Spectral imaging detection and counting of microbial cells in marine sediment

Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using microscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) with nucleic acids. Using this technique, microbial cells could be easily detected in soil and 30-cm deep sediment samples from Tokyo Bay, both of which contain particles other than microbial cells. Total cell density was semiautomatically estimated at 1-6 x 10(9) cells cm(-3) of sediment sampled at different depths in Tokyo Bay, which corresponded to 65-106% (mean 88%) of visual direct counting. This technique may be useful for detecting microbial cells in soil and sediment samples from the deeper subsurface environment.     

Quantification of telomerase activity by direct scintillation counting

An improved telomerase assay was developed that allows direct quantification of the enzyme activity by scintillation counting of the labeled telomerase product. The assay measures the incorporation of ³²P-dGTP into telomeric repeats synthesized at the 3′ end of a biotinylated primer. Telomerase reaction product is separated from the reaction mix by streptavidin-coated magnetic beads and counted. The assay can be used for quantitative studies of human telomerase and its inhibitors.     

Computer-aided cell colony counting

Counting cell colonies is a tedious task when performed with the light microscope. Moreover, unless strict double-blind protocols are adhered to, biased counts are difficult to avoid. Presented here is a computer software application that performs accurate, reproducible cell colony counts with a minimum of user generated bias. The application is based upon the Apple IICX computer system with Image software and AppleScan. Colonies are grown on 24-well plates and prepared in such a way as to permit good quality scanning. The scans are then transferred to Image and the individual colonies in each well are counted. Good correlation with counts done by light microscopy is achieved.     

Automatic cell counting with ImageJ

Cell counting is an important routine procedure. However, to date there is no comprehensive, easy to use, and inexpensive solution for routine cell counting, and this procedure usually needs to be performed manually. Here, we report a complete solution for automatic cell counting in which a conventional light microscope is equipped with a web camera to obtain images of a suspension of mammalian cells in a hemocytometer assembly. Based on the ImageJ toolbox, we devised two algorithms to automatically count these cells. This approach is approximately 10 times faster and yields more reliable and consistent results compared with manual counting.     

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.     

Optimization of dopaminergic neuron counting in substantia nigra of parkinsonian mice

Parkinson’s disease (PD) results from the degeneration of dopaminergic (DA-ergic) neurons of substantia nigra pars compacta (SNc). The disease is modeled in mice by the administration of a neurotoxin precursor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Neurotoxin efficiency is estimated by reduced number of DA-ergic neurons in SNc. Cell counting on serial sections is very laborious and expensive and, therefore, is not widely used in spite of its high precision. The well-known Konigsmark’s formula (KF) allows one to perform counting on sections chosen with a certain interval rather than to utilize serial sections. However, its precision decreases with increasing interval and other parameters. In this paper, we described the mathematical method of approximation (MA) by improving KF. MA maintains counting precision and allows one to reduce the time and expenses for material processing and analysis.     

Optimization of procedures for counting viruses by flow cytometry

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.     

Comparison the cytotoxicity of hydroxyapatite measured by direct cell counting and MTT test in murine fibroblast NIH-3T3 cells

The worldwide growing interest to biomaterials over the last years results from their irreplaceable role in medical clinic. Hydroxyapatite is used in bone reconstruction because of its similar chemical structure compared to the inorganic composition of human bone and it is basic building component of many newly prepared biomaterials. In this study, we evaluated cytotoxic/antiproliferative activity of hydroxyapatite extract using murine fibroblast cell line NIH-3T3 and two in vitro different cytotoxic assays: growth inhibition assay and MTT assay. Hydroxyapatite extract after 72 h of incubation manifested the significant in vitro cytotoxic/antiproliferative effect only at the highest concentration tested (100 %). The antiproliferative effect of hydroxyapatite extract at the other concentrations tested (75 %, 50 %, 25 %, 10 %, 5 % and 1 %) was directly proportional to the concentration and the time of influence. The inhibition of cell proliferation was 86.8 - 0 %. The sensitivity of cell growth inhibition assay (direct counting of viable cells) to the extract influence was higher than that of MTT test.     

A tentative direct microscopic method for counting living marine bacteria.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.     

Just the right size: cell counting in Dictyostelium

The regulation of tissue and organism size plays an essential, but poorly understood, role in multicellular development. Genes have been identified that affect body and organ size in a number of animals. Two recently identified genes, smlA and countin, are required for the proper function of a cell-counting mechanism that regulates organism size in the eukaryotic microorganism Dictyostelium discoideum. The discovery of this process now allows the study of size regulation in a simple multicellular system.     

Development of an adhesive sheet for direct counting of bacteria on solid surfaces

An adhesive sheet was developed for direct counting of microorganisms on solid surfaces. The sheet consists of a polyurethane film base and water insoluble adhesive. SYBR Green II (for total direct counting) or 6-carboxyfluorescein diacetate (6CFDA) (for fluorescent vital staining) was used for fluorescent microscopy of bacteria collected on the adhesive face of the sheet. Adhesive sheet sampling showed a higher recovery rate for microbial enumeration than conventional swab method or stamp agar. This method is simple, rapid, inexpensive and reproducible.     

A direct and simple method for rapidly counting viable chains ofLeuconostoc in batch cultures

Summary Plating determines the concentration in viable cells of bacteria but remains time-consuming and inaccurate. A direct and simple method combining two techniques, haemacytometry and epifluorescence, gives both the viability and the concentration in viable cells. The method was evaluated using Leuconostoc mesenteroides.     

Summary of the National Institute of Standards and Technology and US Food And Drug Administration cell counting workshop: Sharing practices in cell counting measurements

The emergence of cell-based therapeutics has increased the need for high-quality, robust and validated measurements for cell characterization. Cell count, being one of the most fundamental measures for cell-based therapeutics, now requires increased levels of measurement confidence. The National Institute of Standards and Technology (NIST) and the US Food and Drug Administration (FDA) jointly hosted a workshop focused on cell counting in April 2017 entitled “NIST-FDA Cell Counting Workshop: Sharing Practices in Cell Counting Measurements.” The focus of the workshop was on approaches for selecting, designing and validating cell counting methods and overcoming gaps in obtaining sufficient measurement assurance for cell counting. Key workshop discussion points, representing approximately 50 subject matter experts from industry, academia and government agencies, are summarized here. A key conclusion is the need to design the most appropriate cell counting method, including control/measurement assurance strategies, for a specific counting purposes. There remains a need for documentary standards for streamlining the process to develop, qualify and validate cell counting measurements as well as community-driven efforts to develop new or improved biological and non-biological reference materials.     

Application of a rapid direct viable count method to deep-sea sediment bacteria

For the first time, a Live/Dead (L/D) Bacterial Viability Kit (BacLight ) protocol was adapted to marine sediments and applied to deep-sea sediment samples to assess the viability (based on membrane integrity) of benthic bacterial communities. Following a transect of nine stations in the Fram Strait (Arctic Ocean), we observed a decrease of both bacterial viability and abundance with increasing water (1250-5600 m) and sediment depth (0-5 cm). Percentage of viable (and thus potentially active) cells ranged between 20-60% within the first and 10-40% within the fifth centimetre of sediment throughout the transect, esterase activity estimations (FDA) similarly varied from highest (13.3+/-5.4 nmol cm(-3) h(-1)) to lowest values below detection limit down the sediment column. Allowing for different bottom depths and vertical sediment sections, bacterial viability was significantly correlated with FDA estimations (p<0.001), indicating that viability assessed by BacLight staining is a good indicator for bacterial activity in deep-sea sediments. Comparisons between total L/D and DAPI counts not only indicated a complete bacterial cell coverage, but a better ability of BacLight staining to detect cells under low activity conditions. Time course experiments confirmed the need of a rapid method for viability measurements of deep-sea sediment bacteria, since changes in pressure and temperature conditions caused a decrease in bacterial viability of up to 50% within the first 48 h after sample retrieval. The Bacterial Viability Kit proved to be easy to handle and to provide rapid and reliable information. It's application to deep-sea samples in absence of pressure-retaining gears is very promising, as short staining exposure time is assumed to lessen profound adverse effects on bacterial metabolism due to decompression.