Overview

PPLO Broth — Pleuropneumonia-Like Organism Broth — is the foundational broth base for sterol-dependent Mycoplasma cultivation and the lineage ancestor of essentially every commercial mycoplasma broth in use today. Originally developed by D. G. ff. Edward in 1947 at the Wellcome Research Laboratories for Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia, CBPP), PPLO Broth provides the beef-heart infusion + proteose peptone + NaCl base which, when supplemented with heat-inactivated horse serum (sterols), yeast extract (B-vitamins + nucleobases), glucose (carbon), and Penicillin G (selection), supports the majority of veterinary and human Mycoplasma species.

The GMExpression formulation supplies the dehydrated base (21 g/L; matches BD Difco PPLO Broth without Crystal Violet 255420 / Oxoid CM403 / HiMedia M105 / ATCC Medium 243) with a full set of pre-qualified post-autoclave supplements: heat-inactivated horse serum (lot-qualified against M. orale ATCC 23714), 25 % fresh yeast extract solution, 50 % filter-sterilised glucose, Penicillin G stock, and 0.5 % phenol red. The complete medium follows the USP <63> Method I and European Pharmacopoeia 2.6.7 reference formulation for biological-product mycoplasma testing.

We also have

Hayflick Medium (cell-culture mycoplasma testing) · 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 PPLO Broth kit contains:

  • Mixture A — pre-weighed PPLO Broth Base (beef-heart infusion solids 5 g, Bacto Peptone 10 g, NaCl 5 g; total 21 g per 1 L). DAFF EX188M biosecurity-certified bovine source. Triple-foil-pouched for 5 or 10 L final volume. CofA traceable to the beef-heart and peptone supplier lots.
  • Stock HS — heat-inactivated horse serum (56 °C × 30 min), 1 L for the 5 L kit. Lot-qualified against M. orale ATCC 23714 with documented growth-titration certificate. 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).
  • Stock G — 50 % w/v glucose, filter-sterilised 0.22 µm, 50 mL. Dose at 10 mL/L (= 0.5 % w/v final) for glucose-fermenting species.
  • Stock P — Penicillin G sodium, 10,000 U/mL stock, filter-sterilised, 25 mL. Dose at 5 mL/L (= 50 U/mL final) for selection.
  • Stock PR — 0.5 % w/v phenol red, autoclaved, 10 mL. Dose at 2 mL/L (= ~ 10 µg/mL final) as pH indicator.
  • Instruction manual including USP <63> Method I protocol, cell-culture mycoplasma screening protocol, primary isolation from clinical specimens, fried-egg colony identification annex (A5 booklet, v1.0).

Customisation options on request: FBS substitute for horse serum (sterol-equivalent; preferred for some Spiroplasma-adjacent species), arginine-supplemented variant (1 g/L L-arginine for arginine-utilising species), additional Amphotericin B 2.5 µg/mL for fungal selection, Mycoplasma Growth Supplement 10× (MGS) format for compact storage.

Composition — per 1 L equivalent unless stated otherwise

PPLO Broth Base (Edward 1947 / BD Difco 255420 / Oxoid CM403 / ATCC Medium 243; per 1 L)

ComponentConcentrationFunction
Beef heart, infusion from50.0 g raw (yields ~ 5 g infusion solids in dehydrated powder)Cardiac-muscle digest; amino acids, dipeptides, trace lipids native to cardiac tissue
Bacto Peptone (Proteose Peptone No. 3)10.0 gPancreatic / papaic digest of meat; supplies tryptophan and tyrosine
Sodium chloride (NaCl)5.0 gOsmotic balance (86 mM)

Total dry solids: 21 g/L (matches the commercial dehydrated formulation). Pre-autoclaving pH: 7.8 ± 0.2 at 25 °C (overshoot; serum addition drops the final pH by ~ 0.2).

Complete PPLO + supplements (typical sterol-dependent Mycoplasma formulation)

ComponentFinal concentration / volumeFunction
PPLO Broth Base (Mixture A)21 g/L (pre-autoclave)Beef-heart / peptone base
Horse serum, heat-inactivated 56 °C × 30 min (Stock HS)200 mL/L (= 20 % v/v); post-autoclaveCholesterol (~ 400–600 µg/mL contribution), free fatty acids, albumin, lipoproteins
Fresh yeast extract 25 % w/v (Stock YE)100 mL/L (= 2.5 % w/v); post-autoclaveB-vitamins, purines, pyrimidines, NAD precursors
Glucose 50 % w/v (Stock G; optional)10 mL/L (= 0.5 % w/v); post-autoclaveFermentable carbon for glucose-utilising species (M. pneumoniae, M. fermentans, etc.)
Penicillin G 10,000 U/mL (Stock P)5 mL/L (= 50 U/mL final); post-autoclaveSelects against wall-bearing bacterial contaminants; no effect on cell-wall-free Mollicutes
Phenol red 0.5 % (Stock PR)2 mL/L (= ~ 10 µg/mL); pre- or post-autoclavepH indicator (yellow at pH < 7.0; red at 7.4; magenta at > 8.0)
Amphotericin B (optional)2.5 µg/mL final; post-autoclaveFungal selection — recommended for long (≥ 14 d) incubations

Final pH after all supplements: 7.6–7.8 at 25 °C; equilibrates to 7.4 in a 5 % CO2 incubator.

→Culturomics and Media Navigator

Need help matching this medium to your target Mollicute — or starting from a target Mycoplasma species 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 supported Mollicute species, or (ii) start from a target organism (genus, species, or ATCC / DSMZ / NCTC strain ID) and view the panel of GMExpression media for isolation, propagation, and identification. The Navigator integrates PPLO Broth with the wider Hayflick, SP-4, Frey's, Eaton's, A8/U9, MGS-10×, LB, 2×YT, NZCYM, and BHI catalogue.

Use and Applications

  • USP <63> Method I / EP 2.6.7 Mycoplasma testing of biological products. PPLO Broth + 20 % horse serum + 2.5 % yeast extract + Pen G — the regulatory reference broth for direct culture of biological products (cell substrates, vaccines, recombinant proteins, gene-therapy vectors). Incubate 28 d at 35 ± 1 °C, 5–10 % CO2; subculture to PPLO Agar at days 7, 14, 21; positive control M. pneumoniae ATCC 15531 (≤ 100 CFU).
  • Cell-culture mycoplasma contamination screening. Inoculate 0.5 mL of cell-culture supernatant into 5 mL PPLO Broth; 21 d at 37 °C, 5 % CO2; check daily for phenol-red colour shift; subculture to PPLO Agar at days 7, 14, 21 for fried-egg colony confirmation.
  • Veterinary Mycoplasma isolation from bovine, ovine, caprine, and swine specimens — mastitis, pneumonia, arthritis, and reproductive-tract isolates. Standard medium for veterinary diagnostic mycoplasmology.
  • Primary isolation from clinical specimens — urogenital, respiratory, oropharyngeal swabs. Add Amphotericin B 2.5 µg/mL to suppress fungal overgrowth during the long incubation.
  • Reference strain propagation and maintenance — passage M. orale ATCC 23714, M. arginini ATCC 23838, M. hyorhinis ATCC 17981, M. salivarium ATCC 23064, and most veterinary reference strains every 5–10 d.
  • Acholeplasma laidlawii cultivation — sterol-independent, grows in PPLO base alone (serum optional but improves yield).
  • Serum lot qualification — PPLO Broth + candidate serum lot × reference strain titration; the standard method for serum-lot acceptance in mycoplasma laboratories.
  • Cryostock preservation — PPLO broth + 15 % glycerol or 10 % DMSO at −80 °C as the standard cryomatrix for Mycoplasma strains.

Compatible Microorganisms

Reference Mycoplasma strains (USP / EP / cell-culture testing)

  • M. pneumoniae ATCC 15531 (FH strain) — USP <63> positive-control organism
  • M. orale ATCC 23714 — cell-culture contaminant reference; serum-lot qualification
  • M. arginini ATCC 23838 — bovine origin; arginine-utiliser reference
  • M. hyorhinis ATCC 17981 — swine, cell-culture contaminant reference
  • M. salivarium ATCC 23064 — human oropharyngeal
  • Acholeplasma laidlawii ATCC 23206 — sterol-independent reference

Veterinary Mycoplasma

  • M. mycoides subsp. mycoides (CBPP, OIE-notifiable) — bovine
  • M. bovis — bovine pneumonia, mastitis
  • M. ovipneumoniae — ovine pneumonia
  • M. agalactiae — caprine and ovine contagious agalactia
  • M. capricolum, M. mycoides subsp. capri — caprine pleuropneumonia
  • M. canis, M. felis — companion-animal isolates

Human clinical Mycoplasma

  • M. hominis, M. genitalium (slow grower — SP-4 preferred), M. fermentans (slow grower — SP-4 preferred)
  • M. pneumoniae (Eaton's preferred for primary isolation)

Not optimised for: Ureaplasma (use A8 / U9, pH 6.0), avian Mycoplasma (use Frey's), Spiroplasma (use SP-4), M. pneumoniae primary isolation (Eaton's preferred), strictly anaerobic Anaeroplasma (anaerobic-modified PPLO).

Preparation

1Weigh. Use the pre-weighed Mixture A: 21 g per 1 L. Tare a clean autoclavable Schott bottle.
2Suspend & dissolve. Add Mixture A to 680 mL of distilled or deionised water (Type I or II reagent water; sized so that 680 mL base + 317 mL post-autoclave supplements + ~3 mL pH-adjustment q.s. = 1000 mL final). Stir for 5 min at room temperature; heat to gentle boil for 2–3 min if needed to fully dissolve the beef-heart infusion solids.
3Adjust pH. Check pH; target 7.8 ± 0.2 at 25 °C (overshoot; serum addition drops by ~ 0.2). Adjust with 1 M NaOH or 1 M HCl if needed.
4Autoclave the base. Bring to 680 mL final volume of base; do not yet add the 317 mL of supplements. Autoclave 121 °C × 15 min, slow cooling. Cool to < 50 °C.
5Heat-inactivate horse serum. If using BYO serum (Stock HS in the GMExpression kit is supplied pre-inactivated): thaw at 2–8 °C overnight; submerge bottle in 56 °C water bath; incubate exactly 30 min, swirl every 5 min; cool rapidly in ice bath; centrifuge 3000 × g × 10 min; sterile-decant.
6Combine supplements in BSC. In a Class II BSC, aseptically add to the cooled base in order: 200 mL Stock HS (heat-inactivated horse serum), 100 mL Stock YE (fresh yeast extract), 10 mL Stock G (glucose; for glucose-utilising species), 5 mL Stock P (Penicillin G), 2 mL Stock PR (phenol red). Mix gently — avoid serum frothing.
7Adjust final volume and pH. Final volume should be 1000 mL (680 mL base + 317 mL post-autoclave supplements + sterile-water q.s.). Verify final pH 7.6–7.8 at 25 °C; equilibrates to 7.4 in 5 % CO2.
8Optional final sterile filtration. Filter-sterilise complete medium at 0.22 µm PES (low-protein-binding) for cell-culture-testing-grade applications; recommended for USP <63> / EP 2.6.7 use to remove any aggregate carry-over.
9Aseptic dispensing. Typical aliquot: 5 mL in 16 × 100 mm screw-cap tubes for cell-culture screening; 100 mL in 250 mL flasks for primary isolation. Cap screw-tight; store at 2–8 °C in amber bottles with minimal headspace.
10Sterility check. Incubate 10 mL of finished medium at 37 °C × 48 h before release; check for turbidity. Run a positive-control growth check with M. orale ATCC 23714 (expect colour shift within 5–7 d).

Critical control points

  • Serum heat-inactivation is non-negotiable. 56 °C × 30 min inactivates the principal heat-labile complement components C1q (classical pathway initiator) and factor B (alternative pathway), abolishing complement-mediated lysis of cell-wall-less Mollicutes. Skipping this step is the single most common cause of mycoplasma growth failure — viability drops 90 %+ within 24 h.
  • Serum lot qualification. Cholesterol content of horse serum varies from 200 to 700 µg/mL between lots, and anti-mycoplasma antibody titres vary by donor herd. Test every new lot against M. orale ATCC 23714 before bulk purchase; maintain a 12-month reserve from each validated batch.
  • Fresh yeast extract. Yeast extract solution loses growth-promoting activity for Mollicutes over weeks at 2–8 °C. Use within 7 days of autoclave; for regulated workflows, prepare and consume the same week.
  • Amber storage. Cholesterol oxidises to 7-ketocholesterol (toxic to Mycoplasma at sub-µM concentrations) on light + air exposure. Store complete medium in amber bottles, fully filled, headspace minimised; use within 14 d.
  • CO2 equilibration. Most Mycoplasma prefer 5 % CO2; the medium pH drifts from 7.6–7.8 (room-air-loaded) to 7.4 in the CO2 incubator. Do not pre-equilibrate — the CO2 equilibration is part of the standardised growth condition.

Cautions

Phenol red colour change is not specific to mycoplasma. Bacterial contamination produces identical yellow / magenta phenol-red shifts. A colour change must always be confirmed by (i) darkfield or phase-contrast microscopy of the broth (Mollicutes appear as 0.2–0.3 µm pleiomorphic forms), (ii) subculture on PPLO Agar for fried-egg colony morphology, and (iii) species-confirmation by PCR (16S rRNA, tuf, or species-specific gene targets). Single-colour-shift evidence is insufficient for diagnosis.
BSE / TSE prion-risk handling for bovine beef-heart infusion. PPLO Broth Base contains beef-heart infusion solids. Although fully denatured during processing, some jurisdictions (EU, Japan, Canada CFIA) apply customs prion-risk checks. GMExpression uses the Australian DAFF EX188M zoosanitary-certificate pipeline; for jurisdictions requiring porcine-substituted PPLO, ask about the "PPLO-P" custom formulation (porcine-heart-infusion substitute).
Thallium acetate is regulatory-restricted. Historical PPLO formulations included thallium acetate at 0.025 % w/v as an additional Gram-negative selective. Modern GMExpression PPLO does not include thallium acetate (REACH-restricted in EU; TSCA notification in US; OH&S concerns globally). Use gentamicin 25–50 µg/mL or polymyxin B 50 U/mL as modern alternatives where Gram-negative contamination is a recurrent problem.
Sterol oxidation in stored medium. Cholesterol in horse serum oxidises to 7-ketocholesterol and other oxysterols on light + air exposure; oxysterols are toxic to Mycoplasma at sub-µM concentrations. Mitigation: amber bottles, fully filled, 2–8 °C, use within 14 d. For extended storage requirements, bubble N2 over serum before sealing.
Cross-contamination of cell-culture facilities by M. hyorhinis / M. orale. A major problem in research labs — once a cell-culture incubator is contaminated, the mycoplasma persists for months despite cleaning. Use dedicated mycoplasma-test BSCs and incubators; gloves changed between cell-culture and mycoplasma-handling tasks; monthly PCR screening of cell lines.

Storage and Expiry · Safety

  • Dehydrated powder (Mixture A): 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 (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): 2–8 °C, 7 days (prepare and consume fresh).
  • 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 medium (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.
  • Cryostocks of Mycoplasma strains: complete medium + 15 % glycerol at −80 °C or LN2 vapour phase, > 10 years documented for most species.

Safety notes. PPLO Broth supports BSL-2 pathogens (M. pneumoniae, M. hyopneumoniae, M. genitalium are BSL-2 in most jurisdictions; M. mycoides subsp. mycoides is OIE-notifiable and BSL-3 in non-endemic countries). Handle all primary isolation and unknown specimens in a Class II BSC. Beef-heart infusion solids are bovine-derived (DAFF EX188M certified). Horse serum is a biological hazard — handle in PPE; check serology certificate for equine pathogens (EIA, EVA, equine herpesvirus). SDS available on request.

References

  1. Edward, D. G. ff. (1947). A selective medium for pleuropneumonia-like organisms. Journal of General Microbiology 1: 238–243. [Original PPLO formulation]
  2. Hayflick, L. (1965). Tissue cultures and mycoplasmas. Texas Reports on Biology and Medicine 23 (Suppl 1): 285–303. [Cell-culture mycoplasma testing]
  3. Razin, S. (1962). Nutrition and metabolism of pleuropneumonia-like organisms (Mycoplasma). Annual Review of Microbiology 16: 1–18.
  4. Razin, S., Yogev, D., Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiology and Molecular Biology Reviews 62: 1094–1156.
  5. USP <63> Mycoplasma Tests, current revision. United States Pharmacopeial Convention.
  6. European Pharmacopoeia 2.6.7 Mycoplasmas, current revision.
  7. BD Difco & BBL Manual, 12th ed., PPLO Broth and PPLO Agar monographs (BD 255420 / 255410).
  8. Oxoid Manual (Thermo Fisher), 9th ed., Mycoplasma section (CM403).
  9. ATCC Medium 243 (Mycoplasma Broth) specification, current revision.
  10. Carroll, K. C., Pfaller, M. A., Landry, M. L., McAdam, A. J., Patel, R., Richter, S. S., Warnock, D. W. (eds) (2019). Manual of Clinical Microbiology, 12th ed., ASM Press, chapter on Mycoplasma and related organisms.

Frequently Asked Questions

Q1. What is the difference between PPLO Broth, Hayflick Medium, and Mycoplasma Broth with MGS supplement?
All three are functionally related sterol-dependent mycoplasma media built on the PPLO Broth Base lineage. PPLO Broth + 20 % horse serum + 2.5 % yeast extract is the general-purpose veterinary and clinical isolation formulation. Hayflick Medium is essentially the same recipe with a slightly higher glucose load (1 % vs 0.5 %), 100 U/mL Pen G (vs 50), and a defined heritage as the cell-culture-testing reference (USP <63> Method I). Mycoplasma Growth Supplement (MGS) 10× is a concentrated supplement (typically 8 % v/v horse serum + 0.25–0.5 % yeast extract + 0.1–0.5 % glucose + Pen G at 1×) intended for compact storage and direct addition to autoclaved PPLO base at 10 % v/v. Use PPLO + HS + YE for routine veterinary work; Hayflick for USP / EP cell-culture testing; PPLO + MGS-10× for compact / inventory-efficient workflows.
Q2. Why horse serum and not foetal bovine serum?
Horse serum is preferred over FBS for routine Mycoplasma work for two reasons. First, its complement system is more readily heat-inactivated at 56 °C × 30 min (FBS sometimes retains residual lytic activity that requires 56 °C × 45 min for full inactivation). Second, horses are rarely natural Mycoplasma hosts, so horse serum carries a low titre of anti-mycoplasma antibodies; bovine donors (the source of FBS) may carry anti-M. bovis, anti-M. arginini, and anti-M. hyorhinis antibodies that interfere with cell-culture-screening sensitivity. FBS is preferred for Spiroplasma work (where SP-4 specifies FBS) and for some cell-culture-adjacent applications where the FBS lot is already validated; horse serum is the default for PPLO.
Q3. How long does it take to see growth in PPLO Broth?
Highly species-dependent. Acholeplasma laidlawii and most veterinary Mycoplasmas: 2–5 d to visible colour shift. M. orale, M. arginini, M. hyorhinis: 5–10 d. M. pneumoniae: 14–21 d (Eaton's preferred for faster growth). M. fermentans, M. genitalium: 21–42 d (SP-4 strongly preferred for these fastidious species). For USP <63> / EP 2.6.7 cell-culture testing, the regulatory incubation period is 28 days with subcultures to PPLO Agar at days 7, 14, 21; this captures essentially all common cell-culture contaminants.
Q4. How do I recognise mycoplasma growth in PPLO Broth?
Three-step diagnostic workflow. (1) Phenol-red colour shift: yellow indicates acid production (glucose-utilisers like M. pneumoniae); magenta indicates ammonia production (arginine-utilisers like M. hominis, M. arginini). (2) Darkfield or phase-contrast microscopy: Mollicutes appear as 0.2–0.3 µm pleiomorphic forms (cocci, filaments, branched); cytometry alone is insufficient because contaminating bacteria look similar. (3) Subculture to PPLO Agar: incubate 5–14 d; observe at × 20–40 magnification for fried-egg colony morphology (dense central zone with translucent peripheral halo). Definitive identification by PCR (16S rRNA, tuf, or species-specific gene targets).
Q5. Can I use PPLO Broth for primary isolation of M. pneumoniae?
Possible but suboptimal. PPLO Broth supports M. pneumoniae with extended incubation (14–21 d) but recovery rate from clinical respiratory specimens is lower than with Eaton's Medium (M09 in the GMExpression mycoplasma KB), which has pH 7.4 (vs PPLO 7.6–7.8) and 1000 U/mL Pen G (vs PPLO 50) specifically optimised for M. pneumoniae from heavily contaminated specimens. For M. pneumoniae from culture stocks PPLO is acceptable; for diagnostic primary isolation, Eaton's is preferred.
Q6. Why does my PPLO medium turn yellow with no visible turbidity?
Four possible causes. (1) Bacterial contamination (penicillin failure or Gram-negative breakthrough): confirm by Gram stain and darkfield microscopy. (2) Sugar contamination from the serum lot or from glucose carry-over: switch serum lot; verify glucose stock. (3) Genuine mycoplasma growth by a fast-growing species (Acholeplasma, M. mycoides): confirm by subculture to agar and microscopy. (4) Phenol-red degradation from prolonged light exposure (turns toward yellow without organism): store in amber bottles. Run a parallel negative-control tube on every batch to discriminate (1) and (3) from (2) and (4).
Q7. Is PPLO Broth compatible with the USP <63> Method I sterility-test workflow?
Yes — PPLO + HS + YE + Pen G is the USP <63> Method I reference broth. The GMExpression formulation follows the USP and EP 2.6.7 specifications; the kit includes the lot-qualified horse serum with documented growth-titration against M. orale ATCC 23714 (and on request, against M. pneumoniae ATCC 15531 as the USP positive control). Use the kit with the 28-day incubation, subculture-at-7-14-21-days protocol, both Method I (direct broth) and Method II (indicator-cell agar) in parallel as required by the current revision.
Q8. Why do you not include thallium acetate as a selective agent?
Thallium acetate is restricted under EU REACH and requires notification under US TSCA due to mammalian toxicity (LD50 in rat < 30 mg/kg). Historical PPLO formulations used 0.025–0.05 % w/v as an additional Gram-negative selective; modern formulations (GMExpression included) substitute gentamicin 25–50 µg/mL or polymyxin B 50 U/mL, which give equivalent Gram-negative selection with substantially lower OH&S burden. For specialised veterinary applications where thallium acetate is documented as essential, a thallium-supplemented custom formulation can be supplied with full regulatory documentation; contact technical@gmexpression.com.