Elimination of the yeastCandida parapsilosis from lymphoid cells and monolayer cells in culture

Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×10⁵ cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not detectable by light microscopy, can be removed by the addition of macrophages (2×10⁵/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost. This work was supported by Veterans Administration Research Funds.     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

Growth requirements of ferret tracheal epithelial cells in primary culture

In mass cell culture conditions, protease dissociated ferret tracheal epithelial cells (FTE) proliferated in growth factor-supplemented F12 medium to high cell densities (0.5 X 10(5) cells/cm2) with an average population doubling time of 24 hr. The growth factor constituents of the F12 medium included epidermal growth factor (25 ng/ml), insulin (1 microgram/ml), transferrin (10 micrograms/ml), hydrocortisone (18 ng/ml), hypothalamus extract (30-100 micrograms/ml), and conditioned medium from mouse 3T3 fibroblasts. Growth of these cells under clonal conditions was achieved by the partial replacement of F12 medium with M199 medium which was attributed, in part, to the presence of vitamin A in M199 medium. Serum did not stimulate the growth of FTE cells. The epithelial cell nature of these cells in culture was confirmed by ultrastructural features and by immunofluorescent staining for fibronectin.     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Isolation and characterization of a Mr = 38,000 protein from differentiating smooth muscle cells

In culture, vascular smooth muscle cells grow and form a confluent monolayer of cells. Under appropriate conditions, regions of the monolayer can be induced to draw away from the substrate and form multicellular nodules. The ultrastructure of the cells in the nodules appears to be similar to that of differentiated smooth muscle cells. The process of nodulation is associated with the synthesis of a unique protein whose molecular weight is estimated from gradient gel electrophoresis to be 38,000 (38-kDa Protein). The protein is secreted into the culture medium and can be detected either by metabolic labeling or by staining with Coomassie Blue. Partial purification of 38-kDa Protein was achieved using affinity chromatography. The protein is adsorbed to heparin-agarose, but not to gelatin-agarose. The concentration of 38-kDa Protein in nodular conditioned medium is estimated at 1.9 micrograms/ml and less than 0.01 microgram/ml in conditioned medium made from monolayer cells. The presence of 5% fetal bovine serum in the labeling medium does not affect 38-kDa Protein synthesis. Cross-reactivity with fibronectin was evaluated using polyvalent antibodies to 38-kDa Protein. The 38-kDa Protein is not antigenically related to fibronectin. Furthermore, we establish that the protein is not qualitatively influenced by the presence of ascorbate (50 micrograms/ml), beta-aminoproprionitrile fumarate (50 micrograms/ml) heparin (10 ng/ml), or fibronectin (20 micrograms/ml) in the culture medium. We find that the added components neither suppress 38-kDa Protein synthesis in nodular cultures nor enhance 38-kDa Protein synthesis in monolayer cultures. The 38-kDa Protein is not detected in either monolayer or nodular cell layers and appears to be a secreted protein. Its appearance in nodular conditioned medium during nodulation suggests a relationship with that process.     

T-cell-mediated regression of "spontaneous" and of Epstein-Barr virus-induced B-cell transformation in vitro: studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 microgram/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. "Spontaneous transformation" to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 X 10(6)-2.5 X 10(5) cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.     

Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture

Low density vascular smooth muscle (VSM) cell cultures maintained on extracellular-matrix(ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250 micrograms protein/ml); insulin (2.5 micrograms/ml) or somatomedin C (10 ng/ml); and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml). The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence. When cells are plated in the total absence of serum, transferrin (10 micrograms/ml) is also required to induce optimal cell growth. The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed. Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations. However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed. Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations. These experiments demonstrate the influence of the ECM-substrate only in promoting cell growth but also in increasing the longevity of the cultures.     

Effect of thrombopoietin on in vitro production of megakaryocytes from fetal mouse liver cells

Plasma clots containing fetal mouse liver cells (FMLC) were used to study the effects of a thrombocytopoiesis-stimulating factor (TSF) from kidney cell culture medium on the proliferation and maturation of megakarocytes. Cells in the megakaryocytic series were identified by the presence of acetylcholinesterase (AChE) and by their morphological and ultrastructural characteristics. For these experiments, 1 X 10(3) to 1 X 10(5) FMLC were cultured for 1-7 days with 0-5 micrograms of TSF; control cultures were treated with production medium (PMC) in which kidney cells had not been grown. The number of AChE+ cells that were observed depended upon the number of cells plated, i.e., after 6 days of culture with 5 micrograms of TSF, an average of 187 AChE+ cells was found after plating 1 X 10(4) cells and 1020 AChE+ cells were observed after plating 1 X 10(5) cells. In dose-response experiments, the number of AChE+ cells rose with increasing doses of TSF. Significantly elevated numbers of AChE+ cells were observed after the addition of 1-5 micrograms of TSF. The optimum time of culture, based upon the number of AChE+ cells found, was 3-5 days. Ultrastructural analysis of megakaryocytes in plasma clots showed evidence of platelet shedding on Day 5. After the culture of FMLC with TSF, a larger number of AChE+ cells was formed from a given number of cells plated than in previous studies that used adult bone marrow cells. Therefore, because of its greater sensitivity, FMLC may be useful for the assay of low levels of TSF, and may be a valuable tool for studying the effects of megakaryocytic regulatory factors on megakaryocytopoiesis.     

Elimination of mycoplasmas from cultured human cell lines utilizing a combination of two antibiotics, quinolone and a pleuromutilin derivative

Forty three cultured human cell lines were treated with a combination of 2 antibiotics to eliminate contaminant mycoplasmas. One course of treatment was composed of consecutive 3 or 4 cycles. Each cycle grew cells in BM-1 (pleuromutilin derivative; Boehringer Mannheim) containing medium (10 micrograms BM-1/ml culture) for 3 days, alternating with MC-210 (quinolone; Dainihon Pharmaceutical) containing medium (0.625 micrograms MC-210/ml culture) for 4 days. No treatment failure was encountered with this procedure. Before treatments, 18 (90%) of 20 cell line samples were contaminated with mycoplasma, as tested by DNA hybridization method (MYCOPLASMA T.C. RAPID DETECTION SYSTEM; Gen-Probe Inc.). Out of 43 cell lines treated, 7 were reduced in growth and dropped out. Among the other 36 cell lines, 27 became negative, 5 borderline and 4 slightly positive to the mycoplasma detection. All of the latter 9 cell lines, treated with one more similar course, found to be free from mycoplasma. Six of the dropout lines were cured of mycoplasma by a second treatment, under modified culture conditions. The last cell line (NATO) was successfully treated with another lot of FCS. Thus, the procedure proved successful even in treating promiscuously infected cell lines.     

Regulation of connective tissue collagenase production: stimulators from adult and fetal epidermal cells

We have examined the ability of primary adult rabbit skin cells to regulate collagenase production in vitro. Dermal cells constitutively produce collagenase in culture, and enzyme production by these cells can be influenced by epithelial cells. Co-culture with skin epidermal cells resulted in more enzyme production by dermal cells, whereas co- culture with corneal epithelial cells yielded less enzyme activity. Connective tissue cells from a different source, cornea, also produced collagenase when co-cultured with skin epidermal cells, although the stromal cells alone made no enzyme. The drug cytochalasin B had very little influence on collagenase production by dermal cells, either alone or in co-culture with epidermal cells, but did significantly potentiate enzyme production by corneal stromal cells responding to epidermal effector molecules. Epidermal-cell-conditioned medium from both fetal and adult rabbit skin was a potent source of stimulators (apparent mol wt 20,500 and 55,000) of connective-tissue-cell collagenase production. Stimulator production by epidermal cultures was cell density dependent. Optimal production of stimulators occurred in adult cultures containing 10(6) epidermal cells/ml of medium, and in fetal cultures containing 10(5) cells/ml. Inhibitors of connective tissue cell enzyme production were not detected in conditioned medium from either adult or fetal epidermal cells.     

Characterization and serial propagation of mouse prostate epithelial cells in serum-free medium

Epithelial cell cultures derived from the ventral prostate of normal adult mice have been propagated in serum-free medium. The cultures were initiated and maintained in Ham's F-12 nutrient mixture supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), cholera toxin (10 ng/ml), bovine pituitary extract (25 micrograms protein/ml) and antibiotics. The cells exhibited microvilli on cell surfaces, interdigitations and junctional complexes including desmosomes between cells, and cytokeratins in cytoplasm which are characteristic of epithelial cells. In addition, the cells exhibited the tissue-specific markers, prostatic acid phosphatase and prostate-specific antigen.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.     

The effects of peritoneal macrophages on monolayered luteal cell progestin secretion in the rat

Functionally active or regressing luteal cells were obtained from pseudo-pregnant (psp) rats between days 5-8 of psp or on day 15 of psp, respectively. They were monolayer-cultured (10(6)/dish) in the presence of 0.2 micrograms/ml LH 2.0 micrograms/ml PRL and 10 micrograms/ml pregnenolone for 4 days with or without macrophages, although functionally active luteal cells secreted progesterone dominantly during day 1 of culture (Day 1), the amounts of progesterone and 20 alpha-OH-P secreted were inverted on Day 2, and the dominance of 20 alpha-OH-P continued from Day 2 to Day 4. In the functionally regressing luteal cell culture, more 20 alpha-OH-P than progesterone was secreted throughout the culture period. The addition of peritoneal macrophages (2.5 X 10(6] to the active luteal cell monolayer lengthened the dominance of progesterone secretion for an additional day and the inversion occurred on Day 3. The progestin ratio (progesterone/20 alpha-OH-P) on Day 2 was maintained significantly higher. The daily addition of macrophages maintained the progesterone dominance throughout the culture period. On the other hand, macrophages had no effect on luteal cells already functionally regressing. These results indicate that macrophages are effective in maintaining the progesterone secreting activity of luteal cells in vitro.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Studies of fibronectin matrices in living cells with fluoresceinated gelatin

We have used fluorescein isosthiocyanate-conjugated gelatin (FITC- gelatin) (1 mg/ml) to localize cell surface fibronectin in unfixed live cells in cultures. FITC-gelatin stains the fibronectin matrix on primary cultures of rat and chick embryo fibroblasts as well as untransformed, established cell lines. In live cultured cells, fibronectin in many areas of the extracellular matrix is inaccessible to antibody and cannot be visualized by immunofluorescence staining. In contrast, fibronectin in these areas is fully stainable by FITC- gelatin. At a low concentration (20 micrograms/ml), FITC-gelatin stains the fibronectin matrix of primary cultured cells but not of "untransformed" established cell lines. SEM can detect only the matrix stainable with the low concentration of FITC-gelatin, such as that expressed by primary chick embryo fibroblasts. The binding of fibronectin to the extracellular matrix is very stable and FITC-gelatin remained bound to the matrix for at least 10 d in culture. Radioiodinated gelatin has been used to quantitate the level of cell surface fibronectin in living normal and transformed cells. FITC- gelatin appears to be a useful probe for studying the fibronectin of living cells in culture.     

Propagation of differentiating normal human tracheobronchial epithelial cells in serum-free medium

Serial-passage cultures of normal human tracheobronchial (TB) epithelial cells that exhibit functional differentiation have been established in serum-free medium supplemented with bovine pituitary extract (25 micrograms/ml), insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), EGF (5 ng/ml), 10(-6)M each of ethanolamine and phosphoethanolamine, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 80 hours. Further, the passaged cultures retained differentiated morphology as evidenced by secretion of glycoproteins, binding of concanavalin A lectin, and presence of alcian blue and periodic acid Schiff-positive material in their cytoplasm. Ultrastructural observations further supported the functional epithelial nature of the cultures. Most cells exhibited characteristic microvilli on cell surfaces and showed junctional complexes between them. The cytoplasm contained a large number of perinuclear secretory vesicles, a characteristic feature of the differentiated cells. These cultures provide an excellent model to study factors that regulate synthesis and secretion of glycoproteins in normal human TB cells.     

Short-term primary culture of mouse interstitial cells: effects of culture conditions of androgen production

This study examined the effects of culture conditions and hormone treatment on androgen production by mouse interstitial cells in short-term primary culture. Testicular interstitial cells (18-25% 3 beta-hydroxysteriod dehydrogenase-positive) were maintained in serum-free hormone supplemented medium. Basal (nonstimulated) androgen production was found to be plating-density dependent. Androgen production per cell increased dramatically in a time- and cell concentration-dependent manner. This effect was reproduced in low density cultures by addition of charcoal-stripped conditioned medium from high density cultures. The cell anchorage factors, fibronectin and poly-l-lysine, similarly enhanced basal androgen production but did not augment responsiveness to luteinizing hormone (LH). Coating of the culture surface with serum inhibited androgen production. Cultured cells remained responsive to LH for 4 to 5 days and both insulin (5 micrograms/ml) and epidermal growth factor (EGF) (3 ng/ml) augmented LH-stimulated androgen production. There was a transient increase in LH sensitivity and maximum LH-stimulated androgen production for 5 to 72 h in culture followed by a decline in androgen production to low levels after 4 to 5 days in culture. This loss of activity was partially prevented by addition of antioxidants to the medium or by reduction of the ambient O2 concentration to 1%.     

Multiple loop purification method for selective cultivation of Pentatrichomonas hominis

It is always troublesome having protozoan cultures contaminated with other organisms in the laboratory. The method described here produces high efficiencies of purification for fast moving flagellate protozoa. A human strain Pentatrichomonas hominis was employed in the study to examine the effects of multiple loop tubes on the purification of flagellates. Trichomonads were harvested from a trypticase yeast extract iron-serum-33 (TYI-S-33) medium, adjusted to 2 X 10(5) organisms/ml, and mixed with an equal volume of 2 X 10(6) organisms/ml of bacteria. The isolation was performed at 37 degrees C in TYI-S-33 medium containing a suitable amount of antibiotics (1000 U/ml of penicillin, 1000 micrograms/ml of streptomycin, and 4 micrograms/ml of fungizone). Four days later, 10(6) organisms/ml of protozoa, free of bacteria, were observed at the other end of the single loop and the double loop tubes. About the same amount of flagellates could be found at the other end of the triple loop tube six days after incubation. The traditional U-shaped tubes were used as controls and 10(5) cells/ml of flagellates were recovered in the presence of bacteria two days after incubation. An axenic culture of P. hominis was successfully isolated from the feces of a Formosan rock-monkey, Macaca cyclopsis, by this method. Purified trichomonads were recovered from a double loop purification tube five days after incubation.