Plasma preparation from whole blood using ultrasound.

A technique to efficiently separate plasma from human whole blood is described. Essentially, 3-mL samples are held on the axis of a tubular transducer and exposed for 5.7 min to an ultrasonic standing wave. The cells concentrate into clumps at radial separations of half wavelength. The clumps grow in size and sediment under gravity. A distinct plasma/cell interface forms as the cells sediment. The volume of clarified plasma increases with time. The separation efficiencies of transducers of 29-mm and 23-mm internal diameters driven, by test equipment, at radial resonances of 3.4 and 1.5 MHz, respectively, were compared. The average efficiency of separation was 99.6% at 1.5 MHz and 99.4% with the 3.4-MHz system. The cleared plasma constituted 30% of the sample volume at 1.5 MHz and 25% at 3. 4 MHz. There was no measurable release of haemoglobin or potassium into the suspending phase, indicating that there was no mechanical damage to cells at either frequency. A total of 114 samples from volunteers and patients were subsequently clarified in a 1.5-MHz system driven by an integrated generator. The average efficiency of clarification of blood was 99.76% for the latter samples. The clarification achieved is a significant improvement on that previously reported (98.5%) for whole blood exposed to a planar ultrasonic standing wave field (Peterson et al. 1986). We have, therefore, now achieved a six-fold reduction of cells in plasma compared to previous results.

Analytical scale ultrasonic standing wave manipulation of cells and microparticles.

The ultrasonic standing-wave manipulation of suspended eukaryotic cells, bacteria and submicron latex or silica particles has been examined here. The different systems, involving plane or tubular ultrasonic transducers and a range of acoustic pathlengths, have been designed to treat suspension volumes of analytical scale i.e. 5 ml to 50 microliters for both sample batch and 'on-line' situations. Frequencies range from 1 to 12 MHz. The influence of secondary cell-cell interaction forces in determining the cell concentration dependence of harvesting efficiency in batch sedimentation systems is considered. Applications of standing wave radiation forces to (1) clarify cell suspensions, (2) enhance particle agglutination immunoassay detection of cells or cellular products and (3) examine and enhance cell-cell interactions in suspension are described.

Clarification of plasma from whole human blood using ultrasound.

There has been interest for a number of years in the possibility of separating blood into cells and plasma by methods other than centrifugation, so that the plasma can be analysed on-line. Cells in whole blood normally occupy about 45% of the suspension volume. It has been shown with a number of different cell types, such as yeast and bacteria, that for concentrations of this order the cells are not as efficiently harvested by ultrasound as those for lower concentrations. In this study, removal of cells from 3-4 ml whole blood volumes has been examined in ultrasonic standing wave fields from tubular transducers driven at a frequency of 1.6 MHz. Samples of whole human blood (n = 11) from two volunteers have been processed by three tubular transducers where high levels of cell removal, 99.7% on average, have been demonstrated with high reproducibility between samples as well as for different transducers.

Investigation of enhancement of two processes, sedimentation and conjugation, when bacteria are concentrated in ultrasonic standing waves.

Cells aggregate and can be recovered from suspension when exposed to an ultrasonic standing wave field. The acoustic force on individual cells in a standing wave decreases with particle volume. A plane ultrasonic field generated by a transducer driven at 3.3 MHz was used here to investigate the removal of Escherischia coli, cells with dimensions of the order of 1.0 microm, from batch suspension by sedimentation over a range of concentrations (10(3) to 10(10) cells ml(-1)). Cell removal efficiencies greater than 90% were achieved at initial concentrations of 10(10) cells ml(-1). Removal efficiencies decreased gradually to zero, as initial bacterial concentration was reduced to 10(7) cells ml(-1). It was found that, when low concentrations of E. coli (10(3) to 10(5) cells ml(-1)) were added to suspensions of larger particles (i.e. yeast cells) that were of sufficient concentration to form aggregates in the sound field, E. coli could be harvested to an efficiency of 40%. The results imply that the E. coli became trapped and sediment with aggregates of larger particles. Some strains of bacteria are capable of DNA transfer by conjugation. The transfer rate of E. coli RP4 plasmid is order of magnitudes greater when conjugation occurs on solid medium rather than in liquid suspension. We have investigated whether the conjugation rate would also be higher in ultrasonically induced E. coli clumps than in free suspension. The donor strain was mixed with a recipient strain of E. coli, then sonicated in a capillary at 4.6 MHz in a tubular transducer for 5 min. The bacteria aggregated successfully. Results showed a three-fold increase in the rate of conjugation compared to a liquid mating control.

Effect of teat washing on the bacteriological contamination of milk from cows kept under various housing conditions.

The effect of different methods of teat washing on bacterial contamination and sediment levels of the milk of cows kept under 3 housing conditions was studied. By use of an in line milk sampler and steam sterilized clusters the bacterial contamination from the teats after udder preparation was determined directly. The results show that, even under the same housing conditions and teat washing treatments, considerable variation in bacterial counts and sediment level occurred. Overall, washing with hypochlorite (600 mg/l available Cl) and drying with a paper towel gave total counts that were significantly lower than were obtained with the other 4 treatments. The aerobic spore count, in which thermoduric flora predominated, was greatly reduced by drying the teats, but the inclusion of hypochlorite in the wash water had little effect. Coliform contamination was very low (approximately 1/ml) even where the cows' teats were heavily soiled and were not washed. The sediment levels in the milk again showed wide variation and occasional high values (3 mg/l) were recorded for washed cows. In marked contrast to its effect on the bacteriological results, drying had little effect on reducing the sediment.

Effect of psychrotrophic post-pasteurization contamination of the keeping quality at 11 and 5 degrees C of HTST-pasteurized milk in the UK.

The keeping quality of commercial HTST-pasteurized milk and laboratory pasteurized milk from a common bulk raw supply has been investigated for 5 dairies. Spoilage occurred at levels of total bacterial counts around 10(7) colony forming units/ml, but with a slightly higher off-flavour threshold for the commercial milks than the laboratory pasteurized milks. The predominant microflora at spoilage and the type of off-flavour produced differed between the 2 types of milk. Raising the storage temperature from 5 to 11 degrees C caused a slight shift in the spoilage microflora and led to an average reduction in the shelf life of the laboratory pasteurized milk from 28 to 6 d and of the commercial pasteurized milk from 13 to 5 d. Changes in the level of post-pasteurization contamination (PPC) were reflected in changes in keeping quality, particularly at 5 degrees C. However, the greatest improvements were found in the absence of PPC.

Rapid membrane filtration-epifluorescent microscopy technique for direct enumeration of bacteria in raw milk.

Membrane filtration and epifluorescent microscopy were used for the direct enumeration of bacteria in raw milk. Somatic cells were lysed by treatment with trypsin and Triton X-100 so that 2 ml of milk containing up to 5 x 10 somatic cells/ml could be filtered. The majority of the bacteria (ca. 80%) remained intact and were concentrated on the membrane. After being stained with acridine organe, the bacteria fluoresced under ultraviolet light and could easily be counted. The clump count of orange fluorescing cells on the membrane correlated well (r = 0.91) with the corresponding plate count for farm, tanker, and silo milks. Differences between counts obtained by different operators and between the membrane clump count and plate count were not significant. The technique is rapid, taking less than 25 min, inexpensive, costing less than 50 cents per sample, and is suitable for milks containing 5 x 10 to 5 x 10 bacteria per ml.