Overview

SP-4 Medium (ATCC Medium 988) is the gold standard for fastidious Mollicutes — all Spiroplasma species, the difficult-to-culture Mycoplasma species (M. fermentans, M. genitalium, M. pneumoniae clinical isolates, M. penetrans), Entomoplasma, and Mesoplasma. Developed by Tully, Whitcomb, Clark & Williamson (1977, Science 195: 892) for Spiroplasma citri (the corn-stunt and citrus-stubborn pathogen), SP-4's defining innovation was the inclusion of CMRL 1066 tissue-culture medium as a vitamin / co-factor source — bringing the nutritional sophistication of mammalian-cell-culture science to fastidious mycoplasma cultivation.

The GMExpression 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, fresh yeast extract, 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. SP-4 supplies are the highest-cost mycoplasma medium in the catalogue precisely because it is the highest-performing.

We also have

Modified SP-4 (Defined-Supplement Formula) — fresh-YE replaced by GMX-NDS; 24-month supplement shelf-life target · 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 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 (= 1× final). The defining ingredient of SP-4.
  • Stock D — Yeastolate (BD 255752 peptic-digest of yeast) at 2 % w/v in water, autoclaved, 500 mL. Dose at 100 mL/L.
  • Stock E — Fresh yeast extract at 25 % w/v, autoclaved within 7 days of dispatch, 175 mL. Dose at 35 mL/L. Freshness is mandatory — aged yeast extract loses Spiroplasma growth-promoting activity. Note: Due to shelf-life requirements, this item is available for Australian domestic orders only. For international customers, please check the Modified SP-4 Medium, GMNB-SP402.
  • 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 — the high serum load is non-negotiable for Spiroplasma.
  • 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 — aggressive selection for arthropod-tissue isolation).
  • Stock R — 0.5 % w/v phenol red (optional, on request), 20 mL. Dose at 4 mL/L.
  • Instruction manual.

Customisation options on request: M1A variant (modified SP-4 for arginine-utilisers), M1D variant (modified SP-4 for Mesoplasma), insect-tissue extract supplement for arthropod-Mollicute primary isolation, GMP-grade FBS with extended TSE-certified zoosanitary documentation, fresh-weekly mailout shipment plan.

Composition — per 1 L equivalent unless stated otherwise

SP-4 Broth (Tully, Whitcomb, Clark, Williamson 1977 Science 195: 892 / ATCC Medium 988; 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)The defining ingredient. 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, lower nucleotide content than yeast extract
Fresh yeast extract (25 % w/v, ≤ 7 d old)35 mLPurine and pyrimidine bases and ribonucleosides (chemically stable in storage), plus the labile low-molecular-weight reducing fraction — it is the reducing fraction, not the bases, that sets the 7-day limit
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 Gram-positive selection against cell-walled bacteria — arthropod tissues carry a heavy bacterial burden; 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 complete medium (denatures CMRL vitamins, caramelises glucose, denatures serum).

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 brothAdd post-autoclave at 50 °C; pour plates within 10 min of FBS addition; small-dish 35 mm format preferred (USP / mycoplasma-agar standard)

SP-4 variants

  • M1A — SP-4 with arginine substitute for glucose; for M. arginini and other arginine-utilisers.
  • M1D — SP-4 with insect-tissue extract supplement; for Entomoplasma and Mesoplasma.
  • SP-4 + insect-tissue extract — primary isolation from arthropod tissues (ticks, wasps, bees, plant-feeding leafhoppers).

→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 SP-4 with the wider PPLO, Hayflick, Frey's, Eaton's, A8/U9, MGS-10×, LB, 2×YT, NZCYM, and BHI catalogue.

Use and Applications

  • Primary isolation of Spiroplasma from arthropod tissues. Triturate insect tissue in 1 mL SP-4 broth; serial 10-fold dilution into SP-4 broth tubes; incubate 30 °C (for slow growers) or 32 °C (standard) up to 4 weeks. Subculture at first colour shift to fresh broth + SP-4 agar plates. CCU titration to end-point.
  • 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 SP-4 + 15 % glycerol at −80 °C.
  • Cell-culture mycoplasma screening — SP-4 agar plates as the high-sensitivity confirmatory subculture medium for samples that screen negative on Hayflick agar but are clinically suspected of contamination. Incubate 35–37 °C, 5 % CO2, 21 days; examine for fried-egg colonies.
  • Tick / wasp / bee microbiome culturomics for arthropod-borne Mollicutes. Use SP-4 + insect-tissue extract supplement for the most-difficult primary isolations.
  • Plant phloem Mollicute (phytoplasma-associated) isolation — SP-4 supports some plant-associated Spiroplasma (S. kunkelii corn-stunt, S. citri citrus stubborn); true phytoplasmas remain uncultivable.
  • Vaccine antigen production for Spiroplasma-based veterinary vaccines (rare; bee diseases).
  • Definitive species identification by 16S rRNA / RFLP / MLST from SP-4-positive cultures.

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 — critical for the agar variant).
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.
6Fresh yeast extract (Stock E): autoclaved within 7 days of use; do not substitute aged stocks.
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 Stock E → 170 mL Foetal Bovine Serum → 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; final-mix-step contamination is the principal SP-4 spoilage mode. This step controls conventional bacterial/fungal bioburden; 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) degrade at ~ 10 % per month at 2–8 °C in the supplied bottle. Note: CMRL 1066 does not contain cobalamin — the vitamin B12 in SP-4 is supplied entirely by the foetal bovine serum fraction. Use within 12 months of manufacture; lock supplier and lot for reproducibility.
  • Yeast extract freshness. Fresh yeast extract solution (Stock E) must be autoclaved within 7 days of use. Aged yeast extract solutions lose Spiroplasma-growth-promoting activity even though they continue to support routine Mycoplasma. For GMP-grade workflows, prepare and consume in the same week.
  • 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.
  • Filter-sterilisation through low-protein-binding membrane. Standard PES 0.22 µm is the spec for bacterial/fungal bioburden control. Avoid cellulose acetate (CMRL vitamins adsorb), PVDF (FBS protein adsorbs — reduces serum potency by 5–10 %), and 0.45 µm membranes (not a sterilising grade — insufficient bacterial retention). Note that neither 0.45 nor 0.22 µm retains mycoplasmas (wall-less, 0.1–0.8 µm); mycoplasma exclusion is a separate, component-level control — see Cautions and FAQ Q9.
  • 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), so a contaminating mycoplasma would neither be filtered out nor suppressed. Because the Stage-1 base, tryptone, peptone, Yeastolate and the fresh yeast extract are all autoclaved (heat-sterile), the residual mycoplasma risk sits in the non-autoclaved animal components — above all the FBS, with CMRL 1066 a distant second. Control it upstream: use FBS that is γ-irradiated (25–40 kGy) and/or per-lot mycoplasma-NAT-certified (re-qualified for growth promotion in SP-4); for certified reference-strain or quantitative work, add 0.1 µm mycoplasma-grade (A. laidlawii-qualified, PDA TR 75 / ANSI-PDA 05-2021) 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 obligately require. The 56 °C heat-inactivation step targets complement, not mycoplasma, and must not be relied on for mycoplasma control.
SP-4 is expensive and labile. The CMRL-1066 + Yeastolate + 17 % FBS load makes SP-4 the highest-cost mycoplasma medium in the catalogue. Reserve SP-4 for genuinely fastidious primary isolations and for Spiroplasma work; for routine USP <63> / EP 2.6.7 testing where the target organisms are robust (M. orale, M. pneumoniae FH), use Hayflick. For routine veterinary mycoplasmology use PPLO + horse serum. The SP-4 cost premium is justified only by the SP-4-specific applications.
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 herds with full traceability documentation; for jurisdictions requiring origin-restricted FBS (e.g. New Zealand-origin BSE-free) ask about the "SP-4 NZ-FBS" variant.
Cholesterol oxidation in stored medium. Cholesterol oxidises to 7-ketocholesterol and related oxysterols on light + air exposure; oxysterols are reported to be inhibitory to sterol-dependent Mollicutes, with growth depression observed in lots of aged or air-exposed serum. The 17 % FBS load in SP-4 makes the medium especially sensitive to photo-oxidation. Store in amber, fully-filled bottles, 2–8 °C, use within 14 d. Bubble N2 over serum before sealing if extended storage is needed.
Penicillin G at 1000 U/mL is aggressive. The SP-4 Pen G load (1000 U/mL) is 20× the PPLO load (50 U/mL) and 10× the Hayflick load (100 U/mL), specifically because arthropod-tissue isolations and clinical primary isolations of fastidious mycoplasmas carry heavy bacterial burdens. Mycoplasma are cell-wall-free and intrinsically resistant. If contaminating Gram-negative bacteria break through, add gentamicin 25 µg/mL or streptomycin 100 µg/mL.
Arginine-utiliser cross-contamination on SP-4. CMRL 1066 contains L-arginine, which supports growth of arginine-utilising M. arginini (a common cell-culture contaminant). SP-4 is therefore not ideal for differential identification when arginine-utiliser contamination is suspected; switch to the M1A variant (arginine-free) for differential work.
M. genitalium specimen handling. M. genitalium survives < 2 h at ambient temperature in patient urogenital specimens. Transport in SP-4 broth itself on wet ice; inoculate within 4 h of collection. Cold-chain breaks are the principal cause of false-negative M. genitalium isolations.

Storage and Expiry · Safety

  • Dehydrated Stage-1 base (Mixture A): store sealed at 15–25 °C in original packaging away from direct sunlight. 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.
  • Stock E (fresh 25 % yeast extract): 2–8 °C, 7 days maximum.
  • Foetal Bovine 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 100,000 U/mL): −20 °C aliquots, 6 months; 2–8 °C, 2 weeks.
  • Complete SP-4 medium: 2–8 °C in amber bottles, fully filled, 14 days maximum.
  • SP-4 agar plates (poured): 2–8 °C in sealed bags, 7 days maximum.
  • Cryostocks of Spiroplasma / fastidious Mycoplasma: SP-4 + 15 % glycerol at −80 °C or LN2 vapour phase, > 5 years documented.

Safety notes. SP-4 supports BSL-2 pathogens (M. fermentans, M. genitalium, M. penetrans, M. pneumoniae). Some Spiroplasma are arthropod / livestock pathogens but BSL-1 for handling. All test work in a Class II BSC; specimen-handling waste autoclaved before disposal. FBS is bovine-derived; TSE-certified zoosanitary documentation supplied. CMRL 1066 contains glutamine and tissue-culture additives (no cytotoxic components in standard formulation). SDS available on request.

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. 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.
  3. Tully, J. G. & Razin, S. (1996). Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II, Academic Press.
  4. Razin, S. & Hayflick, L. (2010). Highlights of mycoplasma research — an historical perspective. Biologicals 38: 183–190.
  5. Hannan, P. C. T. (2000). Guidelines and recommendations for antimicrobial MIC testing against veterinary mycoplasma species. Veterinary Research 31: 373–395.
  6. ATCC Medium 988 specification, current revision.
  7. JCM Medium 14 specification (Spiroplasma).
  8. Regassa, L. B. & Gasparich, G. E. (2006). Spiroplasmas: evolutionary relationships and biodiversity. Frontiers in Bioscience 11: 2983–3002.
  9. Bébéar, C. M. & Kempf, I. (2005). Antimicrobial therapy and antimicrobial resistance. In Blanchard, A. & Browning, G. (eds), Mycoplasmas: Molecular Biology, Pathogenicity and Strategies for Control. Horizon Bioscience.

Frequently Asked Questions

Q1. When should I use SP-4 instead of PPLO or Hayflick?
Use SP-4 when (i) the target is a Spiroplasma, (ii) the target is a fastidious Mycoplasma (M. fermentans, M. genitalium, M. penetrans, M. pirum) that has failed to grow on PPLO or Hayflick, (iii) the specimen is arthropod tissue or plant phloem material, or (iv) you are running a high-sensitivity confirmatory subculture for cell-culture contamination suspected on a Hayflick-negative sample. For all other applications — routine veterinary Mycoplasma, USP <63> / EP 2.6.7 cell-culture testing, primary isolation of robust species — PPLO or Hayflick is more cost-effective and gives equivalent recovery.
Q2. Why FBS instead of horse serum in SP-4?
Tully et al. (1977) demonstrated that FBS gives better recovery of Spiroplasma than horse serum, likely because Spiroplasma binds horse-serum components differently (the chosen sterol uptake pathway favours FBS-borne lipoprotein-cholesterol over horse-serum-borne LDL-cholesterol). FBS also has a more consistent free-cholesterol content (less variability between lots than horse serum at the cost of higher absolute price). For SP-4 work, FBS is the reference choice; horse serum is an acceptable substitute for some Mycoplasma applications but reduces Spiroplasma recovery by 5–20 %. The GMExpression default is FBS; horse serum is a discount substitute on request.
Q3. Why is fresh yeast extract mandatory in SP-4 but not in PPLO?
Degradation of stored yeast extract is dominated by 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 chemically stable over this timescale and are not what ages out. SP-4 depends on fresh yeast extract as its only source of free purine and pyrimidine bases and ribonucleosides — Yeastolate is a lower-nucleotide peptic digest, and CMRL 1066 supplies deoxynucleosides but no free bases or ribonucleosides — while PPLO Broth carries beef-heart infusion that offsets part of the loss. What the 7-day clock actually tracks is the reducing fraction: composite Spiroplasma-promoting activity falls to roughly half by day 7 and about a quarter by day 14. The decline is smooth, so the 7-day limit is a chosen acceptance margin rather than a biochemical cliff. Robust species such as Mycoplasma orale continue to grow on aged extract because the stable salvage pool is all they require. Prepare and consume in the same week.
Q4. What is CCU titration?
CCU = colour-changing unit. The standard quantification method for Mycoplasma and Spiroplasma in liquid medium. Make a serial 10-fold dilution series of the broth culture into fresh SP-4 broth (with phenol red); incubate; record the last well that shows a colour shift after the standard incubation time (5–14 d depending on species). The titre = reciprocal of the last positive dilution. CCU is the broth analogue of CFU (colony-forming unit) on agar; some fastidious mycoplasmas grow in broth but not on agar, in which case CCU is the only available quantification. For Spiroplasma, CCU and CFU correlate within a factor of 2–5. CCU titration is the GMExpression-supplied SP-4 enumeration protocol.
Q5. Can SP-4 be used for M. pneumoniae in place of Eaton's?
Yes — for slow-growing M. pneumoniae clinical isolates, SP-4 often outperforms Eaton's, particularly from heavily pre-treated patients or chronic infections where the organism load is low and the strain has lost some metabolic capacity. Eaton's is preferred for pH-sensitive M. pneumoniae primary isolation (its pH 7.4 is optimal); SP-4 is preferred for difficult isolates that Eaton's fails to recover. For routine M. pneumoniae reference-strain maintenance, Hayflick is the lowest-cost option.
Q6. Why does the GMExpression SP-4 kit include both Yeastolate and fresh yeast extract?
They are different ingredients. Yeastolate (BD 255752) is a peptic (enzymatic) digest of yeast: yeast cells digested by pepsin, retaining B-vitamins and amino acids but with relatively low free-nucleotide content. Fresh Bacto Yeast Extract (BD 212750) is an autolysate of yeast (self-digestion by endogenous yeast enzymes, followed by water extraction): retains the free-nucleotide pool released by RNA breakdown (adenine, guanine, uracil, cytosine and the corresponding ribonucleosides) alongside B-vitamins. The Tully 1977 SP-4 formulation specifies both because Yeastolate provides amino-acid and B-vitamin support, while fresh yeast extract provides the nucleobases that Spiroplasma nucleotide salvage requires. The two are therefore not interchangeable: Yeastolate cannot supply the free bases and ribonucleosides on which Spiroplasma salvage depends.
Q7. Can I autoclave the complete SP-4 medium instead of filter-sterilising?
No. Autoclaving the complete medium causes (i) denaturation of CMRL 1066 vitamins (especially ascorbate, thiamine, riboflavin), (ii) caramelisation of the 0.5 % glucose into Maillard-reaction products that inhibit Spiroplasma, (iii) denaturation of FBS proteins, and (iv) precipitation of Ca-phosphate from the CMRL salts + serum buffer mix. Filter-sterilisation through 0.22 µm PES is mandatory and is the published Tully 1977 protocol. The Stage-1 base alone is autoclaved; all Stage-2 supplements are sterile-filtered separately; the combined Stage-1 + Stage-2 medium is filter-sterilised as a final mandatory step.
Q8. What temperature does SP-4 cultivation require?
Highly species-dependent. 30 °C: slow-growing Spiroplasma (most plant-associated species; S. mirum; arthropod-borne species); fastidious primary isolation. 32 °C: standard Spiroplasma citri reference. 35–37 °C: M. fermentans, M. genitalium, M. pneumoniae, mammalian-associated species. 26–28 °C: insect-host-adapted Entomoplasma / Mesoplasma. Match the temperature to the host environment of the target organism; when in doubt, run parallel tubes at 30 °C and 37 °C and observe which shows growth first.
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, PDA J Pharm Sci Technol 68:281). The 0.22 µm rating is defined against a larger, walled organism (Brevundimonas diminuta, ASTM F838), so it makes no mycoplasma-retention claim, and SP-4's penicillin G does not touch a contaminating Mollicute (they are β-lactam-resistant). In this protocol the 0.22 µm step controls conventional bacterial/fungal bioburden; it is not a mycoplasma barrier. An actual SP-4 organism illustrates the point: Spiroplasma corruscae is filterable through 220 nm but retained by 100 nm membranes (Hackett et al. 1996). How mycoplasma cleanliness is actually assured: the Stage-1 base, tryptone, peptone, Yeastolate and the fresh yeast extract are all autoclaved (heat-sterile), so the dominant vector is the non-autoclaved serum, with CMRL 1066 a distant second. Source 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 filtration (A. laidlawii-qualified) of the CMRL 1066 fraction plus strain-genotyping release checks. This is more effective — and far more practical — than tightening the terminal filter, since 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 (generic mycoplasma QC cannot distinguish a contaminating Mollicute from your intended strain).