Vitamin B12 Test Kit(Microbiological microwell plate)
REAGEN™Sample and Vitamin B12 culture medium add to the 96-microwell which
has the Lactobacillus delbrueckii subsp. lactis （leichmannii）,if the sample has vitamin
B12 the Lactobacillus delbrueckii 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 Vitamin B12 content.
REAGEN™ Vitamin B12 Test Kit is quantitative analysis of total Vitamin B12 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 (0.06μg/100g)
§ High recovery（90%-105%）
§ High reproducibility
Kit Contents, Storage and Shelf Life
REAGEN™ Vitamin B12 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 12 months when the kit is properly stored.
Vitamin B12 Microwell Plate
1 x 96-well Plate (8 wells x 12 strips)
Vitamin B12 Culture mediu
Vitamin B12 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
§HCl：1mol/L and 0.1mol/L
§NaOH：1mol/L and 0.1mol/L
§NaCNor KCN：1%，100mg NaCN or KCN add 10ml sterile water
§Amylase：z. B. Fluka 86250
§Acetate buffer （pH4.5）,Add 0.66 g anhydrous sodium acetate to 100 mL beaker , add 50 mL sterile water or deionized water, stirring edge to dissolve, then add 0.69 mL of acetic acid (> 99%, such as j. t. Baker, 6052), the soluble liquid pH is 4.5, 0.1 mol/L HCl correction if necessary;Transfer buffer quantitative to 100 ml volumetric flask, with sterile water or deionized water constant volume.2-8 ℃ can store 1 week.
Add the Vitamin B12 detection solid fortified food, need hot water extracted ;Add Vitamin
B12 test liquid samples should be in sterile filtration and sterile water diluted for testing.The
total Vitamin B12 content in samples, the samples must be using NaCN/kau and enzyme
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 B12 samples, sample quantity should be increased to 5 g (mL)
(results should be taken into consideration).The following sample must be sterile filtered or
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 B12) (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 1.2μg/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 = 1.2 u g / 0.12 u g = 10.
So we know the sample need to dilute 1:10 with sterile water.
2) Dilution steps
A)1：10 (100μl sample extraction solution + 900μl 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 Vitamin B12 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
1) 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 sterile water or deionized water and add 500ml 1% NaCN,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, shake, sterile filtration (or the sample in 95 ℃ water bath heating 30 min, and then quickly cooled to below 30 ℃).If the concentration of Vitamin B12 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
20 ml sterile water or deionized water and add 500ml 1% NaCN, shake, and fill sterile water 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 Vitamin B12 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 sterile water or deionized water ,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 Vitamin B12 concentration range, then use of sterile water to filtrate further dilution.
5) Total Vitamin B12
Take 1 g (mL) samples to 50 mL sterile centrifuge tube, add 20 mL sterile water or deionized water and 250 u L 1% NaCN solution , shake,adjust the pH to 4.5 with HCl (or using acetate buffer instead of sterile water extract samples, add 1 g sample to 20 mL pH4.5 acetate buffer, shake and mixed, then add 250 mu L NaCN solution, need not adjust pH).Add 300 mg amylase, shake, 37 ℃ avoid light 1 h incubation, intermittent shake at the same time.Fill with sterile water or deionized water to 40 ml, 30 min, in 95 ℃ water bath heating 30 min,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, according to the
concentration range of vitamin B12, at 1.5 or 2.0 mL sterile sterile water used in the reaction tube of supernatant further dilution.
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.
Prepare standard: Sterile filtration the concentrate standard.
standard curve μg/100g(ml)
Concentrate standard ul
Total volume ul
Vitamin B12 Culture medium preparation
Use tweezers to take out the desiccant medium cap and open and discarded.
Add 10 ml sterile water to Culture medium,lid and mix
Water bath 95℃ 5mins and shake some times,quickly cool to room temperature.
Filter the culture medium to 15ml tupe 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 culture medium to the wells
4. Add 150ul standards or samples 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. Shake the plate and let the Microorganism dissolve in the culture medium.(or press the foil paper and reverse the plate on the desk ,shake to dissolve the Microorganism)
8. Slowly take off the foil paper from the plate wells.not let the solution splash.
9. Break all the foams in the wells by Pipette tips.
10. 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 1 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.Preparation reagent (standard and Culture medium )should be used directly, should be discarded after the completion of the testing.
2) To join in microwell plate sample extract or diluent must be sterile, other consumables for the experiment must also be sterile.
NaCN and kau is highly toxic substances, therefore using NaCN or kau to extract the sample should be done in exhaust hood;Should be used according to the country or local regulations and handle NaCN or kau.
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).