Hayflick Medium

Overview

Hayflick Medium — Leonard Hayflick's 1965 modification of Edward's PPLO Broth — is the single most-cited mycoplasma medium in cell-culture and clinical microbiology. It is the USP <63> Method I reference broth and the European Pharmacopoeia 2.6.7 reference medium for mycoplasma testing of biological products (cell substrates, recombinant proteins, vaccines, gene-therapy vectors, ATMPs). The defining features vs the original PPLO are a slightly higher glucose load (1 % w/v vs 0.5 %), 100 U/mL Penicillin G (vs 50), and a fully documented heritage as the cell-culture-testing standard.

The GMExpression formulation is supplied as a pre-balanced kit: Mycoplasma Broth Base (BD Difco 211458 or PPLO Broth Base BD 255420 equivalent, 21 g/L), heat-inactivated horse serum (lot-qualified against M. orale ATCC 23714 and M. pneumoniae ATCC 15531 and Acholeplasma laidlawii ATCC 23206 — the USP <63> positive-control panel), fresh yeast extract solution, glucose, Penicillin G, and phenol red. Broth and agar (1.4 % w/v mycoplasma-grade agar) formats supplied separately or as the combined kit for the full USP / EP testing workflow (broth + agar subculture at days 7, 14, 21).

We also have

PPLO Broth (foundational mycoplasma base) · SP-4 Medium (Spiroplasma / fastidious Mycoplasma) · Frey's Mycoplasma Broth (avian, NAD-supplemented) · Eaton's Modified Medium (M. pneumoniae) · A8 / U9 Broth (Ureaplasma) · Mycoplasma Growth Supplement 10×

Package Contents

Each GMExpression Hayflick kit contains:

Mixture A — pre-weighed Mycoplasma Broth Base (BD Difco 211458 / Bacto Mycoplasma Broth Base / PPLO Broth Base BD 255420 equivalent; beef-heart infusion + Bacto peptone + NaCl), total 21 g/L. For the agar variant, the base contains 14 g/L mycoplasma-grade agar in addition. Triple-foil-pouched; DAFF EX188M biosecurity-certified bovine source.
Stock HS — heat-inactivated horse serum (56 °C × 30 min), 1 L for the 5 L kit. Triple-lot-qualified: documented growth-titration against M. orale ATCC 23714 (cell-culture contaminant reference), M. pneumoniae ATCC 15531 (USP <63> FH-strain positive control), and Acholeplasma laidlawii ATCC 23206 (sterol-independent reference). Dose at 200 mL/L (= 20 % v/v) post-autoclave.
Stock YE — 25 % w/v fresh yeast extract solution, autoclaved within 7 days of dispatch, 500 mL. Dose at 100 mL/L (= 2.5 % w/v final).
Stock G — 50 % w/v glucose, filter-sterilised 0.22 µm, 100 mL. Dose at 20 mL/L (= 1 % w/v final — 2× the standard PPLO load).
Stock P — Penicillin G sodium, 10,000 U/mL stock, filter-sterilised, 50 mL. Dose at 10 mL/L (= 100 U/mL final — 2× the PPLO Pen load; for heavy clinical specimens dose at 5× for 500 U/mL).
Stock PR — 0.5 % w/v phenol red, autoclaved, 20 mL. Dose at 4 mL/L (= ~ 20 µg/mL final).
Optional positive-control reference strain panel (lyophilised vials of M. pneumoniae FH ATCC 15531, M. orale ATCC 23714, Acholeplasma laidlawii ATCC 23206; quantified ≤ 100 CFU/vial per the USP <63> spec).
Instruction manual including the full USP <63> Method I protocol (28-day incubation; subculture at days 7, 14, 21), EP 2.6.7 protocol, fried-egg colony identification annex, positive-control titration protocol, and a regulatory-documentation pack (A5 booklet, v1.0).

Customisation options on request: FBS substitute for horse serum, arginine-supplemented variant, additional Amphotericin B 2.5 µg/mL, 96-well plate microdilution-format pre-dispense, GMP-grade lot-release certificate with extended traceability documentation.

Composition — per 1 L equivalent unless stated otherwise

Hayflick Broth (Hayflick 1965 / USP <63> Method I reference; per 1 L)

Component	Concentration	Function
Mycoplasma Broth Base (BD Difco 211458 / BD 255420 PPLO equivalent)	21.0 g	Beef-heart infusion + proteose peptone + NaCl
Horse serum, heat-inactivated 56 °C × 30 min	200 mL (= 20 % v/v)	Cholesterol, free fatty acids, albumin — non-negotiable for sterol-dependent species
Fresh yeast extract (25 % w/v stock)	100 mL (= 2.5 % w/v final)	B-vitamins, purines, pyrimidines, NAD precursors
Glucose (50 % w/v, filter-sterilised)	20 mL (= 1 % w/v final — 2× PPLO standard)	Fermentable carbon; drives rapid acidification on positive growth (clear yellow shift within 5–7 d)
Penicillin G (10,000 U/mL stock)	10 mL (= 100 U/mL final — 2× PPLO standard)	Selective; moderate dose for cell-culture screening; raise to 200–500 U/mL for heavy clinical specimens
Phenol red (0.5 % w/v)	4 mL (= ~ 20 µg/mL)	pH indicator
Sterile water	q.s. 1000 mL	—

Pre-autoclaving base pH: 7.8 (overshoot). Final pH after supplements: 7.6 ± 0.2 at 25 °C; equilibrates to 7.4 in 5 % CO₂.

Hayflick Agar (USP <63> Method I + Method II reference; per 1 L)

Component	Concentration	Notes
Mycoplasma Agar Base (BD 211404; same beef-heart / peptone / NaCl + 14 g/L mycoplasma-grade agar)	35.0 g	Total dehydrated solids 35 g/L; agar is 1.4 % (vs 1.5 % for bacterial agar — the softer agar supports fried-egg colony formation)
Post-autoclave supplements	identical to broth	20 % HS + 2.5 % YE + 1 % glucose + 100 U/mL Pen G + 20 µg/mL phenol red; pour plates at 50 °C

→Culturomics and Media Navigator

Need help matching this medium to your regulatory workflow — or starting from a target cell substrate / biological product and finding the right medium? Use the GMExpression Culturomics and Media Navigator: a curated, cross-referenced tool that lets you (i) start from a medium and view supported Mollicute targets, or (ii) start from a regulatory standard (USP <63>, EP 2.6.7, JP 4.07, FDA PTC for cell-substrate testing) and view the panel of GMExpression media for compliance. The Navigator integrates Hayflick Medium with the wider PPLO, SP-4, Frey's, Eaton's, A8/U9, MGS-10×, LB, 2×YT, NZCYM, and BHI catalogue.

Use and Applications

USP <63> Method I Mycoplasma Test (broth + agar testing of biological products). 5 mL Hayflick broth + 1 mL test sample; 5 mL Hayflick broth + 0.5 mL positive control (≤ 100 CFU M. pneumoniae ATCC 15531). Incubate 28 d at 35 ± 1 °C, 5–10 % CO₂. Subculture to Hayflick agar at days 7, 14, 21; examine for fried-egg colonies at × 20–40 magnification.
European Pharmacopoeia 2.6.7 Mycoplasma Test. Same as USP <63> with the additional positive control M. fermentans (EP-specific). The GMExpression EP-panel kit adds the M. fermentans reference strain to the USP panel.
Japanese Pharmacopoeia 4.07 Mycoplasma Testing for cell substrates and biologics — compatible with the Hayflick formulation.
FDA Points-to-Consider Mycoplasma testing for cell-substrate qualification of vaccine, recombinant-protein, and cell-therapy products.
Cell-culture mycoplasma contamination screening (research-grade). Inoculate 0.5 mL of cell-culture supernatant into 5 mL Hayflick broth; 21 d at 37 °C, 5 % CO₂; subculture to Hayflick agar at days 7, 14, 21.
Primary isolation from clinical specimens — respiratory, urogenital, oropharyngeal. Use Pen G 200–500 U/mL + Amphotericin B 2.5 µg/mL for heavily contaminated specimens.
Reference strain maintenance for the USP / EP / JP positive controls: M. pneumoniae FH ATCC 15531, M. orale ATCC 23714, Acholeplasma laidlawii ATCC 23206, M. fermentans ATCC 19989, M. arginini ATCC 23838, M. hyorhinis ATCC 17981.
Indicator-cell co-cultivation (Method II) — Hayflick agar plates co-incubated with Vero or 3T3 indicator cells + test sample; mycoplasma binding detected by Hoechst 33258 DNA stain. The agar serves as the visual confirmatory subculture.

Compatible Microorganisms

Regulatory positive-control reference strains (USP / EP)

Mycoplasma pneumoniae ATCC 15531 (FH strain) — USP <63> Method I positive control; glucose-utiliser; yellow colour shift
Mycoplasma orale ATCC 23714 — USP positive control; arginine-utiliser; magenta colour shift; cell-culture contaminant reference
Acholeplasma laidlawii ATCC 23206 — USP positive control; sterol-independent; bovine origin; cell-culture contaminant
Mycoplasma fermentans ATCC 19989 — EP-additional positive control; glucose-utiliser; slow grower
Mycoplasma hyorhinis ATCC 17981 — cell-culture contaminant; arginine-utiliser; PCR target
Mycoplasma arginini ATCC 23838 — bovine origin; arginine-utiliser; cell-culture contaminant
Mycoplasma salivarium ATCC 23064 — oropharyngeal human commensal

The cell-culture mycoplasma "Big Five"

M. orale, M. hyorhinis, M. arginini, M. fermentans, A. laidlawii — account for > 90 % of all documented cell-culture contaminations in the literature.

Most other human and clinical Mycoplasma

M. hominis, M. genitalium (SP-4 preferred for primary isolation), M. salivarium, M. buccale

Not optimised for: Ureaplasma (use A8 / U9 broth, pH 6.0), avian Mycoplasma (use Frey's, NAD-supplemented), Spiroplasma (use SP-4 / ATCC 988), M. pneumoniae primary isolation from heavily contaminated respiratory specimens (Eaton's preferred at pH 7.4 + 1000 U/mL Pen).

Preparation

1Weigh. Use the pre-weighed Mixture A: 21 g per 1 L (broth) or 35 g per 1 L (agar). Tare a clean autoclavable Schott bottle.

2Suspend & dissolve. Add Mixture A to 700 mL of distilled or deionised water. Stir 5 min; for the agar variant, heat to gentle boil while stirring for 10–15 min until completely dissolved.

3Adjust pH. Target 7.8 ± 0.2 at 25 °C (overshoot; serum drops by ~ 0.2). Adjust with 1 M NaOH or 1 M HCl if needed.

4Bring to 700 mL final pre-supplement volume; autoclave the base. 121 °C × 15 min, slow cooling. Cool to exactly 50 °C for the agar variant (critical — too hot denatures serum, too cool allows agar to set).

5Combine supplements in Class II BSC. Aseptically add to cooled base in order: 200 mL Stock HS (heat-inactivated horse serum — pre-qualified vs USP panel), 100 mL Stock YE (fresh yeast extract), 20 mL Stock G (glucose), 10 mL Stock P (Penicillin G), 4 mL Stock PR (phenol red). Mix gently — minimise serum frothing.

6Verify final pH. 7.6 ± 0.2 at 25 °C. Adjust with sterile 1 N NaOH or HCl if needed.

7Filter-sterilise the complete medium at 0.22 µm PES (low-protein-binding) for cell-culture-testing-grade work — mandatory for USP <63> / EP 2.6.7 lot release; recommended for all regulated workflows.

8Aseptic dispensing. Broth: 5 mL in 16 × 100 mm sterile screw-cap tubes (USP standard). Agar: pour 4 mL per 35 mm Petri dish (USP small-dish standard) or 20 mL per 90 mm dish; let set on a level surface, lids slightly ajar in BSC for 15 min; dry lid-down at 35 °C × 1 h before use.

9Sterility QC. Incubate 10 mL of finished medium at 37 °C × 48 h before release; check for turbidity. Positive-control titration with M. pneumoniae FH (expect colour shift within 7–14 d at ≤ 100 CFU input).

10Storage. Complete medium 2–8 °C in amber bottles, fully filled, ≤ 14 d. Working aliquots in tubes ≤ 7 d.

Critical control points

Triple-strain serum lot qualification. The Hayflick formulation is the regulatory reference, so the serum lot must be qualified against the full USP positive-control panel: M. pneumoniae FH ATCC 15531, M. orale ATCC 23714, Acholeplasma laidlawii ATCC 23206 (and on request M. fermentans ATCC 19989 for EP). The GMExpression Stock HS is pre-qualified; documented growth-titration certificate supplied with each lot.
USP <63> incubation conditions are exact. 28 days, 35 ± 1 °C (not 37 °C), 5–10 % CO₂; subcultures at days 7, 14, 21. Deviations from this protocol invalidate the lot-release test under USP <63>.
Positive-control titration. The USP <63> positive control must contain ≤ 100 CFU of the reference organism; growth in this tube validates the medium and incubation conditions. The negative control (medium alone) must show no growth.
Fried-egg morphology on agar. Confirm under stereomicroscope at × 20–40 (not at × 100–400 — mycoplasma colonies are too small to resolve as fried-egg at higher mag). Image documentation is part of the regulatory submission package.
Method II indicator-cell co-cultivation requires concurrent Hayflick agar use for confirmatory subculture. Method I (broth alone) is acceptable for some submissions but Method II is the EP-recommended Standard.

Cautions

Triple-strain serum lot qualification is regulatory-required. Anti-mycoplasma antibody titres in horse serum vary by donor herd; some lots support M. orale well but fail to recover M. pneumoniae, or vice versa. The USP <63> Method I lot-release test mandates qualification against the full positive-control panel. Skipping or partial lot qualification is the single most common audit finding in regulated mycoplasma-testing workflows.

USP <63> / EP 2.6.7 growth-promotion panel. Both USP <63> and EP 2.6.7 specify a multi-species panel for growth-promotion qualification of the medium — typically A. laidlawii, M. fermentans, M. hyorhinis, M. orale, M. pneumoniae, and M. synoviae, with M. arginini and S. citri added in some workflows. The two pharmacopoeias differ in detail (incubation, subculture timing, indicator-cell choice for Method II) more than in panel membership. Specify the regulatory jurisdiction at order so the correct serum-qualification and growth-promotion certificate is supplied.

Hayflick is NOT optimised for Ureaplasma. Ureaplasma require buffered pH 6.0 and urea-rich medium (A8 / U9 broth, M10 in the GMExpression KB); the Hayflick pH 7.6–7.8 + glucose formulation actively suppresses Ureaplasma growth. If the test sample is expected to contain Ureaplasma, run Hayflick + A8/U9 in parallel.

BSE/TSE handling for bovine beef-heart and bovine serum substitutes. The Mycoplasma Broth Base contains bovine beef-heart infusion; horse serum is equine-derived (low TSE risk). If FBS is substituted for HS in a custom formulation, the FBS must carry full TSE-certified zoosanitary documentation. GMExpression uses the Australian DAFF EX188M-certified pipeline for both bovine and equine raw materials.

Fungal contamination at days 14–21. 28-day USP <63> incubations are highly susceptible to fungal contamination, particularly when working with respiratory or oropharyngeal specimens. Add Amphotericin B 2.5 µg/mL or Nystatin 50 U/mL to specimen-derived tubes; never to the positive-control tubes (false-negative risk).

Phenol-red colour change is not specific. Bacterial contamination (penicillin failure or Gram-negative breakthrough) produces identical yellow / magenta phenol-red shifts. Every colour change must be confirmed by (i) Gram stain or darkfield microscopy, (ii) subculture on Hayflick agar for fried-egg colony morphology, and (iii) species-confirmation by PCR. Single-colour-shift evidence is insufficient for USP / EP positive identification.

Storage and Expiry · Safety

Dehydrated powder (Mixture A, broth or agar): store sealed at 15–25 °C in original packaging away from direct sunlight. Shelf life 36 months from manufacture.
Reconstituted, sterilised base (unsupplemented): 2–8 °C in tightly sealed glass, 12 months.
Stock HS (lot-qualified heat-inactivated horse serum): −20 °C in single-use aliquots, 24 months; 2–8 °C, 4 weeks; ≤ 3 freeze-thaw cycles.
Stock YE (25 % yeast extract, fresh): 2–8 °C, 7 days.
Stock G (50 % glucose): 2–8 °C, 12 months.
Stock P (Penicillin G 10,000 U/mL): −20 °C aliquots, 6 months; 2–8 °C, 2 weeks.
Complete Hayflick broth (with serum): 2–8 °C in amber bottles, fully filled, 14 days maximum.
Working aliquots in 5 mL tubes: 2–8 °C, 7 days maximum.
Poured Hayflick agar plates: 2–8 °C in sealed bags, 7 days maximum (small-volume mycoplasma plates dry rapidly even when sealed).
Positive-control lyophilised reference strains: −80 °C, 5 years; resuscitate per USP protocol within 24 h of use.

Safety notes. Hayflick Medium supports BSL-2 pathogens (M. pneumoniae, M. fermentans, M. genitalium). All test work in a Class II BSC. Sample-handling waste (cell-culture supernatants, clinical specimens, contaminated tubes) must be autoclaved before disposal. The lot-qualification reference strain panel is shipped as BSL-2 lyophilised material; follow institutional biosafety procedures for resuscitation and handling. SDS available on request.

References

Hayflick, L. (1965). Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine 23 (Suppl 1): 285–303. [Original Hayflick Medium]
Hayflick, L. & Stanbridge, E. (1967). Isolation and identification of mycoplasma from human clinical materials. Annals of the New York Academy of Sciences 143: 608–621.
USP <63> Mycoplasma Tests, current revision. United States Pharmacopeial Convention.
European Pharmacopoeia 2.6.7 Mycoplasmas, current revision.
Japanese Pharmacopoeia 4.07 Mycoplasma Testing, current revision.
FDA (2010). Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications. CBER.
Razin, S., Yogev, D., Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews 62: 1094–1156.
Nikfarjam, L. & Farzaneh, P. (2012). Prevention and detection of mycoplasma contamination in cell culture. Cell Journal (Yakhteh) 13: 203–212.
BD Difco & BBL Manual, 12th ed., Mycoplasma Broth Base (BD 211458) and Mycoplasma Agar Base (BD 211404) monographs.

Frequently Asked Questions

Q1. What is the difference between Hayflick Medium and PPLO Broth?

Hayflick Medium is a modification of Edward's original PPLO Broth (1947) with: (i) higher glucose load (1 % vs 0.5 % — drives faster colour shift on positive growth); (ii) higher Penicillin G (100 vs 50 U/mL — more selective for clinical isolation); (iii) Hayflick's documented heritage as the cell-culture-testing reference (USP <63> Method I); and (iv) a defined positive-control strain panel that includes M. pneumoniae, M. orale, and Acholeplasma laidlawii for lot release. Functionally the two media are very similar; use PPLO for routine veterinary mycoplasmology; use Hayflick for USP / EP / JP regulated cell-culture testing.

Q2. What incubation conditions does USP <63> mandate?

28-day incubation at 35 ± 1 °C (not 37 °C), with subcultures from broth to agar at days 7, 14, and 21, examined for fried-egg colony morphology at days 14, 21, and 28. The atmosphere is air + 5–10 % CO₂. Positive control: ≤ 100 CFU of M. pneumoniae ATCC 15531; the positive-control tube must show colour shift and/or fried-egg colonies within the 28-day window. Negative control: medium alone, must show no growth. Deviations from these specifications invalidate the lot-release.

Q3. What is the positive-control strain panel under USP <63> / EP 2.6.7?

Both USP <63> and EP 2.6.7 require growth-promotion qualification of the medium against a multi-species panel of reference Mollicutes — typically A. laidlawii, M. fermentans, M. hyorhinis, M. orale, M. pneumoniae, and M. synoviae (with M. arginini and S. citri added in some workflows). These span the glucose-utiliser, arginine-utiliser, urea-utiliser-adjacent, and sterol-independent metabolic classes and cover the principal cell-culture contaminants. For each actual test the user typically selects a representative positive-control inoculum from this panel (commonly M. pneumoniae ATCC 15531 at ≤ 100 CFU) to validate the run. USP, EP and JP differ more in procedural detail (incubation, subculture timing, indicator-cell Method II implementation) than in panel membership. The GMExpression USP / EP panel kits supply lyophilised reference strains spanning the full growth-promotion panel; specify the regulatory pathway at order.

Q4. Can I use Hayflick Medium for primary isolation of M. pneumoniae from respiratory specimens?

Possible but suboptimal. Hayflick supports M. pneumoniae at pH 7.6 with 100 U/mL Pen G; primary isolation from heavily contaminated respiratory specimens benefits from Eaton's Medium (M09 in the GMExpression KB) at pH 7.4 with 1000 U/mL Pen G + polymyxin B. For reference-strain maintenance and USP <63> positive-control work, Hayflick is the regulatory medium. For diagnostic primary isolation from throat swabs or bronchoalveolar lavage, Eaton's is preferred for higher recovery from contaminated specimens.

Q5. Why is the agar concentration 1.4 %, not 1.5 %?

Mycoplasma colonies grow partially into the agar surface, producing the characteristic fried-egg morphology — a dense central zone where the colony has invaded the agar, and a translucent peripheral halo where the colony has expanded along the agar surface. At 1.5 % w/v (the standard bacterial agar concentration), the agar is too firm to support inversion-style growth and the colonies appear as flat surface colonies, losing the fried-egg morphology that is the diagnostic visual. The 0.1 % softer agar (1.4 % w/v) is also the BD Difco / Oxoid / HiMedia commercial-spec mycoplasma agar concentration. Mycoplasma-grade agar additionally must be low in sulfate and cation content (high sulfate impairs colony growth).

Q6. How do I prepare the ≤ 100 CFU USP <63> positive control?

Resuscitate the lyophilised M. pneumoniae FH ATCC 15531 reference vial in 1 mL Hayflick broth; incubate 7–14 d at 35 °C; verify viability by colour shift. Determine the working titre by serial dilution and CFU enumeration on Hayflick agar. Adjust the working stock so that 0.5 mL contains 10–100 CFU; this is the USP <63> positive-control inoculum. The titration must be performed for every lot of complete Hayflick medium (the cholesterol content of the serum lot affects the recovery limit). Maintain a 12-month reserve of validated working stock at −80 °C in Hayflick + 15 % glycerol.

Q7. Can Hayflick Medium be used for AST of mycoplasma isolates?

Yes, but the CLSI does not formally specify a mycoplasma AST broth (no M11-equivalent for mycoplasma). For AST of M. pneumoniae, M. hominis, and M. genitalium, the published literature primarily uses Hayflick broth or SP-4 broth (Bébéar & Kempf 2005); for Ureaplasma use A8/U9; for M. hyopneumoniae use Friis. The standard format is broth microdilution in 96-well plates with 10⁴–10⁵ CFU/mL inoculum and 48–120 h incubation; MIC read as the lowest concentration with no colour shift. Specify the species at order so the appropriate buffering and indicator concentrations are pre-configured.

Q8. Does GMExpression supply a Hayflick variant for indicator-cell co-cultivation (Method II)?

Yes — Hayflick Agar is the Method II co-cultivation substrate. The standard EP 2.6.7 Method II protocol uses Vero, 3T3, or A549 indicator cells plated on Hayflick agar plates with the test sample; mycoplasma binding is detected by Hoechst 33258 nuclear DNA stain after 5–7 days incubation. The Hayflick agar serves both as the indicator-cell substrate and as the confirmatory subculture medium for any broth-positive Method I result. The GMExpression Hayflick Combination Kit (broth + agar) supports both Method I and Method II in parallel; ask for the indicator-cell protocol annex with order.