Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro.

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250-fold excess concentration of purified GM-CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM-CSF were added to agar cultures stimulated by pokeweed mitogen-stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non-erythroid colonies or the non-erythroid content of mixed erythroid colonies. Although neither GM-CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM-CSF was also able to support the survival in vitro of a small proportion of erythroid colony-forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony-forming cells in similar cultures of adult marrow cells. The results do not support the hypothesis that GM-CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM-CSF und EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM-CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM-CSF is unable to stimulate the formation of colonies of these types.     

Giant eosinophil colonies from cultures of bone marrow cells

Summary To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil colonies and more cells/colony were present in cultures incubated with DSBCS/EL4-CM than in cultures incubated with fetal calf serum/EL4-CM. The ability to generate large numbers of eosinophils in vitro should facilitate study of cloned eosinophils. Supported in part by a grant from the National Institutes of Health, AI 20416, and by the Mayo Foundation. Editor's statement Previous approaches to in vitro culture of eosinophils generally have achieved, at best, mixed cultures of colonies of these cells and granulocyte-macrophage colonies. The improved culture methods described in this report produce more homogeneous eosinophil cultures and larger colonies of these cells. The procedure employs EL4 murine thymoma-conditioned medium, which apparently contains eosinophil colony-stimulating activity in the absence of granulocyte-macrophage colony-stimulating activity. David W. Barnes     

Human granulopoiesis in vitro: an advantage in the use of Iscove's modified Dulbecco's medium

With the aim of determining whether Iscove's Dulbecco's medium (IMDM) provides a growth advantage in the support of granulopoiesis from cultures of human bone marrow in agar, samples from 20 normal subjects were examined in triplicate after 7, 10 and 14 days in parallel cultures containing IMDM or Dulbecco's medium. From every sample, more granulocyte-macrophage colonies were obtained at each culture interval with IMDM. In particular, the number of colonies with IMDM at 14 days (96 +/- 13 per 2x10(5) bone marrow cells) was almost double that with Dulbecco's medium (50 +/- 10). This increment consisted almost entirely of pure granulocyte colonies (P less than 0.001). No significant change in the proportion of eosinophil colonies was observed. These data indicate that IMDM does provide a growth advantage over Dulbecco's medium in the generation of granulocyte (neutrophil and eosinophil) colonies from agar cultures of normal human bone marrow.     

Rapid in situ cytochemical classification of CFU-C colonies in soft agar culture: permanent whole culture preparations

Soft agar culture CFU-C (colony-forming units in culture) are rapidly classified in situ as eosinophil, macrophage, and neutrophil-monocyte types by whole culture staining with luxol fast blue for eosinophil specific granules and acetoorcein for nuclei. Stained colonies may be picked and examined individually as wet mount cover slip preparations, or the agar culture may be air dried and mounted permanently. The whole culture stain has been variously modified for the enzyme markers alpha-naphthyl acetate esterase and peroxidase and for nuclear staining alone with acetoorcein.     

Colony formation in agar by multipotential hemopoietic cells.

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.     

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.     

Ultrastructural aspects of the primary in vitro antibody response of mononuclear cells in human blood

Investigations on the morphology of cells that participate in immune responses in vitro have been limited because the recovery and identification of immunocompetent cells growing dispersely in conventional liquid cultures are technically difficult and allow only the observation of individual antibody-forming cells. Here we used a system in which focal proliferation of antisheep erythrocyte antibody-secreting cells has been induced in semisolid cultures of peripheral blood mononuclear cells. By this method, intact colonies can be observed by light microscopy at the center of each hemolytic area and then processed for electron microscopy without disrupting the connections existing among the cells. Two types of colonies develop: type I colonies which grow deeply into the agar and contain cells that undergo a complete process of differentiation from blast to mature plasma cell, and type II colonies which grow more superficially and do not seem to be directly involved in antibody production.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

A simple plate assay for detection of group A streptococcus proteinase

A simple plate assay based upon the digestion of skim milk contained within Columbia agar base medium has been found to be a sensitive and reproducible method for detection of proteinase production by individual surface grown colonies of group A streptococci. Use of this medium enabled demonstration of proteinase activity by 24-h aerobic cultures, 79% of 72 group A streptococcus M prototype strains being proteinase positive. None of 40 ‘viridans’ streptococcus strains were positive on this medium and of 18 representative strains of different Lancefield serogroups, only the group P streptococcus had proteinase activity similar to that of the group A streptococci. This same medium can also be used for the detection of preformed proteinase present in liquid preparations by a well diffusion assay.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

Functional differences between cells from MLR colonies grown in soft agar cultures

This report describes a method of growing soft agar colonies of human T lymphocytes activated in the MLR. Two types of colonies were demonstrated: lower colonies grew within the agar layer, and upper colonies grew on the surface of the agar layer. Three days of priming the lymphocytes in the MLR and the use of supernatants of day-3 MLR cultures to provide T cell colony growth factor were necessary for optimal colony formation. Lymphocytes obtained from colonies were grown in long-term (2 to 4 weeks) cultures to generate sufficient numbers of cells to be tested in different functional assays. Cells from both types of colonies exhibited PLT activity. Upper colony cells showed considerably higher CML activity than lower colony cells (mean percent cytotoxicity 37 +/- 5 vs 6 +/- 3). Cells from both types of colonies contained radiosensitive suppressor cell activity that inhibited the primary MLR. The suppressor cell effect of lower colony cells was specific for the original stimulator, but upper colony cells displayed nonspecific suppressive effects. For both types of colony cells, it appeared that suppressive effects were unrelated to the CML activity of these cells. These data suggest that the soft agar colony assay offers a promising approach to separate subpopulations of lymphocytes activated in the MLR.     

CHLOROSARCINOPSIS VARIABILIS SP. NOVA FROM A CONNECTICUT CORNFIELD SOIL1

A new and pleomorphic species of Chlorosarcinopsis from a Connecticut cornfield soil is described. Two populations were observed and cultured. One produced mostly smooth colonies on agar and formed few packets; the second produced large percentages of rough colonies on agar and frequently formed packets. In older agar cultures numerous Hormotila-like colonies can be observed in both strains.     

A Rapid Method for Screening Large Numbers of Environmental Microorganisms for Antiviral Activity

A new method for screening microbial colonies endowed with antiviral activity is described. It is based on close contact between microbial agar cultures and agar-covered virus-infected-cell monolayers and allows the screening of large numbers of colonies in just a few months.     

Kinetics of the clonal proliferation of granulocytes and macrophages in cultures of mouse bone marrow cells as supported by two distinct types of colony-stimulating factors

Two different types of colony-stimulating factors (CSF) were used to support the clonal growth of myeloid progenitor cells (CFUc) in semi-solid agar or viscous methylcellulose cultures of mouse bone marrow cells. The cultures stimulated for 5 days with RSP-2-P3 cell CSF (CSFRSP) contained mainly granulocyte colonies, whereas the cultures stimulated for 10 days with human urine CSF (CSFhu) contained mainly monocyte/macrophage colonies. Four lines of study were carried out: 1) a kinetic study using combinations of the two types of CSFs in the same culture; 2) a study of transferring CFUc from the initial 3-day cultures to recipient cultures containing the same or different types of CSF; 3) an examination of the morphology over time of colonies that were confined by glass capillaries plunged in agar; and 4) electron microscopic observations on disintegrating granulocytes. The results of all these lines of study suggest that about one third of the CFUc can be stimulated both by CSFRSP and CSFhu while the other two thirds react specifically either with CSFRSP or with CSFhu. The present study also suggests that granulocytes in the culture stop proliferation and disintegrate while macrophages are still growing there. Thus, mixed-type colonies containing both macrophages and granulocytes later become macrophage colonies.     

An autoradiographic screening test for mycoplasmal contamination of mammalian cell cultures

Summary Autoradiographic detection of incorporation of tritiated thymidine into the cytoplasm of cultured mammalian cells has been evaluated as a test of contamination of the cultures by cell-associated microorganisms, which usually are mycoplasmas. Criteria which indicate the presence of cell-associated mycoplasmas have been established, and the reliability of the standardized autoradiographic method has been assessed by testing the same cultures by two colony formation methods of mycoplasmal detection. The autoradiographic method demonstrated cell-associated microorganisms in all cultures from which characteristic colonies were grown on mycoplasma agar. The autoradiographic method did not produce false positive results, and the outcome of this test was evident in 3 days as opposed to 7 to 14 days by agar culture methods. Some applications of the autoradiographic method are shown, and it is suggested that this method be employed for routine surveillance for mycoplasmal contamination in laboratories where facilities for frequent agar culture tests are not easily available. This research was supported by U.S. Public Health Service Grants CA 12351-02 and CA 12334-01 from the National Cancer Institute.     

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.     

Behavior of transforming growth factors in serum-free media: an improved assay for transforming growth factors

Transforming growth factors (TGFs) are a relatively new category of factors that induce the anchorage-independent growth of non-transformed cells. These factors are usually detected by their ability to induce normal rat kidney (NRK) fibroblasts to grow in soft agar. Until now, this assay has been performed in serum-containing medium (SCM). Unfortunately, the background activity of this assay is variable and dependent on several factors, including passage number of the cells and the serum lot used. Furthermore, the addition of either EGF or TGF-beta alone results in the appearance of additional colonies, which decreases the sensitivity of the assay. To circumvent these problems, serum-free media have been developed that support the growth of the NRK cells at low density in both monolayer culture and soft agar. Long-term growth in monolayer cultures occurs in serum-free medium supplemented with laminin, insulin, transferrin, epidermal growth factor (EGF), fibroblast growth factor (FGF) and high density lipoprotein (HDL). Growth in soft agar occurs when TGFs are added to a serum-free medium, AIG medium, that contains insulin, transferrin, FGF and HDL. In contrast to the background activity observed when the assay is performed in SCM, no colonies form in the AIG medium unless TGFs are added and few, if any, colonies form if EGF or TGF-beta are added alone. Thus, the AIG medium provides an improved assay for TGFs. In addition, the AIG medium should prove useful for examining other factors, including serum factors, for TGF activity.     

Enumeration of Vital and Thermally Stressed Staphylococcus aureus in Foods Using Baird-Parker Pig Plasma Agar (BPP)

Baird-Parker (BP), modified BP medium (egg yolk replaced with pig plasma, BPP) and modified Vogel and Johnson agar (phosphatidyl choline, deoxyribonucleic acid, with catalase added, PCVJ) were equally efficient for enumerating both non-stressed and thermally-stressed populations of Staphylococcus aureus from pure cultures and naturally contaminated foods. Vogel and Johnson agar was inferior. All colonies exhibiting coagulase activity on BPP were subsequently confirmed as Staph. aureus ; this was not the case with presumptive colonies from BP and PCVJ. Based on selectivity, definitive diagnostic characterization of colonies and increased sensitivity (more sample plated), BPP should be considered as the reference method for the routine enumeration of Staph. aureus in foods.     

Detection of Nuclease Activity in Semisolid and Broth Cultures

A method for the detection of deoxyribonuclease activity in semisolid agar cultures was investigated. When cultures were overlaid with an acridine orangedeoxyribonucleate-agar (ADA) mixture, incubated for 1 to 3 hr, and observed under ultraviolet light, clear halos developed around colonies that produced deoxyribonuclease. A variation of the method for use with broth cultures involved impregnation of filter-paper discs with a small portion of the culture and overlaying the discs with the ADA mixture. This alteration has the advantage that the test tube cultures are easily heat-treated prior to assay to determine the heat resistance of the enzyme.