Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls

We designed a rapid assay that assesses the polychlorinated biphenyl (PCB)-degradative competence and congener specificity of aerobic microorganisms, identifies strains capable of degrading highly chlorinated biphenyls, and distinguishes among those that degrade PCBs by alternative pathways. Prior attempts to assay PCB-degradative competence by measuring disappearance of Aroclors (commercial PCB mixtures) have frequently produced false-positive findings because of volatilization, adsorption, or absorption losses. Furthermore, these assays have generally left the chemical nature of the competence obscure because of incomplete gas chromatographic resolution and uncertain identification of Aroclor peaks. We avoided these problems by using defined mixtures of PCB congeners and by adopting incubation and extraction methods that prevent physical loss of PCBs. Our assay mixtures include PCB congeners ranging from dichloro- to hexachlorobiphenyls and representing various structural classes, e.g., congeners chlorinated on a single ring (2,3-dichlorobiphenyl), blocked at 2,3 sites (2,5,2'5'-tetrachlorobiphenyl), blocked at 3,4 sites (4,4'-dichlorobiphenyl), and lacking adjacent unchlorinated sites (2,4,5,2',4',5'-hexachlorobiphenyl). The PCB-degrative ability of microorganisms is assessed by packed-column gas chromatographic analysis of these defined congener mixtures following 24-h incubation with resting cells. When tested with 25 environmental isolates, this assay revealed a broad range of PCB-degradative competence, highlighted differences in congener specificity and in the extent of degradation of individual congeners, predicted degradative competence on commercial PCBs, and (iv) identified strains with superior PCB-degradative ability.     

Anther and tissue culture-induced grain chalkiness and associated variants in rice

Rice, Oryza sativa, plants regenerated from anther culture with and without in vitro selection pressure were evaluated for chalky seed. Progeny evaluated included 21 spontaneously doubled haploids selfed 4 times, progeny from plants regenerated from S-aminoethylcysteine resistant callus selfed 4 times and backcrosses of both types to the parental type. All lines with in vitro histories had higher seed chalkiness than the controls both in the intensity of chalkiness and in the number of seeds expressing the character. The full range of intensity and amount of chalkiness was expressed in the progeny. The average intensity of anther/tissue culture-derived progeny was 4–5, based on a scale of 1 (translucent) to 10 (fully opaque), and the average amount of chalkiness within plants sampled was 50–75 percent. The chalky characteristic is transmitted from parent to offspring into a range of identifiable F₂ segregants. Disclaimer statement Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Kinetic and Physical Studies of Cell Death Induced By Chemotherapeutic Agents Or Hyperthermia

The kinetics of three physical parameters: cell density, relative cytoplasmic viscosity and DNA stability to denaturation have been measured during the period preceding cell death induced by hyperthermia, methylprednisolone and a series of cancer chemotherapeutic agents. This series of measurements employed cultured human lymphoblastoid cells as an experimental system to establish the changes that can be observed in the early stages of cell death, prior to applying such measurements to tissue biopsies from solid human tumours. Cell death, induced by hyperthermia up to 43°C, methylprednisolone, vincristine, 5-fluorouracil, BCNU and melphalan, showed essentially identical and reproducible changes corresponding to those which characterize programmed cell death (apoptosis). Such changes could also be observed following hyperthermia above 43°C, but reproducibility was poor and increasing damage to the cell membranes was evident. In cells treated with adriamycin or methotrexate, cell sub-populations showing an increase in cell density were not detected. Measurements of DNA stability were readily performed by flow cytofluorometry thus allowing rapid quantitation of the fraction of cells in the early stages of cell death. Modified flow cytometric instrumentation would further allow measurement of cytoplastic viscosity as an additional parameter to indicate entry into programmed cell death. This suggests that these measurements could readily be applied to cell suspensions derived from tumour tissue biopsies for a more accurate assessment of tumour growth rate, and to allow monitoring of response to therapy in sequential tumour biopsies.     

The acquired immune deficiency syndrome and epidemic of infection with human immunodeficiency virus: costs of care and prevention in an inner London district.

The epidemic of the acquired immune deficiency syndrome (AIDS) and infection with human immunodeficiency virus (HIV) necessitates early planning of services and allocation of resources. The use of hospital resources by patients with AIDS and the planned additional costs of clinical and preventive services for the epidemic of infection with HIV were calculated for an inner London health district that has treated 18% of the cases in the United Kingdom. Patients with AIDS required on average 50 days of inpatient hospital care each at an estimated current average lifetime cost of pounds 6800. These costs, however, underestimated the additional capital and revenue costs of planned new preventive and treatment services, estimated as being pounds 388,000 revenue and pounds 472,000 capital for 1986-7. It is important to invest now in preventive services throughout the United Kingdom to reduce the future social and financial costs of AIDS.     

Age-related and seasonal variation in the Sertoli cell population, daily sperm production and serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone in stallions

Testes and blood samples were obtained from 201 stallions aged 6 months to 20 years in either December-January (nonbreeding season) or June-July (breeding season) to study the effect of age and season on reproductive parameters. Seasonal differences in the Sertoli cell population of adult (4-20 years old) horses were characterized by a 36% larger number of Sertoli cells in the breeding season than in the nonbreeding season. Seasonal elevation in the Sertoli cell population was associated with an increase in testicular weight and daily sperm production per testis (DSP/testis). Concentrations of luteinizing hormone (LH) and testosterone in serum varied with season. Although follicle-stimulating hormone (FSH) concentrations also tended to be higher in the breeding season, this trend was not statistically significant (P less than 0.08). Sertoli cell numbers averaged over both seasons, like testicular weights, increased with age until 4-5 years of age, but were stabilized thereafter. This age-related difference was also associated with increased concentrations of FSH, LH and testosterone, and with increased DSP/testis. The Sertoli cell population was capable of increasing in the adult horse by fluctuating its size with season. The number of elongated spermatids per Sertoli cell over both seasons increased with age up to 4-5 years of age and was stabilized thereafter. Thus, seasonal and/or age-related differences in DSP/testis were associated with significant elevations in serum concentrations of FSH, LH and testosterone, testicular weights, numbers of elongated spermatids per Sertoli cell and elevation of the Sertoli cell population.