manual of methods for determining micronutrients in fortified foods

1. Inoculate Enterococcushirae ATCC 8043 in 1 mLTrypticase soy broth. Incubate 24 hours at 35-
37°C.
2. Add 1 mL glycerine-15% and store at -70°C.
3. Repeat this procedure during preservation of the culture in Trypticase soy broth with glycerol-15%
from the transference of the strain.
D. Inoculum preparation
1. Transfer cells from the stock culture of Enterococcushirae ATCC 8043 to sterile tube containing 10
mL broth for inoculum (HiMedia 133).
2. Incubate 16-18 h at 35-37 °C.
3. Under aseptic conditions, centrifuge culture at 3,000 rpm for 5 minutes and decant the supernatant.
Wash cells with 10 mL sterile 0.9% saline solution, resuspending the cells in the pellet by agitation.
Repeat this procedure two times more and resuspend the pellet in 10 mL sterile 0.9 % saline solution.
This suspension is the stock inoculum.
a) Appearance of the inoculum after centrifugation for the first time. Bacteria cells form a pellet at the bottom
of the tube. b) After eliminating the media and resuspending the pellet in isotonic saline solution, the liquid in
the tube looks turbid. c) The washed cells form a pellet at the bottom of the tube after the final centrifugation.
4. To obtain the work inoculum, take 10 µL of the stock inoculum and add it to 10 mL sterile isotonic
saline solution.
5. A larger quantity of inoculum might be prepared following the proportion of stock inoculum/isotonic
saline solution. Store it in 5-mL sterile vials.
6. Label the vials with the medium name, lot number and expiration date, and store in the refrigerator.
7. Use the amount of inoculum needed at room temperature the day of the analysis.
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