Distinct volume distribution of viable and non-viable hybridoma cells: A flow cytometric study

Light scattering properties of hybridoma cells were examined with flow cytometry. Viable and dead cells form two distinct populations. The distribution of the two populations changes during a batch culture. the concentration of dead cells measured by flow cytometry correlates well to that measured by hemacytometer. The distribution based on small-angle light scattering is similar to the distribution based on volume as measured by Elzone particle counter. It thus appears that viable cells form the population with a larger mean cell volume. The results also indicate that the volume of viable cells decreases during the cultivation while that of dead cells remains relatively constant.     

A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii.

A standard methodology for quantitatively evaluating neutralizer toxicity against Acanthamoeba castellanii does not exist. The objective of this study was to provide a quantitative method for evaluating neutralizer toxicity against A. castellanii. Two methods were evaluated. A quantitative microtiter method for enumerating A. castellanii was evaluated by a 50% lethal dose endpoint method. The microtiter method was compared with the hemacytometer count method. A method for determining the toxicity of neutralizers for antimicrobial agents to A. castellanii was also evaluated. The toxicity to A. castellanii of Dey-Engley neutralizing broth was compared with Page's saline. The microtiter viable cell counts were lower than predicted by the hemacytometer counts. However, the microtiter method gives more reliable counts of viable cells. Dey-Engley neutralizing medium was not toxic to A. castellanii. The method presented gives consistent, reliable results and is simple compared with previous methods.     

Significance of the surface of immobilized Saccharomyces cerevisiae cells in ethanolic fermentation

S. cerevisiae cells immobilized in alginate beads show in many cases an increase of mean single cell volume during long-time fermentations (successive batch cycles). The biomass loading capacity of the gel beads is characterized by a maximum volume but not by a maximum number of cells occupying the gel volume. In our system this loading capacity, i.e. the maximum volume fraction of cells per volume of beads, amounted to about 0.54. As a more important result it must be stated that the specific product formation rate in the case of fermentations negligibly influenced by diffusion hindrance is related to the total surface of the viable cells but not to their total number, total volume or total dry weight.     

Immunocytochemistry: human neural stem cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only approximately 25 microl of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.     

Adjusting the number of spermatozoa in mini-straws by determination of the operative volume of bovine semen

The importance of the number of sperm per insemination on fertility has been well demonstrated in cattle. This number is usually calculated from the concentration in the extended semen and a theoretical value for the operative volume of semen delivered during insemination. The objective of this experiment was to investigate the usefulness of the measurement of the delivered volume of semen when estimating sperm numbers from frozen-thawed mini-straws, by comparing the results obtained with an analytical balance to the theoretical volume. The density of semen extended with Biociphos Plus and Triladyl was determined to be 1.033 g/ml using the gamma sphere method. This value was used to convert semen weight into operative volume. The effect of semen temperature at the time of weighing (37 degrees C versus 20 degrees C) was investigated on six semen batches, two technicians measuring the operative volume of 50 straws for each combination of temperature and semen batch (a total of 1200 weighings). The temperature effect was found to be insignificant, which allowed warm semen to be weighed before motility was assessed during routine quality control. The operative volume was then measured in straws routinely produced at 17 bovine Al centers (12-105 semen batches per center, mostly three straws from each batch, a total of 1912 measurements). The observed volumes were normally distributed around 198.7 microl, 98% measuring between 180 and 210 microl. The operative volume was significantly different among centers (from 192 to 205 microl, P < 0.0001) and among batches within centers (P < 0.0001). The S.D. among straws within batches was 3.4 microl. Some centers showed high variability in straw volume whereas others were more consistent. Determination of the operative volume of frozen-thawed mini-straws by weighing the delivered contents is an accurate method for estimation of the number of sperm per dose.     

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.     

Cerebrospinal fluid sampling from guineapigs: sample volume--related changes in protein concentration in control animals and animals in the relapsing phase of chronic relapsing experimental allergic encephalomyelitis

Cerebrospinal fluid (CSF) was removed from guineapigs by puncture of the cisterna magna and the total sample volume of 200-360 microl divided into 40 microl aliquots. After determination of albumin and IgG in these CSF aliquots it was found that successive samples gave different results. In general, up to 100 microl CSF could be removed before the protein concentration began to increase. In animals with chronic relapsing experimental allergic encephalomyelitis (CR-EAE) the rise in albumin concentration was accompanied by a corresponding fall in the number of white cells in later samples.     

A rapid chromogenic microtitre assay of arginine aminopeptidase activity in Mycoplasma strains

Arginine-utilizing strains of Mycoplasma can be screened by assay of their arginine aminopeptidase activity. A standardized chromogenic method is described that enables enzyme detection in small volumes of cell suspension in less than 3 h. Cell suspensions (10 microl) in 96-well microtitre plates are incubated at 37 degrees C, pH 8.0, with 0.1 mM arginyl-beta-naphthylamide (100 microl). This is hydrolysed to release beta-naphthylamine, which gives a coloured product on diazotization with fast garnet. M. alkalescens can be detected in this way with as few as 1.1 x 10(5) viable cells and M. fermentans with 2.3 x 10(6) cells. The method has been shown to enable division of 28 strains into three groups of fermentative and arginine-hydrolysing mycoplasmas. This procedure has potential for routine laboratory use.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.     

Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.     

Developmental stages of fetal-type Leydig cells in prepubertal rats

Fetal Leydig cells were studied in rats during and after the perinatal-neonatal period by comparing changes in morphology, number and volume with changes in testicular steroids and serum luteinizing hormone (LH) concentration. Stereologic examination indicated regression of fetal Leydig cells in testis by showing that their total volume as well as the average cell volume decreased between prenatal day 20 and postnatal day 3. The total number and total volume of cells both increased between postnatal days 3 and 11 but the average cell volume did not change during the same time period. Determination of serum LH showed a close correlation between an increase in LH concentration and increases in total number and volume of cells. The combined number of fetal- and adult-type Leydig cells on day 20 was more than 20 times the number of fetal cells at 3 days of age. Electron microscopic analysis showed that fetal Leydig cells after birth formed conspicuous clusters, which were surrounded by a layer of envelope cells and extracellular material. Occasional dividing fetal Leydig cells and possible precursors of fetal or adult Leydig cells were observed. Mitoses of spindle-shaped pericordal cells were frequent during the neonatal period. During and after the second postnatal week fetal Leydig cells again showed signs of regression, indicated by disintegration of the cell clusters, a decrease in cell size, accumulation of collagen between the cells and a decrease in steroid content per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viability of Indigenous Soil Bacteria Assayed by Respiratory Activity and Growth

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The effect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, affected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.     

Effect of Growth Rate and Starvation-Survival on Cellular DNA, RNA, and Protein of a Psychrophilic Marine Bacterium

DNA, RNA, and protein concentrations from starved ANT-300 cell populations grown at different growth rates fluctuated corresponding to the three stages of starvation-survival on total and viable cell bases. During stage 1 of starvation-survival, two to three peaks in the concentration levels for all three macromolecules were characteristic. During stage 2, DNA per total cell dropped to between 4.2 and 8.3% of the original amount for all of the cell populations examined, and it stabilized throughout stage 3. The decrease in DNA per cell was also observed in electron micrographs of cellular DNA in unstarved compared with starved cells. The fluctuations of RNA and protein per total cell concentrations observed during stage 2 coincided in all cases, except for the cells from dilution rate (D) = 0.015 h⁻¹. This ANT-300 cell population showed a decrease in RNA per total cell to only 29.2% and an increase in protein to 129.7% of the original amount after 98 days of starvation. During stage 3, DNA, RNA, and protein concentrations per total cell also stabilized to continuous levels. Cells from the faster-growth-rate cell populations of D = 0.170 h⁻¹ and batch culture had elevated protein per total cell concentrations, which remained primarily residual during the starvation period. Starved cells from D = 0.015 h⁻¹ had estimated nucleoid and cell volumes of 0.018 and 0.05 μm³, respectively, yielding a nucleoid volume/cell volume ratio of 0.40. We consider these data to indicate that slow-growth-rate cells are better adapted for starvation-survival than their faster-growth-rate counterparts.     

The intertubular compartment morphometry in capybaras (Hydrochoerus hydrochaeris) testis

The aim of the present study was to characterize the intertubule element volume density, individual and total Leydig cells volume, Leydig cell number per testis and per gram of testis, and leydigosomatic index in adult capybaras. Eight capybaras from a commercial abattoir were utilized. The intertubular compartment volume density and the Leydig cells were 45.2 and 31.13%, respectively. The individual and total Leydig cell volumes were 8.51 and 2169.41 x 10(-12) mL, respectively. The Leydig cell number per testis was 3.8 billion and the Leydig cell number per gram of testis was 126 million. The leydigosomatic index was 0.037%. In conclusion, this study shows that capybaras have one of the greatest individual and total Leydig cell volume and Leydig cell volume density, and that the Leydig cell number per gram of testis is at least double the mean for mammals previously investigated in its order.     

Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Image analysis method for the rapid counting of Saccharomyces cerevisiae cells.

An image analysis system which incorporates a microscope, video camera, monitor, and Apple computer and which uses image area to count Saccharomyces cerevisiae cells is described and evaluated. Yeast cell suspensions of densities of up to 100 X 10(6) cells per ml can be counted when viewed in the counting chamber of a hemacytometer. The yeast image area measured depends upon the light intensity used to illuminate the yeast cells, the sharpness of the image focused on the monitor, and the grey level selected when scanning the digitized image on the monitor of the Apple computer, all of which can be controlled. The image area also depends upon the yeast strain and medium in which the culture is grown, but it is not affected by the concentration of sugar or ethanol in which the yeast cells are suspended. Yeast growth measured by image analysis can be calibrated to give results similar to those obtained with hemacytometer counting. Yeast cells can be counted in the presence of high cell densities of bacteria by adjusting the grey level at which the digitized image is scanned.     

Image analysis method for the rapid counting of Saccharomyces cerevisiae cells

An image analysis system which incorporates a microscope, video camera, monitor, and Apple computer and which uses image area to count Saccharomyces cerevisiae cells is described and evaluated. Yeast cell suspensions of densities of up to 100 X 10(6) cells per ml can be counted when viewed in the counting chamber of a hemacytometer. The yeast image area measured depends upon the light intensity used to illuminate the yeast cells, the sharpness of the image focused on the monitor, and the grey level selected when scanning the digitized image on the monitor of the Apple computer, all of which can be controlled. The image area also depends upon the yeast strain and medium in which the culture is grown, but it is not affected by the concentration of sugar or ethanol in which the yeast cells are suspended. Yeast growth measured by image analysis can be calibrated to give results similar to those obtained with hemacytometer counting. Yeast cells can be counted in the presence of high cell densities of bacteria by adjusting the grey level at which the digitized image is scanned.