Acetocarmine Staining Research Articles

Karyological leaf squashes in grasses, aided by pectinase digestion at 45C.

Longitudinal sections of rapidly growing leaf shoots were soaked for 2–4 hr in 0.002 M 8-hydroxyquinoline at 25 C, blotted, and fixed in 3:4:1 ethanol, chloroform, acetic acid. A 30 min maceration at 45 C in a pectinase solution (Pectinal 59–L; Rohm and Haas) softened the material for staining and squashing. Excess pectinase was removed and 1% aceto-carmine stain was applied. After locating and gently tapping the cover clip to disperse the cells, heavy pressure was applied with a No. 9 rubber stopper and the heel of the hand. By the use of this procedure, karyotypes could be constructed in several genera of forage grasses. The karyotype of Paspalum notatum Flugge is illustrated.

Biology of a New Cercaria for Posthodiplostomum minimum (Trematoda: Diplostomidae)

Snails, Physa halei Lea, from Madison County, East Texas, shed strigeid cercariae in large numbers. Adult Posthodiplostomum minimum (MacCallum, 1921) Dubois, 1936, were recovered from chicks force-fed metacercariae obtained from both natural and experimental infections of fishes. Trematodes recovered in the present study were identical to type specimens and to those reported by other workers. Cercariae differed from ones previously described mainly in the absence of caudal bodies at any stage of development and in flame cell number. Other differences included body annulations and associated spines, rows of spines on the oral sucker, flagellets on the tail stem, and body size range. Previous studies on metacercarial specificity between fish families and among members of the same family, and three former cercarial descriptions, have suggested the occurrence of several physiological strains or subspecies of P. minimum. Findings in the present study help support this belief. Posthodiplostomum minimum (MacCallum, 1921) Dubois, 1936, has been investigated intensively with plentiful data accumulating in the literature about the metacercaria and adult. During these studies, three different strigeid cercariae have been considered by various workers as the larval type for P. minimum: Cercaria multicellulata H. M. Miller (1923, 1925), C. louisiana E. L. Miller (1935, 1936), and C. minimum J. H. Miller (1954). These cercariae differ in size, arrangement and number of spines and setae, musculature in the tail stem, and presence or absence of caudal bodies. All three types have produced experimentally metacercariae identified as those of P. minimum: Ferguson (1943), J. H. Miller (1954), Hoffman (1958). J. H. Miller considered C. louisiana to be incorrect and C. multicellulata to be confused with another closely related strigeid. Further, C. minimum was regarded as the only cercaria proved experimentally to be the correct one for P. minimum. The present paper describes still another cercaria of Posthodiplostomum minimum, which has been studied experimentally to obtain metacercariae and adults for comparison with type specimens and descriptively for comparison with the three other reported cercariae. MATERIALS AND METHODS One hundred and twenty-five snails, Physa halei Lea, collected in the autumn of 1966 from three small ponds 3 miles west of Madisonville, Madison Received for publication 24 April 1967. * Supported by grants from the Penrose fund of the American Philosophical Society and the Society of Sigma Xi. County, Texas, were isolated in finger bowls where 90% shed strigeid cercariae spontaneously. All larvae were studied intensively while alive under light coverslip pressure both unstained and with supravital neutral red stain. Following live study permanent slides were prepared using either hot 5% formalin or alcohol-formalin-acetic fixative, Semichon's acetocarmine stain, oil of cloves clearing agent, and balsam mountant. Metacercariae and adults obtained during the investigation were studied and made into permanent mounts as described above. A Wild M20 phase-contrast microscope equipped with a camera lucida was employed in all observations and in preparation of illustrations. All larval measurements from specimens heat-killed in 5% formalin are expressed in microns. EXPERIMENTAL STUDIES Eight 21/2-inch fingerlings, four each of Lepomis macrochirus and L. microlophus, from a group of hatchery-reared uninfected fish were placed with about 700 cercariae in an 8-gal aquarium at 25 C. Cercariae were attracted to fishes and actively attempted penetration, causing fish to display violent finfanning and brushing against aquarium walls. Extensive hemorrhaging occurred around the fins of fishes at points of cercarial penetration. One L. macrochirus succumbed on day 20 and two L. microlophus on days 22 and 33, respectively. Controls were unaffected. Postmortem examinations of all four L. macrochirus showed a metacercarial infection of 91 to 147, average 115, and four L. microlophus 5 to 76, average 27. Fish not dead from infection were killed and examined periodically. Morphological changes, but little size change, were present in larvae examined on day 8. By this date, larvae were less tapered posteriorly and were

An aceto-carmine staining technique for the ectocarpales.

THE following technique has been developed for the study of chromosomes in species belonging to the order Ectocarpales and has been used successfully on Ectocarpus, Giffordia and Pylaiella spp.

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.

Staining plant and animal chromosomes by the Feulgen-acetocarmine sequence.

A technic, some fundamentals of which were first worked out on brome grass, has been considerably extended and adapted to the somatic chromosomes of salmon. Fresh salmon eggs were quickly pierced in 45% acetic acid and fixed therein for 4 minutes. The eggs were then placed in N HCl at 60°C. for 8 minutes and thereafter transferred to Feulgen stain for 30 to 45 minutes. Subsequently, each stained embryo was dissected out and divided in two, each half being placed on a slide in a drop of acetocarmine stain. The pieces were well macerated and, after covering with a cover slip, maceration was completed by tapping. Heavy pressure was gradually applied to the cover slip in order to flatten the chromosome complements. A square screw-type laboratory hose clamp was then used to maintain this pressure while a liquid gelatin seal was applied around the edges. The slide, with the clamp on, was placed in the refrigerator overnight. Before the slide was scanned, the clamp was removed permanently. After each scanning pe...

Manipulation of Tissues for Improved Aceto-Carmine Staining

The importance of the study of the structural-mechanic properties of any particular material in order to improve the technic for aceto-carmine smears and to obtain better preparations of that material has not been, perhaps, sufficiently emphasized in the large number of papers on such cytological technic. The usefulness of such a study will be shown here in two cases met by the writer.

Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.

Reaction of Nucleic Acid to Aceto-carmine

THE non-stainability of many plant and animal chromosomes toward the Feulgen reagent1 after prolonged hydrolysis with N hydrochloric acid at 60° C. has recently also been demonstrated in the salivary gland chromosomes of Chirunomus2. Since the Feulgen reaction and aceto-carmine staining are both used for the demonstration of nucleic acid within the chromosome, it would be of interest to know whether acid hydrolysis will also cause the chromosomes to behave negatively toward the aceto-cannine dye. From the experiments thus conducted it was found that in the early period of hydrolysis (generally after 3–5 min.) the salivary gland chromosomes stained with aceto-carmine more distinctly than those unhydrolysed controls, due probably to the removal of other stainable components surrounding the chromosomes. This strong stainability was not changed within fifteen minutes of hydrolysis. Now, if the duration of hydrolysis exceeded this upper limit, a weaker staining capacity of these chromosomes toward the aceto-carmine dye would result ; and finally they might reach a stage practically unstainable if hydrolysis was allowed to continue for 25–30 minutes. This means that the staining reaction of salivary gland chromosomes toward the aceto-carmine dye after hydrochloric acid hydrolysis was entirely parallel to that toward Feulgen reagent.