Method for studying development of colonization and infection of dialysis catheters.

Microbial colonization and infection of dialysis catheters is the major cause of catheter failure. The present study aimed to develop a method to permit the study of microbial biofilm formation and antibiotic prophylaxis and therapy. Standard silicon rubber and silver-impregnated catheters were sectioned into 1 mm slices and placed into 1-cm x 2-cm culture slide chambers. Fresh clinical isolates were obtained from infected patients and suspended in concentrations of 10(3), 10(6), and 10(9) colony forming units (CFU) per milliliter in a variety of liquid suspending culture media, which included serum protein constituents. Antibiotics could be added to the suspending fluid to determine prophylactic activity, or at any time thereafter to determine therapeutic activity. The catheter sections were incubated with the microbial challenge for 6 hours, 12 hours, 24 hours, and 48 hours and then washed in flowing distilled water to remove unattached biofilm. They were then stained with acridine orange, which fluoresces microbial DNA, and were examined by confocal scanning laser microscopy. Biofilm formation, representing colonization and infection, were quantified by comparing the fluorescent pixel analysis of the uninoculated control with the challenged catheter. The method was reproducible and permitted quantitative analysis. Standard silicon rubber catheters demonstrated greater biofilm formation than silver-impregnated catheters. The method examines the factors involved in microbial colonization and infection, and in antibiotic prophylaxis and therapy.

Malaria diagnosis by direct observation of centrifuged samples of blood.

We compared the effectiveness of malaria diagnosis by means of direct observation of centrifuged blood with that by conventional examination of Giemsa stained blood-films in a malaria clinic in Ethiopia. A commercially available, modified hematological apparatus (the QBC tube) was used for centrifugation. Red blood cells infected with diverse stages of Plasmodium falciparum and P. vivax are lighter than noninfected cells and somewhat heavier than granulocytes; thus they can readily be detected by direct inspection of UV-illuminated tubes. About 10% of infections diagnosed by direct centrifugal microscopy in a clinical setting were not detected by conventional examination of stained thick films. Diagnosis by direct centrifugation appears to be at least 8 times as sensitive as conventional microscopy when applied to serially diluted samples of malaria-infected blood. Superior sensitivity, together with the one step, solid state nature of the direct centrifugal procedure, provides important advantages for malaria diagnosis.

Application of 30-MHz acoustic scattering to the study of human red blood cells.

A technique for simultaneously measuring the scattering amplitude of individual particles at two angles is applied to human red blood cells. Using a Rayleigh scattering model, the density and compressibility of the cells may be determined given a priori knowledge of their volume. A calibration method relying on measurements of the bulk properties of particle suspensions is described. Red cell properties in hypotonic and hypertonic hosts are compared with a homogeneous mixture model, and a linear relation between hemoglobin content and scattering amplitude at a 90 deg scattering angle is established.

Quantitative buffy coat analysis of blood collected from dogs, cats, and horses.

Using quantitative buffy coat analysis (QBCA), rapid and accurate measurements can be made of the erythrocyte PCV, total WBC count, and platelet count, and the leukocyte population can be differentiated into total granulocytes (including quantitation of eosinophils), and lymphocytes and monocytes. The QBCA is performed by placing a blood sample (50 to 111 microliters) into a high-precision-bore microhematocrit tube that contains a freely moving, closely fitting, cylindrical plastic float. After centrifugation for 5 minutes, the buffy coat components separate by density. The plastic cylinder floats in the buffy coat, thereby expanding the lengths of the buffy coat layers. The layers are measured in a manner that is similar to that used for measuring PCV. Results of QBCA of blood samples from dogs, cats, and horses indicated that the hematologic values obtained correlated with results obtained by use of conventional methods. The accuracy and ease of use of QBCA and the availability of results while the animal is still being examined make QBCA a useful tool for hematologic evaluation of animals.

Quantitative buffy coat analysis. A new laboratory tool functioning as a screening complete blood cell count.

We have developed a system for the quantitative analysis of the buffy coat in centrifuged whole blood samples. This analysis, performed in a modified microhematocrit tube, provides a hematocrit value, total WBC count, platelet count, and a separation of the leukocyte population into granulocytes and nongranulocytes. All results are available within 15 minutes and correlate well with existing methods. The system is expected to provide a rapid means of performing a complete blood cell count in a physician's office.

An improved turbidimetric method for plasma fibrinogen.

Our experience in the use of Ellis' and Stransky's turbidimetric technique for the determination of plasma fibrinogen is presented. We standardized the test using a plasma pool whose fibrinogen content was determined by a weighed clot technique. The turbidimetric-fibrinogen test is accurate, sensitive, specific, reproducible, convenient, and inexpensive. Correlations between the turbidimetric method and the weighed clot reference method was shown. The assay is sensitive to as little as 25 mg/dl plasma fibrinogen, and the results are not affected by samples with a high content of lipids, bilirubin, fibrin split products, lymphangiogram dye, or therapeutic levels of heparin. Day-to-day replicate determinations on a plasma pool yielded a coefficient of variation of 3.4 percent. Results from as many as 10 plasma samples can be obtained in 30 minutes (10-minutes working time). Reagent cost is less than one cent per test.

High-performance colorimeter with an electronic bubble gate for use in miniaturized continuous-flow analyzers.

We describe a high-performance colorimeter with an electronic bubble gate for use with miniaturized continuous-flow analyzers. The colorimeter has a flow-through cuvette with optically flat quartz windows that allows a bubbled stream to pass freely without any breakup or retention of bubbles. The fluid volume in the light path is only 1.8 mul. The electronic bubble gate selectively removes that portion of the photodector signal produced by the air bubbles passing through the flow cell and allows that portion of the signal attributable to the fluid segment to pass to the recorder. The colorimeter is easy to use, rugged, inexpensive, and requires minimal adjustments.

Device for more economical standby operation of continuous-flow analyzers.

To decrease the reagent-consumption rate of continuous-flow analyzers, I constructed a device that allows standby operation. The device, when actuated by switching off the sampler, decreases the effective pumping rate to less than 1/15 normal. The bubble pattern remains virtually undisturbed, and the system can be immediately restarted for emergency use. During five months of use in our laboratory with a Technicon platelet counter, the device was decreased reagent consumption by 60%, while allowing 24-hour-a-day operation. There have been no analytical errors traceable to the device, and no mechanical problems have arisen. I describe the use of the device in a continuous-flow system for determination of creatinine.