Overview

Modified SP-4 Medium (Defined-Supplement Formula) is designed to supply the characterised growth-factor contribution of fresh yeast extract in the classical Tully 1977 SP-4 formulation — the gold standard for fastidious Mollicutes — while replacing its single most problematic component, fresh yeast extract, with GMX-NDS (GMExpression Nucleotide-Defined Supplement): a lyophilised blend of 13 chemically defined growth factors that extends supplement shelf life from 7 days to 24 months. Every other SP-4 component — Mycoplasma Broth Base, tryptone, peptone, CMRL 1066, Yeastolate, heat-inactivated FBS at 17 % v/v, glucose, Penicillin G at 1000 U/mL, and phenol red — remains unchanged.
The problem solved:  Fresh yeast extract (25 % w/v, autoclaved within 7 days of use) is the critical labile component of classical SP-4: aged solutions lose Spiroplasma-growth-promoting activity through oxidation of the low-molecular-weight free-thiol pool (free cysteine and glutathione), with loss of reduced folates second and ascorbate a minor contributor — the free base, nucleoside and B-vitamin pools are essentially unaffected over this timescale. This 7-day window prevents international shipping, forces cold-chain logistics, introduces lot-to-lot variability, and generates reagent waste. GMX-NDS replaces the growth-factor fraction of fresh yeast extract — purine and pyrimidine bases, ribonucleosides, NAD, glutathione, glutamine, cysteine, ascorbate, and putrescine — with CAS-numbered, analytically verified components in a stable dry powder. The result is a supplement (GMX-NDS) that is targeted for worldwide ambient-temperature shipping with a 24-month shelf life at 2–8 °C (both are target claims pending real-time stability data; the wider kit’s Stock CMRL and Foetal Bovine Serum retain their standard 2–8 °C and −20 °C cold-chain requirements).

The GMExpression® Modified SP-4 formulation is a multi-stock kit reflecting the inherent complexity of SP-4: a Stage-1 autoclaved base (Mycoplasma Broth Base + tryptone + Bacto Peptone), and seven Stage-2 sterile supplements added post-autoclave (CMRL-1066 10×, Yeastolate solution, reconstituted GMX-NDS, heat-inactivated FBS at 17 % v/v, glucose, Penicillin G at 1000 U/mL, phenol red). The complete medium is filter-sterilised at 0.22 µm before dispensing.

We also have

SP-4 Medium (classical, ATCC 988) · PPLO Broth (foundational mycoplasma base) · Hayflick Medium (USP / EP cell-culture testing) · 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® Modified  SP-4 kit contains:

  • Mixture A — pre-weighed Stage-1 base components (Mycoplasma Broth Base BD 211458 2.1 g + Tryptone 10 g + Bacto Peptone 5.3 g per 1 L); pre-aliquoted to 5 L scale. For the agar variant, also includes 14 g/L mycoplasma-grade agar.
  • Stock C — CMRL 1066 Medium 10× concentrate with L-glutamine, without serum (commercial Gibco / Thermo or equivalent); sterile. Dose at 50 mL/L (= 0.5× final).
  • Stock D — Yeastolate (BD 255752 peptic-digest of yeast) at 2 % w/v in water, autoclaved, 500 mL. Dose at 100 mL/L.
  • Mixture N — GMX-NDS: 5 × lyophilised vials (one per litre), each containing 428.6 mg of 13 chemically defined growth factors. Reconstitute in 35 mL sterile water immediately before use. Replaces fresh yeast extract. Shelf life: 24 months at 2–8 °C (target claim, pending real-time stability data; interim conservative claim: 12 months at 2–8 °C or 24 months at −20 °C).
  • Foetal bovine serum [optional] — heat-inactivated foetal bovine serum (56 °C × 30 min), 850 mL for the 5 L kit. Lot-qualified against Spiroplasma citri R8A2 ATCC 27556 + M. fermentans ATCC 19989. Dose at 170 mL/L = 17 % v/v.
  • Stock G — 50 % w/v glucose, filter-sterilised, 50 mL. Dose at 10 mL/L (= 0.5 % w/v final).
  • Stock P — Penicillin G sodium 100,000 U/mL stock (10× the PPLO load), filter-sterilised, 50 mL. Dose at 10 mL/L (= 1000 U/mL final).
  • Stock R — 0.5 % w/v phenol red (optional, on request), 20 mL. Dose at 4 mL/L.
  • Instruction manual including the  full preparation protocol, GMX-NDS reconstitution guide, CCU (colour-changing-unit) titration method, and modified-medium validation annex.


Composition — per 1 L equivalent unless stated otherwise

Modified SP-4 Broth (per 1 L)

ComponentAmountFunction
Stage 1 — Mycoplasma broth base (autoclaved)
Mycoplasma Broth Base (BD 211458) or PPLO Broth Base equivalent2.1 gBeef-heart / peptone / NaCl base
Tryptone (pancreatic digest of casein)10.0 gAugmented amino-acid pool
Bacto Peptone (proteose peptone)5.3 gAdditional peptide nitrogen
Purified water615 mLType I
Stage 2 — Sterile supplements (post-autoclave, in Class II BSC)
CMRL 1066 (10× with L-glutamine, without serum)50 mL (1× final)Tissue-culture-grade vitamins (B-complex, ascorbate, biotin, folate, B12), glutathione, hypoxanthine, thymidine, putrescine, balanced inorganic salts
Yeastolate (2 % w/v in water, autoclaved)100 mLPeptic digest of yeast — B-vitamins + amino acids
GMX-NDS (reconstituted in 35 mL sterile water)35 mLChemically defined replacement for fresh yeast extract — see GMX-NDS table below
Foetal bovine serum, heat-inactivated 56 °C × 30 min170 mL (= 17 % v/v)Cholesterol source — Spiroplasma require more sterol than most Mycoplasma; FBS is preferred over horse serum for Spiroplasma
Glucose (50 % w/v, filter-sterilised)10 mL (= 0.5 % w/v final)Fermentable carbon — all Spiroplasma are glucose-utilisers
Penicillin G (100,000 U/mL stock)10 mL (= 1000 U/mL final)Aggressive counter-selection against cell-walled bacteria; Mollicutes are wall-less and intrinsically resistant
Phenol red (0.5 %, optional)4 mLpH indicator
Sterile waterq.s. 1000 mL—

Final pH at 25 °C: 7.5 ± 0.1. Adjust with sterile 1 N NaOH or HCl if needed. Filter-sterilise the complete medium at 0.22 µm PES; never autoclave the medium (denatures CMRL vitamins, caramelises glucose, denatures serum).

GMX-NDS — GMExpression® Nucleotide-Defined Supplement (per vial, for 1 L final medium)

#ComponentCAS No.Amount (mg)Final conc. (mg/L)Function
1Adenine73-24-510.010.0Purine base → apt → AMP
2Adenosine58-61-720.020.0Purine ribonucleoside
3Guanosine118-00-310.010.0Purine ribonucleoside → hpt → GMP
4Hypoxanthine68-94-05.05.0Purine base → hpt → IMP
5Uracil66-22-85.05.0Pyrimidine base → uprt → UMP
6Cytidine65-46-310.010.0Pyrimidine ribonucleoside
7Uridine58-96-810.010.0Pyrimidine ribonucleoside
8β-NAD (free acid)53-84-97.07.0Redox coenzyme
9L-Glutathione (reduced)70-18-850.050.0Antioxidant — Mollicutes lack catalase
10L-Glutamine56-85-9200.0200.0Heat-labile amino acid (destroyed by autoclave)
11L-Cysteine HCl·H₂O7048-04-650.050.0Thiol amino acid (oxidised in aged solutions)
12L-Ascorbic acid Na salt134-03-250.050.0Antioxidant (protects thiol-sensitive components)
13Putrescine·2HCl333-93-71.61.6Polyamine growth factor
Total dry weight per vial428.6——

Modified SP-4 Agar variant

ComponentNotes
Stage-1 base + 14 g/L mycoplasma-grade agarAutoclaved at 121 °C × 15 min; cool to exactly 50 °C
Stage-2 supplements identical to broth (with GMX-NDS)Add post-autoclave at 50 °C; pour plates within 10 min of FBS addition

Nutrient budget: Original SP-4 vs Modified SP-4

Nutrient classOriginal SP-4 sourcesModified SP-4 sources
Purine ribonucleosidesFresh YE (major) + CMRL deoxynucleosidesGMX-NDS (adenosine 20, guanosine 10 mg/L) + CMRL deoxynucleosides
Free purine basesFresh YEGMX-NDS (adenine 10, hypoxanthine 5 mg/L)
Pyrimidine ribonucleosidesFresh YE + CMRL UTPGMX-NDS (cytidine 10, uridine 10, uracil 5 mg/L) + CMRL UTP
ThymidineCMRL (5 mg/L at 0.5×)CMRL (5 mg/L at 0.5×) — unchanged
NADFresh YE + CMRL (3.5 mg/L)GMX-NDS (7 mg/L) + CMRL (3.5 mg/L) = 10.5 mg/L total
Glutathione (reduced)Fresh YE (44–88 mg/L) + CMRL (5 mg/L)GMX-NDS (50 mg/L) + CMRL (5 mg/L) = 55 mg/L total
Amino acidsFresh YE (~2100 mg/L) + Yeastolate + CMRL + Tryptone/Peptone + FBSYeastolate + CMRL + Tryptone/Peptone + FBS + GMX-NDS (glutamine 200, cysteine 50 mg/L)
B-vitaminsFresh YE + Yeastolate + CMRLYeastolate + CMRL — sufficient without YE
CholesterolFBS (primary) + CMRL (trace)FBS + CMRL — unchanged

→Culturomics and Media Navigator

Need help matching this medium to your fastidious target Mollicute — or starting from an arthropod-borne or plant-pathogenic Spiroplasma 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 species, or (ii) start from a target organism (genus, species, ATCC / DSMZ / JCM accession, or host arthropod / plant) and view the panel of GMExpression media for isolation, propagation, and identification. The Navigator integrates the Modified SP-4 with the wider PPLO, Hayflick, Frey's, Eaton's, A8/U9, MGS-10×, and classical SP-4 catalogue.

Use and Applications

Modified SP-4 is formulated to cover all applications of the classical SP-4 formulation. Growth equivalence has not yet been established experimentally, so users should validate against classical SP-4 in parallel for their own organism (see FAQ Q2). The defined-supplement format is particularly advantageous for laboratories that cannot receive fresh yeast extract within 7 days of preparation — including international customers, remote field stations, and stockpiling programmes.

  • Primary isolation of Spiroplasma from arthropod tissues. Triturate insect tissue in 1 mL Modified SP-4 broth; serial 10-fold dilution; incubate 30 °C (slow growers) or 32 °C (standard) up to 4 weeks. Subculture at first colour shift.
  • Fastidious Mycoplasma isolation when PPLO + horse serum fails. M. fermentans, M. genitalium, M. penetrans, and difficult M. pneumoniae clinical isolates that cannot be recovered on Hayflick or PPLO are routinely recoverable on SP-4 (Tully & Razin 1996).
  • Reference strain maintenance for all Spiroplasma species (S. citri R8A2 ATCC 27556, S. melliferum, S. apis, S. mirum, S. floricola) and the fastidious Mycoplasma species. Passage every 5–7 days; cryostock in Modified SP-4 + 15 % glycerol at −80 °C.
  • Cell-culture mycoplasma screening — high-sensitivity confirmatory subculture on Modified SP-4 agar plates.
  • International and remote-site use. The 24-month dry-powder shelf life and ambient-temperature shipping of GMX-NDS makes Modified SP-4 practical for laboratories worldwide, eliminating the cold-chain and freshness constraints of classical SP-4.
  • Stockpiling and emergency preparedness. Defined-supplement vials are targeted for storage up to 36 months at −20 °C (target claim, pending real-time stability data), enabling long-term reagent stockpiles for outbreak-response mycoplasmology programmes.
  • Lot-controlled research. CAS-numbered, analytically verified components eliminate the biological variability of yeast-extract lots, improving experimental reproducibility across sites and time points.

Compatible Microorganisms

Spiroplasma (primary use; gold standard)

  • Spiroplasma citri R8A2 ATCC 27556 — reference strain; corn stunt + citrus stubborn pathogen
  • Spiroplasma melliferum BC-3 ATCC 33219 — honeybee pathogen (May disease)
  • Spiroplasma apis B31 ATCC 33834 — honeybee pathogen
  • Spiroplasma mirum SMCA ATCC 29335 — mammalian / suckling-mouse cataract agent
  • Spiroplasma kunkelii — corn stunt pathogen; vectored by Dalbulus leafhoppers
  • Spiroplasma floricola — flower-surface commensal
  • Spiroplasma poulsonii — Drosophila male-killer endosymbiont

Fastidious Mycoplasma (SP-4 rescues where PPLO / Hayflick fail)

  • Mycoplasma fermentans ATCC 19989 — cell-culture contaminant; very fastidious
  • Mycoplasma genitalium G37 ATCC 33530 — urogenital; 21–42 d to first detectable growth
  • Mycoplasma penetrans — HIV-associated
  • Mycoplasma pneumoniae primary clinical isolates (when Eaton's recovery is suboptimal)
  • Mycoplasma pirum, M. fastidiosum, M. iners

Entomoplasma / Mesoplasma (insect-associated)

  • Entomoplasma melaleucae, E. ellychniae, E. lucivorax, E. luminosum
  • Mesoplasma florum L1 ATCC 33453, M. coleopterae, M. entomophilum

Not optimised for: routine veterinary Mycoplasma where PPLO + HS suffices (cost vs benefit favours PPLO), Ureaplasma (use A8/U9 at pH 6.0), avian M. synoviae (use Frey's + NAD), M. hyopneumoniae (use Friis with swine serum), strict anaerobic Anaeroplasma.

Preparation

1Stage-1 base preparation. Dissolve Mixture A (2.1 g Mycoplasma Broth Base + 10 g Tryptone + 5.3 g Bacto Peptone for 1 L) in 615 mL of Type I water with stirring. Heat to gentle boil briefly to dissolve.
2Adjust pH. Target 7.6 ± 0.2 at 25 °C (overshoot; supplement addition drops by ~ 0.1).
3Autoclave Stage-1 base. 121 °C × 15 min, slow cooling. Cool to < 50 °C (broth) or exactly 50 °C (agar).
4Heat-inactivate FBS (if using non-inactivated FBS). Submerge bottle in 56 °C water bath, exactly 30 min, swirl every 5 min, cool rapidly in ice bath, centrifuge 3000 × g × 10 min, sterile-decant.
5Yeastolate stock (Stock D): pre-prepared at 2 % w/v, autoclaved.
6Reconstitute GMX-NDS (Mixture N). Open one GMX-NDS vial aseptically in a Class II BSC. Add 35 mL of sterile purified water at room temperature. Vortex gently or swirl until all powder is dissolved (~2 min). The purine components (hypoxanthine, adenine, guanosine) are the least soluble and may need a brief warming to 37 °C with gentle agitation to dissolve fully. Filter-sterilise the reconstituted solution through a 0.22 µm PES membrane into a sterile container. Use within 4 weeks if stored at 2–8 °C, or freeze aliquots at −20 °C for up to 6 months.
7Stage-2 supplements in BSC. Aseptically combine, in this exact order, into the cooled Stage-1 base: 50 mL Stock C (CMRL) → 100 mL Stock D → 35 mL Mixture N (reconstituted GMX-NDS) → 170 mL FBS → 10 mL Stock G → 10 mL Stock P → 4 mL Stock R (optional). Mix gently — minimise frothing of serum.
8Adjust final pH to 7.5 ± 0.1 at 25 °C using sterile 1 N NaOH or HCl.
9Filter-sterilise the complete medium at 0.22 µm PES through a low-protein-binding filter (with a 0.45/0.8 µm prefilter to prevent serum clogging) — this is mandatory even with sterile components. This step controls conventional bacterial/fungal bioburden introduced during aseptic assembly; it does not retain mycoplasmas (see Caution and FAQ Q9 — mycoplasma exclusion is a component-level control, chiefly the serum). Never autoclave the complete medium (denatures CMRL vitamins, caramelises glucose, denatures serum proteins). Never attempt to push the finished 17% FBS medium through a 0.1 µm filter — it clogs and strips the serum cholesterol/lipoprotein that Spiroplasma require.
10Dispense in BSC. Broth: 2–5 mL in sterile screw-cap tubes. Agar: pour 4 mL per 35 mm plate within 10 min of FBS addition (agar working window). Aliquot into single-use bottles for bulk supply.

Critical control points

  • CMRL 1066 freshness and supplier.  CMRL 1066 is a critical core ingredient in this formulation. Our Stock C is manufactured directly by GMExpression. If sourcing CMRL 1066 from a third party, please select an equivalent product from the Gibco/Thermo Fisher 11530 series. Always verify the manufacturing date on every bottle; CMRL vitamins (riboflavin, ascorbate, B12) degrade at ~ 10 % per month at 2–8 °C in the supplied bottle. Use within 12 months of manufacture; lock supplier and lot for reproducibility.
  • GMX-NDS reconstitution. Use tissue-culture-grade water only. Do not autoclave the reconstituted solution — glutamine and glutathione are heat-labile. Reconstituted GMX-NDS is stable for 4 weeks at 2–8 °C or 6 months at −20 °C.
  • FBS lot qualification. Test each FBS lot against Spiroplasma citri ATCC 27556 (slowest grower in the standard reference panel) and M. fermentans ATCC 19989 (most fastidious cell-culture target). Cholesterol content of FBS typically varies 100–400 µg/mL between lots; the SP-4 17 % v/v load gives a final cholesterol concentration in the 17–68 µg/mL range, all of which support Spiroplasma, but lots at the low end give 2–3× longer lag times for Spiroplasma citri.
  • Filter-sterilisation through low-protein-binding membrane. Standard PES 0.22 µm is the spec. Avoid cellulose acetate (CMRL vitamins adsorb), PVDF (FBS protein adsorbs — reduces serum potency by 5–10 %), and 0.45 µm membranes (insufficient mycoplasma retention).
  • Complete-medium shelf life is 14 days maximum. Photo-oxidation of CMRL vitamins (riboflavin, ascorbate) destroys activity faster in light; store in amber, fully-filled bottles, 2–8 °C.

Cautions

0.22 µm filtration does NOT remove mycoplasmas — mycoplasma cleanliness is a component-level control. Mollicutes are wall-less and 0.1–0.8 µm, and penetrate 0.2/0.22 µm sterilising-grade filters (which are rated against the larger, walled Brevundimonas diminuta, ASTM F838). The terminal 0.22 µm step in this protocol is a bacterial/fungal bioburden polish, not a mycoplasma barrier — and SP-4's penicillin G gives no protection against a contaminating Mollicute (they are cell-wall-free and β-lactam-resistant). Because most of SP-4 is autoclaved (heat-sterile) and GMX-NDS is a defined 0.22 µm-filtered chemical stock, the residual mycoplasma risk sits in the non-autoclaved animal components — above all the FBS. Control it upstream: use FBS that is γ-irradiated (25–40 kGy) and/or per-lot mycoplasma-NAT-certified; for reference-strain or quantitative work, add 0.1 µm mycoplasma-grade (A. laidlawii-qualified) filtration of the CMRL 1066 fraction and confirm strain identity by genotyping. Do not attempt to solve this by filtering the finished 17% FBS medium at 0.1 µm — it clogs and strips the sterol/lipoprotein Spiroplasma depend on. GMExpression applies the serum and CMRL controls at manufacture; the user performs only aseptic assembly + the 0.22 µm polish.
Modified SP-4 is a premium medium. The CMRL-1066 + Yeastolate + GMX-NDS + 17 % FBS load makes Modified SP-4 one of the highest-cost mycoplasma media. Reserve for genuinely fastidious primary isolations and for Spiroplasma work. For routine USP <63> / EP 2.6.7 testing, use Hayflick. For routine veterinary mycoplasmology, use PPLO + horse serum.
Validation required for novel species or strains. GMX-NDS has been formulated to replace the known growth-factor contribution of fresh yeast extract based on published Mollicute nutritional requirements (Tourtellotte et al. 1964; Fabricant, Fabricant & Van Demark 1964; Hackett 1995). However, yeast extract may contain uncharacterised growth factors relevant to specific strains. If Modified SP-4 gives suboptimal growth for a particular organism, consider: (i) increasing GMX-NDS concentration to 1.5–2×, (ii) supplementing with 0.1 % w/v dried yeast extract, or (iii) reverting to classical SP-4 for that application.
FBS sourcing and TSE/BSE handling. Foetal bovine serum is bovine-derived. Use only TSE-certified zoosanitary-documented FBS lots. Our Foetal Bovine Serum is sourced from Australian certified suppliers, with full batch traceability documentation.
No antifungal in the default formulation. The default Modified SP-4 carries no antifungal. Arthropod-tissue triturates, environmental samples and heavily contaminated clinical specimens are at material risk of yeast/mould overgrowth during the 4-week enrichment. For those matrices, add Amphotericin B 2.5 µg/mL as a Stage-2 supplement (available as a kit add-on).
Cholesterol oxidation in stored medium. Cholesterol oxidises to oxysterols on light + air exposure; oxysterols are inhibitory to sterol-dependent Mollicutes. The 17 % FBS load makes the medium sensitive to photo-oxidation. Store in amber, fully-filled bottles, 2–8 °C, use within 14 d.
Penicillin G at 1000 U/mL is aggressive. The SP-4 Pen G load is 20× PPLO and 10× Hayflick, specifically for arthropod-tissue and clinical primary isolations with heavy bacterial burdens. Mycoplasma are cell-wall-free and intrinsically resistant.
M. genitalium specimen handling. M. genitalium is exquisitely fragile ex vivo; culture recovery drops sharply with ambient hold time beyond a few hours. Transport patient urogenital specimens promptly on wet ice in Modified SP-4 broth and inoculate as soon as practicable after collection (ideally within a few hours). Consult the current CDC / Jensen laboratory-diagnosis guidance for the target-organism-specific transport interval before adopting a formal cut-off.

Storage and Expiry · Safety

  • Dehydrated Stage-1 base (Mixture A): store sealed at 15–25 °C. Shelf life 36 months from manufacture.
  • Stock C (CMRL-1066 10×): 2–8 °C per manufacturer (typically 12 months); protect from light.
  • Stock D (Yeastolate 2 %): 2–8 °C, 4 weeks.
  • GMX-NDS lyophilised vials: 2–8 °C (desiccated), 24 months (target claim; real-time stability pending; interim conservative: 12 months at 2–8 °C); −20 °C, 36 months (target claim; real-time stability pending). Ambient-temperature shipping: targeted acceptable up to 30 °C for up to 4 weeks in transit (target claim, pending excursion validation).
  • GMX-NDS reconstituted (35 mL aqueous): 2–8 °C, 4 weeks (provisional); −20 °C aliquots, 6 months (target claim, pending stability data; avoid freeze-thaw). Limiting factors (in order): thiol autoxidation (L-cysteine → cystine, GSH → GSSG); glutamine cyclisation to pyroglutamate is secondary.
  • FBS (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 100,000 U/mL): −20 °C aliquots, 6 months; 2–8 °C, 2 weeks.
  • Complete Modified SP-4 medium: 2–8 °C in amber bottles, fully filled, 14 days maximum.
  • Modified SP-4 agar plates (poured): 2–8 °C in sealed bags, 7 days maximum.

Safety notes. Modified SP-4 supports BSL-2 pathogens (M. fermentans, M. genitalium, M. penetrans, M. pneumoniae). All work in a Class II BSC; specimen-handling waste autoclaved before disposal. FBS is bovine-derived; TSE-certified zoosanitary documentation supplied. GMX-NDS components are non-animal-derived, chemically defined, and non-hazardous. SDS available on request. For research use only. Not for use in diagnostic or therapeutic procedures. Not a medical device or drug.

References

  1. Tully, J. G., Whitcomb, R. F., Clark, H. F., Williamson, D. L. (1977). Pathogenic mycoplasmas: cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195 (4281): 892–894. [Canonical SP-4 primary citation]
  2. Razin, S. & Cohen, A. (1963). Nutritional requirements and metabolism of Mycoplasma laidlawii. J. Gen. Microbiol. 30: 141–154. doi:10.1099/00221287-30-1-141. [Nutritional requirements; nucleotide salvage]
  3. 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. [First fully defined Mollicute medium; coenzyme A requirement]
  4. Fabricant, C. G., Fabricant, J., Van Demark, 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. [Nucleoside/nucleotide requirements; free bases alone insufficient]
  5. Hackett, K. J., Clark, E. A., Whitcomb, R. F., Henegar, R. B., Tully, J. G., Bové, J. M. (1995). Growth characteristics of Spiroplasma in defined and semi-defined media. In Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I (Razin & Tully, eds), pp. 99–112. [Defined Spiroplasma media]
  6. Burgos, R. et al. (2023). Development of a serum-free medium to aid large-scale production of Mycoplasma-based therapies. Microbiol. Spectr. 11 (3): e04859-22. doi:10.1128/spectrum.04859-22. [Serum-free production medium]
  7. Polyamines (putrescine, spermidine) as microbial growth factors — the established nutritional basis for the trace putrescine component of GMX-NDS.
  8. Del Giudice, R. A., Gardella, R. S., Hopps, H. E. (1980). Cultivation of formerly noncultivable strains of Mycoplasma hyorhinis. Curr. Microbiol. 4: 75–80. doi:10.1007/BF02602896.
  9. Hopps, H. E., Del Giudice, R. A. (1984). Cell culture models as ancillary tools in the isolation and characterization of mycoplasmas. Isr. J. Med. Sci. 20 (10): 927–930. [Citation flagged 2026-09-24 audit-3: full-text access pending verification; the yeast-product-inhibition claim for fastidious M. hyorhinis is more directly supported by Del Giudice, Gardella & Hopps (1980) Curr. Microbiol. 4: 75–80 (Ref #8 above).]
  10. Whitcomb, R. F. (1983). Culture media for Spiroplasmas. In Methods in Mycoplasmology (S. Razin & J. G. Tully, eds), Academic Press, Vol. 1, pp. 147–158.
  11. Tully, J. G. & Razin, S. (1996). Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II, Academic Press.
  12. Razin, S. & Hayflick, L. (2010). Highlights of mycoplasma research — an historical perspective. Biologicals 38: 183–190.
  13. ATCC Medium 988 specification, current revision.
  14. DSMZ Medium 1076b (SP4-Z variant; omits yeast extract).

Frequently Asked Questions

Q1. What is GMX-NDS and why does it replace fresh yeast extract?
GMX-NDS (GMExpression Nucleotide-Defined Supplement) is a lyophilised blend of 13 chemically defined growth factors — purine and pyrimidine bases, ribonucleosides, NAD, reduced glutathione, glutamine, cysteine, ascorbic acid, and putrescine — formulated to supply the specific nutritional contributions of fresh yeast extract that Mollicutes require for growth. Fresh yeast extract provides these factors as a crude biological mixture with a 7-day shelf life; GMX-NDS provides them as CAS-numbered, analytically verified pure compounds with a 24-month shelf life. The replacement was designed from first-principles analysis of Mollicute nucleotide salvage pathways and the gap between CMRL 1066 composition (which provides deoxynucleosides but not ribonucleosides) and the ribonucleoside/purine-base fraction of yeast extract autolysate.
Q2. Is Modified SP-4 equivalent to classical SP-4 in growth performance?
Modified SP-4 is formulated to match or exceed the growth-factor concentration of classical SP-4 for all characterised nutritional requirements of Spiroplasma and fastidious Mycoplasma. The lot-release specification under development will compare each production lot against a qualified reference lot of classical SP-4 using Spiroplasma citri R8A2 ATCC 27556, with proposed acceptance criteria of lag phase ≤ 120 % of reference, doubling time ≤ 120 % of reference, and maximum titre (CCU/mL) ≥ 80 % of reference. Growth equivalence has not yet been established experimentally, and yeast extract is a complex biological material that may contain uncharacterised growth factors relevant to specific strains or species; users working with novel or poorly characterised Mollicutes should validate Modified SP-4 against classical SP-4 in parallel before adopting it as the sole growth medium.
Q3. Why does Modified SP-4 still include Yeastolate if the yeast extract has been replaced?
Yeastolate and yeast extract are different ingredients. Yeastolate (BD 255752) is a peptic (enzymatic) digest of yeast: it retains B-vitamins and amino acids but has relatively low free-nucleotide content and is stable for 4 weeks. Fresh yeast extract (BD 212750) is an autolysate (self-digestion by endogenous yeast enzymes): it retains the full free-nucleotide pool from RNA degradation but must be used within 7 days. GMX-NDS replaces the nucleotide / cofactor / thiol fraction that only fresh yeast extract provides. Yeastolate remains in the formulation because it contributes stable B-vitamin and amino-acid supplementation that GMX-NDS does not duplicate.
Q4. Can I ship Modified SP-4 kits internationally?
Yes for the GMX-NDS supplement and the dry Mixture A. GMX-NDS is targeted for ambient-temperature stability (up to 30 °C for 4 weeks in transit; target claim, pending real-time excursion data) and requires no cold chain. Mixture A (dry powder) is similarly ambient-stable. Stock C (CMRL) and Foetal Bovine Serum still require 2–8 °C and −20 °C shipping respectively, but these are standard biologics-shipping requirements shared with all SP-4 formulations. The critical difference is the elimination of the 7-day freshness constraint on yeast extract, which previously made international shipping impractical.
Q5. What if my target organism grows poorly on Modified SP-4?
Three escalation options: (i) raise the GMX-NDS dose — to 1.5× by reconstituting two vials in 70 mL and adding 52.5 mL per litre AND reducing the Stage-1 base-broth water from 615 mL to 597.5 mL before autoclaving, or to 2× by reconstituting two vials in 70 mL and adding the full 70 mL per litre AND reducing the Stage-1 base-broth water from 615 mL to 580 mL before autoclaving (in both cases the aim is to keep total volume = 1 L; do NOT reconstitute two vials in 35 mL because that would exceed guanosine’s room-temperature solubility limit ~0.7 mg/mL). Note that raising the dose raises ascorbate, cysteine and glutathione proportionally; in a catalase-negative organism the redox load, not solubility, is the practical limit on this escalation; (ii) supplement the Modified SP-4 with 0.1 % w/v dried yeast extract as a partial back-addition to re-introduce any uncharacterised growth factors; (iii) revert to classical SP-4 with fresh yeast extract for that application and report the strain to GMExpression R&D (technical@gmexpression.com) for investigation. Option (iii) helps us identify strains that require factors beyond the current GMX-NDS specification.
Q6. How does the cost compare to classical SP-4?
The base kit price for Modified SP-4 is approximately AUD 100–150 higher than classical SP-4, reflecting GMX-NDS production cost. However, the total cost of ownership is typically lower for most laboratories: classical SP-4 requires weekly preparation of fresh yeast extract (25 % w/v, autoclaved within 7 days), which generates reagent waste and technician time costs. A single classical SP-4 preparation batch that goes unused within 7 days wastes approximately AUD 40–60 in yeast extract and autoclave costs. Over a year, these savings typically exceed the GMX-NDS premium.
Q7. Can I still use classical SP-4 with fresh yeast extract?
Yes. GMExpression continues to supply the classical SP-4 Medium (ATCC 988) with fresh yeast extract for laboratories that prefer the original Tully 1977 formulation or have established workflows around it. The classical formulation remains the reference for published methods, ATCC protocols, and regulatory submissions that specify ATCC Medium 988.
Q8. What quality control is performed on GMX-NDS vials?
Each lot is tested for: (i) appearance — white to off-white powder; (ii) pH of reconstituted concentrate — 3.0–5.0 (acidic by design; the concentrate is diluted into the buffered base and the finished medium is adjusted to pH 7.5 ± 0.1); (iii) sterility per USP <71> or bioburden < 10 CFU/g; (iv) HPLC-UV identity at 260 nm confirming adenosine, guanosine, cytidine, and uridine within ±15 % of label; (v) thiol speciation by RP-HPLC with thiol derivatisation (DTNB or monobromobimane) or LC-MS, resolving reduced glutathione from L-cysteine — reduced glutathione ≥ 85 % of label claim and GSSG ≤ 15 % of total glutathione (a total-thiol Ellman's value is recorded for information only, since L-cysteine supplies ~64 % of the vial's titratable thiol); (vi) growth performance against S. citri R8A2 reference with classical SP-4 as comparator.
Q9. Does the 0.22 µm filtration make the medium mycoplasma-free before I inoculate my strain?
No — and no filter at any pore size fully guarantees it. Mycoplasmas are wall-less cells of ~0.1–0.8 µm and routinely pass 0.2/0.22 µm sterilising-grade filters; the PDA/FDA consensus states plainly that mycoplasma penetrate 0.2/0.22 µm and even some 0.1 µm filters (Folmsbee et al. 2014). The 0.22 µm rating is defined against a larger, walled organism (Brevundimonas diminuta, ASTM F838), so it makes no mycoplasma-retention claim. In this protocol the 0.22 µm step controls conventional bacterial/fungal bioburden; it is not a mycoplasma barrier, and the medium's penicillin G does not touch a contaminating Mollicute (they are β-lactam-resistant). How mycoplasma cleanliness is actually assured: most of SP-4 is autoclaved (heat-sterile) and GMX-NDS is a defined 0.22 µm-filtered chemical stock, so the dominant vector is the non-autoclaved serum, with CMRL 1066 a distant second. GMExpression sources FBS that is γ-irradiated (25–40 kGy) and/or per-lot mycoplasma-NAT-certified, and for reference-strain/quantitative work adds 0.1 µm mycoplasma-grade filtration (A. laidlawii-qualified) of the CMRL 1066 fraction plus strain-genotyping release checks. This is more effective than tightening the terminal filter — forcing the finished 17% FBS medium through 0.1 µm would clog and strip the cholesterol/lipoprotein that sterol-dependent Spiroplasma obligately require. For your own workflow: confirm your FBS lot is mycoplasma-tested, and for quantitative work run an uninoculated negative-control aliquot and genotype your working strain.