Serum-Free Media (Chapter 9) PDF

# Chapter 9: Serum-Free Media Although many cell lines are still propagated in medium supplemented with serum, in many instances cultures may now be propagated in serum-free media. Historically the need to standardize media among laboratories, provide specialized media for specific cell type, and...

# Chapter 9: Serum-Free Media Although many cell lines are still propagated in medium supplemented with serum, in many instances cultures may now be propagated in serum-free media. Historically the need to standardize media among laboratories, provide specialized media for specific cell type, and eliminate variable natural products led to the development of more complex media. - **M199** of Morgan et al. [1950] - **CMRL 1066** of Parker et al. [1957] - **NCTC109** [Evans et al., 1956] - **Waymouth's MB 572/1** [1959] - **NCTC135** [Evans & Bryant, 1965] - **Birch and Pirt [1971]** for L929 mouse fibroblast cells - **Ham's F10** [Ham, 1963] and **F12** [Ham, 1965] clonal growth media for Chinese hamster ovary (CHO) cells - **Serum-free media** were also developed for HeLa human cervical carcinoma cells [Blaker et al., 1971; Higuchi, 1977] ## Disadvantages of Serum Using serum in a medium has several disadvantages: 1. **Physiological variability**. The major constituents of serum such as albumin and transferrin, are known, but serum also contains a wide range of minor components that can have a considerable effect on cell growth and response to test substances. 2. **Shelf life and consistency**. Serum varies from batch to batch, and at best a batch will last one year, perhaps deteriorating during that time. It must then be replaced with another batch that may be selected as similar, but will never be identical to the first batch. 3. **Quality control**. Changing serum batches requires extensive testing to ensure that the repalcement is as close as possible to the previous batch. This can involve testing against a range of cell types and special functions. ## Advantages of Serum-Free Media All of the problems above can be eliminated by removal of serum and other animal products. Serum-free media have in addition two major positive benefits. ### Definition of Standard Medium Given pure constituents are used, a given medium formulation can be standardized regardless of where it is used and by whom. Not only does this allow easier validation of industrial processes, it also means research labs can replicate conditions to repeat and confirm experimental data. ### Selective Media One of the major advantages of the control over growth-promoting activity afforded by serum-free media is the ability to make a medium selective for a particular cell type. ## Disadvantages of Serum-Free Media Serum-free media are not without disadvantages: 1. **Multiplicity of media**. Each cell type appears to require a different recipe, and cultures from malignant tumors may vary in requirements from tumor to tumor, even with one class of tumors. 2. **Selectivity**. Unfortunately, the tranisition to serum-free conditions, however desirable, is not always as straightforward as it seems. Some media may select a sublineage that is not typical of the whole population, and even in continuous cell lines, some degree of selection may still be required. 3. **Reagent purity**. The removal of serum also requires that the degree of purity of reagents and water and the degree of cleanliness of all apparatus be extremely high, as the removal of serum also removes the proctive, detoxifying action that some serum proteins may have. 4. **Cell proliferation**. Growth is often slower in serum-free media, and fewer generations are achieved with finite cell lines. ## Replacement of Serum The essential factors in serum have been described and include: - **Adhesion factors** such as fibronectin - **Peptides** such as insulin, PDGF, and TGF-β that regulate growth and differentiation - **Essential nutrients** such as minerals, vitamins, fatty acids, and intermediary metabolites - **Hormones** such as insulin, hydrocortisone, estrogen, and triiodothyronine. All these constituents regulate membrane transport, differentiation, and the constitution of the cell surface. Although some of these constituents are included in the formulation of serum-free media, others are not and may require addition and optimization. ## Commercially Available Serum-Free Media Around 50 suppliers now make serum-free media. Some are defined formulations, such as: - MCDB 131 for endothelial cells - LHC-9 for bronchial epithelium Whereas others are proprietary formulations, such as: - BD CHO Medium - Hybridoma AGT Many are designed primarily for culture of hybridomas, when the formation of a product that is free of serum proteins is clearly important, but others are aplicable to other cell types. ## Serum-Free Subculture Propagating attached cells in serum-free conditions may require modification of the subculture protocol as serum contains significant components not necessarily nutrient or growth factor related. - **Adhesion factors**. When serum is removed, it may be necessary to treat the plastic growth surface with fibronectin or laminin. - **Proteins and Polyamines**. The inclusion in medium of proteins such as bovine serum albumin (BSA), 0.5 to 10 mg/mL, or tissue extracts often increases cell growth and survival, but adds undefined constituents to the medium and retains the problem of adventitious infectious agents. - **Protease inhibitors**. After trypsin-mediated subculture, the addition of serum inhibits any residual proteolytic activity. Consequently protease inhibitors such as soya bean trypsin inhibitor or 0.1 mg/mL aprotinin must be added to serum-free media after subculture. Furthermore, because crude trypsin is a complex mixture of proteases, some of which may require different inhibitors, it is preferable to use pure trypsin followed by a trypsin inhibitor. Alternatively, one may wash cells by centrifugation to remove trypsin, although it may still be advisable to include a trypsin inhibitor in the wash. - **Viscosity**. One of the actions of serum is to increase the viscosity of the medium. This is particularly important in stirred suspension cultures. - **Hormones**. Hormones that have been used to replace serum include growth hormone, insulin, and hydrocortisone. - **Growth Factors**. The family of polypeptides that has been found to be mitogenic in vitro is now quite extensive, including: - The heparin-binding growth factors - EGF - PDGF - IGF-I and -II - The interleukins. ## Selecting a Serum-Free Medium If the reason for using a serum-free medium is to promote the selective growth of a particular type of cell, then that reason will determine the choice of medium. ## Replacement of Serum There is increasing pressure from regulatory authorities to remove all animal products from contact with cultured cells used in the production of biopharmaceuticals. Trypsin can be replaced with recombinant trypsin or a nonvertebrate protease, and growth factors with recombinant growth factors. BSA can often be replaced by recombinant growth factors. ## Animal Protein-Free Media There is increasing pressure from regulatory authorities to remove all animal products from contact with cultured cells used in the production of biopharmaceuticals. Trypsin can be replaced with recombinant trypsin or a nonvertebrate protease, and growth factors with recombinant growth factors. BSA can often be replaced by recombinant growth factors. # Table 9.4: Growth Factors and Mitogens | Name and Synonyms | Abbreviation | Mol. mass. (KDa) | Source | Function | |---|---|---|---|---| | Acidic fibroblast gf; aFGF; heparin binding gf 1, HBGF-1; endothelial cell gf (ECGF); myoblast gf (MGF) | FGF-1 | 13h | Bovine brain; pituitary | Mitogen for endothelial cells | | Activin; TGF-ẞ family | AR | 19, 21, 43 | Gonads | Morphogen; stimulates FSH secretion | | Amphiregulin | | 8 | MCF-7 conditioned medium | Autocrine EGF-like gf for keratinocytes | | Angiogenin | | 16 | Fibroblasts, lymphocytes, colonic epithelial; cells | Angiogenic; endothelial mitogen | | Astroglial growth factor-1; member of acidic FGFs | AGF-1 | 14 | Brain | Mitogen for astroglia | | Astroglial growth factor-2; member of basic FGFs | AGF-2 | 14 | Brain | Mitogen for astroglia | | Basic fibroblast gf; bFGF; HBGF-2; prostatropin | FGF-2 | 13h | Bovine brain; pituitary | Mitogen for many mesodermal and neuroectodermal cells; adipocyte and ovarian granulosa cell differentiation | | Brain-derived neurotrophic factor | BDNF | 28 | Brain | Neuronal viability | | Cachectin | TNF-α | 17 | Monocytes | Catabolic; cachexia; shock | | Cholera toxin | CT | 80-90 | Cholera bacillus | Mitogen for some normal epithelia | | Ciliary neurotrophic factor; member of IL-6 group | CNTF | | Eye | | | Connective tissue growth factor; IGFBP8 | CTGF | 38p | Peritoneal mesothelium; mesangial cells | Fibroblast mitogen; angiogenic; matrix production | | Endothelial cell growth factor; acidic FGF family | ECGF | h | Recombinant | Endothelial mitogenesis | | Endothelial growth supplement; mixture of endothelial mitogens | ECGS | | Bovine pituitary | Endothelial mitogenesis | | Endotoxin | | | Bacteria | Stimulates TNF production | | Epidermal growth factor, Urogastrone | EGF | 6 | Submaxillary salivary gland (mouse) human urine; guinea pig prostate | Active transport; DNA, RNA, protein, synthesis; mitogen for epithelial and fibroblastic cells; synergizes with IGF-1 and TGF-β | | Erythropoietin | EPO | 34-39 g | Juxtaglomerular cells of kidney | Erythroid progenitor proliferation and differentiation | | Eye-derived growth factor-1; member of basic FGFs | EDGF-1 | 14 | Eye | | | Eye-derived growth factor-2; member of acidic FGFs | EDGF-2 | 14 | Eye | | | Fibroblast gf-3; product of int-2 oncogene | FGF-3 | 14h | Mammary tumors | Mitogen; morphogen; angiogenic | | Fibroblast gf-4; product of hst/KS3 oncogene | FGF-4 | 14h | Embryo; tumors | Mitogen; morphogen; angiogenic | | Fibroblast gf-5 | FGF-5 | 14h | Fibroblasts; epithelial cells; tumors | Mitogen; morphogen; angiogenic | | Fibroblast gf-6; product of hst-2 oncogene | FGF-6 | 14h | Testis; heart; muscle | Mitogenic for fibroblasts; morphogen | | Fibroblast gf-9 | FGF-9 | 14h | | | | Fibroblast gf-10 | FGF-10 | 14h | | | | Granulocyte colony-stimulating factor; pluripoietin; CFS-B | G-CSF | 18-22 | Recombinant | Trophoblast invasion; stimulate uPA and PAI-1; alveolar epithelial mitogen | | Granulocyte/macrophage colony-stimulating factor; CSA; human CSFa | GM-CSF | 14-35 g | Recombinant | Granulocyte progenitor proliferation and differentiation | | Heparin-binding EGF-like factor | HB-EGF | | Fibroblasts | Epithelial morphogenesis; hepatocyte proliferation | | Hepatocyte gf, HBGF-8; Scatter factor | HGF | h | Breast cancer cells; recombinant | Mammary and other epithelial cell mitogen | | Heregulin; erbB2 ligand | HRG | 70 | Activated lymphocytes | Morphogen; inhibits FSH secretion | | Immune interferon; macrophage-activating factor (MAF) | IFN-y | 20-25 | Ovary | Antiviral; activates macrophages | | Inhibin; TGF-ẞ family | | 31 g | | Morphogen; inhibits FSH secretion | | Insulin | Ins | 6 | ẞ Islet cells of pancreas | Glucose uptake and oxidation; amino acid uptake; glyconeogenesis | | Insulin-like gf 1; somatomedin-C; NSILA-1 | IGF-1 | 7.6 | Liver | Mediates effect of growth hormone on cartilage sulfation; insulin-like activity | | Insulin-like gf 2; MSA in rat | IGF-2 | 7 | BRL-3A cell-conditioned medium | Mediates effect of growth hormone on cartilage sulfation; insulin-like activity | | Interferon-a1; leukocyte interferon | IFNa1 | 18-20 | Macrophages | Antiviral; differentiation inducer; anticancer | | Interferon-a2; leukocyte interferon | IFN-a2 | 18-20 | Macrophages | Antiviral; differentiation inducer; anticancer | | Interferon-ẞ; fibroblast interferon | IFN-B1 | 22-27 g | Fibroblasts | Antiviral; differentiation inducer; anticancer | | Interferon ẞ2; fibroblast interferon; IL-6, BSF-2 (see also IL-6) | IFN-B2 | 22-27 g | Activated T-cells; fibroblasts; tumor cells | Antiviral; differentiation inducer; anticancer | | Interferon y; immune interferon | IFNy | | Activated lymphocytes | Antiviral; macrophage activator; antiproliferative on transformed cells | | Interleukin-1; lymphocyte-activating factor (LAF); B-cell-activating factor (BAF); hematopoietin-1 | IL-1 | 12-18 | Activated macrophages | Induces IL-2 release | | Interleukin-2; T-cell gf (TCGF) | IL-2 | 15 | CD4+ve lymphocytes (NK); murine LBRM-5A4 and human Jurkat FHCRC cell lines | Supports growth of activated T-cells; stimulates LAK cells | | Interleukin-3; multipotential colony-stimulating factor; mast cell growth factor | IL-3 | 14-28 g | Activated T-cells; WEHI-3b myelomonocytic cell lines | Granulocyte/macrophage production and differentiation | | Interleukin-4; B-cell gf; BCGF-1; BSF-1 | IL-4 | 15-20 | Activated CD4+ve lymphocytes | Competence factor for resulting B-cells; mast cell maturation (with IL-3) | | Interleukin-5; T-cell-replacing factor (TRF); eosinophil-differentiating factor (EDF) BCGF-2 | IL-5 | 12-18 g | T-lymphocytes | Eosinophil differentiation; progression factor for competent B-cells | | Interleukin-6; Interferon B-2; B-cell-stimulating factor (BSF-2); hepatocyte-stimulating factor; hybridoma-plasmacytoma gf | IL-6 | 22-27 g | Activated T-cells macrophage/monocytes; fibroblasts; tumor cells | Acute phase response; B-cell differentiation; keratinocyte differentiation; PC12 differentiation | | Interleukin-7; hematopoietic growth factor; lymphopoietin 1 | IL-7 | 15-17 g | Bone marrow stroma | Pre- and pro-B-cell growth factor | | Interleukin-8; monocyte-derived neutrophil chemotactic factor (MDNCF); T-cell chemotactic factor; neutrophil-activating protein (NAP-1) | IL-8 | 8-10 h | LPS monocytes; PHA lymphocytes; endothelial cells; IL-1- and TNF-stimulated fibroblasts and keratinocytes | Chemotactic factor for neutrophils, basophils, and T-cells | | Interleukin-9; human P-40; mouse T-helper gf; mast-cell-enhancing activity (MEA) | IL-9 | 30-40 | CD4 + ve T-cells; stimulated by anti-CD4 antibody PHA or PMA | Growth factor for T-helper, megakaryocytes, mast cells (with IL-3) | | Interleukin-10; cytokine synthesis inhibitory factor (CSIF) | IL-10 | 20 | | Immune suppressor | | Interleukin-11; adipogenesis inhibitory factor (AGIF) | IL-11 | 21 | | Stimulates plasmacytoma proliferation and T-cell-dependent development of Ig-producing B-cells | | Interleukin-12; cytotoxic lymphocyte maturation factor | IL-12 | 40, 35 subunits | Activated T-cell and NK cell growth factor; induces IFN-y | | Keratinocyte gf, FGF-7 | KGF | 14h | Fibroblasts | Keratinocyte proliferation and differentiation; prostate epithelial proliferation and differentiation | | Leukemia inhibitory factor; HILDA; member of IL-6 group | LIF | 24 | SCO cells | Inhibits differentiation in embryonal stem cells | | Lipopolysaccharide | LPS | 10 | Gram-positive bacteria | | | Lymphotoxin | TNF-B | 20-25 | Lymphocytes | Lymphocyte activation | | Macrophage inflammatory protein-1α | ΜΙΡ-1α | 10 | Macrophages | Chemotactic factor for macrophages and monocytes | | Monocyte/macrophage colony-stimulating factor; CSF-1 | M-CSF | 47-74 | B- and T-cells, monocytes, mast cells, fibroblasts | Hematopoietic stem cell inhibitor | | Müllerian inhibition factor | MIF | | Testis | Inhibition of Müllerian duct; inhibition of ovarian carcinoma | | Nerve gf, ẞ | B NGF | 27 | Male mouse submaxillary salivary gland | Trophic factor; chemotactic factor; differentiation factor; neurite outgrowth in peripheral nerve | | Neurotrophin-3 | NT-3 | 28 | Activated T-cells and PMA-treated monocytes | Stimulation of neurite outgrowth | | Oncostatin M; member of IL-6 group | OSM | | | Differentiation inducer (with glucocorticoid); fibroblast mitogen | | Phytohemagglutinin | PHA | 30 | Red kidney bean (Phaseolus vulgaris) | Lymphocyte activation | | Platelet-derived endothelial cell growth factor; similar to gliostatin | PD-ECGF | ~70 | Blood platelets, fibroblasts, smooth muscle | Angiogenesis; endothelial cell mitogen; neuronal viability; glial cytostasis | | Platelet-derived growth factor | PDGF | 30 | Blood platelets | Mitogen for mesodermal and neuroectodermal cells; wound repair; synergizes with EGF and IGF-1 | | Phorbol myristate acetate; TPA; phorbol ester | PMA | 0.617 | Croton oil | Tumor promoter; mitogen for some epithelial cells and melanocytes; differentiation factor for HL-60 and squamous epithelium | | Pokeweed mitogen | PWM | | Roots of pokeweed (Phytolacca americana) | Monocyte activation | | Stem cell factor; mast cell growth factor; steel factor; c-kit ligand | SCF | 31 g | Endothelial cells, fibroblasts, bone marrow, Sertoli cells | Promotes first maturation division of pluripotent hematopoietic stem cell | | Transferrin | Tfn | 78 | Liver | Iron transport; mitogen | | Transforming growth factor α | TGF-α | 6 | | Induces anchorage-independent growth and loss of contact inhibition | | Transforming growth factor ẞ (six species) | TGF-β1-6 | 23-25 dimer | Blood platelets | Epithelial cell proliferation inhibitor; squamous differentiation inducer | | Vascular endothelial gf | VEGF | | Kidney | Angiogenesis; vascular endothelial cell proliferation | # Table 9.5: Selecting a Serum-Free Medium | Cells or cell line | Serum-free medium | Refs. | Commercial suppliers (of specified media or alternatives) | |---|---|---|---| | A549 | PeproGrow-1 | [Rodriguez et al. 2004] | PeproTech | | Adipocytes | HyQ PF CHO; HyG PF CHO | [Pardee et al. 1984] | PromoCell; Stratech; Zen-Bio; Lonza; MP Biomedicals | | BHK 21 | MCDB 201, 202 | [McKeehan & Ham, 1976b] | Biosource; Clonetics (Lonza); PromoCell | | Bronchial epithelium | LHC-9 | [Hamilton & Ham, 1977] | Sigma | | Chick embryo fibroblasts | MCDB 302; PC-1 | [Frame et al. 1980; Freshney, 1980] | Lonza; Sigma; Invitrogen; PeproTech; PromoCell | | CHO | MPS; PC-1 | [Adolphe, 1984] | PromoCell | | Chondrocytes | Supplemented DMEM/F12 | [Waymouth, 1984] | Lonza; Invitrogen; JRH Biosciences; ICN; Sigma | | Continuous cell lines | Eagle's MEM, M199, MB752/1, CMRL 1066, MCDB media, DMEM:F12 + supplements | | Lonza, KGM; Cascade | | Corneal epithelial cells | MCDB 153 | [Doering et al. 2002] | Lonza | | COS-1,7 | MCDB 130, 131 | [Knedler & Ham, 1987; Gupta et al., 1997; Hoheisel et al., 1998] | Cell Applications; Lonza; Cascade; PAA; PromoCell; Sigma | | Endothelial cells | MCDB 110, 202, 402 | [Bettger et al., 1981; Shipley & Ham, 1983] | Lonza; Cascade; PromoCell; Sigma; Stratech; Zen-Bio | | Fibroblasts | Michler-Stucke | [Michler-Stuke and Bottenstein 1982] | Lonza; Invitrogen | | Glial cells | SF12 (Ham's F12 with extra essential and nonessential amino acids) | | Cell Culture Services; Hyclone; Millipore-CellGro; PeproTech | | Glioma | | [Frame et al., 1980; Freshney, 1980] | Lonza; PeproTech | | HEK293 | HEKTOR; HЕК; CDM4HEK293 | [Blaker et al., 1971; Bertheussen, 1993] | Lonza | | HeLa cells | aMEM; Iscove's | [Stanners et al., 1971; Iscove & Melchers, 1978] | Sigma; Roche Diagnostics | | Hematopoietic cells | BD Hepato; Hepatozyme; Hepatocyte Growth Medium | | BD Biosciences; Invitrogen; Promocell | | HL-60 | Ultradoma; Maxicell; Ex-Cell (and many others) | [Li et al., 1997] | Lonza | | HT-29 | | [Oh et al., 2001] | Atlanta Biologicals; BD Biosciences; Invitrogen; Hyclone; Lonza; Sigma | | Hybridomas | MCDB 110, 202; PC-1 | [Bettger et al., 1981; Ham, 1984] | Cascade; Lonza; PromoCell | | Human diploid fibroblasts | Iscove's | [Breitman et al., 1984] | Lonza; Hyclone; MP Biomedicals; Invitrogen; Sigma | | Human leukemia and normal leukocytes | | | | | Human tumors | Brower; HITES; Masui; Bottenstein N3 | [Brower et al., 1986; Carney et al., 1981; Masui et al., 1986b; Bottenstein, 1984; Chopra et al., 1996] | Cell Culture Services; Hyclone; Millipore-CellGro; PeproTech | | Hepatocytes, liver epithelium (human) | Williams E, L15 | [Williams & Gunn, 1974; Mitaka et al., 1993] | Lonza; Sigma | | Hybridomas | Iscove's | [Iscove & Melchers, 1978; Murakami, 1984] | Sigma; Invitrogen; Lonza; MP Biomedicals; Roche; Irvine Scientific; Metachem; PromoCell | | Insect cells | MCDB 153 | [Ikonomou et al., 2003] | Sigma; Millipore-CellGro; Invitrogen; Cascade; Cellntech; Lonza; Millipore; PromoCell; Sigma | | Keratinocytes | MCDB 153 | [Peehl & Ham, 1980; Tsao et al., 1982; Boyce & Ham, 1983] | Sigma | | L cells (L929, LS) | NCTC109; NCTC135 | [Birch & Pirt, 1970, 1971; Higuchi, 1977] | Sigma | | Lymphoblastoid cell lines (human) | Iscove's | [Iscove & Melchers, 1978] | Sigma | | Mammary epithelium | | | | | MDCK dog kidney epithelium | MCDB 170 | [Hammond et al., 1984] | JRH Biosciences; CellGenix; GE Heathcare (Amersham) | | LLC-PK, pig kidney | K-1; PC-1 | [Taub, 1984] | Cascade; Lonza; AthenaES | | Melanocytes | Gilchrest | [Halaban, 2004]; | Lonza | | Melanoma | Gilchrest | [See Protocol 22.21] | Lonza; Cascade; PromoCell | | Mouse embryo fibroblasts; 3T3 cells | MCDB 402 | [Shipley & Ham, 1983; Ham, 1984] | Lonza; Cascade; PromoCell | | Mouse leukemia | | | | | Mouse erythroleukemia | SF12 (Ham's F12 with extra essential and nonessential amino acids); Iscove's | [Murakami, 1984; Freshney, 1980; Iscove & Melchers, 1978; Ham & McKeehan, 1980; Iscove & Melchers, 1978] | Biosource; Invitrogen; Sigma | | Mouse myeloma | MCDB 411; DMEM:F12/N1 | [Murakami, 1984; Bottenstein, 1984] | Sigma; Invitrogen; Peprotech | | Mouse neuroblastoma | DMEM:F12/N3; B27/Neurobasal; N2, WJAC 404; | [Agy et al., 1981; Bottenstein, 1984; Brewer, 1995] | Invitrogen | | Neurons | | | | | Osteoblasts | | [Shiga et al., 2003] | OGM, Lonza; PromoCell | | Prostate | REGm | [Peehl, 2002] | Lonza | | Renal | | | Mesangial, Lonza | | Skeletal myoblasts | | | PromoCell | | Smooth muscle cells | MSGM | [Goto et al., 1999] | Cascade; Lonza; PromoCell; Millipore; Clonagen; CellGenix; Sigma; Stem Cell Technologies | | Stem Cells | ESGRO; CellGro; StemLine; StemSpan (and many others) | | Lonza | | Urothelium | MCDB 153; KSFMC; KGM-2, PFEK-1; PF-Vero; MP-Vero; Ex-cell Vero | [Southgate et al., 2002] | Autogen Bioclear; Clonagen; Hyclone; MP Biomedicals; Sigma | | Vero | | | BD Biosciences; MP Biomedicals; Invitrogen; Biosource; Sigma | # Conclusion However desirable serum-free conditions may be, there is no doubt that the relative simplicity of retaining serum, the specialized techniques required for the use of some serum-free media, the considerable investment in time, effort, and resources that go into preparing new recipes or even adapting exisitng ones, and the multiplicity of media required if more than one cell type is being handled all act as considerable deterrents to most laboratories to enter the serum-free arena. There is also no doubt, however, that the need for consistent and defined conditions for the investigation of regulatory processes governing growth and differentiation, the pressure from biotechnology to make the purification of products easier, and the need to eliminate all sources of potential infection will eventually force the adoption of serum-free media on a more general scale. But first, recipes must be found that are less "temperamental" than some current recipes and that can be used with equal facility and effectiveness in different laboratories.