HBM1202 Anatomy & Physiology 2 Lab Manual 2024 PDF

HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 HBM1202 Anatomy & Physiology 2 Laboratory Manual 2024 Student Use VICTORIA UNIVERSI...

HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 HBM1202 Anatomy & Physiology 2 Laboratory Manual 2024 Student Use VICTORIA UNIVERSITY 1 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 HBM1202 ANATOMY AND PHYSIOLOGY LABORATORY MANUAL FOR STUDENTS *Lab coats are required for all laboratory sessions *Please read the manual before your laboratory class Please also read related risk assessments, watch lab videos and engage in practice lab online worksheets in VU Collaborate. Following your second lab each week (Labs 2, 4 & 6) complete the post lab worksheet by accessing VU Collaborate > Assessments > Quizzes. LIST OF LABORATORIES 1. Week 1- Lab 1- Endocrine System and Homeostasis 2. Week 1- Lab 2- Bones, Joints and Muscles 3. Week 2- Lab 1- Special Senses 4. Week 2-Lab 2- Urinary System 5. Week 3- Lab 1- Acids, Bases and Buffers 6. Week 3- Lab 2- Digestion and Metabolism 2 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PRACTICAL 1: ENDOCRINE SYSTEM AND HOMEOSTASIS WARNING: It has been identified from past experience that a small group of students may experience emotional distress due to finger pricking procedure/sight of blood. These small group of people may experience shivering, fainting, dizziness and/or fall after or during the activity. Although likely hood of this happening is low, the risk is high due to possibility of fall from loss of balance. Please note: ONLY ONE STUDENT FROM THE LAB CLASS WILL DO THE FINGER PRICKING EXPERIMENT. THIS IS A DEMONSTRATION PRAC. PLEASE DO NOT VOLUNTEER FOR THIS ACTIVITY IF YOU ARE UNCOMFORTABLE. INTRODUCTION Homeostasis can be described as a dynamic state of equilibrium in which the internal environment of the body remains relatively constant. An organism is said to be in homeostasis when its extracellular fluid is maintained at optimal volume, temperature, pressure and concentration of gases, nutrients and ions and functional activities are occurring smoothly. In today's class, we will be concerned with the role of hormones in homeostatic mechanisms and demonstrate homeostasis of glucose regulation. While the nervous system communicates using electrical signals, the body’s endocrine system uses chemical signals, called hormones, to regulate body functioning. Hormones are involved in maintaining the body’s homeostasis. These chemical messengers carry signals from one cell to another and regulate many of the body’s functions, including growth and development, metabolism and reproduction. Hormones are secreted by tissues in the body referred to as glands. Endocrine glands secrete hormones directly into the bloodstream while exocrine glands secrete hormones into ducts, or passageways, before they reach their target. Each hormone has a specific list of target tissues, and in many cases these include other endocrine glands. This system of chemical communication, the endocrine system, works with the nervous system to regulate and control all the actions of the human body. OBJECTIVES Demonstrate and explain how the endocrine system functions to regulate blood glucose homeostasis. Using histology to observe and describe the structure and function of endocrine glands. ACTIVITY 1: GLUCOSE REGULATION Glucose is the major carbohydrate fuel source of the body and is transported via the blood to the cells. Normal blood glucose concentrations range between 3-7 mmol/L. Glucose monitoring is a technique whereby the glucose level in the blood can be determined using a simple blood test. The Oral Glucose Tolerance Test (OGTT) involves recording baseline fasting blood glucose levels, the ingestion of a set amount of glucose (75 g) as a challenge, and monitoring of the change in blood 3 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 glucose over a two-hour period. For the normal person, glucose ingestion will stimulate increased insulin secretion. The diabetic with absent or impaired insulin secretion will not have the same response. Other diabetics may secrete insulin but their cells do not respond to it. Hence, diabetics are more likely to exhibit a much greater than normal rise in blood glucose levels, a higher level at two hours and a much slower return to baseline values if tested beyond three hours. Average Results: Fasting: 60 to 100 mg/dL (3.3 to 5.5 mmol/L) 1 hour: less than 200 mg/dL (11.1 mmol/L) 2 hours: less than 140 mg/dL (7.8 mmol/L) If you are participating in the practical exercise: DO NOT EAT FOUR HOURS BEFORE COMMENCEMENT OF THE PRACTICAL! Important Safety considerations for this practical This practical involves the handling of human materials, All care and caution is to be exercised AT ALL TIMES. No one is to handle any sample other than his or her own. Any spillage MUST be reported to the demonstrator IMMEDIATELY. Gloves are to be worn AT ALL TIMES when handling any of the samples. Only one student per bench is needed to complete the test. Maximum of 4 students per lab session is recommended. Do not perform the activity if you have cardiovascular disease, diabetes or other disorders. PROCEDURE: 1. Measure the fasting blood glucose levels as follows: a) Wash your hands, using the antiseptic soap provided b) Warm the finger, sterilising it with an alcoholic swab and air drying thoroughly c) Constrict the venous drainage in the finger by applying pressure with the thumb d) Pierce the finger with the "Redilance" soft touch, allow a large drop of blood to accumulate then transfer this to the end of the Dextrostix 4 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 e) Use the Glucometer (information on how to use this will be provided in the laboratory), and record the result of the glucose determination as time 0. f) Dispose of used "Redilance" tip into the 'sharps' bin. Dispose of all other blood contaminated materials into the biohazard bin. 2. Take careful note of the time. The test is timed from the beginning of the drink. 3. Start the test by drinking a solution of 50 g of glucose in water. You may wash this down with more water to a maximum of 250-300 ml. 4. To avoid contaminating later blood samples with glucose from the test solution, wash your hands thoroughly after drinking the glucose load. 5. Repeat the measurement of fasting blood glucose in point (1) at 20 minute intervals for a period of about 90 minutes ie at 0, 20, 40, 60, and 80 minutes after drinking the glucose load at time 0. RESULTS 1. Record your results in the table below. 2. Make sure to fill in your results on the white board that covers all the students in the class. 3. Graphically represent the data from each student and summary (average) data from your class and try to use the graph to highlight the major findings and any discrepancies. TABLE 1: GLUCOSE REGULATION Group Fast Blood Glucose Level (mmol/L) (Y/N) O min 20 min 40 min 60 min 80 min Student 1 Student 2 Student 3 Student 4 5 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Plot your results on the graph below Oral glucose tolerance test 20 18 16 14 BGL (mmol/L) 12 10 8 6 4 2 0 0 30 60 90 120 Time (min) An example is given below for a study that looked at people with normal glucose or had Type 2 diabetes mellitus or impaired glucose tolerance. Pala, Ciani, Dicembrini, Bardini, Cresci, Pezzatini, Gianinni, Mannucci & Rotella. 2010, Diabetic Medicine, 27, 691-6 6 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 QUESTIONS Feedback Loops: Glucose and Glucagon The control of blood sugar (glucose) by insulin is a good example of a negative feedback mechanism. When blood sugar rises, receptors in the body sense a change. In turn, the control center (pancreas) secretes insulin into the blood effectively lowering blood sugar levels. Once blood sugar levels reach homeostasis, the pancreas stops releasing insulin. Examine the graphic below to understand how this feedback loop works. 1. The image shows two different types of stimuli (1 and 2), but doesn't explain what the stimuli is that causes blood sugar to raise or lower. Based on clues in the graphic, what are the two stimuli? 2. What happens when your blood sugar rises? 3. What is the effect of insulin? 4. What is the effect of glucagon? 5. What is the normal level of glucose in the blood? Why is this called a "set point." 7 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 The following data table shows acceptable minimum and maximum values for the OGTT. Time (minutes) 0 30 60 90 120 Maximum Blood 5.6 9.4 8.9 7.2 5.8 Glucose (mmol/L) Minimum Blood 4.4 8.3 7.8 6.1 4.7 Glucose (mmol/L) 6. Two individuals (Maria and Laura) both complain to their doctor of frequent thirst and frequent urination. Laura has the additional symptom of blurry vision and a “tingling sensation” in her fingers and toes. The doctor suspects diabetes mellitus, a disease in which blood glucose metabolism does not stay within normal ranges. The doctor orders both to undergo an Oral Glucose Tolerance Test to test this prediction. Below are the results of the OGTT. (adapted from POGIL-Murray) Measurements recorded during OGTT Time (minutes) 0 30 60 90 120 Maria: 5.0 7.8 8.3 7.5 6.1 Blood Glucose (mmol/L) Laura: 5.6 9.4 12.2 15.0 16.7 Blood Glucose (mmol/L) a. Which individual is more suspect of having diabetes mellitus? b. Justify your answer comparing each person’s OGTT data to data found above for normal values. 8 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 2: HISTOLOGY OF THE ENDOCRINE GLANDS Endocrine glands significantly vary in structure but they all have some common features as they all secrete hormones into the blood stream. The histological structure of the endocrine glands helps to provide an insight into how these glands function and how they are related to their surroundings. PROCEDURE: 1. Using a low power objective, scan the slides of endocrine glands provided in the laboratory and try and identify their specific features. Histology text books provided in the lab class may help you. 2. Draw and label the slides of the endocrine glands you observed. RESULTS *Draw and Label endocrine gland histology -Pituitary Gland -Adrenal Cortex; Adrenal Medulla -Pancreas Questions: a. Which cells of the pancreas releases insulin? Which cells of the pancreas releases glucagon? b. Which hormones are secreted from the adrenal cortex? Which hormones are secreted from the adrenal medulla? Demonstrators Signature: __________________ Date:_________ 9 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PRACTICAL 2: BONES, JOINTS and MUSCLES BACKGROUND The musculoskeletal system consists mainly of bones, cartilage, joints (i.e. articulations), ligaments and muscles. These structures are responsible for holding the body together and moving it around; support and protection; production of blood cells and heat; and storage of minerals and energy. There are 206 bones and more than 639 muscles in the body. We use about 300 of these muscles just to keep our balance when standing! Skeletal muscles allow the body to perform movements at joints. Muscles have 2 attachments, an origin and an insertion. The origin is on the bone which stays still and the insertion is on the bone that moves. When muscles contract and shorten, they bring the origin closer to the insertion. OBJECTIVES Understand anatomical terminology and identify the regions of the body. Understand how joints allow different body movements. Identify and describe different types of joints including synovial joints. Identify major skeletal muscles of the body and describe their function/actions. Understand the anatomical structure and function of the hip joint. ACTIVITY 1: ANATOMICAL TERMINOLOGY AND BODY REGIONS PROCEDURE: 1. Identify on models or your partner the following body regions; head, neck, thorax, abdomen, pelvis, upper limb, shoulder, arm, forearm, wrist, hand, lower limb, thigh, leg, ankle, foot. 2. Using the descriptions in the table below, identify each of the anatomical terms on the models or your partner and provide examples. For example: The head is superior to the feet. Term Description Example Superior (cranial) Toward the head Inferior (caudal) Toward the feet or tail region Anterior Toward the front of the body (ventral) Posterior Toward the back of the body (dorsal) 10 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Medial Toward (nearer) the midline of the body Lateral Away (farther) from the midline of the body Proximal Toward (nearer) the trunk of the body; toward the attached end of a limb Distal Away (farther) from the trunk of the body; away from the attached end of a limb Superficial Nearer the surface of the body Deep Farther from the surface of the body Peripheral Away from the central axis of the body ACTIVITY 2: BONES OF THE SKELETON PROCEDURE: 1. On the models provided or on your partner identify the following bones of the skeleton The skull: frontal, parietal, occipital, temporal, sphenoid Facial bones: maxilla, mandible Vertebrae: cervical, thoracic, lumbar, sacral, coccygeal Manubrium, sternum, ribs (true, false, floating), pectoral girdle (scapula, clavicle) Humerus, ulna, radius, carpals, metacarpals, phalanges, Pelvis/hip (ilium, ischium, pubis), femur, patella, tibia, fibula, tarsals, metatarsals, phalanges. 11 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 3: BODY MOVEMENTS PROCEDURE: 1. With your partner identify and perform each of the body movements, try to use identify more than one example of each. For example: Eversion is to turn sole of foot outwards Body Examples Movement Gliding Flexion Extension Hyperextension Abduction Adduction Medial rotation Lateral rotation Circumduction Protraction Retraction Depression Elevation Supination Pronation Eversion 12 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Inversion Dorsiflexion Plantar flexion ACTIVITY 4: TYPES OF JOINTS PROCEDURE: 1. With your partner, point out the examples of the joints below while explaining why those joints allow certain movement but not other. For example: On a skull, examine the sutures; note how the bones overlap making a very strong immovable joint. On the skeleton, or the plastic trunk model with one removable vertebrae, examine an intervertebral disc; note that it is composed of cartilage allowing a slight bit of movement. Movement Bones united Term Examples immoveable fibrous tissue fibrous skull sutures slightly cartilage cartilaginous intervertebral discs, symphysis pubis freely joint capsule synovial shoulder, hip, elbow, knee ACTIVITY 5: TYPES OF SYNOVIAL JOINTS PROCEDURE: 1. Identify the following synovial joints on the model, name examples to complete the table and any others you can find. Type Articulating Movements possible Example surfaces Gliding both nearly flat sliding Hinge one convex, one flexion, extension concave Pivot one round, one ring rotation shaped Ellipsoid both are oval shaped flexion, extension, abduction, adduction, circumduction Saddle both are saddle flexion, extension, abduction, shaped adduction, circumduction, rotation (< b & s) 13 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Ball & Socket one ball, one socket flexion, extension, abduction, adduction, circumduction, medial rotation lateral rotation 1. As an example of a gliding synovial joint, use 2 thoracic vertebrae and a rib, to point out the sliding articulating surfaces of a costovertebral joint. 2. As an example of a hinge synovial joint, use the model of the knee to point out the articular surfaces covered in cartilage (menisci), internal ligaments (cruciate ligaments) and external ligaments (co-lateral ligaments). 3. As an example of a pivot synovial joint, use cervical vertebrae 1 and 2 to point out the articulation between the dens of the axis (C2) and the arch of the atlas (C1). 4. As an example of an ellipsoid and of a saddle synovial joint, use a hand to point out the articulating surfaces of the metacarpophalangeal joint of a digit (ellipsoid) and carpometacarpal joint of thumb (saddle). 5. As an example of a ball and socket joint, compare the articulating surfaces of the shoulder with those of the hip. ACTIVITY 6: MAJOR MUSCLES OF THE BODY 1. Examine the models of either the arm or leg, and on your own body, identify each of the muscles from the table below and label their function in the Table. 2. Locate the approximate origin and insertion of the following muscles on the skeleton 3. Name the resulting movement. Groups of muscles or muscle Major Function (s) pectoralis major latissimus dorsi deltoid biceps triceps gluteal muscles quadriceps adductors hamstrings Questions: 1. Consider the knee and describe the type of joint, muscles and movements allowed by this type of joint. Demonstrators Signature: __________________ Date:_________ 14 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PRACTICAL 3: SPECIAL SENSES BACKGROUND A variety of factors in the environment play a significant role in the survival of humans. The body has developed sensory structures, receptors, which detect these stimuli and convert the information into nerve signals. Each type of receptor is highly sensitive to one type of stimulus but virtually non-responsive to other types of sensory stimuli. The nature of the nerve impulse is the same in all nerves, the different sensation experienced being a product of the part of central nervous system where the fibre terminates. Consequently, the interpretation and perception of a particular sensory response is determined at cerebral level no matter what stimulus triggers the impulse in the nerve fibre. Sight is our dominant sense, with receptors in the eyes accounting for about 70 percent of the receptors in the entire body. OBJECTIVES Describe the basic anatomy of the eye and correlate structure to function Explain how light entering the eye is focussed on the retina Outline the major neural pathways carrying visual information to the brain Recognise abnormal results obtained from vision tests Describe the basic anatomy of the ear and relate the main structures to their function. PART A: THE EYE AND VISION The optical system of the eye consists of a lens system and a light-sensitive surface. The function of the lens system is to focus the light reflected from objects onto the back of the eye (retina). The retina consists of cells that respond to different colours and intensities of light. The light-sensitive surface converts the image pattern into nerve impulses for transmission to the brain. ACTIVITY 1: DISSECTION OF A MAMMALIAN EYE In this activity you will observe the structure of the eye by dissection. WARNING: It has been identified from past experience that a small group of students may experience emotional distress in this lab. These small group of people may experience shivering, fainting, dizziness and/or fall after or during the dissection. Although likely hood of this happening is low, the risk is high due to possibility of fall from loss of balance. Please DO NOT participate in this activity if you are uncomfortable. 15 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PROCEDURE: 1. Work in pairs. 2. Examine the external appearance of the eye. Note the adipose tissue which is attached. What is the function of the adipose tissue? ___________________________________________________________________________ 3. Identify the remnants of the extrinsic muscles which may remain attached. What type of muscle are they? ___________________________________________________________________________ 4. Locate the optic nerve. Identify the pupil, iris, and sclera. The sclera is the outer layer of the eye and is a tough connective-tissue layer. It is continuous anteriorly with the cornea, which is transparent and covers the pupil and iris. The cornea and sclera are covered with a thin layer of cells, the conjunctiva, which is also reflected from the eyeball onto the inside surfaces of the eyelids. What is the function of the conjunctiva? __________________________________________________________________________ The middle or vascular layer of the eyeball is the choroid layer. The internal layer is the retina. 5. Remove the muscles and fascia from the sclera, leaving the optic nerve. 6. About 4 mm back from the edge of the cornea, lift the sclera with forceps and cut with scissors parallel to the edge of the cornea for about 2 cm. 7. With scissor tip, pierce the choroid around the eye. 8. Gradually allow the vitreous body with the lens, by its own weight, to detach itself from the dorsal half of the eye, receiving it in a watch glass. (The cornea and lens are clear in the living animal.) 9. Write two words in fine print on a piece of white paper. Place the lens on one word and the vitreous humour over the other. Note the difference in the appearance of the two words. __________________________________________________________________________ __________________________________________________________________________ 10. Study the inner surface of the anterior half of the eye after the removal of the vitreous body. 11. Observe the posterior surface of the iris and the ciliary body. 12. Note on the inner edge of the ciliary body the fibres of the suspensory ligament for the lens. 13. Identify the pupil and the anterior chamber of the eye. What fluid fills this chamber? __________________________________________________________________________ 16 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 14. Study the posterior half of the eye, observing the retina and the area and angle of the optic nerve, central artery, and retinal artery radiating from it. (There is no macula lutea in the calf eye). 15. Lift off the retina and note the choroid. 16. The choroid of animals often shows iridescence (tapetum lucidum); this is lacking in the human eye. What is the function of the choroid? __________________________________________________________________________ ACTIVITY 2: VISION TEST (HYPERMETROPIA AND MYOPIA) Abnormalities in vision can occur if the lens in the eye bends light so that it is not focussed sharply on the retina. For example, if the lens system is too weak or the retinal distance is too short (the eyeball is too short), the sharp image will form behind the retina, causing hypermetropia (farsightedness). On the other hand, if the lens system is too strong or the eyeball is too long, the sharp image will be formed in front of the retina, causing myopia (nearsightedness). PROCEDURE: 1. Work in groups of two to four. 2. Use Hodson's light box to investigate how hypermetropia and myopia can be adjusted using convex and concave lenses. 3. Insert the slide with the three slits, producing three parallel rays. 4. Place the wide bi-convex lens in front of the light source. Imagine that this lens represents the lens in the eye. Note the position of the focus. 5. Now, adjust the position of the focus, by placing a. the thin bi-convex lens, between the light source and the wide bi-convex lens. b. the bi-concave lens, between the light source and the wide bi-convex lens. 6. Use diagrams to show how hypermetropia and myopia can be corrected using convex and concave lenses. Photopupillary reflex: Sudden illumination of the retina by a bright causes the pupil to constrict reflexively. This protective response prevents damage to the delicate photoreceptor cells ACTIVITY 3: VISION TESTS (CHANGES IN PUPIL SIZE) PROCEDURE: 1. Allow a strong light from a window or lamp to fall on your eyes and then look at a dark surface away from the light while your partner observes the pupils of your eyes. What happens to the pupil? __________________________________________________________________________ 17 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 2. Place your hand over one eye for a minute and upon removing it, your partner should observe the reaction of the pupil. __________________________________________________________________________ 3. Cover one eye. Your partner should observe what happens to the pupil of the other eye. Describe what happens to the pupils in these three situations. Why is it useful for this to occur? __________________________________________________________________________ ACTIVITY 4: VISION TEST (VISUAL ACUITY) This can be measured using a Snellen eye chart. The person being tested stands 20 feet (6 m) away from the chart. At the end of each set of letters on the chart is an acuity number in the form of a fraction, which represents the ratio of two distances, that is, the ratio of one’s visual acuity to that of a normal person. If the person can read the letters in the line marked 20/20, from 20 feet way, he /she is said to have normal vision or emmetropic. PROCEDURE: 1. Stand 6 m (20 feet) from a Snellen visual acuity chart placed at eye level. 2. Cover one eye with a small card and measure your visual acuity for that eye. 3. Repeat for the other eye. If glasses are customarily worn, repeat with glasses. 4. Record your results. a. Visual acuity, right eye _______________________________________________________ b. Visual acuity, left eye _______________________________________________________ 18 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 19 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 5: COLOUR BLINDNESS Colour vision is provided by the stimulation of three types of cones, blue, green and red. This refers to the region of visible spectrum (or colour) at which each of these cones absorbs light best. Colour blindness is caused by an inherited lack of one or more type of cone, usually the green or red cones. A person who inherits only 2 fully functioning types of cones have difficulty distinguishing between red and green and is referred red-green colour blindness. PROCEDURE: 1. Look at the colour blindness test book (Ishihara) and try and identify the numbers etc. 2. Compare your results with other students. If your results are different to those obtained by other students, you may have a variation in colour vision. This is not uncommon, particularly amongst males. Some degree of colour blindness is common in the community. For example, red/ green colour blindness which is a sex-linked recessive trait, occurs in 8% of males and 0.5% of females. QUESTIONS 1. Using the Snellen test, what ratio of visual acuity may be regarded as normal? What is the significance of these ratios in physiological terms? What condition may be identified using this test? ________________________________________________________________________ ________________________________________________________________________ 2. How does the eye accommodate to near and distant objects? ________________________________________________________________________ ________________________________________________________________________ 3. What areas and cells of the retina are involved in colour vision? ________________________________________________________________________ 20 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PART B: THE EAR AND HEARING The ear responds to mechanical vibration of sound waves in the air. It can detect differences in frequency, pitch, loudness and direction of sound. Auditory information, collected by the ear, is transmitted in the form of impulses to the central nervous system, where it is processed. ACTIVITY 1: STRUCTURE OF THE EAR PROCEDURE: 1. Examine the model of the human ear. Identify as many structures as possible. ACTIVITY 2: AUDITORY ACUITY Auditory acuity is the ability to perceive sounds (clarity and clearness), to detect differences between frequency or intensity and the ability to recognize the direction from which a sound proceeds. Auditory acuity is a measure of how well a person hears. PROCEDURE: 1. Work in pairs. 2. Close your eyes and plug one ear with cotton wool and/or your finger. 3. Your partner should strike the prongs of a G tuning fork on a piece of wood. 4. Hold the tuning fork by the hand, and be careful not to touch the prongs while it is vibrating. 5. Immediately place the tuning fork close to the subject’s open ear, and move it away in a direct line with the ear until the subject can no longer hear the sound. 6. Note the distance at which this occurs. 7. Repeat the above to give a total of 3 trials, and take an average of the distances. 8. Repeat the above using the other ear, and compare distances. *Care must be taken to strike the tuning fork with equal force and to move the fork away from the subject at the same speed. 9. Tabulate your results for both persons. ACTIVITY 3: LOCALISATION OF SOUND The sense of hearing includes the ability to localise the direction from where sound waves emanate. This ability, called stereophony, is a property of the auditory cortex of the brain. Certain nerve tracts connected to the acoustic nerve cross over within the brain to the opposite hemispheres; the brain is then able to compare the loudness and phase differences of a sound as it is received simultaneously by the two ears. 21 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PROCEDURE: 1. Work in pairs. 2. The auditory acuity of both ears must be fairly similar for this test to function properly. 3. The subject’s eyes should be closed (you might wear a blindfold). Strike a tuning fork and alternately hold the fork near the top, front, back and sides of the head. The subject should attempt to report the location of the tuning fork. 4. Repeat the test with one ear plugged and note any differences in the subject’s ability to localise the sound. 5. Record your observations for both persons. Comment about the subject’s ability to localize sound with both ears or with one ear plugged. Demonstrators Signature: __________________ Date:_________ 22 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 LABORATORY 4: URINARY SYSTEM PLEASE REFRAIN FROM DRINKING AND EATING FOR ONE HOUR PRIOR TO THIS PRACTICAL. Revise your lecture notes on fluids and electrolytes and try to predict the effects on the body following consumption of the 750 ml water. BACKGROUND The urinary system comprises the kidneys, ureters, bladder, urethra and associated blood vessels. The major functions of this body system are: 1. The elimination of metabolic wastes, particularly nitrogenous waste in the form of urea. 2. Regulation of fluid and electrolyte balance in the body, particularly with respect to acid-base balance and the concentration of sodium and potassium ions. 3. Assistance with the regulation of blood volume and pressure. 4. Production of hormones such as erythropoietin which promote the development of red blood cells. The most important organs in this system are the kidneys, which are responsible for urine production. These are located contralaterally to the vertebral column, posterior to the peritoneum and against the posterior wall of the abdomen. They are found between the twelfth thoracic and third lumbar vertebrae. The right kidney is normally lower than the left. The other organs in this system are: the ureters which transport urine from the kidneys, the bladder where urine is stored, and the urethra which transports urine from the bladder to the outside of the body. Homeostasis is the maintenance of a constant internal environment. Some of the parameters which must be kept within limits are the concentration of nutrients and electrolytes, the concentration of wastes, and the volume of body fluids, particularly that of blood. The major organ responsible for the regulation of the composition of body fluids is the kidney. It may act to retain optimal quantities of water and solutes, whilst excreting wastes and solutes which are in excess. Thus the rate of urine formation, its composition and concentration will vary throughout the day dependent upon fluid intake, diet, environmental conditions and the level of activity. The kidneys receive a large blood flow. Any abnormal level of substances in the blood will rapidly be seen in the output from the kidneys, ie the urine. If sodium ion is in excess, it will be excreted by the healthy kidney. If the volume of liquid in the blood is excessive, water will be excreted. Glucose is only excreted when its concentration in blood exceeds a high threshold. Protein is not normally excreted because it is too large to pass through the filtration system. However, if there is an impairment of renal function the kidneys may fail to produce normal quantities and concentrations of urine. Additionally, substances may be released which are not normally found in urine, for example, protein, micro- organisms, and crystalline deposits. Thus urine is routinely analysed for the evidence of disease processes, but can just as easily be analysed to show how the healthy kidney regulates fluids and electrolytes. A urine examination may include: determinations of volume, colour, odour, turbidity, pH, specific gravity, chloride concentration, glucose, protein, ketone bodies, blood, bilirubin, and phenylketone. 23 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 OBJECTIVES Dissect a mammalian kidney. To investigate how the kidneys regulate the composition of body fluids by the formation of urine. Use the questions at the end of the practical to facilitate discussion with your demonstrator regarding the structure and the functions of the urinary system ACTIVITY 1: BODY FLUID REGULATION In this section we will examine the effects on kidney function caused by loading the kidneys with excess water. SAFETY WARNING! THIS EXERCISE SHOULD NOT BE PERFORMED BY STUDENTS WITH RENAL DISEASE, HYPERTENSION, OR CARDIAC DISEASE. IF NAUSEA DEVELOPS DURING THE PRACTICAL, DO NOT CONTINUE WITH THIS SECTION OF THE PRACTICAL. TREAT ALL URINE SPECIMENS AS POTENTIALLY INFECTIOUS. YOU MAY USE GLOVES. ANY SPILLAGE SHOULD BE WIPED AWAY FIRST WITH PAPER TOWELLING AND THEN WITH PAPER AND 70% ALCOHOL. ALL URINE SAMPLES ARE TO BE COLLECTED IN THE NEAREST TOILET BLOCK. WHEN THE TESTING IS COMPLETE, DISCARD URINE IN THE TOILET AND DISCARD THE CONTAINER IN THE BIOHAZARD BIN PROVIDED IN THE LAB. PROCEDURE: 1. Using a sterile specimen jar provided, collect a urine sample. This is Sample 1, collected at the beginning of the practical class. 2. Soon after collection of Sample 1, drink 3 cups (3 x 250 ml) of water, with a liberal amount of cordial added, if you wish. Drink as quickly as is comfortable and take note of the time when you finish drinking. 3. 30 minutes after drinking collect another urine sample. This is Sample 2, collected 40 minutes after the drink of water. 4. 60 minutes after the drink of water (or 30 min. after Sample 2) collect another urine sample. This is Sample 3, collected 60 minutes after the drink of water. 5. Measure the total volume of each urine sample, and record the result in Table 2. 6. Observe the colour of the sample. Record your observation in Table 2. 7. Determine the specific gravity of the sample as demonstrated using a hydrometer or a multi strip and record the result in Table 2. (Specific gravity is the weight of a substance compared with an equal volume of water. Water is represented by 1.000). 24 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 RESULTS TABLE 1: BODY FLUID REGULATION Urine Sample Volume (ml) Colour Specific Gravity Sample 1 (before water load) Sample 2 (30 min after water load) Sample 3 (60 min after water load) QUESTIONS a. What percentage of the water load was excreted in the first 60 min after its consumption? b. Was there any correlation between the colour of the sample and its specific gravity? Explain. c. How would the consumption of 750 ml of water affect the osmolarity of intracellular and extracellular fluids? d. Which receptors are responsible for detecting changes in osmolarity and where are they located? e. Describe key hormones and mechanism involved in regulation of water by the kidneys. 25 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 2: DISSECTION OF THE KIDNEY In this activity you will observe the structure of the kidney by dissection. WARNING: It has been identified from past experience that a small group of students may experience emotional distress in this lab. These small group of people may experience shivering, fainting, dizziness and/or fall after or during the dissection. Although likely hood of this happening is low, the risk is high due to possibility of fall from loss of balance. Please Do NOT participate in this activity if you are uncomfortable. PROCEDURE: 1. Examine the kidney provided and note the fat encasing it, if present. 2. Carefully remove the fat from around the kidney, taking care not to damage the blood vessels near the hilum. 3. Look for a small pyramidal gland embedded in the fat at the superior pole of the kidney. This is the adrenal gland. 4. Examine the shape and colour of the kidney. 5. Examine the shiny, transparent membrane covering the kidney, and remove a small section with forceps. 6. Locate the renal artery, vein, and ureter. Insert probes through these vessels. 7. Use a sharp scalpel to make a coronal section through the kidney as shown but do not completely separate the two halves. 8. Lay the two sides of the kidney open on the dissecting board. 9. Observe where the vessels enter the kidney by examining the probes. 10. Observe the two distinct portions of kidney tissue, the cortex and medulla. 11. Identify the renal pelvis, calyces, pyramids and papillae. 12. Examine the renal blood vessels of the model, then cut away the renal pelvis to expose branches of the renal artery. 13. Dispose of the kidney appropriately. 26 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 3: TESTING OF URINE SAMPLES FOR ABNORMALITIES Normal Urine Values: Volume: The normal 24 hour urine volume ranges from 1200-1500 ml. This may be affected by excessive or reduced intake of fluids and by losses from other sources such as perspiration. Colour: Normal urine is yellow to amber, depending on concentration. Early morning specimens are usually more concentrated and therefore darker. Very dilute urine is pale. Odour: The odour of normal urine should not be unpleasant. A foul odour may indicate infection, whilst a fruity odour may be due to ketone bodies. An unpleasant smell of ammonia may develop when urine samples are left standing. Turbidity: Normal urine is clear when freshly voided. On standing, or refrigeration, mucin and phosphates of magnesium or calcium may precipitate. Turbid, freshly voided urine may indicate cells, or bacteria. Specific gravity: This is the density of urine measured relative to that of water which has a density of 1.000. The more substances which are dissolved, the higher will be the Specific Gravity (S.G.). The normal range for a 24 hour specimen is from 1.015 to 1.025. Individual randomly obtained specimens may vary from 1.001 to 1.040. For example, early morning values are usually concentrated and therefore the S.G. is high. pH: This is a measure of the acidity of urine. A pH of 7.0 indicates neutral urine, values below 7.0 are acidic, and values above 7.0 are alkaline. The pH of urine is normally about 6.1 but varies from 4.6 to 8.0. High protein meals result in the production of acidic urine, whilst vegetarian diets result in alkaline urine. Sodium chloride: This varies tremendously depending on dietary intake. Twenty-four hour values may range from 1 mmol/l to several hundred. Chloride ion may also vary with a typical value of 170- 250 mmol/l for a 24 hour specimen. Glucose: Should not be detected in normal urine. Traces may be found during stress or pregnancy. Significant levels indicate diabetes mellitus. Protein: Normal levels of protein should be undetectable. Significant amounts of protein may leak into the urine when there is renal damage. Ketone bodies: Not normally found. Present in diabetic ketoacidosis. Blood: Not normally found. May indicate damage, infection, malignancy. Bilirubin: Not normally found. Indicates excessive RBC breakdown, or failure to excrete via the liver. 27 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PROCEDURE: 1. Using the test strips supplied, test each of the beakers numbered 1 to 5 for the following: Glucose Ketones Protein Blood pH Specific gravity Note: Follow the instructions on the side of each test strip container. 2. Enter the test strip results into Table 2. RESULTS TABLE 2: URINE TEST RESULTS PROPERTY Beaker 1 Beaker 2 Beaker 3 Beaker 4 Beaker 5 Protein Ketones Glucose Specific gravity pH Odour Colour Turbidity Blood Demonstrators Signature: __________________ Date:_________ 28 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 LABORATORY 5: ACIDS, BASES AND BUFFERS BACKGROUND Acids are substances which release hydrogen ions in solution by dissociating (breaking apart) or by reacting with water. Bases are substances which can accept a hydrogen ion. They usually do this by dissociating to form the hydroxide ion (which subsequently reacts with H+ to form H2O) or by reacting with water to form an H+ acceptor molecule. Weak acids and bases are substances which are incompletely dissociated. This means most of the acid or base is still in its molecular form (and so does not produce much H+ or H+ acceptor). Buffers function to maintain the pH of a solution as constant as possible despite the addition of acids or bases. These consist of weak acid and its conjugate base. When excess acid is added to the buffer solution the conjugate base of the buffer reacts with it and removes it. When excess base is added, the weak acid in the buffer dissociates, producing hydrogen ions which then react with the excess base. Example: The carbonic acid - bicarbonate buffer. This contains carbonic acid (H2CO3, a weak acid), bicarbonate (HCO3- , i.e. the conjugate base) and hydrogen ion (H+): H2CO3 → H+ + HCO3- If base is added, e.g. OH- from NaOH, carbonic acid will keep dissociating (i.e. breaking apart) to produce H+ which reacts with OH-, until the excess OH- is removed. If acid is added, (e.g. H+ from HCl), the bicarbonate will react with the H+ until the excess is removed. Indicators: Often the acidic form and basic form of a weak acid-base pair are of different colour. A change in the relative amounts of these pairs will cause the solution to change colour. For example: H - Phenolphthalein ⇔ H+ + Phenolphthalein - (acid) (conjugate base) In an acidic solution, phenolphthalein is found in its acidic form (with H+ attached) and thus gives a particular colour. In a basic solution, phenolphthalein dissociates so that the hydrogen ion released can react with the base. Therefore the colour of the base is seen. These substances are used as acid-base indicators, as we can deduce the acidity/alkalinity of a solution containing buffer, merely by looking at its colour. The pH at which colour changes occur depends on the individual buffer. Thus indicators can be chosen to show particular pH values. Additionally, indicators which possess more than one detachable H atom may have several coloured forms depending on how many hydrogens are lost. Thus they can indicate more than one pH range. Several indicators may be combined to produce colour changes over a wide range of pH thus producing a universal indicator. Many food colours are due to dyes which are weak acids or bases. These can also be used as indicators. Purple cabbage contains a dye which can be used as a universal indicator. The indicator keeps well if kept frozen, but otherwise decays after about two days at room temperature. 29 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 OBJECTIVES Measure the pH of a number of solutions. Observe a number of buffer systems. Investigate the phosphate buffer system. Use the questions at the end of the practical to facilitate discussion with your demonstrator regarding acids, bases and buffers ACTIVITY 1: TESTING FOR pH USING THE UNIVERSAL INDICATOR PROCEDURE: 1. Look at each of the solutions 1 to 7. 2. Work out the pH of each solution from the colour of the universal indicator (added in the solution). 3. Compare the colour of solution with that in the colour chart. 4. Record the pH of each solution in Table 1 provided RESULTS TABLE 1: TESTING FOR pH USING THE UNIVERSAL INDICATOR Solution pH 1 0.1 M HCl 2 0.001 M HCl 3 0.00001M HCl 4 Distilled Water 5 0.00001 M KOH 6 0.001 M KOH 7 0.1 M KOH ACTIVITY 2: USING CABBAGE DYE AS A UNIVERSAL INDICATOR Purple cabbage contains a dye which can act as a universal indicator. It is a weak acid which can lose several H+ ions in a stepwise fashion. Each time H+ is lost a new coloured substance forms. Intermediate colours are also seen. PROCEDURE: 1. Place 5ml of each solution in separate test tubes. 2. Add 5 drops of cabbage water into each test tube. 3. Observe and record the colours of the solutions in Table 2 provided 30 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 RESULTS TABLE 2: USING CABBAGE DYE AS A UNIVERSAL INDICATOR Solution pH COLOUR 1 1 M HCl 0 2 0.001 M HCl 3 3 cabbage water 5 4 phosphate buffer 7 5 borate buffer 10 6 0.1 M NaOH 13 7 1 M NaOH 14 ACTIVITY 3: USING INDICATOR STRIPS TO TEST COMMON SUBSTANCES PROCEDURE: 4. Use an indicator strip to test some common substances. 5. Place a few ml of each substance in separate test tubes. 6. Dip a test strip into each test tube and read off the pH of each substance using the supplied colour scale. 7. Record the pH of each solution in Table 3 provided. RESULTS TABLE 3: USING INDICATOR STRIPS TO TEST COMMON SUBSTANCES Solution pH lemon juice vinegar milk baking soda (sodium bicarbonate) borax detergent ACTIVITY 4: INVESTIGATING THE PHOSPHATE BUFFER SYSTEM Blood pH is normally maintained within the range 7.3 - 7.5. Plasma pH is kept within 7.35 - 7.45. Death will occur if blood pH falls below 7.0 or rises above 7.9. 31 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Four buffer systems control blood pH. The phosphate buffer in blood is the next most important system after the carbonic acid/bicarbonate system. H2PO4- ⇔ H+ + HPO42- dihydrogen phosphate hydrogen ion monohydrogen phosphate Excess acid in the blood can ultimately be excreted by the lungs as CO2 (via the carbonic acid/bicarbonate buffer system) or by the kidneys as H2PO4- (phosphate buffer). In kidney tubules: HPO42- + HA + --------> H2PO4- + A- excreted in urine (Note: HA represents any acid. A- represents its associated base) In Plasma, 80% of phosphate is in the form of HPO42- but when the urine produced is very acidic almost 100% is in the form of H2PO4- (e.g. at pH 5.4). The digestion and subsequent metabolism of meat results in urine more acidic than normal; because of all the amino acids it contains. In the experiment you are about to do, the behaviour of a phosphate buffer will be investigated. Its ability to absorb excess H+ or OH- will be compared with that of distilled water. PROCEDURE: 1. Label two 100 ml beakers 1 and 2. 2. Add 50 ml distilled water to each. 3. Measure the pH of each beaker and record your results in Table 4. 4. To beaker 1, use a pipette to add 1.0 ml of 0.5 M potassium hydroxide (KOH). 5. Measure and record the pH in Table 4. 6. To beaker 2, use a pipette to add 1.0 ml of 0.5 M hydrochloric acid (HCl). 7. Measure and record the pH in Table 4 provided. 8. Repeat the above steps with buffers 1 and 2 (Add 5 ml of buffer to 50 ml of H2O) Buffer 1: Contains 0.5 M H2PO4- and 0.5 M HPO42- Buffer 2: Contains 0.05 M H2PO4- and 0.05 M HPO42- 32 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 RESULTS TABLE 4: INVESTIGATING THE PHOSPHATE BUFFER SYSTEM Solution pH Beaker 1 Beaker 2 Initial After adding Initial After adding 1ml KOH 1ml HCl Distilled water Buffer 1 Buffer 2 QUESTIONS 1. Do you think cabbage water would make a good indicator? Why? (Refer to your observations from Activity 2) 2. Which common substances are acidic and which are basic? (from the results of Activity 3, see Table 3) 3. What does the addition of a buffer do to the pH of a solution (from the results of Activity 4) 4. Is there any difference between the buffering ability of buffers 1 and 2? Why? (from results of Activity 4) Demonstrators Signature: __________________ Date:_________ 33 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 LABORATORY 6: DIGESTION AND METABOLISM BACKGROUND The digestive system is involved in the progressive breakdown of food into molecules which are small enough to be absorbed into the bloodstream. The gastrointestinal tract is a muscular tube within the body extending from the mouth to the anus. It acts to move substances throughout its length. Along this pathway, it varies in structure, enabling it to carry out storage, digestion, absorption and defecation. These functions are aided by the accessory structures of the digestive system: teeth, tongue, salivary glands, liver, gallbladder and pancreas. One of the goals of metabolism is to extract energy from the food nutrients that we consumed. The other purpose is to chemically breakdown complex food molecules into simple ones that our body can use. Both energy and breakdown food molecules can then be used to make new biological molecules that are required by our body. To carry out these processes, our body needs enzymes to carry out the specific reactions. Enzymes found in the digestive system are extracellular enzymes and they are responsible for degradation of food nutrients in the gastrointestinal tract where intracellular enzymes are responsible for the chemical reactions of the various metabolic pathways that are found in our body. OBJECTIVES Identify the major organs and accessory structures of the digestive system on the models Relate the structures of the gastrointestinal system to their function. Understand how different food nutrients can be identified and the nature of enzyme molecules and their functions. Demonstrate the presence of proteins and reducing sugars and investigate the activities of intracellular and extracellular enzymes. ACTIVITY 1: DETECTION OF CARBOHYDRATES (REDUCING SUGARS) We are used to talking about "energy foods" such as chocolate and meat. But what does this mean? The main chemicals that make up human cells -carbohydrates, lipids and proteins - all contain high levels of energy that our cells can extract. Our cells need this energy for many different purposes: e.g. for moving chemicals around in the cell and between cells (e.g. keeping the blood moving); for contracting specialised cells such as muscle fibres; for heating our bodies; for making other specialised high energy compounds. Even though lipids contain more stored energy per gram, carbohydrates are the preferred energy source, particularly the simple carbohydrate, glucose. Glucose, a monosaccharide, belongs to a group of carbohydrates known as reducing sugars. Reducing sugars contain a free aldehyde or a free ketone group in their structures. Reducing sugars can be monosaccharides or disaccharides. Benedict's test is used to identify reducing sugars. The colour of the resulting precipitate is determined by the amount of reducing sugar present: blue (none) to green (low) to yellow (medium) to reddish orange (high). 34 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 PROCEDURE: 1. Set up 6 test tubes and label them Number 1 to 6. 2. Add the following to each of the 6 test tubes Test Tube 1 1 ml distilled H2O Test Tube 2 1 ml 1% glucose Test Tube 3 1 ml 1% fructose Test Tube 4 1 ml 1% sucrose Test Tube 5 1 ml 1% maltose Test Tube 6 1 ml 1% starch 3. Add 5 drops Benedict's reagent to each test tube and mix well. 4. Put the test tubes in a boiling water bath for 3 minutes only. 5. After 3 minutes have passed, remove the tubes from the water bath and let them cool. 6. Observe any colour change and the amount of precipitate produced. Use (+) to denote small amount of precipitate and (+++) for large amount of precipitate. 7. Record your results in Table 1 provided. 35 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 RESULTS TABLE1: RESULTS OF THE BENEDICT'S TEST ON VARIOUS SUGARS. TEST TUBE CARBOHYDRATE COLOUR AMOUNT OF PRECIPITATE 1 Distilled H2O 2 Glucose 3 Fructose 4 Sucrose 5 Maltose 6 Starch + small amount of precipitate ++ moderate amount of precipitate +++ large amount of precipitate QUESTIONS 1. Can you detect reducing sugar in all samples tested? 2. What result did you observe in the case of sucrose? Did you expect to see this result? 3. Did sucrose and maltose give similar results? Why? 4. What is the difference between sucrose and maltose? 5. What is starch made up of? 36 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 2: DETECTION OF PROTEIN Proteins are a group of chemical substances that are most useful because of their shape. Their basic structure is long and threadlike (chains of amino acids). However, these chains can coil up to assume specific shapes (such as enzymes) or cross-link to provide strong but flexible textures (structural proteins). Structural proteins are used to help build cells and other substances. It is the basic structure of amino acids which can help us identify proteins. Each amino acid contains a carboxyl group and an amine group. When two or more amino acids link together, the carboxyl group of one amino acid bonds with the amino group of another. This bond is called the peptide bond. The amine group of the bond will react with the copper ions in the Biuret Test reagent to produce a colour change. The Biuret reagent is normally blue, but will change to purple in the presence of protein. For this experiment you will perform the Biuret test on some common substances. PROCEDURE: 1. Take 4 test tubes and label them Number 1 to 4. 2. Add the following to the 4 test tubes Test Tube 1 5 ml distilled H2O Test Tube 2 5 ml egg albumin solution Test Tube 3 5 ml chicken stock Test Tube 4 5 ml apple juice * Colour of a positive Biuret test 3. Add 10 drops of Biuret reagent to each test tube and mix well. 4. Note the colour of each tube and record your results in Table 2 provided. RESULTS TABLE 2: RESULTS OF THE BIURET TEST ON VARIOUS SUBSTANCES TEST TUBE SUBSTANCE OBSERVATIONS 1 Distilled H2O 2 Egg Albumin 3 Chicken Stock 4 Apple Juice 37 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 QUESTIONS 1. What is the purpose of using distilled water (in Tube 1)? 2. What conclusion can you draw from your experiment? 3. Do you find any protein in apple juice? ACTIVITY 3: INVESTIGATING AN INTRACELLULAR ENZYME – HYDROGEN PEROXIDASE Enzymes are proteins formed within the cells of living organisms. Each enzyme has a particular shape that allows it to speed up the rate of a particular chemical reaction that occurs in the cell, without the enzyme itself being consumed in the reaction (i.e. enzymes are catalysts). In this exercise you will examine some of the characteristics of the enzyme peroxidase. This catalyses the breakdown of hydrogen peroxide in cells: 2H2O2 → 2H2O + O2 This reaction is important to cells, because hydrogen peroxide is a by-product of some common biological reactions, and it is a very reactive chemical that can easily react with and damage other useful chemicals in the cells. It is therefore important that hydrogen peroxide is rapidly removed from the cell, whenever it forms. Like all enzymes, peroxidase consists of a chain of amino acids delicately wound into a shape that allows it to catalyse the reaction shown above. PROCEDURE: Begin by testing what happens when a supposedly inactive material (fine washed sand) is added to hydrogen peroxide, then liver treated in various ways. Test Tube 1: Fine Sand 1. Pour hydrogen peroxide solution into a test tube to a depth of about 2 cm. 2. Into the test tube sprinkle a small amount (about 0.1 g) of fine sand. 3. Look for any signs of activity; record your observations. You may wish to insert a glowing splint into the tube. (Why?) 38 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 Test Tube 2: Whole Liver 4. Pour fresh hydrogen peroxide solution into a clean test tube to a depth of about 2cm. 5. Using forceps, select a small piece of liver and drop it into the test tube of hydrogen peroxide. 6. Test with a glowing splint, observe for a few minutes. 7. Record your observations in the Table 3 provided. Test Tube 3: Ground Liver 8. Take a piece of Liver about the same size as the one just used and place in a mortar with a little fine sand. 9. Grind the liver. 10. Transfer the ground material to a test tube containing fresh hydrogen peroxide solution. 11. Look for activity as before, and record the results in the Table 3 provided. Test Tube 4: Boiled Liver 12. Take another piece of liver, grind it up as before in a clean mortar and add it to water in a test tube. 13. Boil it well for 5 minutes and let it cool. 14. Pour off surplus water above the boiled liver, then see whether the boiled liver can break down hydrogen peroxide, using the same procedure as before. 15. Again record the results in Table 3. 39 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 RESULTS TABLE 3: EFFECT OF HYDROGEN PEROXIDE ON DIFFERENT MATERIALS MATERIAL OBSERVATIONS Fine Sand Whole liver Ground liver Boiled liver The amount of activity can be recorded using the following scale: + some evidence of activity ++ gas given off but not identified +++ oxygen demonstrated QUESTIONS 1. What is the purpose of inserting a glowing splint into the tube? 2. Do you see any difference in activity in using whole liver and ground liver? 3. What is the aim of boiling the liver tissue? What does this experiment demonstrate about the nature of the enzyme? 40 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 ACTIVITY 4: INVESTIGATING THE ACTION OF AN EXTRACELLULAR ENZYME, PEPSIN. Even though all enzymes are manufactured within cells of living organisms, some of these enzymes work on substances outside the cells. Many enzymes used in the digestive system work extracellularly. One important enzyme used for protein digestion in the stomach is pepsin, which forms part of the gastric juice of the stomach. Pepsin breaks some peptide bonds to form smaller polypeptide fragments from large proteins. Other enzymes that breakdown down proteins in the gastrointestinal tract are trypsin and chymotrypsin. PROCEDURE: This experiment has been set up as a demonstration. Egg white has been treated as shown in test tubes labelled 1 to 6. 1. Observe the contents of test tubes 1 to 6 for any protein digestion that is breakdown of egg white. 2. Record your observations in Table provided. RESULTS TABLE 4: PEPSIN ACTIVITY UNDER VARIOUS pH CONDITIONS T. TUBE MIXTURE OBSERVATIONS 1 2 ml pepsin only 2 2 ml pepsin in HCl 3 2 ml HCl only 4 2 ml pepsin in NaHCO3 5 2 ml NaHCO3 only 6 2 ml distilled H2O only 41 HBM1202 Anatomy and Physiology. Laboratory Manual for Students, 2024 QUESTIONS 1. What is the effect of pepsin on a small piece of boiled egg white (protein) under various conditions? 2. Under which condition is pepsin active? 3. What is the purpose of setting up test tubes number 1, 3, 5 and 6? 4. Explain why control conditions (i.e. experimental controls) were needed in this investigation. Demonstrators Signature: __________________ Date:_________ 42