Fastidious Anaerobe Agar (FAA, ATCC Medium: 2895) | For 7L

FAA is non-selective and broad-spectrum, with its particular strength in solid-phase clinical isolation and in standardised susceptibility testing.

Rule of thumb: FAA for solid-phase clinical isolation, pigment work and EUCAST disk-diffusion AST; GAM / mGAM for broth culture and long incubation; YCFA for SCFA-dependent butyrate producers; GMM for mucin-utilisers; Chopped Meat for long-term hold. FAA is the only medium in the range designed and validated for standardised anaerobe susceptibility testing.

Yes — identical component for component and quantity for quantity, including the 5 g/L yeast extract that distinguishes ATCC Medium 2895 from the unsupplemented base. The only difference is when the pyrophosphate is added: after autoclaving as Stock E, rather than co-autoclaved. The finished medium is the same, so a protocol citing ATCC Medium 2895 is met.

One point if you work from the ATCC sheet directly: its stated suspension mass of 45.7 g/L is the unsupplemented formulation and omits the 5 g/L yeast extract its own formula table specifies. The correct total is 50.66 g/L, and our fraction masses are computed to that figure — a common source of difficulty when reproducing the medium from the sheet.

The short version. The finished medium is identical in composition to ATCC Medium 2895 — same components, concentrations and pH. Only the point of addition differs, and because Stock E is supplied ready-adjusted to pH 7.2 it changes the medium's composition but not its pH. Four benefits, for one extra pipetting step.

1 — It removes a phosphate catalyst from the autoclave. Two well-characterised reactions produce hydrogen peroxide in media of this type, and both are phosphate-catalysed:

• Media containing phosphate together with glucose accumulate H₂O₂ on heating — the reaction needs ≥120 °C for ≥5 min and pH above 6.5, and of the substrates tested only α-hydroxycarbonyl compounds (glucose among them) autoxidised with peroxide accumulation. Strict anaerobes were killed rapidly by as little as 20 µM H₂O₂. (Carlsson et al. 1978)
• Agar autoclaved with phosphate also generates H₂O₂, by a route enhanced by amino and ammonium groups. Colony counts where agar and phosphate were autoclaved together were only 5.7 % of counts where they were autoclaved separately; catalase restored 106 %, but sodium pyruvate only 58 %. (Kawasaki & Kamagata 2017)

FAA contains glucose, agar and abundant peptide amino groups and is autoclaved at 121 °C for 15 min at pH 7.2 — it meets every one of those conditions. Holding the pyrophosphate out removes the catalyst from both reactions. The pyruvate remains as the second line of defence, but as those figures show, prevention is substantially more effective than scavenging.

2 — It delivers pyrophosphate as pyrophosphate. The P–O–P bridge hydrolyses to orthophosphate under heat, and the intact anion is the species that does the work — the Porphyromonas stimulation and the buffering. Co-autoclaving partially converts it before use, so you receive less pyrophosphate and more phosphate than the label states. Post-sterilisation addition delivers the declared 0.940 mmol/L intact.

3 — It removes the catalyst from the pre-adjustment boil too, where Mixture A is dissolved before the pH is set. The protocol also adjusts pH before the boil, so dissolution happens at a controlled near-neutral pH — heating glucose with arginine drives non-enzymatic browning, faster away from neutrality.

4 — Reduced browning. The same chemistry drives Maillard browning; removing it gives a lighter, more consistent prepared appearance.

Why this is good practice generally. Heat-labile and heat-reactive components belong outside the autoclave — already standard for vitamin K₁, which is why Stock D exists. The same reasoning applies to a hydrolysable polyphosphate that catalyses peroxide formation in the components around it.

Yes — specify the reference single-blend presentation at ordering: pyrophosphate in Mixture A at 36.75 g/L, no Stock E. It ships with reference instructions in which the base is made up to 998 mL rather than 978 mL, because Stock D is then the only post-autoclave addition; do not use the Stock E volume budget with it. The finished medium is compositionally identical either way.

Blood is the reference condition, not an optional enhancement: every published QC specification assumes 5–10 % defibrinated blood, and a published comparison found blood improved every medium tested. Without it, expect reduced recovery at low inoculum and no pigment development in the black-pigmented anaerobes. For susceptibility testing it is not optional — the EUCAST method is defined on FAA with 5 % horse blood.

For general isolation, either works. Three cases where it matters: EUCAST disk diffusion specifies mechanically defibrinated horse blood; Haemophilus requires heated horse blood, because ovine NADase destroys V factor; and haemolysis patterns differ between species, so keep the source constant if you read haemolysis.

Yes — that is the medium the method was built on. Prepare as FAA-HB with 5 % v/v mechanically defibrinated horse blood, pour to 4.0 mm ± 0.5 mm (≈24 mL per 90 mm plate), and follow the current EUCAST manual. Zones are depth-sensitive, so measure the pour volume. Cutibacterium acnes grows too slowly for the 16–20 h window. Verifying a given lot under your own quality system remains your responsibility.

Not with intact blood. FAA supplies X factor (haemin) at 10 mg/L, but V factor (NAD) is intra-erythrocytic and must be released by heating — chocolate agar. Use heated horse blood; sheep blood will not work even heated, because ovine NADase destroys V factor. If Haemophilus is the primary target, a dedicated chocolate agar is better.

No — the restriction is deliberate, for three reasons, of which only the first is obvious.

(i) Terminal acidity. Limiting fermentable carbohydrate limits acid accumulation, which concentrates at the colony base rather than equilibrating across the plate. Unrestricted acidification degrades colony morphology, pigment, haemolysis reading and subculture viability, and in susceptibility testing it shifts the activity of several antimicrobial classes.

(ii) Direct monosaccharide inhibition and vitamin-K antagonism — the stronger and more specific reason. Free monosaccharides (glucose, galactose, mannose, fructose) are directly inhibitory to Prevotella melaninogenica at low concentrations, whereas the disaccharides sucrose and lactose are not inhibitory even at high concentrations. The effect is exerted principally during the lag-to-logarithmic transition — exactly the moment of primary isolation from a clinical specimen. Mechanistically, glucose or galactose abolishes vitamin-K-dependent induction of 3-ketodihydrosphingosine synthetase in that organism. So free glucose does not merely acidify the plate; it antagonises the vitamin-K-dependent regulatory programme that FAA's own vitamin K₁ exists to support.

(iii) Peroxide precursor. Glucose is an α-hydroxycarbonyl compound and a substrate for phosphate-catalysed autoxidative H₂O₂ formation under the medium's autoclaving and pH conditions (see Q2).

Hence the 1 g/L glucose + 1 g/L soluble starch pairing: saccharolytic organisms take their carbohydrate from a polysaccharide while free-monosaccharide concentration stays below the inhibitory, peroxide-generating and acidifying threshold. The asaccharolytic majority this medium targets run on peptides, amino acids and organic acids and are unaffected either way.

Yes, but not at equal mass. Menadione's formula weight is 172.2 against phylloquinone's 450.7, so equal masses differ 2.6-fold in molar terms. Our K₃ stock delivers 0.38 mg/L, equimolar to 1.0 mg/L K₁. Menadione is a redox-cycling quinone that can generate superoxide — the last thing wanted in a medium for strict anaerobes. If you make your own stock, dose it on a molar basis.

Pre-reduction is strongly recommended and costs only workstation time. The reason is chemical rather than atmospheric: L-cysteine is a free thiol that autoxidises in air to hydrogen peroxide, which the pyruvate scavenges only incompletely. That is a mechanistic argument rather than a measured claim about this product — but pre-reduction was one of the variables evaluated during development of the EUCAST method. Store sterile medium at 4 °C, dark; use within 2–3 weeks.

Use a CO₂-containing mixture — N₂ 80 % / H₂ 10 % / CO₂ 10 % is ideal. Many clinical anaerobes are capnophilic and the requirement is biosynthetic: Bacteroides and Prevotella fix CO₂ at PEP carboxykinase. Under 100 % N₂ the medium's own 0.4 g/L bicarbonate is the only CO₂ source and is progressively lost from an open plate — workable, but expect reduced capnophile recovery. The hydrogen also serves the palladium catalyst.

No — no antibiotics, dyes, bile salts or azide. It is a maximum-recovery medium. You can add selective supplements, and it is commonly used that way; because the base is non-inhibitory, any selectivity observed is attributable to the supplement rather than the base, which is what a defensible protocol requires.

Bacteroides fragilis ATCC 25285, Clostridium perfringens ATCC 13124 and Peptostreptococcus anaerobius ATCC 27337, at 10–300 CFU on blood-supplemented medium, 35 ± 2 °C anaerobically, read 48–72 h — all three should grow. Add a Prevotella strain to confirm the haemin / vitamin K / succinate module (pigment at 5–7 days is the visible readout) and Eggerthella lenta ATCC 25559 to confirm the arginine, since it grows to barely detectable density without it.

Browning is non-enzymatic (Maillard), driven by heating reducing sugar with amino compounds, accelerated at alkaline pH and catalysed by phosphate — controlled by adjusting pH to 7.0–7.2 before the boil and by keeping the pyrophosphate out of the autoclave via Stock E. If it persists, check the autoclave cycle for excessive time at temperature. Haze is separate: the cause specific to this preparation is calcium pyrophosphate, since pyrophosphate forms a sparingly soluble calcium salt and defibrinated blood is not citrated. Disperse Stock E fully before the blood goes in.

Yes — 0.5 g/L agar for semi-solid; for broth omit the agar and order the Without-Agar SKU. All other quantities, including Stock E at 20 mL/L and Stock D at 2 mL/L, are unchanged. The published performance data and the EUCAST method relate to the solid medium.

At the EUCAST depth of 4.0 mm in a 90 mm dish (≈24 mL per plate), roughly 41 plates per litre — about 290 from the 7 L pack. A conventional 20 mL fill gives about 50 plates per litre (350 per pack) but only ≈3.3 mm depth, which is not suitable for disk diffusion.

Yes. FAA-HB has been evaluated for 36 anaerobic species from faecal samples, most confluent within 16–20 h. The features that make it good for clinical anaerobes — the asaccharolytic-friendly nitrogen base, haem and quinone provision, restricted free sugar and peroxide management — are the same ones that matter for fastidious gut commensals. For butyrate producers see YCFA, for mucin-degraders Gut Microbiota Medium; the Navigator maps a target organism onto the right panel.

The hydrate sets the molar dose: at 0.5 g/L the monohydrate (FW 175.63) delivers 2.85 mmol/L of thiol, whereas the anhydrous salt would deliver 3.17 mmol/L — 11 % more reductant and 11 % more acid. ATCC Medium 2895 specifies the monohydrate, so that is what we supply, stated explicitly so the molar dose can be verified rather than inferred. The separate filter-sterilised solution exists because cysteine is damaged by the very process used to sterilise the medium — autoclaving a free thiol with a reducing sugar, plus oxidation during aerobic handling. Post-sterilisation addition under an anaerobic atmosphere gives the full declared reductant.