NZCYM Broth

Overview

NZCYM Broth is the highest-titre common broth for bacteriophage λ work and the global reference base for λ-derived molecular biology — library plating, screening, packaging extracts, cosmid propagation, and BAC handling. Originally formulated by Blattner, Williams, Blechl et al. in 1977 for the Charon-phage replacement vector series (Science 196: 161), the NZCYM design has three defining features that make it superior to LB and 2×YT for λ work: (i) NZ Amine, an enzymatic casein digest from Sheffield Bio-Science / Kerry with very low free-amino-acid asymmetry, (ii) casamino acids as a supplementary defined amino-acid pool, and (iii) built-in MgSO₄ at 8.1 mM, well above the ~ 2 mM minimum required for stable λ tail-attachment to the LamB outer-membrane receptor, and within the 8–10 mM band that supports maximal adsorption rate without precipitation.

The GMExpression formulation is supplied as a pre-balanced dehydrated base (NZ Amine 10 g/L, casamino acids 1 g/L, yeast extract 5 g/L, NaCl 5 g/L, MgSO₄·7H₂O 2 g/L; total 23 g/L) ready for autoclave-and-use. Optional maltose stock (filter-sterilised 20 % w/v) provides the lamB-operon induction needed for high-efficiency library plating; optional top-agar (0.6 % w/v) and base-agar (1.5 % w/v) packs complete the λ plaque-assay supply chain. Typical liquid lysate titre on the standard λ host LE392: 10¹⁰–10¹¹ PFU/mL — the highest of any common phage broth.

We also have

LB Broth (Miller) · LB Broth (Lennox) · 2×YT Broth (high-density host broth) · Bacteriophage Nutrient Broth (ΦX174 propagation grade) · TSB + 10 % Glycerol (host cryostock) · SM Buffer (phage diluent / storage) · BHI Broth (Gram-positive phage hosts)

Package Contents

Standard pack:

Mixture — pre-weighed dehydrated NZCYM base (NZ Amine 50 g, casamino acids 5 g, yeast extract 25 g, NaCl 25 g, MgSO₄·7H₂O 10 g; total 115 g for 5 L final volume). Sheffield Bio-Science / Kerry NZ Amine A locked supplier. Triple-foil-pouched; CofA traceable.

Optional stock:

Stock Mal (optional) — 20 % w/v D-maltose monohydrate, filter-sterilised at 0.22 µm, sterile-fill 25 mL amber vial; dosed at 10 mL/L for 0.2 % w/v final (= 5.5 mM); pre-induces lamB on the host before λ infection.

Alternative pack:

Top-agar pack (optional) — pre-weighed bacteriological agar (6 g per 1 L) for the 0.6 % w/v soft-agar overlay in λ plaque assays and library plating.
Base-agar pack (optional) — pre-weighed agar (15 g per 1 L) for plaque-assay base plates.

Customisation options on request: NZYM variant (omit casamino acids), NZY variant (omit casamino acids and Mg²⁺), animal-origin-free NZCYM (soybean substitute for NZ Amine), low-endotoxin NZCYM for vaccine-vector packaging, ampicillin-supplemented NZCYM for cosmid selection.

Composition — per 1 L equivalent unless stated otherwise

NZCYM Broth (Blattner et al. 1977 / Sambrook & Russell 2001 / BD Difco 244830; per 1 L)

Component	Concentration	Function
NZ Amine (Sheffield / Kerry enzymatic casein digest)	10.0 g	Primary nitrogen and free amino-acid source with defined low amino-acid asymmetry; the "NZ" in NZCYM
Casamino acids (acid-hydrolysed casein)	1.0 g	Supplementary free amino acid pool; the "C" in NZCYM
Yeast extract	5.0 g	B-vitamins, purines, pyrimidines, NAD precursors; the "Y" in NZCYM
Sodium chloride (NaCl)	5.0 g	Osmotic balance (86 mM)
Magnesium sulfate heptahydrate (MgSO₄·7H₂O)	2.0 g (= 8.1 mM Mg²⁺)	The "M" in NZCYM — stabilises λ tail attachment to LamB; required cofactor for λ DNA replication

Total dry solids: 23 g/L. Pre-autoclaving pH: 7.0 ± 0.2 at 25 °C; typically requires < 0.2 mL of 5 M NaOH per litre to reach pH 7.0. Unlike LB-Mg, NZCYM does not phase-separate or precipitate on autoclaving because the MgSO₄ is balanced against the casamino-acid load.

Optional supplements

Supplement	Final concentration	Use case	Notes
D-Maltose monohydrate (CAS 6363-53-7)	0.2 % w/v (= 5.5 mM)	λ library plating; λ high-titre lysate	Filter-sterilise 20 % w/v stock; add 10 mL/L; pre-grow host to OD₆₀₀ 1.0 in NZCYM-maltose before infection — induces lamB
Additional MgSO₄·7H₂O	+ 1–2 mM (top-up to 10 mM)	Long lysate runs where Mg²⁺ is depleted by host metabolism	Add 1–2 mL of 1 M sterile filtrate per litre post-autoclave
Ampicillin / kanamycin	50–100 µg/mL	Cosmid / BAC selection	Standard antibiotic selection; add post-autoclave
Agar (top agar)	6–7 g/L (0.6–0.7 % w/v)	λ plaque assay overlay; library plating	Add before autoclaving; equilibrate to 50 °C before mixing with phage and host
Agar (base agar)	15 g/L (1.5 % w/v)	λ plaque-assay base plates	Pour 20 mL per 90 mm plate

NZCYM variants

NZYM — omit casamino acids. NZ Amine + yeast extract + Mg²⁺. Slightly cheaper; ~ 20 % lower λ titre on fastidious hosts.
NZY — omit casamino acids and Mg²⁺. NZ Amine + yeast extract only. Used as a substrate-defined base where Mg²⁺ is dosed downstream.
NZCYM + 0.2 % maltose ("NZCYM-Mal") — the standard library-plating base; lamB-induced.

→Culturomics and Media Navigator

Need help matching this medium to your λ-vector / cosmid / BAC workflow — or starting from a packaging-extract specification 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 λ vector classes and host strains, or (ii) start from a vector (Charon, EMBL3/4, λFIX, λZAP, cosmid pJB8, BAC pBeloBAC11) and view the panel of GMExpression media for propagation, plating, and packaging. The Navigator integrates NZCYM with the wider LB, 2×YT, Bacteriophage Nutrient Broth, BHI, SM Buffer, TSB–Glycerol, PPLO, Hayflick, and SP-4 catalogue.

Use and Applications

High-titre λ liquid lysate. Pre-grow LE392 (or another lamB⁺ host) in NZCYM + 0.2 % maltose to OD₆₀₀ 0.5; infect with λ at MOI 0.01; incubate 3–5 h at 37 °C until visible clearing; add 1 % chloroform, vortex, centrifuge, filter. Typical titre 10¹⁰–10¹¹ PFU/mL.
λ genomic library plating and screening. NZCYM + 1.5 % agar plates + maltose-induced host + 50 µL library + 3 mL NZCYM + 0.6 % top agar; pour overlay; 8–18 h at 37 °C. Standard Charon, EMBL3/4, λFIX, λDASH, λZAP library protocol.
λ packaging-extract production. NZCYM as the growth medium for the lysogenic strains BHB2688 and BHB2690 producing the head- and tail-protein fractions of Gigapack-style packaging extracts; standard Stratagene / Promega / NEB packaging extract protocol.
Cosmid library propagation. NZCYM + ampicillin or kanamycin for routine growth of cosmid-containing hosts (pJB8, pHC79, SuperCos, cKM cosmids).
BAC propagation. NZCYM + chloramphenicol 12.5 µg/mL for pBeloBAC11-, pBAC108L-, pCC1BAC-derived BACs; supports single-copy maintenance with low recombination.
λ lysogen induction and excision. NZCYM is the standard broth for thermal induction of λcI⁸⁵⁷ lysogens (shift from 30 to 42 °C) and for mitomycin-C-induced lytic-cycle activation.
Cre/loxP subcloning of λ or cosmid inserts — the Cre recombinase (a P1 phage site-specific recombinase, not a λ recombinase) catalyses excision / integration between loxP sites in NZCYM-grown hosts; routine subcloning, vector retrofitting, and BAC end-clone rescue.

Compatible Microorganisms

Lambda host strains (primary use)

E. coli LE392 (hsdR514, supE44, supF58, lacY1, galK2, galT22, metB1, trpR55) — classic λ-vector plating host; reference NZCYM strain
E. coli Y1090 (r−, m−, supF, lacU169, hsdR, pMC9) — λgt11 expression-library screening host
E. coli MM294 (hsdR−, supE) — cosmid plating host
E. coli Q358, Q359 — Charon-vector plating
E. coli XL1-Blue MRF' (mcrA, mcrB, mcrCB, hsdSMR, mrr) — λZAP and λDASH plating
E. coli SURE, SURE-2 — recombination-suppressing λ-cloning hosts
E. coli BHB2688, BHB2690 — packaging-extract producer strains (cI⁸⁵⁷ lysogens)
E. coli P2392 — Spi-selection λ library plating

Cosmid and BAC hosts

E. coli ED8767, DH1, DH5α-MCR — cosmid propagation
E. coli DH10B, EPI300 — BAC and fosmid propagation; copy-control compatible
E. coli NS3145, NS3622 — recombineering hosts (galK, galETKM positive selection)

Not optimised for: ΦX174 (use LB Lennox + 5 mM Ca²⁺), filamentous M13 (use 2×YT — the Mg²⁺ in NZCYM is unnecessary), Gram-positive phages (use BHI), Φ6-class enveloped phages (use cyanobacterium-derived media), mycobacteriophages (use Middlebrook 7H9).

Preparation

1Weigh. Use the pre-weighed Mixture A: 23 g for 1 L. Tare a clean autoclavable Schott bottle of at least 1.5× final volume.

2Suspend & dissolve. Add Mixture A to 950 mL of distilled or deionised water (Type II reagent water, > 1 MΩ·cm). Stir for 5–8 min until fully dissolved; the MgSO₄ dissolves with mild endothermic cooling.

3Adjust pH. Check pH with a calibrated meter; target 7.0 ± 0.2 at 25 °C. The base typically reads pH 6.8–6.9 before adjustment; titrate with 5 M NaOH (typically < 0.2 mL/L) to bring to pH 7.0.

4Bring to final volume. Make up to 1000 mL with distilled water.

5Dispense. Bulk flasks: leave caps one-quarter turn loose. For λ library plating, partition into 100–500 mL aliquots so the same broth base is used for top agar and bottom agar on each plate.

6Autoclave. 121 °C × 15 min for ≤ 500 mL; 121 °C × 20 min for 1 L bottles. Slow cooling.

7Cool to < 50 °C before adding post-autoclave supplements (maltose, antibiotics).

8Add maltose for λ library plating. Aseptically add 10 mL of 20 % w/v sterile maltose per litre (= 0.2 % final). Pre-grow the host in this NZCYM-maltose to OD₆₀₀ 1.0 before infection — this is the lamB-induction step that gives the 10–100× titre boost.

9Plaque assay overlay (DAL). Mix 50 µL λ library + 200 µL host (OD₆₀₀ 1.0 in NZCYM-Mal) in 3 mL NZCYM top agar (0.6 %, equilibrated to 50 °C); pour over NZCYM base agar (1.5 %); incubate 37 °C 8–18 h.

10Storage. Sealed glass bottles at 2–8 °C, light-protected. NZCYM is unusually stable on storage because the built-in MgSO₄ does not precipitate without phosphate.

Critical control points

Maltose pre-induction is non-negotiable for library work. The lamB-encoded outer-membrane LamB porin is the λ J-protein adsorption receptor. Without maltose induction (or constitutive lamB) the host adsorption rate is 10× slower and the plaque count drops proportionally. Pre-grow the host in NZCYM + 0.2 % maltose for ≥ 2 generations before infection; harvest at OD₆₀₀ 1.0 (not 0.5; library plating uses denser hosts than liquid lysates).
NZ Amine supplier lock. NZ Amine is manufactured by Sheffield Bio-Science (subsequently Kerry). Generic "casein peptone" substitutes are not equivalent — the free amino-acid profile differs, and side-by-side bench comparisons consistently show lower λ titre and reduced library complexity. Lock the supplier on the CofA.
Top-agar equilibration temperature. Soft (0.6 %) NZCYM agar must be held at 50 °C ± 2 in a water bath. Above 55 °C the host suffers heat-shock loss; below 48 °C the agar starts to set during mixing and gives a granular overlay. Use an immersion thermometer rather than the bath setpoint.
Same base for top and bottom agar. Use NZCYM for both layers in the plaque assay. Mixing NZCYM bottom with LB top (or vice versa) causes ion-strength shifts during overlay equilibration that distort plaque morphology and reduce visible plaque count.

Cautions

NZ Amine supplier substitution. Generic "casein peptone" or "NZ Amine Type S/A" lots from non-Sheffield/Kerry sources are not equivalent. The defining feature of NZ Amine is its very low free-amino-acid asymmetry and consistent free-tryptophan content — both of which directly affect λ gene-expression rate during library plating and packaging-extract preparation. Specify Sheffield/Kerry NZ Amine A on every CofA; do not accept generic substitutes for regulated workflows.

Maltose stability. Sterile 20 % maltose stock degrades by ~ 5 % per month at 2–8 °C via slow disproportionation to glucose + reducing sugars. Aliquot in 25 mL single-use vials at −20 °C for long-term storage; once thawed, use within 2 weeks. Do not autoclave maltose with the broth — caramelisation produces inhibitory furans that reduce λ titre.

Mg²⁺ depletion during long lysate runs. Host metabolism consumes free Mg²⁺ into rRNA-bound and ribosome-bound pools; in long (> 6 h) lysate runs the free Mg²⁺ can drop below the 2 mM threshold required for λ tail attachment, prematurely capping the burst. For runs > 5 h, supplement with 1–2 mL of 1 M sterile MgSO₄ per litre mid-run to top up to 10 mM.

Packaging-extract producer strains. BHB2688 (head-protein producer) and BHB2690 (tail-protein producer) carry λcI⁸⁵⁷ temperature-inducible lysogens. Maintain at 30 °C (no induction) on NZCYM + Amp; induce by shift to 42 °C for the packaging-extract protocol. Accidental warming above 32 °C during routine handling causes partial induction and loss of producer-strain stability.

Cosmid / BAC recombination. Long-incubation NZCYM cultures of cosmid- or BAC-bearing hosts can lose insert via RecA-dependent recombination on repeated subculture. Use Rec⁻ hosts (DH10B, SURE, NS3622) for stable BAC propagation; subculture as few times as possible from a frozen stock; verify insert by restriction mapping every 3 subcultures.

Storage and Expiry · Safety

Dehydrated powder (Mixture A): store sealed at 15–25 °C in original packaging away from direct sunlight. Shelf life 24 months from manufacture (slightly shorter than LB due to MgSO₄ hygroscopicity).
Sterilised broth (unsupplemented): 2–8 °C in sealed glass, 6 months; no Mg-precipitation on long storage because phosphate is absent.
Sterilised broth + maltose: 2–8 °C, 2 weeks.
Sterilised broth + antibiotic: antibiotic-dependent; ampicillin 1 week, kanamycin 4 weeks, chloramphenicol 4 weeks.
Maltose 20 % stock: −20 °C aliquots, 12 months; 2–8 °C, 2 weeks.
Poured NZCYM agar plates: 2–8 °C in sealed bags, 4 weeks; bring to room temperature before plating to avoid condensation.

Safety notes. NZCYM is a non-hazardous routine bacterial broth. The principal biosafety concerns are (i) host strain BSL (most λ-host strains are BSL-1), (ii) chloroform vapour during lysate processing (handle in a fume hood; never autoclave chloroform), and (iii) λ packaging extracts (the lytic packaging strains are BSL-1 but should be handled in a Class II BSC to prevent cross-contamination with other λ libraries). SDS available on request.

References

Blattner, F. R., Williams, B. G., Blechl, A. E., Denniston-Thompson, K., Faber, H. E., Furlong, L.-A., Grunwald, D. J., Kiefer, D. O., Moore, D. D., Schumm, J. W., Sheldon, E. L., Smithies, O. (1977). Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science 196: 161–169. [Original NZCYM reference]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., CSH Press. Chapter 2 (Bacteriophage λ).
Frischauf, A. M., Lehrach, H., Poustka, A., Murray, N. (1983). Lambda replacement vectors carrying polylinker sequences. Journal of Molecular Biology 170: 827–842.
Hohn, B. & Murray, K. (1977). Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proceedings of the National Academy of Sciences USA 74: 3259–3263. [BHB2688/BHB2690 strain origin]
Karn, J., Brenner, S., Barnett, L., Cesareni, G. (1980). Novel bacteriophage λ cloning vector. Proceedings of the National Academy of Sciences USA 77: 5172–5176.
Kim, U. J., Birren, B. W., Slepak, T., Mancino, V., Boysen, C., Kang, H. L., Simon, M. I., Shizuya, H. (1996). Construction and characterization of a human bacterial artificial chromosome library. Genomics 34: 213–218.
BD Difco & BBL Manual, 12th ed., NZCYM Broth monograph (BD 244830); Sigma N4019 specification.
Murray, N. E. (2000). Lambda II in molecular biology — a personal view. In Hendrix, R. W. & Roberts, J. W. (eds), Lambda II, CSH Press.

Frequently Asked Questions

Q1. Why does NZCYM give a higher λ titre than 2×YT + Mg²⁺?

Two reasons. First, NZ Amine has a more balanced free amino-acid profile than tryptone, which gives a smoother growth curve and a more reproducible burst-size window. Second, casamino acids provide a defined supplementary pool of free amino acids that the host can immediately drive into λ capsid-protein synthesis during the lytic phase, increasing burst size by 20–40 % vs 2×YT. The built-in 8.1 mM Mg²⁺ is also pre-balanced against the casamino-acid load to avoid the Mg-citrate / Mg-acetate sequestration that occurs in some 2×YT-Mg formulations.

Q2. What is the difference between NZCYM, NZYM, and NZY?

NZCYM = NZ Amine + Casamino acids + Yeast extract + Magnesium (full formulation; reference for library work and high-titre lysate). NZYM = NZ Amine + Yeast extract + Magnesium (omits casamino acids; ~ 20 % lower λ titre on fastidious hosts). NZY = NZ Amine + Yeast extract (omits casamino acids and Mg²⁺; used as a substrate-defined base where Mg²⁺ is dosed downstream). Default to NZCYM unless cost optimisation or upstream Mg²⁺ control dictates otherwise.

Q3. Can I substitute generic casein peptone for NZ Amine?

Not for regulated workflows. The defining feature of NZ Amine (Sheffield Bio-Science / Kerry NZ Amine A) is its very low free-amino-acid asymmetry and consistent free-tryptophan content, both of which directly affect λ lytic-cycle gene expression rate. Generic casein peptone substitutes give measurably lower λ titre and reduced library complexity in side-by-side comparisons; the precise magnitude varies with the specific substitute peptone, with reductions in the order of tens of percent commonly reported. For routine non-regulated work the substitution may be acceptable with a documented titre cost; for λ library plating, packaging-extract production, or any GMP/cGMP workflow lock the NZ Amine supplier.

Q4. Why does library plating use OD₆₀₀ 1.0 host instead of OD₆₀₀ 0.5?

Library plating density is set by the host-cell concentration in the top agar, which determines the lawn density of the overlay. Plaque morphology is best when the host concentration is ~ 5–10 × 10⁸ CFU/mL in the top agar, corresponding to OD₆₀₀ ~ 1.0 from a saturated NZCYM-maltose pre-culture. OD₆₀₀ 0.5 (mid-log) gives a thinner lawn that visualises plaques less crisply and increases the risk of plaque coalescence at high library titres. Liquid lysate work, in contrast, harvests at mid-log for maximum burst-size; the two density rules apply to different steps.

Q5. Why is there no phosphate buffer in NZCYM?

Phosphate would precipitate Mg²⁺ as insoluble Mg-phosphate, removing the 8 mM Mg²⁺ that defines NZCYM's λ performance. The pH of NZCYM is stabilised by the inherent buffering of the peptide / amino-acid pool and by the yeast-extract phosphate (~ 3–5 mM, sub-precipitation threshold against 8 mM Mg²⁺). For very long lysate runs where pH drift is a concern, use Tris-HCl 10 mM as a top-up buffer; never substitute phosphate.

Q6. Can I use NZCYM for BAC propagation, or only for cosmid?

Yes, NZCYM is fully compatible with single-copy BAC propagation. Standard BAC hosts (DH10B, EPI300) grow well in NZCYM + chloramphenicol 12.5 µg/mL; the higher amino-acid content vs LB supports the very low growth-rate demands of single-copy BACs without selecting for high-copy recombinants. For copy-control BAC vectors (pCC1BAC), use NZCYM + chloramphenicol routine maintenance + L-arabinose 0.01 % induction at harvest. Sub-culture as few times as possible to limit RecA-mediated insert loss.

Q7. Why does my λ plaque assay show "halo plaques"?

Halo plaques (small dense centre with translucent halo) usually indicate (i) excess Mg²⁺ driving over-efficient adsorption that overwhelms the host density, or (ii) host strain expressing inhibitory factors. Check the actual Mg²⁺ concentration (8–10 mM is optimal; > 15 mM gives halos); verify the host is LamB⁺ (no malB mutation); confirm the host was maltose-induced before plating. If halos persist, dilute the library 10× further and re-plate — very high plaque densities also look halo-like due to plaque coalescence.

Q8. Is NZCYM the right broth for λ helper-phage rescue of vectors like λ-ZAP?

Yes — λZAP is rescued in NZCYM + maltose using XL1-Blue MRF' or a similar host with the M13K07 / ExAssist helper phage. The NZCYM Mg²⁺ supports both the λ replication phase and the helper-phage rescue phase; the maltose induces lamB for any residual λ activity. Follow the standard Stratagene / Agilent λZAP / ExAssist protocol; NZCYM is the medium-of-record in the kit documentation.