In Vitro Batch-Culture Model to Mimic Colonic Fermentation by Human Gut Microbiota

Abstract

Source: Ahmadi, S., et al. An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota. J. Vis. Exp. (2019).

This video demonstrates a method for modeling gut microbial fermentation by incubating fecal samples with dietary fermentable fiber under anaerobic conditions to study metabolite production by gut microbiota.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

CAUTION: Consult the appropriate Material Safety Data Sheets and follow the instructions and guidelines for appropriate Biosafety Level 2 (BSL-2) training. Follow all the culturing steps as per the standard biosafety rules and use a BSL-2 cabinet using aseptic conditions. Furthermore, fecal samples from different models and human subjects may have a potential risk of spreading microbial-borne diseases. Immediately seek medical aid in the event of any injury or infection. In addition, the use of human and animal stool samples should be approved through institutional ethical committees and must compliant with protocols for using samples and subject information.

1. Preparation of Culture Media

1. Preparation of the culture media, prepare nine types of stock solutions:
   1. Solution A (1,000 mL): Dissolve 5.4 g of sodium chloride (NaCl), 2.7 g of potassium dihydrogen phosphate (KH₂PO₄), 0.16 g of calcium chloride dihydrate (CaCl₂·2H₂O), 0.12 g of magnesium chloride hexahydrate (MgCl₂·6H₂O), 0.06 g of manganese chloride tetrahydrate (MnCl₂·4H₂O), 0.06 g of cobaltous chloride hexahydrate (CoCl₂·6H₂O), and 5.4 g of ammonium sulfate (NH₄)2SO₄, in deionized water to make total volume to 1,000 mL.
   2. Solution B (1,000 mL): Dissolve 2.7 g Potassium Hydrogen Phosphate (K₂HPO₄) in deionized water to make total volume to 1000 mL.
   3. Trace mineral solution (1,000 mL): Dissolve 500 mg of disodium ethylenediamine-tetraacetate dihydrate (Na₂EDTA), 200 mg of ferrous sulfate heptahydrate (FeSO₄·7H₂O), 10 mg of zinc sulfate heptahydrate (ZnSO₄·7H₂O), 3 mg of manganese(II) chloride tetrahydrate (MnCl₂·4H₂O), 30 mg of phosphoric acid (H₃PO₄), 20 mg of CoCl₂·6H₂O, 1 mg of cupric chloride dihydrate (CuCl₂·2H₂O), 2 mg of nickel(II) chloride hexahydrate (NiCl₂·6H₂O) and 3 mg of sodium molybdate dihydrate (Na₂MoO₄·2H₂O) in deionized water to make total volume to 1,000 mL.
      NOTE: This solution is light sensitive, therefore ensure to be stored in dark/black or aluminum wrapped tubes/bottles.
   4. Water soluble vitamin solution (1,000 mL): Dissolve 100 mg of thiamine hydrochloride (Thiamin-HCl), 100 mg of D-pantothenic acid, 100 mg of Niacin, 100 mg of pyridoxine, 5 mg of P-aminobenzoic acid, and 0.25 mg of Vitamin B12 in deionized water to make a total volume to 1,000 mL.
   5. Folate: biotin solution (1,000 mL): Dissolve 10 mg of folic acid, 2 mg of D-biotin, and 100 mg of ammonium bicarbonate (NH₄HCO₃) in deionized water to make a total volume to 1,000 mL.
   6. Riboflavin solution (1,000 mL): Dissolve 10 mg of Riboflavin in 5 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (1.19 g/L) solution to make a total volume to 1,000 mL.
   7. Hemin solution (10 mL): Dissolve 5,000 mg of Hemin in 10 mM sodium hydroxide (NaOH) (0.4 g/L) solution to make total volume to 10 mL.
   8. Short-chain fatty acid mix (10 mL): Combine 2.5 mL of N-valerate, 2.5 mL of isovalerate, 2.5 mL of isobutyrate, and 2.5 mL: DL-α-methylbutyrate.
      NOTE: This solution is recommended to use ina fume hood to avoid smell and fumes.
   9. Resazurin (1,000 mL): Dissolve 1 g of resazurin in deionized water and make the total volume to 1,000 mL.
2. Medium used for in vitro anaerobic fermentation
   1. To prepare this media, mix 330 mL of Solution A, 330 mL of Solution B, 10 mL of Trace mineral solution, 20 mL of Water soluble vitamin solution, 5 mL of Folate: biotin solution, 5 mL of Riboflavin solution; 2.5 mL of Hemin solution, 0.4 mL of Short chain fatty acid mix, 1 mL of Resazurin, 0.5 g of yeast extract, 4 g of sodium carbonate (Na₂CO₃), 0.5 g of Cysteine HCl-H₂O, and 0.5 g of Trypticase, and add 296.1 mL of distilled water.
   2. Check the pH and ensure it is around 7.0; if not, adjust the pH with 1 N HCl or 1 N NaOH. Sterilize by vacuum filtering the media using a bottle filter under the aseptic workstation.
   3. Alternatively, mix all the components (except vitamin and hemin solutions) and autoclave at 121 °C for 20 min and let it cool down to room temperature. Simultaneously, filter-sterilize vitamin and hemin solutions using 0.22 µm membrane filters and add these to the autoclaved and cooled media before dispensing.

2. Preparation of Anaerobic Chamber and Required Materials

1. Keep all the materials, solutions, and tools needed for the fermentation experiment inside the anaerobic chamber at least 48 h before the start of the experiment, to ensure that any residual oxygen associated with tools and soluble oxygen in buffers/solutions is removed and all the materials are acclimatized to the set anaerobic conditions.
   NOTE: Materials needed inside the anaerobic chamber (48 h earlier of experiment to start): (i) fermentation media; (ii) anaerobic solution (prepared according to section 4.1), (iii) vortexer, (iv) weighing balance, (v) muslin cheese cloths, (vi) scissors, (vii) funnel, (viii) 1.5, 2.0, 15, 50 mL tubes, (ix) pipettor (2, 20, 200 and 1,000 µL) and compatible pipet tips, (x) gun pipet and pipets (5 and 10 mL), (xi) paper tissues, (xii) markers, (xiii) tube stands for different tubes, (xiv) waste box, (xv) O2 indicator and (xvi) 70% ethanol spray bottle (disinfectant).

3. Preparation of Tubes and Fibers

1. Weigh 300 mg of inulin and transfer to a 50 mL tube, followed by the aseptic addition of 26 mL of fermentation media already prepared and stored in the anaerobic chamber. Prepare one blank tube for each fecal sample type and experimental tube(s) (according to the number of compounds being tested), in triplicate.
2. Leave these tubes inside the anaerobic chamber for around 24 h to allow hydration of samples before starting the fermentation experiment. Ensure the tube temperature is 37 °C at the time of inoculation, therefore bring the tubes in the incubator enclosed within the anaerobic chamber.

4. Preparation of Inoculum

1. Anaerobic dilution solution (at least 48 h before fermentation experiment): Dissolve 5 g of NaCl, 2 g of glucose and 0.3 g of Cysteine-HCl in deionized water and make total volume to 1,000 mL. Autoclave and store it inside the anaerobic chamber at least 48 h before use.
2. Fecal inoculum preparation (at the day of fermentation experiment): Weigh 5 g of fresh fecal sample in a 50 mL conical tube, add anaerobic dilution solution for a final volume of 50 mL (1:10 w/v) and vortex for 15 min or until completely homogenized. Filter the homogenized mixture through 4 layers of sterile cheesecloth (autoclaved) and use it immediately for inoculation in the tubes containing media.
   NOTE: The fecal samples from a group of subjects can be pooled if the experimental objective is to compare the effect of a given compound/ingredient on overall healthy versus diseased fecal microbiota in general.

5. Fermentation and Sampling

1. Prepare tubes according to the section 4.2 and inoculate blank/control and experimental tubes with 4 mL of diluted and filtered fecal inoculum. Incubate the inoculated tubes at 37 °C inside the anaerobic chamber. Shake the tubes once every hour by inverting gently to re-suspend the fibers and inoculum.
2. Collect samples as frequently as needed, for example, hourly to 0, 3, 6, 9, and 24 h during fermentation by taking a 2 mL aliquot sample out into a 2 mL tube from respective fermenting tubes.
3. Measure the pH of the aliquots using a laboratory pH meter (by directly inserting the pH electrode in the sample); centrifuge the remaining sample at 14,000 x g for 10 min at 4 °C. Immediately freeze the supernatant and pellet in liquid nitrogen, and after snap freezing, store the supernatant for SCFAs (short-chain fatty acids) analysis and pellet for microbiome analysis at -80 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium bicarbonate	Sigma-Aldrich	217255
Ammonium sulfate	TGI	C2388	Toxic
Calcium chloride dihydrate	Sigma-Aldrich	C3306	Irritating
Cobaltous chloride hexahydrate	Sigma-Aldrich	255599
Cupric chloride dihydrate	Acros organics	2063450000	Toxic, Irritating
Cysteine-HCl	Sigma-Aldrich	C121800
D-biotin	Sigma-Aldrich	B4501
D-Pantothenic acid	Alfa Aesar	A16609
Disodium ethylenediaminetetraacetate dihydrate	Biorad	1610729
DL-α-methylbutyrate	Sigma-Aldrich	W271918
Ferrous sulfate heptahydrate	Sigma-Aldrich	F8263	Toxic
Folic acid	Alfa Aesar	J62937
Glucose	Sigma-Aldrich	G8270
Hemin	Sigma-Aldrich	H9039
Hepes	Alfa Aesar	A14777
Isobutyrate	Alfa Aesar	L04038
Isovalerate	Alfa Aesar	A18642
Magnesium chloride hexahydrate	Sigma-Aldrich	M8266
Manganese chloride tetrahydrate	Sigma-Aldrich	221279
Niacin (Nicotinic acid)	Sigma-Aldrich	N4126
Nickel(Ii) chloride hexahydrate	Alfa Aesar	A14366	Toxic
N-valerate	Sigma-Aldrich	240370
P-aminobenzoic acid	MP China	102569	Toxic, Irritating
Phosphoric Acid	Sigma-Aldrich	P5811
Potassium dihydrogen phosphate	Sigma-Aldrich	P5504
Potassium hydrogen phosphate	Sigma-Aldrich	1551128
Pyridoxine	Alfa Aesar	A12041
Resazurin	Sigma-Aldrich	R7017
Riboflavin	Alfa Aesar	A11764
Sodium carbonate	Sigma-Aldrich	1613757
Sodium chloride	Fisher BioReagents	7647-14-5
Sodium hydroxide	Fisher Chemicals	S320
Sodium molybdate dihydrate	Acros organics	206375000
Thiamine hydrochloride	Acros organics	148991000
Trypticase	BD Biosciences	211921
Vitamin B12	Sigma-Aldrich	V2876
Yeast extract	Sigma-Aldrich	70161
Zinc sulfate heptahydrate	Sigma-Aldrich	Z0251
0.22 µm membrane filter
AMPure magnetic purification beads	Agencourt
Anaerobic chamber with incubatore	Forma anaerobic system, Thermo Scientific, USA
Bottle filter	Corning
Cheesecloth
pH meter
Vortex	Thermoscientific