Squash Technique Research Articles

An estimation of proliferative population size in stomach, jejunum and colon of DBA-2 mice.

ABSTRACTLiquid scintillation and autoradiographic techniques have been used to provide quantitative data on the proliferative units, the crypts, of stomach, jejunum and colon of DBA‐2 mice. A slight modification of the crypt squash technique has provided data suggesting that about 50% of the cells of the jejunal crypt are at any given time in the proliferative state. This value is lower in the colon while in the stomach glands only 20% of the cells are involved in cell production. The data provide estimates for cell cycle times of 26·3, 16·0 and 23·2 hr for stomach, jejunum and colon respectively.The size and number of proliferative units have been determined for three regions of the gastrointestinal tract of mice. A review of the literature suggests that considerable strain differences may exist.

A simple and rapid permanent squash technique for bulk‐stained plant material

A simple and rapid permanent squash technique for bulk‐stained plant material

A method of maintaining parenchymal cells from adult rat liver in vitro

ABSTRACT A method for preparing suspensions of adult rat hepatocytes suitable for maintenance in vitro is described. Cultures were established from the cell suspensions by the squash technique. Cells were examined by light and electron microscopy; histochemically for glycogen, bile, lipid and glucose-6-phosphatase; and by autoradiography for DNA, RNA and protein synthesis. Hepatocytes could be maintained in vitro for at least 3 days and began to aggregate after 1 day. Uridine and leucine were incorporated, but not thymidine. Cultures consisted mainly of hepatocytes, though reticulo-endothelial cells were sometimes present.

Deoxyribonucleic acid synthesis in vitro in normal and delayed nidation preimplantation blastocysts of adult rats.

Summary. Blastocysts recovered from adult cycling rats on Day 5 of pregnancy had thirty-four nuclei as determined by the squash technique. The nuclei actively incorporated [3H]thymidine in vitro when examined by autoradiography. In delayed-nidation blastocysts on Day 9 of pregnancy, the number of nuclei observed was seventy-four, and a smaller number of nuclei incorporated [3H]thymidine than on Day 5 under identical experimental conditions. Oestrogen and progesterone treatment 30 hr before autopsy of rats with delayed nidation caused stimulation of DNA synthesis in the nuclei of blastocysts before implantation. This was indicated by a greater number of nuclei incorporating [3H]thymidine than in the controls which did not receive oestrogen.

Karyological studies of Japanese ants (Hymenoptera, Formicidae). 3. Karyotypes of nine species in Ponerinae, Formicinae, and Myrmicinae.

The karyotypes of nine Japanese ants in three subfamilies (Ponerinae, Formicinae, and Myrmicinae) were successfully analysed by the improved squash technique. Three ponerine species had 2n=7 and n=4 (Ponera scabra), 2n=22 (Brachyponera sinensis), and 2n=28 and n=14 (Cryptopone sauteri). Four formicine species had 2n=18 and n=9 (Camponotus sp. and C. tokioensis), 2n=26 and n=13 (Camponotus japonicus), and 2n=30 and n=15 (Lasius niger). Two myrmicine species had 2n=18 and n=9 (Leptothorax congruus), and 2n=37, 38, 39, and n=17, 18, 19, 20 (Pheidole nodus). It was found that the variation of chromosome number observed in P. nodus was caused by Robertsonian type polymorphism.

The Phragmites communis Trin. chromosome complement in the Danube Delta

This study elucidates the caryotypes in two varieties of Phragmites communis of the Danube Delta, by means of an original squash technique. The two varieties studied above coexist in the floating reed biocoenosis, but there exist different masses without any transitory forms. The flavescens and gigantissima varieties have different morphophysiological characteristics, the latter variety flowers later and shows the gigantism characteristics of polyploids. Comparative analysis of the caryotypes of the two varieties shows that the variety gigantissima is probably a natural auto-octopolyploid (2n = 8x = 96) of a relatively recent origin, as the chromosomes can be grouped into units of 4. The origin of the flavescens variety (2n = 4x = 48) is not certain, because the chromosomes can only be grouped in pairs within the caryotype. It seems that this variety, widespread all over the earth, has an older origin, since the rearrangements determining the present configuration of the caryotype occurred at chromosome level.

Cold and chemical pretreatments to aid chromosome counts in a grass leaf squash technique incorporating hot pectinase maceration.

Leaf buds, comprising the basal 3-5 mm of the youngest leaves attached to short stems, were dissected out of fast-growing young tillers of certain grasses, including Festuca, Lolium and Phalaris spp. and various hybrids. They were kept overnight in distilled water at 0-2 C, treated in a mixture of equal parts by volume of saturated aqueous solutions of 5,7-dibromo-8-hydroxyquinoline containing a surfactant (Tween 80), and 1-bromo-naphthalene for 3-4 hr at 0-2 C, and fixed in Newcomer's fluid. The rinsed samples were hydrolysed in 1 N HCl for 8 min at 60 C and Feulgen stained for 1 hr. After rinsing, the buds were macerated in a filtered 3% solution of Pectinol 100-D (Rohm and Haas) in 0.1 M acetate buffer at pH 4.5 for 10 min at 60 C. Squashes were made in 45% acetic acid. The combined cold and chemical pretreatments resulted in strongly contracted, easily counted metaphase chromosomes, while intact cells with full chromosome complements were more readily retained during squashing after enzyme maceration ...

A Technique for the Morphological Examination of Hemopoietic Cells Grown in Agar

Abstract Histological preparations of hemopoietic cells growing in colonies in soft agar were made using a modified squash technique. Colonies were fixed in the Petri dish with 3:1 acetic-alcohol mixture for 1 hour. Individual colonies were pricked out with a microhematocrit tube and placed on a slide. Squash preparations of the cells were then made by the standard method. Preparation for staining the cells was made by immersing the slides in methyl alcohol for 10 minutes. The slides were finally treated with phosphate buffer at pH 6.4 for 45 minutes before staining with MacNeal’s tetrachrome. This technique allowed the recovery and cytological examination of most of the cells growing in a single colony.

The mitotic index in selected red algae in situ. II. A supralittoral species, Porphyra lanceolata (Setchell & Hus.) G. M. Smith

Plants of Porphyra lanceolata (Setchell & Hus.) G. M. Smith, a high intertidal species of red algae, were collected and fixed every 2 h for a 24-h period in November and January. Gross environmental parameters were recorded at the time of fixation in an attempt to correlate them with nuclear division in the vegatative cells. The material was stained with acetocarmine and a squash technique was used in preparing material for cytological observation. Meristematic regions were located and nine distinct stages of mitosis, similar to those described for higher plants and many other algae, were distinguished. A mitotic index was calculated for each 2-h period. In November, nuclear division was initiated and the early stages of nuclear division were most abundant at 16.30h. P. lanceolata has a “phased type” of rhythm in its cell division similar to that of Gonyaulax polyhedra. A periodicity in nuclear division is statistically significant. Synchronous laboratory and in situ preparations were made in January. In the laboratory, plants were maintained under various combinations of light and water cover. In situ conditions were slightly different in January as were the plants, since they had developed extensive reproductive structures; however, nuclear division was initiated at approximately the same time of day as in November. It is suggested that temperature and tidal rhythms do not influence the periodicity of nuclear division: light intensity may be a major factor in the control of nuclear division in vegetative cells of Porphyra lanceolata.

Spermatogenesis in the mosquito, Aedes dorsalis (Meigen).

The chromosome morphology and behavior pattern during spermatogenesis in Aedes dorsalis was studied. The results were compared with the findings of similar investigations in other species of mosquitoes. Chromosome preparations were made from testes-tissue of fourth instar and pupal stages by the squash technique and stained with aceto-lactic-orcein. The two testes of an individual may have cells at different divisional stages. Leptotene and zygotene stages of meiotic prophase were not distinguishable. The unwinding of the homologous chromosomes in later stages of prophase I was found to progress at different rates on either side of the centromere. The separation of the chromosomes during anaphase I was mostly synchronous. The polarization of arms of the prophase II chromosomes was observed. The repulsive forces between the sister chromatids in metaphase II were less apparent and the chromosomes at this period were H-shaped in configuration.

Histopathology of Resistance in the Sorghum bicolor-Sphacelotheca reiliana Interaction

Hypodermic inoculation of five resistant sorghums with compatible monosporidial lines of S. reiliana resulted in three different host-parasite interactions. Early Hegari and F1's having Early Hegari as the male parent were compatible with the pathogen for several days before becoming incompatible. The length of time before the host-parasite relationship became incompatible varied with incubation temp and the genotype of the female parent. The incompatible reaction was characterized by the disappearance of mycelium and the appearance of teliosporelike objects in the host. Lahoma Sudangrass, White Kafir P.I. 48770, and kafir B65L-5427-3 had similar reactions to the monosporidial inoculum. In each entry, hyphal growth or sporidial germ tubes were present in the inoculation wound, but the pathogen did not grow into the host tissue. The reactions were not altered by different temp. Feterita Tx09 and F1's involving this entry were characterized by the complete inhibition of sporidial development and fusion. This reaction was also stable over a range of temp. A squash technique was developed to rapidly identify reaction type

Reinvestigation of meiosis in the male goat, Capra hircus, Linn., with special reference to chiasma formation in the sex and autosomal bivalents.

The chromosome behavior during spermatogenesis of the goat, Capra hircus Linn., has been reinvestigated following hypotonic Earle's treatment technique. The regular process of meiosis in this species has already been reported by Makino (1943) following squash technique, with very little information about chiasmata during division. In this study, a detail account of chiasma formation in each and every autosomal bivalent at diplotene stage and their movement towards terminalisation in different stages of first meiosis have been represented with the remark of average number of chiasmata per cell and per bivalent. Also, this observation differs from previous author, who noticed a spindle fibre attachment between the X and Y during first meiotic division, whereas it has been found here the distinct formation of a chiasma between the X and Y and the homologous part is the minute short arm of X and one of the arms of small metacentric Y.

Mitotic Chromosomes of the Australian King Quail (Excalfactoria Chinensis)

ABSTRACTA simple corneal epithelial squash technique was used to establish the karyotype of the Australian King Quail Excalfactoria chinensis Linne. It was found that the female was the heterogametic sex, and that the sex determining mechanism was the ZZ-ZW type. The karyotype of this species was found to have morphological similarities to the published karyotype of the Japanese Quail Coturnix coturnix japonica, but with certain recognizable differences.

The metaphase chromosomes of Chinese hamster liver cells following partial hepatectomy.

A procedure for preparing and observing metaphase chromosomes of the Chinese hamster is described. The technique involved surgical removal of a constant fraction of the liver to stimulate cell division, injection of colchicine to accumulate metaphase cells and a squash technique for spreading the meta-phase chromosomes. A peak in the mitotic index was observed at 54 hours post-partial hepatectomy. Advantages of scoring metaphase as opposed to telophase and anaphase figures are discussed and examples of normal and 144Ce exposed cells are shown. These techniques can be used in the study of internally deposited radioactive material and other toxic agents which have their major effect on the liver.

Identification and chromomeric interpretation of pachytene bivalents from Canis familiaris.

Squash techniques were used to spread, the 39 pachytene bivalents from spermatogenic Canis familiaris cells collected by testicular biopsy from normal, healthy male collie dogs. Eight cells in which all bivalents could be identified were photographed, enlarged to 4500 × and cutouts prepared for each chromosome in each of the cells. Characterization and identification were based on relative length, mean percentage length, and number of chromomeres in each bivalent.

Chromosome constitution of human endometrium

Chromosome counts have been obtained, with a hypotonic squash technique, from 32 samples of nonmalignant endometrium. Seventy-nine per cent of the total population consisted of diploid cells exhibiting a normal female karyotype. A hypodiploid population of 16.3 per cent and a smaller hyperdiploid population of 2.4 per cent were present. Karyotype analysis indicated that the hypodiploid population consisted of metaphases with simple chromosome loss. No differences were observed in the distribution of chromosome count between proliferative and secretory endometrium or between normal and hyperplastic endometrium.

The Karyology of the Colonial AscidianBotryllus Schlosseri(Pallas)

SUMMARYSet up a squash technique for the chromosomes of Botryllus schlosseri, feature and behaviour of mitotic chromosomes in young embryos and the first stages of meiosis till metaphase have been described.

Chromosome studies on direct and cultured preparations from malignant tissues

Summary Cytogenetic data has been obtained from 16 primary cultures of non-malignant cervical, uterine, and ovarian tissues. Normal diploid cells constituted the major population in these cultures, with a small tetraploid population Forty-eight cultures were set up from biopsies derived from 26 malignant gynaecological tumours. The chromosome constitution of these tumours was also analysed using a direct squash technique. Of the in vitro cultures, 10 cases produced adequate data for comparison with the results from squash preparations. In vitro cultures were characterized by the presence of normal diploid populations in contrast to the marked aneuploidy shown in the squash preparations. In only 1 case was evidence obtained suggesting that malignant cells similar to those present in the in vivo population were present in culture

Cytotaxonomic Notes on Some Neotropical Gentianaceae

Chromosome numbers for 13 species in four genera of neotropical Gentianaceae are reported for the first time. The chromosome numbers suggest a relationship between Symbolanthus, Macrocarpaea, and Chelonanthus and support the separation of Lisianthius and Chelonanthus. The Gentianaceae is a moderate-sized family, comprising approximately 800 species (Engler & Diels, 1936). Their distribution is essentially worldwide. In many cases generic limits within the family are poorly defined, and intergeneric relationships are poorly understood. Particularly troublesome are those approximately 100 neotropical members of the family which are supposedly related to Lisianthius P. Browne. In accordance with the genera accepted by Gilg (1895) this would include the following: Macrocarpaea Gilg, Pagaea Griseb., Helia Mart., Irlbachia Mart., Lehmaniella Gilg, Adenolisianthus Gilg, Calolisianthus Gilg, Chelonanthus Gilg, Purdieanthus Gilg, Lagenanthus Gilg, Rusbyanthus Gilg, Symbolanthus Don, and Lisianthius P. Browne. Taxonomic studies of this group of genera have been hampered not only by the paucity of specimens, but also by their miserable condition. Macrocarpaea is the only genus of moderate size for which a reasonably complete monograph (Ewan, 1948) is available. With the exception of Gentiana and its segregates, little is known about the cytology of the family. There are apparently no published chromosome counts for any of the lisianthioid genera. Counts are reported here for the first time for thirteen species in four of the genera. The standard squash technique was used: Flower buds were fixed in modified Carnoy's solution, stored in 70% ethanol, and the pollen mother cells were subsequently squashed in acetocarmine. The results are listed in Table 1. According to the most comprehensive treatment of the Gentianaceae (Gilg, 1895), Lisianthius and Macrocarpaea belong to the tribe Gentianeae, subtribe Tachiinae, while Chelonanthus and Symbolanthus belong to the tribe Helieae. In Gilg's treatment the Gentianeae and the Helieae, as well as most other major groupings within the family, were delimited on the basis of pollen grain morphology. Those genera whose pollen grains are united into tetrads were referred to the Helieae; all other genera supposedly are characterized by having separate pollen grains. The findings of more recent authors suggest that Gilg's system is probably inadequate. Ewan (1948) argued that the genera most closely related to Macro'Most of the field work involved in this study was supported by National Science Foundation Grant GB-6393. I thank Dr. Robert L. Wilbur and Dr. Donald E. Stone for assistance in the preparation of this manuscript. Cytological vouchers are in DUKE. 2Department of Botany, Duke University, Durham, North Carolina 27706. ANN. MISSOURI BOT. GARD. 56: 439-443. 1969. This content downloaded from 157.55.39.243 on Wed, 05 Oct 2016 04:40:20 UTC All use subject to http://about.jstor.org/terms

Cytogenetic studies of endometrial carcinoma

Chromosome counts have been obtained from 22 cases of carcinoma of the endometrium and 4 cases of atypical cystic hyperplasia. The direct squash technique after hypotonic pretreatment was used. A diploid population of 22.2 per cent was present in the cases of endometrial cardinoma which were analyzed. Eighty-eight per cent of these diploid metaphases exhibited a pseudodiploid karyotype. Ninety-three per cent of the total chromosome counts for endometrial carcinoma were in the range of 35 to 56 chromosomes per cell. The diploid population in the 4 cases of atypical hyperplasia was 66 per cent. Two cases were analyzed karyotypically. Normal female karyotypes appeared exclusively in one case. In the second case pseudodiploid karyotypes predominated, and rearrangement within the chromosome groups was evident in hypodiploid cells.