manual of methods for determining micronutrients in fortified foods

mL standard or sample/tube
mL distilled water to be added
0 (blanks) 5
0.5 4.5
1 4
2 3
3 2
4 1
5 0
3. Add 5 mL agar for folic acid assays (HiMedia 126) to all the tubes and close them.
4. Sterilize in autoclave for 5 minutes at 121-124°C.
5. When the tubes are out of the autoclave, cool them down as rapidly as practicable to keep color
formation at minimum. An ice water bath is useful. Take precautions to keep sterilizing and cooling
conditions uniform throughout assay.
6. Aseptically inoculate each tube adding 25 µL inoculum with an automatic pipette and sterile tips,
except to the blanks without inoculation.
7. Incubate 16 hours at 35-37°C in a water bath.
8. After incubation, check for growth in the blanks. If any growth is obtained, contamination might
have occurred and this invalidates the assay.
The tube to the left shows growth (samples or standards), whereas the solution to the right is clear
showing no growth (blanks).
9. Before reading each tube, agitate for 10 seconds in a vortex mixer.
10. Warm the spectrophotometer up and set the wavelength at 550 nm.
11. After 15-30 minutes, adjust the instrument to 100% transmittance with the blanks without inoculation.
12. Read the % transmittance of the inoculated blanks.
13. Set 100% transmittance with the inoculated blanks and read the % transmittance of the rest of the tubes.
14. Discard the tubes and wash the glassware as described in the glassware cleaning procedure.
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