Biology I E-manual

8/10/2019 Biology I E-manual

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UNIVERSITI TUNKU ABDUL RAHMANCENTRE FOR FOUNDATION STUDIES

(STREAM P)

BIOLOGY

FOR PHYSICAL SCIENCEILABORATORY MANUAL


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Table of Contents:

Practical Experiment Description Page

Practical 1Cell Biology Studies I

Writing of lab reports 1

Practical 2Cell Biology Studies II

Identification of biochemicals 9

Practical 3Cell Biology Studies III

Investigation of action of saliva and HCl in twocarbohydrate solutions

15

Practical 4Cell Biology Studies IV

Effects of various treatments on pieces of stainedpotato cells

19

Practical 5Cell Biology Studies V

Determination of solute potential of potato cell sap 23

Practical 6Cell Biology Studies VI

Investigation of the enzymatic effects of materialson hydrogen peroxide

31

Practical 7Cell Biology Studies VII

Respiration of germinating beans 33

Practical 8Cell Biology Studies VIII

Microscopy 36

Practical 9Cell Biology Studies IX

Microscopic examination of cells at various stagesof plant mitosis and meiosis

48

Practical 10

Cell Biology Studies X

DNA, mitosis and meiosis kit 55


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Lab Manual Version 1.0

Foundation in Science (Stream P)1

Practical 1 Writing of Lab Reports__________________________________________________________________________

hy should I bother writing lab reports in the correct way? The FoundationProgramme is designed to prepare you for undergraduate studies at UTAR whichwill 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 willculminate in an official scientific report. Depending on the quality of your report, the goldenchance remains of publishing your report in a scientific journal. Such recognition may opendoors 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.

Use proper A4 foolscap for all handwritten assignments.

Write neatly and legibly in blue or black ink. Your tutor reserves the absolute right toreject your assignment and ask you to re-do the assignment should he/she consider it tobe below the expected quality.

Submit your assignment on time. Late submissions may entail mark deduction or not begraded at all.

Format of a lab report

Your lab report should be preceded by a cover page which contains the following: Name

Partners name

Group

Date

Program

Unit code

Unit description

Year and semester of study

Title of lab report

Lecturers name

Example:

W


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Your lab report should contain the following sections:

Title

Objective

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

Observations and/or results with Discussion

Conclusion

References

The following guidelines on report writing are those required by the actual internationally-recognized scientific community. The text in quotation marks in the following section is takenfrom Warren D. Dolphin of Iowa State University. Credit has been given to the author by citingthe source. This is good practice as opposed to plagiarism, in which copied material isclaimed 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 reportedin this section. The difficulty in writing this section is to provide enough detail for the reader tounderstand the experiment without overwhelming him or her. When procedures from a labbook or another report are followed exactly, simply cite the work, noting that details can befound in that particular source. However, it is still necessary to describe special pieces ofequipment and the general theory of the assays used. This can usually be done in a shortparagraph, possibly along with a drawing of the experimental apparatus. Generally, thissection 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

ResultsThe results section should summarize the data from the experiments without discussing theirimplications. The data should be organized into tables, figures, graphs, photographs, and soon. 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 explainingany symbols, abbreviations, or special methods used. Figures and tables should benumbered 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 tounderstand them without referring to the text. All columns and rows in tables and axes infigures should be labelled.

This section of your report should concentrate on general trends and differences and not ontrivial details. Many authors organize and write the results section before the rest of thereport.


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2.1 Recording Qualitative DataQualitative experiments include those that require observations of non-quantifiable data suchas observations of colour, slides and whole specimens. Below are guidelines on reporting asegment 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 definitionsas studied in secondary school.

KI solution was added to the starch suspension

emulsion of lipid droplets in water

Amount of light penetrating solutionBe careful to distinguish accurately among transparent, translucent and opaque. It is yourresponsibility to look up the definitions as studied in secondary school.

ColourSome descriptions of colour are unacceptable as they are ambiguous.

Light/pale brown, instead of beige Murky/ cloudy white, instead of milky

If theres a change in colouration, you may choose to report as follows.

The initial blue colouration of the mixture turns green, then yellow and may finallyappear 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 toonly 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 mixturemust be left to settle.

Colour of precipitate - green, yellow, brick red precipitate

Amount of precipitate - a little, moderate amount, abundant

Example:When describing observations involving Benedicts test, one should report that when oneshakes the test tube containing Benedicts solution and precipitate, the entire mixture will takethe colour of the precipitate. This colour upon shaking is recorded and also the amount of lightpenetrating solution (transparent/ translucent/ opaque).

Moderate amount of brick red precipitatesuspended in solution, which bore a tinge ofblue. Solution was opaque.

Note: Particles cannot be regarded as precipitate. (e.g. groundnut particles in water.)


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2.2 Recording Quantitative DataQuantitative experiments include those that require observations of quantifiable data such astime, 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 notbe in the table body)

All processed data related to and required for plotting graph must be shown in thetable. E.g. Averages, rate of yeast respiration in terms of no. of bubbles formed perminute.

Precision and decimal places:One must express data according to the precision afforded by the instrument. E.g., if theinstrument can weigh an item as light as 0.1 g, then do not record it as 0.10 g, so as tocorrectly 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 percentageor 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 ofdecimals (e.g., avoid too many decimals 88.8888888 %). Note that the table and graphbelow 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 placesof the raw data.

Decimals arising from processed data involving multiplication and/ or division shouldbe 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 18.72 8.2 9.3 9.0 8.8

1 5.2 4.5 4.8 4.80.5 2.3 1.8 2.1 2.10.1 0.4 0.3 0.4 0.4


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Graph

Plot agraph that will show the trend of the investigation. Include the following in the plotting ofgraph:

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 ofpoints). Hence, one should not extrapolate otherwise it is a claim that a certain yvalue is predicted for a certain concentration.

Avoid clashing headings with clashing units (e.g., headings with two different units grameggs vs. gram nutrients per gram plain feed)

Mass of eggs laid in a week (g)

Amount of nutrients(g/ g plain feed)

0.30 0.25 0.20 0.15 0.10 0.00

Mean 78.0 74.0 69.3 62.7 59. 7 58.0

Average mass of precipitate of standards at various

concentrations of glucose solutions

0

2

4

6

8

10

12

14

16

18

20

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Concentration of glucose solution (%)

Ave. precipitate mass (g)


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2.3 What if I do not obtain desired results?For the purpose of your UTAR lab report, if you dont obtain the desired results, just recordthem as they are. By right, you should repeat it however, you may be constrained by alimited 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 thepossible reasons for the anomaly.

3 Discussion

This section should not just be a restatement of the results but should emphasizeinterpretation of the data, relating them to existing theory and knowledge. Speculation isappropriate, if it is so identified.

Suggestions for the improvement of techniques or experimental design may also be includedhere.

In writing this section, you should explain the logic that allows you to accept or reject youroriginal hypotheses. You should also be able to suggest future experiments that might clarifyareas of doubt in your results.

3.1 General Comments on Style

1. All scientific names (genus and species) must be italicized. Underlining indicates italicsin a typed paper.

2. Use the metric system of measurements. Abbreviations of units are used without afollowing period.

3. Be aware that the word datais plural while datumis singular. This affects the choice of

a correct verb. The word speciesis used both as a singular and as a plural.

4. Numbers should be written as numerals when they are greater than ten or when theyare associated with measurements

6 mm or 2 g

twoexplanations of sixfactors.

When one list includes numbers over and under ten, all numbers in the list may beexpressed 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 thatindicate the purpose of the paragraph. A report or a section of a report should not beone long paragraph.

6. Every sentence must have a subject and a verb.7. Avoid using the first person, I or we, in writing. Keep your writing impersonal, in the third

person. Instead of saying, "We weighed the frogs and put them in a glass jar," write,"The frogs were weighed and put in a glass jar."

8. Avoid the use of slang and the overuse of contractions.


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9. Be consistent in the use of tense throughout a paragraph--do not switch between pastand present. It is best to use past tense.

10. Be sure that pronouns refer to antecedents. For example, in the statement, "Sometimescecropia caterpillars are in cherry trees but they are hard to find." Does "they" refer tocaterpillars 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 ofprecision:

"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 makereference to the values shown on the graph. Explain all the observations or trend obtainedduring the investigation.

As temperature increases from 25oC to 50

OC, 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 inthe experiment.

4 ConclusionState the general trend obtained through the investigation and provides a concise conclusionabout 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 listingshould be alphabetized by the last names of the authors. Different journals require differentformats for citing literature.

For articles:Fox, J.W. 1988. Nest-building behavior of the catbird, Dumetella carolinensis. Journal ofEcology47: 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 theauthor'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 moreauthors are involved, the Latin et al. (et alia)meaning "and others" may be used. A paper bySmith, 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, usuallylast name preceding initials.


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Practical 2 Identification of Biochemicals__________________________________________________________________________

Objective:To identify the components of the solution in its pure form with various food tests and statethe justifications.

Important notice:Any heating that has to be done in the following tests should be carried out in a water bath at95

oC. Direct heating of test-tubes should not be taking place.

Apparatus & Equipments:Test tubes Test tube rackWater bath, 95

oC Spatula

Test tube holder

Materials:Iodine1 M hydrochloric acidSudan IIIStarch solutionCorn oilEgg albumin1% copper sulphate solution

1% sucrose solution1 M Sodium hydroxide1% glucose solutionAbsolute ethanolDCPIP (dichlorophenolindophenol) solutionAscorbic acid

IntroductionThe nutrients in the food you eat supply your body with energy for growth and repair. Theseprinciple substances include carbohydrates, proteins, fats, minerals and vitamins. We can testfor the presence of these important compounds in food by using chemical reagents that reactin predictable ways in the presence of these nutrients.

Please refer to Practical 1 on:

How to record qualitative data.

What to do if you dont obtain the desired results.


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Procedures:

Part 1: Identification of Carbohydrates

(A) Test for reducing sugarsThe reducing sugars include all monosaccharides, such as glucose and fructose, and some

disaccharides, such as maltose and lactose, use 0.1 1% sugar solutions. Common tests forreducing sugars include Benedicts Test (described below) and Fehlings test (not done here).

See basis of test below for explanation of the following rection:

-C-C=O + 2 CuSO4 + 4 NaOH RCOOH + Cu2O + 2 H2O + 2Na2SO4

(blue) red ppt.

Benedicts solution

Benedicts Test for reducing sugars

Procedure* Basis of test Observation

Reducing sugar test

Add 2 cmof any one

solution of the reducingsugar provided to test-tube.Add an equal volume ofBenedicts solution.

Using a test-tube holder,shake and heat at a hightemperature for one minute(a water bath is provided),shaking continuously tominimize spitting.

Benedicts solution containscopper sulphate. Reducingsugars reduce solublealkaline blue coppersulphate containing copper(II) ions, Cu

2+to insoluble

red-brown copper oxidecontaining copper (I). Thelatter is seen as aprecipitate.

Observe and report characteristics of tube contents before and after precipitate settles tobottom of tube, taking note of liquid, colour and precipitate.


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(B) Test for non-reducing sugarsThe most common non-reducing sugar is sucrose, a disaccharide. If reducing sugars havebeen shown to be absent (negative result in above test), a brick-red precipitate in the testbelow indicates the presence of a non-reducing sugar. If reducing sugars have been shown tobe present, a heavier precipitate will be observed in the following test than with the reducingtest if non-reducing sugar is also present.

The proper procedure for testing for an unknown carbohydrate sample for non-reducingsugars involves:

First test for reducing sugars: Benedicts test on the unknown fresh sampleWhy is this step necessary?What results will one get which will cause this step to be called a negative test?

Second test for reducing sugars: Benedicts test on the acid-hydrolysed unknown sampleWhat results will one get which will cause this step to be called a positive test?

Procedure Basis of test Observation

Non-reducing sugar test

Add 2 cmof fresh sucrose

solution to a test tube. Add 1cm

31 M hydrochloric acid.

Using a test-tube holder,heat at a high temperaturefor one minute.

Carefully neutralize withequal volume (1 cm

3) of 1 M

sodium hydroxide.

Finally, add an equalvolume (4 cm3) ofBenedicts solution to theacid-hydrolysed sugarsolution.

Using a test-tube holder,shake continuously tominimize spitting whenheating at a hightemperature for one minute(a water bath is provided).

A polysaccharide ordisaccharide can behydrolyzed to smallercomponent constituents byboiling with 1 M HydrochloricAcid .

Sucrose is hydrolyzed toglucose and fructose, bothof which are reducingsugars and give thereducing sugar result withthe Benedicts test.

Additional InformationThe mixture is likely to bump violently during heating and extra care should therefore betaken. The test is semi-quantitative, that is, a rough estimation of the amount of reducingsugar present will be possible.

The final precipitate will appear green to yellow to orange to red-brown with increasingamounts to reducing sugar. The initial yellow colour blends with the blue of the coppersulphate solution to give the green colouration.

Is the precipitate that of reducing sugar or copper oxide?


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(C) Test for starchStarch is only slightly soluble in water, in which it forms a colloidal suspension. It can betested as a mainly solid in suspension.


Iodine test

Add a few dropsof 1%

cooked starch solution on awhite tile.

Add a few drops of I2/KIsolution (iodine). Be sure tomix them together on the tilewith an object such as yourpen cover.

A polyiodide complex isformed with starch.

Part 2: Identification of Lipids

Lipids include oils (such as corn oil and olive oil), fats and waxes.


Sudan III

Sudan lll is a red dye. Add 2

cm3

of oil to 2 cm3

of distilledwater in a test-tube. Add afew drops of Sudan III andshake.

Fat globules are stained red

and are less dense thanwater.

Emulsion test

Add 2 cmfat or oil to a

Test-tube containing 2 cm3of

absolute ethanol. Dissolvethe lipid by shakingvigorously. Add an equalvolume of cold (or tap) water.

Lipids are immiscible withwater. Adding water to asolution of the lipid in alcoholresults in an emulsion if tinylipid droplets in the waterwhich reflect light and give a

white, opalescentappearance.


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Part 3: Identification of Proteins

A suitable protein for this test is egg albumen.


Biuret TestAdd 2 cm

protein solutions

to a test tube. Add an equalvolume of 5% sodiumhydroxide solution and mix.

Add 2 drops of 1% coppersulphate solution and mix.No heating is required.

A test for peptide bonds. Inthe presence of dilutecopper sulphate in alkalinesolution, nitrogen atoms inthe peptide chain form apurple complex withcopper(II) ions, Cu

2+.

Biuret is a compoundderived from urea whichalso contains the CONHgroup and gives a positiveresult.

Part 4: Identification of Vitamin C (ascorbic acid)

This test can be conducted on a quantitative basis if required, in which case the volumesgiven below must be measured accurately.


DCPIP test

Using 0.1% ascorbic acidsolution as a standard.

Add 1 cm3of DCPIP

solution to a test-tube.

Fill a 2 cm3pippette with

0.1% ascorbic acid.

Add the acid to the DCPIPdrop by drop until ithe bluecolour of the dye justdisappears.

Note the volume of ascorbicacid solution used.

DCPIP is a blue dye whichis reduced to a colourlesscompound by ascorbic acid,a strong reducing agent.

Additional InformationShaking the solution would result in oxidation of the ascorbic acid by oxygen in the air. Theeffects of shaking could be investigated.


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Practical 3 Investigation of Action of Saliva and Hydrochloric Acid in TwoCarbohydrate Solutions

__________________________________________________________________________

Objective:Students are expected to state the objective of this experiment.

Apparatus & Equipments:Boiling tubes Metal test tube racks BeakerGraduated plastic dropper Water bath, ~ 37

oC Water bath, ~ 95

oC

Materials:Carbohydrate solution A Carbohydrate solution B Benedicts solution3 M Hydrochloric acid 3 M Sodium hydroxide

Procedures:1. Prepare two boiling tubes containing 1 ml solution A and 1 ml solution B respectively.

Add 1 ml Benedicts solution to each tube. Heat both tubes together in the (~95oC)

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

2. Add a few drops of fresh solution A and B separately spaced on a white tile. On eachsolution, add 1-2 drops of iodine solution. Mix with pen cover. Record your observationsin the table 1.

3. Pipette 2 ml solution B into each of four boiling tubes. Label the tubes 1, 2, 3 and 4respectively near mouth of tube. Label your group name.

4. Place tubes 1 and 2 in a water bath of ~37oC. (It doesnt matter how long you put it in at

this stage as no saliva or HCl have been added yet).

5. Salivate into a small beaker till it reaches about 5 ml.

6. Step (6) and (7) is to be done approximately at the same time. Measure out 4 ml of thesaliva prepared in step (4) and pipette 2 ml each into tubes 1 and 4. Shake the contentsof the tubes well to ensure thorough mixing.

7. Measure out 4 ml HCl and pipette 2 ml each into tubes 2 and 3.

8. Let tubes 1, 2, 3 & 4 incubate at their respective temperatures (see Table 2) for 35minutes from this moment.

9. Label 4 more new boiling tubes as follows: 1, 2, 3 and 4.

10. After 5 minutes of incubation of tubes 1 to 4, pour out about half of the contents from allthese tubes into the respective newly labelled tubes (e.g., 1 into 1, 2 into 2 etc.).Straightaway, place back the original tubes (labelled 1 to 4) back into the respectivetemperatures of incubation.

11. Neutralize the acid in each of tube labelled 2 and 3 with 1 ml of sodium hydroxide

(each). Shake each tube (2 and 3) to ensure uniform mixing.

12. Perform Benedict test on the contents of tubes 1 to 4 by pipetting 2 ml of Benedictsolution into each tubes and heating them in 95

oC water bath for one minute. Record

your observations in Table 2.

13. After 35 minutes of incubating tubes 1 to 4, neutralize the acid in each test tube labelled2 and 3 with 1ml of sodium hydroxide. Why is neutralization necessary?

14. Carry out Benedicts test with an equal volume of Benedicts solution (2 ml) for eachtube. Remember to heat your sample. Record your observations in table 2.


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Note:No penalization for unexpected results. Refer to Practical 1 on writing lab reports if you dontobtain the desired results.

Table 1:

Observations Conclusions

Solution A

Benedicts test:

Iodine test:

Solution B

Benedicts test:

Iodine test:

Table 2:

Tube ContentsTemp.

(C)

Benedicts TestColour Observation

After 5 min of incubation(1 4)

After 35 min of incubation(1 - 4)

1 2 ml solution B2 ml saliva

37

2 2 ml solution B2 ml HCl

37

3 2 ml solution B

2 ml HCl

95

4 2 ml solution B2 ml saliva

95


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Guidelines:

ObservationsFor Benedicts test and Iodine tests, please follow guidelines on writing lab report on thefollowing:

o Liquid

mixture, solution, suspension, emulsion? transparent, translucent, opaque?o Colour

state initial and final colours?o Precipitate

colour of precipitate? amount of precipitate?

ConclusionsAbsence/presence of what type of carbohydrate?

Results and Discussions:1. Ensure that the guidelines for constructing tables and recording results for this experiment

are adhered to.

2. The discussion sections of your report should not exceed 2 pages.

3. Discussions should contain:1) Name of enzyme involved.

2) Specific action(s) of enzyme involved.

3) Effect of temperature on enzyme structure (bonds, active site etc.)

4) Effect of HCl on solution B.

5) Product identification : make suggestion(s).

6) Product structure and chemical classification.

7) Chemical bases of tests used.

8) Which carbohydrate is more complex, A or B? Give a reason.

4. Conclusion: Summary of results.


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Flow chart:

1 2 3 4

Solution B2 ml

Solution B2 ml

Solution B2 ml

Solution B2 ml

Incubate at 37C 37C

Mix with 2 ml saliva 2 ml HCL 2 ml HCL 2 ml saliva

Incubate at 37C 37C 95C 95C

After 5 mins Remove tube 1, 2, 3, 4 from water bath.

Pour out forBenedicts test

2 ml 2 ml 2 ml 2 ml

Pour to 1 2 3 4

Place tube 1,2,3,4 back into water bath for continuos incubation

NaOH 1 ml 1 mlBenedictssolution

2 ml 2 ml 2 ml 2 ml

Heat for 1 minRecord observation at Table 2 (After 5thmin)

After 35 mins Remove tube 1, 2, 3, 4 from water bath.

Remainingcontent toperformBenedicts test

2 ml 2 ml 2 ml 2 ml

NaOH 1 ml 1 ml

Benedicts

solution

2 ml 2 ml 2 ml 2 ml

Heat for 1 minRecord observation at Table 2 (After 35thmin)

Results

(After 5 mins)

Results

(After 35 mins)


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Practical 4 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:Pen knife PotatoWhite tile Methylene blue solutionForceps 50% ethanol8 test tubesPetri dishBeakerStop watch

Procedures:

1. Cut five cubes from the potato provided, each approximately 10 mm x 10 mm x 5 mm.Trim off any peel which is still attached.

2. Place the potato cubes in a small beaker, immerse them in methylene blue solution for 10minutes. Use only enough methylene blue to cover them.

3. After 10 minutes, pour off the methylene blue solution and wash the cubes with tap wateruntil the water contains little or no stain. Then cover the cube with tap water.

4. Label four test tubes A, B, C and D. To each of the tubes A, B and C, add 5cm3 of

distilled water, to tube D add 5 cm3of 50% ethanol.

5. Place tube A in boiling water, tube B in a water bath of 38oC to 42

oC and tube C and D at

room temperature.

6. After 5 minutes, add one stained potato cube to each of the four test tubes. Start the stopwatch immediately.

7. After 2 minutes, remove tubes A and B from the water baths and place them in the rackwith tubes C and D. Shake the tubes.

8. Separate the tissue from solutions. Do this by pouring away the liquid into another thecorresponding test tubes labeled A to D.

9. Record your observations of each test tube.

What is the texture and colour of the tissue? What is 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?

Results:

Table 1:

Tube Intensity of blue colouration in liquid(arbitrary unit)

Observation of potato tissue

ABC

D

Arbitrary units: 5 most intense, 1 least intense


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Discussions:From the data you have collected, account fully for the observations you have made and drawclear conclusions, using your knowledge and understanding. Use the following questions asguidelines.

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

2. Why are the potato cubes stained with methylene blue (why is a colour stain chosen)?

3. What is the purpose of having tube C placed in water at room temperature?Why isnt tube D placed at high temperature?

4. What happen to the stained potato cubes when they are placed in water at roomtemperature?

5. What are the effects of temperature on potato cubes in tubes A, B and C?

6. What are the effects of the ethanol on the potato cubes?

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

8. Explain the significance of the 5 minutes incubation before adding the potato cubes.

9. Explain the significance of the 2 minutes incubation after adding the potato cubes.

10. Explain the significance of separating the tissue from solutions after the 2 minuteincubation.


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Name : .........

Group :

Partial Report

Practical 4 Effects of various treatments on pieces of stained potato tissues

Results:

Table 1Tube Intensity of blue colouration in liquid

(arbitrary unit)Observation of potato tissue

AB

CD

Arbitrary units: 5 most intense, 1 least intense

Discussions:

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

2. Why are the potato cubes stained with methylene blue (why is a colour stain chosen)?

3. What is the purpose of having tube C placed in water at room temperature?Why isnt tube D placed at high temperature?

4. What happen to the stained potato cubes when they are placed in water at roomtemperature?

5. What are the effects of temperature on potato cubes in tubes A, B and C?


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6. What are the effects of the ethanol on the potato cubes?

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

8. Explain the significance of the 5 minutes incubation before adding the potato cubes.

9. Explain the significance of the 2 minutes incubation after adding the potato cubes.

10. Explain the significance of separating the tissue from solutions after the 2 minuteincubation.


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Practical 5 Determination of the Solute potential of the Potato Cell Sap__________________________________________________________________________

Objectives:To prepare sucrose solutions of various concentrations from a stock solution of 1.0 M bydilution technique.

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

Introduction:

Water potential,

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

Water potential (denoted by the Greek letter, psi)is used to describe combined effectsof concentration and pressure in a solution on the tendency of water molecules to diffuseacross a membrane.

Water potentialis defined as the net tendency of water to diffuse out of a solutionby osmosis

It is 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

= s

+ p

where = water potential of the cell(its a -ve pressure, i.e., inward force like that created onsolutions or water when the plunger of a syringe is pulledto draw liquid up

1; see picture arrows)

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

arrows)

p = pressure potential, due to wall pressure (its a +ve pressure;outward force like that created on solutions or water whenthe plunger of a syringe is pressed to expel liquid; see picturearrows)

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 negativeits solute potential

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


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Turgor pressure p

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 isdirectlyproportional to pressure.

Physical pressure - pressing the plunger of a syringe filled with water, forexample - 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 waterdue to the presence of solutes or even forcing the water from the solution todiffuse into the compartment with pure water.

It is also possible to create negativepressure, or tension when theplunger of a syringe is pulled up.

If a 0.1 M solution is separated from purewater by a selectively permeable membrane,water will move by osmosis into the solution.

Water will move from the region ofhigher psi (0 MPa) to the region oflower psi (-0.23 MPa). (Fig. 36.3a)

If a 0.1 M solution (psi = -0.23 MPa) isseparated from pure water (psi = 0 MPa) by aselectively permeable membrane, then waterwill move from the pure water to the solution.(Fig. 36.3d)

Application of physical pressure can balanceor even reverse the water potential (Fig. 36.3).

A negative pressure can make water potentialmore negative (Fig. 36.3d).

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

Fig. 36.3. Values for & sin the left and rightarms of the U-tube respectively are given forinitial conditions, before any net movement of

water. (a) The addition of solutes makes waterpotential more ve. (b,c) Application of physicalpressure increases water potential. (d) A vepressure (tension) decreases water potential


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Conclusion:

State the:1. 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).

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

3. Relationship between change in length and sucrose concentration


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Practical 6 Investigation of the Enzymatic Effects of Materials on HydrogenPeroxide Solution

__________________________________________________________________________

Objective:

To investigates the enzymatic effect of various materials in the hydrogen peroxide solution.

Apparatus & Equipment:

Beaker Boiling tubesWater bath Pen knifeGlass rod Parafilm

Materials:

Fresh Liver Potato cubesManganese dioxide Hydrogen peroxide**Wood splints

**Caution: Hydrogen peroxide is formed continuously as a by-product of chemicalreactions in living cells; it is a very toxic (poisonous) substance.

Procedures:Wear gloves when handling liver tissue. One pair of gloves per group to prevent wastage.

1. Label six fresh empty boiling tubes 1, 2, 3, 4, 5, 6.

2. Cut the liver provided into three pieces of roughly 0.8 cm x 0.8 cm x 0.5 cm.

3. Place one piece of liver into tube 1.

4. Place the second piece of liver into the tube 2. Place tube 2 in the boiling or water forabout five minutes. Remove from boiling water and let cool.

5. Put the third piece of liver into test tube. Mash it gently into a pulp with glass rod.

6. Cut potato cubes of roughly 0.8 cm x 0.8 cm x 0.5 cm. Place one cube into a tube 4.

7. Measure two portions of 0.5 g of manganese dioxide powder. Pour each portion into tube5 and tube 6.

8. Put tube 6 in boiling water for five minutes. Remove from boiling water and let cool.

9. Prepare another six fresh empty test tubes. Put 5 cm3

of hydrogen peroxide into each ofthem.

10. Quickly add hydrogen peroxide into the test tubes 1, 2, 3, 4, 5, and 6.

11. Using the parafilm provided, stretch it quickly and seal the mouth of the test tubes.

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

13. Insert a glowing splint (flame extinguished but glow remains) into each tube one at a timeby just penetrating the parafilm with it.

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


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14. Record all your observations in the table. Record your observations on each tubeimmediately after the reaction has started.

Table 1:

TubeContents with 5 cm

3

hydrogen peroxide

Observations

Before using wood splint After using wood splint

1Fresh liver

2 Boiled liver(cooled)

3Pulped liver

4 Potato cube

5 Manganese dioxide

(untreated)

6Boiled manganesedioxide(cooled after heating)

Washing upThoroughly wash and scrubbed all apparatus containing liver pieces with Dettol solutionprovided to rid it of unpleasant odours.

Results and discussions: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 potatocubes? Account for these differences.

6. What were the differences between the effects on the reaction of boiling the liver andheating the manganese dioxide? Account for these differences.


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Practical 7 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 tubeGauze Colour dyeRubber tube Beans

Introduction:

Respiratory quotientMetabolic energy comes primarily from oxidative reactions. As a result, the more highlyreduced 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) ofoxygen consumed during oxidation of a respiratory substrate for a fixed period of time istermed as the respiratory quotient (RQ).

volume of CO2producedvolume of O2consumed

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 completeoxidation of a respiratory substrate, and hence the greater the potential for generating ATPfrom 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 anaerobicrespiration 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 proteinsis too varied for them to give the same RQ. However, most of them have avalue around 0.9

RQ =


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Procedures:

1. You are provided with a syringe in which soda lime has been placed, some germinatinggreen bean seeds and a coloured liquid.(Warning: Do not remove the soda lime from the syringe as it will burn your skin)

2. Place four or five green bean seeds into the barrel of the syringe and carefully replace theplunger.

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 liquidenters the capillary tube. Remove any excess liquid with paper towelling.

5. Place the apparatus on a sheet of white paper alongside a milimeter ruler. Yourassembled apparatus should look like the figure below.

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 the distance the liquid moves in one minute. Repeat the measurement everyminute for the next nine minutes.

9. Record your results.

10. If you do not get any liquid movement after 3 minutes, adjust the apparatus (e.g., add onemore germinating seed, readjust the rubber connecting tube and syringe tip etc.).

Do NOT touch the point of connection between the tube and glass capillary.

Results and Discussions:

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

2. Plot your results on a 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 thecapillary tube can be calculated by using the formula r

2, where = 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 liquidmoves along the capillary tube.


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

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

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

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

8. Discuss the sources of errors and ways to improve the experiment.

Sources oferror

Reasoning Improvement Explanation

This will ensure thatthe change involume of air in thesyringe is due tooxygen absorbed by

the green beanduring theexperiment.

This will ensureconstant rate ofrespiration asoxygen is not alimiting factor.

This will ensure amore accuratemeasurement of therate of respiration of

the green beans inthe specifiedenvironment.

Movement in liquidwill be moresensitively detected.


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Practical 8 Microscopy

Experiment 1 The microscope and Its Uses__________________________________________________________________________

Objective:

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

Introduction: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 becomefamiliar with the parts of the microscope and the procedures in the handling of it. Treatyour microscope well and it will serve you well.

Apparatus and Materials:Binocular Microscope Cover slipsMicroscope slide NewspaperPlastic millimeter ruler

Setting up the Microscope:The microscope when not in use is usually kept in a case. Remove it by grasping thehandle arm while placing one hand under the base. Set it down gently on thelaboratory table and at a reasonable distance from the table edge. Always keep themicroscope upright in the vertical position and never touch any of the lens surfaceswith the fingers since it will deposit a thin film of oil on the glass.

Parts of the Microscope:


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Component Function

Arm For lifting and carrying the microscope.Base To provide stability.Body tube To house the lenses.Eyepieceor ocular lenses

This is a set of lenses that rests loosely at the top end of the body tube. It is obviousthat 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 theocular.

Revolvingnosepiece

Located at the lower end of the body tube, it carries 3 objectives of different lengths.Rotating this part changes the magnification of the objectives.

Objective lenses They are of different magnifications with the following visible properties:

Objectives Magnification Length Lens opening

Scanning lens 4x Shortest Widest

Low power lens 10x short wide High power lens 40x longest Narrowest

Focusingadjustments

These comprise two knobs located on either side of the microscope which are used tochange 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 muchcloser to the object than when it is under low magnification.

Coarse adjustment Made by the large knob beside the body tube forfocusing under low power magnification.

Fine adjustment Made by the small knob, which is for focusing underhigh power magnification and accurate focusing.

Precautions when using the focusing adjustments: Turn both adjustment knobs at the same time. Do not overturn the adjustment knobs (i.e. do not force them to go

beyond their limits) Do not use the coarse adjustment knobs when focussing under the

40x objective lens.

Stage This is the platform for slides and specimens to be viewed under the microscope.Mechanical stage This movable portion of the stage is attached to the specimen holder and allows the

slide to be moved in different directions to facilitate viewing.Specimen holder This holds the glass slide in place.Vertical feed knob Rotating this moves the glass slide in the vertical direction.Horizontal feedknob

This moves the glass slide in the horizontal direction.

Condenser Located just beneath the stage of the microscope, it incorporates a lens whichcollects light on the stage to bear on the object.Iris diaphragm A rotating disk under the stage. This diaphragm is used to vary the amount light that

is projected upward into the slide.Built-in lightsource

This is situated below the iris-diaphragm to provide light for illuminating the object. Itcan be switched on or off.

Brightnessadjustment knob

This provides adjustment to the illumination brightness.

Main switch This ensures that power is turned on or off.


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Preliminaries before Use:1. Use the coarse adjustment to raise the body tube so that the objective can clear the

stage when the revolving nosepiece is turned.

2. Turn the nosepiece until the scanning objective is in-line with the eyepiece. You shouldhear a soft click or else feel a distinct falling into place as the objective moves into

position. If not, the field of view is totally dark or an illuminated crescent instead of acomplete circle.

3. Turn the diaphragm to its largest opening.

4. Look into the eyepiece and make a final adjustment to the light adjustment knob sothat the field of view (i.e., the lit circle which you see) is evenly illuminated. Any glareshould be removed by adjusting the diaphragm.

5. Should either of the lenses appear dirty, wipe it gently with a piece of special lenspaper. Use a circular motion with very light finger pressure.

6. The microscope is now ready for use.

7. Position it so that the stage faces you.

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

9. Ensure that the microscope stage is at its lowest position. This will prevent breaking ofslides and lenses by mistake when adjusting the objectives by moving the stage with thecoarse adjustment knob.

Preparation of Wet Mount:

Materials for microscopic examination are usually placed on the glass slide ofstandard size, the microscope slide. The materials are then covered by small thin

piece of glass, the cover slip. Both microscope slide and cover slip should be very

clean before use.

Cleaning microscope slidesHold the microscope slide by the edges between the index flinger and the thumband dip in water. Then wipe dry using a soft tissue or a clean piece of cloth.

Cleaning cover slipsCover slips are very fragi le and need careful handling. Hold a cover slip by the edgesbetween the index finger and the thumb and then dip in water. To wipe dry insert thecover slip into the fold of a piece of clean cloth or lens paper and apply gentlepressure between the finger and thumb to both surfaces at the same time. Use agentle cir cular wip ing motion for of effective cleaning.

REMEMBERAlways handle glass slides and cover slips bytheir edges, never by their flat surfaces.


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Exercise 1 Focusing the Microscope - e slide

1. Prepare a microscope slide to view the letter e. Cut ou t the letter e from a piece ofnewspaper.

2. Place the tiny piece of newspaper in the centre of the slide with the printed side

up.

3. Add one drop of water onto the newspaper using a dropper.

4. Pl ac e t he cover slip carefully over the newspaper.

Hold the cover slip about 45 to the slide, let it slip down the slide till the lower edge touches thewater, and then slowly lower the cover slip down onto the slide.

If this is done properly, the remaining water should spread out evenly with minimum formation ofair bubble between cover slip and slide.

Some air-bubbles may still be trapped even after the most careful preparation. If so, gentletapping of the cover slip with a pencil point may help remove them.

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

6. Carry out the observations as follows:

Compare the position of image as seen through the eyepiece with that of the printedletter as seen with the unaided eye. Does the image appear to be reversed (i.e. asit would appear if seen in a mirror)?

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

moves. Repeat right to left.

Move the slide away from yourself and describe observe the movement of theimage again.

Exercise 2 Using a higher power objective

1. Great care must be taken when using higher power objectives. DO NOT focus the highpower objectives with the coarse adjustment knob or youll risk breaking the slide andlenses.

2. Most microscopes have parfocal objectives. If one switches from viewing a specimen in

sharp focus under a lower power objective to a higher one, the object shouldautomatically come approximately into focus. Only slight further focussing with the fineadjustment knob is required to see the specimen clearly.

3. When switching to the next higher power objective, look from the side of the microscopeand move the revolving nosepiece slowly till that higher power objective clicks intoposition. Be careful that it does not touch the slide.

4. Take care that the lower end of the high power objective does not touch the cover slip. Ifthis happens, you must repeat the whole procedure focusing again, starting with thescanning objective.


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Exercise 3 Measurement with a Microscope

The unit of length used in nearly all microscopic measurement is the micrometer (um) whichequals 1/1000 mm. A simple way to gauge the size of an object viewed under the microscope is todetermine first the size of the circular field to view. We then use this measurement to approximatethe actual size of the object being viewed.

(A) Estimation of scanning field of view1. Place a small plastic millimeter ruler on the stage.

2. Focus under the scanning objective so that a clear image of the millimeters divisions isobtained.

3. Adjust the ruler so that the marked edge passes through centre of the field view.

4. Count the number of millimeter divisions seen within the field of view from one side to theopposite side. Record of the diameter of the scanning field of view in both millimeters andmicrometers.

Diameter of the scanning field of view = _______ mm

= _______ m

(B) Estimation of low power and high power field of viewWe can find the low power field of view by a simple calculation. Divide the magnificationnumber of the low power objective being used by that of the scanning objective. Next, dividethe diameter of the scanning field (as estimated previously) by this quotient. This gives thediameter of the low power field of view.

Example:Scanning objective magnification = 4 xLow power objective magnification = 10 x

Quotient = 10 4= 2.5

Diameter of scanning (4 x) field = _____ m

Diameter of low power (10 x) field = _____ 2.5

= _____ m

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

2. Replace the slide with the letter e onto the stage and re-examine the letter e. Comparethe height of the letter with diameter of the field of view.

3. Give an estimate of the actual height of the letter in both millimeters and micrometers.


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Exercise 4 Magnification and Resolution

(A) Magnification Power:The total magnification is the magnification of the eyepiece lens multiplied by themagnification of the objective lens. By using different combinations of lenses, differentmagnifications 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 poorillumination. Also, the larger the region of the object viewed, the easier it is to interpret whatyou see.

(B) Resolving Power:This following exercise illustrates to us the resolving power (or resolution) of a microscopewhich is the ability to separate fine details to seen in the object. For most us, for example, twodots separated by less than 0.1 mm will appear as a single dot. The microscope thereforedoes two things for us it magnifies and it allows for finer resolution.

1. Prepare a wet mount using a piece of magazine photograph. Use the same procedure asfor e slide.

2. Examine the wet mount under low power (begin with scanning objective first) and observehow the image compares with the photograph when seen with the unaided eye.

Oil Immersion:If you require a particularly high magnification, immersion oil may be used. Fluid with thesame refractive index as the objective lens is placed between a special objective lens and thecover slip so that it touches both. The fluid permits a larger cone of light rays to enter theobjective from the specimen, and this increases the resolving power obtainable.

Note:If your microscope comes with a 100 x objective, please DO NOT use it. Used the improperway, it will break.

Microscope Care:1. Turn the resolving nosepiece until the scanning objective is in position.2. Adjust the boy tube so that the lower end of the objective is about 1 cm above the stage.3. Ensure that the stage surface is clean and dry.4. Return the microscope in an upright position to its storage case.


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Experiment 2 Preparation of Microscopic Slides__________________________________________________________________________

Objective:To study the microscopic structure of biological samples and to learn the preparation ofbiological samples for microscopic study purposes.

Introduction:Examination of biological materials under the microscope will usually entail long periods oflooking into the eyepiece. It is useful to develop the habit of keeping both eyes open andrelaxed, as though you were looking at a distant object. This will cut out eye-strain caused bycontinual forcing of one eye to remain closed.

Apparatus and Equipments:Binocular Microscope Cover slipsMicroscope slide Soft tissue papers (lens cleaner)Forcept

Materials:Potato OnionHair IodineSafranin

Observation of Onion Cells:The onion scale leaf has generally two major surfaces an outer surface which faces theexterior and an inner surface which faces the interior of the onion. The outer surface mayhave pigmented portions of its outer epidermis while the inner surface may not.

(Mackean, D. G., 1973. Introduction to biology, p. 25.)

Scale leaf

Toward interior toward exterior


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Exercise 1 Preparation of microscopic slides

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

2. Bend the onion scale leaf towards the outer epidermis until it breaks on the uppersurface.

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

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

5. Using a dropper, place 1-2 drops of water on the slide and place the epidermis (~5mm x5mm) on the water.

6. Get rid of air bubble if there is any. Why are bubbles undesirable?

7. Slowly lower the cover slip onto the slide.

Some air-bubbles may still be trapped. If so, gentle tapthe cover slip with a pencil point to remove them.

8. Remove excess water from on top or around the cover slip with a piece of tissue paper.

9. The mounting of a specimen on a slide with solution is called a wet mount. Avoid tiltingthe microscope when using a wet mount.


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Exercise 2 Viewing the slides

1. Place the slide carefully on the stage. Position the specimen in the centre of the hole inthe stage and also in the middle of the circle of light emanating from the lamp throughthe stage hole.

2. Ensure that the scanning objective is in place by moving the revolving nosepiece.(If not, the field of view is totally dark or an illuminated crescent instead of a completecircle.)

3. Slide the eyepieces horizontally to the maximum length away from each other. Place yourhead just above the eyepieces. Slowly, slide the eyepieces towards each otherhorizontally 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 oneilluminated circular field of view. If not, youll see two overlapping illuminated circles.

4. Adjust the brightness adjustment knob to give the right amount of light for viewing theobject clearly.

5. Looking down the eyepiece, slowly adjust the position of stage with the coarseadjustment knob until the object comes into focus. Focus accurately by using the fineadjustment knob.

6. Keep both eyes open when viewing through the eyepiece. Get accustomed to using botheyes otherwise this will strain your eye or give you a headache over time.

7. Once the object is in sharp focus, its time to view it at higher magnification.

8. Never to lower the body tube while looking into the eyepiece and using the coarseadjustment. If you miss the image, look up and repeat the whole procedure of focusing.

9. For viewing under every objective lens, use the fine adjustment to sharpen the focus ofthe specimen.

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

11. Make a drawing of 4 6 cells, each 2 3 cm long. Include only the details you canobserve in your preparation. Label accordingly.

Are all the cells identical in shape and size? Is the nucleus located in the same position in all the cells? Suggest reasons to explain any apparent differences in the shape and size of the cells

as well as the location of the nucleus.

Notes:The lines that form the network between individual cells are non-living cell walls made up chiefly ofcellulose. 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 cellsis taken up by a vacuole filled with a fluid made up mostly of water and various salts. The nucleusappears as a dense body in the translucent cytoplasm.

12. Turn again to the scanning objective and remove the slide from the stage.


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[Additional practice tasks if time permits]

Exercise 3 Observation of Starch Grains

1. Place a small piece of potato in the centre of the slide and rub to distribute the potatojuice 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 ofavoiding 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 theiris diaphragm is decreased. This will increase the contrast between the starch grains andthe surrounding water.

4. Move the slide on the stage until you locate a field in which the grains are well separated.Make a drawing of 4 6 starch grains to illustrate their typical shape.

5. After completing your drawings, turn again to the scanning objective and remove theslide.

6. Stain the grains with iodine using the technique of irrigation.

6. Examine the iodine-stained mount first under the scanning objective and then under lowand high power. Draw 4 6 typical starch grains to illustrate their shape and structure.

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 gentlyrotated 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 preparationcarefully.

What observable changes may be seen in the starch grains exposed to relatively high

iodine concentration? What observable differences are there between these starch grains when compared to

those exposed to lower iodine concentration? Can the internal grain structure better observe in strained grains or unstained ones?

8. Biological materials are often stained before examination under a microscope. Based onyour experience in this exercise suggest reasons for such use of stains.


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Practical 9 Plant Mitosis and Meiosis

Experiment 1 Microscopic Examination of Cells at Various Stages of Plant Mitosis_________________________________________________________________________

Objective:

To examine the cell at various stages of the mitotic cell cycle microscopically.

Equipment:Binocular microscope

Slides provided:Onion mitosis Root tip, Allium l.s.Onion mitosis Root tip, Allium c.s.

Introduction

The primary root system

The meristematic zones radiate from a clump of cells called the quiescent centre situatedimmediately behind the root cap. The significance of the quiescent centreis not as yet fullyunderstood. Its cells divide slowly and it is probably the site from which the other meristematiclayers arise.

Tissue differentiation in the root.

Differentiation of vascular tissue begins near the root apex. Several strands of sieve-tubeelements and companion cells appear near the outside of the procambial strand. Shortlyafterwards a similar number of strands of protoxylem cells alternating with the primary phloemstrands differentiate, Metaxylem cells differentiate last of all at the centre of the procambialstrand. The outermost procambial cells undergo litter change and retain their ability to divide.They become the pericyclewhich may later produce lateral roots.

The apical meristem of the root.

The most obvious differences in appearancebetween longitudinal section of stem and rootapices is the absence of bulges comparable to leafand bud primordial on the root apex.

The root apex is also covered by root cap. Thereis, however, a marked similarly in appearance andbehaviour of the apical cells which constantly divideby mitosis, in most roots it is possible to distinguisha number of zones of cells at the apex.

The outermost zone is called the protoderm. Itproduces cells which differentiate into the root

epidermis and root cap. Inside the protoderm is theground meristem, the derivatives of whichdifferentiate into the root cortex. Just behind theroot apex a single procambialstrand can be seenat the centre of the root. Some roots have anadditional meristematic layer, the calyptrogen,which gives rise to the cells of the root cap.


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Biological Drawing1. Make one detailed drawing showing cells undergoing all mitotic phases.2. Label the mitotic phases.

Discussion:1. From the slides viewed, which is the most frequently observed phase? Why?2. From the slides viewed, which is the least frequently observed phase? Why?3. Figure 1 shows drawings of cell at various stages in the mitotic cell cycle.

Figure 1

a) List the letter shown in Figure 1 in the order in which these stages occur during a mitotic

cell cycle. The first stage has been entered for you.

A _____ _____ _____ _____

Explain what is happening in stage D in Figure 1.

b) Describe in outline what happens to the DNA in the nucleus during stage A in Figure 1.

c) State the importance of mitosis in the growth of a multicellular organism, such as aflowering plant or a mammal.


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Experiment 2 Microscopic Examination of Cells at Various Stages of Plant Meiosis

Slides provided:

Lily Anther early Prophase c.s.

Lily Anther late Prophase c.s.Lily Anther First Meiotic division c.s.Lily Anther second Meiotic division c.s.Lily Anther Pollen Tetrad

Introduction:Unlike asexual reproduction, where only single parent is involved and where offspring areidentical in hereditary characters to the parent, sexual reproduction involves production ofmale and female gametes in specialized organs. Fusion of the nuclei of these gametes resultsin the formation of the zygote that ultimately develops into the offspring showing acombination of characteristics from both parents.

Figure 1: The life cycle of a flowering plant


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Figure 2: Female gametophyte within an ovule

Exercise 1 Lily Flower Bud (TS)

This slide should be examined with the naked eye and then under low power.

Note the following: There are 6 stamens (anthers specifically), each a 4-lobed structure. Note the pollen grains

within each lobe. The 3-loculate (i.e., chamber of three parts) ovary in the centre with the ovules.

Biological Drawing1. Prepare a tissue map.

2. You may draw more than one ovule. However, you need only label one ovule.

Figure 3: Transverse section of flower bud


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Exercise 2 Lily Anther 2-cell Stage (TS)

This is a dehisced stage of the anther. Comparison with the 4-celled stage seen in thepreceding slide should be made. Dehiscence is usually preceded by breakdown of thepartition between the locules of one half.

Note the following:1. The bilayered wall of each locule made up of the outer epidermis and an inner fibrous

layer (endothecium).2. The stomium or opening from the break is slit-like.3. The single vascular bundle (connective) found in between the two lobules.4. The thick exine of each pollen grain. In many of the pollen grains two nuclei will be seen

the vegetative and generative nuclei.

Biological Drawing1. Make a detailed drawing with labels such as the anther filament, wall-tissue, pollen sac

and grains. (Use an appropriate objective lens)

Figure 4: Lily anther Figure 5: Microspore tetrads in lily anther.

Figure 6: Second division in microsporocytes of a lily microsporangium.


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Exercise 2 DNA replication modelling__________________________________________________________________________

Objective:To study and learn to construct a replication bubble using DNA Simulation Student kit

Apparatus and Material:44 Red beads phosphate 13 Yellow beads Thymine/ Uracil44 White beads Deoxyribose sugar 13 Green beads Guanine13 Orange beads Adenine 13 Blue beads Cytosine8 Pink beads Ribose sugar 24 Clear connectors Hydrogen bonds

Introduction:The structure of DNAA nucleotide consists of the pentose sugar deoxyribose, a phosphate, and one of fournitrogenous bases. The nucleotides are linked by covalent bonds to form an alternatingsugar-phosphate backbone. No matter how long the chain may be, the 5 end has a 5 carbonattached to a phosphate and the 3 end has a 3carbon attached to a hydroxyl group.

DNA replicationReplication starts at origins of replication, where the two DNA strands are separated, forminga replication bubble. DNA polymerases add nucleotides only to the free 3 end of a growing

strand or RNA primer. Thus, a new DNA strand can elongate only in the 5 to 3 direction.

Procedure:

1. Group yourselves into 2 to 3 students per group.

2. Construct a single stranded DNA with the base sequence as follows:5 AGCACGTAACGTTCGA 3

3. Construct the complementary DNA strand based on the base sequence shown in Step 2.

4. Join the two strands together by using clear connectors (hydrogen bonds).

5. Slightly twist the DNA molecules to observe the double helix structure of DNA.

6. Lay the DNA molecule flat on your bench.

7. Break open 12 base pairs from the two strands in the middle (origin of replication) to form

a replication bubble.

8. Attach a 2-nucleotide RNA primer (use pink beads to represent ribose sugar) to each ofthe leading strand and lagging strand.

9. Add the respective DNA nucleotides one by one based on the template strand.

10. Before dismantling your completed DNA replication bubble, show and identify thetemplate strand, leading strand, lagging strand, Okazaki fragments and replication fork toyour instructor.


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Discussion:1. Based on the procedure, name the enzymes that participate in:

a) Step 7b) Step 8c) Step 9

2. Predict how long it will take to produce a 100-nucleotides-long DNA if the elongationprocess is done bya) youb) the enzyme involve in Step 9.

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