Direct cell counting

Kim et al. (2002) LNCaP Lyco-Red lO-MCH M Water- dispersible 24, 48, 72h Cell cycle by flow cytometry and cyclins D and E Direct cell counts... 

For direct cell counting, pool eight replicate wells and carry out counts using haemocytometer and Coulter counter. 

Direct cell counting via a microscope can be used to estimate per cell activity (e.g., current per cell) and is a simple and accurate method for low culture densities and monolayers of cells attached to electrode surfaces however, these studies require an optically accessible MBC system [4]. When complex biofilms are established at electrode surfaces (e.g., multiple cell layers and heterogeneous surface coverage), computational tools are required to estimate biovolumes and total cell densities of complex biofilms, which also have some associated errors. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could... 

The direct microscopic count determines the number of viable and dead microorganisms ia a milk sample. A small amount (0.01 mL) of milk is spread over a 1.0 cm area on a microscope sHde and allowed to dry. After staining with an appropriate dye, usually methylene blue, the sHde is examined with the aid of a microscope (oil immersion lens). The number of bacterial cells and clumps of cells per microscopic field is determined and, by appropriate calculations, is expressed as the number of organisms per milliliter of sample. 

Because physiological deterioration is generally accompanied by an increase in bacterial population, as pointed out by Nielsen, Wolford, and Campbell (33), estimation of bacterial numbers might serve as the basis of a test for condition. Obviously, the plate count method is not adaptable because of the time limitations imposed. Direct microscopic count would be much more appropriate, especially if a positive field count were substituted for cell count as suggested by Wolford (39). 

Caekelbergh et al. calculated the direct costs of HIV/AIDS in Belgium from the health care pay perspective. On the basis of 150 patients, they determined the costs of antiretrovirals, outpatient and inpatient resource use for the year 2005. They realize that the costs strongly depend on the CD4- - T-cell count, that is, the annual costs per patient are on average about US 2,900 for a patient with a CD4+ T-cell count >500, US 3,200 (CD4 351-500), US 8,650 (CD4 210-350), US 16,600 (CD4 101-200), US 31,300 (CD4 51-100), and US 49,400 (CD4 0-50), respectively. Consequently, the early detection of an HIV-infection as well as proper management that prohibits disease transition is of high cost-importance. 

Viable cells Direct viable count Detection of viable cells by elongation 123... 

Adverse effects (shortened RBC survival, increased Heinz body formation, increased number of reticulocytes, and reduced blood cell counts) were observed at 747 mg/kg/day in males and 907 mg/kg/day in females (average 827 mg/kg/day) (Bucci et al. 1994). Although not statistically significant, the number of Heinz bodies was increased relative to the controls and to the rats treated at 400 mg/kg/day. The observed effects are consistent with a direct effect on RBC and a decrease in their survival. 

Again, the majority of these parameters are interrelated and highly dependent on the method used to determine them. Red blood cell count (RBC), platelet counts, and mean corpuscular volume (MCV) may be determined using a device such as a Coulter counter to take direct measurements, and the resulting data are usually stable for parametric methods. The hematocrit, however, may actually be a value calculated from the RBC and MCV values and, if so, is dependent on them. If the hematocrit is measured directly, instead of being calculated from the RBC and MCy it may be compared by parametric methods. 

Intermediate-Duration Exposure. Adverse effects have been reported following intermediate-duration occupational exposure to phenol (Baj et al. 1994) and intermediate-duration exposure of humans to phenol in the drinking water (Baker et al. 1978 Kim et al. 1994). The effects reported include decreased red blood cell counts (Baj et al. 1994), gastrointestinal effects, dark urine, and direct skin effects (Baker et al. 1978 Kim et al. 1994). 

Lead exposure Not a substitute for effective abatement of lead exposure. Neutropenia Mild to moderate neutropenia has been observed in some patients receiving succimer. While a causal relationship to succimer has not been definitely established, neutropenia has been reported with other drugs in the same chemical class. Obtain a complete blood count with white blood cell differential and direct platelet counts prior to and weekly during treatment. Withhold or discontinue therapy if the absolute neutrophil count (ANC) is below 1200/mcL and follow the patient closely to document recovery of the ANC to above 1500/mcL or to the patient s baseline neutrophil count. There is limited experience with reexposure in patients who have developed neutropenia. Therefore, rechallenge such patients only if the benefit of succimer therapy clearly outweighs the potential risk of another episode of... 

Monitor white and differential blood cell count, hemoglobin determination, and direct platelet count every 2 weeks for the first 6 months of penicillamine therapy and monthly thereafter. 

Coulter Counter To avoid the tedium of direct microscopic counting, a Coulter counter can be employed. By using this technique, not only the cell number, but the cell size can be measured. The disadvantage of this technique is that it cannot distinguish between cells and any impure particles. The technique is also difficult to use with organisms in chains and is useless with mycelial organisms. 

After both sets of experiments were completed, the ground-water was drained from the cells and selective chemical extractions of the granite cell walls were performed. The cells were filled with 0.5 mol/L CaCl solution and stirred continuously for 72 h to displace exchangeable radionuclides. After a rinse with demineralized water to remove residual CaCl- solution, the cells were filled with KTOX solution and stirred ror 24 h to remove radionuclides associated with oxyhydroxides. All solutions were analyzed by gamma spectrometry to determine the amounts of radionuclides extracted. Residual activity was measured by direct gamma counting of the cells. 

The above results demonstrated hematological effects in animals following CDD exposure however, the observed changes in the red and white blood cell counts were nonspecific and were probably due to the broad systemic toxicity of 2,3,7,8-TCDD rather than to a direct effect on the hematological system. 

Because the cells are viewed directly, this method enables a judgement to be made about the quality of the cell suspension, i.e. clumped or broken cells present. By diluting the cell suspension with a solution of a vital dye, e.g. trypan blue at a final concentration of 0.2 g/1 PBS-A (Appendix 1), and counting only the unstained cells a measure of the viable cell count is obtained. (N.B. Trypan blue is toxic and should not be allowed to come in contact with the skin.)... 

Surrogate endpoint A laboratory finding or physical sign that may not, in itself, be a direct measurement of how a patient feels, functions, or survives, but nevertheless is considered likely to predict therapeutic benefit. An example would be CD4 cell counts, used to measure the strength of the immune system in AIDS. 

Laboratory tests showed that hemoglobin was 7.5 g/dL (normal range is 12-15 g/dL), and the reticulocyte count was 262 x 109/L (normal range is 50-100 X 109/L). The tests also indicated neutrophilia, a white cell count of 34 X 109/L (normal range is 4-11 x 109/L), and a platelet count of 328 X 109/L (normal range is 150-400 X 109/L). Renal function was normal. Malarial parasite screen and direct Coombs test were negative. Blood film showed nucleated red blood cells and anisocytosis with bite cells ... 

In this guidance the FDA offers some direction for the demonstration of comparability. They request that the manufacturer provide evidence that the methods, facilities, and controls that were used to manufacture previous products conformed to cGMPs and to other applicable regulatory requirements. In addition they request the submission of validation summaries, as well as product characteristics such as total nucleated cell count, viable CD34 cell count, and number of colony-forming units. Stability data and information from the scientific literature can also be used. Clinical outcomes can be part of the comparability demonstration. 

Figure 4 A photograph of the Selec H robot built by The Technology Partnership, Cambridge, UK. This robot maintains between 1 and 50 cell lines, performs passaging, cell counting and viability measurements, and direct plating into microtitre plates for bioassay...

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