Zoo 120 Laboratory General Laboratory Instructions 1. Read the protocol in advance. Understand clearly the instructions especially for the use of laboratory apparatus or equipment. 2. Each group will be responsible for bringing the fresh/live specimens and the following materials: Dissecting set Alcohol Plastic/small Cleaning rags Household bleach garbage bags (separate for (chlorox) Matches cleaning glass and Detergent / soap Sponge for wiping mess) Masking tape small Ruler Cotton Pentel pen Pencil 3. A group will be assigned as monitor for each experiment. The assigned group monitor will be responsible for the requisition of needed reagents and for checking the room after the experiment. 4. Observe cleanliness and orderliness in the laboratory. 5. Work carefully and follow precautionary measures. 6. Avoid conducting unauthorized experiments. Maintenance of Laboratory Facilities 1. Always wash and clean each item used in the experiment. 2. Return all laboratory materials/ chemicals to their proper places before leaving the room. 3. Living animals used for experimental work must be treated humanely with with utmost consideration given to minimizing any discomfort that an experiment might entail. 4. Dispose animals or parts of it properly. 5. All reusable equipment such as pipettes must be washed and disinfected with a 1:10 1:10 solution of household bleach. 6. Working area of each each group must be clean of debris, spilled items and other non essential or disposable materials 7. A laboratory disinfectant should be used to clean laboratory surfaces. 8. Before leaving, check all electrical electric al and lighting fixtures in order to avoid accidents. Also make sure that the group’s assigned locker is properly closed and secure.
Schedule of Experiments Experiment 1. Permeability Permeabilit y of the Cell Membrane 2. Reflex Activity of the Frog 3. 4. 5. 6. 7.
Sensory Pathways in Man Human Chorionic Gonadotropin Blood Studies, Pulse and Blood Pressure Determination Human Respiration
8. Measuring Carbon dioxide Production
Date to be Date of Submission of Performed Laboratory Reports April 17, 2012 April 19, 2012 (Wednesday) (Monday) April 18 April 23 (Monday) (Wednesday) April 19 (Thursday) April 24 (Tuesday) April 23 (Monday) April 25 (Wednesday) April 26 (Thursday) April 30 (Monday) May 3 (Wednesday) May 4 (Thursday)
May 7, 2012 (Monday) May 8 (Tuesday)
and Oxygen Consumption of Selected Test Animals 9. Osmoregulation 10. Urinalysis 11. Characterization of Digestive Enzymes of Killed Animals
May 7 (Monday) May 8 (Tuesday) May 10 (Thursday)
May 9 (Wednesday) May 10 (Thursday) May 14 (Monday)
Laboratory Report Format I. Introduction Objectives Brief background of the experiment II. Materials and Methods Summary of procedures In narrative form III. Results and Discussion Presentation of data thru figures (graphs, pictures) and tables Explanation of results Include proper citation of references IV. Answers to Questions V. Summary VI. References
PERMEABILITY OF THE CELL MEMBRANE OBJECTIVE In this experiment, you will learn about 1. Osmosis, a very fundamental process to cell life 2. Selective permeability property of the cell 3. Factors that determine permeability INTRODUCTION The cell membrane bears the primary responsibility of determining what materials come in and out of the cell and at what rate. This regulation is very crucial especially for the proper nutrition, maintenance of irritability of the cells and homeostasis. The cell membrane is highly selectively permeable. It’s basic structure and composition as illustrated by the fluid mosaic model explains the behavior. The cell membrane transports molecules through it by both passive and active processes. Permeation of substances may occur through the lipid layer or through protein channels or pumps. The passive mechanisms of transport depend on available energy from combined chemical and electrical gradients or potentials. The active means move substances against their gradients and require metabolic energy expenditure by the cells. The species-specific proteins and lipids in the cell membrane spell the differences in behavior of cells from different organisms. The RBC especially of humans is a convenient specimen for studying the permeability of the cell membrane. How the cell controls the flow of water is shown in hemolysis experiments. The osmotic content of the human RBC is about 0.9% sodium chloride. In dilute or hypotonic solutions, the cells swell due to influx of water. Eventually the cells hemolyze. During hemolysis, the cell contents including the red colored protein, hemoglobin, are released into the surrounding medium. Hemolysis is indicated by the change in appearance from opaque red cell suspension to a clear pink solution. Increasing the osmotic concentration of the red blood cells by suspending them in a medium containing permeating solute particles results to observable changes. Thus, the relative rates of permeation of substances can also be determined using rates of hemolysis as basis. MATERIALS AND METHODS Reagents: 0.9% NaCl 0.2M NaCl 0.1 M NaCl 0.02 M NaCl Other materials : Test tubes Test tube rack Sterile blood lancet
0.3M Glucose 0.2M Glucose 0.1M Glucose 0.2 M KCl
0.1 M KCl 0.05M KCl Distilled water Neutral red
Cotton Paper with prints Alcohol
Technique for drawing blood 1. Clean one fingertip of donor with cotton wet with alcohol; and allow to dry. 2. Unwrap cover of sterile blood lancet making sure that the pointed tip is untouched. 3. Hold firmly 3/4 of the body of the lancet with your thumb, index and middle fingers. 4. With your other hand, hold fingertip of donor. 5. With a swift and forceful stroke, prick the fingertip with the blood lancet. 6. To make a red cell suspension, allow 10 drops of blood to drip freely into the test tube filled with isotonic saline solution or until the saline suspension’s color becomes reddish pink.
HEMOLYSIS 1. Get 2 test tubes. Fill the first test tube with 5 ml distilled water. To the second tube, add 5 ml of 0.2 M NaCl. 2. Add 5 drops of RBC suspension to each of the 2 test tubes. 3. Mix by gently inverting the test tube from side to side several times. 4. Place the test tubes against a proper background. 5. Observe hemolysis by looking at the prints through the liquid at the sides of the tube. Hemolysis is indicated when prints finally turn clear or when the suspension becomes translucent to the naked eye. 6. Establish the hemolyzing concentration of glucose, NaCl and KCl. For each tube, use 5 ml of the test solution with 5 drops of blood suspension. Do hemolysis reading at a fixed time interval. 7. Use the values you obtained to calculate the isotonic coefficients of the salts. The isotonic coefficient is the apparent number of ions released upon ionization of the salt in solution. It can be determined from it’s hemolytic pint (or osmotic activity) relative to the non electrolyte Isotonic coefficient = Hemolytic point of electrolyte (molar concentration) Hemolytic point of non electrolyte PENETRATION OF ALKALI 1. Add 10 ml of neutral red solution to 15 ml of yeast suspension. Take note of any changes in color that may occur over a period of 5 min. Neutral red is red at pH below 7 but yellow at pH between 7 and 8. 2. Measure 2 ml of the mixture and place in 4 labeled tubes. Add the ff: Test tube A—0.5 ml OF 0.01 N NH4OH Test tube B—0.5 ml of 0.01 N KOH Test Tube C—0.5 ml of 0.01 N NaOH Test Tube D 0.5 ml of distilled water 3. Filter each mixture and observe color of yeast cells and the filtrate under the microscope. Question for Research 1. Cite and explain the physical and chemical factors that affect the process of diffusion. 2. What are the factors responsible for the difference in concentration of substances (used in the experiment required to produce hemolysis? 3. Discuss the factors affecting the permeablilty of the plasma membranes. 4. Discuss the penetration of Alakali I relation to their degree of ionization.
REFLEX ACTIVITY IN TOAD INTRODUCTION The reflex arc is the basic unit of an integrated neural activity and consist s of the following: (a) sensory receptors (b) afferent neurons to transmit impulses to the central nervous system (c) one or several synapses (d) efferent neurons that sends messages from the CNS and (e) an effector organ. In toads, reflexes in response to different stimuli may be described into three general types (1) simple coordinated movement involving contraction of a relatively small number of muscles (2) complex coordinated movements involving contraction of a large number of muscles acting in due sequence to produce definite purposeful motions of a complicated nature and (3) uncoordinated or convulsive reflexes involving contraction of several muscles acting often in opposition to another. The exercise aims to provide the opportunity to observe the different reflex activities of the toad. METHODOLOGY SPINAL REFLEXES: The following observations should be made on normal, spinal and double pithed toads. Record the observations. A. Posture: Place the experimental animal on the desk. Take note of the position of the head, eyes, forelimbs, hind limbs and the belly. Draw the posture for each of the experimental animals. Determine the respiration rate by counting pulsation caused by the raising and lowering of the throat area per unit time. Also, take note of the closing and opening of the nostrils if any. B. Righting Reflex: Place the animal on its dorsal side. Observe the movements of the body and the limbs and the manner in which it turns to it ’s normal position. Observe swimming reflex by placing the animal gently in an aquarium or large glass vessel filled with water room temperature. C. Withdrawal Reflex: Suspend the animal on an iron stand by a wire hook passed through the lower jaw and pinch the left toe with forceps. Observe which leg flexes and extends. D. Scratch Reflex: Get a piece of filter paper and moisten with 80 % acetic acid. With the animal still suspended by it’s lower jaw, place filter paper on the surface of the belly. Take note o0f the reaction of the legs toward the irritated spot. REFLEX TIME: Use the spinal animal utilized earlier. Prepare beakers, each filled with about 100 ml of the different available concentrations of HCL. Also prepare a 100 ml beaker with 1% NaHCO3 solution and another beaker of tap water. Place the long toe in 1 % HCL. Make sure you do not let any part of the foot come in contact with the walls of the dish or acid. Likewise, do not allow the toe to remain in the acid for more than 1.5 min. Note how many seconds it takes before the foot is withdrawn. Immediately bathe the foot in 1% NaHCO3 solution and the in tap water. Remove excess water from the foot and toes with coarse filter paper. Let the animal rest for about a minute. Repeat the experiment using the different concentrations of HCL prepared. Make sure that the same toe is used and that it is immersed to the same extent in each trial. Record all results. INHIBITION OF REFLEXES: Use the spinal toad utilized earlier. Suspend it by it’s lowe r jaw. Determine the reflex time with0.3% HCl by following the procedure in the previous experiment using the toe in the other foot. If this solution gives a reflex time of less than 3 seconds, use a more dilute solution. Determine the reflex time. Bathe the foot in 1 percent NaHCO3 and then in tap water. Allow the toad to rest for at least
3 minutes. Again dip the left toe in the acid, at the same time pinch the toes of the right foot. Take note of the time the toad moves the left toe. What is the reflex time of the left toe? Questions for Research 1. What is the physiological significance of reflexes? Discuss also the need for an intact nervous 2. system for coordinated activity 3. What is decerebrate rigidity and spinal shock? 4. Discuss the mechanism of reflex inhibition. SENSORY PATHWAY IN MAN INTRODUCTION During the process of evolution, the higher brain level (cerebral cortex) in man developed into a more advanced nerve center. It is responsible for motor and sensory functions previously performed by less developed brain of lower animals. It also serves primarily as a storage area for information and as an organ of associative memory both essential components enabling the animal to profit from past experience. Likewise, a parallel development and specialization of sensory receptors were attained since an advanced central nervous system would be highly dependent on such structures as man, effective contact point between the animal and its environment. These highly specialized receptors can be characterized by its sensitivity to a certain stimulus and/or sensation and its ability to translate such into a nerve impulse, which are in turn transmitted to the appropriate nerve centers in the brain. The exercise aims to identify the location and functions of some sensory receptors. METHODOLOGY A. Olfactory Sensations Connect one end of the rubber tubing to the stem of a funnel and place funnel over a container with an odoriferous substance like perfume or cologne. Insert the other end of the tubing into the lower posterior chamber of the nares. Remove the tubing, rest for a minute. Place tubing again inside the nasal chamber but make sure this time that it is in the upper anterior chamber. Compare the strength of the sense of smell in the lower and upper chambers. B. Gustatory Sensations Taste Zones: Dry the tongue of your subject using a clean filter paper and place sugar crystals on the tip of the tongue. Take note of the number of seconds before the sweet taste is sensed. Repeat test but this time, use a drop of sugar solution. Again record how long it takes for the subject to taste the sugar. Prepare different solutions: 0.5, 1.0, 5, 10, 25 and 50%. Take a small amount of each solution and place a few drops on the mouth of the subject. Start with the weakest solution. What is the lowest concentration of sugar solution that evoked the sweet taste in the mouth? C. Visual sensations 1. Blind spot On a piece of paper make two figures. On the left side make a circle with a 5 mm diameter and on the other side a small cross measuring 1 mm. The two figures should be 60 mm apart. Hold the paper at arm’s length in front of the eye with the figures arranged directly in front of the eye. Close the left eye and fix the right eye upon the cross at the right hand mark. Move the paper towards the face gradually.
At a certain distance, the circle will disappear. Measure this distance and record it. 2. Pupillary reflex Instruct the subject to stand in a place where the lighting is relatively dim or an area far from the window. Note the size of the pupil in each eye. Then for about 5 seconds, shine a light (use a pen light) into one eye, six inches away from the eye. Observe. Remove light; ask the subject to close his/her eyes for two minutes. As soon as subject opens eyes, note the size of his pupil. Note if there is constriction or dilation of aperture of eye. 3. Near point accommodation Hold a pencil approximately 50 mm from your eyes. Close the right eye and gradually move it slowly towards the left eye. At a certain distance, the print of the pencil will be blurred and will not produce a clean image. Measure the distance. Test the other eye and also measure the distance. D. Auditory Sensations 1. Sound Localization Conduct the following test in a quiet room. Instruct the subject to close his eyes and plug the left ear with cotton. Hold a watch a few feet away from him, but in line with his right ear and slowly bring the watch towards his ear. The subject must indicate when he first hears the clicking of the watch. Measure the distance of the watch from the ear. 2. Equilibrium Ask the subject to place his? Her right foot in front of the left foot with the big toe of the latter is touching the back of his right foot. Subject must stand upright and be as till as possible. Observe the body movement. Direct the subject to place his/her feet together with the sides touching. Observe the body movements with (a) eyes closed and (b) eyes open. E. Cutaneous sensations 1. Referred pain Place elbow in a large shallow pan of ice cold water and note the progression of sensation experienced. Initially, pain will be felt in the elbow. Later, pain will be felt elsewhere. 2. Identifying receptors Draw one cm sq on the back of the hand. Divide it into 16 equal parts. Take a straight pin and gently touch each square. Whenever a sensation is felt, mark and record the chart with a “p”. Repeat the same experiment this time using a dull pencil point gently touching the square. Record on the chart “t” tactile sensations when you feel pressure. Straighten one end of a paper clip. Hold the straightened end in an ice cube for not less than five min. Again repeat experimentally by touching each square gently. Record your reaction to cold on the chart with a “c” . Rest hand for 5 min. Carefully, place the straight end of the paper clip in a beaker of hot water for not less than 3 min. Repeat the experiment. Record your reaction to hot on the chart with an ‘h’. Questions for Research 1. Make a diagram of the different sensory pathways 2. What is the role of the sense of smell and taste? 3. Diagram the pathway of salivary reflex and discuss mechanism involved. What is the significance of this type of reflex? 4. Differentiate between nerve and bone conduction deafness. How can bone conduction
remedy abnormalities? 5. Of what importance are vestibular reflexes? Give other factors involved in equilibrium. 6. Define blind spot. Of what importance is the field of vision? Illustrate a perimeter chart 7. Discuss the different types of nerve endings in the skin and their differences in terms of stimuli, sensitivity and location.
HUMAN CHORIONIC GONATROPIN INTRODUCTION Human chorionic gonadotropin (HCG) is a glycoproteinhormone produced in pregnancy that is made by the developing embryo soon after conception and later by the syncytiotrophoblast (part of the placenta). Its role is to prevent the disintegration of the corpus luteum of the ovary and thereby maintain progesterone production that is critical for a pregnancy in humans. HCG may have additional functions; for instance, it is thought that HCG affects the immune tolerance of the pregnancy. Early pregnancy testing, in general, is based on the detection or measurement of HCG. Human chorionic gonadotropin is a glycoprotein composed of 244 amino acids with a molecular mass of 36.7 kDa. It interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly-negative charge, HCG may repel the immune cells of the mother, protecting the fetus during the first trimester PROCEDURE 1. Add the same volume of diethyl ether to your urine sample. The urine should be from a pregnant female preferably in her first trimester. 2. Using a burette, extract the urine. 3. Place in a container, cover and store in the refrigerator while not in use. 4. To 1 sexually immature female mouse, inject ½ ml of the urine subcutaneously twice a day for three days. The other mouse will serve as control. Use a 1 ml syringe 5. Rest on the fourth day. 6. On the fifth day, sacrifice both mice. Dissect and observe the uterus and the ovary. The uterus of the mouse Questions for Research 1. What is the function of HCG? 2. How does HCG induce ovulation in mice?
BLOOD STUDIES, PULSE AND BLOOD PRESSURE DETERMINATION The Blood A.
ABO System of blood typing The agglutination reaction is the basis for classifying blood into different blood types: 1. Draw two circles on a microscope slide. Mark them A & B. 2. Clean your left hand ring finger with a cotton ball dipped in alcohol. Dry. 3. With a sterile lancet, make a puncture at the tip of the finger. The lancet would be able to pierce around 1/8 inch so that only one puncture is needed 4. Place a drop of blood on each of the circles on the slide, then add a drop of anti-A serum to the blood in the circle A and anti —B serum to that in the B circle. Stir the blood with separate toothpicks. What did you observe? For confirmation, observe under the microscope. Obtain the blood types of all members of the group. Get the results of the class. How do the percentages compare with national percentages? With those of other races?
B.
Clotting time 1. Follow numbers 2 & 3 of the agglutination procedure. Put a drop of blood at the center of a clean slide. Take note of the time when the blood was shed from the finger. 2. Get a needle or pin and draw the blood from the center to the periphery to observe if fibrin will cling to the pin. Do this every minute. 3. Once a fibrin thread is observed to cling to the pin, take note of the time . The time when blood leaves the vessel up to the time fibrin is formed is the clotting time.
C.
Factors affecting coagulation 1. Arrange test tubes in the ff series: a) Empty test tube for normal coagulation b) Small amount of cotton fibers at the bottom of the tube c) To be placed on an ice bath d) To be placed in a hot water bath e) 1/2 pinch EDTA f) To be continuously stirred 2. Collect blood. Without delay, add 2 ml of blood to each tube. Remove the needle when transferring blood. 3. Observe what happens in each tube. Take note of the clotting time in each tube.
D.
Pulse Rate The pulse may be counted in the jugular but conveniently, the radial artery of the wrist is used. Establish a basal pulse rate with the subject lying in a flat position. Make three trials with an interval of one minute in between. Regard this as the normal pulse. Determine the average pulse rate of three of the following subjects. Record and compute for the average. AGE: A. Children 5 to 10 yrs B. Teenagers 13 –18 C. Adults 30 and above
GENDER: must be 17-22 year-old and non-athletes A. Male B. Female ACTIVITY: 17-22 year-old A. Athletes w/ regular training B. Untrained subjects C. Subjects after 10 min of exercise OTHERS: A. Pregnant on first trimester B. Pregnant on last trimester E.
Blood Pressure Wrap and secure the rubber cuff of the sphygmomanometer on the left arm of the subject. Place a stethoscope on the artery below the cuff. Listen for the pulse. Press the bulb until there is no sound heard on the stethoscope. Deflate the cuff gradually. Take note of the reading in the meter when the first sound is heard in the stethoscope. This is the systolic blood pressure. As cuff pressure is further reduced, the sound suddenly becomes faint. Check the reading and record this as the diastolic blood pressure. Record the systolic/diastolic pressure. Repeat blood pressure determination but this time asks the subject to do exercises for 30 min. Get the blood pressure as soon as subject stops and also 30 min after complete rest.
Questions for Research 1. Why is the red cell number more in males than in females? More in infants than in adults? Give factors that cause deviation from the normal value. Do you consider these normal mechanisms? When do you consider a deviation pathological? What is polycythemia? Oligocythemia? 2. Is there a difference between white blood cell number in males and females. What is the term for increase/ decrease in WBC?Does increase or decrease denote a proportional increase or decrease in the different types of WBC? 3. Can type O blood be transfused to type B? How about the antibodies of type O as donor, will these not clump the RBC of the recipient? 4. If you are Rh positive, do you have antibodies for Rh? How about if you are negative? If a man is Rh positive and the wife is RH negative, is there a chance for a normal child? When and how will it happen physiologically? And medically? 5. Is coagulation the same as agglutination? How does temperature affect clotting time? Why do dentists advise their patients to take something cold after a tooth extraction 6. What buffers are found in blood plasma? 7. Discuss the effect of the different conditions tested on pulse rate. 8. Discuss other factors that can influence pulse rate. 9. Explain the principle behind blood pressure determination. 10. What is pulse pressure?
HUMAN RESPIRATION INTRODUCTION The respiratory center in the medulla oblongata is where information for the need of ventilation is received and coordinated. It is sensitive to the concentration of carbon dioxide in the blood. A slight increase in carbon dioxide in the blood produces deeper and faster breathing, permitting more carbon dioxide to the lungs and consequently the removal of the gas. This stabilizing process continues until the carbon dioxide level returns to normal. Respiration may be divided into three phases: (1) pulmonary ventilation wherein gases (CO 2) in the lungs are exchanged for gases (O 2) in the atmosphere, (2) internal respiration or exchange of gases between pulmonary capillaries and alveolus and (3) external respiration wherein tissue cells give up CO2 in exchange for O2 from the capillaries. All three phases depend on the partial pressure of gases such that simple diffusion occurs. The aim of the exercise is to observe different breathing patterns in man. METHODOLOGY For parts A-C, each group should choose subjects of the same sex and with similar body sizes. Do the procedure in part D on a male and female subject. A. Normal Respiration Rate The normal respiration rate, also known as quiet respiration, is measured by counting the number of breaths per minute while the subject is in a sitting (resting) position. Note the depth of breathing as shallow, moderate or deep. After recording the normal respiration rate, instruct the subject to hold tightly a paper bag over nose and mouth. Let him / her breathe into the bag while recording the respiration rate and taking note of any changes in the depth of breathing that may occur. Record all data. B. Breath Holding Time Immediately after inhaling, have the subject hold his /her breath for as long as she/he can. Determine time, in seconds, the subject can hold his/her breath. This is the breath holding time. Note the depth of breathing and number of breaths while at the same time observing any changes in the depth of breathing. Record all data. C. Hyperventilation Instruct the subject to breathe deeply with the mouth open (in order to over ventilate the lungs) for as long as he / she can. The subject may experience difficulty in breathing and may force himself to continue. Ultimately, cessation of breathing will take place for 10-20 seconds. Record the length of time the subject can force oneself to hyperventilate. Take note also of the duration or period the subject temporarily ceases to breathe. Likewise, record the respiration rate and depth of breathing immediately after the recovery period. Record all data. CAUTION: “HEAVY hyperventilation may cause dizziness or fainting. Limit over ventilation from 30 to 60 seconds only. D. Recording of Respiratory Patterns Record the respiratory movements of subjects following activities: swallowing water, reading aloud, laughing, mentally multiplying 789 by 234, coughing and yawning. For the effects of exercise, have the subject perform 15 to 20 deep knee bends and then immediately return to his sitting position to record his respiratory movement. Take note of the time elapsed before respiration was increased and the length of time retained at an elevated state.
Questions for Research 1. Discuss the respiratory center, its control of breathing and factors that may influence it. 2. What is the average normal human respiration rate? What are the factors affecting respiration rate in humans. 3. What is the effect of forced breathing on the length of time one can hold his breath?
MEASURING CARBON DIOXIDE PRODUCTION AND OXYGEN CONSUMPTION OF SELECTED TEST ANIMALS INTRODUCTION Respiration in aquatic animals is different from that of land animals basically because of the amount of oxygen available in the water. Concentration of dissolved oxygen is dependent on factors such as properties of water and the solubility of gases. Essentially, oxygen in the aquatic environment is less than that found in the air. It is the objective of this exercise to determine the factors affecting respiration rate in aquatic animals. METHODOLOGY Label six 50 ml beakers E1 to E6 and one similar bottle C (CONTROL). Fill up all the bottles with 100 ml of dechlorinated water and add two drops of phenolphthalein indicator. A. Respiration rate of fingerlings Place in each E bottle 2 fingerlings. The C bottle will not contain any animal. Note the start of the experiment and record the time. Observe all activities of experimental animals like mobility inside the vessel, gill movements, etc. from beginning to end. After half an hour, remove all animals from the bottle and measure collective weight of the animals. Enter all values in a table. While other members of the group are weighing the subjects, the rest of the group can proceed and determine the carbon dioxide content of the water in each of the bottles including the control. Using a base burette, slowly add 0.4% NaOH to the water while gently shaking or swirling the bottle to ensure thorough mixing. Continue this procedure until enough NaOH solution turns the water to a light pink color. Record the amount of NaOH used. Enter all data in a table. Following the same procedure, repeat the experiment using the same animals in E1 to E3 but at a temperature 10°C below room temperature. Likewise do the same for E4 to E6 at a temperature above 10°C. Separate control bottles should be used for separate conditions. Enter all data in the table. Take note of the activity of the animals in the bottles subjected to different temperatures. Table 1 Carbon dioxide (μm oles) produced is equivalent to ml NaOH used.
Bottle Weight (g) NaOH used (ml) CO 2 present Respiration rate (μmoles) (μmoles/g/hr)
B. Measuring Oxygen Uptake Preparation of the Scholander Respirometer The Scholander respirometer is a simple respiration or metabolic chamber adapted for use with small terrestrial animals. Prepare the set up as illustrated below. Glass jar Cork Pipette
Record the weight of the mouse. Place it inside the airtight glass jar. Make sure the mouse doe not touch the NaOH placed underneath the wire mesh as it will cause burns. Introduce a drop of water on the outer end of the graduated pipette. As the oxygen is consumed by the mouse, CO 2 is given off, but this is absorbed by the NaOH. This results in the decrease in pressure inside the jar causing the drop of water to move inward. Determine the oxygen uptake for one hour at ten minute intervals by measuring the movement of water in the pipette. Express readings as ml oxygen uptake per gram body weight per hour. Questions for Research 1. What are the factors affecting respiration rate of aquatic animals? Terrestrial animals? 2. What is the effect of temperature on respiration rate?
OSMOREGULATION PREPARATION Collect at least 20 earthworms for this experiment. The best time to hunt for earthworms is on a damp night when it is not too cold. Use a flashlight to help locate them in patches of grass, in parks, lawns or fields. You may have plenty in your garden. Usually, you will find them stretched out on the surface of the ground with their posterior end inside their burrows. They may pop back into the ground when you appear. If you collect during the day, you may dig for them preferably at places where the soil is loose. Digging for worm however, is not equally successful in the different parts of the garden or field. It is preferable to dig for worms after a heavy rain. Heavy rain drives worms to the surface where they may be seen crawling on the ground. Actually, worms come up from their flooded burrows below the ground. Bring the worms to the laboratory in an uncovered can or glass jar containing some damp soil and some moist leaves or grass. When you reach the laboratory, tumble them on paper towels to remove excess soil. In groups of four, place them in separate containers with rain or aged tap water. PROCEDURE 1. Prepare 5 (NaCl) solutions of the following concentrations: 0.03 M, 0.06 M, 0.09 M, 0.12 M and 0.15 M. 2. Weigh the worms in groups of four and place them in separate containers with aged tap water. 3. Weigh at the end of 15 and 30 minute. If the weights are fairly constant already, determine the volume of each group by volume displacement in a graduated cylinder containing water. 4. After you have determined the weight and volume of each group, transfer each group to a different dish containing one of the salt solutions.
5. Weigh the worms at 20 mine intervals within 40 to 100 minutes of immersion. At the end of the experiment, determine their volume. 6. Record your results in tabular form, then, (a) plot on graph paper the weight and volume changes against time and (b) plot the percentage gain or loss in weight and volume against time and osmotic value of the salt solutions. QUESTIONS: 1. What determines the salt content f the soil? What is the effect of rainfall on the soil? On the behavior of worms? What mechanisms are of survival value to the earthworms in variable environments? 2. What response in the weight and volume of the earthworms do the different salt solutions evoke? 3. Compare the water exchanges in the worms exposed to varying saline media. What is the regulatory role of these exchanges? 4. Why ionic regulation, volume regulation, and osmoregulation are inextricably associated with each other. 5. Aside from osmoregulation, what is the other reason for the earthworm to crawl to the surface at night? Do they have eyes?
URINALYSIS INTRODUCTION The basic function of the excretory system is to regulate and maintain the chemical attributes of the blood. It is also considered a mechanism for maintaining the homeostasis of the internal medium. Different organs eliminate different substances and the by-products of metabolism. The lungs remove carbon dioxide while water and salts are removed through the skin and kidneys. Salts of heavy metals and blood pigment products are excreted mainly via the kidneys in urine form. The examination of urine’s composition by various tests can more or less determine the physical state of an animal. It also serves as an index for determining the presence of metabolites in small or large amounts in the urine. Normal urine is a highly complex solution of organic and inorganic compounds representing large waste products derived from the metabolic processes. Urine constituents are chiefly in the form of nitrogen containing and nitrogen free substances as well as inorganic salts. The principal dissolved substances are urea, uric acid, ammonia, chlorides, phosphates and sulfates. The exercise will examine the different physical and chemical characteristics of urine samples. METHODOLOGY Collect urine samples. A.
Physical characteristics Take note of the following characteristics: color, odor, pH, transparency, odor, amount. Then vigorously shake the samples and take note of any changes. Enter all data in the data sheet.
B.
Chemical Characteristics of the urine 1. Albumin Test: Filter the urine through coarse filter paper to clean any turbid specimens that may be present. Heat 5 ml of urine in a test tube to boiling. Note the color of the
precipitate formed, which is caused by albumin and phosphates. If no precipitate is formed, the sample is negative for albumin. Acidify the resulting hot mixture by slowly adding 3-5 drops of acetic acid that will result in the clearing of the precipitation caused the presence of phosphates. If the samples remain clear, albumin is absent, otherwise the precipitate will persist and the mixture may become more flocculent. 2. Glucose Test: If albumin is present in appreciable amount, it should be removed by acidifying the urine with dilute acetic acid, boiling and then filtering. The test is then performed on the filtrate. Place 5 ml of Benedict’s solution in a test tube and add to it 0.5 ml of urine sample. Boil vigorously for 1-2 min over an open flame and allow it to cool slowly. If the urine remains clear or white turbidity develops (presence of ureates or phosphates) no sugar is present. But in the presence of glucose, the entire solution becomes opaque and filled with precipitate colored green to red indicating the amount or quantity of sugar present. Positive result may be recorded based on the following: 0.1 to 0.25% glucose indicated by green turbidity (weak) 0.5 to 1 % glucose indicated by yellow to orange precipitate (moderate) > 1 % glucose indicated by brick red precipitate (strong) 3. Urea Test: Evaporate about 3 ml of urine to complete dryness, finishing the process in a hot water bath. Remove the sample from the water bath by rubbing the residue with enough acetone (using a stirring rod on a watch glass) Allow cooling. Crystals appearing as crystal needle or the like indicate the presence of urea. If necessary, examine a drop of the treated samples under the microscope in order to determine the presence of urea crystals. 4. Uric Acid: Treat 10 ml of urine with 2 drops of strong NH 4OH. Then saturate the solution with powdered NH 4Cl in order to settle the mixture. Pour off the resulting supernatant liquid into another beaker. Take note of the precipitate that may indicate ammonium urate and transfer it to a clean evaporating dish. Add 2-3 drops of strong nitric acid and evaporate to dryness in a water bath. Take note of the color of the resulting residue. Add 2 ml of 1% NH 4OH. Again take not of the resulting color. The presence of the uric acid positively indicated by the resulting pink residue upon the addition of strong nitric acid, then turns purple upon addition of 1% NH 4OH. 5. Indican Test: Fill a test tube with 10 ml sa mple and add 5 ml Obemayer’s reagent. Warm the mixture slightly and put 3 ml of chloroform. Mix the resulting solution by inverting the test tube from time to time. Normal amount of indican will give a faint blue color while excess amount of indican will show an indigo-blue color (chloroform) which sinks to the bottom of the tube. Questions for Research 1. Give the normal physical characteristics (color, transparency, volume, odor, Ph) of urine. What are the substances responsible for these? 2. What pathological conditions are prevailing if the test samples show significant differences in amount? Composition? 3. What is hemodialysis? Peritoneal dialysis?
CHARACTERIZATION OF DIGESTIVE ENZYMES OF KILLED ANIMALS Enzymes are needed for the chemical digestion of food. There are specific enzymes for different types of food. It is thus easy to demonstrate enzyme activity using different substrates. Methodology: I. Preparation of Substrates A. Amylase Substrate Mix 200 mg soluble starch with a little water and make up a volume of 100 ml. Heat the mixture until it boils and then slowly adds 2 g agar powder. After cooling to about 50 –60°C, pour into Petri dishes and add 2 drops strong iodine solution, stirring to ensure even distribution of the iodine and the formation of a uniformly bluecolored gel.
II.
B.
Protease Substrate Mix 100 ml of milk powder with 2 grams agar powder and slowly heat the mixture until it boils. Pour into petri dish. Solidify.
C.
Lipase Substrate Mix 4 g mayonnaise with 5 ml water and add 1 ml of 1 molar NaOH. Prepare the agar (2%) and allow to cool to 50-60 C. Add 1 ml mayonnaise mixture, stirring to ensure even distribution. Pour into petri dish. Determining the Presence of Enzyme Activity After allowing the gels to cool and harden, cut 3 uniformly sized wells in each plate. Into each well place uniform lengths of the stomach, small intestines and large intestines of a freshly killed mouse. Add a little water into each well. If specific enzymes are present, their diffusion from the wells into the surrounding agar causes digestion of substrate resulting in the formation of transparent or pale colored zones around the perimeter of the wells. The total area of a zone developed around each well in unit time is directly proportional to the concentration of the enzyme, provided factors like temperature and rigidity of the gel are controlled. A. Amylase: Digestion of starch from the plates leads to the formation of transparent zones around the perimeter of wells. Plate containing starch-iodine complex need not be incubated. Satisfactory results can be obtained after 6 hrs at room temperature. B. Protease: Protease digests casein (milk protein) from the gel, forming transparent zones. Plates should be incubated at room temperature for about 12 hours before they are examined. C. Lipase: Plate should be incubated at 37 C before being examined. Lipase will digest the substrate to form transparent zones, but this can be seen more distinctly by flooding the plates with 10% copper sulfate solution which should be left for 30 min to penetrate the gel. Zones containing fatty acids stain blue green after this treatment.
Preparing the animals: Always treat your lab animals humanely. All animals used for this study must be freshly killed. After killing the animal. Transfer it to a dissecting pan and dissect
out the digestive system as fast as soon as you can. The digestive mucosa degenerates very fast so it is important that you work rapidly. Dissections should be made dry, since enzymes diffuse through water to spread to regions of the gut where they are not normally present. Questions for research: 1. What are the different factors that affect enzyme activity? 2. What substances were digested in which part of the digestive tract?
