Culture Harvest Research Articles

Diploid Chromosome Number and Chromosomal Variation in the White Rhinoceros (<italic>Ceratotherium simum</italic>)

Chromosomal studies were conducted on 38 white rhinoceroses representing both the northern and southern subspecies and one subspecies hybrid. Improvements in tissue culture methods and harvesting techniques have made it possible to obtain a highly repeatable diploid number of 82 chromosomes for both subspecies. Comparison of G-banded karyotypes from the two subspecies failed to indicate a difference in banding pattern, but did reveal size polymorphisms involving short arm additions in five individuals. Chromosomal polymorphism, resulting in three individuals with a diploid number of 2n = 81, was noted in northern white rhinoceroses.

Cytogenetic studies of epithelial ovarian carcinoma

We performed cytogenetic studies of 36 human epithelial ovarian carcinomas using in situ culture and robotic harvest. We obtained analyzable metaphases of all 36 tumors (100%). One or more chromosomally abnormal clones were observed in 80% of tumors. Common clonal chromosome gains (each occurring in six or more cases) included +1, +2, +3, +6, +7, +9, and +12. Common clonal chromosome losses (occurring in 12 or more cases) included −X, −4, −8, −11, −13, −15, −17, and −22. Common clonal structural abnormalities (occurring in four or more cases) involved regions 1p36, 1q32, 1q42, 3p13→p26, 3q26→q29, 7p22, 9q34, 11p13–p15, 17q1→q23, 19p13.3, and 19q13.3. Trisomy 12 was noted as the sole anomaly in three of five borderline and grade 1 tumors. Two grade 2 tumors contained i(1q), −14, −15 and −22. The results suggest that the pathogenesis of borderline and low-grade tumors may differ from that of higher grade tumors. Two high-grade tumors had an apparent translocation between 17q21 and 19p13.3, two chromosome regions believed to be critical to ovarian carcinogenesis.

Newcastle Disease Virus Passage in MDBK Cells as an Aid in Detection of a Virulent Subpopulation

Newcastle disease virus strains (NDV) La Sota, Texas GB (GB), and mixtures of the two strains were serially passaged in embryonated chicken eggs or the MDBK cell line, a more restrictive culture system than eggs for NDV. The two culture systems were compared by evaluating culture harvests for pathogenicity in inoculated chickens; the harvests were identified by hemagglutination-inhibition tests against monoclonal antibodies that can differentiate La Sota and GB cultures. Both viruses, inoculated alone or as mixtures, were propagated by passage in chicken eggs. La Sota strain failed to propagate by continuous passage in MDBK cells, and only GB was identified in culture harvests propagated in MDBK cells that had been inoculated with GB or mixtures of GB and La Sota. The results indicate that the MDBK cell line is a more selective substrate than chicken eggs and suggest that passage in MDBK cells may aid in selecting for more virulent subpopulations of NDV in a mixed culture. Other reference NDV strains, pigeon NDV isolates, and recent lentogenic NDV isolates from chickens were also passaged in MDBK cells; all strains except those that are classified as lentogens like La Sota could be serially propagated in MDBK cells.

Pseudomosaicism, true mosaicism, and maternal cell contamination in amniotic fluid processed with in situ culture and robotic harvesting.

This study was designed to test the usefulness of the common definitions for maternal cell contamination, true mosaicism, and pseudomosaicism for amniotic fluid specimens processed by in situ culture and robotic harvesting. We prospectively studied 4309 consecutive amniotic fluid specimens processed with these methods and found that 0.84 per cent had maternal cell contamination, 0.28 per cent had true mosaicism, and 5.4 per cent had pseudomosaicism. Although the frequencies of maternal cell contamination and true mosaicism were comparable to those in similar published studies, the frequency of pseudomosaicism was more than twice as high as that in previous reports. This finding is most likely not due to the method, but rather to a more accurate estimate of the actual frequency of pseudomosaicism in amniotic fluid cultures than reported heretofore. Follow-up clinical information was available on 72 per cent of the cases. In three cases of true mosaicism involving structural anomalies, the results of cytogenetic follow-up studies on the neonates were normal. None of the pseudomosaic cases involving trisomy 8, 13, 18, or 21; triple X; or monosomy X were associated with newborns who had birth defects.

Prognostic value of cytogenetic analysis in human cerebral astrocytomas.

Cytogenetic analysis has become an important part of the diagnostic evaluation of most hematological neoplasms. However, there is limited information on the value of cytogenetic analysis in most solid tumors, including cerebral astrocytomas. This report summarizes a prospective cytogenetic study of 99 human cerebral astrocytomas, 16 mixed oligoastrocytomas, and 2 gliosarcomas. The cytogenetic procedure involved in situ culture and robotic harvesting techniques. Correlative clinical and survival data were available on all patients. We successfully cultured and obtained suitable metaphases in 107 of the 117 tumors. One or more chromosomally abnormal clones were observed in 72 tumors, and nonclonal or normal karyotypes were observed in 35 tumors. In a multivariate analysis, survival time was significantly better in patients whose tumors had normal or nonclonal karyotypes on cytogenetic analyses than in those whose tumors had clonal abnormalities (p = 0.0071). Our study demonstrates that cytogenetic analysis provides independent prognostic information in patients with cerebral astrocytomas.

Comparison of sensitivity for human cytomegalovirus of the polymerase chain reaction, traditional tube culture and shell vial assay by sequential dilutions of infected cell lines

Although traditional tube culture (TTC) is still considered by many as the ‘gold standard’ for the laboratory diagnosis of human cytomegalovirus (HCMV), the shell vial assay (SVA) offers greater speed of detection. This technique utilizes immuno-fluorescence (IF) to detect early or immediate early nuclear antigens (IEA). The detection capabilities of these two tests were compared with the polymerase chain reaction (PCR), a technique that amplifies enzymatically selected DNA target sequences. Serial dilutions of crude culture harvests from 2 HCMV strains, Towne and a clinical urine isolate, were made up to 1:1 000 000. Ten-μl aliquots of the original sample and each dilution were tested by PCR, TTC and SVA. For PCR, the nested-primer approach was used. Outer primers delimited a 721-bp sequence contained within the 2nd to 4th exons of the immediate-early protein. Inner nest primers delimited a 167-bp sequence in the third exon, detected by a 32P-labelled probe. The results show that: (1) control samples which contained all PCR reagents but no DNA were uniformly negative; (2) radiolabelled-probe detection (RPD) of PCR products is, on average, 100 × more sensitive than detection by ethidium bromide; (3) PCR is, on average, 100 × more sensitive than evaluation of cytopathic effect (CPE) in the TTC; (4) the predictive value of a negative SVA result is low compared to PCR.

Diagnostic Relevance of Clonal Cytogenetic Aberrations in Malignant Soft-Tissue Tumors

Malignant soft-tissue tumors often present substantial diagnostic challenges. Chromosome aberrations that might be diagnostic have been identified in some types of soft-tissue tumors, but the overall frequency and diagnostic relevance of these aberrations have not been established. We attempted to determine the karyotypes of a series of 62 consecutive, unselected malignant spindle-cell or small round-cell soft-tissue tumors (from 46 adults and 16 children) after direct harvesting of cells or short-term culture. All tumors were examined independently by immunohistochemical staining in addition to routine light-microscopical evaluation, and all but two tumors were examined by electron microscopy. Metaphases were obtained from 61 of the 62 tumors, and clonal chromosome aberrations were identified in 55 (89 percent). In the six tumors that yielded metaphases but lacked apparent clonal aberrations, the normal metaphases were found to originate from non-neoplastic stromal elements within the tumor specimens. Thus, all tumors in which karyotyping was successful contained clonal chromosome aberrations. Forty of 62 tumors (65 percent) contained clonal chromosome aberrations that either suggested or confirmed a specific diagnosis; in 15 of these tumors (24 percent of all tumors), the aberrations were important in establishing the final diagnosis. Cytogenetic analyses were particularly informative about small round-cell tumors from children: 8 of 14 round-cell tumors contained diagnostically important chromosome aberrations. Using the combined approaches of light and electron microscopy, immunohistochemistry, and cytogenetics, we established an unambiguous diagnosis for 60 of 62 tumors. Cytogenetic analyses reveal clonal chromosome aberrations in virtually all malignant soft-tissue tumors. These clonal chromosome aberrations, particularly in small round-cell tumors in children, often have diagnostic relevance.

Improved efficiency ofin vitropropagation ofCamellia reticulatacv. Captain Rawes

SummaryEffects of various factors were studied on the in vitro multiplication and rooting of a clone of Camellia reticulata cv. Captain Rawes established in vitro from shoots of an adult tree. Multiplication rates were increased by repeated harvesting of cultures in which shoots were subcultured in a horizontal rather than a vertical position. Best rooting responses were obtained with 175.3 mM sucrose in the rooting medium using 8 week old shoots cultured in 500 ml glass jars; these shoots contained more anthocyanin than those from test-tube cultures. The orientation and repeated harvesting of the mother shoots had no effect on rooting response.

Manipulation of yeast for transport studies: Critical assessment of cultural and experimental procedures

In the manipulation of Saccharomyces cerevisiae and other yeasts for transport studies, several factors must be taken into account. These include growth and cultural procedures, harvest, and suspension in an appropriate medium with a suitable substrate concentration. Of particular importance is suspension density, since if this is high, it may limit the availability of exogenous solute for transport and thus affect the rate of accumulation and intracellular/extracellular solute concentrations. This has been demonstrated with respect to the uptake of 2-deoxy-D-glucose (DOG) by S. cerevisiae, where a low suspension density of 0.27 μl (cell volume) ml (medium volume) -1 resulted in a high initial rate of uptake, a higher accumulation ratio, and negligible disappearance of DOG from the external medium, contrasting with a high suspension density of 20 μl (cell volume) ml (medium volume) -1, where the initial rate of uptake and accumulation ratio were lower, coupled with a substantial decrease in external DOG concentration. Such factors have a significant influence on the analysis and interpretation of transport data and should always be considered. Published reports of yeast transport studies should contain sufficient relevant, detailed information so that these effects can be fully evaluated .

Acetylcholinesterase in the human erythron. III. Regulation of differentiation

Acetylcholinesterase (AChE) is present in both primitive and mature erythroid cells, but a role for the enzyme in human hematopoiesis has not been defined. This prospect represented the primary objective of the following study. In clonal culture of normal human bone marrow cells, a "wave" of AChE activity was demonstrated, rising from undetectable levels to a peak (of 1.48 femto-moles per min per cell) at 10 days in the course of progressive erythroid clonogenesis. At concentrations of enzyme inhibitor that clearly reduced AChE activity in a dose-dependent fashion, there was no overall effect on erythropoiesis in vitro, but the clones were generally smaller and significantly more often multi-focal than in control cultures. Furthermore, in the presence of AChE inhibitors, a concentration-dependent increase in the myeloid-erythroid ratios of the culture harvests was observed. Likewise, a clear reduction in hemoglobination was revealed, in cells of 10 day cultures, from a mean hemoglobin concentration of 35.0 pg per cell in controls to 20.1 pg per cell in the presence of the maximal concentration of the inhibitor (10(-6) M eserine). These data point to a role for AChE in the regulation of differentiation in the human erythron.

‘Ozark Pink’ VF Tomato

Abstract ‘Ozark Pink’ VF is an indeterminate, pink-fruited tomato (Lycopersicon esculentum Mill.) developed at the Univ. of Arkansas to replace ‘Traveler 76’ (2), which is susceptible to verticillium wilt. ‘Ozark Pink’ provides the excellent flavor quality of traditional Arkansas pink tomatoes with improved fruit size and disease resistance over ‘Traveler 76’. Fruits also are larger and firmer than ‘VF Pink’ (3), which was grown primarily in southeastern Arkansas. ‘Ozark Pink’ is adapted to stake culture and vine-ripe harvest for local market, shipping short distances and for home gardens throughout Arkansas and the Ozark Mountain area. Pink cultivars remain the most popular type sold as bedding plants and at roadside stands in Arkansas, although part of the commercial acreage now grows red-fruited cultivars.

A cytogenetic study of 53 human gliomas

Cytogenetic studies were performed on human glioma samples obtained by stereotactic biopsy, stereotactic craniotomy, or routine craniotomy. Using in situ culture and robotic harvesting techniques, we obtained suitable metaphases in 50 (94%) of 53 tumors, including 28 diffuse astrocytomas, four juvenile pilocytic astrocytomas, two gliosarcomas, three other miscellaneous astrocytomas, eight oligodendrogliomas, four mixed oligodendroglioma—astrocytomas, and four ependymomas. Cytogenetic studies were performed only on primary cultures; the mean culture time was 9.6 days (range 1–31 days). One or more chromosomally abnormal clones were observed in 35 (66%) tumors. Eleven (21%) other specimens had random nonclonal chromosome abnormalities. In four (8%) specimens, no chromosome abnormalities were noted. The results of this study suggest that grade 3 and 4 tumors are more likely to contain an abnormal clone than tumors of grade 1 or 2 ( p < 0.01). The most common numeric chromosome abnormalities were −6, +7, −10, −13, −14, −15, −18, and −Y. The most common structural abnormalities involved 1p, 6q, 7q, 8p, 9p, 11p, 11q, 13q, and 19q. Four tumors had two or more independent clones and ten contained subclones demonstrating karyotype evolution. With in situ culture and robotic harvesting techniques, cytogenetic studies can be successful on nearly all human gliomas, including those derived from small stereotactic biopsies.

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.

Rapid detection of bovid herpes virus 1 antigens by counter immunoelectrophoresis and agar-gel immunodiffusion

Using hyperimmune rabbit sera to BHV1, ID and CIEP tests detected one antigen in BHV1 infected cell culture harvests. In nasal and ocular secretions collected from experimentally infected cattle during the early course of clinical disease two and one BHV1 antigens were detected by the ID and CIEP tests respectively. In specimens collected from eight cattle for 10 days after the onset of pyrexia the ID test demonstrated precipitating antigens in 22/35 ocular secretions and 43/60 nasal secretions, whereas the CIEP detected antigens in 40/64 ocular secretions and 44/69 nasal secretions. The antigens were thermostable, being detectable by CIEP 14 days and seven days after storage at 4 degrees C and room temperature respectively.

Techniques for the laboratory culture of mysidopsis species (crustacea: Mysidacea)

AbstractThe objectives of this study were to evaluate existing techniques for culturing three species of the genus Mysidopsis that are routinely used as toxicity test organisms. A comparison of the life history, ecology and distribution of M. bahia, M. bigelowi and M. almyra is presented. Culture systems are described, supplemental foods are suggested, and guidelines are given for salinity, temperature, dissolved oxygen and nitrogen levels that will maintain optimum laboratory culture conditions. Methods are outlined for harvesting juveniles and controlling contaminant organisms, and general feeding procedures are described. Culture management is stressed as the most significant factor in maintaining productive mysid cultures. Specific recommendations are made regarding culture density, harvesting frequency, sex ratio and introduction of feral animals.

FLOCCULENCE OF BREWERY YEASTS AND THEIR SURFACE PROPERTIES: CHEMICAL COMPOSITION, ELECTROSTATIC CHARGE AND HYDROPHOBICITY

The flocculence of a top and a bottom fermentation brewery yeast was studied through the cell surface properties: surface composition (X-ray photoelectron spectroscopy), surface charge (microelectrophoresis) and hydrophobicity (adhesion tests). Aerobic Cultures and fermentation simulating industrial conditions were performed. The flocculence (tendency to flocculate in favourable conditions) has been determined by a test which separated the occurrence of flocculation and the measurement of its manifestation (suspension clearing); the flocculating agents were ethanol and calcium. The top fermentation strain does not flocculate with calcium alone but is very sensitive to ethanol (0.01% (v/v)). The lower flocculence observed for cells cultured on malt extract as compared with cells cultured on yeast extract is accompanied by a more negative zeta potential. The bottom fermentation strain which has a more negative surface flocculates only in the presence of calcium but ethanol enhances the process. The variation of flocculence according to the culture medium and harvesting time is correlated to the variation of the phosphate surface concentration. This is not paralleled by a variation of the zeta potential which does not decrease below a value of about-35 mV as the phosphate surface concentration increases. The increase of flocculence as a function of culture time correlates also with an increase of the cell hydrophobicity; this is more obvious for the cultures than for the fermentations.

The effect of Ca 2+ on the thermal stability of trypsin in phosphate-buffered saline solution used for harvesting of human embryonic lung fibroblast cultures

The stabilizing effect of Ca 2+ (264·9 mg CaCl 2 · 2H 2O ml −1) on a 0·5% solution of twice-crystallized bovine trypsin in phosphate-buffered saline (used for harvesting human embryonic lung fibroblasts) was studied at 7–37 °C and at −20 and −70 °C. It can be concluded that storage of the enzyme in the buffer (with or without Ca 2+) is not advisable at temperatures ⩾20 °C. At 7 °C, on the other hand, trypsin can be stored for some weeks in the calcium-containing phosphate-buffered saline if a moderate loss of activity is acceptable. At −20 °C and −70 °C the stability of the enzyme was good. In the presence of Ca 2+ about 90% of the activity remained after 18 weeks. Without Ca 2+ the activity was approximately 10% lower.

A pipette method for rapid karyotyping in prenatal diagnosis.

The "pipette method' is introduced as a method of prenatal diagnosis which is in competition with the "in situ' and the "trypsinization' technique. It is sufficiently standardized for routine diagnosis and the banding techniques currently used in prenatal diagnosis (G, Q, C-banding and NOR) have been adapted for it. In 180 cases from 27 different centres, the "pipette method' was employed for chromosomal harvesting in order to save time. An average of 6 X 6 days was taken to achieve a result. There was a pathological karyotype in 28 cases (16.1 per cent) and this high proportion can be related to cases where ultrasound scan has led to a diagnosis of "suspected chromosomal abnormality.' This technique is also of use in advanced stages of pregnancy. The early recognition of the fetal karyotype can contribute to the future management of the pregnancy. The "pipette method' can also be used in chromosomal harvesting of tumour cells and fibroblast cultures.

A rapid one-step multiwell tray test for release of soluble mediators

A simple one-stage bioassay for testing the release of diffusible mediators from cells has been developed in a modified standard 96-well tissue culture tray. The method is based on connecting the adjacent wells containing the producer and the indicator cells, so as to allow free passage of the culture supernatant, but not of the cells, between the connected wells. This method obviates the use of semipermeable membranes, requires minimal numbers of producer cells, and allows the harvesting of replicate cultures by using standard cell harvesting equipment.

An indirect sandwich enzyme labelled immunosorbent assay for the detection of foot and mouth disease virus immunizing antigen in tissue culture harvests

The optimum conditions for an indirect sandwich enzyme-linked immunosorbent assay for foot and mouth disease virus 140S antigen assay are described. Factors which could contribute to the variation in the test were investigated and a calibration coefficient for the conversion of ELISA values to antigen concentration in micrograms of 140S antigen per millilitre was calculated. Antigen mass in nine tissue culture harvests was estimated and these correlated well with estimates made by sucrose density gradients (r = 0.95).