A simple fluorescence of staining technique for in situ soil microorganisms.

A simple, rapid straining technique using the magnesium salt of 1-anilino-8-naphthalene sulfonic acid is described. Treatment of soil with an aqueous, membrane-filtered solution (3.5 mg/ml) of the salt causes the soil microorganisms to fluoresce when examined with light from a mercury arc light source.     

A silver impregnation technique to demonstrate muscle-bone-cartilage relationships in fishes

A modification of the Winkelmann and Schmitt (1957) technique originally designed to investigate patterns of peripheral nervous innervation is given for demonstration of developing bone growth centers and associated muscle origins and insertions in larval and small fishes. This technique offers a simpler and faster way of obtaining such ontogenetic information than the standard method of double staining with alizarin red S and Alcian blue followed by clearing with potassium hydroxide. Muscles also stain, facilitating the location of their origins and insertions.     

The use of a simple haematoxylin and eosin staining procedure to demonstrate micronuclei within rodent bone marrow

In the pharmaceutical industry, the majority of drug-safety evaluation studies are carried out preferentially in the rat. Consequently, drug absorption, distribution, metabolism and excretion profiles are available for this species. Such data usually have to be generated independently in the mouse, to allow validation of any micronucleus tests carried out in this species. Unfortunately, at the present time, the rat is not ideal for use in the micronucleus test due to the presence of large numbers of contaminating mast cell granules. Such granules are stained blue by the most commonly accepted staining procedure (May-Grunwald-Giemsa), and can be erroneously scored as micronuclei when they overlay erythrocytes. A simple haematoxylin and eosin staining procedure was evaluated in the micronucleus test using rats and mice. With this procedure, micronuclei stained blue-black and were readily distinguishable from cell inclusions resembling micronuclei such as mast cell granules, which remained unstained. Essentially similar quantitative data for micronucleus incidence and erythrocyte distribution were obtained in mice using this staining technique when compared to the use of the more established May-Grunwald-Giemsa staining procedure. However, unlike the use of the May-Grunwald-Giemsa procedure, the use of the haematoxylin and eosin stains allowed the accurate estimation of micronucleus incidence within the marrows of treated rats in the presence of contaminating mast-cell granules. Furthermore, unlike alternative procedures using fluorescent stains, the haematoxylin and eosin stained preparations are stable, constitute a permanent record of the experiment, and can be analysed at the convenience of the investigator. Therefore, this staining procedure may offer a useful alternative, for example, when evaluating rat bone-marrow smears within which considerable mast cell contamination can occur.     

A simple molecular technique for identifying marine host fish by sequencing blood-feeding parasites

Gnathiid isopods are common ectoparasites of fish on the Great Barrier Reef, Australia. While screening for appropriate markers for phylogenetic studies of gnathiids, we found that primers for 12S and 16S rDNA preferentially amplified the host fish DNA instead of gnathiid DNA. This amplification occurred even when using gnathiids that were not engorged with host blood and adult gnathiids that do not feed on fish blood. This method could be used in host-parasite studies to identify hosts without having to sample parasites directly from the host (which can be costly and requires considerable skill in a marine environment). Target ribosomal DNA sequences can be amplified from total DNA extracted from parasites that are captured in funnel traps or plankton tows. Sequence data from these can be used to identify the hosts that gnathiids were feeding on before capture.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

A simple fluorescence staining technique for the differentiation of human tissue transplanted into nude mice.

Human and mouse nuclei can be distinguished by differences in the constitutive heterochromatin when stained with quinacrine dihydrochloride. With the staining method described, mouse heterochromatin during interphase appears as brilliant fluorescent chromocenters. By replacing the commonly used aqueous buffer mounting medium with a xylene-diluted synthetic resin, the haziness of the nuclear fluorescence is eliminated thus allowing identification of the heterochromatin pattern in histological preparations. A requirement for the definite identification of cells of human or murine origin in the nude mouse is the knowledge that the heterochromatin arrangements changes according to the stage of differentiation of the cell of the position of a particular nucleus within the cell cycle.     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Modification of the original technique of Champy: a simple procedure for staining melanocytes

A simple osmium-potassium-iodide post-fixation procedure for brightly and specifically visualizing melanocytes of human epidermis on semithin sections is described.