Correction for the inherent error in optical density readings.

Except at very low levels, uncorrected photometric determination of bacterial cell densities showed a decreasing proportionally to actual cell density or dry weight. A standard curve was prepared to convert photometric readings to truly proportional optical density values. With one dry weight determination, optical density values may be converted to absolute dry weight values.     

Measurement of the Growth of a Floc Forming Bacterium <Emphasis Type="Italic">Pseudomonas putida</Emphasis> CP1

Pseudomonas putida CP1 formed clumps of cells when grown on mono-chlorophenols but not on phenol or glucose. An increase in cell numbers for the organism grown on mono-chlorophenols was accompanied by a decrease in the dry weight. The change in shape of the bacterium from rod shape to coccus shape coupled with a reduction in cell size when the organism was grown under nutritional stress was found. This result together with cell aggregation affected the measurement of growth parameters in the system by conventional methods (optical density measurements, dry weight measurements and the plate count technique). Monitoring growth of Pseudomonas putida CP1 by a direct microscopic count technique was found to be more representative than conventional methods including optical density measurements, dry weight measurements and the plate count technique when grown on phenolics.     

A flow cell photometer for bacterial growth monitoring

A flow cell photometer is described with automatic cleaning of the photometric cell, denasimetric separation of air bubbles and precipitates, and a constant sensitivity from 0 to 10 mg/ml of bacterial dry weight.     

Monitoring of Saccharomyces cerevisiae in commercial bakers' yeast fermentation

In the highly competitive market of commercial bakers' yeast, fermentations are operated for maximum efficiency and minimum production cost. In order to maintain competitiveness, the fermentations must be highly consistent with minimum variation in yeast performance, maximum yield on raw materials, and minimum production of undesirable side products. The use of advanced instrumentation is of critical importance to achieving these goals by the production engineer. An in situ optical density probe was used to determine the yeast cell density in full-scale commercial bakers' yeast fermentations. The optical density probe results were compared with oxygen uptake rate analyses, packed cell volume, and off-line measured cell dry weights. The most accurate measurement of cell density was found to be the optical density probe. This instrument allowed the on-line determination of cell density with highly consistent results from fermentation batch to batch and with out the need for intermittent recalibration. (c) 1995 John Wiley & Sons, Inc.     

Method of determining the concentration of glycogen, DNA, 3H-thymidine label and dry weight in one and the same cell]

A method is proposed for determination of glycogen, DNA, 3H-thymidine incorporation and dry weight in the same cell, the technique being based on successive discovery and measuring of each of these indices. Cells are obtained from animals, previously injected with 3H-thymidine, to be charted on preparation, made pictures and measured in square units. Then on preparations embedded into glycerine or vaseline oil, the optical path difference of rays for the nucleus and cytoplasm of selected cells is measured with the interferencial microscope. This is followed by the fluorescent PAS reaction and the content of glycogen is registered microfluorimetrically in the same cells. Preparations after that are treated with a freshly prepared water solution of 0.025% borohydride sodium, stained with the routine or fluorescent Feulgen reaction, and DNA content is determined in the same cells in which glycogen and delta delta were previously measured. The stained nuclei are photographed, their areas are measured and the dry weight of the nucleus and cytoplasm of marked cells is calculated from the values of the nuclear areas and of delta delta. Eventually the preparations are covered by emulsion and exposed, and 3H-thymidine-containing nuclei are determined, the index of marked nuclei and the marking intensity over the nucleus are calculated. As a result, a precise and reliable determination of glycogen, DNA, dry weight and 3H- or 14C-thymidine incorporation is made in either of the marked cell.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Determination of low concentration of Paracoccus denitrificans encapsulated in polyvinyl alcohol LentiKat’s pellets

The aim of this work was to compare three methods to determinate low concentrations of Paracoccus denitrificans encapsulated in polyvinyl alcohol pellets, which is important for evaluation and optimization of pellet production as well as for monitoring of biomass growth. Pellets with different and well-defined biomass concentrations were used for experiments. The following fast and simple methods were tested: (1) dissolution of polyvinyl alcohol in hot water followed by dry weight estimation, (2) dissolution of polyvinyl alcohol in hot water followed by optical density measurement, (3) and extraction and quantification of proteins. Dry weight estimation proved to be problematic as it was difficult to separate biomass from polymeric carrier. Optical density measurement showed good linearity of dependence of optical density on biomass content, but determined limits of detection and limits of quantification were not within the range necessary for intended application. The only tested method meeting the requirements for sensitivity was determination of protein concentration after protein extraction.     

Effect of inclusion body production on culture turbidity and cell dry weight in growing bacterial cultures.

An approach to the optimization of product yield in an inducible inclusion body-producing system is presented. Following induction by indoleacrylic acid (IAA) of a trpLE-HIVgp41 fusion protein, we found a large increase in culture turbidity and single-cell dry weight. After an initial transition phase, new and constant values for specific growth rate, single-cell light turbidity, and single cell dry weight were achieved, allowing for the determination of optimal conditions for product formation.     

Growth kinetics of Apple plant cell cultures

Summary Aerobic batch cultures of Apple fruit cells were carried out using sucrose as the sole carbon source. The determination of the growth parameters was used in order to estimate the maintenance coefficient m and the maximum growth yield Y_G; their values at 25°C are respectively 0.32. 10^-2 g sugar/g cell dry weight.h and 0.57 g cell dry weight/g sugar. Then, a mathematical expression of the cell growth is discussed, with respect to the specific growth rate evolution during cultivation.     

Significance of fresh weight to dry cell weight ratio in plant cell suspension cultures

Summary The ratio of fresh weight to dry cell weight (FW/DCW), an index of cell water content, is important in determining the theoretical maximum cell concentration in high density culture of plant cell suspensions. Theoretical maximum cell density for Thalictrum rugosum was estimated to be as high as 137.2 g/l. The FW/DCW values were found to vary from species to species and it was related to medium osmolarity. When the cells were placed in a high osmolarity environment, decrease of FW/DCW was very rapid, taking place within 24 hours.     

Comparison of four methods for quantification of biofilms in biphasic cultures

Three methods for determining the total biofilm amount in biphasic cultures have been compared: dry weight by filtration after solvent treatment, optical density with a biomass probe and protein content. The activity of the biofilm was estimated through mineral nitrogen consumption. Calculation of the coefficients of variation shows that these parameters could be used to characterise such a biofilm. The optical density by biomass probe was the most reliable one (repeatability <0.5%) to quantify total biofilm and a linear relation was verified against dry weight. © Rapid Science Ltd. 1998     

A method of measuring protein dry weight in solutions of unknown salt concentration and an application to lipoproteins

The dialysis bag dry weight method was developed for the measurement of dry weights of lipoproteins subfractionated on density gradients where total recovery of lipoprotein in the gradient was to be determined. Use of the conventional method for dry weight determination was precluded because of inconsistent concentration changes which would occur in the dialysis step due to differences in both the lipoprotein and salt concentration among gradient fractions. The method described consists of transfer of measured undialyzed samples into previously weighed bags followed by dialysis against water, lyophilization of the protein-bag combination and calculation of the protein dry weight as the difference between the bag weight and the total weight.Since the method described incorporates dialysis in the assay, it is capable of giving an accurate protein dry weight measure of a non-predialyzed sample, whereas the conventional dry weight method gives an accurate value only of a previously dialyzed sample. The increase in the standard deviation of the overall dialysis bag method was shown to be less than double that of the conventional method for a sample of known salt concentration and there is no distinguishable difference in the central values obtained by the two methods.The particular usefulness of this method for lipoprotein solutions was presented.     

Optical properties of Nannochloropsis sp and their application to remote estimation of cell mass

The absorption and scattering coefficients and reflectance spectra of ultra-high density Nannochloropsis occulata cultures were investigated in detail to identify the optical properties of the cultures and devise algorithms for remote estimation of dry cell mass in ultra-high cell density cultures. High-spectral resolution measurements of apparent absorption and attenuation as well as reflectance from 400 to 900 nm were carried out in relation to the dry weight, cell count, and pigment concentration in outdoor cultures. Indices calculated as (R(NIR) - R(red))/(R(NIR) + R(red)) and R(NIR)/R(red), in which R(NIR) is reflectance in the range from 750 to 800 nm and R(red) is reflectance in the range 670-680 nm, were used for remote assessment of dry cell mass. Remote estimation in the range 1 to 8 g/L was accomplished with an error of less than 0.66 g/L. A different index, i.e., (R(NIR) - R(red)) was employed for estimation of cell-chlorophyll concentration. This is the first report of in vivo specific absorption coefficient of chlorophyll-a and specific scattering coefficient per dry algal weight of Nannochloropsis sp., providing a basis for remote monitoring of dense phytoplankton masses.     

Cell mass of Mycobacterium bovis BCG estimated by gas chromatography

The presence of additives and large cellular aggregates in freeze-dried BCG vaccines precludes accurate measurement of total cell content by traditional methods. The possibility that extraction and quantitation of a cell membrane fatty acid may provide a suitable means of cell mass determination was tested. The palmitic acid methyl ester peak area determined by gas chromatography was directly proportional to the wet weight of freshly grown Tice-, Pasteur-, and Glaxo-substrain BCG, as well as the dry weight of the ampoule contents after removal of soluble material. Extraction of palmitic acid from Tice BCG vaccine was not appreciably affected by lyophilization and the calculated dry cell mass values of freeze-dried vaccine samples correlated well with particle number. This method, therefore, may be useful in measuring BCG cell mass during all stages of vaccine manufacture and storage.     

Modelling and Operation of a Turbidity-Meter for On-Line Monitoring of Microbial Growth in Fermenters

The PROIMI-2 prototype turbidity-meter for measuring cell concentrations in stirred tanks, has been designed and constructed using theories based on kinetic laws and the general principles of cell growth. Laboratory assays were carried out with batch cultures of Bacillus amyloliquefaciens, Zymomonas mobilis and Saccharomyces cerevisae. The signals showed a good correlation with optical density and biomass. In the latter case up to 4·0 g per dm³ expressed as dry weight could be monitored successfully.     

Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.     

Dense autotrophic cultures of Alcaligenes eutrophus.

Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter.     

Determination of the mass of viruses by quantitative electron microscopy.

The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.