Overview

TPY (Trypticase-Peptone-Yeast Extract) is the reference medium for the cultivation, enumeration, and identification of Bifidobacterium species — the principal genus of beneficial Gram-positive anaerobic gut commensals and the genus underlying the human-milk-oligosaccharide-fermenting probiotic industry. Designed by Vittorio Scardovi (Bologna, 1960s–80s) as part of the systematic description of the Bifidobacterium genus, TPY is documented in Scardovi 1986 Bergey's Manual of Systematic Bacteriology, ATCC Medium 1058, and DSMZ Medium 56.

TPY differs from generic anaerobic media (BHI, GAM) in three ways: (a) pH 6.5 ± 0.2 optimum (lower than the 7.0–7.4 of BHI / mGAM), matching the colonic pH at which Bifidobacterium grows optimally; (b) defined trace-cofactor matrix (Mg2+, Zn2+, Ca2+, Fe3+) for the genus-specific fructose-6-phosphate phosphoketolase pathway (Scardovi & Trovatelli 1965 Ann Microbiol 15:19); (c) Tween 80 (polysorbate 80) supplying oleic-acid esters that enhance exopolysaccharide production. No bile, no antibiotics in the base formulation — TPY is non-selective "rich-but-Bifidobacterium-friendly". For selective recovery from mixed gut or dairy cultures, add mupirocin 50 mg/L (post-autoclave filter-sterilised) to make TPY-Mup; Bifidobacterium is intrinsically mupirocin-resistant while most other Gram-positives are sensitive.

TPY is the ISO 29981:2010 reference medium for Bifidobacterium enumeration in milk products — the regulatory standard for probiotic-strain quality control by Yakult, Danone, Chr. Hansen, Morinaga, Nestlé, and the global dairy industry. The Australian customer base includes the Australian Dairy Council, Saputo Dairy Australia, and Bega — all of whom require TPY-based Bifidobacterium CFU certificates of analysis on probiotic-supplemented dairy and infant-formula products.

We also have

YCFA Modified Medium · YCFA Full Recipe · YCFA Base · Modified Chopped Meat Broth (ATCC 1490) · Chopped Meat with Carbohydrates · Beef Granules

Package Contents

Each GMExpression TPY kit contains:

  • Mixture A — pre-weighed TPY base (Trypticase 10 g/L + Phytone Peptone 5 g/L + yeast extract 2.5 g/L + glucose 5 g/L + MgCl2 0.5 g/L + ZnSO4 0.25 g/L + CaCl2 0.15 g/L + K2HPO4 2 g/L) for 5 L final volume.
  • Mixture B — L-cysteine·HCl·H2O 2.5 g, N2-flushed PP bag.
  • Stock Fe — Ferric chloride (FeCl3) stock 10 mg/mL in 0.01 M HCl, amber vial. Light- and air-protected.
  • Stock T — Tween 80 (polysorbate 80, Sigma P1754 grade) in original-packaging amber bottle, 5 mL × 5.
  • Vial Mup (optional) — Mupirocin stock 10 mg/mL in 70 % ethanol, filter-sterilised, for the selective TPY-Mup variant (50 mg/L final = 5 mL per litre).
  • 5 × airtight PP storage bags + 5 × heat-resistant rubber bands.
  • Instruction manual (A5 booklet, v1.0) with Scardovi 1986 protocol, ISO 29981 enumeration annex, dairy / infant-formula QC workflow, and a current LPSN taxonomy reference (the Bifidobacterium genus has been substantially reorganised 2020–2024).

Customisation options on request: agar variant (TPY agar, 15 g/L agar) for plate counts; ISO 29981 ready-to-use 9 mL tube format for dairy QC laboratories; resazurin-supplemented variant (1 mg/L; YCFA-style visual O2 indicator); pre-supplemented TPY-Mup for selective Bifidobacterium from mixed cultures; LP-MRS / BSM selective variant (with lithium chloride 3 g/L + propionic acid 5 mL/L pH-adjusted).

Composition — per 1 L equivalent unless stated otherwise

TPY Broth (Scardovi 1986; ATCC Medium 1058; DSMZ Medium 56; per 1 L)

ComponentConcentrationFunction
Trypticase (pancreatic digest of casein)10.0 gPrimary peptide source; BBL Trypticase or Difco Trypticase Peptone
Phytone Peptone (papain digest of soybean meal)5.0 gPlant-derived peptide; supports aromatic-amino-acid biosynthesis
Yeast extract2.5 gB-vitamins (especially riboflavin, niacin)
D-Glucose5.0 gPrimary fermentable carbohydrate; substrate for F6P phosphoketolase
L-Cysteine·HCl·H2O0.5 gReductant; Eh < −150 mV
Magnesium chloride hexahydrate (MgCl2·6H2O)0.5 gTrace cofactor (enzymatic Mg2+)
Zinc sulphate heptahydrate (ZnSO4·7H2O)0.25 gTrace cofactor (DNA polymerase Zn2+)
Calcium chloride dihydrate (CaCl2·2H2O)0.15 gTrace cofactor (cell-wall stabilisation)
Ferric chloride (FeCl3)0.1 gIron source (electron transport in some Bifidobacterium)
Tween 80 (polysorbate 80)1.0 mLOleic-acid ester; enhances exopolysaccharide
Dipotassium hydrogen phosphate (K2HPO4)2.0 gBuffer
For agar: Agar15.0 gSolidifying agent

Pre-autoclaving pH: 6.5 ± 0.2 at 25 °C (lower than BHI / mGAM; matches Bifidobacterium colonic optimum).

Selective TPY-Mup variant

Additional componentConcentrationFunction
Mupirocin (post-autoclave, filter-sterilised)50 mg/LSelective: Bifidobacterium intrinsically resistant; most other Gram-positives sensitive (especially LAB co-isolates)

Alternative LP-MRS / BSM selective variant

Additional componentConcentrationFunction
Lithium chloride (LiCl)3.0 g/LSuppresses LAB
Propionic acid (pH-adjusted to 6.5)5.0 mL/LSuppresses LAB; Bifidobacterium tolerates short-chain organic acids

→Culturomics and Media Navigator

Need help matching this medium to your microorganism — or starting from a target organism and finding the medium that fits? Use the GMExpression Culturomics and Media Navigator: a curated, cross-referenced tool that lets you (i) start from a medium and view the bacterial taxa it is recommended for, or (ii) start from a target organism (genus, species, or DSMZ/ATCC strain ID) and view the panel of GMExpression media that will support it. The Navigator integrates TPY Bifidobacterium Medium with the wider YCFA, Chopped Meat Broth, BHI-S, Wilkins-Chalgren, Modified GAM, Anaerobic Columbia Blood Agar, GMM, M2GSC, BBE, KVLB, CCFA, and Egg Yolk Agar catalogue.

Use and Applications

  • Probiotic strain QC and enumeration for commercial Bifidobacterium products (Yakult, Danone Actimel, Chr. Hansen BB-12, Morinaga BB536, Nestlé Bifidus). TPY is the ISO 29981 reference medium for Bifidobacterium enumeration in dairy products.
  • Infant-formula and follow-on-formula probiotic QC — TPY-based plate counts of B. animalis subsp. lactis, B. infantis, B. breve, B. longum per Codex Alimentarius standards. Australian regulatory market (FSANZ).
  • Antimicrobial-susceptibility testing of Bifidobacterium — TPY-base broth is the EFSA-recommended medium for Bifidobacterium AST (EFSA FEEDAP 2018 guidance).
  • Research and isolation of novel Bifidobacterium species — the genus is undergoing rapid expansion (2020–2024 new species B. samirii, B. felinis, B. choloepi) with TPY as the standard isolation medium.
  • Microbiome research on infant gut development — B. infantis (now B. longum subsp. infantis) is the dominant infant-gut commensal whose human-milk-oligosaccharide-utilisation locus (HMO-cluster I and II) is best studied on TPY.
  • Faecal Bifidobacterium enumeration in adult populations (use TPY-Mup for selectivity in faecal background).
  • Veterinary probiotic QC — B. animalis, B. pseudolongum ruminant and porcine probiotic strains.
  • Yogurt / fermented-dairy QC when Bifidobacterium is part of the starter culture alongside Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

Compatible Microorganisms

Bifidobacterium genus — designed targets

  • Bifidobacterium animalis subsp. lactis (ATCC 27536; JCM 10602) — the BB-12 / Lafti-B94 commercial probiotic lineage (Chr. Hansen). Most commercially important probiotic Bifidobacterium.
  • Bifidobacterium animalis subsp. animalis (ATCC 25527) — animal-gut isolate.
  • Bifidobacterium adolescentis (ATCC 15703) — adult human-gut commensal; type species of the B. adolescentis phylogenetic group.
  • Bifidobacterium bifidum (ATCC 29521; JCM 1255) — genus type species; infant-gut isolate; extracellular endo-β-galactosidases for mucin glycans.
  • Bifidobacterium breve (ATCC 15700; JCM 1192) — infant-gut isolate; M-16V Morinaga probiotic strain.
  • Bifidobacterium longum subsp. longum (ATCC 15707) — the BB536 Morinaga commercial probiotic.
  • Bifidobacterium longum subsp. infantis (ATCC 15697; JCM 1222; formerly B. infantis) — infant-gut HMO specialist.
  • Bifidobacterium dentium (ATCC 27534) — adult-gut and oral cavity.
  • Bifidobacterium pseudocatenulatum (ATCC 27919) — adult-gut.
  • Bifidobacterium ruminantium — bovine-rumen isolate.

Related Bifidobacteriaceae (also supported)

  • Gardnerella vaginalis (recently placed in Bifidobacteriaceae per genomic taxonomy) — vaginal microbiome relevance.
  • Scardovia spp.
  • Parascardovia denticolens.

Non-Bifidobacterium lactic-acid bacteria (also grow; use TPY-Mup for selectivity)

  • Lactobacillus sensu lato (multiple genera per Zheng et al. 2020 reclassification): L. acidophilus, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus.
  • Pediococcus pentosaceus, P. acidilactici.
  • Lactococcus lactis subsp. lactis and subsp. cremoris.
  • Streptococcus thermophilus.

Preparation

1Weigh Mixture A and disperse. Use the pre-weighed Mixture A (~25.5 g per litre, dry basis). Combine with 950 mL distilled water in a clean autoclavable Schott bottle of at least 1.5× final volume. The trace elements (ZnSO4, MgCl2, CaCl2) dissolve readily; K2HPO4 may take a moment to disperse.
2Add FeCl3 from Stock Fe. 10 mL of Stock Fe (10 mg/mL in 0.01 M HCl) = 100 mg/L final. The medium will have a light-yellow tinge from the ferric chloride. Critical: add FeCl3 last among the trace elements and immediately before pH adjustment, since FeCl3 precipitates as Fe(OH)3 above pH 4.5 unless cysteine is present to maintain reduction.
3Add Tween 80 from Stock T. 1 mL of Stock T per litre. Tween 80 is viscous; pipette slowly with a positive-displacement pipette or rinse the pipette tip with warm medium to ensure complete delivery.
4Heat-dissolve gently. 50–60 °C with stirring for 10 min. Do not overheat. FeCl3 at high temperature can precipitate as Fe(OH)3 through local pH spikes; the inclusion of cysteine and the slight medium acidity (pH 6.5) help maintain iron in solution but are not infinite buffers.
5Add Mixture B (L-cysteine). Open the N2-flushed Mixture B bag inside the AAE or use an oxygen-purged needle-and-septum technique. Add 0.5 g L-cysteine·HCl·H2O per litre and stir until dissolved (rapid).
6Adjust pH. Target 6.5 ± 0.2 at 25 °C. The medium is mildly acidic by formulation; usually only minor adjustment is needed. Use 1 M NaOH or 1 M HCl. Note: the pH 6.5 specification is tighter than BHI-S or mGAM; the phosphate buffer at 2 g/L gives ~ 20 mM/pH-unit buffering capacity, sufficient for post-autoclaving slight drift.
7Bring to final volume. 1000 mL with distilled water.
8Dispense. Broth tubes: 10 mL per Hungate or screw-cap tube under N2 flush for PRAS preparation. ISO 29981 dairy enumeration format: 9 mL per 16 × 125 mm tube.
9Autoclave. 121 °C × 15 min (the Scardovi 1986 original; some heat-sensitive variants use 115 °C × 15 min). The medium darkens to honey-amber from glucose-peptone Maillard during autoclaving — this is expected; excessive browning indicates over-autoclaving.
10Cool and reduce. Transfer to AAE while still warm. Allow 24-h equilibration in AAE before use. Note: TPY does not contain resazurin; for visual O2 indication, add 1 mg/L resazurin (custom-order variant).
11Optional: add mupirocin for TPY-Mup. After cooling to ≤ 50 °C inside the AAE, add 5 mL of Vial Mup (10 mg/mL mupirocin in 70 % ethanol, filter-sterilised) per litre = 50 mg/L final. Gently swirl.
12For TPY agar. Add 15 g/L agar pre-autoclaving. Pour at 50 °C; allow to set; bag for storage. ISO 29981 plate-count format: 10 mL per 60 mm Petri dish.

Critical control points

  • Iron precipitation control. FeCl3 in solution can precipitate as Fe(OH)3 above pH 4.5; the inclusion of cysteine and slight medium acidity (pH 6.5) help maintain iron in solution but require disciplined preparation order. Mitigation: dissolve at moderate temperature (50–60 °C); add FeCl3 last among trace elements; immediately before pH adjustment; use freshly-prepared FeCl3 stock (the supplied Vial Fe is stored at 4 °C and is light-protected); pre-warm the agar to 55 °C before iron addition.
  • Tween 80 oxidation. Polysorbate 80 contains an unsaturated oleic-acid moiety that auto-oxidises slowly during storage of the dehydrated medium. Storage of dehydrated TPY powder at 4 °C in a sealed container with desiccant extends useful shelf life from 12 to 24 months. Use a single Tween 80 supplier (Sigma P1754 is the de-facto reference); validate with B. animalis subsp. lactis BB-12 growth curve when changing lots.
  • pH measurement. Measure pH at 25 °C; the 6.5 ± 0.2 specification is tighter than BHI-S or mGAM. Bifidobacterium grows optimally at pH 6.5–7.0 (colon pH range); growth is significantly slower at pH 7.4 (the BHI / mGAM range).
  • Glucose-peptone Maillard. The 5 g/L glucose + peptone combination produces moderate Maillard browning during autoclaving — the medium darkens to honey-amber. Excessive browning (dark coffee colour) indicates over-autoclaving and is associated with reduced Bifidobacterium growth.

Cautions

Tween 80 brand and grade matter. Different commercial Tween 80 preparations contain different oleic / stearic / palmitic ester ratios and different residual peroxide content. Lot-to-lot variation can affect Bifidobacterium growth kinetics. Mitigation: use a single supplier (Sigma P1754 reference); validate each new lot with B. animalis subsp. lactis BB-12 OD600 growth curve.
TPY is non-selective in basic form. TPY supports both Bifidobacterium and lactic-acid bacteria (LAB) growth. For mixed-product QC (e.g., a yogurt containing S. thermophilus + L. delbrueckii subsp. bulgaricus + B. animalis subsp. lactis), use TPY-Mup (with mupirocin) for selective Bifidobacterium enumeration, OR use differential incubation at 45 °C to suppress Bifidobacterium growth (most strict Bifidobacterium optimum is 37 °C) and count the LAB fraction separately.
Aerotolerance varies by species. Most Bifidobacterium are strict anaerobes; B. animalis subsp. lactis BB-12 is slightly aerotolerant and can grow on un-reduced TPY agar inside a CO2 incubator. For ISO 29981 enumeration of dairy probiotics, anaerobic incubation is the standard; do not substitute CO2 incubation for compliance work.
Iron precipitation interfering with colony reading. If FeCl3 precipitates during agar cooling, brown / orange flecks can obscure colony morphology. Mitigation: careful pH control; freshly-prepared FeCl3 stock (10 mg/mL in 0.01 M HCl); pre-warm agar to 55 °C and add iron after dissolution of all other components.
Probiotic strain confidentiality. Many commercial Bifidobacterium probiotic strains (BB-12, BB536, M-16V, A1) are proprietary intellectual property. Customers' QC laboratories sometimes ask for strain-specific recovery validation — GMExpression cannot supply or validate against unreleased proprietary strains; only publicly-available type-strain depositions (ATCC, DSMZ, JCM) are appropriate reference materials.
Taxonomic flux. The Bifidobacterium genus has been substantially reorganised in 2020–2024 with new species (B. samirii, B. felinis, B. choloepi) and re-elevation of subspecies (e.g., B. longum subsp. infantis sometimes treated as a separate species). For product labelling, refer to current LPSN (lpsn.dsmz.de) for accepted species names.

Storage and Expiry · Safety

  • Dehydrated Mixture A: 4 °C in original packaging with desiccant. Shelf life 24 months (the Tween 80 component is the limit; without desiccant, reduce to 12 months).
  • Mixture B (L-cysteine, N2-flushed): 4 °C in sealed original packaging. Shelf life 18 months sealed; use within 14 days of opening (cysteine oxidises rapidly once exposed to air).
  • Stock Fe (FeCl3): 4 °C, light-protected (amber vial). Shelf life 12 months sealed; 6 months after opening.
  • Stock T (Tween 80): 4 °C, light-protected. Shelf life 12 months. Once opened, use within 6 months.
  • Vial Mup (Mupirocin in 70 % ethanol): 4 °C for 6 months; −20 °C for 12 months. Aliquot to single-use portions to avoid freeze-thaw cycling.
  • Prepared broth, aerobic 4 °C: 4 weeks routine.
  • Prepared broth, anaerobic (vacuum-sealed + O2 absorber): 4–6 months for strict-anaerobe use.

Safety notes. Tween 80 is a non-hazardous food-grade material but is a mild eye irritant. Ferric chloride solution is corrosive (HCl carrier); use eye protection when handling Stock Fe. Mupirocin is a topical antibiotic — handle with skin protection; avoid inhalation of dry powder if preparing from scratch. SDS available on request.

References

  1. Scardovi V. (1986). Genus Bifidobacterium. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG (eds), Bergey's Manual of Systematic Bacteriology vol. 2, pp. 1418–1434. Williams & Wilkins, Baltimore. [Primary reference for TPY formulation]
  2. Scardovi V, Trovatelli LD. (1965). The fructose-6-phosphate shunt as a peculiar pattern of hexose degradation in the genus Bifidobacterium. Annales di Microbiologia 15: 19–29. (Underlying metabolic basis for TPY's fructose / glucose content.)
  3. ISO 29981:2010. Milk products — Enumeration of presumptive Bifidobacteria — Colony count technique at 37 °C. [TPY-based enumeration standard for the dairy industry]
  4. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP). (2018). Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA Journal 16(3): 5206. (TPY for Bifidobacterium AST.)
  5. Goldin BR, Gorbach SL. (1992). Probiotics for humans. In: Fuller R (ed), Probiotics — The Scientific Basis. Chapman & Hall. (TPY in probiotic context.)
  6. Zheng J et al. (2020). A taxonomic note on the genus Lactobacillus: description of 23 novel genera. IJSEM 70: 2782–2858. (Reclassification relevant to TPY co-cultivable LAB.)
  7. ATCC Microbiology Catalogue: Medium 1058 (TPY).
  8. DSMZ Microbiology Catalogue: Medium 56 (Bifidobacterium medium).

Frequently Asked Questions

Q1. Is TPY the only medium for Bifidobacterium enumeration in dairy products?
ISO 29981:2010 specifies TPY as the reference medium for Bifidobacterium enumeration in milk products. Alternative media in different applications include BSM (Bifidus Selective Medium, with mupirocin and propionic acid) and MRS-NNLP (de Man Rogosa Sharpe + nalidixic acid + neomycin + lithium chloride + paromomycin). ISO 29981 mandates TPY for compliance work; for non-ISO applications other media are acceptable. The GMExpression TPY Kit is ISO 29981-conforming.
Q2. Can I use MRS broth instead of TPY for Bifidobacterium?
MRS (de Man Rogosa Sharpe) is the reference medium for Lactobacillus and most lactic-acid bacteria; it also supports Bifidobacterium growth but with characteristically lower density and slower growth. For strict Bifidobacterium work — especially ISO 29981 compliance enumeration — use TPY. MRS is acceptable for screening but not for compliance-grade enumeration. The pH 6.5 of TPY versus 6.2 of MRS is part of the difference.
Q3. Why is the medium pH so much lower than for other anaerobes (6.5 vs 7.0–7.4)?
Bifidobacterium grows optimally at pH 6.5–7.0 (the colon pH range); growth is significantly slower at pH 7.4 (the BHI / mGAM range). The Scardovi 1986 specification of 6.5 ± 0.2 is the genus-optimum and is well-validated. Adjusting upward to 7.0 is acceptable but not preferred; the resulting OD600 at 48 h is typically ~30 % lower than at pH 6.5.
Q4. Can I add mupirocin or other antibiotics for selectivity?
Yes — for selective Bifidobacterium recovery from mixed cultures, use TPY-Mup (TPY + 50 mg/L mupirocin, post-autoclave filter-sterilised). Bifidobacterium are intrinsically mupirocin-resistant; most other Gram-positives (especially LAB that otherwise co-grow on TPY) are mupirocin-sensitive. Mupirocin stock: 10 mg/mL in 70 % ethanol (Vial Mup); add 5 mL per litre at 50 °C. Alternative selective: LP-MRS (lithium chloride 3 g/L + propionic acid 5 mL/L).
Q5. Do I need an anaerobic chamber for TPY work?
For Bifidobacterium primary isolation and accurate enumeration, yes — most Bifidobacterium are strict anaerobes (with the partial exception of B. animalis subsp. lactis, mildly aerotolerant). For routine subculture of established lab strains of aerotolerant variants, microaerophilic conditions (5–10 % CO2 + 5–10 % O2) are acceptable. The GMExpression Anaerobic Preparation Kit (APK) workflow applies to TPY identically to YCFA.
Q6. How does TPY perform for Akkermansia muciniphila?
Poorly. Akkermansia requires mucin substrate as a glycan source; TPY lacks mucin. For Akkermansia, use GMM (Gut Microbiota Medium, § B1) which contains 4 g/L porcine gastric mucin, or BHI-Mucin (BHI + 4 g/L porcine gastric mucin). TPY supports the Bifidobacterium genus but not the broader mucin-degrading commensal community.
Q7. Can TPY be used for enumeration of Bifidobacterium in human faecal samples?
Yes, but with caveats. TPY supports all human-gut Bifidobacterium species and is therefore appropriate for genus-level enumeration from faeces. However: (a) faeces contain very high backgrounds of competing anaerobes (especially Bacteroides); use TPY-Mup for selectivity; (b) the relationship between TPY plate counts and 16S amplicon-based abundance estimates is not 1:1 due to differential culturability of Bifidobacterium species; (c) for population studies, qPCR of Bifidobacterium-specific 16S regions is increasingly preferred over plate counts.
Q8. Is TPY compatible with the GMExpression Anaerobic Preparation Kit (APK)?
Yes. The APK vacuum-deoxygenation workflow (Hungate-tube vacuuming + oxygen-absorber-equipped vacuum-sealed bag storage) is medium-agnostic and applies to TPY broth identically. The standard YCFA Modified Medium Instructions v2.8 Steps 8–11 apply. Note: TPY does not contain resazurin (the YCFA visual oxygen indicator); add 1 mg/L resazurin if visual indication is desired (custom-order variant available).