Vitamins AACC Method 86-47

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Total Folate in Cereal ProductsMicrobiological Assay Using

Trienzyme Extraction
First approval November 8, 2000

Objective
This is a microbiological method employing the organism Lactobacillus casei
subsp. rhamnosus (ATCC no. 7469) to determine the amount of folate present in
foods and in vitamin concentrates. This method is semiautomated through the
use of automated dilution and turbidity reading instruments or, optionally, the
96-well microtiter plate and reader system. See Note 1. Samples, with water
added, are autoclaved to break up particles, gelatinize starch, and denature pro-
teins to enhance enzymatic attack and make folate more extractable. Folate
(pteroylglutamic acid in various forms) occurs naturally in foods bound to glu-
tamic acid residues of varying chain lengths. Most naturally occurring folates
cannot be utilized by the assay organism. Folic acid (pteroylglutamic acid) is
extracted from the sample using a triple enzyme system. A protease and an
amylase are used to digest the food matrix and aid in the release of folates. Des-
iccated chicken pancreas conjugase is used to hydrolyze folylpolyglutamates to
folyldiglutamates, which, along with folic acid, can be utilized by the assay
organism. The freed folates are extracted and diluted with basal medium con-
taining all required growth nutrients except folate, and the turbidity of the L.
casei subsp. rhamnosus growth response for the samples is compared quantita-
tively to that of known standard solutions. The method is applicable to cereal
grains and cereal grain foods containing added folate (folic acid) or naturally
occurring folates with levels from 5 g/100 g to 100% folate.

Apparatus. See Note 2.

1. Centrifuge, clinical, to accommodate 20- 150-mm test tubes.
2. Spectrophotometer, to read 20- 150- (or 18- 150-mm) test tubes.
3. Covered water bath, 37 0.2, with rotary shaker.
4. Disposable glass test tubes, borosilicate glass, 20- 150-mm (or 18- 150-
mm).
5. pH meter, with long combination electrode.
6. Vortex mixer.
7. Volumetric flasks, class A, clear, low-actinic; 25, 50, 100, 250, 500, and
1000 ml.
8. Pipetter, Eppendorf repetitive, with 50-ml capacity.
9. Disposable tips, Eppendorf, 1.25 ml.
10. Adjustable digital pipet, Eppendorf, 1001000 l.
11. Pipetting machine(s), to deliver 1- and 5-ml aliquots. Optional: Pipetter,
12-channel, to use with 96-well microtiter plates.
12. Pipets, volumetric, class A; 1, 2, 3, 4, 5, 10, 20, 25, and 50 ml.
13. Autoclave, for sterilizing at 15 psi and 121123.
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Total Folate in Cereal ProductsMicrobiological Assay Using

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14. Refrigerator, set to 28; freezer, set to 18.

15. Centrifuge tubes, Oak Ridge type, 28.6 106.1 mm, polypropylene, reus-
able.
16. Centrifuge tubes, disposable.
17. Test tube racks, 4 12 in., to hold 20- 150-mm test tubes.
18. Large rack, with cover, to hold four or more test tube racks.
19. Filter paper, Whatman no. 2V, 12.5 cm.
20. Balance, analytical, reading to at least four places.
21. Balance, top loading, three-place.
22. Hot plate stirrer.
23. Distilling apparatus for water. (Use distilled or double-distilled water
throughout.)
24. Syringe, fitted with long needle, sterilizable, capable of delivering 50 l.
25. Desiccator.
26. Inoculating loops and straight wire.
27. Bunsen burner
28. (Optional for tube assay) Sample changer, automated, assay-tube reading,
modified with an air agitation system and connected to a spectrophotometer with
a flow cell and either a printer or a computer, or a diluter and reader.
29. (Optional microtiter plate system) Microtiter plate reader, 96-well, and
microtiter plates, 96-well. A reader with appropriate filter(s) for 570630 nm and
efficient software for calculation is suitable. See Note 3. Use of the microtiter
plate systems requires filter sterilization of solutions, using 0.22-m sterilization
filter units (15-ml, 500-ml, and 1-liter sizes).

Reagents. See Note 4.

1. Acetic acid, glacial.
2. Adenine sulfate.
3. Agar, Bacto.
4. Agar, Lactobacilus casei.
5. -Amylase.
6. p-Aminobenzoic acid.
7. Ammonium hydroxide.
8. Antifoam. Mix 1.5 ml antifoam with 100 ml water. Shake before using.
9. Ascorbic acid.
10. L-Asparagine monohydrate.
11. Biotin.
12. Calcium pantothenate.
13. Casein hydrolysate, vitamin-free, acid-hydrolyzed.
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Total Folate in Cereal ProductsMicrobiological Assay Using

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14. Celite (optional as filter aid).

15. Conjugase source, chicken pancreas, desiccated.
16. L-Cysteine-HCl.
17. Dextrose (glucose), anhydrous.
18. Ferric sulfate heptahydrate.
19. Folic acid reference standard.
20. Glutathione.
21. Guanine hydrochloride.
22. Liver.
23. Magnesium sulfate heptahydrate.
24. Manganese sulfate monohydrate.
25. Niacin.
26. Octanol.
27. Potassium phosphate, dibasic.
28. Potassium phosphate, monobasic.
29. Protease, pronase E.
30. Pyridoxine hydrochloride.
31. Riboflavin.
32. Sodium acetate, anhydrous, and sodium acetate-3H2O.
33. Sodium ascorbate.
34. Sodium chloride.
35. Sodium hydroxide.
36. Thiamin hydrochloride.
37. Toluene.
38. D,L-Tryptophan.
39. Uracil.
40. Xanthine.
41. Acetic acid 0.02N. Dissolve 1.2 ml of glacial acetic acid in ~500 ml water.
Dilute to 1 liter with water.
42. Ammonium hydroxide (2+3), NH4OH (2+3). Carefully mix 2 volumes
concentrated ammonium hydroxide with 3 volumes water.
43. Hydrochloric acid (1+1). Carefully mix equal volumes of concentrated
HCl and water. See Note 4.
44. Phosphate buffer, pH 7.8 (extract buffer). Dissolve 1.42 g sodium phos-
phate dibasic and 1.0 g ascorbic acid in water and dilute to 100 ml. Adjust pH to
7.8 with 4N NaOH. Prepare fresh on day used. Assay requires approximately 35
ml of buffer for each sample and standard.
45. Phosphate buffer, pH 6.8 (assay buffer). Dissolve 1.42 g sodium phosphate
dibasic and 1.0 g ascorbic acid in 85 ml water and dilute to 100 ml. Adjust pH
to 6.8 0.02 with 4N NaOH. Prepare fresh on day used. Assay requires 1215
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Total Folate in Cereal ProductsMicrobiological Assay Using

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ml of buffer for each sample and standard tube for test tube assay and 2.5 ml of
buffer for each sample and standard for microtiter plate assay option.
46. Phosphate buffer, 0.1M, pH 7.0 (buffer for making standard solutions).
Dissolve 13.61 g potassium phosphate monobasic in water and dilute to 1 liter
with same. Adjust pH to 7.0 with 4N potassium hydroxide.
47. Potassium hydroxide, 4N. Dissolve 224 g of potassium hydroxide in ~500
ml water. See Note 4. Cool; dilute to 1 liter with water.
48. Sodium hydroxide, 4N. Dissolve 160 g of sodium hydroxide in ~500 ml
water. See Note 4. Cool, dilute to 1 liter with water.
49. Sodium hydroxide, 0.01N. Pipet 2.5 ml of 4N sodium hydroxide into a 1-
liter volumetric flask. Dilute to volume with water.
50. Adenine-guanine-uracil solution. Dissolve 1.0 g each of adenine sulfate,
guanine hydrochloride, and uracil in 50 ml warm HCl (1+1); cool; and dilute
with water to 1 liter.
51. Xanthine solution. Suspend 1.0 g xanthine in 150200 ml water. Heat to
~70; add 30 ml NH4OH (2+3); and stir until solid dissolves. Cool, and dilute to
1 liter with water.
52. Asparagine solution. Dissolve 10 g L-asparagine monohydrate in water and
dilute to 1 liter.
53. Vitamin solution for folate. Dissolve 10 mg p-aminobenzoic acid, 40 mg
pyridoxine hydrochloride, 4 mg thiamin hydrochloride, 8 mg calcium pantothen-
ate, 8 mg niacin, and 0.2 mg biotin in ~300 ml water. Add 10 mg riboflavin dis-
solved in ~200 ml 0.02N acetic acid. Add a solution containing 1.9 g anhydrous
sodium acetate and 1.6 ml acetic acid in ~40 ml water. Dilute to 2 liters with
water. (Not necessary to prepare if using commercial basal medium preparation
in reagent 58.)
54. Saline, sterile. Dissolve 9 g NaCl in 1 liter water. Dispense 10-ml portions
to 20- 150-mm test tubes capped with plastic tops. Sterilize 15 min at 121
123 and store in refrigerator.
55. Salt solution B. Dissolve 20 g MgSO4-7H20, 1 g NaCl, 1 g FeSO4-7H2O,
and 1 g MnSO4-H2O in water. Dilute to liter. Add 1 ml HCl.
56. PABA-vitamin B6 solution. Dissolve 50 mg p-aminobenzoic acid and 120
mg pyridoxine hydrochloride in 200 ml water. Add 0.95 g sodium acetate and
0.8 ml acetic acid to ~40 ml water. Combine the two solutions and dilute to 500
ml with water.
57. Agar maintenance medium. Into 1 liter hot water containing 10 ml of 100
ng/ml vitamin B12, dissolve 48 g Lactobacilli agar and 3 g Bacto agar. After
agars dissolve, dispense 10-ml portions to 20- 150-mm test tubes; plug with
cotton; and cap. Cover tubes to prevent contamination; sterilize 15 min at 121
123; and store in refrigerator.
Vitamins AACC Method 86-47
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Total Folate in Cereal ProductsMicrobiological Assay Using

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58. Folic-acid-free, double-strength basal medium. Prepare as shown in Table

I. (Alternatively, commercially available folic acid casei medium can be used.
Prepare as directed, i.e., suspend 9.4 g casei medium and 50 mg ascorbic acid in
100 ml water; boil for 12 min; cool.)
59. Standard solutions. Use low actinic glassware.
a. Stock solution (100 g/ml). Accurately weigh 50 mg USP folic acid that
has been dried to constant weight and dissolve in 0.1M, pH 7.0 phosphate
buffer in 500-ml volumetric flask. Dilute to volume with 0.1M phosphate
buffer. Top with enough toluene to keep surface covered (usually 35
ml). Store in refrigerator. Check purity of standard, and verify concentra-
tion of stock solution by pipetting 10 ml stock solution to 100-ml volu-
metric flask and diluting to volume with 0.1M, pH 7.0 phosphate buffer.
Measure absorbance of solution at 282 and 346 nm, using 0.1M, pH 7
phosphate buffer as blank. Folic acid concentration (FA) in stock solu-
tion:
FA (g/ml) = absorbance/absorptivity DF 1000 MW

TABLE I
Preparation of Folic-Acid-Free, Double-Strength Basal Medium (reagent 58)
Milliliters of Basal Medium to Prepare
250 500 1000
Add in order listed (ml)
Adenine-quanine-uracil solution 2.5 5 10
Xanthine solution 5 10 20
Asparagine solution 15 30 60
Vitamin solution for folate 50 100 200
Salt solution B 5 10 20
PABA-vitamin B6 solution 2.5 5 10
Add ~100 ml water and the following solids (g)
Vitamin-free casein, hydrolyzed 2.5 5 10
Dextrose 10 20 40
Potassium phosphate, dibasic 0.25 0.5 1
Potassium phosphate, monobasic 0.25 0.5 1
Sodium acetate-3H 2O 16.6 33.2 66.4
Glutathione 0.00125 0.0025 0.005
Dissolve the following solids (g) in dilute HCl and
add to above solution
L-Cysteine-HCl 0.125 0.25 0.5
D,L-Tryptophan 0.05 0.1 0.2
Mix well, adjust to pH 6.8 with NaOH, and dilute to
1 liter with water
Vitamins AACC Method 86-47
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Total Folate in Cereal ProductsMicrobiological Assay Using

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where absorptivity = 27,600 at 282 nm and 7200 at 346 nm, DF = dilu-

tion factor, and molecular weight (MW) = 441.4.
b. Working standard solution (1 g/ml). Dilute 5 ml stock solution to 475
ml with water and adjust pH to 7.5 with HCl. Dilute to 500 ml with
water. Prepare fresh on day of use. This will be diluted further when
standards are run in parallel with samples.
c. Diluted stock solution (100 ng/ml) (for use in working inoculum). Dilute
10 ml intermediate stock solution to 90 ml with water and adjust pH to
7.5 with HCl. Dilute to 100 ml with water. Top with enough toluene to
keep surface covered (usually 35 ml). Store in refrigerator. Prepare
fresh; discard after use.
60. Liquid culture medium (50% basal medium with 0.4 ng/ml folic acid and
10 g/ml solubilized liver). Suspend 0.1 g liver in 100 ml water. Hold mixture
for 1 hr at 50 and filter. Top with enough toluene to keep surface covered
(usually 35 ml). Store in refrigerator. Pipet 20 ml to a 1-liter volumetric flask.
Add (via pipet) 8 ml of diluted folic stock solution (100 ng/ml). Dilute to volume
with water. Mix equal volumes of solution with basal medium solution. Dispense
10-ml portions of diluted medium to 20- 150-mm screw cap test tubes; auto-
clave 15 min at 121123; and cool tubes rapidly. Store in refrigerator. (A com-
mercially available bacto micro inoculum broth has also been found satisfactory
as liquid culture medium.)
61. Conjugase solution: chicken pancreas solution (5 mg/ml). Weigh 0.5 g
chicken pancrease and add 100 ml pH 7.8 buffer. Stir vigorously for 10 min.
Transfer to 20- 150-mm test tubes and centrifuge for 10 min at ~2000 rpm.
Decant supernatant through glass wool pledget into beaker; cover with parafilm;
and store in refrigerator. Prepare fresh on day of use. Each sample and standard
requires 45 ml of conjugase solution.
62. -Amylase solution (20 mg/ml). Dissolve 0.5 g -amylase in 25 ml water.
Store in refrigerator. Prepare fresh on day of use. Each sample and standard
requires 1 ml -amylase solution.
63. Protease solution (2 mg/ml). Dissolve 0.05 g protease in 25 ml water. Fil-
ter through glass wool pledget, if necessary, and store in refrigerator. Prepare
fresh on day of use. Each sample and standard requires 1 ml protease solution.

Procedure
Preparation of inoculum
1. Test organism is lyophilized L. casei rhamnosis (ATCC 7469).
2. Cryprotected inoculum. Dissolve 4.7 g liquid culture medium (reagent 60)
or commercial folic acid casei medium in 50 ml distilled water. Heat to boiling;
cool in ice; then add 50 ml water and 25 mg sodium ascorbate. Add 0.5 ml
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Total Folate in Cereal ProductsMicrobiological Assay Using

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diluted folic stock solution (reagent 59c), mix, and sterile filter. Suspend 1 vial
lyophilized L. casei in 1.0 ml of the above medium, using sterile techniques.
Transfer 0.15 ml of this suspension to the culture medium. Incubate at 37 for 18
hr. Mix 120 ml glycerol and 30 ml water, autoclave, and cool in ice bath. Cool
incubated bacterial culture in ice bath, and add 100 ml cold 80% glycerol. Mix
gently but well. Aliquot 2.0-ml samples into sterile tubes (stir suspension occa-
sionally to maintain even suspension). Tubes can be stored at 20 for up to three
months or at 70 for up to six months.
3. Working inoculum. For tube cultures, dilute 2.0-ml tube of frozen suspen-
sion to 50 ml with sterile saline. Vortex mix. Use this suspension as working
inoculum. For 96-well plates, add 5 ml sterile saline to 2.0 ml solution direct
from freezer. Vortex mix. Use this suspension as working inoculum.

Sample preparation
All samples must be ground to ensure homogeneity and should be stored in
air-tight containers, free from light exposure.

Sample hydrolysis and extraction

1. Use distilled water unless otherwise indicated.
2. Standard (10 ng/ml). Accurately pipet 1.0 ml working standard solution (1
g/ml) to 125-ml Erlenmeyer flask containing 20 ml pH 7.8 phosphate buffer;
mix; and add 30 ml water.
3. Samples.
a. Accurately weigh an amount of sample equal to 0.251.0 g (dry basis)
solids containing ~1 g folic acid into a 125-ml Erlenmeyer flask.
b. Add 20 ml pH 7.8 phosphate buffer and mix thoroughly. (If sample is
low in folate, do not take >1.0 g. To compensate for the lower levels, use
a larger aliquot in the second serial dilution below.)
c. Add enough water to bring total volume to 50 ml. Add 0.11.0 ml octa-
nol (antifoam) to all flasks.
d. Cover flasks with 50-ml beaker; autoclave 15 min at 121123 and cool.
e. Add an additional 10 ml buffer to each flask.
f. Add 1 ml protease solution and incubate 3 hr at 37.
g. Inactivate enzyme by autoclaving 3 min at 100 and cool.
g. Add 1 ml -amylase solution to each flask; cover; and incubate 2 hr at
37.
h. Add 4 ml conjugase solution; cover; and incubate 16 hr at 37.
i. Inactivate enzymes by autoclaving 3 min at 100 and cool.
j. Adjust samples and standards to pH 4.5 with HCl (1+1), and dilute to 100
ml with water.
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Total Folate in Cereal ProductsMicrobiological Assay Using

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k. Filter approximately 20 ml through 2V filter paper (use of Celite is

acceptable to obtain clear filtrate). See Note 5.
l. Options: For standard tube/spectrometer assay, proceed to next step, i.e.,
Diluted standards and sample extracts. If using 96-well microtiter plate,
filter sterilize approximately 5 ml of filtered solution, using 0.22-m fil-
ter system, into sterile screw cap vials and store in dark at 4 until ready
to assay. Proceed directly to Optional 96-well microtiter plate assay.

Diluted standard and sample extracts (Test tube method only)

1. Standard (0.3 ng/ml). Pipet 15 ml standard extract into a 500-ml volumetric
flask.
2. Secondary standards (0.2 and 0.4 ng/ml). Pipet 10 ml and 20 ml of standard
extract into respective 500-ml volumetric flasks.
3. Samples. Pipet 10 ml of each sample extract into a 500-ml volumetric flask.
4. Add an equal volume of pH 6.8 phosphate buffer to volume pipetted into
each standard and sample flask. (If additional serial dilutions are necessary,
always add an equal volume of buffer to volume pipetted to flask). Dilute to vol-
ume with water. If concentration of folate is low, use a larger aliquot; if too high,
use a smaller aliquot or larger volumetric flask.

AssayTest Tube Method

See Note 6.
1. Prepare 21 tubes (20- 150-mm) for standard solutions (0, 0, 0.02, 0.04,
0.06, 0.08, and 0.10 ng/ml). In triplicate, place 0, 0, 1, 2, 3, 4, and 5 ml of diluted
standard extract (0.3 ng/ml) into tubes. (Three of the 0-ml tubes will be used as
uninoculated blanks.)
2. Prepare 15 tubes (20- 150-mm) for each secondary standard. In triplicate,
place 1, 2, 3, 4, and 5 ml of diluted secondary standard extracts into tubes.
3. Prepare 12 tubes (20- 150-mm) for each sample. In triplicate, place 1, 2,
3, and 4 ml of diluted sample extracts into tubes.
4. Add water to each tube for a total volume of 5 ml. Add 5.0 ml of folic-acid-
free double-strength basal medium (reagent 58) to each tube.
5. Cover tubes to prevent bacterial contamination. Autoclave 5 min at 121
123. Cool tubes as rapidly as possible to <40 to minimize browning reactions
between amino acids and sugars in the basal medium, which darken color and
reduce availability of essential amino acids, and to assure against injury to
inoculum in step 6. Treat all standards and samples the same.
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Total Folate in Cereal ProductsMicrobiological Assay Using

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6. Aseptically inoculate each tube, except for one set of triplicate blank tubes
(containing 0 ml standard extract), with 50-l drop of working inoculum deliv-
ered from a sterile syringe fitted with a long needle, assuring that drop falls onto
surface of tube contents. Angle syringe slightly to assure that complete drop
reaches surface.
7. Incubate at 37 for 22 hr.
8. Add one drop antifoam to each tube and mix. Place uninoculated 0-ml tubes
(should be clear, or else tubes were dirty or sterility was not attained) in pho-
tometer set to 550 nm and adjust to 100% T when steady state is achieved. Read
%T on remaining tubes at steady state. If %T for inoculated blank is less than
72.5, discard run. High inoculated blank (low %T) is indicative of contamination
of basal medium or culture. If 5.0-ml standard level is greater than 50% T, dis-
card run. (High %T indicates inadequate growth).

Optional 96-Well Microtiter Plate Assay

See Note 7.
1. Using 0.22-m filter system, filter sterilize a sufficient quantity of pH 6.8
phosphate buffer (reagent 45) and double-strength basal medium (reagent 58),
i.e., 2.5 ml of each solution per sample plus 70 ml extra.
2. Using 12-channel pipetter, pipet 150 ul of pH 6.8 buffer to wells A1H12 of
a sterile microtiter plate.
3. Pipet 150 ul standard extract (from Sample Hydrolysis and Extraction
above) to wells G1G2.
4. For each sample extract (from Sample Hydrolysis and Extraction above),
pipet 150 ul in duplicate into wells in row G, i.e., 150 ul of sample 1 extract to
each well G3 and G4, 150 ul of sample 2 extract to G5 and G6, etc., up to G12.
(Therefore, you can run five sample extracts and one standard extract per
microtiter plate.)
5. Mix contents of each well to homogeneity by using a 12-channel pipetter
and repeating aspiration and delivery steps.
6. Make serial dilutions (2) of standard and samples by transferring 150 ul
from wells G1G12 to F1F12 and mixing, then from F1F12 to E1E12 and
mixing, etc. For samples A1A12, withdraw 150 ul from each well after mixing
and discard.
7. Add 1 ul working inoculum per 1 ml folic-acid-free basal medium (reagent
58). Mix well. See Note 8.
8. Add 150 ul inoculated basal medium to wells A1H12. (H is the inoculated
blank row.)
9. Put plate into a plastic bag and seal. Incubate 22 hr at 37. Place pan of
water in incubator to assure adequate humidity in incubator to inhibit evaporation
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of water from outer wells. Remove plate and allow to stand 30 min to equili-
brate to room temperature.
10. Mix contents of each well using a 12-channel pipetter by repeating aspira-
tion and delivery steps until bacterial suspension becomes homogeneous.
11. Read optical density of all wells on microtiter plate reader at 595 or 600
nm using inoculated blanks (H1H12) as reference blanks.

Calculations
Basic method
1. Prepare standard concentration response curve by plotting average %T
reading for each level of standard solution used against amount of standard folate
contained in the respective tubes.
2. Determine amount of folate per ml for each sample tube by interpolation
from standard curve. Discard any %T value for samples equivalent to <0.5 or
>4.5 ml of standard solution.
3. Calculate concentration of folate in extract solution for each sample. Aver-
age the values obtained and calculate average 10%. Accept tubes within the
range. If number of acceptable values is >2/3 of original number of tubes used in
the four levels of sample assay solution, calculate folate content in original sam-
ple from average of acceptable tubes.
4. Alternatively, computer programs designed and validated for the calculation
can be used.

Optional Microtiter Plate Reader

Reduce data by plotting results and determining folate concentration in wells
as above, or use appropriate commercial data analysis software. Average results
of duplicates.

Notes
1. Folates are light and oxygen sensitive. Use of yellow lighting and low
actinic glassware is recommended. Preparation and storage of samples under
subdued lighting is essential.
2. For quality assurance, glassware must be low-actinic and must be cleaned
meticulously and heated 12 hr at 250 to destroy any folic acid residues present.
Folic acid should be stored in a desiccator prior to preparation of stock standard
solution. Potential sources of error are shown in Table II.
3. For some plate reader systems, blackwall microtiter plates may be necessary to
assure against deviations in readings between edge-row wells and center-row wells.
4. Caution. Ammonium hydroxide, hydrochloric acid, and potassium or so-
dium hydroxide are extremely caustic and can cause severe burns. Protect skin
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TABLE II
Potential Sources of Error
Cause Type of Resulta
Incomplete wetting of sample at extraction L, random
Sample splattered during mixing L, random
Incorrect preparation of extraction buffers L, bias
Weighing error H/L, random
Balance out of calibration H/L, bias
Air bubbles in diluter L, random
Diluter not in calibration L, bias
Failing power board on spectrophotometer H/L, random
pH electrode calibration error L, bias
Sample spilled L, random
Stress on assay organism L, bias
Incorrect assay medium preparation L, bias to no growth in assay tubes
a A random error can be high (H) or low (L) but of indeterminate magnitude. A high or low bias
affects every individual analysis in the same way, at the same magnitude.

and eyes. When using flammable liquids, perform operations behind a safety
barrier when using steam or electric mantle heating. Use an effective fume
removal device to remove flammable vapors as produced. Leave ample head-
room in flasks, and add boiling chips before heating is begun. All controls,
unless vapor sealed, should be located outside of vapor area. For toxic liquids,
use an effective fume removal device to remove vapors as produced. Avoid
contact with skin.
5. Filtered solutions can be set aside in the dark at 4 overnight.
6. Use of automatic pipetting machine (apparatus 11) is recommended.
7. To assure against contamination of sterile microtiter plates and solutions,
assure that bench area is clean and not susceptible to airborne contaminants.
Alternatively, use a biosafety hood, if available, as this will reduce the risk of
contamination.
8. A minimum of 15 ml inoculated media (i.e., 15 ul inoculum in 15 ml
medium) is needed per plate (i.e., one standard and five samples). Prepare
amount needed plus 70 ml extra.

Reference
DeVries, J. W., Keagy, P. M., Hudson, C. A., and Rader, J. I. 2001. Collaborative study on
determination of total folate in cereal products by microbiological assay using trienzyme
extraction (AACC Method 86-47). Cereal Foods World 46:(in preparation).