manual of methods for determining micronutrients in fortified foods

Where the parameter of the equations are:
Parameter Explanation Value
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X New average after discarding outlier results ?
w s
Sample weight (g) ?
V1 Initial volume (volume 1) 200 mL
V2 Aliquot volume (volume 2) 5 mL
V3 Volume 2nd dilution (volume 3) 100 mL
C std
Actual concentration stock solution of folic acid (~1 ng/mL) ?
X. Optimum incubation time
1. Prepare two tubes with 0 mL folic acid standard and 5 mL distilled water.
2. Prepare two tubes with 5 mL folic acid standard solution-1 ng/mL.
3. This makes 4 tubes per incubation time. It is recommended to determine the % transmittance of
tubes at 16, 18, 20, 22 and 24 hours after inoculating the tubes. Prepare 20 tubes in total.
4. Add distilled water to complete the final volume to 5 mL in the blank tubes.
5. Add 5 mL medium for folic acid analysis (HiMedia 126). Sterilize, cool down and add 25µl inoculum
and incubate at 37 °C.
6. Mix and set the wavelenght at 550 nm. Set the 100% transmittance with the tube with 0 mL standard
solution. Read the % transmittance of the tubes containing 5 mL standard solution.
7. Repeat this procedure to complete 24 hours. When the change in % transmittance between two
different incubation times is lower than 3 units, that is the optimum incubation time.
XI. Spectrophotometer verification
A. Preparation
1. Wash a porcelain crucible with soap, tap water and rinse it with distilled water.
2. Dry the crucible in an oven at 110°C until constant weight.
3. Cool the crucible down in a dessicator and weigh it before using it.
4. Prepare the inoculum as described in the procedure.
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