General Rules in Pharmacology Practicals
Most of the pharmacology practicals are regarding the study of drug action on biological systems. These are mostly done using organ preparations isolated from some of the commonly used laboratory animals such as Frog's, Guinea pigs, Albino Rats, Mice etc.
Following are some rules or general procedure that a student should follow in order to carry out the experiment successfully.
* All equipments should be checked for their working condition before sacrificing the animal.
* The organ bath should be thoroughly cleaned and should be filled with the appropriate physiological solution. Aeration should be provided continuously so as to keep the tissue alive. Temperature of the organ bath should be consistently monitored and regulated.
* The writing lever should be adjusted for the desired degree of magnification and tension as per the protocol followed.
* Animals like frog's are usually killed by a head blow (Stunning), while rats and mice by spinal traction/cervical dislocation. Soon after this step they are immediately dissected and the organs to be studied are isolated and immersed in the physiological salt solution so as to preserve the biological properties of the preparation.
* The tissue should be mounted and allowed to stabilize for about 30 to 60 minutes. If the preparation is found to relax, the lever should be readjusted to horizontal position. Some times the tissue fails to relax, then it is necessary to increase the tension on the lever by increasing weights. (care should be observed such that the tissue doesn't get teared off)
* The fluid in the organ bath is to be replaced with fresh solution through out the experiment at regular intervals so as to prevent the alteration of PH due to prolonged aeration which in turn changes the tone of the preparation. (Depending on the nature of experiment)
* The drum is started at a slow speed. The volume of the drug solution is to be measured accurately (0.1 to 0.5mL) with the help of a 1mL graduated pipette or a tuberculin syringe fitted with needle and then by dipping its tip into the bath fluid, the drug is added with a uniform speed (Care should be taken such that the drug is not added onto the walls of the organ bath). Simultaneously with the addition of the drug a stop watch is started.
* The drug is allowed to act till the response reaches a steady level or up to a fixed time (say, 30 to 60 sec) depending on the nature of the experiment after which the bath fluid containing the drug is washed out and fresh solution is allowed to fill the bath. The washing is repeated two or three times, if necessary, in order to allow the lever to come back to original base line.
* At the time of washing, the drum is stopped and any contractions due to the exposure of the tissue to air are recorded as vertical line so that they can be readily distinguished from true contractions. This can, however, be avoided by emptying the organ bath by overflow method, that is by displacing the fluid by fresh solution from the bottom without actually emptying the bath. After some definite interval depending on the tissue as well as the drug, another dose of drug is added and the stop watch is restarted, thus completing a cycle which is strictly followed and repeated throughout the whole experimental period.
* Sensitivity of the preparation may sometimes be improved by raising the temperature or by increasing the dose intervals.
* Every time a drug is added into the bath, an arrowhead is put at the point of addition on the drum near the baseline and labeled appropriately indicating the drug and its dose. At the end of the experiment, full details such as the date, tissue employed, bath fluid and its volume, temperature, nature of the experiment etc. are recorded on the smoked surface before fixing the tracings permanently.
* The experiment should start with a low dose of drug (sudden exposure of the tissue to a high concentration of the drug may affect it for a prolonged period making it unsuitable for further testing) which produces slight or no response and then repeating after each washing a dose greater than the preceding concentration until the activity range is found.
* It is a good practice to try two or three standard drugs (e.g. Acetylcholine, Histamine, 5-Hydroxytrypamine, etc.) before and after each addition of the unknown drug so as not to lose sight of the latter's inhibitory property, if any. In the case of an inhibitory drug, a very high concentration will have a nonspecific antagonism against a number of agonists and thus its true specific nature will not be detected.
Fundamentals of Experimental Pharmacology- By M.N Ghosh, 2nd Edition, Scientific Book Agency.