Project Work

Sample preparation and its series dilution

30 gm of sample was mixed with 270 ml peptone physiological salt solution for sample preparation.

For series dilution, 9 ml of peptone physiological salt solution was transferred into each test tube by using sterile pipette. Then, 1ml of sample was added into 1^st test tube using another sterile pipette. So, its dilution was 10^-1. Then, 1ml solution was taken from first test tube using another sterile pipette and transferred into 2^nd test tube which was containing 9 ml PPS. Its dilution became 10^-2. This process was repeated to make several dilutions (10^-3, 10^-4, 10^-5, 10^-6 and 10^-7).

Fehling solution (A): 69.29 gm to copper sulphite was dissolved in water dilute to 1000 ml if necessary filter through No.4 whatman paper.

Fehling solution (B): 346 gm of Rochelle salt (Potassium sodium tartarate, KNaC₄H₄O₆. 4H₂O) was dissolved and 100 gm NaOH in water and made up to 1000 ml.

Methylene blue indicator: 1mg of methylene blue was dissolved in 100 ml of water.

45% Neutral lead acetate solution:  225 gm of neutral lead acetate was dissolved in water and dilute to 500 ml.

Determination of Crude Fiber

The method is based on the procedure developed by Henneberg, Stohman and Rautenberg in the agricultural experiment station at Weende bei Gottingen in Germany in 1864. Only slight modifications have been made in the Weende method since its development.

Reagents

a) 0.255N sulfuric acid solution: Concentrated sulfuric acid (3.54 ml) was added in distilled water and the volume was made up to 500 ml.

b) 0.313N sodium hydroxide solution: Sodium hydroxide (6.26g) was dissolved in distilled water and the volume was made up to 500 ml.

Crude fiber

Procedure

Moisture and fat free sample was taken into a 500 ml beaker. 0.255N sulfuric acid (200 ml) was added in the beaker and boiled for 30 minutes, keeping the volume constant by addition of distilled water at frequent intervals. At the end of this period the mixture was filtered through a muslin cloth and the residue washed with hot water till free form acid. The material was then transferred to the same beaker and 0.313N  boiling NaOH (200 ml) was added after boiling for 30 minutes (keeping the volume constant as before) the mixture was filtered through muslin cloth, the residue was washed with hot water till free from alkali followed by washing with  some alcohol and diethyl ether. It was then transferred into a crucible, dried overnight at 105⁰ C in an oven and weighed. Then the crucible was heated in a muffle furnace of 600⁰ C for 3-5 hours. Cooled and weighed again. The differences in the weights represent the weight of crude fiber.

Calculation

% of crude fiber (g)   =