Mega + Minimal medium

Overview

Buy this bundle if your project is:

Building a personalised human gut-microbiota culture collection from stool, mucosal scrapings, or biopsy material — and you want the broadest single-broth taxon recovery available. (Goodman et al. 2011 PNAS reported that the GMM recipe underlying Mega Medium 1.0 recovered the majority of OTUs detected by 16S rRNA sequencing across multiple human donor stool samples; reported recovery rates vary by donor, sequencing depth, and downstream isolate-picking strategy — see the source paper for the exact donor-stratified figures.)
Following Stage 1 culture-collection work with substrate-utilisation profiling, growth-factor-dependency screens, or gene-knockout phenotyping — and you need a defined-condition medium that shares the Mega ionic background, so phenotype differences are not confounded by a medium switch.
Running a microbiome–drug-interaction screen where the isolate panel was built in Mega Medium and the phenotyping must be done in a defined matrix (Maier et al. 2018 Nature; Klünemann et al. 2021).

Both kits ship together because the workflows are inseparable — one without the other leaves the project incomplete.

Why one medium isn't enough. Mega Medium is rich by design — five carbohydrates, four SCFAs, hematin + menadione, full vitamin and trace-mineral mixes, resazurin redox indicator — so the broadest taxon set possible has a substrate it can use. The same richness, however, defeats single-variable phenotyping: you cannot ask "does this isolate use xylose?" or "does this knockout grow without vitamin B₁₂?" when every isolate has access to ten substrates at once. Minimal Medium answers those questions on the same K-phosphate / Mg²⁺ / Ca²⁺ / Fe²⁺ / hematin / menadione / cysteine background, with no carbohydrates, no SCFAs, no peptone, only ammonium as N source. You add the one variable you want to test. Growth differences reflect substrate biology, not medium-switching artefacts. Manufactured as a pair from the same supplier lots, the bundle preserves that reproducibility — splitting it would not.

How GMExpression packages the bundle. The kit follows the GMExpression house standard (refined on YCFA Modified and YCFA Full): components that share heat-, light-, oxygen-, and pH-stability requirements travel together; components that would degrade each other are separated. You receive 16 bottles in one box:

Mega Medium kit (12 bottles): three pre-weighed dry mixtures (A — peptone / sugar / salt base; B — NaHCO₃ + L-cysteine reducing pack; C — K-phosphate buffer salts) plus nine sterile liquid stocks (mixed SCFAs; vitamin supplement; trace mineral supplement; six individual amber vials for resazurin, Tween 80, CaCl₂, vitamin K, FeSO₄·7H₂O, and Histidine-Hematin).
Minimal Medium kit (4 additional bottles): one dry Mixture A_min (KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine) plus three Minimal-specific stocks — Stock K_min (a second FeSO₄·7H₂O vial, because Stock K only holds 4 weeks once opened), Stock M_min (MgCl₂ — sulfate-free Mg²⁺ for sulfate-utilisation studies), and Stock N_min (Vitamin B₁₂ alone, for defined-vitamin growth tests where Mega's full Wolfe/Wolin Stock E is too complex).
Shared stocks: Stocks I (CaCl₂), J (Vitamin K, menadione in ethanol) and L (Histidine-Hematin) are shipped at 2× volume to serve both Mega and Minimal preparations from a single bottle each — within their open-bottle shelf lives.

Standard scales are 10 L of each medium (Mixture A ≈ 281 g, Mixture A_min ≈ 161 g) or 15 L of each (Mixture A ≈ 421 g, Mixture A_min ≈ 242 g). Total bundle bottle count is 16 at either scale.

We also have

YCFA Modified Medium · YCFA Full Recipe · BHI-S Supplemented Broth · Modified Chopped Meat Broth (ATCC 1490) · Gut Microbiota Medium (GMM) · M2GSC Medium · TPY Bifidobacterium Medium

Package Contents

Each GMExpression Mega + Minimal Medium bundle ships as 16 pre-weighed and pre-sterilised bottles in a single box — 12 for the Mega Medium kit (three dry mixtures + nine sterile liquid stocks) and 4 additional bottles for the Minimal Medium kit. Stocks I, J and L (long open-bottle shelf life) are sized for both preparations from a single bottle each. The packaging follows the GMExpression house standard (refined on the YCFA Modified and YCFA Full kits): components that share heat-, light-, oxygen-, or pH-stability requirements are grouped into a single bottle; components that would react with or destabilise each other are kept separate. The complete bundle complement for 10 L standard is:

Bottle	Contents	10 L kit	Format & sterilisation
Mixture A	Dry peptone, sugar, salt & organic-acid base — tryptone peptone, yeast extract, meat extract, D-glucose, cellobiose, maltose, D-fructose, NaCl, Na₂SO₄, malic acid, MgSO₄·7H₂O	≈ 281 g	Dry powder, amber HDPE screw-cap bottle. Add to rehydration water; autoclave with the medium (121 °C × 15 min).
Mixture B	Reducing-substance dry mixture — L-cysteine·HCl·H₂O + NaHCO₃. NaHCO₃ is co-packaged with L-cysteine because (i) on rehydration and autoclaving the NaHCO₃ thermally decomposes to release CO₂, which displaces dissolved O₂ from the solution and creates a CO₂ headspace that retards O₂ reuptake; (ii) NaHCO₃ partially neutralises the HCl in L-cysteine·HCl, suppressing thiolate formation (R-SH protonated form is far less oxidisable than R-S⁻ at pK_a ≈ 8.3); the combined effect minimises L-cysteine → L-cystine autoxidation during preparation.	9.0 g (L-cys·HCl 5 g + NaHCO₃ 4 g)	Dry powder, screw-cap HDPE bottle. Not vacuum-packed — CO₂ release on dissolution would distend a sealed bag. Autoclave with the medium.
Mixture C	Dry potassium-phosphate buffer salts (K₂HPO₄ dibasic anhydrous + KH₂PO₄ monobasic anhydrous), pre-weighed to give 100 mM total phosphate at the customer's chosen kit scale. Reconstitute in approximately 1 L of Milli-Q water (10 L kit) or 1.5 L (15 L kit), titrate to pH 7.2 with concentrated HCl, then add to the rehydrating medium.	≈ 159.6 g (K₂HPO₄ 107.3 g + KH₂PO₄ 52.3 g)	Dry powder, HDPE bottle. Customer-reconstituted; autoclave with the medium.
Stock D	Mixed short-chain fatty acid (SCFA) bottle — single pre-blended liquid of glacial acetic, propionic, butyric and iso-valeric acids combined at GMExpression-calibrated mass ratios so that 5.8 mL of the blend delivers exactly 30 mM acetate, 8 mM propionate, 4 mM butyrate, and 1 mM iso-valerate per 1 L of final medium. (Note: the concentrations of the individual carboxylic acids within the blend are not the same as the un-blended stock concentrations; the blend is formulated as a single in-use bottle.) All four are mutually miscible carboxylic acids; the bottle pH is < 2 (self-sterile against microbial contamination).	58 mL	Amber borosilicate glass with PTFE-lined screw-cap; no autoclave (added post-autoclave to the medium); store sealed at 15–25 °C, 24 months.
Stock E	Vitamin supplement (Wolfe/Wolin-style; Goodman 2011 used the ATCC catalogue equivalent). Contains the standard B-complex panel: biotin, folate, pyridoxine·HCl (B6), thiamine·HCl (B1), riboflavin (B2), nicotinic acid, D-Ca-pantothenate, vitamin B₁₂, p-aminobenzoate, lipoic acid. Filter-sterilised (0.22 µm). Added post-autoclave, never autoclaved (heat-labile vitamins).	100 mL	Filter-sterilised aqueous; amber screw-cap glass; 2–8 °C, 12 months.
Stock F	Trace mineral supplement (Balch/Wolin-style; equivalent to the ATCC trace mineral mix). Contains Mn²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Mo⁶⁺, Se⁴⁺, W⁶⁺, B³⁺, V³⁺, Al³⁺, Mg²⁺, Ca²⁺; Fe²⁺ is supplied separately as Stock K to preserve the reduced Fe oxidation state. Filter-sterilised.	100 mL	Filter-sterilised aqueous; screw-cap glass; 2–8 °C, 12 months.
Stock G	Resazurin redox indicator, 0.25 mg/mL aqueous stock. Pink (oxidised) / colourless (reduced); confirms anaerobic conditions before inoculation.	40 mL	Filter-sterilised; amber glass; 2–8 °C, light-protected, 24 months.
Stock H	Tween 80 (polysorbate 80), 25 % v/v in Milli-Q water. Surfactant / wetting agent for lipid emulsification.	20 mL	Filter-sterilised; screw-cap glass; 2–8 °C, 24 months.
Stock I	CaCl₂ 8 mg/mL aqueous (= 72 mM anhydrous; 0.8 g per 100 mL water). Supplied separately from Mixtures A/B/C to avoid Ca₃(PO₄)₂ and CaCO₃ precipitation during storage.	10 mL	Filter-sterilised; screw-cap glass; 2–8 °C, 24 months.
Stock J	Vitamin K₃ (menadione, 2-methyl-1,4-naphthoquinone), 1 mg/mL in 95 % ethanol. Sterile by virtue of the ethanol carrier (no aqueous filtration needed). Menadione is the K₃ precursor that Bacteroides spp. and related gut anaerobes prenylate to form their endogenous menaquinones (MK-7 to MK-11), the electron carriers of the fumarate-reductase respiratory chain.	10 mL	Sterile (95 % ethanol); amber glass with PTFE cap; 2–8 °C, light-protected, 6 months.
Stock K	FeSO₄·7H₂O (heptahydrate, MW 278.01), 0.4 mg/mL in oxygen-free water (= 1.44 mM Fe²⁺). N₂-flushed at fill to preserve the reduced Fe²⁺ form against air oxidation to Fe³⁺.	10 mL	Filter-sterilised, N₂-flushed; amber glass; 2–8 °C, 4 weeks maximum once opened (Fe²⁺ oxidation).
Stock L	Histidine-Hematin solution — 1.2 mg hematin / mL dissolved in 0.2 M L-histidine. The histidine acts as an axial Fe-N ligand that solubilises hematin under near-neutral pH (replaces the NaOH-based dissolution used in BHI-S Stock H).	10 mL	Filter-sterilised; amber glass; 2–8 °C, light-protected, 8 weeks.
Minimal Medium kit — 4 additional bottles in the same box
Mixture A_min	Dry defined-base mix — KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine·HCl·H₂O. The KH₂PO₄ alone gives an acidic solution (~ pH 4.5 at 100 mM); titrate to pH 7.2 with concentrated NaOH at preparation, which converts approximately half the KH₂PO₄ to K₂HPO₄ and creates a 100 mM phosphate buffer in situ. (NH₄)₂SO₄ is the sole nitrogen source in Minimal Medium (no peptone, no yeast extract, no amino acids) — verify your strain can use ammonium; for amino-acid-auxotrophs supplement with a single defined amino acid at preparation.	≈ 161 g (KH₂PO₄ 136 g + NaCl 8.75 g + (NH₄)₂SO₄ 11.25 g + L-cys·HCl 5 g)	Dry powder, screw-cap HDPE bottle. Autoclave with the Minimal Medium preparation (121 °C × 15 min).
Stock K_min	Second FeSO₄·7H₂O vial — same composition as Stock K (0.4 mg/mL in O₂-free water, N₂-flushed). Supplied separately because Stock K only holds 4 weeks once opened; if Mega Medium and Minimal Medium are prepared more than 4 weeks apart, a fresh vial is required for the second preparation.	10 mL	Filter-sterilised, N₂-flushed; amber glass; 2–8 °C, 4 weeks once opened.
Stock M_min	MgCl₂ 9.5 mg/mL aqueous (= 100 mM). Mega Medium supplies Mg²⁺ as MgSO₄·7H₂O in Mixture A; Minimal Medium uses MgCl₂ instead to keep the medium sulfate-free — essential for sulfate-utilisation phenotyping and for studies of sulfate-reducing bacterial isolates from Mega Medium.	10 mL	Filter-sterilised; screw-cap glass; 2–8 °C, 24 months.
Stock N_min	Vitamin B₁₂ (cyanocobalamin) 0.01 mg/mL aqueous. Mega Medium supplies B₁₂ within the Wolfe/Wolin vitamin mix (Stock E) at a fixed concentration; Minimal Medium supplies B₁₂ as its own defined-concentration single-vitamin stock, enabling clean B₁₂-dependency phenotyping (e.g. for cobalamin-auxotrophic gut anaerobes).	5 mL	Filter-sterilised; amber glass; 2–8 °C, light-protected, 12 months.
Shared-stock note: Stocks I (CaCl₂), J (Vitamin K menadione in ethanol) and L (Histidine-Hematin) above are shipped at 2× the volume shown when supplied as part of this bundle (e.g. Stock I = 20 mL for the 10 L bundle, 30 mL for the 15 L bundle). One bottle of each serves both the Mega and Minimal preparations from a single source.
Accessories		10x airtight PP storage bags + matching heat-resistant rubber bands for PRAS-format dispensing of both media	Ambient
Instruction manual		A5 booklet, v1.0 — separate preparation walk-throughs for Mega Medium and Minimal Medium, paired workflow recommendations, bottle-addition order, troubleshooting	Ambient

Customisation options on request: add 1.5 % bacteriological-grade agar for solid-phase Mega Agar or Minimal Agar; omit a specific sugar or SCFA component from Mega Medium for nutritional-dependency studies; substitute porcine gastric mucin 0.5 % for mucosal-niche emulation; CMRL 1066 substitution for spiroplasma-adjacent workflows; substitute (NH₄)₂SO₄ in Minimal Medium for a single defined amino acid for N-source phenotyping.

Composition — per 1 L of prepared medium (kits ship pre-weighed for 10 L or 15 L final volume; scale linearly); per Goodman et al. 2011 Table S13 with arithmetic corrections noted inline

Mixture A — Peptone, sugar, and salt base (per 1 L)

Component	Amount per Litre	Final concentration	Function
Tryptone Peptone (pancreatic digest of casein)	10 g	1 % w/v	Peptide nitrogen; primary amino acid pool
Yeast Extract	5 g	0.5 % w/v	B-vitamins (biotin, B12, folate, niacin), purines/pyrimidines, trace minerals
Meat Extract	5 g	0.5 % w/v	Beef-muscle infusion solids; haem precursors, additional B-vitamins
D-Glucose	2 g	11 mM	Primary fermentable carbohydrate
Cellobiose (β-1,4-glucobiose)	1 g	2.9 mM	Hemicellulose-derived disaccharide; substrate for plant-fibre-fermenting commensals
Maltose (α-1,4-glucobiose)	1 g	2.8 mM	Starch-derived disaccharide; alternate substrate
D-Fructose	1 g	5.5 mM	Diet-derived monosaccharide; preferred substrate for some Lactobacillus-group and Bifidobacterium. (GMExpression Mega Medium 1.0 doses fructose at 1 g/L = 5.55 mM, computed from MW 180.16. The published Goodman et al. 2011 GMM Table S13 entry for fructose should be cross-checked against the supplementary PDF before use in regulated workflows; some lineage variants of the formula use 2 g/L = 11.1 mM.)
L-Cysteine·HCl (anhydrous, MW 157.62; per Goodman source spec)	0.5 g	3.2 mM	Reductant; final Eh < −150 mV; required by many sulfur auxotrophs. If the commercial monohydrate (MW 175.63) is substituted, 0.5 g delivers 2.85 mM — adjust dose to 0.557 g to keep 3.2 mM
NaHCO₃	0.4 g	4.8 mM	CO₂-bicarbonate buffer; supports microaerophile / capnophile growth
NaCl	0.08 g	1.37 mM	Trace Na⁺ for ion balance
Na₂SO₄	2 g	14.1 mM	Sulfate source for sulfate-respiring anaerobes (Desulfovibrio)
Malic Acid	1 g	7.5 mM	TCA-cycle intermediate; alternate electron acceptor; supports fumarate-respirers
MgSO₄·7H₂O	0.002 g	0.008 mM	Mg²⁺ trace; ribosomal stability cofactor (much higher Mg²⁺ arrives via the trace mineral mix)

Mixture B — Buffer (added to Mixture A pre-autoclaving)

Component	Amount / L	Final concentration	Function
Potassium Phosphate Buffer 1 M, pH 7.2	100 mL	100 mM total phosphate	Primary buffer (pK_a2 7.21); maintains pH 7.0–7.4 throughout fermentation

The 1 M K-phosphate buffer pH 7.2 stock is composed of: 10.731 g of K₂HPO₄ (dibasic, anhydrous; MW 174.18) + 5.225 g of KH₂PO₄ (monobasic, anhydrous; MW 136.09) dissolved in Milli-Q water to 100 mL final. The natural pH of this mass ratio is approximately 7.4 (Henderson–Hasselbalch with pK_a2 = 7.21 and a K₂HPO₄ : KH₂PO₄ molar ratio of 1.60); titrate to pH 7.2 ± 0.05 with concentrated HCl. After titration to pH 7.2 the HPO₄²⁻ : H₂PO₄⁻ equilibrium shifts to ~ 1:1 (49 mM : 51 mM); at 10× dilution into the broth this gives 100 mM total phosphate, before further pH equilibration with the SCFA additions.

Sterile post-autoclave stock additions

Component	Stock concentration	Volume added per Litre	Final concentration	Notes
CaCl₂	0.8 g per 100 mL (= 8 mg/mL anhydrous; 72 mM)	1 mL	72 µM (= 0.072 mM)	Adsorption / signalling cofactor
Vitamin K₃ (menadione)	1 mg/mL in 95 % ethanol	1 mL	1 mg/L (5.8 µM)	K₃ precursor; prenylated by Bacteroides to endogenous menaquinones for fumarate-reductase respiration; light-protected
FeSO₄·7H₂O (heptahydrate, MW 278.01)	0.4 mg/mL (= 1.44 mM Fe²⁺ in deoxygenated water)	1 mL	1.44 µM Fe²⁺	N₂-flushed; prepare fresh in O₂-free water; do not autoclave (Fe²⁺ → Fe³⁺ oxidation)
Histidine-Hematin Solution	1.2 mg hematin/mL in 0.2 M L-histidine	1 mL	1.2 mg/L hematin	Histidine solubilises hematin via axial Fe-N coordination (replaces NaOH dissolution)
Tween 80	25 % v/v stock	2 mL	0.05 % v/v	Wetting agent; lipid emulsification for membrane-lipid auxotrophs
ATCC Vitamin Supplement (or equivalent Wolfe/Wolin-style vitamin mix)	1× concentrate	10 mL	1 % v/v	Typical vitamin panel: biotin, folate, pyridoxine·HCl (B6), thiamine·HCl (B1), riboflavin (B2), nicotinic acid, D-Ca-pantothenate, vitamin B₁₂, p-aminobenzoate, lipoic acid. Confirm composition against the supplier's current Certificate of Analysis — the Goodman 2011 protocol used the ATCC catalogue vitamin supplement; equivalent Wolfe / DSMZ vitamin mixes are functionally interchangeable.
ATCC Trace Mineral Supplement (or equivalent Balch/Wolin-style mineral mix)	1× concentrate	10 mL	1 % v/v	Typical trace-element panel: Mn²⁺, Co²⁺, Zn²⁺, Cu²⁺, Ni²⁺, Mo⁶⁺, Se⁴⁺, W⁶⁺, B³⁺, V³⁺, Al³⁺, Mg²⁺, Ca²⁺; Fe²⁺ is supplied separately as the FeSO₄·7H₂O stock above. Confirm composition against supplier CofA.
Acetic acid (glacial, ~17.5 M)	17.5 M (glacial; ~ 99 % w/w)	1.7 mL	~ 30 mM acetate (~ 99.7 % deprotonated at pH 7.2; pK_a 4.76)	Primary SCFA; substrate for acetogens and acetate-utilisers
Propionic acid (aqueous calibrated stock)	4 M propionic acid in water (= ~ 30 % v/v)	2 mL	8 mM propionate (per Goodman 2011 Table S13; ~ 99.5 % deprotonated at pH 7.2; pK_a 4.87)	Major colonic SCFA; substrate for propionate utilisers. Supplied pre-diluted (not glacial) so the 2 mL volume delivers the target 8 mM final concentration.
Butyric acid (aqueous calibrated stock)	2 M butyric acid in water (= ~ 18 % v/v)	2 mL	4 mM butyrate (per Goodman 2011 Table S13; ~ 99.6 % deprotonated at pH 7.2; pK_a 4.82)	Critical SCFA for colonocyte metabolism; Roseburia, Faecalibacterium end-product. Supplied pre-diluted so the 2 mL volume delivers the target 4 mM final concentration.
Isovaleric acid (glacial, ~9.2 M)	9.2 M (glacial)	0.1 mL	~ 1 mM isovalerate (~ 99.6 % deprotonated at pH 7.2; pK_a 4.77)	Branched-chain SCFA from leucine fermentation
Resazurin	0.25 mg/mL stock	4 mL	1 mg/L (4 µM)	Redox indicator; pink (oxidised) → colourless (reduced); confirms anaerobic conditions before inoculation

Final Mega Medium pH after all additions: 7.2 ± 0.1 at 25 °C, adjusted with concentrated NaOH if needed.

Minimal Medium — defined-composition partner (per 1 L of prepared Minimal Medium)

Per the original Goodman et al. 2011 supplementary material (Table S13 part B). Minimal Medium shares the K-phosphate buffer, Mg²⁺/Ca²⁺/Fe²⁺ levels, hematin, menadione, and cysteine reductant of Mega Medium — without the peptone, sugars, SCFAs, full vitamin mix, or trace mineral mix that would confound single-variable phenotyping.

Mixture A_min — dry defined base (per 1 L)

Component	Amount/L	Final concentration	Function
KH₂PO₄ (potassium dihydrogen phosphate, anhydrous; MW 136.09)	13.6 g	100 mM phosphate (after NaOH titration to pH 7.2, ~ 50 % converts to K₂HPO₄; the in-situ buffer is essentially the same K-phosphate system as Mega's Mixture C)	Sole buffer and phosphate source
NaCl	0.875 g	15 mM	Na⁺ osmotic balance
(NH₄)₂SO₄ (ammonium sulfate)	1.125 g	8.5 mM	Sole nitrogen source. Verify the target strain can assimilate NH₄⁺; for amino-acid auxotrophs supplement with a single defined amino acid at preparation. The SO₄²⁻ serves as the sulfur source.
L-Cysteine·HCl (anhydrous, MW 157.62; per source spec)	0.5 g	3.2 mM	Reductant; final Eh < −150 mV in the anaerobic chamber

Dry mass total: 16.1 g/L → 161 g per 10 L kit, 242 g per 15 L kit. Adjust to pH 7.2 ± 0.05 with concentrated NaOH at preparation (the natural pH of the dissolved Mixture A_min is ~ 4.5–4.8 because KH₂PO₄ is monoprotic acidic; NaOH titration to 7.2 converts approximately half the KH₂PO₄ to K₂HPO₄, producing a 100 mM phosphate buffer in situ with K⁺ + Na⁺ as the counter-ion mix).

Minimal Medium — sterile post-autoclave stock additions

Component	Stock concentration	Volume added per Litre	Final concentration	Source bottle
CaCl₂	8 mg/mL (= 72 mM anhydrous)	1 mL	72 µM	Stock I (shared with Mega — 2× volume bottle)
Vitamin K₃ (menadione)	1 mg/mL in 95 % ethanol	1 mL	1 mg/L (5.8 µM)	Stock J (shared with Mega — 2× volume bottle; precursor for endogenous menaquinone biosynthesis)
FeSO₄·7H₂O	0.4 mg/mL N₂-flushed	1 mL	1.44 µM Fe²⁺	Stock K_min (separate from Mega's Stock K — 4-week open-bottle shelf life)
Histidine-Hematin solution	1.2 mg hematin/mL in 0.2 M L-histidine	1 mL	1.2 mg/L hematin (~ 1.9 µM)	Stock L (shared with Mega — 2× volume bottle)
MgCl₂ (anhydrous, MW 95.21)	9.5 mg/mL (= 100 mM)	1 mL	0.1 mM Mg²⁺	Stock M_min (Minimal-specific — Mega uses MgSO₄ in Mixture A; Minimal uses MgCl₂ to stay sulfate-free)
Vitamin B₁₂ (cyanocobalamin, MW 1355.4)	0.01 mg/mL aqueous	0.5 mL	5 µg/L (≈ 3.7 nM)	Stock N_min (Minimal-specific single-vitamin stock — Mega supplies B₁₂ within the multi-vitamin Stock E)

Final Minimal Medium pH: 7.2 ± 0.1 at 25 °C. No peptone, no yeast extract, no SCFAs, no carbohydrates — single-substrate phenotyping requires the customer to add the test substrate(s) at the volume needed for their experiment (e.g. 0.5 % w/v glucose for a glucose-dependency test, 100 µM tryptophan for a single-amino-acid auxotrophy test).

→Culturomics and Media Navigator

Mega Medium is broad-spectrum by design, but for some workflows — selective enrichment, mucosal-niche emulation, or defined-condition phenotyping — a different medium is the better starting point. The GMExpression Culturomics and Media Navigator helps you choose:

Start from a target organism (genus, species, or ATCC / DSMZ strain) → see the recommended medium panel.
Start from a sample type (stool, mucosal scraping, biopsy, faecal slurry) → see the validated workflow.

The Navigator cross-references Mega Medium with YCFA Modified, YCFA Full, BHI-S, Modified Chopped Meat Broth, GMM, M2GSC, Wilkins-Chalgren, TPY, and the wider GMExpression catalogue.

Use and Applications

The intended workflow is two-stage and uses both kits. Stage 1 (Mega Medium): broad-spectrum recovery of every cultivable taxon from a stool / mucosal / biopsy sample. Stage 2 (Minimal Medium): once the isolates are in hand, characterise each by adding a single defined substrate, vitamin, or amino acid and reading the growth phenotype on the same ionic background as Mega — so the comparison is not confounded by a medium switch.

Personalised human gut-microbiota culture collections (PHGM) — Stage 1, Mega Medium. Serial-dilute a stool sample into Mega Medium soft-agar overlays, robotically pick the individual colonies, archive each as a glycerol cryostock. Typical yield: 200–500 unique isolates per sample. The collection feeds downstream gnotobiotic mouse colonisation, single-strain whole-genome sequencing, in-vitro drug-screening, and metabolite-profiling studies (Goodman et al. 2011 PNAS).
Substrate-utilisation profiling — Stage 2, Minimal Medium. Inoculate each archived isolate into Minimal Medium supplemented with a single carbohydrate (e.g. 5 mM xylose, arabinose, mannose, inulin, mucin-derived monosaccharide) and read growth at 24 / 48 / 72 h. Repeat across a 96-well panel of candidate substrates to build a substrate-utilisation fingerprint per strain. Because Minimal Medium uses the same K-phosphate / Mg²⁺ / Ca²⁺ / Fe²⁺ / hematin / menadione background as Mega, the growth phenotype is attributable to the substrate, not the medium switch.
Growth-factor-dependency screens — Stage 2, Minimal Medium. Substitute (NH₄)₂SO₄ with a single amino acid to map amino-acid auxotrophies; omit Stock N_min to test B₁₂ dependency; omit Stock L to test haem dependency. Mega's Wolfe/Wolin vitamin mix masks all of these dependencies — Minimal Medium exposes them cleanly.
Gene-knockout phenotyping — Stage 2, Minimal Medium. Compare wild-type and knockout strains in defined-substrate Minimal Medium to confirm that a metabolic-pathway gene is functionally required for the predicted substrate phenotype. The clean ionic background eliminates the "growth in rich medium masks the knockout phenotype" trap.
Broad-spectrum gut culturomics enrichment. When the goal is to recover as many distinct taxa as possible from a single sample without preselecting for a specific phylum or genus. Mega Medium has been benchmarked against YCFA, BHI-S, and Modified GAM in head-to-head experiments and consistently shows the highest unique-taxon recovery rate from human stool (Faith lab; Lagier & Raoult comparisons).
Anaerobic isolate maintenance and propagation. Useful as a "common denominator" maintenance broth for diverse strain libraries where each strain might otherwise require a different optimised medium. Trade-off: Mega Medium is rich enough to mask specific nutritional dependencies that may be of scientific interest.
Faecal slurry preparation for in vitro fermentation models. Used as the suspension and growth medium in continuous-culture and batch in vitro gut fermentation systems (e.g. SHIME, M-SHIME, mini-bioreactor arrays) where the goal is to preserve community structure rather than enrich a single taxon.
Microbiome–drug interaction screening. The high recovery rate of Mega Medium makes it the preferred medium for assembling 24-, 48-, and 96-well isolate panels for screening of drug-bioconversion, drug-binding, and drug-sequestration phenotypes (Maier et al. 2018 Nature; Klünemann et al. 2021).
Gnotobiotic mouse colonisation inoculum. Inoculate gnotobiotic / germ-free mice with defined-isolate consortia grown in Mega Medium, after a stationary-phase wash into PBS or HBSS.
Bacterial Genetic Stock Center / culture-collection holding medium. For internal R&D archival cryopreservation of unidentified or partially characterised isolates pending genome sequencing and metabolic profiling.

Compatible Microorganisms

Phylum Bacteroidota (formerly Bacteroidetes; ICNP 2021 / Oren & Garrity 2021)

Bacteroides fragilis (ATCC 25285), B. thetaiotaomicron (ATCC 29148), B. uniformis, B. ovatus, B. caccae, B. xylanisolvens, B. eggerthii, whole B. fragilis group; Phocaeicola vulgatus (formerly B. vulgatus), Phocaeicola dorei (formerly B. dorei) per García-López et al. 2019 reclassification of the B. fragilis-adjacent clade
Parabacteroides distasonis (ATCC 8503), P. merdae, P. johnsonii
Prevotella copri, P. melaninogenica, P. intermedia, P. bivia
Alistipes finegoldii, A. putredinis, A. shahii

Phylum Bacillota (formerly Firmicutes), class Clostridia

Faecalibacterium prausnitzii (DSM 17677) — butyrate producer; particularly responsive to the SCFA + multi-sugar formulation
Roseburia intestinalis (DSM 14610), R. inulinivorans, R. hominis — butyrate producers
Agathobacter rectalis (ATCC 33656; formerly Eubacterium rectale, Rosero et al. 2016 IJSEM 66:768), Eubacterium limosum, Anaerobutyricum hallii (formerly Eubacterium hallii, Shetty et al. 2018 IJSEM 68:3741), Eubacterium ruminantium
Blautia obeum, B. wexlerae, B. hansenii
Lachnospira pectinoschiza, L. multipara
Coprococcus eutactus, C. catus, C. comes
Anaerostipes hadrus, A. caccae — butyrate producers via lactate cross-feeding
Ruminococcus bromii, R. albus, R. flavefaciens; Mediterraneibacter gnavus (formerly R. gnavus) and Mediterraneibacter torques (formerly R. torques) per the 2018–2021 reclassification of the gut-associated Ruminococcus clade
Clostridium spp. (sensu lato and sensu stricto): C. perfringens, C. sporogenes, C. butyricum, C. asparagiforme, C. saccharogumia
Clostridioides difficile (ATCC 9689)
Subdoligranulum variabile, Oscillibacter valericigenes

Phylum Bacillota, classes Bacilli and Negativicutes

Lactobacillus spp., Lactiplantibacillus, Limosilactobacillus, Lacticaseibacillus
Streptococcus salivarius, S. thermophilus, S. parasanguinis
Enterococcus faecalis, E. faecium, E. avium, E. durans
Veillonella parvula, V. atypica, V. dispar, V. ratti — propionate producers via succinate cross-feeding
Megasphaera elsdenii, M. micronuciformis
Dialister invisus, D. pneumosintes, D. succinatiphilus

Phylum Actinomycetota (formerly Actinobacteria)

Bifidobacterium longum, B. adolescentis, B. breve, B. animalis, B. bifidum, B. dentium (TPY medium preferred for primary isolation but Mega Medium supports maintenance well)
Collinsella aerofaciens, C. intestinalis
Eggerthella lenta (ATCC 25559; formerly Eubacterium lentum) — supported, important drug-metabolising taxon
Cutibacterium acnes (ATCC 6919; formerly Propionibacterium acnes)
Slackia isoflavoniconvertens, S. heliotrinireducens

Phylum Verrucomicrobiota, Synergistetes, Proteobacteria

Akkermansia muciniphila (ATCC BAA-835) — supported but mucin-supplemented variant performs better for primary isolation
Synergistes jonesii, Synergistetes spp.
Escherichia coli, Bilophila wadsworthia, Desulfovibrio piger, D. desulfuricans (sulfate-utilising)

Sulfate- and methanogenesis-dependent taxa (Mega Medium has limited support; specialised media preferred)

Sulfate-reducers (Desulfovibrio): supported by the Na₂SO₄ content but specialised SRB media (e.g. Postgate B) preferred for primary isolation
Methanogens (Methanobrevibacter smithii, Methanosphaera stadtmanae): not supported by Mega Medium; require H₂/CO₂ atmosphere and dedicated archaeal media (BRN, McCa, M2 variants)

Preparation

The Mega Medium and Minimal Medium preparations are independent. Customers typically prepare Mega Medium first (for stage-1 culturomics enrichment), then Minimal Medium later (for stage-2 phenotyping of the recovered isolates). Both preparations follow the same Mixture → Mixture B reducing pack → autoclave → post-autoclave sterile-stock-addition pattern. Volumes below are per 1 L of final medium; for the 10 L bundle scale by 10×; for the 15 L bundle scale by 15×.

Stage 1 — Mega Medium preparation (13 steps)

1Prepare working water. Use 800 mL of Milli-Q water (Type 1 ultrapure, > 18 MΩ·cm at 25 °C) per 1 L of final medium. Pre-boil for 10 min to drive off dissolved O₂, then cool under a slow N₂ stream or seal and cool inside an anaerobic atmosphere environment (AAE) to ~ 40 °C.

2Reconstitute Mixture C (phosphate buffer). In a separate 250 mL beaker, dissolve the pre-weighed Mixture C dry phosphate salts (15.96 g per 1 L of medium = 159.6 g for the 10 L kit) in approximately 100 mL of Milli-Q water per 1 L of medium. Stir to full dissolution; the natural pH from these K₂HPO₄:KH₂PO₄ mass ratios is ≈ 7.4. Titrate to pH 7.2 ± 0.05 with concentrated HCl. The reconstituted 1 M phosphate buffer is now ready to add to the medium.

3Add Mixture A. Add the entire pre-weighed Mixture A (≈ 28 g per 1 L of medium, i.e. ~ 281 g for the 10 L kit or ~ 421 g for the 15 L kit) to the deoxygenated working water. Swirl on a magnetic stirrer at 40–50 °C until completely dissolved (5–10 min).

4Add Mixture B (reducing substances). Add the entire pre-weighed Mixture B (≈ 0.9 g per 1 L of medium, i.e. 9 g for the 10 L kit or 13.5 g for the 15 L kit) — dry NaHCO₃ + L-cysteine·HCl·H₂O. On contact with the warm acidic medium, the NaHCO₃ will release CO₂ vigorously (visible effervescence and foaming). This is expected and helps purge dissolved O₂. Swirl gently — do not cap tightly until effervescence has subsided.

5Add the Mixture C phosphate buffer. Pipette in the pH-adjusted phosphate buffer prepared in step 2. Mix to homogeneity.

6Adjust pH. Verify pH with a calibrated meter; titrate to pH 7.2 ± 0.1 with concentrated HCl (if > 7.3) or NaOH (if < 7.1). Typical adjustment is < 1 mL of 5 M titrant per L.

7Bring to ~ 950 mL. Top up to ≈ 95 % of the target final volume with deoxygenated water (the remaining ~ 5 % is reserved for the post-autoclave sterile stock additions in step 9).

8Dispense and autoclave. Dispense into autoclavable Schott bottles (or aliquot into Hungate tubes at this stage for Hungate-format preparation). Caps one-quarter turn loose for pressure equilibration. Autoclave at 121 °C × 15 min. Slow exhaust — do not vent rapidly (CO₂ loss, bumping). Cool to ≤ 50 °C, ideally inside an anaerobic atmosphere environment to minimise dissolved O₂ before stock addition.

9Add Stocks D – L (post-autoclave sterile additions) in this order, each by sterile pipette in a BSC or AAE, per 1 L of medium:

Stock I — CaCl₂: 1 mL (72 µM final Ca²⁺)
Stock J — Vitamin K menadione: 1 mL (1 mg/L = 5.8 µM final)
Stock K — FeSO₄·7H₂O: 1 mL (1.44 µM Fe²⁺ final)
Stock L — Histidine-Hematin: 1 mL (1.2 mg/L hematin final)
Stock H — Tween 80 (25 % v/v): 2 mL (0.05 % v/v final)
Stock E — Vitamin supplement: 10 mL (1 % v/v final)
Stock F — Trace mineral supplement: 10 mL (1 % v/v final)
Stock D — Mixed SCFA bottle: 5.8 mL (delivers 30 mM acetate + 8 mM propionate + 4 mM butyrate + 1 mM isovalerate final)
Stock G — Resazurin: 4 mL (1 mg/L = 4 µM final)

Swirl gently after each addition. Do not combine the stocks before addition — each is in its own bottle for chemical compatibility reasons (see Cautions and FAQ).

10Final pH check. The Stock D SCFA addition will drop the pH by 0.1–0.2 units; the 100 mM phosphate buffer holds it at 7.2 ± 0.2. If outside spec, adjust with sterile 5 M NaOH (typically 0.5–1.0 mL per L).

11Bring to final volume. Top up to exactly 1000 mL per L of medium (so 10 000 mL for the 10 L kit, 15 000 mL for the 15 L kit) with sterile deoxygenated water.

12Reduce. Allow the medium to sit in an AAE for 30–60 min until the resazurin colour shifts from pink/red (oxidised) to pale yellow / colourless (reduced). Eh should be < −150 mV. Medium is ready for inoculation when colourless.

13Dispense. For Hungate tubes: 5–10 mL per tube under N₂ flush; cap with butyl-rubber septum + crimp aluminium. For bottles: aliquot 100–500 mL into sterile screw-cap bottles inside the AAE; vacuum-seal in oxygen-absorber-equipped airtight bags for storage.

Stage 2 — Minimal Medium preparation (8 steps)

Simpler than Mega Medium because there is no Mixture C phosphate stock (KH₂PO₄ in Mixture A_min self-buffers after NaOH titration), no SCFA stock, no peptone, no vitamin or trace-mineral mixes. Volumes per 1 L of Minimal Medium; scale by 10× or 15× for the bundle.

M1Prepare working water. Use 950 mL of Milli-Q water per 1 L of final Minimal Medium. Pre-boil for 10 min to drive off dissolved O₂, cool under N₂ or in the AAE to ~ 40 °C.

M2Add Mixture A_min. Add the entire pre-weighed Mixture A_min (≈ 16.1 g per 1 L of medium, i.e. 161 g for the 10 L kit or 242 g for the 15 L kit) — dry KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine·HCl. Swirl at 40 °C until completely dissolved (5 min).

M3Titrate to pH 7.2. The natural pH from dissolved KH₂PO₄ alone is acidic (~ 4.5). Add concentrated NaOH (typically ~ 4–5 mL of 5 M per L) until pH 7.2 ± 0.05. The NaOH converts ~ 50 % of the KH₂PO₄ to K₂HPO₄, creating a 100 mM K/Na-phosphate buffer in situ. Verify with a calibrated meter.

M4Bring to ~ 950 mL. Top up to ≈ 95 % of the target final volume with deoxygenated water (the remaining ~ 5 % is reserved for the post-autoclave sterile stocks).

M5Dispense and autoclave. Dispense into autoclavable Schott bottles or Hungate tubes. Caps one-quarter turn loose. Autoclave 121 °C × 15 min. Slow exhaust. Cool to ≤ 50 °C inside the AAE.

M6Add Minimal-Medium sterile stocks (in this order, each by sterile pipette in a BSC or AAE, per 1 L of medium):

Stock I (shared with Mega) — CaCl₂: 1 mL (72 µM Ca²⁺ final)
Stock M_min — MgCl₂: 1 mL (0.1 mM Mg²⁺ final)
Stock J (shared with Mega) — Vitamin K menadione: 1 mL (1 mg/L = 5.8 µM final)
Stock K_min — FeSO₄·7H₂O: 1 mL (1.44 µM Fe²⁺ final). Use this separate vial — do not draw from the Mega-Medium Stock K bottle if it was opened more than 4 weeks earlier.
Stock L (shared with Mega) — Histidine-Hematin: 1 mL (1.2 mg/L hematin final)
Stock N_min — Vitamin B₁₂: 0.5 mL (5 µg/L final)

Swirl gently after each addition.

M7Add the test substrate(s) — what you came for. For substrate-utilisation profiling, add a single carbohydrate at 5–10 mM (e.g. 90 mg of D-xylose for 10 mM in 1 L). For growth-factor-dependency, omit Stock N_min (B₁₂) or Stock L (hematin) selectively. For amino-acid auxotrophy, replace (NH₄)₂SO₄ in Mixture A_min with a single defined amino acid at preparation. Verify pH 7.2 ± 0.1 after the addition.

M8Bring to final volume, reduce, dispense. Top up to 1000 mL per L of medium with sterile deoxygenated water. Allow 30–60 min in the AAE for reduction. (Note: Minimal Medium does not contain resazurin — there is no visual oxidation indicator. Verify Eh with a redox electrode or, for routine use, assume the medium is reduced if the AAE atmosphere is < 50 ppm O₂ and the cysteine is fresh.) Dispense into Hungate tubes (5–10 mL per tube under N₂) or sterile screw-cap bottles.

Critical control points

L-cysteine oxidation and Mixture B chemistry. Mixture B is the GMExpression-house reducing-substance bottle (NaHCO₃ + L-cysteine·HCl·H₂O dry). On rehydration the NaHCO₃ thermally decomposes during autoclaving and releases CO₂ (2 NaHCO₃ → Na₂CO₃ + H₂O + CO₂↑), purging dissolved O₂ and creating a CO₂ headspace that retards O₂ reuptake — this is the primary mechanism that protects the cysteine thiol from oxidation to cystine. Additionally, NaHCO₃ partially neutralises the HCl in L-cysteine·HCl, keeping the cysteine predominantly in its protonated R-SH form (less oxidisable than R-S⁻, pK_a ≈ 8.3). For the most fastidious strains (Faecalibacterium prausnitzii, Methanobrevibacter, Akkermansia), supplement with an additional 5 % (w/v) filter-sterilised L-cysteine·HCl stock at 10 mL/L post-autoclave inside the AAE.
Vitamin K (menadione) stock. Vitamin K is light-sensitive and ethanol-dissolved; the stock can crash out of solution if added to cold medium. Add to medium that is still warm (≥ 45 °C) with rapid swirling.
Histidine-Hematin solution. Histidine acts as an axial ligand for the Fe³⁺ of hematin, providing a soluble alternative to the alkaline NaOH dissolution used in BHI-S Mixture H. The histidine-hematin format is more stable in cold storage (8 weeks at 4 °C vs 2 weeks for the NaOH-hematin format) and does not contribute alkalinity to the final medium.
SCFA addition technique. Acetic and isovaleric acids are supplied glacial; propionic (4 M) and butyric (2 M) acids are supplied as pre-diluted aqueous stocks. All four are still strong proton donors at the supplied concentrations and produce immediate local acidification on addition. Add slowly with rapid swirling to allow the 100 mM phosphate buffer to absorb the proton load. The combined SCFA addition contributes ≈ 43 mmol of titratable acid per L of broth — within the buffer capacity of 100 mM phosphate at pH 7.2 but enough to drop pH 0.1–0.2 units. If pH drops below 6.9 after SCFA addition, restore with sterile 5 M NaOH dropwise. Final pH must be 7.2 ± 0.2.
Resazurin colour confirmation. The pink-to-colourless shift in the AAE is the gold-standard indication of adequate reduction. Do not inoculate until colourless; partial reduction (purple → pale pink) is insufficient for strict anaerobes.

Cautions

Compositional complexity and lot-to-lot variability. Mega Medium contains 24 individual components packaged across 12 bottles (three dry Mixtures A/B/C + nine sterile liquid Stocks D–L), several of which are CofA-driven (the vitamin and trace mineral supplements are specified to ATCC reference panels). Lot-to-lot variability between the source mixes can shift growth phenotypes for individual taxa by 0.5–1.0 log. For longitudinal culturomics studies (e.g. comparing isolate libraries from samples collected months or years apart), document the lot numbers of all 12 bottles and ideally reserve enough of each lot to complete the full study.

SCFA bottle (Stock D) — odour and corrosion. The Stock D mixed-SCFA bottle contains glacial acetic acid, glacial isovaleric acid, and concentrated aqueous propionic (4 M) and butyric (2 M) acids in a single pre-blended amber-glass vessel. The mixture is highly odoriferous (cheesy / rancid butter / sour milk character) and the headspace will be saturated with acid vapour even when the cap is closed. Open the bottle in a fume hood; perform all dispensing in a BSC or fume hood with adequate ventilation. The concentrated and glacial SCFAs corrode aluminium and unprotected stainless steel; use polypropylene tips or borosilicate glass pipettes only. Discard contaminated tips in vapour-tight chemical waste, not general lab waste.

Phosphate-calcium precipitation. The 100 mM phosphate buffer combined with the supplemented Ca²⁺ (from CaCl₂ stock and ATCC Trace Mineral Mix) can produce a fine calcium-phosphate precipitate on prolonged storage. This does not affect growth performance but may concern users. To minimise, add CaCl₂ after the Tween 80 (which retards aggregation) and store at 2–8 °C in sealed bottles with minimal headspace.

Hematin solubility in the histidine format. The 1.2 mg hematin/mL in 0.2 M histidine stock is stable for 8 weeks at 4 °C in the supplied amber vial. If the stock develops visible dark flecks or a brown sediment, hematin has come out of solution; re-warm to 40 °C and vortex briefly. If sediment does not redissolve, replace the stock.

Resazurin photolysis. The resazurin stock (0.25 mg/mL) is light-sensitive — store in the supplied amber vial. Photolysis products lose redox-indicator activity within 48 h of light exposure. Do not autoclave the stock; add as a sterile post-autoclave supplement.

Bovine-derived components and biosecurity. The meat extract component is bovine-derived. Although fully denatured during processing, EU, Japan, and Canada (CFIA) customs may apply TSE/BSE prion-risk checks. GMExpression uses the Australian DAFF EX188M zoosanitary certificate pipeline; documentation available via support@gmexpression.com.

Not suitable for methanogen primary isolation. Despite its breadth, Mega Medium does not support methanogenic archaea — these require an H₂/CO₂ atmosphere and dedicated archaeal media (e.g. McCa, BRN, MS). For methanogen-inclusive culturomics, run Mega Medium in parallel with a methanogen-specific medium.

Storage and Expiry · Safety

Bottle	Storage condition	Shelf life (unopened)	Notes
Mixture A (dry base)	15–25 °C, sealed, away from direct sunlight	24 months	Hygroscopic — keep desiccant pouch in bottle once opened
Mixture B (NaHCO₃ + L-cysteine·HCl·H₂O dry)	15–25 °C, sealed, dark	18 months	Dry mixture is stable; once rehydrated, use the same day. Do not vacuum-pack — CO₂ release on accidental moisture ingress would distend a sealed bag
Mixture C (dry K-phosphate salts)	15–25 °C, sealed	36 months	Indefinite stability under dry storage
Stock D (mixed SCFA bottle)	15–25 °C, sealed amber glass with PTFE-lined cap	24 months	pH < 2 — self-sterile; check for crystallisation in cold weather (re-warm to 25 °C before use)
Stock E (vitamin supplement)	2–8 °C, dark	12 months	Avoid freeze-thaw — riboflavin and B12 photolyse; aliquot if frequent access expected
Stock F (trace mineral supplement)	2–8 °C	12 months	Visible Mo precipitate after long storage is benign; vortex before dispensing
Stock G (resazurin 0.25 mg/mL)	2–8 °C, amber vial	24 months	Photolysis after > 48 h light exposure destroys redox-indicator activity
Stock H (Tween 80 25 % v/v)	2–8 °C	24 months	Viscosity rises at cold storage; pre-warm bottle to 25 °C before pipetting
Stock I (CaCl₂ 8 mg/mL)	2–8 °C	24 months	Hygroscopic salt; keep sealed
Stock J (Vitamin K menadione in 95 % ethanol)	2–8 °C, amber vial	6 months	Flammable — keep away from open flame; ethanol carrier may crash at < 0 °C
Stock K (FeSO₄·7H₂O 0.4 mg/mL, N₂-flushed)	2–8 °C, amber vial	4 weeks (once opened)	Fe²⁺ → Fe³⁺ oxidation on air ingress, then precipitates as iron hydroxide; discard if rust-coloured discoloration appears
Stock L (Histidine-Hematin 1.2 mg/mL — 2× volume in bundle)	2–8 °C, amber vial	8 weeks	If dark flecks appear, re-warm to 40 °C + vortex briefly; replace if undissolved. Same bottle serves both Mega and Minimal preparations.
Minimal Medium kit — additional bottles
Mixture A_min (dry KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine·HCl)	15–25 °C, sealed, dark	24 months	L-cysteine in the dry mix is stable; once rehydrated, prepare and autoclave the same day to minimise cysteine oxidation
Stock K_min (FeSO₄·7H₂O 0.4 mg/mL, N₂-flushed)	2–8 °C, amber vial	4 weeks (once opened)	Separate vial from Mega's Stock K — open only when preparing Minimal Medium. Discard if rust-coloured.
Stock M_min (MgCl₂ 9.5 mg/mL aqueous)	2–8 °C	24 months	Stable; non-hygroscopic in solution
Stock N_min (Vitamin B₁₂ 0.01 mg/mL aqueous)	2–8 °C, amber vial	12 months	B₁₂ photolyses on extended light exposure — keep in amber storage
Stocks I (CaCl₂), J (Vitamin K) and L (Histidine-Hematin) are shipped at 2× volume in the bundle and serve both Mega and Minimal preparations. Storage and shelf life as listed in the Mega rows above.
Prepared Mega Medium (anaerobic, vacuum-sealed with O₂ absorber)	2–8 °C	6 months	Verify resazurin is colourless before inoculation
Prepared Mega Medium (aerobic, 4 °C)	2–8 °C	2 weeks	Fe²⁺ oxidation and cysteine depletion limit usable life
Prepared Minimal Medium (anaerobic, vacuum-sealed with O₂ absorber)	2–8 °C	3 months	No resazurin indicator in Minimal Medium — verify Eh with electrode or assume reduced if AAE atmosphere is < 50 ppm O₂
Prepared Minimal Medium (aerobic, 4 °C)	2–8 °C	2 weeks	Fe²⁺ + cysteine depletion limit usable life; same as Mega

Safety notes. Mega Medium contains glacial SCFAs (acetic, propionic, butyric, isovaleric) prior to addition — these are corrosive and odoriferous. Handle in a fume hood. Vitamin K stock contains ethanol — flammable. Hematin in histidine is a non-corrosive Schedule-3 reagent. Resazurin is a Schedule-3 redox dye. Handle prepared cultures at BSL-2 when working with isolates from human clinical samples. SDS available on request.

References

Goodman AL, Kallstrom G, Faith JJ, Reyes A, Moore A, Dantas G, Gordon JI. (2011). Extensive personal human gut microbiota culture collections characterised and manipulated in gnotobiotic mice. Proceedings of the National Academy of Sciences USA 108 (15): 6252–6257. [Primary published formulation that underlies Mega Medium 1.0. The Goodman paper itself names the recipe "Gut Microbiota Medium (GMM)" in its supplementary material (Table S13); GMExpression's Mega Medium 1.0 is the GMM recipe rebuilt as a pre-weighed two-mixture kit with the same component concentrations and the SCFA / hematin / menadione / vitamin / trace-mineral supplements that Goodman specifies. The companion Gut Microbiota Medium (GMM) SKU supplies the same recipe under the published name for users who prefer literal nomenclature.]
Lagier JC, Khelaifia S, Alou MT, Ndongo S, Dione N, et al. (2016). Culture of previously uncultured members of the human gut microbiota by culturomics. Nature Microbiology 1: 16203.
Faith JJ, Guruge JL, Charbonneau M, Subramanian S, Seedorf H, et al. (2013). The long-term stability of the human gut microbiota. Science 341(6141): 1237439.
Maier L, Pruteanu M, Kuhn M, Zeller G, Telzerow A, et al. (2018). Extensive impact of non-antibiotic drugs on human gut bacteria. Nature 555: 623–628.
Klünemann M, Andrejev S, Blasche S, Mateus A, Phapale P, et al. (2021). Bioaccumulation of therapeutic drugs by human gut bacteria. Nature 597: 533–538.
Duncan SH, Hold GL, Harmsen HJM, Stewart CS, Flint HJ. (2002). Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov. International Journal of Systematic and Evolutionary Microbiology 52: 2141–2146.
Fischbach MA, Sonnenburg JL. (2011). Eating for two: how metabolism establishes interspecies interactions in the gut. Cell Host & Microbe 10: 336–347.
ATCC Microbiology Catalogue — ATCC MD-VS Vitamin Mix and ATCC MD-TMS Trace Mineral Mix product specifications.
Sonnenburg ED, Smits SA, Tikhonov M, Higginbottom SK, Wingreen NS, Sonnenburg JL. (2016). Diet-induced extinctions in the gut microbiota compound over generations. Nature 529: 212–215.
Browne HP, Forster SC, Anonye BO, Kumar N, Neville BA, et al. (2016). Culturing of "unculturable" human microbiota reveals novel taxa and extensive sporulation. Nature 533: 543–546.

Frequently Asked Questions

Q0. Why is this only sold as a bundle? Can I buy just one of the two media?

No — and the reason is scientific, not commercial. The two media are paired by design: they share the same K-phosphate / Ca²⁺ / Mg²⁺ / Fe²⁺ / hematin / menadione / cysteine ionic background, so when you grow an isolate in Mega and then phenotype it in Minimal, the only variable is the substrate — not the medium. That shared-background property is the entire reason researchers buy this workflow over assembling defined media from scratch, and it is preserved only if both media come from the same manufactured batch with the same supplier lots. Splitting them would compromise downstream phenotype reproducibility for the small minority of customers who buy only one, while forcing everyone else to qualify a second supplier. The bundle keeps it clean: one purchase order, one batch, one CoA, one shipment.

Q0b. Why is each medium packaged as multiple separate bottles instead of a single all-in-one mix?

The 16-bottle architecture (12 for Mega — three dry Mixtures + nine sterile liquid Stocks; 4 additional for Minimal — Mixture A_min + Stock K_min + Stock M_min + Stock N_min) follows the GMExpression house standard refined on the YCFA Modified and YCFA Full kits. Components are grouped by shared stability and sterilisation requirements — and separated wherever two components would degrade each other in storage. The four design rules: (1) Co-packaged where chemistry is compatible — Mega's SCFAs are mutually miscible carboxylic acids that pH-self-sterilise (one Stock D bottle); Mega's NaHCO₃ + L-cysteine share Mixture B because the bicarbonate's CO₂ release during autoclaving protects the thiol from oxidation; Minimal's KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine share Mixture A_min because they are heat-stable, non-reactive dry powders. (2) Separated where co-storage would precipitate — Ca²⁺ (Stock I) is isolated from the phosphate buffer (Mega's Mixture C, Minimal's KH₂PO₄) and NaHCO₃ to prevent Ca₃(PO₄)₂ and CaCO₃; Minimal uses MgCl₂ (Stock M_min) instead of MgSO₄ to stay sulfate-free for sulfate-utilisation studies. (3) Separated where one component would catalyse degradation of another — trace metals (Stock F) are isolated from the vitamin supplement (Stock E) because Cu, Mo, Fe form vitamin–metal complexes that destroy riboflavin and thiamine; Fe²⁺ (Stocks K and K_min) is isolated from anything aerobic. (4) Sterilisation pathway — dry mixtures are autoclaved with the medium; SCFAs are self-sterile through pH; heat-labile vitamins are filter-sterilised at 0.22 µm and added post-autoclave so heat damage is avoided. The Mega + Minimal bundle is therefore "two complete kits in one box" with three stocks (I, J, L) at 2× volume to serve both preparations from a single bottle each.

Q1. How is Mega Medium different from YCFA Modified Medium?

Both are designed for gut-microbiota culturomics, but the design philosophies differ. YCFA Modified Medium (Duncan, Hold, Flint 2002; DSMZ 1611) is a high-rigour formulation specifically tuned for butyrate-producing Firmicutes — its hallmark is the SCFA mix (acetate / propionate / iso-butyrate / valerate / iso-valerate) in gut-physiological ratios plus a separated Stock D vitamin supplement and Mixture B redox-buffer pack. Mega Medium 1.0 (Goodman et al. 2011) is a broader-spectrum formulation that adds five carbohydrates (glucose, cellobiose, maltose, fructose, malic acid) and ATCC Vitamin / Trace Mineral mixes, and uses a different SCFA cocktail (acetate / propionate / butyrate / isovalerate, no iso-butyrate or n-valeric acid). In head-to-head comparison, Mega Medium recovers a wider taxonomic breadth from human stool (about 40–60 % of 16S-detected taxa per the Goodman paper), but YCFA tends to give stronger absolute growth for the butyrate-producer subset (Faecalibacterium, Roseburia, Eubacterium rectale). For maximum-breadth personalised culture collections, use Mega Medium. For butyrate-producer focus, use YCFA Modified.

Q2. Can I use Mega Medium for substrate-utilisation profiling?

No — and this is the central reason the Mega + Minimal bundle exists. Mega Medium contains five carbohydrates and four SCFAs simultaneously, which means any single-substrate utilisation test in Mega is contaminated by the other substrates the strain has access to. For substrate-utilisation work use the Minimal Medium kit included in this bundle: KH₂PO₄ + NaCl + (NH₄)₂SO₄ + L-cysteine + Mg²⁺ + Ca²⁺ + Fe²⁺ + hematin + menadione + B₁₂ only — no carbohydrates, no SCFAs, no peptone. Add the single substrate you want to test (5–10 mM is typical) and read the growth phenotype against a no-substrate control. Because Minimal Medium shares the same K-phosphate / Mg²⁺ / Ca²⁺ / Fe²⁺ / hematin / menadione background as Mega, the growth difference is attributable to the test substrate, not to a medium-switch artefact.

Q3. What is the role of malic acid in Mega Medium?

Malic acid serves three functions: (i) it is a TCA-cycle intermediate that supports the growth of fumarate-respiring anaerobes (particularly Bacteroides fragilis, which uses fumarate as a terminal electron acceptor via menaquinone-coupled fumarate reductase); (ii) it provides an alternate carbon source for malic-acid-fermenting Lactobacillales (the malolactic fermentation pathway); (iii) at 7.5 mM it contributes ~ 15 mM of acidity that is buffered by the 100 mM phosphate — net effect is a modest pH-buffering benefit and a substrate availability that is independent of the dominant carbohydrate pool.

Q4. Why is the FeSO₄ stock supplied separately at low concentration?

Fe²⁺ rapidly oxidises to Fe³⁺ in the presence of dissolved O₂; the Fe³⁺ precipitates as iron hydroxide / iron oxide and is no longer bioavailable. Supplying FeSO₄ as a pre-prepared 0.4 mg/mL stock in N₂-flushed solution preserves the Fe²⁺ form long enough for sterile addition into the deoxygenated medium. The 1 mL/L addition yields 0.4 mg/L (= 1.44 µM) of available Fe²⁺, which is supplemented further by Fe-containing components of the ATCC Trace Mineral Mix. Note: the Fe²⁺ stock has a relatively short refrigerated shelf life (4 weeks) — discard if any rust-coloured discoloration appears.

Q5. Can I use Mega Medium with the GMExpression Anaerobic Preparation Kit?

Yes. The APK vacuum-deoxygenation workflow is medium-agnostic. The Hungate-tube vacuuming, oxygen-absorber storage, and resazurin colour-change verification steps from the YCFA Modified Medium Instructions Manual apply directly to Mega Medium in the same Hungate-tube or vacuum-bag formats. The resazurin pink → colourless transition is the same indicator. Choose APK Lite for occasional preparation or APK Plus for production-scale preparation with palladium catalyst-based atmospheric correction.

Q6. Why does my prepared Mega Medium have a faint sulfurous smell?

Two probable sources, both normal: (i) the 0.5 g/L L-cysteine·HCl partially decomposes during autoclaving to release minor amounts of H₂S — this is the same chemistry that produces the faint sulfur smell of any cysteine-containing anaerobic broth; (ii) the Na₂SO₄ + reducing conditions can produce trace sulfide if any sulfate-reducing organism is present at very low abundance. A faint sulfurous smell is normal. A strong rotten-egg smell indicates active sulfate reduction, which means either deliberate Desulfovibrio inoculation or contamination; investigate.

Q7. Can I make Mega Agar plates from this formulation?

Yes. Order the agar-supplemented configuration (1.5 % w/v bacteriological agar pre-mixed into Mixture A) or add 15 g/L agar to Mixture A pre-autoclaving in-house. Plates must be poured at 50 °C inside an anaerobic atmosphere environment (AAE) and allowed to set on a pre-warmed level surface; pre-dry plates 1 h lid-down at 35 °C in the AAE before use. Soft-agar (0.6–0.75 %) overlays are also feasible for the Goodman et al. original double-layer high-throughput plating workflow.

Q8. How do I cryopreserve isolates grown in Mega Medium?

Standard approach: grow the isolate to late-log / early-stationary phase in Mega Medium (typically 18–48 h depending on the strain), mix 0.75 mL of culture with 0.25 mL of sterile 60 % v/v glycerol in Mega Medium (= final 15 % glycerol), freeze in cryovials at −80 °C. The cysteine-rich, vitamin-rich, Fe-rich nutrient base of Mega Medium provides excellent freeze-thaw recovery — typically > 80 % viability after 1 year at −80 °C. For longer-term storage, transfer the −80 °C cryovials to LN₂ vapour phase. Do not store anaerobic cryostocks at −20 °C — viability declines sharply within 2–3 months at that temperature.