Incubation In Salt Solution Research Articles

New C-band protocol by heat denaturation in the presence of formamide.

C-banding techniques detect the presence of constitutive heterochromatin, which is usually located in centromeric regions of chromosomes in the majority of analysed species. The common method for C-banding used over the last 30 years involves treatment with a mild alkali barium hydroxide 5% Ba(OH)2 at 50 degrees C for 5-15 min and subsequent incubation in salt solution (2 x SSC at 60 degrees C for 1 h). We here present a new, easy and reliable technique for C-banding, which basically involves heat denaturation of chromosomal DNA in the presence of formamide and incubation in 2 x SSC at room temperature.

Surface Texture of Poly(styrenesulfonate sodium salt) and Poly(diallyldimethylammonium chloride) Micron-Sized Multilayer Capsules: A Scanning Force and Confocal Microscopy Study

Ultrathin polyelectrolyte capsules fabricated by stepwise assembly of poly(styrenesulfonate sodium salt) and poly(diallyldimethylammonium chloride) are examined by confocal laser scanning microscopy and scanning force microscopy. The capsule surface is composed of grains of 124 ± 10 nm in diameter with a roughness of ∼13 nm. These grains are formed by laterally segregated polyelectrolyte complexes. Osmotically and annealing-induced capsule swelling facilitated further grain segregation together with a capsule surface area increase. Incubation in salt solution induced capsule shrinking and grain aggregation.

Role of sulfolipds in grana thylakoid stacking

In higher plants and algae, the thylakoid membranes are organized into closely appressed or stacked regions (grana), and a network of single interconnecting unstacked regions (stroma lamellae). The components and forces responsible for adhesion between thylakoids have been the subject of intense studies. Although these studies have added significantly to our knowledge of chloroplast membrane structure and function, they have failed to provide a clear rationale for grana membrane stacking. Completedestacking of grana occurs when chloroplasts are suspended in low-salt (10 mM NaCl) solution. Normal levels of stacked and un-stacked regions can be restored by incubation in salt solutions (150 mM NaCl).

Silver-coated nylon fiber as an antibacterial agent.

A blend of nylon fiber and silver-coated nylon fiber (the latter known as X-static) was used in these experiments. This fiber was bactericidal when bacteria were exposed to it directly or to an extract derived from its prior incubation in salt solution. At ambient temperatures, a rapid exponential decrease of survival occurred, usually after a delay of approximately 1 h. The rate of killing (decrease of survival) increased with an increase in X-static percentage of the fiber blend, temperature of fiber extraction, concentration of Tris buffer present during extraction, and temperature at which bacteria were exposed to the extract. When bacteria were exposed to the extract at 37 degrees C as opposed to ambient temperature, there was no delay in onset of killing. Escherichia coli was generally the indicator organism tested, but comparable results were also found for Pseudomonas, Klebsiella, Staphylococcus, and Streptococcus species. The rate of killing increased with increasing silver ion concentration of the fiber extract, as determined through atomic absorption spectrophotometry. The rate of killing was greater and the onset was earlier with an extract containing silver ions from fiber than with a salt solution containing the same concentration of silver ions from silver nitrate. Studies of the kinetics of ion release suggested that X-static may be an effective, sustained-release antibacterial agent.

The effects of reducing conditions, medium, pH, temperature, and time on in vitro excystation of Cryptosporidium.

Whereas excystation of sporozoites from oocysts of most coccidian species requires exposure to reducing conditions followed by pancreatic enzymes and bile salts, sporozoites of a bovine isolate of a bovine isolate of Cryptosporidium excysted without exposure to either reducing conditions or to pancreatic enzymes and bile salts. Without prior exposure to reducing conditions, a high percent excysted after incubation in a mixture of trypsin and bile salts in Ringer's solution; fewer excysted after incubation in tap water, even fewer after incubation in salt solutions, and none after incubation in saliva. Excystation, generally greater at pH 7.6 than at pH 6.0 and at 37 degrees C than at 20 degrees C, was observed as early as 1 h after incubation in water or the trypsin-bile mixture. These findings provide circumstantial evidence that oocysts of Cryptosporidium can excyst in extraintestinal sites and liberate sporozoites that can initiate autoinfection.

In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence.

In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.

Properties of membrane-bound hexokinase in rat brain.

Abstract— —‐(1) The total hexokinase activity present in the mitochondrial fraction can be solubilized completely by incubation with salt and Triton X‐100. This activity cannot be entirely released by washing with sucrose or by freezing and thawing.(2) A part of the particle bound hexokinase exists in a latent form. The latent form is apparent after incubation with high salt concentrations, detergents or by freezing and thawing.(3) Solubilization of membrane bound hexokinase is pH‐dependent. Incubation in salt solutions increases the specific activity ten‐fold. The salt concentration and pH are con‐current. At pH 7.0 part of the hexokinase is solubilized. The lower the pH the less salt is required to release the same amount of activity.(4) Triton X‐100 solubilizes particle‐bound hexokinase, but to a less extent than salts. The activation of hexokinase is greater with Triton X‐100 than with salt.(5) The possible nature of the bonds between hexokinase and mitochondrial membranes is discussed.

The Development of Haemonchus contortus to the Fourth Stage In vitro

Exsheathed infective larvae of Haemonchus contortus developed to the fourth stage in a salt solution under carbon dioxide. The changes in morphology were similar to those seen in worms from sheep, but development was slower. Greatest numbers of fourth-stage larvae were obtained from solutions gassed with 40% carbon dioxide. Small numbers of fourth-stage worms were present after 48 hr, and as many as 90% were in the fourth stage after 72 hr. Once larvae had commenced to develop carbon dioxide could be withdrawn, and development proceeded readily under air. Serum labeled with a fluorochrome was not detected in the intestine of third-stage worms cultivated in vitro, but was present in fourth-stage worms. It is concluded that under these conditions third-stage larvae of H. contortus were unable to ingest the medium. Stoll (1940) and Sommerville (1964) have shown that exsheathed infective larvae, i.e., third-stage larvae, of Haemonchus contortus will develop to the fourth stage after incubation in salt solutions at 38 to 39 C. Stoll's experiments demonstrated that more fourthstage larvae developed when an aqueous extract of liver was added to the salt solution and when the supply of oxygen was restricted. Silverman (British Patent 894603) has shown that development of H. contortus to the fourth stage can take place in Earle's salt solution together with hydrolysates of liver and casein. Sommerville (1964) suggested that either dissolved gaseous carbon dioxide or carbonic acid, or both, provided a stimulus which induced development. He failed to demonstrate any effect from the addition of liver extract. This paper reports the results of further experiments on the development of the thirdstage larva to the fourth stage in vitro. MATERIALS AND METHODS Infective larvae of H. contortus were harvested from cultures of sheep feces 7 to 14 days old, and stored in tap water at 5 C for about 1 week before use. They were prepared for culture by passage through three layers of lens tissue in a sterile Baermann apparatus which contained 0.4% sterile sodium chloride at 38 C. Larvae were subsequently washed twice by suspension in 0.4% sterile sodium chloride and centrifuged at 350 g. They were exsheathed by rinsing for 10 min in 20 ml of 0.4% sterile sodium chloride to which had been added 1 ml of "Milton" (1% sodium hypochlorlite: Received for publication 20 April 1965. * Present address: Department of Zoology, University of Adelaide, Adelaide, South Australia. Milton Pharmaceuticals Ltd., London). They were then washed six times in sterile 0.4% sodium chloride and counted. These procedures were essentially those followed by Weinstein and Jones (1956). In some experiments, exsheathment was achieved by gassing with 100% carbon dioxide. The larvae were suspended for 21/ hr at 40 C in a sterile solution of sodium bicarbonate, the concentration of which was adjusted to give pH 6 when gassed with 100% carbon dioxide (Umbreit, Burris, and Stauffer, 1957). Larvae were incubated in screw-capped roller tubes which contained either 2 or 3 ml of the medium and 1,000 larvae, i.e., either 500 or approximately 330 larvae per ml of medium. The roller drum was kept in an incubator in the dark and rotated 12 times each hour. The temperature was 40 ? 0.5 C unless otherwise indicated. The temperature inside the tubes was checked periodically with thermocouples inserted in the tube and connected to an automatic recorder. The medium contained the following components in grams per liter: NaCl, 8.23; KCI, 0.42; CaC12, 0.33; MgS04-7H20, 0.34. Sodium bicarbonate was added to give the required pH with the particular gas mixture used, usually pH 6. The amount of bicarbonate required was calculated from data supplied by Umbreit, Burris, and Stauffer (1957). All components were sterilized by filtration before use, and the final medium contained 500 mg streptomycin and 500 units penicillin G (sodium salt) per ml. Commercially prepared gas mixtures were used. These contained either 5, 20, 40, 60, or 80% carbon dioxide, 10% oxygen, and the balance nitrogen. In some experiments, 100% carbon dioxide or 50% carbon dioxide in air was used. Each gas mixture was passed through sterile cotton wool, a d cultures were gassed for 2 min in a water bath at the same temperature as for subsequent incubation. Tubes were sealed with a screw top containing a silicone rubber lining. Unless otherwise stated, larvae were incubated under the gas mixtures for 72 hr. At the termination of each experiment, sterility tests were made