Lysis In Distilled Water Research Articles

An efficient and reliable DNA extraction method for preimplantation genetic diagnosis: a comparison of allele drop out and amplification rates using different single cell lysis methods

To evaluate methods of DNA extraction from single cells for their suitability to amplify and provide a correct diagnosis of target disease genes. Experimental study. University hospital laboratory. Two normal adult male and female blood donors. Exon 51 of the dystrophin gene and the ZFX/ZFY gene were amplified from single lymphocytes using nested PCR. Five different methods of DNA extraction were tested (lysis in distilled water with freezing and thawing using liquid nitrogen, lysis in distilled water, alkaline lysis buffer, Proteinase K/sodium dodecyl sulfate (SDS) buffer, and N-lauroylsarcosine salt solution). Allele drop out and amplification rate. The amplification efficiency from single unaffected lymphocytes was 89.0% using the liquid nitrogen method, 88.1% with the distilled water lysis method, 97.5% with the alkaline lysis buffer method, 91.5% with the Proteinase K/SDS lysis buffer method, and 84.8% using the N-lauroylsarcosine salt solution method. The mean allele drop out rate was 16.7%, 43.9%, 2.0%, 9.8%, and 18.9%, respectively, for each lysis method using single male lymphocytes as a template. Based on these results, DNA extraction using an alkaline lysis buffer results in more efficient rates of DNA amplification and less allele drop out than the other methods of DNA extraction tested. This method is suitable for the lysis of single cells in clinical preimplantation genetic diagnosis.

The “Spanning Protocol”: A new DNA extraction method for efficient single-cell genetic diagnosis

We evaluated methods of preparation of DNA from single cells for amplification and preimplantation genetic diagnosis (PGD), including our "spanning protocol." Dystrophin gene exons 45 and 51 were amplified by nested polymerase chain reaction (PCR) from a single lymphocyte or blastomere. Amplification efficiencies were compared between DNA extraction by (A) lysis in distilled water with freeze-thawing and boiling; (B) two-step lysis involving potassium hydroxide and dithiothreitol; and (C) the spanning protocol, using N-lauroylsarcosine. With method A, amplification efficiency was 66/120 (55%) and false-positive such as amplification failure or allele drop out was 42/120 (35%); with B, 96/120 (80%) and 21/120 (17.5%); and with C, 111/120 (92%) and 5/120 (4.2%), using single blastomeres and unaffected lymphocytes from male. Occurrence of false-negative such as contamination of another DNA with method A was 4/120 (3.3%); with B, 10/120 (8.3%); and with C, 2/120 (1.7%) from using single lymphocytes from affected males. The spanning protocol was most efficient for extracting DNA from a single cell and should be particularly useful for preimplantation genetic diagnosis.

Common methodology is inadequate for studies on the microbicidal activity of neutrophils

Microbicidal activity of neutrophils is usually measured by colony-counting techniques after cell lysis in distilled water. While studying the effect of the reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitor diphenyleneiodonium (DPI) on the staphylocidal activity of neutrophils, we obtained inconsistent results: various degrees of inhibition in some experiments and no effect in others. The lysis step, i.e., dilution of neutrophils in distilled water, was the source of error. Cell-associated microorganisms were not dispersed effectively by this treatment. We overcame this problem by using water at pH 11 for cell lysis. Under these conditions, killing was inhibited completely and reproducibly by DPI. Here, we show that cell lysis in distilled water is incomplete and leads to an overestimate of microbial killing. This hinders identification of partial defects and makes complete defects appear as partial. We found that DPI-treated neutrophils and chronic granulomatous disease neutrophils were completely defective in killing of Staphylococcus aureus and Candida albicans and partially defective in killing of Escherichia coli after lysis with water pH 11, whereas after lysis in distilled water, killing of S. aureus and C. albicans was approximately 60% and approximately 70% of control killing, respectively, and killing of E. coli was normal. Likewise, killing of S. aureus by myeloperoxidase-deficient neutrophils was severely impaired after lysis in water pH 11 but appeared normal after lysis in distilled water. As most studies about neutrophil microbicidal activity have been performed using distilled water, our findings indicate that previous data about killing defects and the effects of agents that modulate microbicidal activity of neutrophils should be re-evaluated.

A simple method for measuring the F-actin content of human polymorphonuclear leukocytes in whole blood.

We have developed an improved method for measuring the filamentous (F) actin content of human blood polymorphonuclear leukocytes (PMNs). The essential feature of the method is the immediate fixation of the F-actin cytoskeleton. Fresh whole blood (100 microliters) is shock-cooled by the addition of 1.0 ml of a mixture of 18.75% glycerol and 5% formaldehyde in phosphate buffer pre-cooled to -8 degrees C and subsequently fixed at 4 degrees C for 15 min. After lysis in distilled water and removal of the red blood cells by centrifugation, the F-actin cytoskeleton of the PMNs is stained with fluorescein isothiocyanate (FITC)-phalloidin and quantified by means of flow cytometry. In healthy test subjects, PMN stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) for 20 s resulted in a significantly increased F-actin assembly, while in patients with multiple organ failure, two subpopulations arose: one with greater F-actin content and a second with lower F-actin content in comparison with the unstimulated blood sample. This simple and fast method may be a useful tool in basic and clinical research.

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.

Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt).

The localization of two carohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the "spherulous cells". After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water. Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the meshwork of the spongin fibres of the central axis. The are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.

Production of spore spheroplasts of Clostridium botulinum and DNA extraction for density gradient centrifugation.

A method is described whereby Clostridium botulinum 33A and 51B spore spheroplasts are produced in two sequential steps: (1) by reducing spore coats for 3 h with 50% mercaptoacetic acid – 8 M urea – 0.01 M EDTA, and (2) by lysis of the cortex for 2 h with 500 μg/ml lysozyme in 0.01 M Tris-HCl (pH 8.0) – 0.01 M EDTA – 1 mg/ml dithiothreitol and 25% lactose added as osmotic stabilizing agent.Optical density measurements (absorption spectra) of ultraviolet (UV)-absorbing material released after submitting spheroplasts to osmotic lysis in distilled water showed absorption maxima around 270 nm (dipicolinic acid) and 260 nm (nucleic acids). Radioactivity profiles of 3H-labeled TCA-insoluble material from centrifugation of alkaline sucrose gradients showed that the above schedule for obtaining spore spheroplasts resulted in optimal DNA extraction and did not cause extensive breakage of DNA.Phase-contrast microscopy indicated that spore appearance is not visibly affected by chemical coat reduction; however, spores become phase dark after treatment with lysozyme. Electron microscopy showed that after chemical reduction coat layers are less compact, of less electron density, and dissolved in some areas. Furthermore, lysozyme treatment disintegrates and to a large degree removes the cortex and structural details become visible in the core.

Inhibition of cell division in Micrococcus lysodeikticus dis-II.

A strain of Micrococcus lysodeikticus (designated dis-II) has been isolated by nutritional selection using aspartic acid as the major source of carbon, nitrogen, and energy. This organism is inhibited from dividing when grown in a defined medium in the presence of D-serine, penicillin, mitomycin c or under conditions of suboptimal magnesium concentration. All of the division-inhibiting compounds decrease growth yields and induce varying amounts of lysis. L-Lysine, when added to D-serine-containing media, allows some increase in growth yields, but division inhibition appears more pronounced. Pantoyl lactone, D- and L-alanine, and to a lesser extent Carbowax 400 (hypertonic conditions) and glycine, but not spermine, will significantly prevent but not reverse division inhibition in D-serine-lysine-containing media. Thin-slicing and electron microscopic observation of non-dividing cells shows that a relatively thick layer of porous-appearing cell wall mucopeptide is synthesized when cells are grown in the presence of D-serine. These cells as well as those grown in the presence of penicillin or mitomycin c are not lysed in distilled water. Amount of cell wall mucopeptide is greatly decreased in cells grown under conditions of suboptimal magnesium concentration; these cells undergo extensive lysis in distilled water.

Antagonistic effect of monovalent cations in maintenance of cellular integrity of a marine bacterium.

The susceptibility of a marine bacterium, designated isolate c-A1, to lysis in distilled water and in salt solutions has been found to be a function of Na(+) concentration. Optical densities of cells pre-exposed to 0.05 m MgCl(2) were maintained in 1.0 m KCl, whereas those of cells pre-exposed to 1.0 m NaCl were not maintained at any KCl concentration tested. Cells transferred from MgCl(2) to low concentrations of NaCl underwent more extensive lysis than did those transferred to distilled water. The degree of disruption of cells transferred to distilled water from mixtures of 0.05 m MgCl(2) and NaCl (0 to 1.0 m) was dependent on the concentration of NaCl; similar results were obtained with LiCl, but not with KCl. In electron micrographs of thin sections, c-A1 cell envelopes consisted of two double-track layers which fractured and peeled apart on lysis after pre-exposure to NaCl-MgCl(2) mixtures. Envelope eruptions or "hernias" occurred only in lysed cells pre-exposed to NaCl alone. No evidence for a functional lytic enzyme was found. Comparative studies on a terrestrial pseudomonad with a multilayered envelope indicated that preexposure to NaCl did not enhance the susceptibility of this cell to lysis in distilled water. The lytic susceptibility of the marine bacterium is considered to be the consequence of competition between specific monovalent cations and Mg(++) for electrostatic interactions with components of the cell envelope of this organism.

Comparative studies of immunogenic properties of Trypanosoma brucei inactivated with beta-propiolactone and with some other inactivating agents.

Success in immunizing animals with dead trypanosomes depends on both the method of inactivating trypanosomes and the strain used for immunization. Comparative studies with various inactivating agents showed that β-propio-lactone and formalin are superior to phenol, heat at 56 °C, and lysis in distilled water. A vaccine containing Trypanosoma brucei with frequent passages through mice and not exposed to antibodies could elicit in animals protective antibodies and resistance to a challenge with a homologous strain. Passive immunity was shown in young mice born of vaccinated mothers. On the other hand, no active or passive immunity was demonstrated when animals were immunized with a vaccine prepared from a variant of T. brucei after passage through rabbits or sheep.

THE LYSIS OF A PSYCHROPHILIC MARINE BACTERIUM AS STUDIED BY MICROELECTROPHORESIS

Non-growing cells of a psychrophilic marine bacterium lyse if subjected to temperatures above 21 °C, to low ionic strengths, or to pH values below 6.0. Microelectrophoretic techniques were used to help determine the surface effects of cellular breakdown. During lysis, however induced, the cell populations became heterogeneous in mobility. This was apparently due to the adsorption of intracellular material, with a higher negative charge than intact cells, onto the cell surface. The mobilities of cells lysed in water or at acid pH reverted to the control values after repeated washings, which were thought to remove adsorbed intracellular material. Lysis induced by these latter treatments, therefore, did not involve the external cell surface. Water-lysed cells were stable at temperatures where both whole cells and mechanically prepared cell envelopes were subject to breakdown. After temperature-induced lysis, the washed cells possessed a higher negative mobility than intact cells, which was not due to the presence of adsorbed intracellular material, but rather to an irreversible surface change. Lysis in distilled water at 25 °C left the cell surface unchanged, and the "temperature effect" of a permanently increased negative charge was only evident when lysis at 25 °C preceded exposure to distilled water.

Effect of spermine on lysis and reproduction by bacteriophages phi-X174, lambda, and f2.

Groman, Neal B. (University of Washington, Seattle), and Grace Suzuki. Effect of spermine on lysis and reproduction by bacteriophages phiX174, lambda, and f(2). J. Bacteriol. 92:1735-1740. 1966.-A test was made of the hypothesis that lysis by all bacteriophages shares as a common and critical step an alteration in the osmotic stability of the infected cell. This was done by examining the effect of spermine on lysis. Spermine is one of a number of compounds which can stabilize spheroplasts and protoplasts to lysis in distilled water. Spermine stabilized both phiX174- and f(2)-infected cells at concentrations ranging from 2 x 10(-3) to 4 x 10(-2)m, but failed to stabilize lambda-infected cells at concentrations up to 8 x 10(-2)m. Stabilization was reflected both in optical density measurements and in the retention of mature phage in structures sedimentable at low speeds. At optimal concentration, over 90% of the phage was retained in these structures. These data suggest that the mechanism of lysis by phiX174 and f(2) differs sharply from that caused by lambda, and other observations suggest that there are differences in the lytic process of phiX174 and f(2) as well. Spermine also displayed a differential effect on phage reproduction. The reproduction of lambda and f(2) was inhibited by spermine, though the data do indicate that maturation occurs in its presence. The reproduction of phiX174 was enhanced by spermine.

The use oe dilute alkali in haemoglobinometry

Although it has long been known that lysis of blood in dilute alkali gives clearer solutions than lysis in distilled water, many alternative explanations have been offered. A common view is that alkali prevents the precipitation of plasma globulins. The present investigation shows that this explanation is at best only partly correct. When human blood is lysed in water, up to 10% of the haemoglobin may remain inside the ghosts. Few, if any, ghosts are actually destroyed and the scattering of light by the suspended ghosts causes considerable turbidity. In dilute alkali, however, the ghosts are almost completely destroyed and all the haemoglobin is liberated. When plasma is diluted with water, the turbidity is usually negligible under the conditions used for oxyhaemoglobin determinations and no significant reduction of turbidity occurs in alkali. Thus precipitation of substances from the plasma is not important in this case and the main function of alkali is to ensure complete destruction of ghosts and liberation of haemoglobin. When coal-gas containing acid impurities is bubbled through blood lysed in water, considerable additional turbidity develops. Pure carbon monoxide is without effect. The additional turbidity is due to the precipitation of substances from the plasma, and this can be prevented by sufficient alkali. Hence, both incomplete lysis and plasma flocculation are important causes of turbidity when laboratory coal-gas is used for carboxyhaemoglobin determination. Both factors can be eliminated by using alkali. The nature of the precipitable material is unknown. Coal-gas is without action on solutions of various plasma globulins, albumins and re-constituted human dried plasma. There is some evidence that lipids are involved and it appears possible that the chylomicra onto which plasma globulin may be adsorbed, arc responsible.