The 96-well twin-layer system: a novel approach in the cultivation of microalgae

A novel system for the growth and maintenance of microalgae has been developed that allows the cultivation of a large number of strains with little manual effort. The system is based on a 96-well microtiter plate in which a membrane filter constitutes the bottom of each well. Algal strains are immobilised on the membranes and provided with culture medium through contact with layers of glass fibre located beneath the membranes in a special cultivation chamber. The configuration effectively separates culture medium from algal cells which allows the simultaneous exchange of the culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary, algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of microalgal strains including delicate flagellates can be reliably grown in the system under axenic conditions and without cross-contamination. As an array system, the 96-well twin-layer system using immobilised algae is also amenable to high-throughput and massively parallel applications increasingly sought after in algal bio- and environmental technology.     

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.     

Monoxenic and axenic cultivation of carrier and patient strains of Entamoeba histolytica.

All of five strains of Entamoeba histolytica, isolated from symptomatic cases of amoebiasis, could be adapted to axenic growth on the TP-S-1 medium of Diamond (1968). Four axenic strains were started from amoeba-Crithidia cultures; one could be axenized directly after isolation from a case of cutaneous amoebiasis. Attempts to monoxenize, resp. axenize strains, isolated from Dutch, asymptomatic carriers, were less successful. Only three out of ten strains could be submitted to bacteria-free growth. These three strains, however, originated probably from a recent case of intestinal amoebiasis. The results, suggesting that highly virulent strains can be easier cultivated bacteria-free than those with low or no virulence, are further discussed. The yield of axenic amoebae per tube fluctuates largely depending on many factors such as the strain, the number of transfers (i.e. degree of establishment), the quality of Panmede liver digest and serum in the TP-S-1 medium, and the care of manipulating the cultures. For optimal growth, a more acid medium was required in an amoeba-Crithidia culture than in an axenic culture. Multinucleated, giant amoebae were frequently observed in axenic cultures.     

Effect of phosphatidylcholine-cholesterol liposomes on Entamoeba histolytica virulence

Trophozoites of Entamoeba histolytica HM-1:IMSS become less virulent after long-term maintenance in axenic cultures. The factors responsible for the loss of virulence during in vitro cultivation remain unclear. However, it is known that in vitro cultivation of amoeba in culture medium supplemented with cholesterol restores their virulence. In this study, we analyzed the effect of adding phosphatidylcholine-cholesterol (PC-Chol) liposomes to the culture medium and evaluated the effect of this lipid on various biochemical and biological functions of E. histolytica HM-1:IMSS in terms of its virulence. The addition of PC-Chol liposomes to the culture medium maintained the virulence of these parasites against hamster liver at the same level as the original virulent E. histolytica strain, even though these amoebae were maintained without passage through hamster liver for 18 months. The trophozoites also showed increased endocytosis, erythrophagocytosis, and carbohydrate residue expression on the amoebic surface. Protease activities were also modified by the presence of cholesterol in the culture medium. These findings indicate the capacity of cholesterol to preserve amoeba virulence and provide an alternative method for the maintenance of virulent E. histolytica trophozoites without the need for in vivo procedures.     

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoeba spp.

A total of 36 strains belonging to 19 different species of Acanthamoeba were compared for temperature tolerance, ability to grow in an axenic medium, cytopathic effect in Vero cell culture, and virulence in mice. Pathogenic strains appeared to belong to different species, whereas pathogenic and nonpathogenic strains occurred in one species. Although growth at high temperatures and readiness to grow axenically indicated a potential for pathogenicity, each such strain had to be tested in cell cultures or laboratory mice to determine whether or not it was virulent. This study was not intended to differentiate Acanthamoeba spp., but to provide methods to be used for the specific isolation and identification of pathogenic Acanthamoeba strains.     

Virulence of bacteria-associated, Crithidia-associated, and axenic Entamoeba histolytica: experimental hamster liver infections with strains from patients and carriers.

Experimental infections of the hamster liver were carried out with five strains from patients with clinical amoebiasis and ten strains from asymptomatic carriers. Inocula of comparatively small size (12000-36000 amoebae) were injected under the liver capsule. 1. The virulence of the patient strains varied from 21-96% (see article) and declined sharply within 7-15 weeks after elimination of the associated bacterial flora. The virulence of the carrier strains varied from 0-100%, probably fluctuating with changes in the concomitant bacterial flora (Table 1). 2. The interrelation between size of inoculum, period of bacteria-free growth, and virulence was demonstrated with a Crithidia-associated patient strains (Table 2). 3. A patient strain showed a faster decrease of virulence during axenic than in Crithidia-associated cultivation (Table 3). 4. Two successive passages through hamster liver resulted in a marked increase of virulence of two bacteria-free strains, lasting for several months (Table 4). 5. A significant enhancement of virulence of Crithidia-associated and axenic amoebae by reassociation with a mixed bacterial flora during two weeks, followed by elimination of the bacteria, was demonstrated with two strains. The restored virulence was lost again within a few weeks (Table 5). 6. The virulence of an attenuated patient strain did not become manifest by adding large numbers of dead amoebae to the inoculum (Table 6). 7. The pathology of the different lesions caused in the hamster liver by the amoebae is described, including one of a granulomatous type, frequently found after inoculation with bacteria-free amoebae. 8. In an attempt to explain the occurrence of strains differing in pathogenicity an hypothesis is put forward based on the idea of selection of virulence and avirulent amoebae.     

我国沿海四爿藻的室内培养

在水产养殖中,四爿藻是一种重要的饵料资源,从我国沿海(包括广东、浙江、山东等省)采集到的海洋单细胞绿藻四爿藻,用毛细管法挑取进行单种培养,然后在固体培养基中稀释划线分离得到无菌纯培养物,利用改良的Guillard &; Ryther(Guillard F)培养基、海洋Ⅲ号培养基和改良的海洋Ⅲ号培养基对四爿藻进行了实验室培养。结果发现,改良的海洋Ⅲ号培养基中四爿藻的生长速率最快,最适pH范围为7．0～8．0,加入维生素B12(10μg·L^-1),也可以适当增加四爿藻的生长速率。这种改良的海洋Ⅲ号培养基配制简单,适合于水产养殖中四爿藻的规模生产。     

Recurrent senescence in axenic cultures of Physarum polycephalum

When subcultures of the aux-2 and aux-4 strains of Physarum polycephalum, which had been grown for more than four years in axenic shake culture, were transferred to non-axenic surface culture they displayed progressively shorter lifespans (older axenic surface cultures yield shorter lived non-axenic cultures). Similar subcultures transferred to axenic agar medium also underwent senescent-like events. These subcultures, after a period of vigorous growth, displayed a slower growth rate, reduced cytoplasmic streaming, loss of yellow pigment, and eventually they fragmented into a number of small spherical structures with the concomitant lysis of most of the plasmodium. In non-axenic culture these structures quickly degenerated (and disappeared from the culture); however, in axenic culture they revived and after several days produced new vigorous plasmodia. Following a period of vigorous growth the plasmodium again underwent senescent-like events. This cycle of senescence and growth was repeated a number of times before death finally occurred.     

Growth and antimicrobial activity of an association of an actinomycete and a green alga

During joint cultivation of the actinomycetes Streptomyces griseus (strains 65 and 744) isolated from the soil and the green algae Chlorella vulgaris larger amount of biomass as compared with solitary axenic culture have been shown. The relation of biomass of actinomycetes S. griseus strain 65 and S. griseus strain 744 and algae in the lichen-like experimentally formed thallom make up 42:1 and 40:1 relatively, i. e. the mass of actinomycetes forms 97-98% from the mass of thalloms. Actinomycetes in the associations with the algae accumulate larger amount of biomass that in the axenic cultures on corresponding medium, whereas the algae produce the same amount of the biomass as the axenic culture under the same conditions. The associations have the antimicrobic properties differed from the axenic cultures established.     

Characterization of an Axenic Strain of Hartmannella vermiformis Obtained from an Investigation of Nosocomial Legionellosis

ABSTRACT. A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila . This strain of H. vermiformis , designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.     

Axenic Mass Cultivation of Entamoeba invadens and Cell Membrane Isolation

SYNOPSIS. A method for axenic mass cultivation of Entamoeba invadens is described. A known monophasic culture medium for E. histolytica was modified by mixing it with a medium for Acanthamoeba castellanii and adding ferric chloride to provide more particulate material. This results in a high number of cells per ml after a relatively short culture period.
A cell membrane fraction of such a mass culture of E. invadens can be prepared by simple disruption of the cells and by low speed centrifugation. Owing to the lack of membrane-bounded organellae in the amebic cytoplasm there are only 2 kinds of membranes to be separated: the plasmalemma and the vacuole membranes.     

Lipid Auxotrophy and the Effect on Lipid Composition of Entamoeba invadens*†

SYNOPSIS. A medium for the axenic cultivation of Entamoeba invadens has been developed. Serum, an essential constituent of conventional media, has been replaced by a mixture of albumin, unsaturated fatty acids, Tween, and cholesterol to control the lipid composition of the medium. Entamoeba invadens requires both cholesterol and unsaturated fatty acids for growth. The fatty acid composition of the phospholipids of the ameba reflects that of the medium to a great extent, especially with regard to the unsaturated fatty acids. The amount of membrane bounded cholesterol depends on the cholesterol concentration in the medium.     

Axenic Culture of the Heterotrophic Dinoflagellate Pfiesteria shumwayae in a Semi-Defined Medium

ABSTRACT. A semi-defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae . Maximum cell yields and division rates in the semi-defined medium ranged from 0.1 × 10⁵ to 4.0 × 10⁵ cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi-defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae , needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate.     

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

A New Method For Axenic Mass Cultivation of Paramecium Tetraurelia*

SYNOPSIS A method is described for the axenic mass cultivation of Paramecium tetraurelia strains 51s and 299s. the ciliate is grown in an enriched axenic medium developed by Soldo, Godoy & van Wagtendonk. Under continuous shaking on a rotary shaker, cultures were grown in one-liter Erlenmeyer flasks with 330 ml medium yield cell densities of 32,000 cell/ml and 20,000 cells/ml for strains 299s and 51s respectively. Doubling time is considerably shorter under these conditions than in the conventional static cultures. A 20-liter airlift bioreactor is described in detail which can be used successfully to otain up to 100 g wet weight of Paramecium in a single run; in this reactor the cell density reaches 38,000 cells/ml for strain 299s. and 23,000 cells/ml for 51s. This technic should facilitate the study of minor protein components of the ciliate.     

Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis

A free-living amoeba identified as Hartmannella vermiformis was isolated from a water sample obtained during an investigation of nosocomial legionellosis. Hartmannella vermiformis is known to support the intracellular multiplication of Legionella pneumophila. This strain of H. vermiformis, designated CDC-19, was cloned and established in axenic culture to develop a model for the study of the pathogenicity of legionellae. Isoenzyme patterns of axenically-cultivated strain CDC-19 were compared with two strains of H. vermiformis derived from the type strain, one axenic (ATCC 50236) and the other grown in the presence of bacteria (ATCC 30966). Enzyme patterns suggested that all three strains are assignable to the species H. vermiformis. Axenic H. vermiformis strain CDC-19 has been deposited with the American Type Culture Collection (ATCC 50237) and should prove useful in the study of protozoan-bacterial interaction.     

Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

The axenic cultivation of insect forms of Trypanosoma (Duttonella) vivax and development to the infective metacyclic stage

A method for axenic cultivation of epimastigote and metacyclic forms of Trypanosoma (Duttonella) vivax at 27 degrees C in vitro is described. Iscove's medium was supplemented with specific concentrations of foetal bovine serum, L-proline, L-glutamine hypoxanthine, adenosine, pyruvate, and 2-mercaptoethanol. Bloodstream form parasites rapidly transformed into epimastigote forms that grew as surface-adherent colonies in plastic culture flasks. Transformation of epimastigotes to metacyclic forms was first observed 9-12 days after initiation of cultures. Percentages of metacyclics varied: East African T. vivax ranged up to 40% and West African T. vivax ranged up to 24%. Subcultures were made at two-week intervals and maintained for several months. Transformation of bloodstream forms to epimastigotes depended on initial attachment to the bottom of culture flasks and the presence of L-proline. The number and maturity of metacyclic forms was influenced by the concentrations of foetal bovine serum, L-proline, L-glutamine, and 2-mercaptoethanol. Trypanosomes from cultures were cryopreserved, revived, and used to re-establish fresh axenic cultures. These results represent a significant advance in cultivation of T. vivax insect forms that should enable studies to be accomplished on metabolism, differentiation, and pharmacology of this parasitic protozoan, free from the influence of extraneous cells.