about 850 ml of distilled water. Then add 25 mg of pyrogallol red dye, and mix
well to dissolve it completely. Make thefinal volume to 1 litre with distilled
water. Store in an amber bottle.
2.Working standard: Prepare 1 g/dl BSA stock. Then prepare dilutions of BSA
with 20, 40, 60, 80, and 100 mg/dl in normal saline.
34.2.1.3 Procedure
Take a set of seven test tubes, and label as blank, standard (S 1 – S 5 ), and test for CSF
(Ts). Add 1 ml of standard with concentrations 20, 40, 60, 80, and 100 mg/dl in tubes
labelled as S 1 to S5.To blank tube, add 1 ml of normal saline. In test (Ts), add 1 ml of
CSF. Then add 3 ml of pyrogallol red in each tube. Mix well and incubate at 25C for
15 min. Read absorbance at 600 nm.
34.2.1.4 Calculations
Plot a graph between amount of protein at x-axis and absorbance at y-axis. Extrapo-
late protein concentration of given CSF sample from the graph. The protein concen-
tration can also be calculated by using equation
Amount of protein inCSF mgðÞ¼=dlODof testamount of standard 100
ODof standardvolume of sample mlðÞ34.2.2 Turbidimetry Method
34.2.2.1 Principle
Proteins present in CSF are precipitating with sulfosalicylic acid, and protein amount
is measured by comparing the absorbance of the turbidity at 640 nm with that of
protein standards.
34.2.2.2 Reagents
1.Normal saline: Prepare 0.9% (w/v) NaCl.
2.Sulfosalicylic acid(3 g/dl): Dissolve 3 g sulfosalicylic acid in afinal volume of
100 ml distilled water. Reagent is stable for 6 months if stored at 25– 35 Cinan
amber-colored bottle.
3.Stock standard: Prepare 1 g/dl BSA.
4.Working standard: Prepare dilutions of BSA with 20, 40, 60, 80, and 100 mg/dl
in normal saline.
34.2.2.3 Procedure
Take a set of seven test tubes, and label as blank, standard (S 1 – S 5 ), and test for CSF
(Ts). Add 1 ml of standard with concentrations 20, 40, 60, 80, and 100 mg/dl in tubes
labelled as S 1 to S5.To blank tube, add 1 ml of normal saline. In test, add 1 ml of
CSF. Then add 4 ml of sulfosalicylic acid in each tube. Mix and incubate the
136 34 Analysis of Cerebrospinal Fluid for Proteins and Sugars