Vitamin B9 Test Kit(Microbiological microwell plate)
REAGEN™Sample and Folic acid culture medium add to the 96-microwell which has
the Lactobacillus Rhamnose subsp. lactis （leichmannii）,if the sample has vitamin B9
the Lactobacillus Rhamnose will grow,after culture 37℃,44-48h, read the OD by microplate
reader 610 - 630 nm (or540 - 550 nm),The sample of the turbidity compared with
standard curve can get folic acid content.
REAGEN™ Folic acid Test Kit is quantitative analysis of total Folic acid in food ,feed and drug.The unique features of the kit are:
§rapid, and cost-effective extraction methods,the same as other Vitamin
§The kit provide positive control and serial standards
§High sensitivity (0.05μg/100g) and low detection limit (0.0125μg/100g)
Kit Contents, Storage and Shelf Life
REAGEN™ Folic acid Test Kit has the capacity for 96 determinations or testing of 42
samples in duplicate (assuming 12 wells for standards). Return any unused microwells
to the foil bag and reseal them with the desiccant provided in the original package. Store
the kit at 2-8℃ .The shelf life is 6 months when the kit is properly stored.
Vitamin B9 Microwell Plate
1 x 96-well Plate (8 wells x 12 strips)
Vitamin B9 Culture mediu
Vitamin B9 standard
Required Materials Not Provided With the Kit
§Laminar flow bench
§Microtiter plate reader：610 - 630 nm （540 - 550 nm）
§Water bath 95℃
§Sterile Pipette 20-200ul,100-1000ul
§Sterile centrifuge tubes (1.5-2ml ,15 and 50 mL)
§The 500 mL glass bottles and flasks (100 and 1000 mL), 100 mL beaker
§Sterile syringe, 0.2 um sterile membrane
§Sterile water or deionized water
§NaOH：2mol/L，take 8g NaOH add 100ml Sterile water or deionized water
§NaOH：1mol/L and 0.1mol/L
§Phosphate buffer（0.05 mol/L，pH7.2）,add sodium dihydrogen phosphate 7.8 g in 1L sterile water or deionized water, adjust pH 7.2, buffer needs on the day of preparation
§The pig pancreatic enzyme
§Chicken pancreatic enzyme
Add the Folic acid detection solid fortified food, need hot water extracted ;Add Folic
acid test liquid samples should be in sterile filtration and sterile water diluted for testing.The total Folic
acid content in samples, the samples must be using enzymes for processing.Samples should be stored at
4 ℃ avoid light.Standard and samples shall be in triplicate.Samples of unknown samples should be extract
twice dilution.Take liquid should day using samples, and should be stored away from light.
The Sample Extract Preparation
Add 1g sample to 40 ml sterile water or deionized water or extract solution ,the diluted multiples is 40.Diluted multiples directly included in the standard curve.For the lower concentration of vitamin samples, sample quantity should be increased to 5 g (mL) (results should be taken into consideration).The following sample must be sterile filtered or sterile extraction：
1) The sample extraction without heating, such as juice or health drinks (in 95 ℃ water bath heating except for 30 min sample).
2) Samples containing traditional Chinese medicine (TCM) and seasoning and honey and tea
3) Vitamin mixture, premix or tablets (sample higher levels of vitamin B9) (in 95 ℃ water bath heating except for 30 min sample).
4) The vitamin content low dark sample (filtration process can remove color).
5) If the sample contains solid particles or sample turbidity effect filter, in front of the sterile filtration process should be in more than 8000 g centrifugal 5 min.
Note: if the sample in the 95 ℃ heating 30 min, do not need to be sterile filtration.But the sample extract kit provides no bacteria of water must be used for dilution.
The Sample Extract Diluted
1) Dilution ratio calculation
To a concentration of 160μg/100g as an example of a solid sample, with the concentration of divided by the concentration of the standard 2 get diluted multiples.
Dilution multiples = 160 u g / 0.4 u g = 400( or about 400).
So we know the sample need to dilute 1:400 with sterile water.
2) Dilution steps
A)1：10 (100μl sample extraction solution + 900μl sterile water)
B) 1：10(100μl A + 900μl sterile water)
C) 1:4 (250 μl B + 750μl sterile water)
Note: Each step dilution need fully blending and then take the next step diluted, extract the diluent should now match with now.
The Sample Processing
1) Liquid Samples
Add 1ml sample to 50ml sterile centrifuge tupe,add 40ml 1x sample extraction buffer,vortex,sterile filtration.(or 95℃ water bath 30mins,quickly cool to below 30℃).If the concentration is high,dilute in the 1.5-2ml sterile tupe.
2) Pectinose and Sweets
Take 15 to 20 g pectinose or sweets to 50 ml sterile centrifuge tube, add about 40 ml sterile water or deionized water, dissolve the sample in 95 ℃ water bath.Quickly cooling below 30 ℃, with sterile water or deionized water transferring quantitative extraction solution into 100 ml flask, with sterile water or deionized water constant volume.Take about 1 g sample extraction solution to 50 mL sterile centrifuge tube, fill with sterile water or deionized water to 40 mL, oscillation, sterile filtration (or the sample in 95 ℃ water bath heating 30 min, and then quickly cooled to below 30 ℃). If the concentration of Folic acid is high,dilute in the 1.5-2ml sterile tupe.Such as pectinose samples of 17 g , then transferred to the solution of sterile centrifuge tube (1 g sample corresponding to extract solution) for volume: 100 mL/17g = 5.88 mL/g
3) Capsules, pills and vitamin mixture
The first ,determine the weight of each capsule or pill (weighing 5 capsules or pills, average), and then put the pills in a mortar or blender crush (capsules can be extracted directly when cut) .
n1 g sample preparation and extraction
Take 1 g pills, vitamins mixture or cut the capsule to 500 ml glass bottles, add about 400 ml phosphate buffer (0.05 mol/L, pH7.2),shake .In 95 ℃ water bath extraction 30 min, there should be at least 5 times, and then rapidly cooling below 30 ℃.With sterile water or deionized water transferring extraction solution to 1000 ml flask and constant volume with sterile water or deionized wate.Transferring extraction solution 1 mL to 50 mL sterile centrifuge tube, fill with sterile water or deionized water to 40 mL, oscillation, sterile filtration (or the sample in 95 ℃ water bath heating 30 min, and then quickly cooled to below 30 ℃).If the concentration of Folic acid is high,dilute in the 1.5-2ml sterile tupe.
Note: the results of calculation should be considered when diluted multiples is1000, dilution steps will fill 1 ml to 40 ml has been included in the standard curve.
n0.2 g sample preparation
Take 0.2 g pills, vitamin mixture or cut the capsule to 50 ml sterile centrifuge tube, add about 30 ml phosphate buffer (0.05 mol/L, pH7.2), shake, and fill phosphate buffer to 40 ml.In 95 ℃ water bath extraction 30 min, there should be at least 5 times (make sure that the centrifugal tube seal), and then quickly cooling below 30 ℃.Centrifugal 5 min under greater than 8000 g, if the concentration of Folic acid is high,dilute in the 1.5-2ml sterile tupe.
4)Grain, baby food, bread, flour and dairy products
Take 1 g sample to 50 ml sterile centrifuge tube, add 40 ml phosphate buffer (0.05 mol/L, pH7.2),shake.During the 95 ℃ water bath extraction in 30 min, should be at least 5 times (make sure that the centrifugal tube seal), and then quickly cooling below 30 ℃.Using samples of 0.2 um sterile membrane filtration to 1.5 or 2.0 mL sterile reaction tube.According to the folic acid concentration range, then use of sterile water to filtrate further dilution.
5)Total Folic acid
1g (mL)sample and 20 mg pig pancreatic enzyme (grain, vegetables, fruits, yeast, the yeast products and liver recommended 10 mg of Chicken pancreatic enzyme) to a homogeneous sample 50 mL centrifuge tube, with about 30 mL phosphate buffer (0.05 mol/L, pH 7.2), shake well, add phosphate buffer to 40 mL.2 hours at 37 ° C under the condition of dark incubation (oscillation) from time to time.Grains and the liver We recommend that the incubation time not less than 12 hours or overnight.Later, in 95 ° C water bath extraction for 30 minutes.In extraction tube oscillation must be at least five times, and ensure the test tube is closed, rapid cooling to under 30 ° C.Under the condition of centrifugal (greater than 8000 g centrifugal 5 minutes), according to the concentration range, determine whether in 1.5 mL (2.0 mL) or sterile tubes on further diluted solution..
All the reagents need to return to room temperature.shake the reagents before use. Don't pollute the kit’s reagents.
Add 2 ml sterile water to standard , cover cap and shake points dissolved with standard
is the standard concentrate 2.4ug/100g. Filter the standard concentrate to 15ml tube by 0.2um
sterile filter Prepare standard: Sterile filtration the concentrate standard.
standard curve μg/100g(ml)
Concentrate standard ul
Total volume ul
400ul standard concentrate
Vitamin B9 Culture medium preparation
Use tweezers to take out the desiccant medium cap and open and discarded.
Add 10 ml sterile water to Culture medium,then lid and mix.
Water bath 95℃ 5mins and shake some times,quickly cool to room temperature.
Filter the culture medium to 15ml tube by 0.2um sterile filter.
1. All the protocols’ water must be sterile
2. Get out some wells ,put back the left wells to the foil bag and seal.
3. Get 150ul standards or samples to the wells
4. Add 150ul culture medium to the wells
5. Get out Aluminum foil paper ,remove the protect layer of the foil paper,seal the wells By Pressing the foil paper on the wells(must be sealed).
6. Incubation 37℃,44-48h,in dark.
7. Slowly take off the foil paper from the plate wells.not let the solution splash.Mix each well with pipette tips and let the Microorganism dissolve in the culture medium.
8. Read OD value by micro plate reader 610-630nm(or 540-550nm) .
Notice:It is better read the OD after incubation. This plate can be store at 2-8℃ just for 48h but not open the foil paper.
Blank well OD
STANDARD 5 OD>0.6
Standard curve :vertical coordinate: OD;Horizontal coordinate :concentration.
Read the sample concentration on the curve. If extra dilute the sample ,Multiply the dilution factor.
Warnings and Precautions
1)Kit should be 2-8 ℃ storage, do not use when expired.
2) To join in microwell plate sample extract or diluent must be sterile, other consumables for the experiment must also be sterile.
3) Test medium can stimulate eye, skin and mucous membrane, should be used with caution.
4) After the completion of the test should be carried out in accordance with the regulations on waste treatment (such as high pressure sterilization).
5) Enzyme during sample preparation should be a blank,make sure the folic acid reagent (enzyme) will not affect the final result.