BioI Cell Bio LabManual SL Version 6 201505

Lab manual version 6_201505 FHSB1214 Biology I & FHSC1214 Fundamentals of Cell Biology

1

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


2

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:


3

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?


4

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


5

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


6

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.


7

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.)


8

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


9

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)


10

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.


11

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.


12

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/


13

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.


14

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:


15

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?


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.


16

(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.


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.


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.


17

Part 3: Identification of Proteins A suitable protein for these tests is egg albumin.


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.



18

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.


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.



19

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?


20

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.


21

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.


22

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?


23

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?


24

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.


25

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.


26

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.


27

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.


28

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?


29

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.


30

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.]


31

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).


32

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.


33

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


34

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.


35

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.


36

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.


37

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


38

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.


39

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.


40

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


41

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.


42

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.


43

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


44

[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.


45

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.


46

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


47

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.


48

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

BioI Cell Bio LabManual SL Version 6 201505

Documents

Transcript of BioI Cell Bio LabManual SL Version 6 201505