Development of a lysis-filtration blood culture technique

Abstract

A lysed-blood culture system that quickly lyses patients' blood near neutrality and is relatively noninjurious to more delicate pathogens such as Haemophilus influenzae and Bacteroides fragilis is reported. The lysing solution includes culture medium, 0.004 M sodium carbonate and bicarbonate, 0.04% Triton X-100,and 0.6% Rhozyme (a mixture of proteases). Most of the pathogens tested multiplied in the lysing solution. The lysed blood normally is immediately filtered. The membrane is transferred to culture broth. The greatest advantage realized from this blood culture technique is separation of pathogens from antibiotics, bactericidal antibodies, complement, opsonins, and phagocytic systems. Another advantage is the concentration of organisms into a small volume of clear medium for faster growth and visualization of growth. It was observed that both gram-negative and -positive organisms were attracted during filtration to the filter material and were not removed from it by backwashing with buffer. Thus, filter membranes with porosities much larger than would nominally be expected to retain bacteria retained all or part of light and heavy Escherichia coli and Staphylococcus aureus suspensions. Advantage may be taken of this phenomenon to use filters with larger pore sizes and avoid filter clogging by poorly lysed specimens. Porr lysis may result from addition of too much blood to the lysing solution, blood with elevated numbers of erythrocytes or leukocytes, or blood from some people whose blood is naturally more resistant to lysis.

Technical note: A comparison between rehydrating solutions in the pretreatment of mummified and corified skin for forensic microscopic examination

“…It is therefore conceivable that different rehydrating substances may have a different impact on preserving the hemorrhagic infiltrate in the skin. In particular, as far as Sandison’s solution, the presence of Na 2 CO 3 can cause the osmotic lysis of red blood cells [ 31 , 32 ]; if confirmed, this could undoubtedly be a limitation for the application of Sandison’s solution, since it could deprive the forensic pathologist of crucial information, especially about the vitality of mummified or corified skin lesions or associated with underlying bone fractures. Therefore, further studies on the interaction between rehydrating substances and hemorrhagic infiltrate are necessary.…”

Bacterial Extraction and Concentration from Blood through Filtration Processes

“…The need to decrease the BSI diagnostic time has promoted development of novel technologies and devices to directly collect bacteria from blood [10]. Our proposed process described herein improves upon direct blood-lysis filtration technologies [11][12][13][14]. The basis of this method is the lysis of formed blood elements (red blood cells (RBC), white blood cells (WBC), and platelets) through the use of detergents, salts, and/or proteases followed by the filtration of the solution for isolation and concentration of bacteria.…”

“…However, if only 10 of 20 mL blood-lysis blend were passed before clogging the filter, then the ratio would be 2.5 mL/cm 2 of whole blood per filter area. Initial publications regarding this technique described whole blood filtration after dilution with and lysis by a solution of Triton X-100 and Na 2 CO 3 [13,14]. The best results were 0.288 mL/cm 2 of blood per filter area.…”

“…The best results were 0.288 mL/cm 2 of blood per filter area. When proteases were incorporated into the solution to further improve the process, the ratio improved to 0.865 mL/cm 2 [14,15]. However, this lysis solution was toxic to some bacteria if not filtered within minutes.…”

“…There are other studies that investigated altering the filter membrane surface to ensure bacterial collection, [18] but more importantly for rapid identification is subsequent removal of bacteria from the filter. Zierdt examined bacterial removal from filters and found only 12% of the bacteria were removed by backflushing using 5 mL of phosphate buffer [14]. Detergent was needed to remove bacteria and only 45% of bacteria could be removed with a single backflush [19].…”