Freeze-Fracturing of Biological Material With A New Specimen Table

1978 ◽  
Vol 113 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Elvira Nickel ◽  
Gertrud Oebel ◽  
Peter Pscheid
Author(s):  
T. Müller ◽  
S. Moser ◽  
M. Vogt ◽  
B.E. Brooker ◽  
N. Terren

Freezing has turned out to be the only method capable of immobilizing biological material in its natural state. Freeze-fracturing and replication complete this preparation protocol of specimens for transmission elctron microscopy (TEM) which is based on purely physical methods. With a propane-jet, specimens of a thickness up to 20 μm can be frozen without detectable ice crystal formation, Cell and macromolecule suspensions, emulsions, liquids and polymers in a solvent can be “sandwiched” between two copper foils and kept in place while a coolant is moved very rapidly against the opposite surfaces. In the present jet freezing device (JFD 030) the temperature of the specimen, while residing in a thermostatically controlled chamber, can be monitored between 10 and 90°C immediately prior to freezing. Thus physiologically interesting situations can be immobilized and any precooling of the specimen is avoided. A special device enables the abslutely simultaneous exposure of the specimen to the double-sided jet of the coolant. High cooling rates with an exceptional reproducibility are achieved routinely (Fig.1).


Author(s):  
G. R. Mackay ◽  
M. L. Mead

Color contrasting of 1 to 2 micron sections of plastic embedded biological material is an important adjunct to electron microscopy. The procedures in general use today are simple and rapid giving monochromatic results, e.g., toluidine blue. Although many di- and polychromatic histologic staining techniques have been modified to obtain a counterstaining effect with plasticembedded tissue, the methods are usually undesirable for routine work because they are time consuming, complicated and often defy good reproducibility.


Author(s):  
K. J. Böhm ◽  
a. E. Unger

During the last years it was shown that also by means of cryo-ultra-microtomy a good preservation of substructural details of biological material was possible. However the specimen generally was prefixed in these cases with aldehydes.Preparing ultrathin frozen sections of chemically non-prefixed material commonly was linked up to considerable technical and manual expense and the results were not always satisfying. Furthermore, it seems to be impossible to carry out cytochemical investigations by means of treating sections of unfixed biological material with aqueous solutions.We therefore tried to overcome these difficulties by preparing yeast cells (S. cerevisiae) in the following manner:


Author(s):  
J. L. Farrant ◽  
J. D. McLean

For electron microscope techniques such as ferritin-labeled antibody staining it would be advantageous to have available a simple means of thin sectioning biological material without subjecting it to lipid solvents, impregnation with plastic monomers and their subsequent polymerization. With this aim in view we have re-examined the use of protein as an embedding medium. Gelatin which has been used in the past is not very satisfactory both because of its fibrous nature and the high temperature necessary to keep its solutions fluid. We have found that globular proteins such as the serum and egg albumins can be cross-linked so as to yield blocks which are suitable for ultrathin sectioning.


Author(s):  
J. D. McLean ◽  
S. J. Singer

The successful application of ferritin labeled antibodies (F-A) to ultrathin sections of biological material has been hampered by two main difficulties. Firstly the normally used procedures for the preparation of material for thin sectioning often result in a loss of antigenicity. Secondly the polymers employed for embedding may non-specifically absorb the F-A. Our earlier use of cross-linked polyampholytes as embedding media partially overcame these problems. However the water-soluble monomers used for this method still extract many lipids from the material.


Author(s):  
N. Seki ◽  
Y. Toyama ◽  
T. Nagano

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.


Author(s):  
A.J. Verkleij

Freeze-fracturing splits membranes into two helves, thus allowing an examination of the membrane interior. The 5-10 rm particles visible on both monolayers are widely assumed to be proteinaceous in nature. Most membranes do not reveal impressions complementary to particles on the opposite fracture face, if the membranes are fractured under conditions without etching. Even if it is considered that shadowing, contamination or fracturing itself might obscure complementary pits', there is no satisfactory explanation why under similar physical circimstances matching halves of other membranes can be visualized. A prominent example of uncomplementarity is found in the erythrocyte manbrane. It is wall established that band 3 protein and possibly glycophorin represents these nonccmplanentary particles. On the other hand a number of membrane types show pits opposite the particles. Scme well known examples are the ";gap junction',"; tight junction, the luminal membrane of the bladder epithelial cells and the outer membrane of Escherichia coli.


2016 ◽  
Vol 52 ◽  
pp. 171-176
Author(s):  
M. Palkina ◽  
O. Metlitska

The aim of the research – adaptation, optimization and using of existing DNA extraction methods from bees’ biological material with the reagent «Chelex-100" under complex economic conditions of native laboratories, which will optimize labour costs and improve the economic performance of DNA extraction protocol. Materials and methods. In order to conduct the research the samples of honey bees’ biological material: queen pupae exuviae, larvae of drone brood, some adult bees’ bodies (head and thorax) were selected. Bowl and drone brood were obtained from the experimental bee hives of Institute of Apiculture nd. a. P. I. Prokopovich of NAAS. DNA extraction from biosamples of Apis mellifera ssp. was carried out using «Chelex-100®» ion exchange resin in different concentrations and combinations. Before setting tests for determination of quantitative and quality indexes, dilution of DNA samples of the probed object was conducted in ratio 1:40. The degree of contamination with protein and polysaccharide fractions (OD 260/230), quantitative content of DNA (OD 260/280) in the extracted tests were conducted using spectrophotometer of «Biospec – nano» at the terms of sample volume in 2 µl and length of optical way in 0,7 mm [7]. Verification of DNA samples from biological material of bees, isolated by «Chelex-100®», was conducted after cold keeping during 24 hours at 20°C using PСR with primaries to the fragment of gene of quantitative trait locus (QTL) Sting-2 of next structure [8]:  3' – CTC GAC GAG ACG ACC AAC TTG – 5’; 3' – AAC CAG AGT ATC GCG AGT GTT AC – 5’ Program of amplification: 94 °C – 5 minutes – 1 cycle; 94 °C – 1 minute, 57°C – 1 minute, 72 °C – 2 minutes – 30 cycles; elongation after 72°C during 2 minutes – 1 cycle. The division of obtained amplicons was conducted by gel electrophoresis at a low current – 7 µÀ, in 1,5 % agarose gel (Sigma ®) in TAE buffer [7]. The results. At the time of optimization of DNA isolation methods, according to existing methods of foreign experts, it was found optimal volume of ion exchange resin solution was in the proposed concentration: instead of 60 µl of solution used 120 µl of «Chelex-100®», time of incubation was also amended from 30 minutes to 180 minutes [9]. The use of the author's combination of method «Chelex-100®» with lysis enzymes, proteinase K and detergents (1M dithiothreitol), as time of incubation was also amended, which was reduced to 180 minutes instead of the proposed 12 hours [10]. Changes in quality characteristics of obtained DNA in samples after reduction in incubation time were not found. Conclusions. The most economical method of DNA isolation from bees’ biological material is 20% solution of «Chelex-100» ion exchange resin with the duration of the incubation period of 180 minutes. It should also be noted that the best results can be obtained from exuviae, selected immediately after the queen’s exit from bowl, that reduces the likelihood of DNA molecules destruction under the influence of nucleases activation, but not later than 12 hours from release using the technology of isolated obtain of queens.


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