manual of methods for determining micronutrients in fortified foods

3. Intermediate standard (1)-1mg/L: Transfer 1 mL stock solution to a 100-mL volumetric flask
with around 50 mL distilled water. Adjust pH between 7-8 with HCl or NaOH-0.1 N drop by drop,
and using a potenciometer. Bring to volume with distilled water. Store in a dark container with a
2-mm toluene layer. Solution is stable for two weeks.
4. Intermediate standard (2)-100 ng/mL: Transfer 10 mL intermediate standard solution-1 to a 100
mL volumetric flask and bring to volume with distilled water. Prepare a fresh soluton every time is
needed.
5. Working solution-1 ng/mL: Transfer 1mL intermediate solution-2 to a 100-mL volumetric flask
and bring to volume with distilled water treated with activated carbon.
C. Culture medium for HiMedia brand
According to the experience in the laboratory, only the amount of medium needed is prepared every
time. Follow the instruction in the container to adjust the amount to be weighed according to the
volume to be used.
1. Trypticase soy broth: Weigh 30 g broth and suspend in 1L distilled water. Heat until all the solids
have dissolved. Adjust pH to 7.3±0.2 (25°C) with HCl 0.1N or NaOH 0.1 N. Sterilize in an
autoclave at 121-124°C for 15 minutes. Dispense in tubes. Incubate 24 hours at 35-37°C. If no
microbial growth is observed, use it or store it. For storage, seal the tubes with parafilm, store at
2-8°C. The medium expires after two months of preparation.
2. Medium for maintenance of strains used in vitamin microbiological assays (M132D): Weigh
11.1 g agar, suspend in 1L distilled water and boil 2-3 minutes. Cool down rapidly in a cold water
bath. When the medium is at room temperature, the final pH should be 6.7±0.2 (25°C). If not,
adjust it, but it should not be necessary. Dispense 10 mL medium in tubes 16-20 mm diameter and
sterilize in autoclave at 121-124°C for 15 minutes. Cool down at room temperature on top of an
inclined surface to get a slant. Incubate 24 hours at 35-37°C. It can be used if no microbial growth
is observed. Seal the tubes with parafilm, store at 2-8°C. The medium expires after two months of
preparation.
3. Inoculum broth for vitamin microbial assays (HiMedia 133): Weigh 52.1 g medium, suspend
in 1 L distilled water. When the medium is at room temperature, the final pH should be 6.7±0.2
(25°C). If not, adjust it, but it should not be necessary. Dispense 10 mL in tubes 16-20mm diameter
and sterilize in an autoclave at 121-124°C for 15 minutes. Cool down rapidly in a cold water bath.
Incubate 24 hours at 35-37°C. Use it provided that no microbial growth was observed. Seal the
tubes with parafilm and store at 2-8°C and discard after two months of preparation.
4. Medium for folic acid analysis (HiMedia M126): Weigh 11.1 g medium and suspend in 100 mL
distilled water. Let boil 2-3 minutes. Cool down rapidly in a water bath. When the medium is
at room temperature, the final pH should be 6.7±0.2 (25°C). If not, adjust it, but it should not be
necessary. Prepare the amount needed for each run only.
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