Effect of anticoagulants on the activity of trypsin-like proteinases in human blood plasma

Influence of some anticoagulants (heparin, sodium citrate, their mixture) on blood trypsin-like proteinases activity was examined. The activity was determined using synthetic substrate N-alpha-benzoyl-L-arginine-p-nitroanilide. It was shown that heparin greatly activated blood trypsin-like proteinases (at heparin concentration 5 unit/ml of blood, the enzyme activity in plasma was about 10 times higher than the activity in serum). Heparin added to serum caused the activation effect too, maximum of activation was reached at heparin concentration in serum 800 unit/ml, following increase of heparin concentration did not led to the activity change. Sodium citrate had no significant effect both on the trypsin-like proteinases activity and on the activation effect of heparin. It was found that investigated anticoagulants did not affect blood antitryptic activity.     

Effect of temperature on mineralization by heterotrophic bacteria.

When pure cultures of the bacteria Pseudomonas fluorescens (a psychrotroph), Escherichia coli (a mesophile), and SRL 261 (a thermophile) were shifted away from temperatures to which they were adapted, the percentage of substrate mineralized increased (percent mineralized = [substrate respired to CO2]/substrate respired to CO2 + substrate incorporated into biomass] X 100). The increase in the percent mineralized was larger for larger temperature shifts. Similar responses were observed when natural heterotrophic bacterial populations from sediments of Lake George, N.Y., and a thermophilic algal-bacterial mat community at the Savannah River Plant, Aiken, S.C., were subjected to temperature shifts. These results suggest that an increase in the percent mineralized may be an indication of thermal stress in bacterial populations.     

Effect of growth temperature on cellular fatty acids in sulphate-reducing bacteria

The effect of growth temperature on the cellular fatty acid composition of sulphate-reducing bacteria (SRB) was studied in 12 species belonging to eight genera including psychrophiles and mesophiles. Most of these species were of marine origin. The investigated SRB with the exception of four Desulfobacter species exhibited only a minor increase in the proportion of cis-unsaturated fatty acids (by < or = 5% per 10 degrees C) when the growth temperature was decreased; psychrophiles maintained their typically high content of cis-unsaturated fatty acids (around 75% of total fatty acids) nearly constant. The four Desulfobacter species, however, increased the proportion of cis-unsaturated among total fatty acids significantly (by > or =14% per 10 degrees C; measured in late growth phase) with decreasing growth temperature. The ratio between unsaturated and saturated fatty acids in Desulfobacter species changed not only with the growth temperature, but also with the growth state in batch cultures at constant temperature. Changes of cellular fatty acids were studied in detail with D. hydrogenophilus, the most psychrotolerant (growth range 0-35 degrees C) among the mesophilic SRB examined. Desulfobacter hydrogenophilus also formed cis-9,10-methylenehexadecanoic acid (a cyclopropane fatty acid) and 10-methylhexadecanoic acid. At low growth temperature (12 degrees C), the relative amount of these fatty acids was at least threefold lower; this questions the usefulness of 10-methylhexadecanoic acid as a reliable biomarker of Desulfobacter in cold sediments.     

Influence of temperature on blood coagulation in vitro (author's transl)]

The influence of different temperatures between 13 degrees C and 45 degrees C on coagulation factors in vitro was studied by measuring clotting time with the recalcification time, partial thromboplastin time (PTT), and thromboplastin time test. In all three tests the shortest clotting times were measured at a temperature of 40 degrees C. The relation between temperature and clotting time was similar in fresh plasma and in plasma which had been stored at a temperature of --20 degrees C before examination. However, in all tests stored plasma showed shorter coagulation times. Prolongation of coagulation time at 45 degrees C is caused by irreversible reduction of coagulation activity in the plasma. At the same time thromboplastin- and PTT-reagent are imparied in their coagulation acitvity by a temperature of 45 decrees C. In comparison to plasma obtained from healthy persons plasma from patients with hemophilia A or B or with v. Willebrand's disease reacted more sensitive to changes in temperature in the PTT test. The coagulation defect was definitely more pronounced at 27 degrees and 17 degrees C than at 37 degrees C. It was not possible to differentiate these three coagulopathies with the PTT test at 27 degrees and 17 degrees C.     

Effect of temperature on growth parameters of psychrophilic bacteria and yeasts

Three bacterial (Pedobacter heparinus, Pedobacter piscium, Pedobacter cryoconitis) and three yeast strains (Saccharomyces cerevisiae, Leucosporidiella creatinivora, Rhodotorula glacialis) of different thermal classes (mesophiles and psychrophiles) were tested for the effect of temperature on a range of growth parameters, including optical density, viable cell numbers, and cell dry mass, in order to determine the temperature conditions under which maximum biomass formation is obtained. Maximum values of growth parameters obtained at the stationary growth phase of the strains were used for statistical calculation. Temperature had a significant (P ≤ 0.05) effect on all growth parameters for each strain; correlations between the growth parameters were significant (P ≤ 0.05–0.01). The maximum growth temperature or the temperature at which microbial growth was fastest was in no case the temperature at which the investigated strains produced the highest amount of biomass. All tested psychrophilic bacteria and yeast strains produced highest amounts of cells (as calculated per mg cell dry mass or per OD₆₀₀ unit) at 1°C, while cell numbers of mesophiles were highest at 20°C. Thus, cultivation temperatures close to the maximum growth temperature are not appropriate for studying psychrophiles.     

Effect of dilution on sedimentational separation of bacteria from blood

Bacteria must be separated from septic whole blood in preparation for rapid antibiotic susceptibility tests. This work improves upon past work isolating bacteria from whole blood by exploring an important experimental factor: Whole blood dilution. Herein, we use the continuity equation to model red blood cell sedimentation and show that overall spinning time decreases as the blood is diluted. We found that the bacteria can also be captured more efficiently from diluted blood, up to approximately 68 ± 8% recovery (95% confidence interval). However, diluting blood both requires and creates extra fluid that end users must handle; an optimal dilution, which maximizes bacteria recovery and minimizes waste, was found to scale with the square root of the whole blood hematocrit. This work also explores a hypothesis that plasma backflow, which occurs as red cells move radially outward, causes bacterial enrichment in the supernatant plasma with an impact proportional to the plasma backflow velocity. Bacteria experiments carried out with diluted blood demonstrate such bacterial enrichment, but not in the hypothesized manner as enrichment occurred only in undiluted blood samples at physiological hematocrit.     

Effect of anticoagulants in vitro on the viability of lymphocytes and content of free fatty acids in plasma

Summary These authors attempted to test the effect of anticoagulants on lymphocytes viability by reproducing the procedure used for lymphocyte isolation for various immunologic tests in which blood specimens are allowed to stay at room temperature for 2 h before lymphocytes are isolated. Blood was obtained with three different anticoagulants i.e. heparin, citrate, and CPDA (citrate, phosphate, dextrose, and adenine). Plasma was lyophilized and extracted with ethanol. Dried ethanol extracts were suspended in medium (RPMI 1640+10% fetal bovine serum) and incubated with a lymphocyte cell line (MOLT-4). After 24 h of incubation the viability of cells was examined. The following death rates of the cells were observed: heparin −63±4.6% (mean±SEM), citrate −27±6.7%, and CPDA 6.2±0.6% (P<0.0005). A significant correlation was found between these results and changes in the concentrations of free fatty acids in the extracts. These results emphasize the importance of choosing the right anticoagulant when the viability of lymphocytes is obligatory.