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MANAGEMENT AND SCIENCE UNIVERSITY (MSU)

LABORATORY MANUAL

BIOLOGY

FBD 0044

FOUNDATION IN HEALTH SCIENCE

Name ID

: ____________________________ : ____________________________

Lecturer : ____________________________

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Practical
No Practical Introduction to practical 1 2 3 Introduction to biology lab Introduction to light microscope Detection test for carbohydrate, protein and lipids 4 5 6 7 8 Osmotic fragility Enzyme Respiration Photosynthesis Biodiversity 14 16 19 21 23 Page 3 5 7 10

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Introduction to Practical
Laboratory safety There are few guide lines that each students need to follow:a) Students can only be in the laboratory with the presence of a lecturer/tutor assigned. b) Please do not run, play around or do any indecent behavior while in the lab. c) In the case of an accident, injury of illness immediately informs the lecturer or tutor in the laboratory. d) Please do not eat, drink, smoke or handle contact lenses in the laboratory. e) Be aware of the location and operation of all emergency equipment and how to call for help if needed. f) Know the potential hazards, precautions and safety procedures before conducting a practical exercise. g) Please wear comfortable, inexpensive clothing and most importantly a LAB COAT. h) Please wash your hands after handling chemicals, animals or after removing your gloves. i) j) Please do not intentionally sniff any chemicals. Please promptly report any faulty equipment, damage specimens, water or gas leaks to the lecturer or tutor in the laboratory.

k) Ask the lecturer or tutor if you are unsure of any part in the practical procedure before the practical starts. l) Please clean work surfaces, switch off all electrical outlets after each practical exercise.

m) If there is any fire or a fire alarms rings, immediately leave the lab in an orderly manner to the designated safe area.

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How to perform well in a practical class Some of the tips to help you optimize your work in the lab are: a) Try to arrive early to the practical class so that you are calm and in a clear mind. b) Take notes on the instruction given at the beginning of the practical. c) Ensure that you have all the necessary equipment and specimens for your work. d) Check that the equipment and specimens are in proper condition. Check any broken or defective parts and if anyone is detected please inform your lecturer or tutor. e) Microscope will be prepared for you during practical. Please remember they are expensive and delicate instruments. Therefore it is your responsibility to handle them properly. f) Do not hesitate to call and ask the lecturer or tutor for guidance or if you are having problems. g) Do your work promptly and avoid idle conversation. h) When working with other students try to work quietly and efficiently so as not to disturb others. i) Carefully handle and examine the specimens given without removing them from the worktable. Remember that the specimens are there for other students as well as yourself. Special care and consideration have to be given if you are working with permanent slides. Please ask your lecturer or tutor for correct handling of slides.

j)

The criteria of a good report are as follows: a) Facts and content is complete and well written. b) Data obtained from practical is correct and concisely written. c) Diagrams are neatly drawn and labeled correctly with eligible writing. d) Presentation of your report is neat and appealing with contents in the right order and sentences concise. e) Give complete and correct answers to all questions in the practical manual.

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1.0. Introduction

INTRODUCTION TO BIOLOGY LAB

There are a number of general rules that expects to be followed when you are in the Laboratories: y Do not eat or drink in the laboratory y Report all spills and accidents to your demonstrator immediately y Always wash your hands at the end of a laboratory y Upon completion of laboratory exercises, place all materials in the area or container designated by your demonstrator y Leave the lab clean and organized at the end of each lab period. Lab 1.1 : Introduction to biology lab

Objectives To explain and expose first semester students the basic instruments which will be use in biology lab.

Materials and Methods 1. Identify the following instruments and lab apparatus: i. ii. iii. iv. v. vi. vii. viii. ix. x. 2. Centrifuge swing-out rotor Bunsen burner Conical flask Test tube Measuring cylinder Tripod stand Test tube rack Burette Pipette Micropipette

Discuss the functions of the instruments and lab apparatus as above with lab demonstrator and make a report. Your report must include the picture and functions of the lab apparatus.

3.

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Results eg. Lab instruments/ apparatus Centrifuge swing-out rotor Functions

1.

2.

Bunsen burner

3.

Conical flask

4.

Test tube

2.0.
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INTRODUCTION TO LIGHT MICROSCOPE 6

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Introduction Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye, using a microscope or other magnification tool. It is often used more specifically as a technique of using a microscope. Microscopy has evolved with the development of microscopes. Hence there are three main branches of microscopy; optical, electron and scanning probe microscopy. Optical and electron microscopy involves the diffraction, reflection, or refraction of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. This process may be carried out by wide field irradiation of the sample (for example standard light microscopy and transmission electron microscopy) or by scanning of a fine beam over the sample (for example confocal microscopy and scanning electron microscopy). Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest. The development of microscopy revolutionized biology and remains an essential tool in that science, along with many others.

Lab

2.1

: Introduction to Light Microscope 7

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Objectives To expose and teach students the steps in using light microscope.

Materials Light microscope Lens tissue Staphylococcus aureus slide Bacillus subtilis slide

Methodology 1. Carry the microscope with two hands. Keep one hand underneath the microscope and the other on the arm. Never touch any lens with your fingers. This leaves oil which is hard to clean and particles which may damage the lens. If a lens needs cleaning, use lens tissue, a lens cloth or a lens pen and be gentle. Do not use your shirt or a towel. Learn the parts of your microscope. Rotate the objectives on the nosepiece of the microscope until the shortest objective is over the slide and make sure the objective clicks into place. Always start with the low power objective. Normally, light microscope have three power of objective; 10x, 40x and 100x. Set the light control. Start with plenty of light, but once you have focused and found your specimen in the field of view, start reducing light until you see the most amount of detail. Place the slide on the stage of the microscope, and secure with the stage clips. Under a different power of objectives, starting with 10x, 40x and 100x, observe the following prepared slides: a) b) Staphylococcus aureus Bacillus subtilis

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3. 4.

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6.

Lab

2.2

: Observation of Animal Cell under Light Microscope 8

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Objectives To prepare and observe animal cell under a microscope and to identify cell membrane, cell wall, and nucleus. Materials Light microscope Glass microscope slides Cover slips Water with eye dropper Methylene Blue stain Toothpicks Methodology 1. Scrape cells from the inside of their cheek. This can be done with the side of a toothpick. Smear the cells onto a slide by wiping the toothpick across the slide. Air dry the cells onto the slide by gently blowing on the slide. Place a small drop of methylene blue stain in the center of the slide (where the cells were smeared). Cover the stain with a cover slip. Place a fairly large drop of water adjacent to one side of the cover slip and using a paper towel as a wick, draw the water under the cover slip towards the other side. This will remove excess stain, leaving behind the cheek cells stained blue. Place the slide (cover slip up) onto the stage of the microscope. Observe cheek cells at different magnifications. Draw what you see under the microscope, labeling cell membrane and nucleus. Also state the magnification used (remember that the total magnification is the ocular x objective).

2. 3. 4.

5. 6.

7. 8. 9.

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3.0. Lab

DETECTION TEST FOR CARBOHYDRATES, PROTEIN AND LIPIDS 3.1 : Carbohydrates Detection Test

Objectives To detect the presence of carbohydrate in a food sample. Materials Test tubes Molisch reagent Benedicts reagent Sugar solution Concentrated sulphuric acid Waterbath Methodology Molisch Test 1. Add 2 drops of Molisch reagent to 2 ml of the sugar solution and mix thoroughly. Incline the tube, and GENTLY pour 5 ml of concentrated H2SO4 down the side of the test tube. A purple color at the interface of the sugar and acid indicates a positive test. Disregard a green color if it appears. Benedicts test 1. Add 1 ml of the solution to be tested to 5 ml of Benedict's solution, and shake each tube. Place the tube in a boiling water bath and heat for 3 minutes. Remove the tubes from the heat and allow them to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing sugars 3.2 : Protein detection test

2.

3.

2. 3. 4. Lab

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Objectives To detect the presence of protein in a food sample. Materials Test tubes Millon reagent 0.1% ninhydrin solution Casein 2% egg albumin 0.1 M tyrosine 0.1 M glycine Water bath Methodology Millons Test 1. Place 1 mL of casein, 2% egg albumin, and 0.1 M tyrosine into separate, labelled, 12 x 75 mm test tubes. Add 3 drops of Millon's reagent and immerse the tubes in a boiling water bath for 5 minutes. Cool the tubes and record the colors formed.

2.

3.

Ninhydrin Test 1. 2. Place 1 mL of of casein, 2% egg albumin, and 0.1 M glycine into separate, labelled, 12 x 75 mm test tubes. Add 4 drops of 0.1% ninhydrin solution. (CAUTION: NINHYDRIN IS A CARCINOGEN - AVOID DIRECT CONTACT) Add a boiling chip to each test tube and heat to boiling in a hot-water bath. Record the results.

3. 4.

Lab

3.3

: Lipid detection test

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Objectives To detect the presence of lipid in a food sample. Materials Sample solution Filter paper Methodology Oil Spot Test 1. 2. 3. Put a drop of sample solution on the filter paper. Let it dry for 5 minutes. If the spot remain, it is a positive result for lipid.

4.0.

OSMOTIC FRAGILITY

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Lab

4.1

: Osmotic Fragility Test

Objectives To determine the fragility of the erythrocyte membrane against the haemolytic effect of NaCl solutions of varying concentrations.

Materials Test tubes NaCl (0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10%) Distilled water Blood Pipette Spectrophotometer Cuvettes

Methodology 1. 11 test tubes prepared and labeled as follows: 0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10% and 0% NaCl. 5 ml of NaCl pipetted in each test tube according to the label and 5 ml distilled water pipetted in test tube labeled 0% NaCl. 10 L blood (with added heparin or sitrat which act as anti coagulant) was pipetted at each test tube. The content of the test tube mixed carefully and the test tube sentenced to resting period of 20 minute at room temperature. Test tubes shaked carefully and centrifuged for 9 minute at the velocity of 2000 rpm. Supernatant transferred to labeled cuvettes. Using supernatant from the test tube which is labeled 0.85% NaCl as reference (0% haemolysis) OD of each supernatant calculated using spectrophotometer with the wavelength of 540nm. Percentage of haemolyis calculated using the following formula: OD Sample/ OD of the sample with 100% lysis x 100 8. OD recorded and percentage of NaCl converted to mole/L.

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5. 6.

7.

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9.

A graph percentage of haemolysis vs. concentration of NaCl plotted. (This graph is known as osmotic fragility curve).

5.0. Lab 5.1

ENZYME

: Preparation of a standard curve graph 14

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Objectives To guide and explain to students the importances of standard curve graph in quantitative experiments. Materials Spectrophotometer Cuvettes 10uM p-nitrophenol Distilled water 0.2 M NaOH

Methodology 1. Series of test tube as shown in the table below are prepared. Test tube 10uM p-nitrophenol (mL) Distilled water (mL) 0.2M NaOH (mL) 2. 3. 1 0 8.4 1.0 2 0.5 7.9 1.0 3 1 7.4 1.0 4 1.5 6.9 1.0 5 2 6.4 1.0 6 3 5.4 1.0 7 4 4.4 1.0

Mix the solution as mentioned above and read the absorbance at 410nM Plot the graph with the amount of p-nitophenol that is produced in each tube vs absorbance.

Lab

5.2

: Effect of temperature on the enzymes activity

Objectives
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To show how temperature can affect enzyme activity.

Materials Spectrophotometer Cuvettes 5mM nitrophenol phosphate Distilled water 0.2 M NaOH Buffer pH10 Waterbath Methodology 1. Prepare water at the following temperatures: 5oC, 29oC (room temperature), 37oC, 55oC and 70oC. Prepare the test tubes as the following: Test tube Buffer pH10 (mL) 5 mM nitrophenol phosphate (mL) 2. 1 1.0 0.2 2 1.0 0.2 3 1.0 0.2 4 1.0 0.2 5 1.0 0.2

Prepare a control group test tube by adding 1.0 mL buffer solution, 0.2 mL substrate and 0.2 mL of water, soak it in the 37oC water. Add the contents of the test tube and put it in the water as shown below (1 in 5oC, 2 in 29oC, 3 in 37oC, 4 in 55oC and 5 in 70oC). After 5 minutes, add 0.2 mL of alkaline phosphataste in the test tube except for the control group based on the times set. Keep it in the water for precisely 15 minutes After 15 minutes, add 8 mL 0.02 M NaOH quickly based on the time shown above. Take the optical reading at 410nm. Determine the enzyme activity of every test tube using the calibration graph. Plot a graph of enzyme activity vs. temperature.

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6.

Lab

5.3

: Effect of pH on the enzymes activity

Objectives
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To show how pH can affect enzyme activity.

Materials Spectrophotometer Cuvettes 5mM nitrophenol phosphate Distilled water 0.2 M NaOH Buffer pH4, pH6, pH9, pH10, pH13

Methodology 1. Prepare the test tubes as the following Test tube Buffer (mL) 5 mM nitrophenol phosphate (mL) 2. 1 (pH4) 1.0 0.2 2 (pH6) 1.0 0.2 3 (pH9) 1.0 0.2 4 5 (pH10) (pH13) 1.0 1.0 0.2 0.2

Prepare one test tube for control group by adding 1.0 ml of buffer solution pH 10, 0.2 ml substrate and 0.2 ml water. Put all the test tubes in the water at the temperature of 37oC for 5 minutes. Add 0.2 ml alkaline phosphatase to the test tube except for the control group based on the time set as shown below: Keep it in water for precisely 15 minutes. After 15 minutes, immediately add 8 ml 0.02M Na oH. Take the reading at optical density at 410 nm. Determine the enzyme activity of every test tube using the calibration graph. Plot a graph of enzyme activity vs. pH

3. 4.

4. 6. 6. 8.

6.0. Lab 6.1

RESPIRATION

: Respiration (reaction of yeast with methylene blue) 17

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Objectives To differentiate the yeast respiration process at different situation.

Materials Test tubes 10 cm3 of yeast suspension Methylene blue Water bath Cork or rubber stopper Methodology 1. 2. 3. 4. Label 3 test tubes with A, B and C. Fill each tube with 10 cm3 of yeast suspension. Boil tube C for each 5 minutes. Add 10 drops of methylene blue into each of the tubes. Shake and let the colour evenly distributed.

5. 6. 7. 8.

Heat all tubes in the water bath with 38OC - 42OC for 15 minutes. After 15 minutes, observe the colour changes in all tubes. Place tube B in boiling water for 5 minutes. Do not shake the tube. Plug tube A with cork or rubber stopper. Press it with your thumb and shake the tube vigorously for more a less 10 times. Observe the colour changes. Remove the stopper and place tube A into the water bath again. Repeat step 8 using tube B and C. Observe again the colour of the suspension precipitated in each tube. Record your observations in a table as below: Boiling tubes A After 15 minutes Light blue Colour of the suspension While shaking After shaking Blue green White 18

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B C

Light blue Blue

Light blue Blue

Light blue Blue

7.0.

PHOTOSYNTHESIS

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Lab

7.1

: Photosynthesis (separation of pigments by chromatography)

Objectives To detect the presence of different pigments that involve in photosynthesis in healthy leaves.

Materials 20g leaves Acetone Buchner funnel Pin Chromatography strip Cork

Methodology Chloroplast extracts preparation 1. Cut 20g of healthy leaves using a cutter; grind/blend the leaves in 50cm3 acetone. Leave it for 10 minutes. Grind again and add more acetone. Extract chlorophyll using Buchner funnel into a beaker.

2.

Paper chromatography 1. Using the head of small pin as a dropper, place a drop of the chloroplast extract on the chromatography strip. Let the drop dry. Repeat the process several times (5 times) to build up a small area of concentrated pigment.

2.

Attach the paper strip to the stopper with a pin. Suspend the strip straight into the boiling tube that contains 10cm3 solvent. The bottom edge of the paper should dip into the solvent, but make sure that the pigments spot is not immersed in the solvent. Let the solvent rise until its front reaches 1 cm from the top of the strip. Remove the strip and draw a pencil line to mark the solvent front. Also mark the pigmented area. 20

3. 4.

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5.

Calculate the Rf value for each pigment using the following formula. Rf = Distance moved by the pigment from its original Distance moved by the solvent from the origin

6.

Record your results in the table given below: Pigment 1. Chlorophyll a 2. Chlorophyll b 3. Carotene 4. Xantophyll Colour Rf value

8.0. Lab 8.1

BIODIVERSITY

: Biodiversity in Malaysia 21

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Objectives To expose students the biodiversity in Malaysia by visiting FRIM at Kepong.

Methodology 1. Form a group of 5 and follow the nature guide to walk through the forest for about 1 hour. Get the explanation from your nature guide about biodiversity and species which can be find at FRIM, Kepong. In your group, identify and draw a minimum 3 species of living organisms from kingdom fungi, plantae and animalia. Make a report.

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3.

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