Cell Biology LabManual Version 8.0 May 2012 1 TRUNCATED

UTAR

FHSC1214 Fundamentals of Cell Biology

Trimester 1

Lab Manual Version 8.0_May 2012

Foundation in Science 1

E-lab manuals: towards saving the earth More and more people all over the world and in Malaysia are getting involved in saving the planet – electric cars, wind and solar energy, and paper-less projects. What can YOU do? Beginning January 2011, CFS PJ Biology will pilot test e-lab manuals in an attempt to join global conservation efforts to save the planet by saving trees. Less paper means more trees will be left standing to absorb carbon dioxide and reduce the greenhouse effect. Don’t you & your future loved ones deserve to enjoy a cooler planet? Another advantage is that full-colour biology pictures will be accessible to students for the first time. Important rules on tests and lab assignments Summary:

• No MCs or any valid reasons accepted for late/missed assignments and tests.

• It is the student’s responsibility to submit another assignment in-lieu or sit for a replacement test when announced (on different topic).

• The lecturer will NOT remind students to submit late/missed assignments nor attend replacement tests. (Treating you as adults.)

Details:

• Unacceptable: MCs, valid reasons (chicken pox, met with an accident, menstrual cramps, stomach ache, dog died etc, non-valid reasons (forgetfulness; lateness; server/ IT problems). This is to be fair to everyone as fake MCs and liars are present in Malaysia.

• 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 to choose one other assignment to be submitted later in the semester when all students are so busy with tests and assignments.

• 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 there’s 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. If you’re late, a link for ‘Late submissions’ is available if you don’t mind getting a 50% discount, or you may choose to submit another assignment which the lecturer will not bother to remind you about.

I acknowledge reading the above & agree to be bound by terms therein. Your signature:

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How YOU can do well in BIOLOGY Follow the 4A’s and you can expect A’s. 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 won’t 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 can’t 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 don’t you understand about this page? Student: EVERYTHING. Professor: But I already went through this during lecture. Student: Oh, I didn’t 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: I’m sorry, I can’t help you. Student: (Hmmmph, HE’S 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 lecturer’s 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, you’ll 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.

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Writing 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

• Partner’s name

• Group

• Date

• Program • Unit code

• Unit description • Year and semester of study

• Title of lab report • Lecturer’s name

Example:

W

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Your lab report should contain the following sections:

• Title

• Objective

• Apparatus, materials and methods (if your assignment is submitted online, this step may be omitted)

• Observations and/or results with discussion

• Conclusion 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 methods “As the name implies, the materials and methods used in the experiments should be reported in this section. The difficulty 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 Observations and/or results with discussion 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 text 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.

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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.”

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, emulsion etc. Often, “mixture” is a safe descriptive term to employ. 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 transparent, translucent and opaque. 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 there’s a change in colouration, you may choose to report as follows.

• The initial blue colouration of the mixture 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 Benedict’s test, one should report that when one shakes the test tube containing Benedict’s 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 opaque.”

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

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 don’t obtain desired results? For the purpose of your UTAR lab report, if you don’t 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 “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.” “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.” 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. 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.

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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.

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.

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5 Literature Cited 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. 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 Literature Cited, all names would be listed, usually last name preceding initials.

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Practical 2 Investigation of Action of Saliva and 3 M Hydrochloric Acid in Two Carbohydrate Solutions

__________________________________________________________________________ Objective: Students are expected to state the objective of this experiment. Apparatus & Equipments: Boiling tubes Metal test tube racks Pipette filler Graduated glass pipette, 10ml Water bath, 37-40

oC Water bath, ~90-95

oC

Beaker Pasteur pipette Materials: Carbohydrate solution A Carbohydrate solution B Benedict’s solution 3 M Hydrochloric acid 3 M Sodium hydroxide (or potassium hydroxide) Procedures: This experiment is to be done in pairs. To avoid congestion, each pair should collect the following before beginning the experiment:

• 8 ml NaOH

• 16 ml Benedict’s Solution

• 2ml Solution A

• 42ml Solution B

• 2ml HCl

• 1 pipette and 1 rubber teat (to be washed with distilled water each time before reuse)

• 5 ml measuring cylinder (to be washed with distilled water each time before reuse)

• Metal test tube racks (not wooden) 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? Flowchart Students will be allowed to proceed with the experiment only if they have come into the laboratory with a flowchart of the day’s experiment. Carry out your investigation as follows. 1. Prepare two test tubes containing 2 ml solution A and 2 ml solution B respectively. Add

2 ml Benedict’s solution to each test tube. Heat both tubes together in the hotter (~90-95

oC) water bath for two minutes. Record the results in table 1.

2. Pipette 10 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. Place tubes 1 and 2 in a water bath of ~37

oC. (It

doesn’t matter how long you put it in at this stage as no salive or HCl have been added yet).

3. Salivate into a separate test-tube till it reaches a height of about 1 cm - 1.5 cm. Dilute

the saliva with an approximately equal volume of distilled water. 4. Ensure that the following two steps (5 and 6) 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?) Use a 5 ml measuring cylinder to measure out 2 ml of the diluted saliva prepared in (3) and pipette 1 ml each into tubes 1 and 4. Shake the contents of the tubes well to ensure thorough mixing.

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5. Use a measuring cylinder to measure out 2 ml HCl and pipette 1 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 ~37

oC), 3 & 4 (recently in water bath of

~95oC) incubate at their respective temperatures (see Table 2) for 35 minutes from this

moment. 6. 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 one-third 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.). Ensure that the volume in each of the tubes 1’-4’ is approximately the same (why is this important?). Straightaway, place back the original tubes (labelled 1-4) back into the respective temperatures of incubation.

7. Neutralize the acid in each of tube labelled 2’ and 3’ with 2ml of sodium hydroxide (or potassium hydroxide) (each). Shake each tube (2’ and 3’) to ensure uniform mixing.

8. Remove 1ml of the solution from each tube (1’ to 4’) into new tubes and label

appropriately as you wish as long as you don’t get confused. To carry out Benedict’s test, add an equal volume of Benedict’s solution (1 ml) for each tube. Using a test-tube holder, shake and heat at a high temperature for one minute (use the hotter water bath provided), shaking continuously to minimize spitting. Record your observations in table 2.

9. Wash the test tubes 1’- 4’. After 35 minutes of incubating tubes 1-4, pour out about one-

third of the total volume from test samples from all the tubes into the respective tubes labelled 1’- 4’.

10. Neutralize the acid in each test tube labelled 2’ and 3’ with 1ml of sodium hydroxide (or

potassium hydroxide). (Why is neutralization necessary?) Remove 1ml of solution from each tube 1’- 4’ and carry out Benedict’s test with an equal volume of Benedict’s solution (1 ml) for each tube. Remember to heat your sample (please see previous . Record your observations in table 2.

11. 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 an object such as your pen cover. Record your observations in the table 1.

Note: no penalization for unexpected results. Please refer to Practical 1 “Writing lab report”. Table 1:

Observations Conclusions

Solution A

Benedict’s test: Iodine test:

Solution B

Benedict’s test: Iodine test:

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Table 2:

Tube Contents Temp (°C)

Benedict’s Test—Colour Observation

After 5th min (from tubes 1 – 4 into

1’ – 4’)

After 35th min (from tubes 1 – 4 into 1’

– 4’)

1 10 ml solution B 1 ml saliva

37

2 10 ml solution B 1 ml 3 M HCl

37

3 10 ml solution B 1 ml 3 M HCl

95

4 10 ml solution B 1 ml saliva

95

Guidelines Observations For Benedict’s test and Iodine tests, please follow lab manual guidelines for students on writing lab report on the following:

o Liquid � mixture, solution, suspension, emulsion? � transparent, translucent, opaque?

o Colour � state initial and final colours?

o Precipitate � colour of precipitate? � amount of precipitate?

Conclusions • Absence/presence of what type of carbohydrate? Results and Discussions: 1. The results and discussion sections of your report should not exceed 2 pages. 2. Ensure that the guidelines for constructing tables and recording results for this experiment

are adhered to (see introduction). 3. If you’re required to write a discussion straight-to-the-point, follow the numbering below. If

your report is full-length, write your discussion in prose form. Discussion should contain: 1) Name of enzyme involved 2) Specific action(s) of enzyme involved 3) Effect of temperature on enzyme structure (bonds, important sites etc.) 4) Effect of HCl on substances (e.g., Solution B) 5) Effect of temperature on substances (e.g., Solution B, saliva content) 6) Product:

a. Identification (make suggestion(s)/ educated guesses) b. Structure (e.g., chemical classification etc.)

7) Chemical bases of tests used 8) Which carbohydrate is more complex, A or B? Give a reason. 9) Conclusion: Summary of results

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Practical 4 Investigation of the Enzymatic Effects of Materials on Hydrogen Peroxide Solution __________________________________________________________________________ Objective: To investigates the enzymatic effect of various materials in the hydrogen peroxide solution. Apparatus & Equipment: Beaker Test tubes Either: water bath (95

oC) or Bunsen burner

Materials: Fresh Liver Potato cubes Manganese dioxide Hydrogen peroxide** Wood splints **Caution: Hydrogen peroxide is formed continuously as a by-product of chemical

reactions in living cells; it is a very toxic (poisonous) substance. Procedures: 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. Wear gloves when handling liver tissue, so as not to be contaminated by any pathogen associated with the liver tissue used. Please stick to using one pair of gloves per person to prevent wastage. 1. Label six fresh empty test tubes 1, 2, 3, 4, 5, 6 and stand them in a rack. 2. Using a razor blade, cut the provided liver into several pieces of roughly 0.8 cm x 0.8 cm

x 0.5 cm. 3. Place one piece of liver into tube 1. 4. Boil 100 cm

3 of water in a beaker. (If you’re using a water bath set at 95

oC, this step is not

necessary). 5. Place the second piece of liver into the bottom of tube 2. Using a wooden splint, gently

spread the liver, without mashing it, over as wide an area as possible of the bottom of the test tube. Place tube 2 in the boiling or water bath (95

oC) for about five minutes.

6. Using the weighing balance, measure out two 0.5 g portions of manganese dioxide

powder each onto a weighing boat. Pour each portion into tube 5 and tube 6. 7. Put tube 6 in the beaker of boiling water or water bath (95

oC) for five minutes.

8. After five minutes let cool tube 2 and 6. 9. Now put the third piece of liver onto a white tile. With a mortar and pestle, mash it gently

into a pulp. Scoop the pulp into tube 3. 10. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4. 11. Prepare another six fresh empty test tubes and stand them in a rack. Put 5 cm

3 of

hydrogen peroxide into each of them. 12. Next, quickly add hydrogen peroxide into the test tubes 1, 2, 3, 4, 5, and 6. If needed, you

may push down some materials with one end of the wood splints provided.

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13. Using the parafilm provided, stretch it quickly seal the mouth of the test tubes by stretching the film over it. Step 12 and 13 are to be done quickly.

14. Leave for 20 minutes or till when you see quite a lot of gas being produced in some test tubes as evidenced by the bulging of parafilm from the test tube mouths.

15. Once enough gas has accumulated in some test tubes, insert a glowing splint (flame

extinguished but glow remains) into each tube one at a time by just penetrating the parafilm with it. You may use the same splint.

Why is it important to test each test tube at about the same time or at least without too much difference in the duration of sealing among the tubes?

16. Record all your observations in the table. Record your observations on each tube

immediately after the reaction has started. Table 1:

Test Tube

Contents with 5 cm3

hydrogen peroxide Observations before and after using wood splint

1

Fresh liver

2

Boiled liver (cooled)

3

Pulped liver

4 Potato cubes

5

Manganese dioxide (untreated)

6

Boiled manganese dioxide (cooled after heating)

Washing up Thoroughly wash and scrubbed all apparatus containing liver pieces with detergent or Dettol solution provided to rid it of unpleasant odours. Results and discussion: The results table and discussion should not exceed 2 pages. In your report, be sure to address the following: 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. 5. What were the differences between the reactions with fresh liver and with fresh potato

cubes? Account for these differences.

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6. What were the differences between the effects on the reaction of boiling the liver and

heating the manganese dioxide? Account for these differences.

Assignments Please check with your tutor which option is required of you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of qualitative data Option 3: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet.

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Practical 5 Experiment 1 The Microscope and Its Uses_________________________________ Objective: To study the uses of microscope aTo learn microscopic techniques such as different power of magnifications. Introduction: The microscope is a basic tool of the biologist. It is a valuable easily damaged by careless usage. It is very important familiar with the parts of the microscope and your microscope well and it will Apparatus and Materials: Binocular Microscope Microscope slide Plastic millimeter ruler Setting up the Microscope:The microscope when not in use is usually kept in a case. Remove it by handle arm while placing one hand under laboratory table and at a reasonable distance from the microscope upright in the vertical position with the fingers since it will deposit Parts of the Microscope:


2012

The Microscope and Its Uses __________________________________________________________________________

microscope and its maintenances. To learn microscopic techniques such as focus the object with correct illumination under different power of magnifications.

The microscope is a basic tool of the biologist. It is a valuable precision optical instrumenteasily 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.

Cover slips Newspaper (1 page) Wash bottle

: The microscope when not in use is usually kept in a case. Remove it by grasping the

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 with the fingers since it will deposit a thin film of oil on the glass.

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_________________________________________

focus the object with correct illumination under

optical instrument for the student to become

the procedures in the handling of it. Treat

grasping the down gently on the

table edge. Always keep the and never touch any of the lens surfaces

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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 60x longest narrowest

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 60x 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.

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

stage when the revolving n 2. Turn the nosepiece until the scanning objec

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 ancomplete circle.

3. Turn the diaphragm to its largest opening. 4. Look into the eyepiece and make a final adjustment to the

that the field of view (i.e., the lit circle which you see)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. You should any other type of paper or cloth. Discard the lens paper after use.

6. The microscope is now ready for 7. If you’re using a binocular compound light microscope like the diagram above

so that the stage faces you. 8. Connect the microscope to the power supply and tur 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

small thin piece of glass, the cover slip

be very clean before use.

Cleaning microscope slides Hold the microscope sl ide by the edges and dip in water. Then wipe dry using a soft tissue or a clean piece of cloth. Dirty handkerchiefs will not do. Cleaning cover slips Cover slips are very fragile and need careful handling. between the index finger and the thumb and cover slip into the fold of a piece of pressure between the finger and gentle circular wiping motion for of effective cleaning.


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the coarse adjustment to raise the body tube so that the objective can clear the stage when the revolving nosepiece is turned.

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

If not, the field of view is totally dark or an illuminated crescent instead of a

Turn the diaphragm to its largest opening.

Look into the eyepiece and make a final adjustment to the light adjustment knob(i.e., the lit circle which you see) is evenly illuminated. Any glare

should be removed by adjusting the diaphragm.

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. You should never use

er or cloth. Discard the lens paper after use.

The microscope is now ready for use.

compound light microscope like the diagram above, position it so that the stage faces you.

Connect the microscope to the power supply and turn on the built-in light.

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


microscope slide. In most cases, the materials are then covered by

cover slip. Both microscope slide and cover slip should

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. Dirty

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 cover slip into the fold of a piece of clean cloth or lens paper and applpressure between the finger and thumb to both surfaces at the same time. Use a

tion for of effective cleaning.

REMEMBER Always handle glass slides and cover slips their edges, never by their flat surfaces.

20

can clear the

the eyepiece. You should hear a soft click or else feel a distinct falling into place as the objective moves into

illuminated crescent instead of a

light adjustment knob so minated. Any glare

Should either of the lenses appear dirty, wipe it gently with a piece of special lens never use

, position it

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


covered by

microscope slide and cover slip should

between the index flinger and the thumb and dip in water. Then wipe dry using a soft tissue or a clean piece of cloth. Dirty

Hold a cover slip by the edges insert the

clean cloth or lens paper and apply gentle thumb to both surfaces at the same time. Use a

Always handle glass slides and cover slips by

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Focusing the Microscope - ‘e’ slide

1. Prepare a microscope slide to view the letter “e”. Using a pair of scissors, cut a piece of newspaper about 3 mm square that includes a tiny sharp-lined letter (an “R” or "e" is best).

2. Place this square piece of newspaper in the centre of the slide with the printed

side up.

3. Add one or two drops of water onto the newspaper using a dropper. The water should be sufficient for the newspaper to absorb and st i l l leave some remaining around i t .

4. Place the cover slip carefully over the newspaper. If this is done properly, the

remaining water should spread out evenly with minimum formation of air bubble between cover slip and slide.

This may take bit of practice. One effective method is to hold the cover slip about 45º 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. Use a mounted needle if necessary. 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. Anyway, a few bubbles should not hinder most observations to be made.

5. Make a drawing of the image under 4x magnification.

How to Mount an Object for Microscopic Examination

Carry out the observations as follows: 1. 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?

2. Slowly move the slide from left to right, observe and describe the way the image moves.

Repeat right to left. 3. Move the slide away from yourself and describe observe the movement of the image

again.

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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. 2. Most microscopes have parfocal objectives. This means that 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 some slight further focussing with the fine adjustment knob is required to see the specimen clearly. Therefore, if you’re using the higher power objectives, do not use the coarse knob to refine focus or you’ll risk breaking the slide and lenses.

If the objectives are not parfocal, adjust the stage such that it is about 1cm from the low-power objective. Change to the high-power objective and then adjust the stage with the coarse adjustment knob until it is about 1mm away from the objective. This is determined by looking from the side of the microscope. Using the fine adjustment knob and looking through the eyepiece now, slowly bring the object into focus. Repeat the procedure carefully if the first attempt at finding an object under high-power magnification is unsuccessful.

3. When changing from one objective to another, you will hear a ‘click’ when the objective is

set in position. 4. You are now ready to switch from the scanning objective to a higher power objective after

obtaining a sharp focus of the object. 5. If required, adjust the fine adjustment knob to see the specimen clearly. 6. If you’re going to switch 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 (normally it shouldn’t unless the specimen is too thick and also covered by a thin cover slip).

7. 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.

Trouble-shooting Below are some common problems associated with not being able to find and focus on an object under high-power magnification.

• Is the objective lens in position?

• Is the cover slip on the slide facing upwards?

• Is the object in the centre of the stage?

• Are the lenses clean and free from dirt and moisture?

• Is the condenser adjusted and focused?

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Activity: Manipulation Skill practice task Note to lecturer: this activity may be graded. Any mistake will result in subtraction of 1 mark.

Observed Yes No Skill: Manipulation

1. Position compound light microscope so that the stage faces you. 2. Ensure that the microscope stage is at its lowest position. 3. Position the specimen holder such that it is roughly in the middle

of the stage and not at either left or right extremes.

4. Secure the slide in position correctly with the specimen holder 5. Ensure that the scanning objective is first employed. 6. Ensure that the field of view is a complete circle and not totally

dark or an illuminated crescent.

7. Both eyes open and used to look through the eyepieces. 8. 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 object’s finer details).

9. Focus on image accurately and sharply by using the coarse and fine adjustment knobs.

10. When using the next higher power objective, look from the side of the microscope to ensure that it does not touch the slide.

11. When using higher power objectives (e.g., 40 X onwards), only the fine adjustment knob is used (i.e., not the coarse adjustment knob).

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. Estimation of scanning field of view 1. Place a small plastic millimeter ruler on the stage and focus under the scanning objective so

that a clearly defined image of the millimeters divisions is obtained. 2. Adjust the ruler so that the marked edge passes through centre of the field view. 3. 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 = _______ µm Estimation of low 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:

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Scanning objective magnification = 4 x Low power objective magnification = 10 x Quotient = 10 ÷ 4

= 2.5 Diameter of scanning (4 x) field = _____ µm Diameter of low power (10 x) field = _____ ÷ 2.5

= _____ µm

1. Using this simple method of calculation, determine the diameter of the high power field of you own microscope.

2. Replace the slide with the letter “e” onto the stage and re-examine the letter “e”. Use

scanning and compare the height of the letter with diameter of the field of view. Give an estimate of the actual height of the letter in both millimeters and micrometers.

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. Resolving Power: 1. Remove the wet mount from the stage, carefully lift off the cover slip, discard the piece of

newspaper and dry both slide and cover slip. 2. Prepare another wet mount this time using a piece of magazine photograph. Use the

same procedure as for piece of newspaper. 3. Examine the wet mount under low power (begin with the scanning objective first) and

observe how the image compares with the photograph when seen with the unaided eye. Notes This simple 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. Oil Immersion: If your microscope comes with a 100 x objective, please do NOT use it. Used the improper way, it will break. 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. Microscope Care: Like all laboratory instruments, the microscope needs proper care for best service. Observe the following: 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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Practical 5 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. 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. (The final image is theoretical focused at infinity as any book on optical instruments will tell us). This will cut out eye-strain caused by continual forcing of one eye to remain closed. Apparatus and Equipments: Binocular Microscope Cover slips Microscope slide Soft tissue papers (lens cleaner) Plastic millimeter ruler Wash bottle Forcept Materials: Potato Onion Hair Iodine Safranin Observation of Onion Cells: The onion scale leaf (see figure below) 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. 2. Bend the onion scale leaf towards the outer epidermis until it bre

surface. 3. Although broken, there is some thin tissue layer of the inner epidermis still intact.

appears as a transparent paperthe leaf.

4. With your fingers, pull the inner epidermis 5. You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2 small

sized portions of the inner epidermis (roughly 5 mm X 5 mm). Make sure that it is not too big so as to reach the width of the cove

6. Using a dropper, place 1-2 drops of water on the slide and place the epidermis on the water. Make sure that there is some water covering the specimen and surrounding it.

7. If there are any bubbles, try to get rid of them by pricking them with a mounting needle

provided. Why are bubbles undesirable?

8. Referring to the diagram below, hold a coverthat one edge touches the water droplet.


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Preparation of microscopic slides

Cut an onion bulb into quarters. Remove one of its fleshy scale leaves.

end the onion scale leaf towards the outer epidermis until it breaks on the upper

Although broken, there is some thin tissue layer of the inner epidermis still intact.appears as a transparent paper-thin skin with a ragged edge along the broken edge of

With your fingers, pull the inner epidermis gently away from the scale leaf.

You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2 smallsized portions of the inner epidermis (roughly 5 mm X 5 mm). Make sure that it is not too big so as to reach the width of the cover slip or of such a size that it rolls up easily.

2 drops of water on the slide and place the epidermis on the ure that there is some water covering the specimen and surrounding it.

any bubbles, try to get rid of them by pricking them with a mounting needle Why are bubbles undesirable?

Referring to the diagram below, hold a cover slip at about 45° to the slide and lower it so that one edge touches the water droplet.

26

aks on the upper

Although broken, there is some thin tissue layer of the inner epidermis still intact. It thin skin with a ragged edge along the broken edge of

You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2 small-sized portions of the inner epidermis (roughly 5 mm X 5 mm). Make sure that it is not too

r slip or of such a size that it rolls up easily.

2 drops of water on the slide and place the epidermis on the ure that there is some water covering the specimen and surrounding it.

any bubbles, try to get rid of them by pricking them with a mounting needle

to the slide and lower it so

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9. Slowly lower the cover slip onto the slide using the points of a pair of fine forceps, pencil

or a mounted needle. 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. Anyway, a few bubbles should not hinder most observations to be made.

10. Remove excess water from on top or around the cover slip with a piece of tissue paper provided – be careful not to absorb all the water from under the cover slip. Removal of excess water ensures that when the slide is viewed under the microscope, water will not spill onto the stage.

11. The mounting of a specimen on a slide with solution is called a wet mount. Avoid tilting

the microscope when using a wet mount. Exercise 2 Viewing the slides 1. Place the slide carefully on the stage and position such that the specimen is 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. The

revolving nosepiece is in correct position if the objective lens is felt to ‘click’ to fit an unseen internal groove that aligns the lens’s field of view with the eyepieces. If not, the field of view is totally dark or an illuminated crescent instead of a complete circle.

3. If the bifocal compound light microscope you’re using has eyepiece lenses which can be

slid horizontally, slide the eyepieces to the maximum length away from each other first.

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, you’ll see two overlapping illuminated circles.

4. Adjust the brightness adjustment knob to give the right amount of light for viewing the object clearly. Some materials are best viewed in dim light, others in bright light.

A common cause of poor definition of (i.e., not being able to see details clearly) the image is that the object is over-illuminated. Best definition is often obtained by cutting down the amount of light and not increasing it. If the condenser is not adjusted and focused, the specimen may appear too dark or too bright, obscuring the object’s finer details.

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. If the details of the specimen are not clear, adjust the brightness adjustment knob and/or

condenser. 8. Once the object is in sharp focus, it’s time to view it at higher magnification (i.e., the 10X

objective).

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9. Never to lower the body tube while looking into the eyepiece and using the coarse adjustment. If for some reason you miss the image, look up and repeat the whole procedure of focusing.

10. For viewing under every objective lens, get a sharp focus first. Use the fine adjustment to sharpen the focus of the specimen.

11. Count the number of cells you see at 10X magnification.

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.

12. Make a drawing of 4 – 6 cells, each 2 – 3 cm long. Include only the details you can

observe in your preparation. Label accordingly. Answer these questions:

1. Are all the cells identical in shape and size? 2. Is the nucleus located in the same position in all the cells? 3. Suggest reasons to explain any apparent differences in the shape and size of the cells as well as the location of the nucleus.

13. Turn again to the scanning objective and remove the slide from the stage. 14. Stain the grains by the technique of irrigation. This is done by placing 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

15. Examine first under low power (begin with the scanning objective first) and then under

high power. Answer these questions:

1. What are the effects of the iodine stain on the cells? 2. Can you observe any changes in the cells? If so, describe them. 3. Are there starch grains in the cells? 4. How can you identify the starch grains if they are present?

Tissue paper

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16. Prepare another slide of the onion epidermis. This time add a drop of safranin onto the epidermis instead of distilled 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.

17. Examine first under low power (begin with the scanning objective first) and then under

high power. Answer these questions:

1. What are the effects of the safranin stain on the cells? 2. 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. Exercise 3 Observation of Starch Grains (Additional practice tasks if time permits) 1. Place a clean glass slide on the white tile. Place a small piece of potato in the centre of

the slide and rub the piece of the potato in a circular pattern 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. Examine them under low or high power. Make a drawing of 4 – 6 starch grains to illustrate their typical shape with any other observable details.

5. After completing your drawings, turn again to the scanning objective. Remove the slide

from the stage and place it on a white tile. Stain the grains with iodine using the technique of irrigation earlier mentioned.

6. Examine various aspects of the iodine-stained mount first under the scanning objective

and then under low and high power. Note the effects of different iodine concentration on the starch grains. Draw 4 – 6 typical starch grains to illustrate their shape and structure.

Answer these questions: 1. What observable changes may be seen in the starch grains exposed to relatively high iodine concentration? 2. What observable differences are there between these starch grains when compared to those exposed to lower iodine concentration? 3. Can the internal grain structure better observe in strained grains or unstained ones?

7. 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.

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 (Additional practice tasks if time permits) 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.

Answer these questions:

1. While shifting the focus with the fine adjustment, what changes in the image can be observed? Explain why these changes take place.

2. Does higher-power magnification allows greater detail to be seen? 3. Is the depth of focus as great with higher power as with low power?

4. Is the resolving power increased or decreased when magnification is increased? Using the procedure outlined in Experiment 1, Exercise 3, estimate the width of the hair. State his measurement in millimeters as well as in micrometers.

4. 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. The differences and similarities could be represented by diagrams based on your observations

5. The exercise in step 4 above can be further extended by using hair from different animals,

e.g. dog, cat, mouse, rabbit, guinea pig, etc.

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Practical 6 Experiment 1 Extraction of Cell Organelles by Differential Centrifugation __________________________________________________________________________ Objective: To show how organelles can be purified from homogenated liver tissue by differential centrifugation. 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 are sedimented. 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 (rpm

2) 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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Lab Manual Version 8.0_May 2012 32

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 bottom of the tube.

Golgi

Mitochondria

Nuclei

4.8 Cell Fractionation. The organelles can be separated from one another after cells are broken open and centrifuged.

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Differential centrifugation schemes (Figure 1) involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated froand the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is recan be done on it. The fractionation An important thing to note is that there is cross contamination between the second and third pellets. Mitochondria show up in Pellet 3 and lysosomes show up in Pellet 2. This shows that the separations made by this technique aren't absolute purifications, but relative enrichments of organelles. In order to develop a differential centrifugation scheme to isolate a particular organelle, a marker must be used to follow its isolation. The marker can be theis confined to that organelle. For example, enzymes of the electron transport chain are membrane bound and confined to the inner membrane of the mitochondria. Therefore, after a centrifugation to isolate mitochondria, both the pwhich part has more of the activity associated with these enzymes. The fraction with more of the activity has been "enriched" with mitochondria. Purification of the organelle is accomplished by following the enr Another way to follow enrichment is by binding a radioactive label to protein on the organelle. For example, cell surface membranes can be isolated by first binding a radioactive drug that has its protein receptor in the remains there, so the fractions that contain the most radioactivity are enriched for cell surface membranes.

Figure 1 Centrifugation Scheme. separate larger cell structures and then higher centrifugation force to separate smaller organelles.


2012

Differential centrifugation schemes (Figure 1) involve stepwise increases in the speed of centrifugation. At each step, more dense particles are separated from less dense particles, and the successive speed of centrifugation is increased until the target particle is pelleted out. The final supernatant is removed, the pellet is re-suspended, and further study or purification can be done on it. The fractionation of rat liver is an example of how this process works

An important thing to note is that there is cross contamination between the second and third pellets. Mitochondria show up in Pellet 3 and lysosomes show up in Pellet 2. This shows that

made by this technique aren't absolute purifications, but relative enrichments

In order to develop a differential centrifugation scheme to isolate a particular organelle, a marker must be used to follow its isolation. The marker can be the activity of an enzyme that is confined to that organelle. For example, enzymes of the electron transport chain are membrane bound and confined to the inner membrane of the mitochondria. Therefore, after a centrifugation to isolate mitochondria, both the pellet and supernatant can be analyzed to see which part has more of the activity associated with these enzymes. The fraction with more of the activity has been "enriched" with mitochondria. Purification of the organelle is accomplished by following the enrichment through successive steps.

Another way to follow enrichment is by binding a radioactive label to protein on the organelle. For example, cell surface membranes can be isolated by first binding a radioactive drug that has its protein receptor in the cell membrane. The drug binds tightly to the receptor and remains there, so the fractions that contain the most radioactivity are enriched for cell surface

Figure 1 Centrifugation Scheme. Homogenized liver tissue is subjected to low centrifugation force to separate larger cell structures and then higher centrifugation force to separate smaller organelles.

34

Differential centrifugation schemes (Figure 1) involve stepwise increases in the speed of m less dense particles,

and the successive speed of centrifugation is increased until the target particle is pelleted out. suspended, and further study or purification

of rat liver is an example of how this process works.

An important thing to note is that there is cross contamination between the second and third pellets. Mitochondria show up in Pellet 3 and lysosomes show up in Pellet 2. This shows that

made by this technique aren't absolute purifications, but relative enrichments

In order to develop a differential centrifugation scheme to isolate a particular organelle, a activity of an enzyme that

is confined to that organelle. For example, enzymes of the electron transport chain are membrane bound and confined to the inner membrane of the mitochondria. Therefore, after a

ellet and supernatant can be analyzed to see which part has more of the activity associated with these enzymes. The fraction with more of the activity has been "enriched" with mitochondria. Purification of the organelle is

Another way to follow enrichment is by binding a radioactive label to protein on the organelle. For example, cell surface membranes can be isolated by first binding a radioactive drug that

cell membrane. The drug binds tightly to the receptor and remains there, so the fractions that contain the most radioactivity are enriched for cell surface

gation force to separate larger cell structures and then higher centrifugation force to separate smaller organelles.

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Procedures: 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. 1. Obtain an uncut piece of liver (~ 3 cm x 3 cm). Be sure to remove any associated fat or

surrounding foreign (non-liver) tissue. 2. Using a mortar and pestle, grind the liver piece into a pulp. This is called homogenization

and the product the homogenate. 3. Add 10 ml or more isotonic solution as you grind, to increase the volume and dilute the

pulp. [Use distilled or tap water if no isotonic solution if provided.] (Why is isotonic solution needed?)

4. Remove and dispose any unground tissue or fat. 5. Using labelling paper (or masking tape), label of the 50 ml graduated plastic tube

provided with your group’s name. 6. If the pulp is very thick, pour about 10-15 ml into the 50 ml graduated plastic tube and top

up to 20 ml of the tube with isotonic solution.

If the pulp is not too thick, pour 20 ml of the pulp (homogenate) into the 50 ml graduated plastic tube. The graduated tube has markings to guide you. Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning.

7. Wipe off any spillage on the tube exterior so as not to contaminate the centrifuge. 8. With the support of lab technical staff, spin the tube for 5 minutes.

While waiting, prepare for the next experiment. 9. After 5 minutes, collect your centrifuged sample. Note: Do NOT shake the tube. 10. Use one hand to hold the 50 ml graduated plastic tube and another to hold a glass

pipette. 11. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the

supernatant, being careful not to mix up the contents. 12. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15 ml

graduated plastic tubes in roughly equal volumes. 13. Unless there is enough supernatant, top up just one of 15 ml graduated plastic tubes to

10 ml with distilled H20.

Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning.

14. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate the

centrifuge. 15. Centrifuge the graduated plastic tube for about 20 minutes. While waiting carry on with

the next experiment. 16. After 20 minutes, collect your centrifuged sample. Note: Do NOT shake the tube. 17. Use one hand to hold the 15 ml graduated plastic tube and another to hold a glass

pipette.

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18. Press the air out of the rubber bulb of the pipette. Slowly insert the pipette tip into the

supernatant, being careful not to mix up the contents. 19. By slowly releasing the pressure on the bulb, aspirate out the supernatant into one 15 ml

graduated plastic tube. 20. Unless there is enough supernatant, top up just one of the 15 ml graduated plastic tubes

to 10 ml with distilled H20.

Note: It is extremely IMPORTANT that each tube is topped up to this volume as precisely as possible, so that the centrifuge will not be damaged during spinning.

21. Label the tube and wipe off any spillage on the tube exterior so as not to contaminate the centrifuge.

22. Centrifuge the labelled 15 ml graduated plastic tube for about 1 hour. While waiting carry

on with the next experiment. 23. After 1 hour, retrieve the tube and take a look at it. Due to shortage of time and limitations

of the centrifuge model used, this experiment will stop here. 24. Dispose of the excess liver pulp or pieces into the given plastic bag (not the dustbin).

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Practical 6 Experiment 2 Micrometer Calibration __________________________________________________________________________ Objective: To measure dimensions of stained cells of the plant material supplied.

Apparatus and Materials: A pair of forceps 1 dropper Onion (1 per bench) Microscope & micrometer A pair of scissors I white tile A bottle of distilled water 2 watch glasses 1 glass slide 1 mounting needle 1 cover slip Methylene blue A bottle of tissue paper Introduction Quantitative observations under the microscope – making microscopic measurements A more exacting type of observation introduces the idea of amount and is called quantitative. How big? How fast? Measuring instruments such as rulers, balances and thermometers must be used to extend the range and accuracy of the senses. The microscope can be used for the dual purpose of measuring very small objects (too small to be measured with an ordinary ruler) and observing them accurately. Magnification When looking through the microscope, it is important to know how much the object being observed is magnified. To find the degree of magnification, multiply the magnification on the objective being used by that on the eyepiece. For example, if the magnification of the eyepiece is 10x, and that of the objective is 40x, then the total magnification is 10x40, or 400x. Objects viewed under the microscope will be seen in three-dimensions. For a given object, the distance that allows it to be in focus should thus be investigated using the fine adjustment knob. This distance is the focal depth of the object being observed. The focal depth decreases with increasing magnification.

Determining the diameter of the field of view by means of a stage graticule/ micrometer or an eye-piece graticule/ micrometer

A graticule/ micrometer is a ‘micro-ruler’. It is a piece of plastic or glass slide which is finely calibrated to 0.1mm or even smaller (0.01mm). It is used for measuring microscopic structures. There are two types of graticule/micrometer:

1. Stage graticule/ micrometer

This is placed on the stage over the specimen slide. When viewed under the microscope, the finely calibrated division marks can be seen clearly and are magnified according to the magnification of the two lenses used. Since it is more finely calibrated than a ruler, it more accurately measures the size of a specimen under the microscope.

2. Ocular/ Eyepiece graticule/ micrometer

This is placed in the body tube together with the eyepiece lens. It is also used to measure microscopic structures under the microscope. However, it is magnified differently from that of an object on the stage. The object on the stage is magnified by both the objective lens and the eyepiece lens, while the

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eyepiece graticule is only magnified by the eyepiece lens. Thus, there is a need to further calibrate the scale when different objective lens is used.

(a) Calibrating the stage graticule/ micrometer

Each graticule/ micrometer, whether it is the stage type or the eyepiece type; must be calibrated before it can be used.

(i) Place a stage micrometer on the microscope stage, and using the lowest magnification (4X), focus on the grid of the stage micrometer.

(ii) Rotate the ocular micrometer by turning the appropriate eyepiece. Move the stage until you superimpose the lines of the ocular micrometer upon those of the stage micrometer. With the lines of the two micrometers coinciding at one end of the field (align the start (0 mm) of divisions on the stage graticule with the start (0mm) of divisions on the eyepiece graticule).(Figure 1)

(iii) Count the spaces of each micrometer to a point at which the lines of the micrometers coincide again (Figure 2) (i.e. find the division /scale of eyepiece graticule which is parallel with a particular division/scale of the stage micrometer).

Since each division of the stage micrometer measures 10 micrometers (µm)/0.01mm, and since you know how many ocular divisions are equivalent to one stage division, you can now calculate the number of micrometers in each space of the ocular scale.

**Alternatively: Suppose the full length of the ocular/eyepiece graticule covered 250 divisions of the stage micrometer. Then the full length of the ocular/eyepiece graticule is equivalent to (250 x 0.01mm) = 2.5mm long. For an ocular/eyepiece graticule with 100 divisions, each division will measure 25µm at the stage for this magnification.

(vi) Record your calculations as shown below.

(using the lowest magnification (4X objective / total magnification 40x)

_____number of divisions on eyepiece graticule corresponds to_____divisions of stage micrometer.

Value for each division of the stage micrometer = __________mm

= __________µm

Thus value/size for each division of eyepiece micrometer =

____________µm (give your answer in µm)

(vi) Repeat your measurements for 10x, 40x/60x, and 100 x objectives.

(vii) Fill in your calibrated values for 1 ocular/eyepiece graticule scale at various magnifications in Table 1.

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The diagram below illustrates the calibration of the eye-piece/ ocular graticule/micrometer.

Figure 1

Note: the display of scales of stage and ocular micrometers shown here may be different from the actual ones provided in your laboratory)

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Figure 2

Table 1 : Value of eyepiece graticule/micrometer division for various objective magnifications

Objectives magnification Calibrated values of eye piece graticule

1 4X 1 graticule unit = ___________ mm = __________ µm

2 10X 1 graticule unit = ___________ mm = __________ µm

3 40X/60x 1 graticule unit = ___________ mm = __________ µm

4 100x 1 graticule unit = ___________ mm = __________ µm

Note: all values to have precision up to 4 decimal places for mm ,and 2 decimal

places for µm)

(b) Determine the size of a given specimen.

Name of specimen: Magnification power:

(Draw your specimen and show the measurement/size of specimen in µm)

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Procedures : 1. Cut an onion bulb in half. Remove one of its fleshy scale leaves. 2. Share one onion scale leaf with your partner. 3. Bend the onion scale leaf with which you have been supplied towards the inner epidermis

until it breaks on the upper surface. 4. Although broken, there is some thin tissue layer of the inner epidermis still intact. 5. With your fingers, pull the inner epidermis gently away from the scale leaf. 6. You may use forceps, scissors or a scalpel (used against a white tile) to pull out 2 small-

sized portions of the inner epidermis (about roughly 5 mm X 5 mm is enough; no need to follow precisely). Make sure that it is not too big so as to reach the width of the cover slip or of such a size that it rolls up easily.

7. Place the cut pieces in a small dish and cover them with methylene blue solution using a

dropper. Put this aside for about 2 minutes. 8. After about 5 minutes, remove one of the pieces of epidermis, wash it carefully in

water in a small dish. 9. Using a dropper, place 1-2 drops of water on the slide and place the epidermis on the

water. You may decide to add another drop of water on the specimen to ensure that there is some water covering the specimen and surrounding it. If there are any bubbles, try to get rid of them by pricking them with a mounting needle provided. Why are bubbles undesirable?

10. Hold a coverslip at about 45° to the slide and lower it so that one edge touches the water droplet. Then slowly lower the coverslip onto the slide using a mounted needle or the points of a pair of fine forceps. A gentle tapping with the point of the needle will usually remove any air bubbles that may be present.

11. Remove excess water from on top or around the coverslip with a piece of tissue paper

provided – be careful not to absorb all the water from under the cover slip. Removal of excess water ensures that when the slide is viewed under the microscope, water will not spill onto the stage.

12. Measure the length and width of 10 cells that you can see under 10X magnification.

Calculate the average.

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Practical 7 Determination of the Solute potential of the Potato Cell Sap_________________________________________________________ Objectives: To find out the solute potential of potato cell sap asof sucrose solution and in the length of potato strips. To prepare sucrose solutions of various concentrations dilution technique for the determination of the solute potential Introduction You are advised to read the following carefully before writing your lab report.

Water potential, ΨΨΨΨ

• The tendency of water molecules to diffuse across a membrane is affected by:

• the concentration of the solution on either side of the membra

• the resultant pressure in the solution on each side of the membrane

• Water potential (denoted by the Greek letterof concentration and pressure in a solution on the tendency of water molecules to diffuse across a membrane.

• Water potential is defined as the net tendency of water to diffuse by osmosis

• It is measured in pressure units such as kPa (kilopascals) and MPa (megapascals).

• Water potential of a solution = effect of the solute coneffect of pressure on that solution

where Ψ = water potential of the cell

Ψs = solute potential of the cell

Ψp = pressure potential, due to wall pressure (it’s a +ve pressure;

• Solute potential is the tendency of a solution to

• the potential of a solution to gain water is always negative

• The more concentrated a solution,

1 Campbell (2002). Chp 36. (6th Ed


2012

Determination of the Solute potential of the Potato Cell Sap ____________________________________________________________________

To find out the solute potential of potato cell sap as a function of changes in the concentration of sucrose solution and in the length of potato strips.

To prepare sucrose solutions of various concentrations from a stock solution of 1.0 M dilution technique for the determination of the solute potential.

You are advised to read the following carefully before writing your lab report.

The tendency of water molecules to diffuse across a membrane is affected by:

the concentration of the solution on either side of the membrane

the resultant pressure in the solution on each side of the membrane

(denoted by the Greek letter Ψ, psi) is used to describe combined effects of concentration and pressure in a solution on the tendency of water molecules to diffuse

is defined as the net tendency of water to diffuse out of a solution

measured in pressure units such as kPa (kilopascals) and MPa (megapascals).

Water potential of a solution = effect of the solute concentration of that solution + effect of pressure on that solution

ΨΨΨΨ = ΨΨΨΨs

+ ΨΨΨΨp

= water potential of the cell (it’s a -ve pressure, i.e., inward force like that created on solutions or water when the plunger of a syringe is pulleto draw liquid up

1; see picture arrows)

= solute potential of the cell (it’s a -ve pressure also, i.e., inward force; see picture arrows)

= pressure potential, due to wall pressure (it’s a +ve pressure; outward force like that created on solutions or water when the plunger of a syringe is pressed to expel liquid; see picture arrows)

Solute potential is the tendency of a solution to gain water

the potential of a solution to gain water is always negative

The more concentrated a solution, the more negative its solute potential

6th Ed.).

42

________________

a function of changes in the concentration

from a stock solution of 1.0 M by

is used to describe combined effects of concentration and pressure in a solution on the tendency of water molecules to diffuse

of a solution

measured in pressure units such as kPa (kilopascals) and MPa (megapascals).

centration of that solution +

ve pressure, i.e., inward force like that created on solutions or water when the plunger of a syringe is pulled

ve pressure also, i.e., inward force; see picture

= pressure potential, due to wall pressure (it’s a +ve pressure; solutions or water when

the plunger of a syringe is pressed to expel liquid; see picture

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• For the purposes of comparison, potential of 0. If a solute, such as sugar, is added to this water, effectively decreased or lowered.

• water concentration in a given space is decreased

• water molecules are attracted to the solute molecules and so move less freely

• Water potential of a solution is always

• The more concentrated thnegative is its water potential.

• In pure water, water molecules are free to move about at random. • In a solution, solute particles attract water • thus, water molecules cannot leave a solution so easily • the presence of solute particles lowers the water potential. • The movement of water molecules across a selectively

solution with a higher water potential to a solution with a lower water potential is known as osmosis.

2 Never use the term ‘semi-permeable

and no other solutes.


2012

For the purposes of comparison, pure water at atmospheric pressure has a water If a solute, such as sugar, is added to this water, its water potential is

effectively decreased or lowered. This is because

water concentration in a given space is decreased

water molecules are attracted to the solute molecules and so move less freely

Water potential of a solution is always negative owing to the presence of solutes

The more concentrated the solution, the lower its water potential i.e., the more negative is its water potential.

(Pic. Hoh, 2003. A Level biology)

In pure water, water molecules are free to move about at random.

In a solution, solute particles attract water molecules and restrict their movement

thus, water molecules cannot leave a solution so easily

the presence of solute particles lowers the water potential.

The movement of water molecules across a selectively-permeable membrane higher water potential to a solution with a lower water potential is known as

permeable’ membrane as it means that only water gets through

43

pure water at atmospheric pressure has a water its water potential is

water molecules are attracted to the solute molecules and so move less freely

owing to the presence of solutes.

e solution, the lower its water potential i.e., the more

(Pic. Hoh, 2003. A Level biology)

molecules and restrict their movement

permeable membrane 2 from a

higher water potential to a solution with a lower water potential is known as

as it means that only water gets through

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• For example, if a plant cell is immersed in a solution with a higher water potential than the cell, then osmotic uptake of water will cause the cell to swell.

• By moving, water can perform work. • Therefore the potential in water potential refers to the potential energy that can be

released to do work when water moves from a region with higher psi to lower psi. • Plant biologists measure psi in MPa, where one MPa is equal to about 10 atmospheres of

pressure. 3

3 Campbell (2002). Chp 36. (6th Ed.).

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For your understanding only: the concept of turgor pressure ΨΨΨΨp

• Any solution at atmospheric pressure has a negative water potential. • For instance, a 0.1-molar (M) solution of any solute has a water potential of -0.23 MPa.

4

• In contrast to the inverse relationship of psi to solute concentration, water potential is

directly proportional to pressure.

• Physical pressure - pressing the plunger of a syringe filled with water, for example - causes water to escape via any available exit.

• If a solution is separated from pure water by a selectively permeable membrane,

external pressure on the solution can counter its tendency to take up the water due to the presence of solutes or even forcing the water from the solution to diffuse into the compartment with pure water.

• It is also possible to create negative

pressure, or tension when the plunger of a syringe is pulled up.

• If a 0.1 M solution is separated from pure

water by a selectively permeable membrane, water will move by osmosis into the solution.

• Water will move from the region of

higher psi (0 MPa) to the region of lower psi (-0.23 MPa). (Fig. 36.3a)

• If a 0.1 M solution (psi = -0.23 MPa) is

separated from pure water (psi = 0 MPa) by a selectively permeable membrane, then water will move from the pure water to the solution. (Fig. 36.3d)

• Application of physical pressure can balance

or even reverse the water potential (Fig. 36.3).

• A negative pressure can make water

potential more negative (Fig. 36.3d).

4 Pic. and text from Campbell (2002). Chp 36. (6th Ed.).

Fig. 36.3. Values for ΨΨΨΨ & ΨΨΨΨs in the left and right arms of the U-tube respectively are given for initial conditions, before any net movement of water. (a) The addition of solutes makes water potential more –ve. (b,c) Application of physical pressure increases water potential. (d) A –ve pressure (tension) decreases water potential

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Essential knowledge for practical


2012

nowledge for practical: Osmosis in living plant cells

46

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The figure below shows the changes in water potential, pressure potential and solute potential of a plant cell as it takes up or loses water and so changes in volume.

At full turgor

• The plant cell is fully turgid

• The cell wall is stretched fully

• The pressure of the cell contents resists the uptake of water

• Turgor pressure (Ψp) is at a maximum

• There is no net tendency of water to move in or out of the cell, i.e., water potential (Ψ) = 0, because the rate of water movement into the cell equals that of water out of the cell.

Incipient plasmolysis

• Is when the shrinkage of the protoplasm reaches the point where the cell surface membrane is just about to pull away from the cell wall

• No pressure is exerted by the protoplast (= cytoplasmic contents + membrane) against the cell wall, i.e. (Ψp) = 0, i.e. (Ψ)= (Ψs).

• The cell is flaccid. Full plasmolysis

• Occurs when the cell surface membrane is pulled away from the cell wall with the cytoplasm contracted.

The curves for water potential (Ψ) and solute potential (Ψs ) are the same when the water content of this cell falls below 50%. As the cell contents shrink, there is no more force pushing on the cell contents. i.e., the pressure potential (Ψp) = 0. Hence, water potential (Ψ)= solute potential (Ψ s) [Note: This is a very important equation.] In your lab report, be sure to use the proper terms (e.g., plasmolysis, the various ‘potentials’ etc.) when explaining the concepts herein.

Ψs

Ψp = Ψs Ψ = 0

Ψs

0 kPa

- ve kPa

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Apparatus: Boiling tubes (5 per pair) Boiling tube rack Ruler Petri dish Knife White tile Forceps Graduated glass pipette, 10cm

3

Glass rod Materials: Sucrose solution, 1.0 M Distilled water Potato Tissue paper Graph paper Procedures: 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. 1. Each group should have 35cm

3 of sucrose. In labelled boiling tubes, prepare a series of

20 cm3 of sucrose solutions at the concentrations of 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.5

M from the stock solution using dilution technique. Record the volumes of solution and of distilled water used in the table below.

You may find this formula helpful: M = molarity V = volume M1V1=M2V2 Existing solution: 1 M (M1) of sucrose Desired solution: 0.1M (M2) of 20cm

3 sucrose

what is the volume of 1 M sucrose in cm3?

(1M) (V1) =(0.1M) (20 cm

3)

V1 = ?

Molarity

0.1 M

0.2 M

0.3 M

0.4 M

0.5 M

Volume of 1.0 M sucrose solution (cm

3)

Volume of distilled water (cm

3)

2. Prepare 15 strips of potato tissue of about equal length (choose any single length

between 4 cm to 6 cm) with a cross-section of about 0.5 cm x 0.5 cm. Using a table, record the average length of the potato strips.

(Note: Don’t spend too much time cutting – dimensions can be approximated instead of being identical. Ideally, it is important for each strip to be of equal dimensions. Why?)

3. Using a ruler and simple eye estimation, measure the initial level of 0.1 M sucrose

solution in the boiling tube before adding the potato strips. Record this in your manual.

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Take 3 potato strips, record the length of each strip, and place the strips into the boiling tube. Repeat these steps using 0.2 M, 0.3M, 0.4 M

and 0.5 M

sucrose solutions.

4. After 30 minutes, remove the strips with the forceps provided. Wipe them gently with

tissue paper and record the final length and change in length of each potato strip in your table.

5. In your manual, record the final level of the sucrose solution in each boiling tube and

record any changes to the physical condition of the potato strips. 6. Calculate the average change in the length of the potato strips and record it in your table.

7. On a piece of graph paper, plot a standard graph of solute potential against the

concentration of the sucrose solution to determine the solute potential of the potato cell sap.

Concentration

(M)

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

Solute potential (atm)

1.3 2.6 4.0 5.3 6.7 8.1 9.6 11.1 12.6 14.3 16.0

8. Plot a second graph of the change in average length of the potato strip against the

concentration of the sucrose solution. Use a best fit curve/ line. Assignments Please check with your tutor which option is required of you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data Your table should have the following: 1. initial, final and change in lengths of each strip and their averages 2. initial, final and change in levels of

sucrose solution in the boiling tubes before the

addition of potato strips 3. physical condition of the potato strips 4. Adherence to the specifications mentioned in the introduction of this manual.

Length of potato strips (mm)

Sucrose solution (M)

0.1M 0.2M 0.3M 0.4M 0.5M

Initial length

Average initial length

Final length

Average final length

Change in average length

The above table is just a suggested format. You may present in another way if suitable. Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel) of the change in average length of the potato strip against the concentration of the sucrose solution. Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table, write a discussion or conclusion. Option 4: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded.

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• Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet.

General Note: Students must relate results with theory (no separate paragraphs on theory without reference to expected results) Definition of relevant terms:

a. Osmosis

b. Water potential

c. Solute potential

d. Turgor pressure Terms above used correctly below.

Positive change in length

Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water?

Zero change in length Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water?

e. Relationships among ψw plant cell , ψs plant cell , ψw sucrose, ψs plant cell,

ψs sucrose

Negative change in length (shortening of strip)

Comment on: a. Change in length b. Tonicity (e.g., is solution hypertonic? etc.) c. Condition of cells (e.g., turgid, etc.)? d. (Net) movement of water?

Conclusion State the:

a. The concentration of sucrose solution at which the water potential of the potato tissue is equal to the water potential of sucrose solution (determine from graph).

b. The solute potential in atm at which the water potential of the potato tissue is equal to the water potential of sucrose solution (determine from graph).

c. Relationship between change in length and sucrose concentration

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Practical 8 Effects of various treatments on pieces of stained potato tissues __________________________________________________________________________ Objective: To investigate the effects of various treatments on pieces of stained potato tissues Apparatus and Materials: 1 pen-knife or scalpel (if provided) 1 Potato rod (if provided) 1 white tile Methylene blue solution A pair of forceps 50% ethanol A thermometer (if no incubator used) A plastic ruler 10 test tubes 3 syringe (5ml) 2 beakers 1 stop watch Procedures: 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. Unless otherwise instructed by your tutor, you may conduct this experiment in pairs. 1. Use a sharp scalpel, cut four cubes from the potato rod provided, each approximately 1

cm (breadth) x 1 cm (length) x 2 to 5 mm thick (select one size of thickness. Ensure all cubes are the same size and thickness). Trim off any peel which is still attached.

Why should the surface area be kept constant for each piece of tissue?

2. Place them in a 50mL size small beaker (or petri dish or specimen tube), immerse them in

methylene blue solution for 10 minutes. Use only enough methylene blue (maximum 10mL) to cover them (Swirl the contents to ensure that all surfaces of the cubes are exposed to the stain).

Always read through your lab notes once before starting and look out for waiting time. What can you do while waiting?

3. After 10 minutes, pour off the methylene blue solution and wash the cubes with tap water

until the water contains little or no stain. Then cover the cube with tap water.

Why is a coloured stain chosen?

4. Label four test tubes A, B, C and D. To each of the tubes A, B and C, add 5cm

3 of

distilled water, to tube D add 5 cm3 of 50% ethanol and to tube

State the reason for using equal volumes of liquids in all tubes.

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5. Place tube A in boiling water or water bath (95

oC), tube B in a water bath of 38

oC to 42

oC

and tube C and D at room temperature.

What purpose does having tube C placed in water at room temperature? Why isn’t tube D placed at high temperature?

6. After 2 minutes, add one stained potato cube to each of the four test tubes. Start the stop

watch immediately.

Explain the significance of the 2 minutes of incubation before adding the tissue in.

7. After 2 minutes, remove tubes A and B from the water baths and place them in the rack

with tubes C and D. Shake the tubes.

Explain the significance of the 2 minutes of incubation.

8. Immediately separate the tissue from solutions. For each tube, quickly pour away the

liquid into another the corresponding test tubes labeled A’ to D’. Immediately record

your observations of each test tube. [Some guidelines (non-exhaustive): What’s the

texture (i.e., soft/ flaccid, hard/ turgid?) and colour of the tissue? What’s the colour of the liquid and how much light can pass through? How will you record the difference in intensity of colouration of the liquid?]

Explain the reason for separating the tissue from solutions after adding the tissue in.

9. Repeat the experiment again using fresh potato cubes.

Why repeat the experiment again?

Results and Discussions: Table of Observations Data to tabulate:

• Tube A, B, C

• Observations:

• Texture of tissue in arbitrary units: Eg (but not confined to): 5 – most hard/turgid, 1 – least hard/turgid OR 5 – least soft/flaccid, 1 – most soft/flaccid

• Colouration of tissue in arbitrary units: Eg (but not confined to): 5 – most intense, 1 – least intense

• Compile at least 2 sets of data. Unless otherwise instructed by tutor, you may calculate average where necessary.

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Assignments Please check with your tutor which option is required of you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of qualitative data Option 3: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet. From the data you have collected in the practical, account fully for the observations you have made and draw clear conclusions, using your knowledge and understanding. You will need to use the following questions as guidelines. 1. Why are the potato cubes stained with methylene blue? 2. What happens to the stained potato cubes when they are placed in water at room

temperature? 3. What are the effects of temperature on potato cubes in tubes A, B and C? 4. What are the effects of the ethanol on the potato cubes? 5. State the reason for using equal volumes of liquids in all tubes. 6. Explain why the potato cubes added after the tubes are left at the respective conditions

for 2 minutes. 7. What is the purpose of repeating the experiments by doing 2 sets?

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Practical 9 Respiration of Germinating Beans __________________________________________________________________________ Objectives: To investigate the aspects of respiration in germinating mung bean seeds. To understand the concept of respiratory quotient. Apparatus and Materials: Syringe capillary tube Gauze colour dye rubber tube beans Introduction: Respiratory quotient Metabolic energy comes primarily from oxidative reactions. As a result, the more highly reduced a respiratory substrate, the higher potential it has for generating biological energy. When a respiratory substrate (eg. glucose) is oxidized for energy, carbon dioxide is produced. The volume (or moles) of carbon dioxide produced with reference to the volume (or moles) of oxygen consumed during oxidation of a respiratory substrate for a fixed period of time is termed as the respiratory quotient (RQ). volume of CO2 produced volume of O2 consumed RQ gives indication of the � type of respiration, � nature of respiratory substrate, and hence � amount of metabolic energy that can be produced.

For example, the complete oxidation of glucose is represented by the following equation: C6H12O6 + 6O2 6CO2 + 6H2O + energy RQ for glucose = 6/6 = 1.0 In general, the lower the respiratory quotient, the more oxygen is required for complete oxidation of a respiratory substrate, and hence the greater the potential for generating ATP from that respiratory substrate.

The table given illustrates some common values and their significance.

When RQ is

>1.0 Carbohydrates are used as a respiratory substrate with some anaerobic respiration occurring simultaneously.

1.0 Carbohydrates are used as the respiratory substrate.

0.7 Mainly fats are being used as the respiratory substrate.

0.8 – 0.9 A mixture of carbohydrates, lipids and proteins are used as respiratory substrate

0.85 A mixture of carbohydrates and lipids are used as respiratory substrates.

0.9 Proteins are the respiratory substrates. Note that the composition of proteins is too varied for them to give the same RQ. However, most of them have a value around 0.9

RQ =

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You are required to investigate some aspects of respiration in germinating green bean seeds, using the apparatus shown in Fig. 1.

Fig. 1

You are provided with a syringe in which soda lime has been placed. Warning: Do not remove the soda lime from the syringe as it will burn your skin. Procedures: 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. 1. You are provided with some germinating green bean seeds and a coloured liquid. 2. Place four or five of these green bean seeds into the barrel of the syringe and carefully

replace the plunger. 3. Attach the length of glass capillary tube to the syringe, using the rubber tubing provided. 4. Dip the end of the glass capillary tube into the coloured liquid so that a drop of liquid

enters the capillary tube. Remove any excess liquid with paper towelling. 5. Place the apparatus on a sheet of white paper alongside a milimeter ruler. Your

assembled apparatus should now look like that shown in Fig. 1. 6. Wait until the drop of coloured liquid starts to move. 7. Mark the position of the coloured liquid on the capillary tube with a marker pen. 8. Measure how far the liquid moves in one minute. Repeat the measurement every minute

for the next nine minutes. 9. If you do not get any liquid movement after 3 minutes, adjust the apparatus (e.g., add one

more germinating seed, readjust the rubber connecting tube and syringe tip etc.). Do NOT touch the point of connection between the tube and glass capillary.

10. If you’ve obtained reasonable readings, you may dismantle the apparatus and dispose the used soda lime (without touching it) in the syringes into the beakers provided.

11. Record your results also on the whiteboard. Results and Discussions: If you are required to prepare an individual report, complete with a write up on procedures, results and discussion. The results and discussion section will be your answers to questions ‘a’ to ‘h’. Questions ‘c’ to ‘h’ not exceed 2 pgs of your report. A conclusion is not required.

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1. Construct a table and record your results of how far the liquid moves in one minute over ten minutes.

2. Plot your results on a piece of graph paper. Use a best fit line. 3. From your table, calculate the mean distance travelled in mm min

-1. Show your working.

4. Assume the diameter of the capillary tube hollow is 0.2 mm. The area of the end of the

capillary tube can be calculated by using the formula πr2, where π = 22/7. Calculate the

volume of gas that is absorbed by the seeds in one hour. Show your working. 5. With reference to respiration of the green bean seeds, explain why the drop of liquid

moves along the capillary tube. 6. The formula used for calculating the RQ (respiratory quotient)is given as follows:

RQ = Vol. of carbon dioxide evolved during respiration Vol. of oxygen absorbed during respiration

Explain how you would use or modify the apparatus in our experiment to calculate the RQ of the seeds.

7. Experiments of this kind are very susceptible to changes in temperature. Explain how you could compensate for any temperature changes during the experiment.

8. Discuss the sources of errors and ways to improve the experiment. You may provide answers other than the ones below as long as you follow this tabulated format.

Sources of error

Explanation Improvement Explanation

This will ensure that the change in volume of air in the syringe is due to oxygen absorbed by the green bean during the experiment.

This will ensure constant rate of respiration as oxygen is not a limiting factor.

This will ensure a more accurate measurement of the rate of respiration of the green beans in the specified environment.

Movement in liquid will be more sensitively detected.

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Assignments Please check with your tutor which option is required of you. Option 1: (please refer to WBLE/ Turnitin for instructions which may incorporate other options below) Option 2: Skills-Based Assessment: Tabulation of quantitative data Tabulate a table on how far the liquid moves in one minute over ten minutes Option 3: Skills-Based Assessment: Graphing of quantitative data Present your graph (pasted from Excel). Use a best fit curve. To get full marks, please observe the guidelines given on pp6-7 as well. No need to write procedure, draw table, write a discussion or conclusion. Option 4: Skills-Based Assessment: Discussion Write your discussion in prose form and without numbering. Excluding your cover page, your discussion and conclusion should NOT exceed ONE A4 page of Word document (standard/ default size). Anything in excess will NOT be graded. • Font Arial, size 11. • Margins: 1 inch from top, bottom, left and right (no need to change if you’re using the standard/ default size when MS Word opens). • Theory to apply: Refer to relevant information from lecture topics which may or may not have been covered yet.

Documents

Cell Biology LabManual Version 8.0 May 2012 1 TRUNCATED