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.     

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures

Ziehl–Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein–Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.8 and 100%, respectively). The overall percentages of mycolic acid detection for both sample types were 43.2 and 91.3%, respectively. The mycolic acid analysis of the Middlebrook 7H9 cultures had the fewest false negative detections with respect to the LJ cultures. The analysis of fluorescent derivatives of mycolic acids, using HPLC, is useful for concentrated sputum samples with large number of bacilli (3+) and is preferred for Middlebrook 7H9 cultures, even for clinical specimens with a low number of bacilli. Furthermore, with this analytical method, the simultaneous detection and identification of mycobacteria is usually possible.     

Screening of terrestrial and freshwater halotolerant cyanobacteria for antifungal activities

Cyanobacterial cultures tolerating 200 mmol l⁻¹ sodium chloride isolated from terrestrial and freshwater habitats of North Maharashtra region of India were evaluated for antifungal activity. Aqueous, methanol, n-propanol, and petroleum ether extracts of 40 cyanobacterial isolates belonging to nine genera were examined for inhibitory activity against five fungal pathogens. Eighteen isolates belonging to genus Oscillatoria dominated the population of halotolerant cyanobacterial cultures. Four antifungal bioassays viz. double layer agar method, disc diffusion assay, silica gel method, and minimum inhibitory concentration (MIC) were used to screen the cultures for antifungal activity. Among the solvents used, methanol extracts showed 34.9% inhibition followed by n-propanol, petroleum ether, and water exhibiting 30.2%, 18.6% and 16.2% inhibition, respectively. The double agar layer method was found to be a suitable method in preliminary screening for handling large number of cultures without extraction of compounds. However, in later screening experiments, silica gel method was seen to be advantageous over MIC and agar disc diffusion methods.     

A method for the rapid production of fine plant cell suspension cultures

A method has been devised which allows the rapid production of fine suspension cultures of small aggregate size from suspension cultures of large average aggregate size, such as those of Capsicum frutescens. The method, which uses a Waring blender for aseptic homogenisation of cultures, has also been shown to be effective in rapidly producing suspension cultures from callus cultures. The suspension cultures so produced are particularly useful for immobilisation, such as in porous polyurethane foam matrices.     

The use of 5-bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

A method for killing dividing cells (Puck and Kao, '67) was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10(-4) M for 3 days and then irradiated with long UV light. The selective elimination of dividing cells led to a loss of myosin Ca2+-activated ATPase in the cultures. This indicates the presence of dividing cells which contain myosin. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages.     

Cryopreservation and Plant Regeneration from Embryogenic Cultures of Larch (Larixxeurolepis) and Black Spruce (Picea mariana)

A method has been developed for the routine cryopreservationof embryogenic cultures of hybrid larch (Larixxeurolepis) andblack spruce (Picea mariana Mill.). The method involves growingthe cultures in the presence of sorbitol and then briefly exposingthem to DMSO followed by controlled cooling to –40°C.The cultures were then submerged and stored in liquid nitrogen.Growth of the embryogenic cultures was monitored for 14 d afterrapid thawing and plating on to media. The highest relativeincrease in the tissue fresh weight, after storage in liquidnitrogen, was observed when embryogenic cultures of both specieswere pregrown for 24 h in a medium with 0·4 M sorbitoland then treated with 10% DMSO. This pretreatment also ensuredthe shortest lag phase in resuming the growth. The post-thawcultures gave rise to mature somatic embryos which developedinto plants Key words: Larixxeurolepis, Picea mariana, cryopreservation, embryogenic tissue, plant regeneration     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Simultaneous quantitative measurement of luciferase reporter activity and cell number in two- and three-dimensional cultures of hepatitis C virus replicons

Hepatitis C virus (HCV) is a global health problem and an important human pathogen. The development of cell culture models for HCV infection has been difficult to accomplish, primarily because HCV is very sensitive to the host cell state. Future models will require the use of three-dimensional (3D) cultures that model the host cell state and environment more accurately. Higher information content screens for anti-HCV therapeutics will also involve 3D cell cultures. Here we report a method for screening cell models for HCV replication that involves normalizing luciferase reporter activity based on cell number in two-dimensional (2D) and 3D HCV replicon cultures. Human hepatoma cells stably replicating luciferase-containing HCV replicons were cultured in 2D monolayer culture and 3D spheroid culture. Optimization of cell lysis was performed so that cell lysates could be used to quantify both luciferase activity and cellular DNA content. Cellular DNA content was quantified using Hoechst 33258 dye and was converted to cell number. The method is straightforward, reproducible, and sensitive down to 5000 cells. This method enables low-throughput but high-information content screening of HCV replicons, with the potential for high-throughput screening in a variety of 3D cultures and cocultures.     

Comparison of titration results of diphtheric antitoxic antibodies obtained by means of Jensen's method and the methods of tissue cultures and haemagglutination.

In 33 human sera the determination of diphtheric antitoxic antibodies was performed in a double blind test using Jensen's method, the method of tissue cultures and the haemagglutination method. In the method of tissue cultures the antibody levels in the sera were determinated in the first and second experiment with the precision of +/- half dilution of the geometrical progression. In Jensen's method, the difference between the first and second measurements slightly exceeded +/- 1 dilution. In the haemagglutination method the error considerably exceeded the binary step dilution. In most cases, the determination fluctuated up to seven times the actual value. Differences among the mean values of examination results obtained by Jensen's method and the method of the tissue cultures are statistically insignificant. The differences between the haemagglutination method and both the other methods are statistically significant.     

Two-Hour Embedding Procedure for Intracellular Detection of Viruses by Electron Microscopy

An embedding method requiring only 2 h to complete and giving excellent ultrastructural preservation has been used for the rapid detection of viruses in tissue cultures. The method has also been applied successfully to mammalian tissue. It provides a rapid technique for identifying viruses isolated in tissue cultures, for screening cultures for adventitious agents, and for examining tissue biopsies for viruses.     

Differential rates of cytokine production and apoptosis in venipuncture and finger-stab derived blood cultures

The collection of finger-stab (FS) blood is a convenient and non-invasive method of rapidly acquiring human blood and is becoming increasingly popular for use in human biomonitoring studies. This study compared whole blood (WB) and peripheral blood mononuclear cell (PBMC) cultures derived from venipuncture (VP) and FS blood, to determine whether they respond similarly under culture conditions. The rates of spontaneous- and radiation-induced apoptosis and pro-inflammatory cytokine production were monitored over 72 h in each of four culture conditions. In non-irradiated WB cultures, the spontaneous rate of apoptosis was significantly lower in cultures from FS-derived blood than from VP-derived blood. However, FS- and VP-derived cultures responded similarly to radiation-induced apoptosis. PBMC cultures, regardless of the source, were the most responsive to radiation. When the levels of pro-inflammatory cytokines were measured, a significant time-dependent increase in TNF-alpha, IL-6 and IL-1beta production was observed in FS-derived cultures, but not in VP-derived cultures. While VP and FS blood cultures were found to respond similarly to radiation-induced apoptosis, there was a significant difference in the rate of spontaneous apoptosis in non-irradiated WB cultures and in the in situ production of pro-inflammatory cytokines between VP- and FS-derived blood cultures.     

Cell cultures derived from embryos and melanoma of poeciliid fish

In poeciliid fish, melanoma of different degrees of malignancy can be produced by crossing specific genotypes. For a detailed investigation of the processes leading to proliferation or differentiation of the melanoma cells, it is necessary to establish cell cultures. The aim of the present study was to find out the optimal conditions for initiating and culturing poeciliid fish cells for the purpose of establishing cell cultures of melanoma. The optimal method was developed by using small pieces of late embryos as starting material and includes: (a) dispersion of tissue by mild stepwise treatment with a trypsin-EDTA mixture at low temperature; (b) culture of cells in the complex medium 199; (c) supplementation of medium with high percentage (20%) of fetal bovine serum; and (d) stabilization of pH by buffering the medium with HEPES. Under these conditions, primary and secondary cultures of embryonic cells have been initiated. An epithelial-like cell line has been subcultured for more than 80 passages. The method developed for embryonic tissues was used to start cell cultures from melanoma of platyfish-swordtail hybrids. Until now, only cells of rapidly growing malignant albino melanoma could be maintained in primary cultures. Secondary cultures could not be initiated since the melanoma cells tended to differentiate and stopped growing before a confluent monolayer was formed.     

Growth of Boletus variegatus in Surface and Shake Cultures

A method of growing the mycelium of Boletus variegatus Fr. (Suillus variegatus Kuntze) in shake cultures is described. The growth curves of the fungus in stationary surface cultures and shaken submerged cultures have been compared. Growth in stationary cultures comprised ( 1 ) an acceleration phase, characterized by an approximately linear cube root plot, ( 2 ) a linear phase of constant increase of dry weight per day, and ( 3 ) a deceleration phase. In the shake cultures, two growth periods, both characterized by linear cube root plots, were observed. The growth rate constant was higher for the first than for the second period. During the second period, enlarged rounded cells with dense contents but normal cell walls were formed. The carbon source was exhausted and growth stopped after nine days in the agitated cultures and after about three weeks in the stationary ones. The economic coefficient for glucose utilization was higher in the agitated than in the stationary cultures.     

Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures

A new radiometric method for the automatic detection of bacterial growth in blood cultures has been compared with conventional methods. A total of 2,967 cultures from 1,280 patients suspected of having bacteremia were studied. A 2-ml amount of blood was inoculated into culture media in which the glucose was labeled with carbon-14. The release of (14)CO(2) by bacterial metabolism was checked hourly for 18 to 24 hr, daily for the next 2 days, and, on the 12th day, with an automated instrument. A 10-ml amount of blood was studied by conventional bacteriological techniques. In 125 cultures from 50 patients, there was bacterial growth in at least one of the routine media. Of these, the radiometric method detected 102 cultures from 40 patients. In 111 cultures from 48 patients, there was radiometric detection of bacterial growth. In all of these cultures, there was detection of bacterial growth in subcultures from the radioactive medium. Of these, the routine laboratory detected 98 cultures from 40 patients. Neither method detected all patients with bacteremia. Among the 57 patients positive by one or both methods, routine techniques detected bacteria in 87% and the radiometric method detected bacteria in 85%. Seventy per cent of the cultures were detected first by the radiometric method, 65% on the day of inoculation. Our results suggest that the radiometric method is faster than conventional techniques and comparable in accuracy. Its great advantage is that it is simple, automatic, and can be extended to automatic detection of bacterial sensitivity to antibiotics.     

Effects of long-term preservation on growth and productivity of Panax ginseng and Catharanthus roseus cell cultures

Cell cultures of Panax ginseng and Catharanthus roseus producing secondary metabolites were preserved in liquid nitrogen or under mineral oil for six months. The growth behaviour and the ability of the cultures to produce ginsenosides or indole alkaloids were measured after a recovery period and compared with cultures maintained by frequent subcultivation during the same period. Neither growth kinetics nor the degree of vacuolization during growth were affected by the long term preservation. Some changes in secondary metabolism were however found, indicating that preservation under mineral oil does not preserve the productivity of cell cultures whereas the cryogenic method does.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulphoxide - HPLC high performance liquid chromatography     

Microinjection of endogenous and exogenous proteins into primary cultures of rat hepatocytes and the degradation of the injected proteins

1. A method to microinject proteins into cells through packaging proteins to erythrocyte ghosts (erythrocyte-mediated microinjection) was modified partially in order to apply the method to primary cultures of rat hepatocytes. 2. Degradation of the microinjected proteins was examined employing the improved method. The mean half-life of the injected endogenous liver protein was 20 hr. The data suggested that the injected proteins are degraded through both lysosomal and non-lysosomal proteolytic pathways probably depending on their structure. 3. The present method to microinject exogenous proteins into primary cultures of rat hepatocytes can be employed usefully for the investigations of protein metabolism in liver.     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

Selective enrichment and purification of cultures of Methanosaeta spp

A simple method for the isolation of axenic cultures of members of the obligately acetotrophic methanogenic genus Methanosaeta is described. To overcome the competitive advantage obtained by faster growing acetate-utilizing Methanosarcina spp. in batch enrichment cultures, acetone and isopropanol are used as the growth substrates for the enrichment step. Acetone- and isopropanol-utilizing bacteria slowly ferment these substrates to acetate, which allows Methanosaeta spp. to maintain the acetate concentration at levels below the threshold required for growth of Methanosarcina spp. These enrichments eventually develop dense populations of Methanosaeta spp., which can then be separated from contaminating microorganisms to yield axenic cultures.     

Freeze-drying of unfixed monolayer cultures for electron microscopic autoradiography

A method has been developed for freezing, drying and embedding of unfixed monolayer cultures for electron microscopic autoradiography (EM ARG). Experimental results showed: a) Aclar 33 C was a more suitable substrate than the plastic of petri dishes, b) cultures pressed rapidly against the polished face of a large copper cylinder chilled in liquid nitrogen had better cellular morphology than did cultures dipped in Freon 12 chilled in liquid nitrogen, and c) cultures embedded in Epon alone had finer extracellular ice spaces and lower background grain densities than did cultures embedded in Epon with 1% silicone. This method has been used to evaluate the effect of fixation on the localization of the neurotransmitter, 3H-gamma-aminobutyric acid (3H-GABA), in neurons of dispersed cell cultures. EM ARG results showed that the neuronal cell bodies and vesicle elements were present in similar numbers in both glutaraldehyde fixed and freeze-dried cultures.     

A simple, accurate method for determining wet and dry weight concentrations of plant cell suspension cultures using microcentrifuge tubes

Summary A simple method using microcentrifuge tubes for determining fresh and dry weights, and collecting cell-free supernatant from plant suspension cultures is described. This method offers improvements in accuracy, precision, and time efficiency over traditional filtration methods. Using 4-day-old Nicotinia tabacum cultures, the centrifuge method was shown to remove 25% more of the interstitial water from cell aggregates compared to a suction filtration method, with significantly less variation in fresh weight data.