PPLO Broth Modified (GMExpression® Formula with Defined-Supplement) | For 5L

Overview

The GMExpression® Modified PPLO formulation is a multi-stock kit: a dehydrated PPLO Broth Base (Mixture A), and the post-autoclave supplements — heat-inactivated horse serum at 20 % v/v, reconstituted GMX-NVDS (replacing the fresh yeast extract), glucose, Penicillin G at 50 U/mL, and phenol red. For cell-culture-testing-grade applications, an optional terminal 0.22 µm filtration of the complete medium can be performed in your laboratory (Preparation step 8); it controls bacterial/fungal bioburden and does not retain mycoplasmas (see Cautions).

Package Contents

Each GMExpression Modified PPLO Broth kit (5 L) contains:

• Mixture A — pre-weighed PPLO Broth Base (beef-heart infusion solids 6 g (from 50 g raw beef heart) + proteose peptone 10 g + NaCl 5 g per 1 L; total 21 g/L; the classical PPLO Broth base composition, cf. BD Difco PPLO Broth, cat. 255420). Contains animal-derived ingredients, including bovine beef-heart infusion solids. For export, we can arrange an Australian Department of Agriculture, Fisheries and Forestry (DAFF) Declaration and Certificate as to Condition for the consignment where your destination requires one. DAFF issues the certificate on application, and its content and any endorsements follow the importing country’s requirements (see the BSE/TSE note under Cautions). Please send us your destination, and any certification requirement or import permit from your authority, so that we can confirm the documents and lead time. Triple-foil-pouched.
• GMX-NVDS — 5 × lyophilised vials (one per litre), each containing ~443 mg of chemically defined growth factors across three modules (purine/pyrimidine salvage substrates, B-vitamin complex, reducing agents). Reconstitute in 35 mL sterile water when you prepare the medium (Preparation step 5); the reconstituted solution can be stored as described under Storage and Expiry. Replaces fresh yeast extract (Stock E). Shelf life currently claimed: 12 months at 2–8 °C; 24-month target pending real-time data at 2–8 °C and 36-month target pending real-time data at −20 °C.
• Horse Serum [optional] — heat-inactivated horse serum (56 °C × 30 min), 1 L for the 5 L kit. Lot-qualified by growth titration against reference strains (a glucose fermenter, for example M. pneumoniae ATCC 15531, and an arginine hydrolyser, for example M. orale ATCC 23714), with a documented growth-titration certificate. Dose at 200 mL/L (= 20 % v/v) post-autoclave. (An alternative serum, for example foetal bovine serum (FBS), is available on request; it has not been validated for this medium, so qualify it with your own organism before use.)
• 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 for a 10,000 U/mL stock (250,000 U per kit = 25 mL of stock). Dose at 5 mL/L (= 50 U/mL final) for selection against wall-bearing bacteria. Australian orders: supplied as a filter-sterilised 25 mL liquid stock. International orders: supplied as pre-measured Penicillin G sodium powder (250,000 U; approximately 150 mg, adjusted to lot potency), because the liquid stock is short-lived and depends on a cold chain. Store the dry powder sealed, as labelled. Shortly before use, dissolve it in 25 mL sterile water and filter through 0.22 µm to give the 10,000 U/mL stock (storage of the prepared stock: see Storage and Expiry).
• Stock R — 0.5 % w/v phenol red, autoclaved, 10 mL. Dose at 2 mL/L (= ~10 µg/mL final) as pH indicator.

Composition — per 1 L equivalent unless stated otherwise

Use and Applications

Modified PPLO is intended for the same applications as classical PPLO Broth, with a defined supplement in place of the 7-day fresh yeast extract. It is especially advantageous for laboratories that cannot receive the fresh-yeast-extract stock of classical PPLO — international customers, remote sites, and stockpiling programmes. Growth equivalence to classical PPLO has not yet been established experimentally; validate against classical PPLO in parallel for your own organism (see FAQ Q2).

• Candidate broth for the culture-based (agar and broth) procedure of USP <63> / Ph. Eur. 2.6.7 mycoplasma testing of biological products (cell substrates, vaccines, recombinant proteins, gene-therapy vectors), subject to your own qualification. Medium: PPLO base + 20 % horse serum + GMX-NVDS + glucose + Penicillin G + phenol red. Follow the incubation and subculture schedule of the current chapter. As an example, USP <63> specifies:
  • liquid media in tightly stoppered containers at 36 ± 1 °C for 20–21 d, with 0.2 mL subcultured onto solid medium on days 2–4, 6–8, 13–15 and 19–21;
  • solid media incubated microaerophilically (for example nitrogen with 5–10 % CO₂) for at least 14 d (7 d for plates from the final subculture);
  • positive controls of no more than 100 CFU, including at least one dextrose (glucose) fermenter, such as M. pneumoniae ATCC 15531, and one arginine hydrolyser, such as M. orale ATCC 23714.
  Ph. Eur. 2.6.7 was revised with effect from 1 April 2026, so always work to the current official text of each chapter. Modified PPLO is supplied for research use only and is not a pharmacopoeial-grade product; before compendial use it must be shown, in your own laboratory, to meet the chapter’s nutritive-properties and inhibitory-substances criteria (see FAQ Q7).
• Cell-culture mycoplasma contamination screening (research use; example laboratory schedule, not the compendial one). Inoculate 0.5 mL culture supernatant into 5 mL broth and incubate for 21 d at 37 °C in 5 % CO₂, subculturing to agar at days 7, 14 and 21. This is not the USP <63> schedule described in the first item above (tightly stoppered containers; subcultures on days 2–4, 6–8, 13–15 and 19–21). For compendial testing, follow the current USP <63> or Ph. Eur. 2.6.7 text instead. Adapt and validate this schedule in your own laboratory.
• Veterinary Mycoplasma isolation from bovine, ovine, caprine and swine specimens (mastitis, pneumonia, arthritis, reproductive-tract).
• Isolation from research specimens — urogenital, respiratory and oropharyngeal swabs collected under approved research protocols (not for diagnostic use); add Amphotericin B 2.5 µg/mL for long incubations.
• Reference-strain propagation and maintenance — M. orale, M. arginini, M. hyorhinis, M. salivarium and veterinary reference strains (growth not yet confirmed for these; validate each strain against classical PPLO, see FAQ Q2). M. hyorhinis ferments glucose, so growth typically turns the phenol red orange-yellow (acid). M. orale, M. arginini and M. salivarium do not ferment glucose; they obtain energy by hydrolysing arginine, so growth typically shows as an alkaline shift (phenol red turning a deeper red-magenta) rather than an acid one. The arginine-supplemented variant (1 g/L L-arginine) is available on request for these arginine-utilising species.
• Acholeplasma laidlawii cultivation — sterol-independent, so the horse serum is not needed as a sterol source; however, the organism still needs a long-chain fatty-acid supply (an absolute requirement in chemically defined medium; Tourtellotte et al. 1964), which the serum provides in this formulation. Prepare the complete medium. Growth in the base alone, or with reduced or no serum, has not been established for this product; if you wish to omit or reduce the serum, validate this for your own strain against the complete medium.
• International and remote-site use. Replacing the fresh-yeast-extract stock of classical PPLO, which is supplied to Australian customers only, with lyophilised GMX-NVDS removes that Australian-domestic-only restriction. GMX-NVDS is designed for ambient-temperature shipping (target claim, pending excursion validation); the optional horse-serum stock still ships at −20 °C.
• Defined supplement composition. GMX-NVDS is made from CAS-numbered, chemically defined components, so its composition is set by the formulation and not by a yeast-extract lot (the planned lot-release specification is in FAQ Q10). The PPLO base (beef-heart infusion and proteose peptone) and the horse serum remain biological materials and can vary between lots.

Intended Organisms

Growth in Modified PPLO has not yet been confirmed experimentally for the organisms listed below. Validate it alongside classical PPLO Broth for your own organism (see FAQ Q2).

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

• Mycoplasma pneumoniae ATCC 15531 (FH strain) — USP <63> / EP 2.6.7 reference strain (glucose fermenter)
• Mycoplasma orale ATCC 23714 — cell-culture contaminant reference; arginine hydrolyser (does not ferment glucose; score growth by titre rather than by colour), used with a glucose fermenter for serum-lot qualification
• Mycoplasma arginini ATCC 23838 — arginine-utiliser reference
• Mycoplasma hyorhinis ATCC 17981 — swine; cell-culture contaminant reference
• Mycoplasma salivarium ATCC 23064 — human oropharyngeal
• Acholeplasma laidlawii ATCC 23206 — sterol-independent reference

Veterinary Mycoplasma

• M. mycoides subsp. mycoides (contagious bovine pleuropneumonia, CBPP; WOAH-listed disease; cultures subject to national biosecurity regulation) — bovine; the first mycoplasma to be isolated (1898) and the “pleuropneumonia organism” after which PPLO (pleuropneumonia-like organisms) were named
• M. bovis (pneumonia, mastitis), M. ovipneumoniae, M. agalactiae, M. capricolum, M. mycoides subsp. capri, M. canis, M. felis

Human clinical Mycoplasma

• M. hominis, M. pneumoniae (Eaton's preferred for primary isolation)

Not optimised for: fastidious Spiroplasma, M. genitalium and M. fermentans (use Modified SP-4); Ureaplasma (use A8/U9, pH 6.0); avian Mycoplasma (use Frey's); strictly anaerobic Anaeroplasma.

Note for compendial and veterinary-biologics testing: the strains a nutritive-properties test must cover depend on the method and the product type, and can include organisms in the “Not optimised for” list above. For example:

• USP <63> accepts M. fermentans as an alternative D-glucose-fermenting test strain to M. pneumoniae, and lists M. gallisepticum and M. synoviae where avian material is used in production or the product is intended for poultry.
• VICH GL34 includes M. fermentans in its culture-validation panel and requires M. synoviae where avian-origin materials are used.

Check the current text of the method that applies to your product (USP <63>, Ph. Eur. 2.6.7 or VICH GL34), and show that your complete medium supports each required strain before using it for release testing (see FAQ Q7).

Preparation

The amounts in steps 1–7 are for 1 L of medium. Each kit prepares 5 L of complete medium, so for a full kit multiply each of these amounts by five: base water, Mixture A, horse serum, Stock G, Stock P and Stock R (the final volume in step 7 then becomes 5 L). For GMX-NVDS, use all five vials (one per litre) and reconstitute each vial separately in 35 mL (step 5). Do not multiply the aliquot and sterility-check volumes in step 9.

Critical control points

• Serum heat-inactivation (recommended). Heating serum at 56 °C for 30 min destroys the heat-labile complement components (including C2 and factor B). This avoids complement-mediated lysis of the wall-less Mollicutes, which can occur when a serum lot contains anti-Mycoplasma antibody. The GMExpression horse serum is supplied heat-inactivated. Both practices are in established use. The Hayflick medium in USP <63> specifies unheated horse serum, whereas ATCC Medium 243 specifies serum heat-inactivated at 56 °C for 30 min. Follow your validated method. Qualify each serum lot for growth support, and treat every lot the same way, because heating can alter the growth-supporting properties of serum.
• Serum lot qualification. Horse-serum total cholesterol varies between lots and suppliers (published equine serum reference intervals span roughly 0.5–1.5 mg/mL, i.e. about 100–300 µg/mL in the finished medium at 20 % v/v), and lots also differ in how well they support growth. Before bulk purchase, check the lot certificate and run a growth titration of each new lot with your own working strain or with reference strains: ideally one glucose-fermenting strain (e.g. M. pneumoniae ATCC 15531, read as an acid shift of the phenol red) and one arginine-hydrolysing strain (e.g. M. orale ATCC 23714, scored by titre; it does not ferment glucose, so it gives no acid shift in this medium).
• GMX-NVDS reconstitution. Use tissue-culture-grade water; do not autoclave the reconstituted solution; sterilise it by 0.22 µm filtration only (Step 5). Glutamine, NAD and ascorbate are heat-labile, and cysteine and glutathione oxidise rapidly when heated. Provisional storage limits, pending real-time stability data: 4 weeks at 2–8 °C, or 6 months at −20 °C in single-use aliquots (avoid freeze–thaw).
• Amber storage. Light and air promote oxidation of serum lipids and sterols (for example, cholesterol to 7-ketocholesterol and polyunsaturated fatty acids to lipid hydroperoxides). In addition, the riboflavin-5′-phosphate in GMX-NVDS is itself photolabile and, when illuminated, acts as a photosensitiser that generates reactive oxygen species; these can oxidise ascorbate, thiols (cysteine, glutathione) and folinate, and so erode the reducing capacity the supplement provides. Store complete medium in amber, fully-filled bottles, 2–8 °C, use within 14 d.
• CO₂ equilibration. Some Mycoplasma strains grow better at 5 % CO₂, and CO₂ requirements vary by species and strain. The medium has no added bicarbonate, so its pH drifts downward from 7.6–7.8 as it equilibrates, but only in vessels that exchange gas with the incubator atmosphere, such as loosened or gas-permeable closures. Tightly stoppered containers, as USP <63> specifies for liquid media, do not exchange gas. The size of any change is not specified here, so record the pH and phenol-red colour of an uninoculated control under your own incubation conditions and use it as the baseline. Do not pre-equilibrate the bulk medium; it is part of the standardised growth condition.

Cautions

Storage and Expiry · Safety

• Dehydrated base (Mixture A): 15–25 °C, sealed, away from sunlight. Shelf life 36 months.
• GMX-NVDS lyophilised vials: store desiccated at 2–8 °C. Shelf life currently claimed: 12 months at 2–8 °C; 24-month target pending real-time data at 2–8 °C and 36-month target pending real-time data at −20 °C. Ambient-temperature shipping (target claim, pending excursion validation): targeted to tolerate up to 30 °C for up to 4 weeks in transit.
• GMX-NVDS reconstituted (35 mL): 2–8 °C, 4 weeks (provisional); −20 °C aliquots, 6 months (avoid freeze-thaw). Expected limiting factors (mechanism-based; real-time stability data pending): oxidation of ascorbate, cysteine and glutathione. Cyclisation of glutamine to pyroglutamate (acid-catalysed below about pH 5) is expected to be secondary at these storage temperatures.
• Horse serum (heat-inactivated): −20 °C in single-use aliquots, 24 months; 2–8 °C, 4 weeks; ≤ 3 freeze-thaw cycles.
• 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, up to 7 days.
• Complete Modified PPLO medium: 2–8 °C in amber bottles, fully filled, 14 days maximum.

Safety notes. PPLO Broth supports BSL-2 pathogens (for example M. pneumoniae). M. mycoides subsp. mycoides, the agent of contagious bovine pleuropneumonia, is a WOAH-listed pathogen whose possession and culture are regulated by national biosecurity authorities, so permits and containment above BSL-2 may apply. Handle all primary isolation and unknown specimens in a Class II BSC. Beef-heart infusion solids are bovine-derived (for export, we can apply to DAFF for a Declaration and Certificate as to Condition for your consignment where your import authority requires one; see Cautions); horse serum is a biological hazard — handle in PPE, check the serology certificate. GMX-NVDS is a blend of chemically defined components, each listed with its CAS number in the GMX-NVDS composition table. The Safety Data Sheet (SDS), available on request, gives the hazard classification of GMX-NVDS; handle the powder as a laboratory chemical and follow the SDS. If your import authority requires a statement on the origin of the components, please send us the requirement before ordering so that we can confirm whether it can be provided. For research use only. Not for use in diagnostic or therapeutic procedures. Not a medical device or drug.

References and Further Reading

1. Edward, D. G. ff. (1947). A selective medium for pleuropneumonia-like organisms. J. Gen. Microbiol. 1: 238–243. [Edward selective medium for PPLO: penicillin and thallium acetate selection; enriched with fresh yeast extract]
2. Razin, S. & Knight, B. C. J. G. (1960). A partially defined medium for the growth of Mycoplasma. J. Gen. Microbiol. 22: 492–503. [Partially defined medium for Acholeplasma laidlawii strain A (then Mycoplasma laidlawii): riboflavin & nicotinic acid reported essential; yeast extract needed to match Edward-medium growth]
3. Razin, S. (1962). Nutrition and metabolism of pleuropneumonia-like organisms. In: Klieneberger-Nobel, E., Pleuropneumonia-like Organisms (PPLO): Mycoplasmataceae, pp. 91–104. London & New York: Academic Press. doi:10.1016/B978-1-4832-3244-7.50011-9.
4. Tourtellotte, M. E., Morowitz, H. J., Kasimer, P. (1964). Defined medium for Mycoplasma laidlawii. J. Bacteriol. 88 (1): 11–15. doi:10.1128/jb.88.1.11-15.1964. [Coenzyme A requirement]
5. Fabricant, C. G., Fabricant, J., VanDemark, P. J. (1964). Studies on the nutrition and growth requirements of Mycoplasma gallisepticum. J. Gen. Microbiol. 35 (1): 135–144. doi:10.1099/00221287-35-1-135.
6. Mitchell, A., Sin, I. L., Finch, L. R. (1978). Enzymes of purine metabolism in Mycoplasma mycoides subsp. mycoides. J. Bacteriol. 134 (3): 706–712. doi:10.1128/jb.134.3.706-712.1978.
7. Rylance, H. J., Marr, W., Malcolm, M. G., Stewart, S. M., Marmion, B. P. (1979). Growth factors for Mycoplasma pneumoniae in yeast and tissue extracts. J. Appl. Bacteriol. 47 (2): 341–345. doi:10.1111/j.1365-2672.1979.tb01764.x. [Low-molecular-weight, water-soluble, heat-stable growth factor for M. pneumoniae in yeast and tissue extracts; reported to decompose on storage, presumably by oxidation]
8. Razin, S., Yogev, D., Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiol. Mol. Biol. Rev. 62 (4): 1094–1156. doi:10.1128/MMBR.62.4.1094-1156.1998. [Most cofactor-biosynthesis genes lost; culture media must supply essentially all vitamins]
9. Wang, L., Hames, C., Schmidl, S. R., Stülke, J. (2010). Upregulation of thymidine kinase activity compensates for loss of thymidylate synthase activity in Mycoplasma pneumoniae. Mol. Microbiol. 77 (6): 1502–1511. doi:10.1111/j.1365-2958.2010.07298.x. [Thymidine-salvage basis for the thymidine component]
10. Breuer, M. et al. (2019). Essential metabolism for a minimal cell. eLife 8: e36842.
11. Ras, T. A., Strauss, E., Botes, A. (2021). Evaluating the genetic capacity of mycoplasmas for coenzyme A biosynthesis in a search for new anti-mycoplasma targets. Front. Microbiol. 12: 791756. doi:10.3389/fmicb.2021.791756.
12. Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362: 709–715. doi:10.1038/362709a0. [Spontaneous hydrolytic and oxidative decay of the DNA polymer]
13. Levy, M. & Miller, S. L. (1998). The stability of the RNA bases: implications for the origin of life. Proc. Natl. Acad. Sci. USA 95 (14): 7933–7938. doi:10.1073/pnas.95.14.7933. [Aqueous decomposition half-lives of free nucleobases, extrapolated to 0 °C from high-temperature measurements]
14. USP <63> Mycoplasma Tests; European Pharmacopoeia 2.6.7 Mycoplasmas; VICH GL34 Biologicals: Testing for the detection of Mycoplasma contamination (EMA/CVMP/VICH/463/2002); current revisions.
15. Morton, H. E., Smith, P. F., Leberman, P. R. (1951). Investigation of the cultivation of pleuropneumonia-like organisms from man. Am. J. Syph. Gonorrhea Vener. Dis. 35 (4): 361–369. [Early PPLO cultivation medium]
16. BD Difco & BBL Manual of Microbiological Culture Media, PPLO Broth monograph (BD cat. no. 255420).

Frequently Asked Questions