Semisolid Agar Medium Research Articles

Erwinia chrysanthemi EC16 Produces a Second Set of Plant-Inducible Pectate Lyase Isozymes

The enterobacterium Erwinia chrysanthemi causes soft-rot diseases involving extensive tissue maceration in a wide variety of plants and secretes multiple pectic enzymes that degrade plant cell walls and middle lamellae. An E. chrysanthemi mutant with directed deletions or insertions in genes pehX, pelX, pelA, pelB, pelC, and pelE, which encode exo-poly-alpha-d-galacturonosidase, exopolygalacturonate lyase, and four isozymes of pectate lyase, respectively, was constructed by the marker exchange of a cloned pehX::TnphoA fragment into E. chrysanthemi CUCPB5010, a Delta(pelA pelE) Delta(pelB pelC)::28bp Delta(pelX)Delta4bp derivative of strain EC16. This mutant, E. chrysanthemi CUCPB5012, no longer caused pitting in a standard pectate semisolid agar medium used to detect pectolytic activity in bacteria. Nevertheless, the mutant still macerated leaves of chrysanthemum (Chrysanthemum morifolium), although with reduced virulence. The mutant was found to produce significant pectate lyase activity in rotting chrysanthemum tissue and in minimal media containing chrysanthemum extracts or cell walls as the sole carbon source. Activity-stained, ultra-thin-layer isoelectric focusing gels revealed the presence in these preparations of several pectate lyase isozymes with pIs ranging from highly acidic to highly alkaline. Sterile culture fluids containing these isozymes were able to macerate chrysanthemum leaf tissue. Unlike the products of the pelA, pelB, pelC, and pelE genes in E. chrysanthemi EC16, these plant-inducible pectate lyase isozymes were not produced in minimal medium containing pectate. The results suggest that E. chrysanthemi produces two sets of independently regulated pectate lyase isozymes that are capable of macerating plant tissues.

Fractal morphogenesis by a bacterial cell population.

Many species of bacteria have been found to form fractal colonies. Environmental (physicochemical) and biological factors for this fractal morphogenesis have been examined for their roles in the genesis of fractal and pattern diversity. Morphology of a bacterial colony on a solid agar medium depends on the nutrient diffusion field (two-dimensional). When concentrations of nutrients are low, point-inoculated bacteria (e.g., Bacillus subtilis) exert diffusion-limited growth. A self-similar fractal colony formed slowly under such a condition has the same morphology as one made by the diffusion-limited aggregation (DLA) model. The value of fractal dimensions (ca. 1.72) and the appearance of specific phenomena (screening and repulsion effects) are consistent with computer simulations of the DLA fractal model. On the other hand, a round colony recognized on an agar-rich medium was considered to be the product of reaction-limited growth and was simulated by the Eden model. When motile bacteria are point inoculated onto semi-solid agar media, bacterial spreading behavior also is morphogenic. Branching patterns with various morphologies (e.g., dense-branching morphology) have been recognized and examined for factors responsible for pattern changes. By microscopic inspection of the extending branch, multicellular behavior of bacteria has been observed in the structured cell distribution. Besides cell division and translocation activities, wetting agents produced by some species of bacteria (e.g., serrawettins produced by Serratia marcescens) are considered to be important microbial factors for efficient space occupation and specific cell transpositions in various surface environments.

In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10(6) protoplasts per g fresh weight using 2.0% Cellulase 'Onozuka' R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH4NO3. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 μM BAP and 9.0 μM 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 μM zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK − cells

L929TK − cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK + transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK + transformation. Southern blot analysis of chromosomal DNA of TK + transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK − cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occured in chromosomal DNA of TK + clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimmensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK + transformant clones was noticed.

Variations in cytotoxicity and isoenzyme patterns of uncloned and cloned cultures of axenic Entamoeba histolytica

Five clones of axenic Entamoeba histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. The clone HMI-C121 was more cytotoxic to the cultured Baby Hamster Kidney (BHK) cells while all other clones were significantly (P < 0.001) less cytotoxic as compared to the cloned HMI-C121 and uncloned E. histolytica (HMI). The uncloned Indian axenic E. histolytica (KCG:0986:11) as well as E. histolytica (NIH: 200) cultures were significantly (P<0.001) less cytotoxic to cultured BHK cells. No difference in the electromobility of maleate NADP oxidoreductase (ME) or glucophosphate isomerase (GPI) isoenzyme in the lysates of all the cloned and uncloned cultures of E. histolytica was observed. The clones HMI-C121, HMI-C131, HMI-G143 and HMI-C144 had three bands of hexokinase (HK) while all uncloned cultures and one of clones, HMI-C145 had only two bands. Though cloned and uncloned cultures had a single PGM band, the relative electromobility (rf) of phosphoglucomutase (PGM) for clone HMI-C131, HMI-C143 HMI-C144 was relatively less (rf 0.075) and these were also significantly (P < 0.001) less cytotoxic to BHK cells as compared to clone HMI-C121. It is felt that axenic E. histolytica culture consists of several populations (clones) and expression of isoenzymes pattern or cytotoxic potentials would depend upon the population which predominantly multiples and outgrows other populations in the culture system.

Variations in cytotoxicity and isoenzyme patterns of uncloned and cloned cultures of axenicEntamoeba histolytica

Five clones of axenic Entamoeba histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. The clone HMI-C121 was more cytotoxic to the cultured Baby Hamster Kidney (BHK) cells while all other clones were significantly (P less than 0.001) less cytotoxic as compared to the cloned HMI-C121 and uncloned E. histolytica (HMI). The uncloned Indian axenic E. histolytica (KCG:0986:11) as well as E. histolytica (NIH:200) cultures were significantly (P less than 0.001) less cytotoxic to cultured BHK cells. No difference in the electromobility of maleate NADP oxidoreductase (ME) or glucophosphate isomerase (GPI) isoenzyme in the lysates of all the cloned and uncloned cultures of E. histolytica was observed. The clones HMI-C121, HMI-C131, HMI-G143 and HMI-C144 had three bands of hexokinase (HK) while all uncloned cultures and one of clones, HMI-C145 had only two bands. Though cloned and uncloned cultures had a single PGM band, the relative electromobility (rf) of phosphoglucomutase (PGM) for clone HMI-C131, HMI-C143 HMI-C144 was relatively less (rf 0.075) and these were also significantly (P less than 0.001) less cytotoxic to BHK cells as compared to clone HMI-C121. It is felt that axenic E. histolytica culture consists of several populations (clones) and expression of isoenzymes pattern or cytotoxic potentials would depend upon the population which predominantly multiples and outgrows other populations in the culture system.

Nodulation competitiveness of Tn5-induced mutants of Rhizobium fredii USDA208 that are altered in motility and extracellular polysaccharide production

The role of motility and extracellular polysaccharide production in nodulation competitiveness of Rhizobium fredii was examined. Transposon Tn5 was used to mutagenize strain USDA208, and mutants with reduced motility on semisolid agar medium were isolated. One such mutant, 208M3, migrated to only one-seventh the distance of the parental strain. Solid medium amended with triphenyltetrazolium chloride was used to identify mutants altered in extracellular polysaccharide production. Type 1 colonies, typified by mutant 208T13, were heavily mucoid, while type 2 colonies, represented by mutant 208T3, were dry and nonmucoid. Compared with strain USDA208, these mutants produced 4- to 5-fold more extracellular polysaccharide and 20% as much extracellular polysaccharide, respectively. Marker exchange of 208T3 genomic DNA containing Tn5 into strain USDA208 resulted in a mutant, 208K1, that produced extracellular polysaccharide levels similar to mutant 208T3. Mutants 208M3, 208T3, and 208T13 contained single Tn5 insertions. All formed pink nodules on 'Peking' soybean that were structurally indistinguishable and contained proteins with similar profiles. Rates of nodulation were similar in the mutants and the parental strain. Mutants 208M3 and 208T13 were as competitive against an isolate of Bradyrhizobium japonicum serogroup 123 as was strain USDA208. In contrast, mutants 208T3 and 208K1 were competitively superior. Key words: nodulation competition, motility, extracellular polysaccharide, Rhizobium.

Enumeration of interleukin-1 producing monocytes from human peripheral blood mononuclear leukocytes by agar plating technique

An agar plating technique was developed for enumeration of IL-1-producing monocytes based on the principle that when IL-1-producing monocytes were cocultured with mouse thymocytes and PHA in semisolid agar medium in a plate, mouse thymocytes proliferated around IL-1-producing monocytes resulting in the clusters or colonies of cells. The IL-1-produced clusters or colonies of cells can be counted under a dissecting microscope. Optimal conditions were established for induction and development of IL-1-producing monocytes. The numbers of IL-1-producing monocytes ranged from 819 to 1930 cells/10 5 monocytes, with mean ± SEM = 1344 ± cells/10 5 monocytes; the IL-1 activity ranged from 11.7 to 85.9 U/10 5 monocytes/ml, with mean ± SEM = 42.8 ± 11.2 U/10 5 monocytes/ml in seven normal subjects. The IL-1 activity per one monocyte ranged from 12.7 to 86.5 mU, with mean ± SEM = 33.5 ± 9.8 mU. The mean numbers of IL-1-producing monocytes and the mean IL-1 levels produced by monocytes from the same normal subjects were highly correlated ( r = 0.981). The numbers of IL-1-produced colonies resulting from IL-1-producing monocytes could be completely abolished by incorporation of rabbit anti-human IL-1 in the semisolid agarose but not by rabbit anti-human IL-6 or anti-human TNF-α.

APPROACHING MECHANIZATION OF PLANT MICROPROPAGATION

ABSTRACT, Investigations of materials and methods for growing plant tissue in a continual-flow, liquid nutrient medium as an alternative to semisolid agar medium have been made. Enhanced growth of plant tissue on microporous polypropylene membranes floating on a liquid nutrient has been demonstrated. Moreover, in vitro plantlets on the microporous membrane are free from entanglement with the support matrix and readily available to mechanized handling. Trained growth of plantlets through polypropylene netting shows potential for mechanization by mass handling (separation, singulation, and transfer) of plant tissue cultures.

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.

An in situ technique to measure bacterial chemotaxis in natural aquatic environments

A simple and reliable technique to study bacterial chemotaxis in natural aquatic environments is reported. This technique uses the test chemicals in known volumes of semi-solid agar media placed in double layered, highly porous, polyester tubes. Following in situ incubation, bacteria attracted by the test chemicals are enumerated with fluorescence microscopy following acridine orange staining. Studies in an eutrophic reservoir showed that significant numbers of bacteria were attracted to D-glucose and glycine; no significant effects were observed with L-serine, sodium succinate, or sodium chloride.

Newly established human retinoblastoma cell lines exhibit an “immortalized” but not an invasive phenotype in vitro

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta.

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.

Влияние трансформирующего фактора роста-&amp;beta;, эпидермального фактора роста и инсулина на пролиферацию клеток в среде с полужидким агаром

Влияние трансформирующего фактора роста-&amp;beta;, эпидермального фактора роста и инсулина на пролиферацию клеток в среде с полужидким агаром

Cryopreservation of Coleus blumei Suspension and Callus Cultures

Callus and suspension cultures of Coleus blumei Benth. were cryopreserved and stored for various periods of time in liquid nitrogen. Sorbitol was the only additive used during preculture and during cryoprotection treatments. The best results were obtained using cells from the early growth period. The duration of storage was 3, 6, 12 and 15 months, respectively. Cell viability could be improved considerably either by thawing the samples rapidly in a 60 °C water bath (instead of 45 °C) or by thawing them in a microwave oven. After a short period of recovery growth on semi-solid agar medium, the cells were cultivated under standard conditions. The growth characteristics and the accumulation of rosmarinic acid, a compound of pharmaceutical value, were found to be in the range of untreated controls.

Enhancement of cis-diamminedichloroplatinum and mitomycin C-induced sister chromatid exchanges by dibutyryl cyclic AMP in a human salivary gland adenocarcinoma cell line in culture.

A human salivary gland adenocarcinoma cell line was cultivated in the presence of dibutyryl cyclic AMP (dB-cAMP), cis-diammine dichloroplatinum (cisplatin) or mitomycin C (MMC) only, or of the combination of dB-cAMP and each of the antineoplastic drugs. Then the treated cells were examined for the induction of sister chromatid exchanges (SCEs), colony-forming efficiency (CFE) in semisolid agar medium and cell survival as measured by counting for viable cells. It has been found that the frequency of SCE induction is significantly increased by treatment of cells with cisplatin or MMC under the presence of dB-cAMP, when compared to that in the cells treated with cisplatin or MMC only. Moreover, marked reduction in CFE and cell survival was found to occur in the cells treated with the combination of dB-cAMP and cisplatin or MMC. These finding indicate that treatment of a human salivary gland adenocarcinoma cell line with dB-cAMP results in modification of SCE induction, CFE and cell survival in the cells exposed to cisplatin or MMC.

Subject index for volume 163

Mammalian reoviruses exhibit a large host range and infected cells are generally killed; however, most studies examined only a few cell types and host species, and are probably not representative of all possible interactions between virus and host cell. Many questions thus remain concerning the nature of cellular factors that affect viral replication and cell death. In the present work, it was observed that replication of the classical mammalian reovirus serotype 3 Dearing in a bat epithelial cell line, Tb1.Lu, does not result in cell lysis and is rapidly reduced to very low levels. Prior uncoating of virions by chymotrypsin treatment, to generate infectious subviral particles, increased the initial level of infection but without any significant effect on further viral replication or cell survival. Infected cells remain resistant to virus reinfection and secrete an antiviral factor, most likely interferon, that is protective against the unrelated encephalomyocarditis virus. Although, the transformed status of a cell is believed to promote reovirus replication and viral “oncolysis”, resistant Tb1.Lu cells exhibit a classical phenotype of transformed cells by forming colonies in semisolid soft agar medium. Further transduction of Tb.Lu cells with a constitutively active Ras oncogene does not seem to affect cell growth or reovirus effect on these cells. Infected Tb1.Lu cells can produce low-level of infectious virus for a long time without any apparent effect, although these cells are resistant to reinfection. The results suggest that Tb1.Lu cells can mount an unusual antiviral response. Specific properties of bat cells may thus be in part responsible for the ability of the animals to act as reservoirs for viruses in general and for novel reoviruses in particular. Their peculiar resistance to cell lysis also makes Tb1.Lu cells an attractive model to study the cellular and viral factors that determine the ability of reovirus to replicate and destroy infected cells.

Invasion Test

In cases of malignant brain tumors, the histological diagnosis may be of prognostic value, but the clinical course is sometimes unpredictable. Malignant brain tumors are destructive mainly through their invasive tendency and the pressure they exert as they grow. It is difficult to evaluate a tumor's invasive capacity in a two-dimensional culture system such as monolayer culture. Although threedimensional culture systems have been used for testing the invasive patterns of some malignant tumors, little is known about the invasive mechanisms of neurogenic tumors. Using a modified “invasion test, ” the authors examined the invasive characteristics of four neurogenic cell lines (T1, 9L, T1A, and T1B). Multicellular tumor spheroids (MTS) were produced from tumor cell colonies consisting of 20 to 30 cells by shake culture. With this method, MTS of constant shape and size were obtainable. Pulsating heart tissue obtained from 9-day-old chick embryos was minced and also shake cultured. The MTS and cultured embryonic chick heart (ECH) fragments were placed together in semisolid agar medium for 2 hours, then cultured in a gyratory shaker for 7 days. Tumor invasiveness was examined by hematoxylin-eosin staining and anti-ECH antibody immunostaining. All cell lines examined were able to form MTS and all proved highly invasive.

Visualisation of oxidizing power of rice roots and of possible participation of bacteria in iron deposition

AbstractThe oxidizing power of rice roots was observed in narrow transparent root boxes containing different media. Plants precultivated in nutrient solution were embedded in semisolid agar medium to observe oxidation of ferrous iron cations and leuco methylene blue as well as solubilization of ferrous sulfide. In the presence of ferrous sulfate reddish brown coloration due to formation of ferric oxide/hydroxide was observed around the roots and on the root surface during one day of incubation. When agar medium blackened by ferrous sulfide was used, the root zone became transparent. Within a few hours leuco methylene blue was oxidized to methylene blue on and near the roots. Furthermore, seedlings were grown in agar medium containing ferrous sulfide inoculated with soil filtrate. Besides diffuse ferric iron precipitation, iron was also deposited on spherically shaped structures in the rhizosphere and near the agar surface as well as in slimy layers appearing on the root surface. The spherical structures and slimy layers were obviously bacterial colonies extending with time. As the roots grew old, parts of them turned black. In the rhizosphere, black spots occurred resembling colonies of sulfate‐reducing bacteria. Rice was also grown in sand supplemented with nutrients and iron sulfide. While root growth was straight in agar, it was twisted in the sand medium. Again, heavy ferric iron deposition occurred on the root surface. On older root parts the lateral roots became blackish. The results suggest participation of bacteria in ferric iron deposition in the rhizosphere of rice.

Colony formation by subpopulations of human T lymphocytes: VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4 + cells (inducer/helper phenotype) and 20% of CD8 + cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10–200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7 + and CD16 + were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4 + or CD8 + cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4 + cells were easily recognized, but approximately one-third of the CD4 + colonies could not be distinguished from colonies composed of CD8 + cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.