Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 × 10⁷/ml after 10 days of incubation at 33 C, and 5 × 10⁷/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 × 10⁷/ml after 12 days of incubation.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Growth of Peptococcus and Peptostreptococcus: effect of variations of culture media on efficiency of recovery.

Reference strains and clinical isolates of Peptococcus and Peptostreptococcus spp. were evaluated for their growth response in supplemented thioglycolate-yeast extract media. Supplements used included various combinations of hemin, menadione, sodium bicarbonate, and Tween 80. Parallel studies were done to compare the efficiency of recovery of viable cells grown in thioglycolate-based media and Wilkins-Chalgren broth and agar. In addition, the effects of age of the medium and medium storage on viable cell yields for reference strains were determined. Reference strains grown in freshly prepared thioglycolate-yeast extract medium supplemented with sodium bicarbonate produced a 10-fold greater increase in the number of viable cells recovered after 24 h of incubation than did the same organisms cultivated in Wilkins-Chalgren medium. The efficiency of recovery of organisms when either mid-logarithmic- or mid-stationary-phase cells were used to prepare standardized inocula was similar. The results suggest that thioglycolate-yeast extract medium supplemented with sodium bicarbonate is more productive than Wilkins-Chalgren medium for the cultivation of anaerobic gram-positive cocci and may represent a suitable alternative for antimicrobial susceptibility testing of these organisms.     

Trypanosoma cruzi: cultivation in macromolecule-free semisynthetic and synthetic media.

A macromolecule-free semi-synthetic medium (F-81) was devised to culture Trypanosoma cruzi serially at room temperature. F-81 contains only one undefined substance, trypticase, which consists primarily of short-chain polypeptides. In F-81 medium T. cruzi will grow to a density of 35 to 43 × 10⁶ organisms/ml, a density comparable to that obtainable in a serum-containing medium such as F-69. High concentrations of water-soluble vitamins appear to have a serum-replacing effect in the F-81 medium. A completely synthetic medium (F-84) was prepared by replacing trypticase in F-81 with Trager's amino acid mixture. T. cruzi epimastigotes could be serially cultured in F-84, with a maximum yield of 9.2 × 10⁶ organisms/ml of medium after 3 to 4 weeks of incubation at 27 C.     

Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild-type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures. Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild-type viruses, were 5 to 1,000-times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild-type virus decreased mouse neurovirulence.     

Evidence of two inapparent nonoccluded viral infections of Culex tarsalis

Culex tarsalis from a mosquito colony maintained at Bakersfield, California, were infected with two noninclusion cytoplasmic viruses. Both viruses were polyhedral with average diameters of ca. 56 nm and 33 nm for C. tarsalis virus 1 and 2 (CTV1 and CTV2), respectively. CTV1 particles had an average prevalence in the colony of 24% over a 2-year period and CTV2 had a prevalence of 5%.The viruses were first observed in negatively stained preparations of crushed heads using sodium phosphotungstate. Both viruses were seen in thin sectioned material from the salivary glands, fat body, and nervous tissue of infected mosquitoes.CTV1 was retained through six serial passages by needle inoculations of infected mosquito suspensions into virus-free C. tarsalis adults. An estimated total dilution of >;10¹⁸ indicated CTV1 had multiplied in C. tarsalis. The coincident appearance of CTV2 in some of the inoculated mosquitoes through the 5th passage suggested that this virus also multiplied on serial passage.CTV1 had a median incubation period of ca. 6.6 days in adult C. tarsalis kept at 25°C. Preliminary data indicated that the median survival period of male and female C. tarsalis injected with CTV1 was reduced 2 or 3 days over that of inoculated control insects when incubated at 25°C. Infection may cause a degeneration of salivary glands in adult females.CTV1 did not infect Culex pipiens and did not produce plaques in Vero or duck embryo cell cultures.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth of Trypanosoma cruzi in Cultures of Chick Embryo Cells, and Effects of Furazolidone and Tris (p-Aminophenyl)Carbonium Chloride

SYNOPSIS. Monolayers of cells of coverslips were produced by culturing known numbers of trypsinized chick cells in growth medium (solution 199 plus 20% calf serum) at 37 C for 2 days. The fluid was then replaced with maintenance medium (solution 199 plus 5% calf serum) containing various known numbers of T. cruzi and the preparations were incubated at 33 C for 5 days; fresh maintenance medium was substituted on the 2nd or 3rd day. The inocula of parasites were obtained from T. cruzi -cell cultures, supplemented with 2% sterile NNN overlay, or from NNN cultures.
The numbers of extracellular parasites, proportions of infected cells, and percentage distribution of infected cells relative to the number of intracellular leishmanial bodies were determined on days 2 or 3 and 5 of parasite cultivation in many experiments. Analyses of the data gave the following results. Extracellular parasites increased 2- to 14-fold during the first 2 or 3 days, depending upon the source and size of the inocula, and 10- to 20-fold during the last 2 or 3 days. Cell infection continued throughout incubation at daily rates of 1.4-4.5%; 8-22% of the cells became infected during the 5 days of incubation. Intracellular growth was reflected most clearly by increases in the proportion of cells having >10 leishmanial bodies. This increase was about 5% daily during the last 2 or 3 days of incubation.
A useful test procedure for assessing the antiparasitic action and chick embryo cell toxicity of drugs is illustrated by data obtained with furazolidone and tris ( p -aminophenyl)carbonium chloride.     

Clostridium perfringens: I. Sporulation in a Biphasic Glucose-Ion-Exchange Resin Medium

A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH(4))(2)SO(4) and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media.     

Adaptation of Leishmania to In Vitro Cultivation at 37 C*

SYNOPSIS Promastigotes of Leishmania donovani 3S grown in Tobie's modified medium (Tm) at 25 C multiplied at 37 C after short periods of growth at 32 and 35 C. Only inocula from logarithmic phase cultures grew when placed at 32 C. Amastigotes placed at 32, 35, or 37 C became promastigotes but did not multiply upon transfer. The shortest period required for adaptation of 3S promastigotes to 37 C was 44 days (∽66 generations requiring 18 serial transfers). Addition of chick embryo extract to the medium was unnecessary for growth at elevated temperatures. Promastigotes of the Khartoum strain could not be acclimated to temperatures above 35 C. The long-lasting nature of the adaptation was indicated by (a) ability of 37 C-acclimated promastigotes to grow at this temperature after 14 serial transfers (∽100 generations) at 25 C, and (b) immediate growth of promastigotes at 37 C in cultures inoculated with homogenized hamster spleen previously infected with 37 C acclimated cells. The ability of the temperature-adapted promastigotes to grow at 37 C was lost after 30 serial transfers (224 generations) at 25 C. Since sexual reproduction has not been demonstrated in Leishmania, it was impossible to ascertain whether acclimation was a consequence of mutation or represented a dauermodification.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

A Macromolecule-Free Partially Defined Medium for Trypanosoma cruzi*

SYNOPSIS. A macromolecule-free medium, containing in its defined part 3 salts, glucose, hemin, 21 amino acids, 3 lipids, and some undefined components obtained by dialysis of liver infusion, was developed for serial cultivation of Trypanosomacruzi at 28 C. The medium allows prolonged cultivation of T. cruzi by serial transfers and growth comparable to that obtained in more complex media, including those containing blood serum.     

L form of Neisseria gonorrhoeae

Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609-1614. 1966.-L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO(2) tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium.     

Effects of synthetic lysophosphatidylcholines on suspension cultures of the Chinese hamster ovary DG44 cells in protein-free media

This study described the effects of synthetic lysophosphatidylcholines on the growth of recombinant CHO-DG44 cells in suspension. Overall, cell growth characteristics were improved when cultivated in suspension in a protein-free medium supplemented with natural soybean lysophosphatidylcholines. To substitute synthetic lysophosphatidylcholines for the naturally occurring lysophosphatidylcholines, we implemented a systematic approach in which twelve synthetic lysophosphatidylcholines were grouped into three lipid mixtures according to the length of their acyl chains. We found that synthetic lysophosphatidylcholines with medium acyl chain lengths (C14-C18), including oleoyl lysophosphatidylcholine (C18:1) could increase cell growth in the protein-free media. The fortified protein-free medium with medium acyl chain length lysophosphatidylcholines (C14-C18) maintained growth of CHO-DG44 cells over five consecutive passages, whereas the cell growth in a CHO protein-free medium was decreased gradually after four passages. We also observed that the restorative effect of oleoyl lysophosphatidylcholine was comparable to that of natural lysophosphatidylcholine in batch and long-term cultivation. These results show that synthetic lysophosphatidylcholines can be used as lipid supplements in either protein-free media or chemically defined media for CHO cell suspension cultures.     

Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.     

Effects of long-term serial cell passaging on cell spreading,migration, and cell-surface ultrastructures of cultured vascular endothelial cells

The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.     

Trypanosoma cruzi: morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C.Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C.No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr.Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.