ESTIMATION OF SERUM CALCIUM (TRINDERS METHOD) PROCEDURE take three bigger centrifuge tubes and label them T for

r test, B for blank and S for std and make following additions:S. No. 1 2 3 4 Reagents Distilled Water Calcium standard (conc. 0.1 mg/ml) Serum Calcium reagent T 0.2 ml 5.0 ml B 0.2 ml 5.0 ml S 0.2 ml 5.0 ml

Mix the contents of the tubes by rotating between palms. Allow all the three tubes to stand at room temperature for 30 mts and then centrifuge for 10 mts. Decant the supernatant carefully by inverting the tubes and immediately placing them on a pad of filter paper to drain off the supernatant before restoring them to upright position. Then make the following addition. 5 EDTA solution 1.0 ml 1.0 ml 1.o ml Shake well suspend the precipitate and then keep the tubes in boiling water bath for 10 mts with occasional mixing to dissolve the precipitate completely. Cool the tubes by keeping in cold water and then add 6 Colour reagent 3.0 ml 3.0 ml 3.0 ml Mix well by inverting the tubes. Read the absorbance for T, S and S in a colorimeter by using blue filter or transmission at 450 nm. Calculations 1. Mgms of serum calcium per 100 ml T B x 10 SB 2. Serum calcium (Meq/L) = mg/dl of calcium x 10 x valency Atomic weight of calcium Normal Range Total serum calcium = 09 11 mg/dl 4.5 = 5.5 meq/L Concentration of standard 0.1 mg/ml (mg/dl of calcium X 10 X 2) 40

ESTIMATION OF BLOOD UREA (Diacetyl monoxime method) PROCEDURE I. Preparation of protein free filtrate: Distilled Water 07 ml Blood or Plasma 01 ml 10% Sodium Tungstate 01 ml 2/3 N H2SO4 01 ml Mix and keep at room temperature for 5 mts and filter. II. Colour development: Label three test tubes as Blank, Standard and Unknown and make following additions

Reagents Blank (B) Standard (S) Unknown (U) Distilled Water 0.5 ml Protein free filtrate 0.5 ml Urea Standard 0.5 ml Urea colour reagent 5.0 ml 5.0 ml 5.0 ml Keep in boiling water bath for 20 mts & cool to room temperature

Read colorimetrically at 530 nm / green filter. Calculations Blood Urea mg/dl ODU - ODB x 30 ODS - ODB Total Blood Urea (mg/dl) = Blood Urea nitrogen (mg/dl) x 2.14 Concentration of standard 0.03 mg/ml

ESTIMATION OF TOTAL PROTEINS (Biuret Method) PROCEDURE Take three bigger centrifuge tubes and label them as T for Test, B for Blank and S for standard and make the following additions. S. No. 1 2 3 4 Reagents Normal Saline Protein standard ( 05 mg/ml) Diluted Serum Biuret reagent T 1.0 ml 5.0 ml B 1.0 ml 5.0 ml S 1.0 ml 5.0 ml

Mix the contents by inverting the tubes and allow it to stand for 10 mts at room temperature. After 10 mts find out the optical density for test, Blank and standard by using a green filter or transmission at 530 nm in a colorimeter. Calculations Gms of total protein per 100 ml TP B x 10 SB Dilution of serum: Take 0.2 ml of serum sample and add 3.8 ml of Normal Saline and mix. Concentration of standard 5 mg/ml