ZOO502 Lab Manual

Contents

S. No.

Practical

P. No. Determination of haemoglobin content, determination of haematocrit and cell counting Preparation of blood smears Nerve muscle preparation, Muscle twitch, Comparison of muscle and nerveirritability, effect of stimulus strength, effect of stimulus frequency (tetany), effect ofload or stretch, effect of prolonged activity (fatigue), neuromuscular fatigue, stimulationof motor points in frog. Recording of action potential by oscilloscope and demonstration of its variousfeatures. Experiments to demonstrate characteristic of reflex arc, experiment in human (students themselves) to demonstrate some aspect of sensory physiology. Normal cardiac activity, effect of temperature, effect of drug, heart block, tetanization of heart. Measurement of blood pressure. Oxygen consumption in fish and effect of temperature (by dissolved oxygen meter) and terrestrial animal (mouse). Oxygen consumption (by respirometer) Study of stages in estrous cycle. Locomotory behavior of small animals, earthworm, garden snails etc. Ear pinna reflex responses in domestic cats Preparation of skinner box or maze for study of mouse or rat behavior Mother-pup bond in mice and rats, infant killing behavior Pecking behavior of chickens and hiding behavior of chicks Observation of birds’ nests and study of parental behavior Altruistic behavior in monkeys

Practical No: 1 Determination of hemoglobin content, determination of hematocrit and cell counting

General information

Hemoglobin is a conjugated protein composed of the basic protein globin linked to 4 heme molecules. Ninetyeight percent of all the iron found in the blood is contained in hemoglobin. Hemoglobin transports oxygen and carbon dioxide. This important substance reacts with oxygen to form oxyhemoglobin. In the tissues, oxygen is released and reduced hemoglobin is formed. Hemoglobin can react with acids, bases, and oxidizing and reducing agents. It also can exist in a variety of forms. The normal values for hemoglobin determinations are as follows: Fig 1.1: The volume of hemoglobin in male and female

Compounds of hemoglobin

• Oxyhemoglobin. Oxygen combines loosely with iron (ferrous state) in hemoglobin. The loosely attached oxygen diffuses into the tissues for oxidative processes. The hemoglobin then binds carbon dioxide and exists as reduced hemoglobin.
• Carboxyhemoglobin. Hemoglobin combines with carbon monoxide to form carboxyhemoglobin. Carbon monoxide has an affinity 200 times greater for hemoglobin than oxygen does. Hemoglobin in this combination is incapable of oxygen transport.
• Methamoglobin. This compound is formed when the ferrous state of the heme is oxidized to the ferric state. This compound is incapable of oxygen transport.
• Sulfhemoglobin. This compound results from the combination of inorganic sulfides and hemoglobin. This compound is incapable of oxygen transport. This is an irreversible reaction.
• Cyanmethemoglobin. This compound results when methemoglobin combines with the cyanide radical. This compound is used in hemoglobinometry.

Hemobloginometry

The hemoglobin concentration is directly proportional to the oxygen-combining capacity of blood. Therefore, the measurement of the hemoglobin concentration in the blood is important as a screening test for diseases associated with anemia and for following the response of these diseases to treatment. There are four basic ways to measure the hemoglobin concentration:

1. Measurement of the oxygen-combining capacity of blood (gasometric)
2. Measurement of the iron content (chemical method)
3. Colorimetric measurement of specific gravity (gravimetric method)
4. The cyanmethemoglobin method is the method of choice and is most widely used

Cyanmethemoglobin (hemoglobin-cyanide) method for estimation of hemoglobin

This is the method of choice for estimation of hemoglobin and is recommended by International Committee for Standardization in hematology. This is because i. All forms of hemoglobin are converted to cyanmethemoglobin (except sulfhemoglobin) ii. A stable and reliable standard is available.

Principle

Blood is mixed with a solution of potassium ferricyanide, potassium cyanide and a non-ionic detergent (Drabkin’s solution). Erythrocytes are lysed producing an evenly distributed hemoglobin solution. Potassium ferricyanide converts hemoglobin to methemoglobin, and methemoglobin combines with potassium cyanide to form cyanmethemoglobin (hemiglobincyanide). All forms of hemoglobin present in blood are completely converted to a single compound, cyanmethemoglobin. When the reaction is completed, absorbance of the solution is measured in a spectrophotometer at 540 nm. At this wavelength, cyanmethemoglobin has a broad absorbance peak. To obtain the amount of hemoglobin in the unknown sample, its absorbance is compared with that of the standard cyanmethemoglo-bin solution (the hemoglobin concentration of which is known) by using a formula or a previously prepared graph/table.

Equipment

1. Photoelectric colorimeter or spectrophotometer
2. Sahli’s pipette marked at 20 μl (20 cmm)
3. Pipette 5 ml

Reagents

1. Drabkin’s solution (ph 7.0-7.4): Potassium ferricyanide 200 mg Potassium cyanide 50 mg

Potassium dihydrogen phosphate 140 mg Non-ionic detergent 1 ml Distilled water to 1000 ml

2. Cyanmethemoglobin standard solution with known hemoglobin value.

Specimen: EDTA-anticoagulated venous blood or blood obtained from skin puncture.

Method

1. In a test tube, take 5 ml of Drabkin’s solution and to it add 20 μl of blood (1:251 dilution). Stopper the tube, mix by inverting several times, and allow standing for at least 5 minutes. This time is adequate for conversion of hemoglobin to cyanmethemoglobin.
2. Transfer the test sample to a cuvette. Read the absorbance in a spectrophotometer at 540 nm or in a photoelectric colorimeter using a yellow-green filter. Also take the absorbance of the standard solution. Absorbance should be read against reagent blank (Drabkin’s solution).
3. Hemoglobin value is derived from the formula given below or from the previously prepared graph or table.

Preparation of graph and table: If a graph or a table is prepared which correlates absorbance with hemoglobin concentration, result can be obtained quickly. This is particularly suitable when a large number of samples are regularly processed on the same instrument. Diluted cyanmethemoglobin standards are available commercially for preparation of a calibration graph. Alternatively, standard cyanmethemoglobin solution is diluted serially with Drabkin’s solution. On a linear graph paper, hemoglobin concentration (horizontal axis) in each dilution is plotted against the absorbance (vertical axis). A straight line joining the points and passing through the origin is obtained. From this graph, a table can be prepared relating absorbance to hemoglobin concentration.

Determination of hematocrit in blood sample

The hematocrit or packed cell volume (PCV) determines the percentage of red blood cells (RBCs) in whole blood. The normal hematocrit value for men is 42% to 52%; for women, 37% to 47%; and for newborns, 53% to 65%. When hematocrit determinations are below normal, medical conditions such as anemia and leukemia may be present. Above-normal hematocrit determinations indicate medical conditions like dehydration, such as occur in severe burn cases.

Fig 1.2: Detail of blood parts Currently, automated hematology analyzers supply most hematocrits. However, when hematology analyzers are not available, hematocrit determinations can be manually performed by two methods,