MF011 Fhs Lab May10

MF011 General Biology 2 Mr.
Veerapen, MK May August 2010 Semester
FACULTY OF APPLIED SCIENCES
Laboratory Practicals Manual
MF 011 General Biology 2

Prepared by: Mr. Veerapen, MK and Mr. Jayaraj VK Edited by: Mr. Andrew Morgan Tennant
Lecturer : Laboratory technician: Lab Session : Semester :
Mr. Veerapen, MK Ms. Lawrene d/o Ratnasingam Wednesdays, 2 5 pm (subject to change) May - August 2010
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MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester
Content Page
Content
Guidelines : Practical 1 : Practical 2 : Report Writing, submission and lab conduct An Investigation of the carbohydrates metabolised by yeast Observation of mammalian kidney model and tissue slides Analysing kidney filtration using simple filtration system Observation of Digestive System in a Murine model Observation of Reproductive System in a Murine model Comparison of transport system between mammalian and amphibian models The Simple Muscle Twitch (SWT) Experiment Chi-squared (2) test and its Application on Mendelian ratios Testing the Hardy-Weinberg Equilibrium DNA Extraction from Human Buccal Cells Digestion of Lambda () DNA with a restriction enzyme (EcoR I endonuclease)
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2 5 7
Practicals 3 &4 : Practical 5 :
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Practical 6 : Practical 7 : Practical 8 : Practical 9 : Practical 10:
28 32 36 41 43
Guidelines for writing laboratory reports Laboratory reports should be written according to the format below (failure to do so will result in marks being deducted): Formatting Font : Arial size 12 Spacing: 1.5, justified Pages : 3 (minimum) - 7 (maximum) [must be numbered]
Cover page You are required to use the cover page available on the eAdvantage and are to include these details: lab no., title of experiment, students names and student numbers, and date of experiment. Indicate also your group number. Introduction This section serves to acquaint the reader with the subject and justify the objective(s) of the experiment. There should be two parts to the introduction: first, a clear description of the principle(s) underlying the experiment; and second, a statement of how you will conduct the experiment to study the underlying principle(s). About 0.5 page would do. Objective(s) The objective(s) addressed in the experiment must be clearly stated and numbered. Materials List only the important materials used. Commonly used apparatus or glassware (e.g. clamp, forcep, beaker, measuring cylinder) need not be mentioned. Do measure magnitude/volume of apparatus used, when applicable. Methods The description of experimental methods must contain sufficient information to allow another person to duplicate the experiment. All methods should be written in past tense, passive voice and should be written in your own words. Some lab sessions may have several parts and as such, you may use subheadings for these parts ( i.e. Practical 2 which has 2 parts A. Kidney model examination and B. Nephron kidney slide observation). Results Results may be combined with discussion in order to provide a more coherent flow. This section must contain sufficient information (and calculations if necessary) to fully describe the outcome of the experiment. Where necessary, the uses of tables is encouraged. Tables must contain enough information within them and with their respective titles or legends to be understandable without referring to the text. Drawings must be done using pencil. A maximum of 2 drawings per A4 size paper is advised. Discussion This section contains an explanation of the meaning of the results. The principles, relationships, and general truths shown by the results should be presented without reiterating the results. Use text to emphasize important points in your results, to connect results with one another, and to restate the trend of the idea ( the objective already mentioned in the Introduction) while connecting with textbooks or other reference materials. Exceptions or lack of correlation and important precautionary measure(s) 2
should be pointed out and unsettled points (if any) explained. The theoretical or practical implications of the experiment should be discussed. Conclusion The conclusion should be stated briefly. Questions All questions should be answered regardless of whether the calculation or answer has been mentioned, either directly or indirectly (if indirectly, please underline answers), in the Results and/or Discussion sections. Keep answers short and precise (these are not mini-essay questions). References Please obtain a copy of the FOAS Thesis Guideline 2010 to guide you along the way. Failure to follow this referencing method will result in deduction in marks. Submission of laboratory reports Students are required to conduct experiments in groups of 4 persons. Only 1 report is submitted per group for every experiment. Students are required to submit laboratory reports within 2 weeks after each day of laboratory session (unless stated otherwise). Failure to do so will result in an automatic 0. Plagiarism is strictly forbidden. If found, students will be automatically awarded a 0. Each laboratory report is to be submitted together with a copy of the Laboratory Report Submission Form. Only if you require an acknowledgement receipt of your laboratory report, please prepare a duplicate copy of the form. The form is downloadable from eAdvantage. Laboratory Practical Rules
Questions are HIGHLY encouraged in lab sessions and will be rewarded in terms of marks. All students are to ensure proper attire (long pants, covered shoes, clipped/tied hair (if long), lab coat, no shorts, slippers and skirts) at all times during the lab session. Failure to do so will result in the removal of penalised student from the laboratory and the student will be awarded 0. No caps/hats, sunglasses, food, drinks, mobile phones and headphones allowed in the laboratory. Be serious in the lab. Laughing is allowed but no horse-play is allowed. All students are not allowed to wear lab coats outside the lab.
Attendance is COMPULSORY. It is the responsibility of the student to ensure that his/her chosen laboratory session schedule does not clash with other course(s) undertaken during the semester. All students are required to read and understand the procedures of the corresponding experiment(s) before coming for their laboratory sessions. Flow charts are necessary to submit before starting the experiment. Failure to do so will result in expulsion and 0 mark awarded for the particular lab. Students are expected to be proactive during the lab sessions. All students must be be punctual for their laboratory sessions. Those absent for any laboratory session must produce medical certificates or written letters stating their reasons for being absent. If a valid reason is not shown, the student will be given an automatic 0 for that session. Acceptance of reasons will be subject to the sole discretion of the lecturer. All students are to enter and leave the laboratory only with the permission of the lecturer or laboratory technician. All students are to ensure to equip themselves with felt tip permanent marker pens for labelling, and camera(s) (optional) to photograph work and data. The lab benches should not have personal belongings other than essential stationary during the lab sessions (in the lab bench area). Students are to be seated according to group number and they are responsible to clean up their respective apparatus and lab bench after each lab session. Failure to do so will result in deduction of marks. Safety measures are to be observed (i.e. gloves, masks etc.) at all times especially when handling dangerous materials such as carcinogens (i.e. ethidium bromide). Do not wear gloves to touch the door of the laboratory, take off your gloves before leaving. Students are to report if accident occurs at any point during the session to either the lecturer or lab staff for further assistance. All students are to wash hands thoroughly before and after practical sessions (in the lab).
Practical 1
An Investigation of the Carbohydrates Metabolised by Yeast
1.0
Background information
Yeast, Saccharomyces cerevisiae, can metabolise carbohydrates under two different conditions. When oxygen is present, aerobic respiration occurs yielding a large amount of energy for the organism and producing carbon dioxide and water as waste products. However, in anaerobic conditions, when oxygen is in short supply the yeast will break down the carbohydrate into ethanol and carbon dioxide with a much reduced energy output (alcoholic fermentation). Specific enzymes catalyse both of these forms of respiration in addition to most metabolic processes where the efficiency of the yeast is in metabolising different carbohydrates can be monitored by observing the time taken for Methylene blue to be discoloured. In this experiment, you will be investigating the relative efficiency with which different carbohydrates can be metabolised by yeast.
2.1
Materials
Boiling tubes Carbohydrates (0.5M glucose, 0.5M fructose, 0.5M lactose, 0.5M sucrose) Graduated pipettes Distilled deionised water 10% (w/v) Bakers yeast Methylene blue (0.005% v/v) o Water bath (40 C)
2.2
Methodology
1. Prepare and label 5 duplicate boiling tubes and fill as table 1.1 below:TABLE 1.0 Yeast metabolism boiling tube composition Component (volumes in mL) Tube number 1 0.5M glucose 0.5M fructose 0.5M lactose 0.5M sucrose 0.5M Starch ddH2O 10 % (w/v) yeast suspension (10g bakers yeast per 100mL of 10mM KH2PO4 buffer) 2. Place all the boiling tubes in the water bath (40C) for 5 minutes. 2.0 1.0 2 2.0 1.0 3 2.0 1.0 4 2.0 1.0 5 2.0 1.0 6 2.0 1.0
3. Then, add 2 mL of methylene blue (0.005% (v/v)) and vortex the boiling tubes. Start the timer immediately for the 30 minute incubation period. 4. After 30 minutes, gently remove the boiling tubes and place the rack back on bench-top. A thin film of blue at the surface of the tube can be ignored. Observe and compare the intensity of the blue colour formation. Score the results based on the intensity of blue colour formation (using + sign). 5. Record results obtained in a table 1.1. TABLE 1.1 Tabulation of Carbohydrate Usage Results using methylene blue indicator. Ensure that observation is taken after the 30 minutes incubation. Tube number Blue colour intensity (after 30 minutes) (- : no presence, + : least intense, ++ : intense, +++ : most intense ) Trial 1 Trial 2 1 2 3 4 5
3.0 Questions 1. Explain your findings in terms of:a. enzymes activity, b. carbohydrate structure c. affinity of yeast to a particular carbohydrate and d. the glycolysis pathway. 2. Assess the reliability of the results obtained and suggest any modifications you could make to improve the experiment.
Practical 2
Observation of mammalian kidney model and tissue slides Analysing kidney filtration using simple filtration system 1.0 Background information
The excretory system of mammals centres on a pair of kidneys. In humans, each kidney tubules are about 10 cm long and is supplied with blood by a renal artery and drained by a renal vein. Blood that flows through the kidneys is voluminous. The kidneys account for less than 1 % (w/w) of the human body mass but receive roughly 25% (v/v) of the blood exiting the heart. Various important structures that exist in the kidney perform its main function in homeostasis for osmoregulation. In this experiment, you will be investigating the general structure of a mammalian kidney while analysing a simple filtration system.
2.1
Materials
Microscope Mammalian kidney model Kidney nephron slides Whatman filter paper (8 nm pore size) Conical flask Filter funnel Biuret solution Benedicts solution Iodine in potassium iodide (KI) solution Test tubes
2.2
Methods
a) Observation of major structures in a mammalian kidney 1. Observe the kidney model presented to you and identify the various important structures in the kidney that is vital for osmoregulation. 2. Draw these main structures and label them accordingly. Indicate the position of nephrons and tubules in your figure based on your understanding. b) Examination of tissue slides under a microscope 1. Observe the slides under a light microscope under low power to determine the plan of general tissue distribution. Examine the detailed structures under high power by observing the form of the cells and other features. 2. Make two drawings of each slide:a. Plan drawing: Drawing of the outline as seen under low power microscope. Do not draw any cells. b. High power drawing: Detailed drawing as seen under high power microscope showing accurate cell/tissue characteristics. Draw only a few cells.
3. Each drawing must have a complete title which gives the following information: a. Name of organ b. Type of section c. Magnification 4. Observe the capsule (connective tissues), pelvis, cortex and medulla. Note that the Malphigian corpuscle consists of glomerulus and Bowmans capsule, the cuboid epithelial cells of the proximal convoluted tubules have brush borders while the distal convoluted tubules have bigger lumen and the cells lack brush borders. c) Analysing kidney filtration (Note: You would have to troubleshoot accordingly. Explore and enjoy errors/ambiguities in your results) 1. Add 1 mL test solution as table 2.1 below. Label them accordingly:TABLE 2.0 Content (in mL) Protein Glucose Starch ddH2O Kidney filtration experiment test tube composition Test tubes 1 2 3 4 1.0 1.0 1.0 1.0
2. Add 1 mL Biuret solution to the test tube labelled protein. Record your observations. 3. Add 1 mL of Benedicts solution into the test tube labelled glucose. Place the test o tube in the water bath (100 C) and leave for 5 minutes, shaking. Record your observations. 4. Add 1 drop of Iodine in potassium iodide solution into test tube labelled starch. Record your observations. 5. Repeat steps 2 3 for test tube 4. 6. Perform steps 2 4 for tube 4. 7. Discard the tested solutions and rinse the test tubes clean. 8. Re-prepare the solutions as table 2.1, but this time, prepare for a volume of 2.0 mL instead. 9. Prepare a filter paper on a filter funnel and a conical flask. Pour content of each test tube to the filter paper and collect the filtered solution in the conical flask. 10. Repeat steps 2 - 4 onto the filtrate. 11. Record your observations accordingly in a table 2.1. TABLE 2.1 Tube number Results tabulation of the kidney filtration simulation. Ensure that observation is taken before and after filtration. Colour intensity Colour intensity (before filtration test tube) (after filtration - filtrate) (- : no presence, + : least intense, (- : no presence + : least intense, ++ : intense, +++ : most intense ) ++ : intense, +++ : most intense )
1 (Biuret) 2 (Benedicts) 3 (Iodine) 4 (All 3)
3.0
Questions 1. Which compounds passed through the filter paper? 2. Was there a difference in the intensity of the colour that was observed from your initial tests? If so, why? If not, why? Suggest a method to quantify your method. 3. Explain why some of the compounds did not pass through the filter paper. 4. What is the function of test tube 4? 5. How does the filtration in this activity resemble the activity of the kidney? 6. Explain the difference in the roles played by the different tubules present in the kidneys nephronic system. 7. Why do mammals need to get rid of their excretory products?
Suggested reference Histology Text books
Practicals 3 and 4
Observation of Digestive System in Rats Observation of Reproductive System in Rats 1.0 Background Information
The rat you are working with has a scientific name of Rattus norvengicus and belongs to the family Muridae. The external anatomy of a rat (Figure 3.0) consists of a head (cranial region), neck (cervical region), body and tail; 2 pairs of legs hind and front; covered with short fur except in the ear and feet with miniscule amounts on the tail (along with scales). The dissection would be referring to several anatomical terms as seen in Figure 3.0.
Posterior
Anterior
FIGURE 3.0 External anatomy of a rat. Note: Cranial/Anterior towards the head; Caudal/posterior towards the tail; ventral towards the belly; and dorsal towards the back. The sharp head on the rats anterior is where the round circular ears are located on both sides. The eyes of a rat are small. The mouth is located below its nose and is surrounded by a pair of lips. Whiskers on its nasal region known as vibrisa, are part of the rats touch sensor system. As for the neck and body, a rat has a short neck with its body consisting of a thorax (thoracic region) on its anterior and an abdomen/belly (pectoral region) on its posterior. The pectoral region refers to the area where the front legs attach. The ventral surface of the pectoral region consists of the rats nipples or mammary gland openings. Its tail is long with its anus located on the ventral region of the base of the tail. The pelvic region is the area where the hind legs are attached. On a male rat, the penis is located on the colop . Both of its rather large scrotal sac covers both of the testicles. The opening of the genitalia is observed at the end of the penis. However, on a female rat, the genitalia opening would be observed as a slit that would be the vulva. The clitoris would look like a rod on the ventral wall. The urethra would be at the end of the clitoris. In this practical, you will be dissecting a rat and observing the digestive and reproductive systems.
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2.1
Materials
Lab rat (Rattus norregicus) Cotton Chloroform Dissecting tray Dissecting kit Pins Gloves
2.2
Methods (based on a right hander) 1. Pin the rat that you have euthanized, onto the dissecting tray. The ventral side of the rat is to be facing upwards. Note the genitalia, nipples and anus. Note the gender of the rat (Figure 3.1).
FIGURE 3.1 Ventral surface anatomy Female (left) and male (right) Note: Pins are to be pierced slanting into the wrist of the rats paws and onto the tray while ensuring that the rat is stretched as far as it can go on each paw (Figure 3.2).
FIGURE 3.2 Slanting pin pierce onto dissection board of rat paw. 2. Pull the upper epidermal layer a few cm cranial of the genitalia of the rat along the middle ventral area (Figure 3.3).
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FIGURE 3.3
Cut (--->) skin along the lines for a male and female (inset box).
3. Make an incision using a pair of scissors. Ensure only the upper epidermal layer is cut and no deeper. 4. Using the 1-blunt sided scissors (blunt end facing down), start cutting upwards (cranial) towards the mouth and downwards (caudal) towards the posterior. Ensure no external genitalia is cut. 5. Hold the skin flap with a pair of forceps. 6. Using a scalpel blade, slice through the intermediary tissue between the skin and bodys muscle wall. Go as far as possible while ensuring no/minimal bleeding. 7. Absorb blood with cotton, if bleeding occurs. 8. Pin onto the upper epidermal layer onto the dissecting tray. Note the muscles and glands (Figure 3.4).
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Mammary glands Pectoral muscles Rib cage and intercostals muscles
Xiphoid cartilage
Femoral artery and vein
Mammary gland
FIGURE 3.4
Cut along the abdominal region as shown by the dotted lines in the diagram
9. Carefully, pull the thin abdominal peritoneal wall using a pair of forceps. Ensure no organs are pulled along. 10. Using the 1-blunt sided scissors (blunt end facing downwards towards the abdominal cavity), make an incision at the pulled layer (Figure 3.4). 11. Using techniques of steps 8 and 9, start cutting upwards towards the mouth and downwards towards the tail. Be careful not to pierce any abdominal visceral organs (Figure 3.5).
FIGURE 3.5 Cutting along the abdominal wall 12. Using techniques of steps 8 and 9, make a horizontal cut on the abdominal peritoneal wall, slightly posterior of the last rib. 13. Pin down the excised abdominal peritoneal wall layer. 14. You have now exposed the abdominal cavity (Figure 3.6). Note that there may be a fair bit of adipose tissue. Remove it carefully with a pair of forceps. 15. Absorb blood with cotton, if bleeding occurs.
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FIGURE 3.6
Serosal membrane of the abdominal cavity of a female rat
16. For the digestive system - Carefully, with a pair of forceps, lift the liver forward and the visceral organs (organs within the abdominal cavity) moved to the left to ensure proper visual of the gastrointestinal (GI) tract. 17. Be careful with the small intestines and colon for it is highly coiled. Hence, it has to be released from the coils by carefully cutting along the mesentery with a pair of scissors and forceps. Be careful not to cut along any of the blood vessels on the mesentery. 18. Arrange the mesentery to be on the left of the rat. Note: Move the duodenum slightly to reveal the pancreas. You may use a probe to move the organs around carefully. 19. Identify organs as per table 3.1 and figure 3.7 below:TABLE 3.1 Digestive system. Organs to be identified. Pancreas and Liver and Sphincter pylori Appendix ducts middle, left and right lobes Duodenum Stomach Oesophagus Caecum Jejunum Colon Bile duct Anus Ileum Rectum Cardiac Spleen sphincter
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liver Pyloric vein Anterior pancreas-duodenum vein Liver-Pancreas duct Liver-pancreas duct pore Posterior pancreas-duodenum vein Duodenum Posterior mesenteric vein Mid-colon vein Posterior mesenteric vein Posterior colon vein Limp node Mesentery Colon Colon-ileum vein Rectum Caecum Appendix Bile duct stomach Pyloric vein Anterior mesenteric vein Lineal vein Jejunum Ileum Intestinal vein
Mesentery
FIGURE 3.7 The Digestive system of the rat and its v enous supply. Please highlight the organs in order to different them from the veins. 20. Snap photos of the full dissection and of, if possible, organs in table 3.1 for records and drawing purposes. 21. Show lecturer your dissection for evaluation. 22. Cut the gastrointestinal tract by snipping at the posterior end of the rectum. Do this carefully. 23. Remove the cut GI tract and place it on a white tile. 24. For the male reproductive system - Look for the scrotal sac and cut along the left ventral surface (figure 3.8), similar to techniques in step 8 9.
Placental symphysis
FIGURE 3.8 Groin region. Cut along the dotted lines. 25. For both reproductive systems Remove muscles around the middle of the pelvic curve (placental symphysis figure 3.8) to reveal the groin area. 26. Carefully, cut around the groin area to remove the middle of the groin area. 27. Move the bladder and the penis/clitoris carefully to the right of the rat. You may use a probe to move the organs around carefully.
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28. Identify organs as per table 3.2a and figure 3.9a or table 3.2b or figure 3.9b below:TABLE 3.2a Reproductive system (male). Organs to be identified. Cowpers gland Prostate gland Penis Vas deferens Testicles and Epididymis scrotum Urethra Seminal vesicle
Right adrenal gland Right adrenal vein Right kidney Right renal vein Right ureter Posterior vena cava
Dorsal aorta Artery selom Anterior mesenteric artery Left adrenal artery Left renal artery
Left seminal vein Left seminal artery Rectum
Pampinoform plexus
Seminal vesicle Coagulating gland
Urinal bladder Urethra Cut surface of the pelvic curve Cowpers gland Penis Scrotal sac with testis Colop gland Penis head Urethral pore
Ampullary gland Prostate gland Ventral prostate gland Vas deferens Caput epididymis Testis Corpus epididymis Caudal epididymis Gubernaculums
FIGURE 3.9a Urigenitalial system of the rat (Male). Highlight important organs to observe as stated in table 3.2a.
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TABLE 3.2b
Reproductive system (female). Organs to be identified. Left and right Uterus ovary Clitoris Vagina Vulva Uterine horn Fallopian tubes (left and right)
Dorsal aorta Artery selom Anterior mesenteric artery Left kidney Left renal artery Left renal vein Vein & artery of left ovary Left Fallopian tube Ovary without periovarian pouch Left ovarian artery Left ovarian vein Left uterus Left uterine vein Left uterine artery
Right adrenal gland Right adrenal vein Right adrenal vein Posterior vena cava Right ureter Fat layer Periovarian pouch with ovary Right lumbar ilial artery Right lumbar ilial vein Rectum
Urinal bladder Urethra Cut surface of the pelvic curve Colop gland Clitoris Colop Rectum
Vagina
Vulva
Anus
FIGURE 3.9b Urigenitalial system of the rat (Female). Highlight important organs to observe as stated in table 3.2b. 29. Snap photos of the full dissection and of, if possible, organs in table 3.2 for records and drawing purposes. 30. Show lecturer your dissection for evaluation. 31. DO NOT discard the rat. View section a) Practical 5 to begin exploring the thoracic region. NOTE: Photos can be optionally included as results. However, it is essential that plan/detailed drawings be included.
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3.0
Questions 1. How many ureters does a rat have? 2. What is the organ related to digestive system that is not listed in Table 1 but can be found in humans? 3. Describe one function of the liver. 4. Name the two ring-like muscles that are found at the beginning and end of the stomach. 5. Why are these two ring-like muscles important? 6. The fragile tissue that connects the small intestines together is called the mesentery. The mesentery is full of arteries that are helping the small intestine with one of its primary functions. What function do these arteries serve? 7. Name and describe one disorder associated with the pancreas. 8. One job of the spleen is to produce white blood cells. Why would rats need more white blood cells than humans?
Adapted from nd Lum, H.K., 1982. Kursus Amali Biologi: Pra Universiti Buku 2. 2 Ed. Longman: Malaysia, pg 128 139.
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Practical 5
Comparison of Transport System in Amphibian and Mammalian models
1.0
Background Information
The transport system, being the area of focus in this practical session, is an important bodily biological system used for transport of oxygen and food to cells while removing carbon dioxide and metabolic wastes. Commonly known to most would be the mammalian transport system. But what about the amphibians? The most notable difference is of course based on the circulatory loop system and the heart chambers. For this laboratory session, you will be comparing the transport system between an amphibian and mammalian model. The mammalian model used is that of the rat (Practical 3 & 4) whereas the amphibian model used is the common frog, Bufo melanostictus. The external anatomy of a frog (Figure 5.0) consists of a head (cranial region), body, and 2 pairs of legs hind and front. The skin is smooth. The dissection refers to several anatomical terms as seen in Figure 5.0.
FIGURE 5.0 External anatomy of a frog. Note: Cranial/Anterior towards the head; Caudal/posterior towards the tail; ventral towards the belly; and dorsal towards the back. The frogs posterior consists of an orifice called the cloacae that is situated together with its anus and urinogenitalial organs. Its forelimbs (front legs) consist of the feet (with 4 toes), wrists and forearm (that is jointed with the upper arm at the elbow). The hindlimbs (hind legs) are comparatively longer and consist of the feet (5 toes), ankle, and shank. The mouth of the frog is on its anterior end with a strikingly large nose consisting of 2 nostrils - near the mid line of the frog. The very apparent and popping eyes are slightly above the nose. Posterior to the eyes are the tympanic membranes for hearing.
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2.1
Materials
Lab rat (Rattus norregicus) Frog (Bufo melanostictus) Cotton Chloroform Dissecting tray Dissecting kit Pins Thread Gloves 2.2 Methods a) Rat Transportation system Continuation from step 31 (Practical 3 and 4) 1. Remove the thoracic membrane layer (similar to removing the peritoneal layer) by holding the layer with a pair of forceps, start cutting upwards towards the mouth. Ensure that the diaphragm and internal organs are not damaged. 2. Using the 1-blunt sided scissors, pierce the rib cage and cut through and around the rib cage and intercostals (Figure 5.2).
FIGURE 5.2 Removal of the ventral thoracic wall. 3. Lift the rib cage and pin the xyphoid cartilage away from the thorax.
Intercostals muscles Rib bone Left costal artery and vein Sternum
Pectoral muscle Thymus gland The 4 lobes of the right lung
Right auricle Left ventricle Left lung
Posterior vena cava Dorsal aorta Right frenal nerve Diaphragm muscles
Right ventricle Oesophagus
Xyphoid cartilage
FIGURE 5.3
in situ of the thoracic visceral organs.
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4. Carefully remove the thymus gland (by cutting it) to expose the heart from the pericardium. 5. Cut the heart out and place on the white tile. 6. Remove traces of fats surrounding the blood vessels. 7. Remove the pectoral muscles from the rib cage wall. Be careful not to disrupt the veins below the heart and around the thoracic region. 8. You should be able to differentiate between the veins and arteries with the veins bring comparatively darker in colour. Identify the organs listed in table 5.1:TABLE 5.1 Thoracic organs. Rat circulatory system organs are to be identified Left and right Pulmonary ventricles artery and vein Left and right Left and right auricles jugular veins Vena cava Left and right (posterior and lungs anterior) Aorta Diaphragm .
Epiglottis Larynx Thyroid gland Trachea
Outer left carotid artery Inner left carotid artery Linked left carotid artery Left cervical artery Left vertebraic artery Left axillal artery Left brachial artery
Right shoulder artery Anterior frenal artery Anterior intercostals artery Brachiocephalic artery brachiocephalic Arteriosus duct Ventricle Auricle Right lung Left lung
Inner left costal artery Left shoulder artery Aortic curve Left pulmonary artery Intercostal artery Dorsal aorta Oesophagus Posterior vena cava
Diaphragm
FIGURE 5.4
Thoracic arterial supply in rats.
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Outer right carotid artery
Anterior left facial vein Posterior left facial vein Outer left posterior jugular vein Outer left jugular vein Inner left jugular vein Left cephalic vein Left verterbraic vein Left shoulder vein
Inner right carotid artery Linked right carotid artery Trachea Right brachial plexus
Right brachial vein Right axillal vein Brachiocephalic artery Aortic curve Arteriosus duct Left pulmonary artery Left lung Right lung lobes Diaphragm Mid tendon diaphragm region Xyphoid cartilage Intercostal vein Intercostal artery
Linked carotid artery Inner costal vein Anterior left vena cava Left shoulder artery Anterior left intercostals vein Intercostal artery Azygus vein Intercostal vein Dorsal aorta Oesophagus Posterior vena cava
FIGURE 5.5
Thoracic venous supply in rats.
9. Snap photos of the full dissection and of, if possible, organs in table 3.2, figure 5.4 and figure 5.5 for records and drawing purposes. 10. Show lecturer your dissection for evaluation. 11. Examine and sketch the heart. Label accordingly (Figure 5.6)
Right anterior vena cava Aortic curve Left anterior vena cava Arteriosus duct Pulmonary artery Left auricle
Right auricle Right ventricle
Left ventricle Posterior vena cava
FIGURE 5.6
Ventral view of the rats heat.
NOTE: Photos can be optionally included as results. However, it is essential that plan/detailed drawings be included.
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b) Frog Dissection and Transport System 1. Neatly pin down a freshly anesthetised frog onto the dissection board with its ventral side facing upwards. This may need some force. 2. Starting from in between the hind legs, start cutting the upper epidermal layer upwards in the middle towards the jaw of the frog with a 1-blunt sided scissors (Figure 5.7).
FIGURE 5.7
Cut along the dotted lines (--->)
3. By using a seeker/probe, space the area below the upper epidermal layer. Note that the skin is neatly stuck on the dermal layer below it in certain areas. 4. Now, using a scalpel blade, start removing the skin like how you performed in during the dissection of the rat. Be careful not to cut through any veins (Figure 5.8). 5. Pin the skin/upper epidermal layer onto the dissection board (Figure 5.8).
FIGURE 5.8
Separating and pinning down the upper epidermal layer.
6. Observe the anterior abdominal vein that would be found along the midline of the frog that is slightly below the ventral abdominal wall. Pinpoint the vein at about halfway in between the pelvic curve and the sternum. 7. Using a pair of scissors, make two small incisions of about 0.5cm long, on the left and right of the pinpointed area in step 6 (Figure 5.9 (1)). 8. Make a knot on the top and bottom (points A and B) of this incision to tie the pinpointed area of the abdominal anterior vein (Figure 5.9 (1)). 23
9. Make a horizontal cut, in between the knots made on the vein (Figure 5.9 (2)).
Anterior abdominal vein
FIGURE 5.9 Disrupting the anterior abdominal veins flow. 1. Pull the thread horizontally to tie the vein at points A and B; 2. Horizontal cut in between the 2 points A and B. 10. Cut upwards/vertically through the abdominal muscle wall on both sides of the anterior abdominal vein (Figure 5.10). Be very mindful that you maintain a straight line all the way while ensuring that you do not pierce any of the blood vessels below or the organs.
Cut
FIGURE 5.10
Cutting the abdominal wall.
11. Now, lift the tissue strip and the bone that was cut. Proceed to releasing and removing it from the organs and the anterior abdominal vein (Figure 5.11).
Anterior
abdominal vein
FIGURE 5.11
Releasing the anterior abdominal vein from the bodys wall .
12. In a similar manner to step 11, make vertical cuts (dotted lines C and D) horizontal cuts (dotted lines E, F, G, and H) as Figure 5.11 and pin this layer onto the board. This will reveal the visceral organs in the abdominal cavity (Figure 5.12).
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Anterior abdominal vein Liver
Heart
Oviduct Intestines Ovary Stomach Duodenum Urinal bladder Rectum
FIGURE 5.12 In situ observation of frogs visceral organs. Remember, the focus is on the transport system, not the digestive system! 13. Carefully remove the thin layer, called the pericardium surrounding the heart with the help of a pair of scissors and forceps. 14. Snap photos of the heart for records and drawing purposes. Label accordingly ( Table 5.2 and Figure 5.13). You would be able to differentiate between the veins and arteries where the veins are comparatively darker in colour.
Carotid curve
Right aortic curve
Coetaneous-pulmonary curve
Systemic curve
Left aortic curve

Right auricle
Left anterior vena cava Left auricle
Right anterior vena cava Right pulmonary vein
Arteriosus trunk
Left pulmonary vein Auricle-ventricle pathway
Sinus venosus
Ventricle
Posterior vena cava
FIGURE 5.13
The frog heart. A. and B. indicate the anatomical planes/angles.
25
TABLE 5.2 identified.
Thoracic organs. Frog circulatory system organs that need to be Ventricle Left and right auricles Vena cava (posterior and anterior) Aorta Pulmonary artery and vein Left and right jugular veins Left and right lungs Diaphragm
Laryngeal artery Outer carotid artery Inner carotid artery carotid labyrinth Carotid curve Vertebraic-occipital artery Shoulder artery Systemic curve Systemic curve Hepatic artery Coelom mesenteric artery Coelomic artery Gastric artery Mesenteric artery Splenetic artery Coetaneous-pulmonary curve Arteriosus trunk Pulmonary artery Coetaneous artery
Seminal artery Dorsal aorta Renal artery
Anterior mesenteric artery Ilial artery Haemorrhoidal artery Posterior mesenteric artery Sciatic artery
FIGURE 5.14
Systemic arterial supply of a frog.
26
Mandibular vein Lingual vein Brachiocephalic veins Anterior vena cava
Outer jugular vein Inner jugular vein Brachiocephalic veins Shoulder vein Limbic artery Coetaneous-muscular vein
Dorsal Sinus venosus Posterior vena cava Hepatic vein Adipose body Seminal vein Testis Renal vein Dorsal lumbar vein Kidney Renal hepatic vein Anterior abdominal vein
Pulmonary vein Gall bladder Liver lobes Portal hepatic vein Gastric vein Bile duct Splenetic vein Stomach Pancreatic duct Liver-pancreas duct Pancreas Duodenum Ileum Mesentery Intestinal veins
Hip vein
Femural vein Sciatic vein Rectum
FIGURE 5.14
Systemic venous supply of a frog.
NOTE: Photos can be optionally included as results. However, it is essential that plan/detailed drawings be included. 3.0 1. 2. 3. 4. 5. Questions What is the largest blood vessel in the amphibian and the mammal? Explain the difference between different blood vessels. What is the point in cutting the anterior abdominal vein in the frog? Which animal bled less when cut? Why? Give details of the main anatomical differences of the transport system, that you observed between the mammal and amphibian. 6. Explain how essential molecules (i.e. oxygen) get transported in an amphibian. 7. Make academically sound inference(s) as to why there is a noted difference between and amphibian and a mammal.
Adapted from nd Lum, H.K., 1982. Kursus Amali Biologi: Pra Universiti Buku 2. 2 Ed. Longman: Malaysia, pg 115 122.
27
Practical 6
The Simple Muscle Twitch (SMT)
1.0
The primary function of the nervous system is to conduct impulses from a receptor to a specific effector in order to coordinate bodily activities, inadvertently supporting homeostasis as well. For most animals, the nervous system comprises of 2 systems - the central nervous system (CNS) which is made up of the brain; and the peripheral nervous system (PNS) which connects the CNS with the corresponding receptors (sensory cells and organs to receive stimuli) and effectors (muscles and glands that react to nerve impulses). An example of an effector that frequently responds in both autonomic and mostly voluntary manner is the shank/calf or the gastrocnemius muscle of a frog that is comprised of muscle fibers from 1 40 mm in length. The sciatic nerves innervates this muscle.
Gastrocnemius
FIGURE 6.1 Muscles of the frog hind leg. As highlighted, the gastrocnemius muscle that will be used for this experiment. In this experiment, you are going to explore the effects of direct stimulation of electrodes onto the gastrocnemius muscle.
28
2.1
Materials
Stimulator (BSLSTM; includes AC100A power) Force Transducer Assembly (SS12LA includes S-hooks) Ring stand Tension adjuster (HDW100A) Weights for calibration (must attach to S-hook) Needle Electrodes (ELSTM2) Thread (non-stretch nylon or equivalent) Live frog Amphibian Ringers solution Gloves Dissection board Dissection Kit
2.2
Methods
a) Isolation of the gastrocnemius muscle Note: Moisten the leg with amphibian Ringers solution so you don't lose function from drying out. 1. Take the blunt end of the forceps and place it under the belly of the gastrocnemius muscle to free it from the surrounding fascia.
2. Place a probe under the Achilles tendon and exit from the other side. Note: The Achilles tendon, located at the distal (near to the foot) end of the gastrocnemius muscle, wraps around the posterior part of the ankle and is inserted into the foot.
29
3.
Leaving the probe between the muscle and surrounding fascia of the leg, loop some thread around the Achilles tendon and tie a knot at the distal end of the muscle which will be cut and attached to the transducer. The proximal end of the muscle (attached at the knee) will remain attached to the frog.
4. Cut the gastrocnemius muscle at the distal end (distal to the knot on the Achilles
tendon) so it can be attached to the transducer and elicit a response. Note: Try to get as much tissue distal to the knot as possible in case the knot slips.
b) Attaching the muscle to the transducer 1. Place the frog knee just beneath the transducer hook. 2. Pin the knee in place to stabilize its position.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Use the muscle of the anterior thigh. Use muscles of the anterior leg on the other side of the knee. Lay the thread from the muscle over the transducer hook. Pull the thread so there is a little tension on the thread. Position the muscle so the thread is vertical, so that the muscle is not pulling at an angle (you want an accurate reflection of the muscle's contractile force). Use a double square knot to ensure a firm hold when you start stimulating the muscle. Remove any additional thread. Moisten the frog with Ringers Solution. Adjust the ring stand height so the connection between the muscle and the transducer is relatively tight. Use the fine adjuster knob (at the top of the stand) to remove any remaining slack without stretching the muscle.
30
c) Direct stimulation 1. Set duration at 2 msec and the frequency at 1/sec. 2. Stimulate the hooked up muscle with voltages ranging from 0.1 25 v. Choose 10 voltages within that range. Perform duplicates of your chosen voltages. Allow 5-10 sec intervals before stimulating the muscle again. Record your results in table 6.1. 3. Note the threshold stimulus (the minimum amount needed for stimulation) and the maximum stimulus (the plateau effect). 4. Using the averaged maximum stimulus obtained, record a single twitch on a fast chart speed. Record the duration of the twitch. Perform a duplicate and note the duration as well. Record results in table 6.2. 5. Spray the muscle with ice-cold Ringers solution. 6. Repeat step 4. Note differences. TABLE 6.1 Data tabulation on threshold and maximum level needed for muscle stimulation Voltage (v) 0.1 0.5 1.0 3.0 5.0 7.0 10.0 15.0 20.0 25.0 Average Threshold stimulus Maximum stimulus Data tabulation of time taken during the muscle twitch. Muscle is stimulated with the averaged maximum stimulus in voltage, obtained from table 6.1 Stimulation (using the averaged maximum stimulus) Duration of twitch (room temperature) Duration of twitch (cold Ringers solution) Questions 1. What would be different if you stimulated the sciatic nerve with the electrodes? 2. Explain the difference between threshold level and maximum stimulus. 3. Why was there a difference between room temperature twitching and upon spraying the muscle with cold Ringers solution? 4. What would be the cause of the muscle twitching? Explain in relation to actin and myosin? 5. Describe the possible neural route that would have occurred during the twitching. Seconds nd 2 trial
Tick where applicable
TABLE 6.2
1 trial
st
Average
Adapted from BIOPACs BSL Pro Lessons A01 A02.
31
Practical 7
Chi-squared ( ) test and its Application on Mendelian ratios
2
1.0
The basic rules governing inheritance were first discovered in 1866 by Gregor Mendel. As the father of genetics, he characterised 7 characteristics of pea plants and how they were inherited. Alleles are alternative forms of the gene controlling a characteristic [i.e brown eye color - B and b (A dominant allele is usually written as capital and recessive allele as lower case)]. When a pair of alleles exist in a heterozygous form (i.e. Bb), one of them expresses its phenotype effect fully (dominant allele) while suppressing the expression of the other (recessive allele). Alleles can also exist in homozygous forms that is homozygous dominant (BB) or homozygous recessive (bb). The recessive allele will only express its phenotype in homozygous (bb) form. The dominant allele, on the other hand, always expresses its phenotype (irrespective of homozygous (BB) or heterozygous (Bb) form). A monohybrid cross is a cross that involves only a pair of alleles (one characteristic). If 2 pair of alleles are considered, it is known as a dihybrid cross (2 characteristics). The genetic composition of a person is known as genotype and the expression/physical presence is known as phenotype. A monohybrid cross ratio 3:1 and a dihybrid cross ratio 9:3:3:1 are hypothetical estimations based on (1) dominance/recessive; (2) segregation; (3) independent assortment; and (4) random fertilisation. The last 3 are influenced by chance and are subjected to normal deviation. To assess a genetic hypothesis, one would need a test that can change the deviation from the expected ratio/value to the probability that chance alone could be responsible for the deviation. Furthermore, this test must also take into account the sample size and the number of parameters (degree of freedom (df) number of parameters (n) minus 1). In genetics, df is one less than the number of phenotypes. For example, in a monohybrid cross of wrinkled and non-wrinkled bean, 2 phenotypes, meaning n = 2, df = n 1 = 2 1 = 1. In a dihybrid of perhaps 4 traits, wrinkled, non-wrinkled, green and white beans, 4 phenotypes, n = 4, df = n 1 = 4 1 = 3. Hence, the 2 test would take into account all these factors. In this laboratory practical, you will get to know the calculations for the ratio of heritable traits which obeys Mendels first and second laws by using the product of monohybrid and dihybrid crosses. Verification is by the 2 test.
2.1
Materials
Special type of corn (with different colours) 2 table of critical values
32
TABLE 7.1: Critical Values
33
2.2 Methods a) Monohybrid cross Background The kernels you are using have a colour trait of being either red or yellow. fter carrying out a few crosses, it was discovered that the yellow seeds is dominant to red. Using A for dominant allele and a for recessive, a cross can be presented as below. P: (Yellow) F1: (Yellow) AA x aa (Red) Aa x Aa (Red) Self-fertilise
F2:
A : aa (Yellow) (Red)
1. Examine the corn. Count all the yellow and red kernels. 2. Record data in table 7.2 and individually track each count (i.e. 1. Yellow, 2. Red, 3. Yellow, so on and so forth). 3. From total number of corn kernels (both red and yellow), calculate the expected number of each seed colour. 4. Calculate the value of 2 from table 7.1 and draw a conclusion about your results based on Mendels First Law. TABLE 7.2 Monohybrid cross: student 2 test Expected number of count (e) Divergence / expected no. of count 2 (o e) /(e)
2
Phenotype Yellow Red Total
Expected ratio 3 1
Observation (o)
Divergence (o - e)
Divergence 2 (o e)
n=
34
a) Dihybrid cross Background The kernels you are using have a colour trait of being either red or yellow and either white striped or white spotted. After carrying out a few crosses, it was discovered that the yellow seeds are dominant to red; and the white striped is dominant to white spotted. Using A and B for dominant allele and a and b for recessive, a cross can be presented as below. P: AABB x aabb (Yellow, striped) (Red, spotted) AaBb x AaBb (Yellow, striped) (Red, spotted) Self-fertilise
F1:
F2: 9/16 A-B- : 3/16 A-bb : 3/16 aaB- : 1/16 aabb (Yellow, (Yellow, (Red, (Red, Striped) spotted) striped) spotted) 5. Examine the corn. Calculate all the striped yellow, spotted yellow, striped red and spotted red kernels. 6. Record data in table 7.3 and individually track each count (i.e. 1. Yellow, 2. Red, 3. Yellow, so on and so forth). 7. From total number of corn kernels (striped yellow, spotted yellow, striped red and spotted red), calculate the expected number of each seed colour. 8. Calculate the value of 2 from table 7.1 and draw a conclusion on your results based on Mendels second Law. TABLE 7.3 Dihybrid cross: student 2 test Expected number of count (e) Divergence / expected no. of count 2 (o e) /(e)
2
Phenotype Yellow striped Yellow spotted Red striped Red spotted Total 3.0
Expected ratio 9 3 3 1
Observation (o)
Divergence (o - e)
Divergence 2 (o e)
n=
Questions 1. Did your data follow the Mendelian predicted ratios? If they did, why? If they didnt, why not? 2. How accurate is the Chi-squared test? Can you prove its accuracy? 3. Does every inheritance follow Medelian Laws? If not, what are these exceptions?
Adapted from STPM Biology: Students Manual 2003/2004. Pg. 48 52. 35
Practical 8
Testing the Hardy-Weinberg Equilibrium
1.0
In 1908, G.H. Hardy and W. Weinberg separately introduced a mathematical model that shows the relationship between the frequency of an allele and the frequency of a genotype in a hypothetical population that has reached genetic equilibrium. Collectively this is commonly used population genetics equilibrium is known as the Hardy-Weinberg equilibrium. It states that under certain conditions, after one generation of random mating, the frequency of dominant alleles and the frequency of recessive alleles at a single gene locus in a population remain constant from generation to generation under certain conditions. These conditions are: A large population (so that any change in allelic frequency is negligible, or to minimize genetic drift) No natural selection Random mating (all individuals are sexually active and equally fertile) No mutation (in case of mutation, the rate of forward mutation is the same as the rate of backward mutation) No migration/gene flow (no immigration or emigration) Based on the equilibrium and conditions, Hardy and Weinberg developed an equation based on the theory that the total frequency of alleles of a gene is 100% (generally represented as 1.0). Hence, the Hardy-Weinberg equation Equation (8.1):p + q = 1.0 where, p = frequency of dominant allele; q = frequency of recessive allele However, in diploids, alleles occur in pairs. Thus a 2 Hardy Weinberg population genotype frequency) Equation (8.2):p + 2pq + q = 1.0 where, p = frequency of homozygous dominant allele genotype; 2pq = frequency of heterozygous genotype; 2 q = frequency of homozygous recessive genotype.
2 2 2 nd
derivation (commonly known as the
Hence, the Hardy-Weinberg equilibrium allows us to predict the populati ons genotype frequency from its allelic frequencies. The application of the Hardy-Weinberg principle in relation to an X-linked gene (i.e. the one controlling eye colour in Drosophila melanogaster and colour blindness in humans) is slightly different. The allelic frequencies are estimated from the frequencies of genotypes in males; and the frequencies of the genotypes in females are obtained by applying the Hardy-Weinberg principle to these estimated allelic frequencies (assumption: allelic frequencies are the same in the 2 sexes). For example, in human colour blindness (X-linked recessive mutation), if the frequency of normal vision ( C) is p = 0.88 and the frequency of colour blindness (c) is q = 0.12, then under the Hardy-Weinberg assumptions, refer to table 8.1:-
36
TABLE 8.1 Allelic frequencies in colour blindness Sex Genotype Frequency Phenotype C p = 0.88 Males Normal vision (single allele C c q = 0.12 Colour blind because either X Y c or X Y - hemizygous) 2 CC p = 0.77 Normal vision Cc 2pq = 0.21 Females Normal vision 2 cc q = 0.02 Colour blind Drosophila melanogaster (common fruit fly) is a commonly used model organism for genetic studies because it is easily cultured in the lab, has a short generation time, and can produce many offspring. In this laboratory practical, you will be looking at the sex-linked (X-linked mutation) inheritance of the eye colour inheritance in Drosophila melanogaster (fruit flies) where the wild + + type/normal phenotype is red (allele denoted by w or X ) and the mutant phenotype is white w (allele denoted by w or X ). Remember this gene is only found on the X chromosome. The males in this fruit fly only have one X chromosome, similar to human sex chromosomes. This is as shown in the cross between a homozygous mutant female and a hemizygous wild-type male below:P: X X (White eyed female) X X (Red eyed female)
+ w w
X Y (Red eyed male) X Y (White eyed male) Self-fertilise

w + w
F1:
X X X X X Y X Y (White eyed) (Red eyed) (White eyed) (Red eyed) (FEMALE) (MALE) Note: An organism that has only one copy of a gene is called a hemizygote (i.e. males that have only a single X chromosome, and in this case, carries the gene controlling eye colour) 2.1 Materials Ice (crushed) Petri dish Fly culture media Magnifying glass Forceps Culture bottle (flask) 1 vial of wild type flies 1 vial of mutant flies
F2:
37
2.2 Methods a) Sexing and selection of required Drosophila melanogaster flies 1. Work in pairs - one pair working on the wild-type flies and another on the mutant flies. 2. Obtain a vial of wild-type/mutant flies and anesthetize your flies by placing into the freezer for about 20 minutes (or until still). 3. While waiting, construct a simple chilling plate where you will adding crushed ice on top of a Petri dish. Then place the bottom of the Petri dish on top. This will keep the flies chilled long enough for sexing and selection. 4. Once your flies are anesthetized, use a pair of forceps and pick 5 flies at random and perform fly sexing (Figure 8.1). Use a magnifying glass to view. Ensure that the flies are placed on ice (in vials) or on the chilling plate when you are sexing.
Ventral view
B Dorsal view
FIGURE 8.1 Drosophila melanogaster surface anatomy : Male (A) and Female (B). For the male flies, they have "sex combs" (arrow) on their front legs. The posterior part of the abdomen is quite dark in males and considerably lighter in females. The tip of the abdomen in more rounded in males than in females. In general the male is smaller than the female. As for the dorsal view, it can be seen that they have external genitalia. For the female flies, the posterior part of the abdomen is considerably lighter in females compared to male. The tip of the abdomen is more pointed. In general, females are larger than males. As for the dorsal view, it can be observed that they have a somewhat striped abdominal region with no external genitalia. 38
5. Repeat step 4 until you are able to pick 5 male red eyed flies and 5 female white eyed flies. 6. Obtain the culture bottle provided. Place it in a horizontal position on your bench. 7. Place the selected flies into the horizontally positioned culture bottle. Ensure that the flies are not pushed towards, or near, the culture medium. 8. Close with bung provided. Ensure it is tight so that your flies do not fly out of the bottle. 9. Label the bottle carefully with your group name and date. 10. Lift the bottle to a vertical position once the flies are awake and store at room temperature. th 11. Carry out your observation on the 10 day at the time allocated by the laboratory technician. b) 10 day observation 1. Anaesthetize and select the flies as steps 2-5 in section a). This time, make piles of male and female flies. From those piles, record their phenotypes (either red or white eyes) into table 8.2 (F1 generation column). 2. From your piles created, pick 5 red eyed virgin females (newly born and alive for about 8 hours) and 5 white eyed males and perform a second breeding in a brand new culture flask. Repeat steps 6-10 from section a). c) 20 day observation 1. Anaesthetize and select the flies as steps 2-5 in section a). Again, make piles of male and female flies. From those piles, record their phenotypes (either red or white eyes) into table 8.2 (F2 generation column). TABLE 8.2 Parent 5 0 0 5 Phenotype data tabulation F1 generation Predicted Actual
th th
Sex
Phenotype Red eyed
F2 generation Actual Predicted
Male
White eyed Red eyed
Female
White eyed
Hint: To obtain predicted values, refer to the crossing on page 37 and predict using Mendellian ratios learnt in Practical 7.
39
Sex
TABLE 8.3 Possible allelic (males) or genotypic (female) frequency F1 generation F2 generation Allele/ Variables Parent Genotype + w p w w w
+ + +
Phenotype Red eyed White eyed
Males
q p
2
N/A
Red eyed Red eyed White eyed
Females
w w ww
2pq q
2
Hint: To obtain the allelic frequency, you would only have to observe the male flies. Assume that the allelic frequencies are the same in the 2 sexes. Use that allelic frequency in order to predict the genotypic frequencies by using the variables provided. Remember, p + q = 1.0 and 2 2 p + 2pq + q = 1.0! 3.0 Questions 1. Based on table 8.2, does your count follow the predicted phenotype using the crossing? Explain. a. BONUS: Connect this with the 2 test to prove your results statistically. 2. At the end of your experiment, what is the probability of obtaining red-eyed flies? 3. What can be concluded regarding the inheritance of the red and white eye colour in this fruit fly? Explain, using your results as proof. 4. Perform crossings and state the probability of obtaining each phenotype at every generation as follows:a. Red-eyed homozygous female and white-eyed male (Perform cross till F2 generation where F1 generation is self-fertilised) b. Red-eyed heterozygous female and white-eyed male Note: Observe the similarity in F1 generation between what you have done in this practical and items 2a and 2b 5. Why is it important to use virgin female flies? 6. Did you perform a reciprocal cross? Explain reciprocal crossing and the point of conducting this type of crossing, in relation to your experiment.
Adapted from nd Lum, H.K., 1982. Kursus Amali Biologi: Pra-Universiti Buku 2. 2 ed. Longman: Malaysia. Pg. 87. Reference th Snustad, P., and Simmons, M.J., 2006. Principles of Genetics. 4 Ed. John Wiley and Sons: Asia. Pg. 87 89, 738 741.
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Practical 9
DNA Extraction from Human Buccal Cells
1.0
With the advent of gene technology, it is important to understand not only the phenotype of the organism but also the genotype. Previously, you should have learnt the analysis of genetic traits and the various ways where they can be transmitted from parents to children (by phenotype analysis). Each chromosome is divided into different sections called genes. Genes are the basis of inheritance where traits like hair colour and blood type are controlled by the production of proteins by these genes. Genes contain coded instructions that the body uses to assemble hundreds of different types of proteins that make an individual unique! These amazing trait controllers (genes) are made up of molecules called deoxyribonucleic acid (DNA). DNA is a double-helical polymer bound together by hydrogen bonds between complementary base pairing nucleotides (A to T, G to C). A particular gene is a set of coded instructions made up of a particular order of nucleotides. The variation of which allows the myriad of codes to exist in an organism for it to be unique. This is what controls the genotype of an organism and henceforth, the extraction and isolation of an organisms DNA is imperative, in order to allow further insight into the organism using different molecular-based methods. In this experiment, you will be taking a closer look at this DNA molecule. You will be extracting your own DNA using buccal/cheek cells as the starting material. 2.1 Materials
Saline 15 ml centrifuge tube Paper cup Drinking water Vortex Centrifuge 10% SDS Protease Ice cold Ethanol Graduated pipettes
2.2
Methods
IMPORTANT NOTE: Ensure that you have not eaten in the past 1 hour before conducting this experiment (if you are the DNA donor). Ensure that gloves are worn at all times in the experiment. 1. Swish you mouth with about 100 mL drinking water, for about 20 seconds, to remove any food particles. Discard this wash into the sink. 2. Using a permanent marker pen, label your group name onto the paper cup and 15 mL centrifuge tube containing 10mL saline. 3. Pour all the 10mL saline solution into your mouth and vigorously swish for 60s. Do not discard the centrifuge tube. 41
4. Expel the saline mouthwash into the labelled paper cup. 5. Carefully, pour the saline mouthwash from the paper cup, back into the 15 mL centrifuge tube from step 2. Tightly cap the tube. 6. Pass the capped tubes to the laboratory technician in order to be centrifuged (13000 rpm, 5 min). 7. Upon centrifuging, you should be able to see your buccal cell pellet ( the whitish lower solid layer at the bottom of the tube). Gently, pour away the supernatant ( the liquid upper layer). 8. Place the tube on ice. 9. Add 2 mL saline into the tube and vortex for 5-10 seconds. 10. Add 1 mL 10% (w/v) sodium dodecylsulphate (SDS) solution ( active component in detergents). 11. Gently tap the tubes several times (~8 times) to gently mix the contents. 12. On ice, add 2 3 drops of the lab supplied protease enzyme/meat tenderizer into the tube. 13. Gently tap the tubes several times (~8 times) to gently mix the contents. o 14. Cap the tube and place it is a 50 C for 10 minutes. 15. With a clean pipette, gently pipette in 10 mL ice cold ethanol (95% v/v) slowly into the tube. Tip: Place the filled pipette with its tip against the inside wall of the test tube. Slowly allow the ethanol to dribble down the inside of the tube. 16. Cap and place the tube on ice for 10 minutes. DO NOT mix, shake, or bump the test tube during this period. 17. The ethanol is lighter than the contents of the tube. When added according to the directions, the ethanol will form a clear layer ABOVE the suspension. 18. Observe the test tube for 5 minutes. The DNA will gradually separate from the suspension and rise into the ethanol layer. Describe the appearance of the DNA. 19. Take a photo as proof of your observation. 20. To remove the accumulated DNA from the tube, follow the directions for DNA spooling as below:a. Gently insert the glass rod through the ethanol layer into the clumped/accumulated DNA. b. Carefully, twirl the rod between your fingers, winding the DNA strands onto the rod. c. Slowly remove the rod. Describe the appearance of the spooled DNA. d. Take a photo as proof of your observation. 3.0 Questions 1. Which one of the following do you think will contain DNA? Explain your reasoning. Bananas; concrete; fossils; meat; metal; spinach; strawberries. 2. What effect would the SDS have on the cell membranes and cold ethanol on DNA? 3. What type of enzyme would be needed to separate the DNA into smaller pieces? 4. Is the DNA extracted pure enough for further applications (i.e. PCR)? 5. If you were to repeat the experiment with an equal number of red blood cells, the amount of DNA collected would either: increase / decrease / stay the same ( choose one). Explain your answer. Adapted from:Bres, M., Weisshaar, A., 2008. Thinking about Biology: An Introductory Laboratory Manual. rd 3 Ed. Pearson Prentice Hall: New Jersey, USA. Pg. 333 - 338. Teaching AS Biology Practical Skills. University of Cambridge: International Examination. Pg. 74 78.
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Practical 10
Digestion of Lambda () DNA with a Restriction Enzyme ( EcoR I endonuclease)
1.0
Restriction enzymes (nucleases) are enzymes that cleave the phosphodiester bonds on the sides of deoxyribonucleic acid (DNA). These nucleases recognize specific DNA sequences in the double-stranded DNA, which is usually a four to six base pair sequence of nucleotides, and digests the DNA at these sites, resulting in the DNA becoming fragmented into various lengths. Some restriction enzymes cut cleanly through the DNA double helix while some produce uneven or sticky ends. By using the same restriction enzyme to cut DNA from different organisms, the sticky ends produced will be complementary and the DNA from the two different sources can be recombined. In humans, no two individuals have the exact same restriction enzyme pattern in the DNA except for identical twins. Restriction enzymes are named based on a system of nomenclature where the first letters represents the genus name of the organism whereas the next two letters come from the species name. If there is a fourth letter, it stands for the strain of the organism. Finally, if there are Roman numerals, it represents whether that particular enzyme was the first or second etc. isolated in that category.
FIGURE 10.1
Cartoon of how EcoR I recognises the restriction site and cleaves the DNA.
The second technique used in this practical is the separation and analysis of DNA fragments. Agarose gels are commonly used for this where the gels that have been prepared with a suitable nucleic acid stain in it, have wells for the samples of DNA to go into. The agarose gel is covered in a suitable buffer so that the DNA is in a neutral pH solution. That way, the DNA moves one direction because of its charge. Since the phosphate groups on the skeleton of DNA are negatively charged, the whole molecule takes on the negative charge. Hence, when the DNA is placed inside the gel and the electricity is turned on, the poles are drawing the DNA toward the positive side, where it will then move through the gel and separate according to the size of the fragments. This technique is called electrophoresis. Results are obtained with the help of UV light that is refracted by the nucleic acid stain that sticks onto the DNA fragments.
43
In this experiment, you will be using the EcoR I restriction endonuclease to digest a known DNA called phage lambda () and analysing your sample using agarose gel electrophoresis. 2.1 Materials Pre-laboratory work Computer/laptop LambdaDNA.docx (Word document file) Laboratory work Micropipette Sterile pipette tips Microcentrifuge tube (1.5 mL capacity) EcoR I (20 U/L) and buffer Distilled deionised sterilised water Agarose gel (prestained with EtBr) 1x TAE buffer Agarose gel electrophoresis set UV Transilluminator 2.2 Methods
Pre-laboratory work (this is to be done at home and to be discussed before beginning the experiment!) 1. Download and save the lambda DNA sequence from the class website. 2. Save the file onto your computer or flash drive. 3. Open the saved file in Microsoft Word. It contains the entire phage Lambda DNA sequence or genome. Browse and notice how the file is filled with a long string of letters A, C, G, and T. 4. To determine how many letters (nucleotides) there are in the genome:a. On your keyboard, press CTRL+A in order to select all. b. Under the TOOLS menu or on the 2007 version, the REVIEW tab, choose WORD COUNT. c. Note the number of characters. d. Click OK to close the window. 5. To simulate digestion of EcoR I (recognition site G AATTC) (Arrow representing the place where the DNA will be cut by the enzyme upon encountering the recognition site sequence):a. Place the cursor at the beginning of the Lambda sequence. b. On your keyboard, press CTRL+F and click on the REPLACE tab. c. In the Find what: box, type gaattc d. In the Replace with: box, type in G^p^pAATTC e. Select REPLACE ALL. f. Click YES or OK to close the window. Note: This cuts the Lambda DNA at all the Eco R I restriction sites by placing 2 paragraph breaks (^p^p) at each point where the sequence has been cut. It also converts the 6 nucleotide sequence to all UPPERCASE letters so that you can see them more easily. Scroll through your newly edited document. 6. To determine the number of fragments produced - repeat step 4 (a-b). Note the number of words. Record results in table 10.1. 7. Click OK to close window.
44
8. To determine the size of each of the fragments - Go to the top of the document and highlight the first paragraph (the first fragment produced from the cutting) by clicking and dragging the cursor from the beginning till the end of the paragraph. 9. Repeat step 4 (b-d). Note the number of characters and enter results in table 10.1. 10. Click OK to exit window. 11. Repeat step 10 on the consecutive paragraphs, one at a time till the end of the document. 12. Now, to simulate gel electrophoresis, use Figure 10.1. Make believe that you have placed in your EcoR I digested Lambda DNA into the well (marked by a - ). Label your lane as shown in the figure. 13. Draw a rough sketch of how your electrophoresis gel would look like after you ran it for a while. Draw in terms of bands (the thick highlighted ) of the predicted DNA fragments based on Table 10.1. 14. Remember that the top most band, closest to the well is the largest fragment and the lowest is the smallest fragment. Using the size marker lane as your DNA fragment size guide (your ruler); arrange your bands according to the size order.
Size of phage Lambda DNA genome: a. in bases (base pair - bp)= _____________ b. in kilobases (kb) (Note: 1 kb = 1 000 bases) = _____________
TABLE 10.1 Fragment sizes (in bases) resulting from a restriction digest of phage Lambda DNA using EcoR I Restriction Size of each fragment produced (in number of Total Number of Enzyme characters/bases) Fragments EcoR I
45
FIGURE 10.1
Agarose gel electrophoresis prediction. Depict your predicted electrophoresed EcoR I digested Lambda DNA fragments using table 10.1.
46
NOTE: Ensure gloves are worn at all times! Be careful while loading your sample into the agarose gel wells. The stain used is a carcinogen (cancerous). Laboratory work 1. Set up the digestion reaction as prepared for you by the laboratory technician in a small microcentrifuge tube. 2. For your information the reaction is prepared as in table 10.2 TABLE 10.2 Restriction enzyme reaction tube mix Components Volume (L) Lambda () DNA (150 ng/L) 1.0 10x restriction enzyme buffer 2.0 Restriction enzyme, EcoR I 2.0 (20 u/L) Water (ddH20, sterilised) 15.0 Total volume 20.0 3. Remove the reaction tubes from ice and place them onto a floatie. o 4. Place the collected tubes in floaties in an incubation oven (dry heat) at 37 C for 1 - 3 hours. 5. Once incubation period is up, quick spin the tubes in a microcentrifuge (minispin) to allow all evaporation to sink down. o 6. Remove the floaties and place in a water bath at 65 C for 10 minutes (to keep the fragmented DNA strands from rejoining). During this period, listen carefully to the briefing provided by the lecturer regarding the usage of a micropipette. Ensure proper usage of this sensitive and expensive apparatus. 7. Remove the floatie from the water bath and place it on the bench nearby the electrophoresis bench. 8. Collect your own reaction tube and add in 5 L of DNA loading dye (6x) into the reaction tube. Pipette up and down gently to resuspend the mixture. 9. Note the prepared 1% (w/v) agarose gel in the electrophoresis chamber and listen to the precautionary steps. Remember, the gels contain ethidium bromide (a carcinogen) that can be very harmful. 10. Follow the steps shown by the lecturer regarding loading your samples into the well. 11. Do not forget to load the DNA size marker as well. 12. The laboratory technician will then close the tank top and run electricity through the gel (80V for 1 hour). 13. When the run has ended, the laboratory technician will remove the gel and place it into the UV transilluminator (which is a chamber that allows UV illumination. This is so that you DNA bands can be seen). 14. Using the machine, focus your gel and take a photo. Scan and attach this to your results. 15. Based on your gel photo, fill in table 10.3 using the measurements of the size marker. The size of the sequence (bp) per band is set by the manufacturer whereas the measurements (mm) is measured by the individual band from the well itself. 16. Use table 10.3 to sketch a standard curve of size (bp) versus measured distance (mm) using Microsoft Excel. Use the option of creating a formula based on the curve obtained. 17. Repeat step 15 for the digested Lambda DNA with EcoR I and fill in the Measured distance (mm) column in table 10.4 accordingly, where band 1 is the largest fragment (closest to the well) and band 8 is the smallest (furthest from the well). 18. To obtain the Actual sequence size column in table 10.4, use the formula obtained from the curve that you sketched (based on table 10.3) and plug in values to obtain the actual size. 47
19. Compare and analyse your obtained laboratory work data with your pre-laboratory work data. 20. Sketch a restriction map based on your obtained and concluded data. TABLE 10.3 Measurement of size marker tabulation. Measurements (mm) taken based on the band position from the well. Sketch your standard curve using this table. Size (kb) Measured distance (mm) 10.0 8.0 6.0 5.0 4.0 3.0 2.0 1.5 1.0 0.5
TABLE 10.4 Bands 1 2 3 4 5 6 7 8
Measurement of Lambda DNA digested with Eco R I. Measurements (mm) taken based on the band position seen on gel from the well. Measured distance Estimated Sequence Actual Sequence (mm) Size (bp) Size (bp)
48
3.0
Questions 1. From your pre-laboratory work, what does each of the letters represent? 2. What is the source of restriction enzymes? What is their function in nature? 3. Describe in electrophoresis, the function of a. electricity b. agarose gel 4. Explain why you might not see all of the predicted fragments that were produced from table 10.1, figure 10.1 when you actually ran the gel (In answering this, indicate the probable sizes of the fragments HINT!). 5. Use the graph prepared from the lab data to predict how far (in mm) a fragment of 8000 base pairs would migrate. 6. If a mutation occurs on the Lambda DNA sequence along one of the EcoR I restriction site, how can it be detected by gel electrophoresis? 7. Predict the number of DNA fragments and their sizes if Lambda phage DNA were incubated and cleaved with Hind III (refer to the map below). Hind III (7 restriction sites in -DNA)
8. Provide an application of restriction enzyme usage in forensic science. Adapted from Foglia, K., n.d. AP Biology 1 Lab 12. Pg. 1 6. Edvotek, n.d. Restriction Enzyme Cleavage of DNA and Electrophoresis (AP Biology Lab 6B) . References DNA Sequences and Maps, n.d. New England Biolabs [Online] Available from: <http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dna_sequences_maps. nd asp> [Accessed on April 22 , 2010]
~ THE END ~ Hope you enjoyed the lab sessions! 49

MF011 Fhs Lab May10

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MF011 General Biology 2 Mr.

Veerapen, MK May August 2010 Semester

FACULTY OF APPLIED SCIENCES

Laboratory Practicals Manual

MF 011 General Biology 2

Lecturer : Laboratory technician: Lab Session : Semester :

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Practicals 3 &4 : Practical 5 :

Practical 6 : Practical 7 : Practical 8 : Practical 9 : Practical 10:

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

1 (Biuret) 2 (Benedicts) 3 (Iodine) 4 (All 3)

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Suggested reference Histology Text books

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Mammary glands Pectoral muscles Rib cage and intercostals muscles

Femoral artery and vein

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Serosal membrane of the abdominal cavity of a female rat

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Left seminal vein Left seminal artery Rectum

Seminal vesicle Coagulating gland

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

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Right auricle Left ventricle Left lung

Right ventricle Oesophagus

in situ of the thoracic visceral organs.

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Epiglottis Larynx Thyroid gland Trachea

Thoracic arterial supply in rats.

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Outer right carotid artery

Thoracic venous supply in rats.

Right auricle Right ventricle

Left ventricle Posterior vena cava

Ventral view of the rats heat.

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Cut along the dotted lines (--->)

Separating and pinning down the upper epidermal layer.

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Cutting the abdominal wall.

Releasing the anterior abdominal vein from the bodys wall .

MF011 General Biology 2 Mr. Veerapen, MK May August 2010 Semester

Anterior abdominal vein Liver

Oviduct Intestines Ovary Stomach Duodenum Urinal bladder Rectum

Left aortic curve

Left anterior vena cava Left auricle

Right anterior vena cava Right pulmonary vein

Left pulmonary vein Auricle-ventricle pathway

Posterior vena cava