peroxide decomposition. Introductory
instructions:
You may perform this experiment in groups of 2-3. Apparatus
& Equipment: Beaker Boiling tubes & test tubes Water bath
(95oC) Pen knife/ blade Glass rod Parafilm Materials: Fresh Liver
Potato cubes Manganese dioxide 10% hydrogen peroxide** Wooden
splinters **Caution: Hydrogen peroxide is formed continuously as a
by-product of chemical reactions in living cells; it is a very
toxic (poisonous) substance. Flowchart Students will be allowed to
proceed with the experiment only if they have come into the
laboratory with a flowchart of the days experiment. Procedures: 1.
Label six clean empty boiling tubes as 1, 2, 3, 4, 5 and 6. 2. Cut
the provided liver into three pieces of roughly 0.8 cm x 0.8 cm x
0.5 cm. 3. Place one piece of liver into tube 1. 4. Place the
second piece of liver into tube 2. Place tube 2 in water bath
(95oC) for about
five minutes. Remove it from the water bath and let it cool. 5.
Put the third piece of liver into tube 3. Mash it gently into a
pulp with a glass rod.
6. Cut the potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm.
Place one cube into tube 4. 7. Measure two portions of 0.5 g
manganese dioxide powder. Pour each portion into tube
5 and tube 6.
8. Put tube 6 in the water bath (95oC) for five minutes. Remove
it from water bath and let it cool.
9. Prepare another six clean empty test or boiling tubes. Put 5
cm3 of hydrogen peroxide
into each of them.
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10. Quickly add hydrogen peroxide into tubes 1, 2, 3, 4, 5, and
6 respectively. **Step 9 and 10 are to be done quickly. 11. Stretch
the parafilm and seal the mouth of the boiling tubes quickly.
In order to prevent the parafilm from being displaced if a lot
of gas is produced, secure the parafilm covering the side of the
test or boiling tube with another piece of parafilm.
12. Record your observations in Table 1.
13. Leave the tubes for 20 minutes or till when you see quite a
lot of gas being produced in some boiling tubes as seen by the
bulging of parafilm from the test or boiling tube mouths.
14. Insert a glowing wooden splinter (flame extinguished but
glow remains) into each tube
one at a time by just penetrating the parafilm with it.
Why is it important to test each boiling tube at least without
too much difference in the duration of sealing among the tubes?
15. Again, record all your observations in Table 1. REPORT
GUIDELINES Results/ observations: Table 1: (title)
Test Tube
Contents with 5 cm3
hydrogen peroxide
Observations
Before inserting glowing splinter
After inserting glowing splinter
1
Fresh liver
2
Boiled liver (cooled)
3
Pulped liver
4 Potato cubes
5
Manganese dioxide (untreated)
6
Boiled manganese dioxide (cooled after heating)
[Note: be sure to use the following terms correctly: glowing
splinter glowed brighter, flame rekindled, effervescence (bubbles)
observed, reference to sound, etc.]
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Discussion: 1. What is the equation of the reaction observed? 2.
What plant or animal organelle is involved? 3. What effect does
pulping the liver have upon the reaction? Account for this. 4. What
effect does boiling the liver have upon the reaction? Account for
this (include
reference to enzyme structure (bonds, molecular motion, shape,
active site). 5. What were the differences between the reactions
with fresh liver and with fresh potato
cubes? Account for these differences (include reference to
enzyme structure (bonds, molecular motion, shape, active site)
6. What were the differences between the effects on the reaction
of boiling the liver and
heating the manganese dioxide? Account for these differences
(include reference to susceptibility (sensitivity) to heat, enzyme
shape, bonds etc).
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Practical 5 (FHSC 1214 Cell Biology); Practical 7 (FHSB 1214
Biology I) Microscopy
Experiment 1 Microscope and Its Uses Introductory
instructions:
Individual experiment Objective: To study the uses of microscope
and its maintenances. To learn microscopic techniques such as focus
the object with correct illumination under different power of
magnifications. Introduction: The microscope is a basic tool of the
biologist. It is a valuable precision optical instrument easily
damaged by careless usage. It is very important for the student to
become familiar with the parts of the microscope and the procedures
in the handling of it. Treat your microscope well and it will serve
you well. Apparatus and Materials: Binocular Microscope Cover slips
Microscope slide Newspaper Plastic millimeter ruler Setting up the
Microscope: The microscope when not in use is usually kept in a
case. Remove it by grasping the handle arm while placing one hand
under the base. Set it down gently on the laboratory table and at a
reasonable distance from the table edge. Always keep the microscope
upright in the vertical position and never touch any of the lens
surfaces with the fingers since it will deposit a thin film of oil
on the glass.
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Parts of the Microscope:
Component Function
Arm For lifting and carrying the microscope.
Base To provide stability.
Body tube To house the lenses.
Eyepiece or ocular lenses
This is a set of lenses that rests loosely at the top end of the
body tube. It is obvious that if the microscope is tilted while
being carried, the lens may fall out and be ruined. The
magnification of the eyepiece (given as 10X) is printed on the
metal part of the ocular.
Revolving nosepiece
Located at the lower end of the body tube, it carries 3
objectives of different lengths. Rotating this part changes the
magnification of the objectives.
Objective lenses They are of different magnifications with the
following visible properties:
Objectives Magnification Length Lens opening
Scanning lens 4x Shortest Widest Low power lens 10x short wide
High power lens 40x longest Narrowest
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Focusing adjustments
These comprise two knobs located on either side of the
microscope which are used to change the distance between the object
being viewed and the objective lens. Changing the distance
determines the focus. For the object to be viewed in focus under
high magnification, the lens must be much closer to the object than
when it is under low magnification.
Coarse adjustment
Made by the large knob beside the body tube for focusing under
low power magnification.
Fine adjustment Made by the small knob, which is for focusing
under high power magnification and accurate focusing.
Precautions when using the focusing adjustments: Turn both
adjustment knobs at the same time. Do not overturn the adjustment
knobs (i.e. do not force them to go beyond their limits) Do not use
the coarse adjustment knobs when focussing under the 40x objective
lens.
Stage This is the platform for slides and specimens to be viewed
under the microscope.
Mechanical stage This movable portion of the stage is attached
to the specimen holder and allows the slide to be moved in
different directions to facilitate viewing.
Specimen holder This holds the glass slide in place.
Vertical feed knob
Rotating this moves the glass slide in the vertical
direction.
Horizontal feed knob
This moves the glass slide in the horizontal direction.
Condenser Located just beneath the stage of the microscope, it
incorporates a lens which collects light on the stage to bear on
the object.
Iris diaphragm A rotating disk under the stage. This diaphragm
is used to vary the amount light that is projected upward into the
slide.
Built-in light source
This is situated below the iris-diaphragm to provide light for
illuminating the object. It can be switched on or off.
Brightness adjustment knob
This provides adjustment to the illumination brightness.
Main switch This ensures that power is turned on or off.
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Preliminaries before Use: 1. Use the coarse adjustment to raise
the body tube so that the objective can clear the
stage when the revolving nosepiece is turned. 2. Turn the
nosepiece until the scanning objective is in-line with the
eyepiece. You
should hear a soft click or else feel a distinct falling into
place as the objective moves into position. If not, the field of
view is totally dark or an illuminated crescent instead of a
complete circle.
3. Turn the diaphragm to its largest opening. 4. Look into the
eyepiece and make a final adjustment to the light adjustment
knob
so that the field of view (i.e., the lit circle which you see)
is evenly illuminated. Any glare should be removed by adjusting the
diaphragm.
5. Should either of the lenses appear dirty, wipe it gently with
a piece of special lens
paper. Use a circular motion with very light finger pressure. 6.
The microscope is now ready for use. 7. Position it so that the
stage faces you. 8. Connect the microscope to the power supply and
turn on the built-in light. 9. Ensure that the microscope stage is
at its lowest position. This will prevent breaking
of slides and lenses by mistake when adjusting the objectives by
moving the stage with the coarse adjustment knob.
Preparation of Wet Mount: Materials for microscopic examination
are usually placed on the glass slide of standard size, the
microscope slide. The materials are then covered by small thin
piece of glass, the cover slip. Both microscope slide and cover
slip should be very clean before use. Cleaning microscope slides
Hold the microscope slide by the edges between the index flinger
and the thumb and dip in water. Then wipe dry using a soft tissue
or a clean piece of cloth. Cleaning cover slips Cover slips are
very fragile and need careful handling. Hold a cover slip by the
edges between the index finger and the thumb and then dip in water.
To wipe dry insert the cover slip into the fold of a piece of clean
cloth or lens paper and apply gentle pressure between the finger
and thumb to both surfaces at the same time. Use a gentle circular
wiping motion for of effective cleaning.
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Exercise 1 Focusing the Microscope - e slide 1. Prepare a
microscope slide to view the letter e. Cut out the letter e from a
piece
of newspaper.
2. Place the tiny piece of newspaper in the centre of the slide
with the printed side up.
3. Add one drop of water onto the newspaper using a dropper. 4.
Place the cover slip carefully over the newspaper.
Hold the cover slip about 45 to the slide, let it slip down the
slide till the lower edge touches the water, and then slowly lower
the cover slip down onto the slide. If this is done properly, the
remaining water should spread out evenly with minimum formation of
air bubble between cover slip and slide. Some air-bubbles may still
be trapped even after the most careful preparation. If so, gentle
tapping of the cover slip with a pencil point may help remove
them.
5. Make a drawing of the image under 4x magnification.
6. Carry out the observations as follows:
Compare the position of image as seen through the eyepiece with
that of the printed letter as seen with the unaided eye. Does the
image appear to be reversed (i.e. as it would appear if seen in a
mirror)?
Slowly move the slide from left to right, observe and describe
the way the image moves. Repeat right to left.
Move the slide away from yourself and describe observe the
movement of the image again.
REMEMBER Always handle glass slides and cover slips by their
edges, never by their flat surfaces.
REMEMBER Always handle glass slides and cover slips by their
edges, never by their flat surfaces.
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Exercise 2 Using a higher power objective 1. Great care must be
taken when using higher power objectives. DO NOT focus the
high power objectives with the coarse adjustment knob or youll
risk breaking the slide and lenses.
2. Most microscopes have parfocal objectives. If one switches
from viewing a specimen
in sharp focus under a lower power objective to a higher one,
the object should automatically come approximately into focus. Only
slight further focussing with the fine adjustment knob is required
to see the specimen clearly.
3. When switching to the next higher power objective, look from
the side of the
microscope and move the revolving nosepiece slowly till that
higher power objective clicks into position. Be careful that it
does not touch the slide.
4. Take care that the lower end of the high power objective does
not touch the cover slip.
If this happens, you must repeat the whole procedure focusing
again, starting with the scanning objective.
Exercise 3 Measurement with a Microscope The unit of length used
in nearly all microscopic measurement is the micrometer (um) which
equals 1/1000 mm. A simple way to gauge the size of an object
viewed under the microscope is to determine first the size of the
circular field to view. We then use this measurement to approximate
the actual size of the object being viewed. (A) Estimation of
scanning field of view 1. Place a small plastic millimeter ruler on
the stage. 2. Focus under the scanning objective so that a clear
image of the millimeters divisions is
obtained. 3. Adjust the ruler so that the marked edge passes
through centre of the field view. 4. Count the number of millimeter
divisions seen within the field of view from one side to the
opposite side. Record of the diameter of the scanning field of
view in both millimeters and micrometers.
Diameter of the scanning field of view = _______ mm
= _______ m (B) Estimation of low power and high power field of
view We can find the low power field of view by a simple
calculation. Divide the magnification number of the low power
objective being used by that of the scanning objective. Next,
divide the diameter of the scanning field (as estimated previously)
by this quotient. This gives the diameter of the low power field of
view.
Example: Scanning objective magnification = 4 x
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Low power objective magnification = 10 x
Quotient = 10 4 = 2.5
Diameter of scanning (4 x) field = _____ m
Diameter of low power (10 x) field = _____ 2.5
= _____ m
1. Using this simple method of calculation, determine the
diameter of the high power field
of the microscope. 2. Replace the slide with the letter e onto
the stage and re-examine the letter e.
Compare the height of the letter with diameter of the field of
view. 3. Give an estimate of the actual height of the letter in
both millimeters and micrometers. Exercise 4 Magnification and
Resolution (A) Magnification Power: The total magnification is the
magnification of the eyepiece lens multiplied by the magnification
of the objective lens. By using different combinations of lenses,
different magnifications can be obtained. Do not use higher power
than is necessary. More can be made out under lower power with good
illumination than under higher power with poor illumination. Also,
the larger the region of the object viewed, the easier it is to
interpret what you see. (B) Resolving Power: This following
exercise illustrates to us the resolving power (or resolution) of a
microscope which is the ability to separate fine details to seen in
the object. For most us, for example, two dots separated by less
than 0.1 mm will appear as a single dot. The microscope therefore
does two things for us it magnifies and it allows for finer
resolution. 1. Prepare a wet mount using a piece of magazine
photograph. Use the same procedure
as for e slide. 2. Examine the wet mount under low power (begin
with scanning objective first) and
observe how the image compares with the photograph when seen
with the unaided eye.
Oil Immersion: If you require a particularly high magnification,
immersion oil may be used. Fluid with the same refractive index as
the objective lens is placed between a special objective lens and
the cover slip so that it touches both. The fluid permits a larger
cone of light rays to enter the objective from the specimen, and
this increases the resolving power obtainable.
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Note: If your microscope comes with a 100 x objective, please DO
NOT use it. Used the improper way, it will break. Microscope Care:
1. Turn the resolving nosepiece until the scanning objective is in
position. 2. Adjust the boy tube so that the lower end of the
objective is about 1 cm above the
stage. 3. Ensure that the stage surface is clean and dry. 4.
Return the microscope in an upright position to its storage
case.
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Experiment 2 Preparation of Microscopic Slides
______________________________________________________________________
Objective: To study the microscopic structure of biological samples
and to learn the preparation of biological samples for microscopic
study purposes. Introductory instructions:
Individual experiment Introduction: Examination of biological
materials under the microscope will usually entail long periods of
looking into the eyepiece. It is useful to develop the habit of
keeping both eyes open and relaxed, as though you were looking at a
distant object. This will cut out eye-strain caused by continual
forcing of one eye to remain closed. Apparatus and Equipment:
Binocular Microscope Cover slips Microscope slide Soft tissue
papers (lens cleaner) Forceps Materials: Onion Iodine Potato
(optional) Hair (optional) Safranin (optional) Observation of Onion
Cells: The onion scale leaf has generally two major surfaces an
outer surface which faces the exterior and an inner surface which
faces the interior of the onion. The outer surface may have
pigmented portions of its outer epidermis while the inner surface
may not.
(Mackean, D. G., 1973. Introduction to biology, p. 25.)
Scale leaf
Toward interior toward exterior
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Exercise 1 Preparation of microscopic slides 1. Cut an onion
bulb into quarters. Remove one of its fleshy scale leaves. 2. Bend
the onion scale leaf towards the outer epidermis until it breaks on
the upper
surface. 3. Although broken, there is some thin tissue layer of
the inner epidermis still intact. It
appears as a transparent paper-thin skin with a ragged edge
along the broken edge of the leaf.
4. With your fingers, pull the inner epidermis gently away from
the scale leaf.
5. Using a dropper, place 1-2 drops of water on the slide and
place the epidermis (~5mm
x 5mm) on the water. 6. Get rid of air bubble if there is any.
Why are bubbles undesirable? 7. Slowly lower the cover slip onto
the slide.
Some air-bubbles may still be trapped. If so, gentle tap the
cover slip with a pencil point to remove them.
8. Remove excess water from on top or around the cover slip with
a piece of tissue paper. 9. The mounting of a specimen on a slide
with solution is called a wet mount. Avoid tilting
the microscope when using a wet mount.
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Exercise 2 Viewing the slides 1. Place the slide carefully on
the stage. Position the specimen in the centre of the hole
in the stage and also in the middle of the circle of light
emanating from the lamp through the stage hole.
2. Ensure that the scanning objective is in place by moving the
revolving nosepiece.
(If not, the field of view is totally dark or an illuminated
crescent instead of a complete circle.)
3. Slide the eyepieces horizontally to the maximum length away
from each other. Place
your head just above the eyepieces. Slowly, slide the eyepieces
towards each other horizontally so that they fit the position of
the eyes on your head.
If the eyepieces are in correct position, you should be able to
observe only one illuminated circular field of view. If not, youll
see two overlapping illuminated circles.
4. Adjust the brightness adjustment knob to give the right
amount of light for viewing the object clearly.
5. Looking down the eyepiece, slowly adjust the position of
stage with the coarse
adjustment knob until the object comes into focus. Focus
accurately by using the fine adjustment knob.
6. Keep both eyes open when viewing through the eyepiece. Get
accustomed to using
both eyes otherwise this will strain your eye or give you a
headache over time. 7. Once the object is in sharp focus, its time
to view it at higher magnification. 8. Never to lower the body tube
while looking into the eyepiece and using the coarse
adjustment. If you miss the image, look up and repeat the whole
procedure of focusing. 9. For viewing under every objective lens,
use the fine adjustment to sharpen the focus
of the specimen. 10. Count the number of cells you see at 10X
magnification. 11. Make a drawing of 4 6 cells, each 2 3 cm long.
Include only the details you can
observe in your preparation. Label accordingly.
Are all the cells identical in shape and size? Is the nucleus
located in the same position in all the cells? Suggest reasons to
explain any apparent differences in the shape and size of the
cells as well as the location of the nucleus. Notes: The lines
that form the network between individual cells are non-living cell
walls made up chiefly of cellulose. This cell wall is the outermost
part of the cell and immediately surrounds the cell membrane, also
called plasma membrane, which in turn enclose the cytoplasm. The
central part of most plant cells is taken up by a vacuole filled
with a fluid made up mostly of water and various salts. The nucleus
appears as a dense body in the translucent cytoplasm.
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12. Turn again to the scanning objective and remove the slide
from the stage. 13. Stain the specimen by the technique of
irrigation. Place a drop of iodine at one edge of cover slip. A
small piece of filter paper is brought into contract with the water
at the opposite edge of the cover slip. As water is absorbed the
iodine from the other side will be drawn under the cover slip.
Continue this until the iodine is drawn halfway across the space
beneath the cover slip. The iodine will then slowly spread
throughout the mount.
The Technique of Irrigation
14. Examine first under low power (begin with the scanning
objective first) and then under high power.
What are the effects of the iodine stain on the cells? Can you
observe any changes in the cells? If so, describe them. Are there
starch grains in the cells? How can you identify the starch grains
if they are present?
15. Prepare another slide of the onion epidermis. This time add
a drop of safranin onto the
epidermis instead of water. Allow the stain to take for 10
minutes before drawing it off with tissue paper. Use the irrigation
technique to dilute and wash off the excess free stain. Finally put
on a cover slip.
16. Examine first under low power (begin with the scanning
objective first) and then under
high power.
What are the effects of the safranin stain on the cells? How is
this preparation different from the previous one observed in step
14?
Add to your drawing any additional details you may observe with
this second preparation.
Tissue paper
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[Additional practice tasks if time permits] Exercise 3
Observation of Starch Grains 1. Place a small piece of potato in
the centre of the slide and rub to distribute the potato
juice in an even layer. Discard the piece of potato. 2. Add a
drop of water and then a clean cover slip to the slide. Take the
usual precaution
of avoiding air-bubbles. 3. Examine the preparation under low
power (begin with the scanning objective first).
The starch grains in the mount can be more readily observed if
sized of the opening in the iris diaphragm is decreased. This will
increase the contrast between the starch grains and the surrounding
water.
4. Move the slide on the stage until you locate a field in which
the grains are well separated. Make a drawing of 4 6 starch grains
to illustrate their typical shape.
5. After completing your drawings, turn again to the scanning
objective and remove the
slide. 6. Stain the grains with iodine using the technique of
irrigation. 7. Examine the iodine-stained mount first under the
scanning objective and then under
low and high power. Draw 4 6 typical starch grains to illustrate
their shape and structure.
8. Prepare another slide of starch as outlined in step no. 1 but
do not add the cover slip
yet. The grains are stained first by adding a drop of iodine
onto them and the slide gently rotated by tilting to-and- fro so
that the whole area of grains is evenly covered by iodine. Excess
stain is drained off before a cover slip is added. Examine this
preparation carefully.
What observable changes may be seen in the starch grains exposed
to relatively high iodine concentration? What observable
differences are there between these starch grains when compared to
those exposed to lower iodine concentration? Can the internal grain
structure better observe in strained grains or unstained
ones? 9. Biological materials are often stained before
examination under a microscope. Based
on your experience in this exercise suggest reasons for such use
of stains.
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Exercise 4 Observation of Hair 1. Mount a small portion of your
own hair in a drop of water on a slide. Add a cover slip,
taking the usual precautions not to trap air beneath it. 2.
Adjust the diaphragm of the microscope to its largest opening and
bring the hair into
sharp focus under low power (begin with the scanning objective
first). Reduce light gradually by progressively closing the
diaphragm. In this way, determine the diaphragm setting that
provides the clearest image of the hair. As you further examine the
hair, shift the focus by slowly turning the fine adjustment back
and forth.
3. Move the hair to the centre of the scanning field and shift
to higher power
magnifications. Note any changes in the brightness of the field
of view. Bring the hair image into the sharpest possible focus and
examine carefully.
4. Estimate the width of the hair. State his measurement in
millimeters as well as in
micrometers. (Refer to the procedure outlined in Experiment 1,
Exercise 3)
While shifting the focus with the fine adjustment, what changes
in the image can be observed? Explain why these changes take
place.
Does higher-power magnification allows greater detail to be
seen? Is the depth of focus as great with higher power as with low
power?
Is the resolving power increased or decreased when magnification
is increased? 5. As an interesting corollary of this exercise you
could examine hair from different
members of the class and try to determine differences between
fine and coarse hair, curly and straight hair, and between hair of
different shades or colours.
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GUIDELINES On-site Assessment
Each student will be assessed on-the-spot identification of 3
structures within certain minutes (10 marks) (The duration will be
decided by the tutor). This section may comprise 10 marks out of 20
marks. Any mistake will result in subtraction of 1 mark. Checklist
for on-site slide structure identification
Observed
Yes No
Skill: Manipulation
1. Position compound light microscope so that the stage faces
you and
ensure that the microscope stage is at its lowest position.
2. Position the specimen holder such that it is roughly in the
middle of
the stage and not at either left or right extremes.
3. Ensure that the scanning objective is first employed.
4. Ensure that the field of view is a complete circle and not
totally dark
or an illuminated crescent.
5. Both eyes open and used to look through the eyepieces.
6. Adjust the brightness adjustment knob to give the right
amount of light
for viewing the object details clearly (i.e., instead of either
too dark or
too bright, obscuring the objects finer details).
7. When using the next higher power objective, look from the
side of the
microscope to ensure that it does not touch the slide.
8. When using higher power objectives (e.g., 40 X onwards), only
the
fine adjustment knob is used (i.e., not the coarse adjustment
knob).
9. Focus on image accurately and sharply by using the coarse and
fine
adjustment knobs.
Skill: Identification
10. Able to name the specimen from the slide or identify two -
three
structures from the slide.
Total marks
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Practical 6 (FHSC 1214 Cell Biology ONLY) Extraction of Cell
Organelles by Cell Fractionation
___________________________________________________________________
Objective: To show how organelles can be purified from homogenated
liver tissue by differential centrifugation. Introductory
instructions:
You may perform this experiment in groups of 5-6. Introduction:
Cell fractionation A. Homogenization Cells or tissues are ground
up/ blended in such a way that its consistency is even. This is to
destroy the cell membrane so that the cytoplasmic components flow
out. B. Centrifugation Principle: Different cell components are of
a certain size and density, and descend to the bottom of the
centrifuge tube at different speeds. The faster the rotation of the
centrifuge, the smaller the particles is sediment. Components can
be separated from larger to smaller ones based by using a series of
increasing speeds. This is called differential centrifugation. A
cell component can be designated 70S. S is Svedberg unit or
sedimentation coefficient. It refers to how fast a substance
/particle sediments in an ultracentrifuge, based on its size and
shape. The greater the S number, the greater the rate of
sedimentation. The process of differential centrifugation is based
on the fact that organelles have differences in size, shape and
density. As a result, the effect of gravity on each is different.
We can use this principle to separate an organelle from a
homogenous solution of particles by artificially controlling the
gravity of a solution. This is done by putting the solution in a
variable speed centrifuge and rotating them at a high rate of
speed. This creates a force that can be much greater than the force
of gravity, and particles that would normally stay in solution will
fall out and form a pellet at the bottom of the tube. The relative
centrifugal force can be calculated by the following equation:
R.C.F. = 1.119 x 10 -5 (rpm2) r Where rpm is the revolutions per
minute of the rotor and r is the distance (in cm) of the particle
from the axis of rotation. The radius used is the distance from the
center of the axis of rotation to the middle of the centrifuge
tube. The forces created at low speeds are small (e.g. 600 X g) and
only very large or dense particles will fall out of solution
(nuclei, whole cells and large cellular debris). At high speeds,
the force created can be quite great (e.g. as much as 300,000 X g).
At these speeds, most particles will fall out of solution and only
very small, highly soluble molecules will remain in solution.
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Figure 1 Cell Fractionation. The organelles can be separated
from one another after cells are broken open and centrifuged.
Diagram: Life, the science of biology (6th Ed.). William K. Purves,
David Sadava, Gordan H. Orians, and H. Craig Heller (2001)
A piece of tissue is homogenized by physically grinding it.
The cell homogenate contains large and small organelles.
A centrifuge is used to separate the organelles based on size
and density.
The heaviest organelles can be removed and the remaining
suspension re-centrifuged until the next heaviest organelles reach
the