Overview of Discussion Objective Principle Requirements Experimental specifications (conditions) Preparation of histamine standard solution Preparation of Tyrode solution (PSS) Procedure Kymograph recording of contractions Observation table Calculation Result and interpretationTo determine unknown concentration of histamine using isolated guinea pig ileum by matching bioassay.Matching Bioassay It is the simplest type of bioassay. In this type of bioassay, the response of the test substance is taken first and the observed response is tried to match with the response that is obtained with the standard drug. Several responses of the standard drug are recorded till a close matching response to that of test substance is observed and the corresponding concentration is thus calculated. This assay is employed when the sample size is very small. Since the assay does not involve the recording of concentration response curve (CRC), the sensitivity of the preparation is not taken into consideration. Therefore, the precision and reliability of such bioassay is not good.Histamine is an autocoid having profound physiological effect in the body. Besides the triple response caused, it has spasmogenic response on the intestinal smooth muscle. By acting on H1- histamine receptors, it causes contraction of intestinal smooth muscle. Guinea pig is highly sensitive to histamine. The guinea pig ileum preparation is very commonly used for isolated tissue experiments. It is sensitive to even ng/ml concentrations of histamine. Overnight fasted guinea pigs are used to record better response of histamine on intestinal smooth muscle.Animal: Guinea pig (400-600 g, overnight fasted) Physiological solution: Tyrode solution. Drug- Histamine (Stock solution: 1 mg/ml), Histamine std. (1 or 10 ug/ml) Chemical- Fixing solution. Instruments: Sherrington recording drum , Student organ bath, Aerator, Insulin or tuberculin syringe to inject drugs in small fractions, Dissecting board and various dissecting instruments. Simple straw lever or frontal writing lever and stand, Pipette, Stop watch etc. Miscellaneous: Kymograph paper, plasticin, clips, and thread.Experimental specifications (conditions) = Isolated tissue- Isolated guinea pig ileum preparation = Drug- Histamine (Stock solution: 1 mg/ml), Histamine std. (1 or 10 ug/ml) = Physiological salt solution (PSS)- Tyrode solution. = Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact time- 90 seconds, Washing period- 3 minutes = Applied load/tension- 0.5 g = Bath capacity- 40 ml = Bath temperature- 32- 35°C = Speed of rotation of drum- 0.25 mm/ second = Magnification value (Mf) = d (F-W)/ d (F-T) = Aeration- Normal air (1- 2 bubbles/ second)Preparation of histamine standard solution Stock solution- 1 mg/ml or 1000 pg/ml * Dissolve 1 mg of histamine in 1 ml of distilled water > 1000 pg/ml Standard solution * Dilute 0.1 ml of 1000 ug/ml solution up to 10 ml with distilled water — 10 pg/ml * Dilute 1 ml of 10 ug/ml solution up to 10 ml with distilled water — 1 ug/mlPreparation of Tyrode solution (PSS) Prepare 1 litre of Tyrode solution by dissolving NaCl (8.0 g), KCI (0.2 g), MgCl2 (0.1 g), NaHCO3 (1.0 g), NaH2PO4 (0.05 g) and glucose (2.0 g) in distilled water. MgcCl2 should be added at last. CaCl2 (0.2 g) should be dissolved separately in distilled water to avoid chances of precipitation of salt. Mix CaCl2 solution to the higher volume of PSS.Procedure = Guinea pig is sacrificed by a blow on the head and carotid bleeding. =" Cut open the abdomen and lift the caecum to trace the ileocecal junction. Cut and remove a few centimeter long of the ileal portion and immediately place it in the watch glass containing Tyrode solution. Trim the mesentery and with gentle care clean the contents of the ileum by pushing the Tyrode solution into the lumen of the ileum. Utmost care should be taken to avoid any damage to the gut muscle. Cut the ileum into small segments of 2-3 cm long. = Take a piece of ileum of 2-3 cm long and tie the thread to the top and the bottom ends without closing the lumen, and mount the tissue in the organ bath containing Tyrode solution maintained at 32- 35°C and bubbled with O2 or air. A tension of 0.5 g is applied and the tissue is allowed to equilibrate for 30 minutes before adding the drugs to the organ bath.Record the contraction due to histamine test dose (T) and selected dose of standard (S) (of either 1 or 10 ug/ml) using frontal writing lever. Contact time of 90 seconds and 5 minutes of total time cycle may be followed for recording of response of Tands. Observe the doublet of response (T and S) and decide whether to increase or decrease the dose of S till a close matching response of S to that of T is obtained. Matching of responses should be ensure simply by measuring the height of the responses (cm/ mm). Record the doubled dose response of the doses of standard and test which shows the perfect match. Properly label and fix the kymograph recording of responses with the help of fixing solution. Calculate the corresponding concentration of histamine in test dose (T) is then using the formula- Dose of the Standard Cone. of Unknown = ———________ X Cone. of St. Dose of the TestKymograph recording of contractions Fig. Bioassay of histamine using guinea pig ileum by matching method Perfect Match Loretto tell blot Reon Leo ce ll Mole Cease OD Leite lac] Ceo) mV CL ena a ede 2Observation table- Example Sr. Drug Dose Response in No. (Conc.) (ml) Height (cm/mm) 1 T 1.0 2.5 2 | S(10 ug/ml) 0.2 1.8 3 T 1.0 2.5 4 | S(10 ug/ml) 0.4 3.0 5 T 1.0 2.5 6 | S(10 ug/ml) 0.3 2.5 7 T 2.0 35 8 [S(10 ug/ml) 0.6 3.5Calculation Magnification value (Mf) = d (F-W)/ d (F-T) Where- d (F-W) — distance between fulcrum and stylus (writing tip) d(F-T) — distance between fulcrum and point of attachment of tissue Unknown concentration of Histamine T (ug/ml) | Dose of the Standard Cone. of Unknown = ——_______ X Cone. of Std. Dose of the Test Conc. of Unknown (T) = (0.3/1) X 10 = 3.0 ug/ml OR Conc. of Unknown (T) = (0.6/2) X 10 = 3.0 ug/mlResult and interpretation * The unknown concentration of histamine (T) using isolated guinea pig ileum by matching bioassay was found to be sone ig/ml. = The magnification value was found to be-.........Thank You... _ Any Questions ???