An improved technique for visualization of Neisseria gonorrhoeae pili by SEM

1990 ◽  
Vol 48 (3) ◽  
pp. 796-797
Author(s):  
Nusi P. Dekker ◽  
Claudia J. Lammel ◽  
Geo. F. Brooks
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Culture Medium ◽  
Endometrial Adenocarcinoma ◽  
Calf Serum ◽  
Uranyl Acetate ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Critical Point Drying ◽  
Cacodylate Buffer ◽  
Improved Technique

Piliated Neisseria gonorrhoeae (gc) are virulent and attach to human cells more readily than nonpiliated gc . Pili have been difficult to visualize in the scanning electron microscope (SEM), appearing as thick, branched structures, possibly because of artifacts or distortion associated with critical point drying (CPD) . The use of hexamethyldisilazane (HMDS), as an alternative to CPD with bacterial or tissue culture cells, as reported here, improved preservation and visualization of pili and other delicate surface structures.The methods were: Human endometrial adenocarcinoma (HEC-l-B, ATCC HTB 113) cells were grown in cell culture medium containing fetal calf serum on round thermanox coverslips. Suspensions of piliated and nonpiliated strains of gc (FA1090, F62-SF, and g1412) at dilutions of about 2 × 107 cfu were added to the cells and allowed to attach. Unattached gc were rinsed off and replaced with fresh media after 30 min. After 1 or 8h incubation, the cells were fixed in 2.5% gluteraldehyde in 0.1M cacodylate buffer for 4h; rinsed well in the buffer; post-fixed in 1% OsO4 in cacodylate buffer for lh; rinsed 3 × 10 min in buffer and 2 × 5 min in water; placed in 1% tannic acid in water for 30 min; rinsed 3 × 10 min in water; placed in 2% uranyl acetate for lh; rinsed 3 × 10 min in water; dehydrated in graded ethanols (50%, 70%, 95%, 2 × 5 min each; 100% 4 × 5 min; 100% 10 min); placed in HMDS for 10 min.

Fish kidney cell line in response to heat shock

1992 ◽  
Vol 50 (1) ◽  
pp. 704-705
Author(s):  
Li-Chu Tung ◽  
Yung-Reui Chen ◽  
Shiu-Nan Chen ◽  
Guang-Hsiung Kuo
Keyword(s):  
Heat Shock ◽  
Cell Line ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Uranyl Acetate ◽  
Ultrastructural Changes ◽  
Heat Shock Treatment ◽  
Acanthopagrus Schlegeli ◽  
Cacodylate Buffer ◽  
Fish Kidney

In the present study, the ultrastructural changes of BPK cells, a fibroblast-like cell line, derived from the kidney of juvenile black porgy Acanthopagrus schlegeli, under heat shock treatment are described.The BPK cells were maintained in L-15 medium supplemented with 10% fetal calf serum and 0.15 M NaCl at 28|C2. The heating was carried out in precalibrated water baths. Monolayers of cells, grown on coverslips in parafilm-sealed petri dishes were submerged under water for 30 min at 40|C treatments. Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer supplemented with 6.6% sucrose, postfixed in 1% OsO4 and flat embedded in Spurr’s resin. Silver section were cut parallel to the substratum, stained with uranyl acetate and Reynold’s lead citrate, and examined in a Hitachi H-600 electron microscope at 75 KV.

A plunge-freezing method for imaging mammalian tissue culture cells

1991 ◽  
Vol 49 ◽  
pp. 58-59
Author(s):  
E.T. O'Toole ◽  
J.R. McIntosh
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Freezing Method ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Humidity Chamber ◽  
Cellular Components ◽  
Coated Carbon

Ultrarapid freezing of tissue culture cells followed by freeze substitution has been used a method for the optimum fixation of cytoskeletal components that are often sensitive to routine chemical fixation. This is due to the fact that freezing methods such as plunge freezing, result in the almost instantaneous fixation of all cellular components without alteration of the cell's morphology. In addition, we have found that the plunge freezing method is useful for obtaining thin frozen cells for direct cryoimaging. Here we describe how the plunge freezing method can be applied both for freeze substitution analysis and for direct cryoimaging of frozen tissue culture cells.PTK, cells were grown to confluence on formvar coated, carbon stabilized gold grids. Prior to freezing, the grids were blotted in a 37°C, high humidity chamber so that a minimum of culture medium remained on the grid. This blotting step was critical to obtain a sample thin enough for optimum cryopreservation and subsequent cryoimaging.

GACH-prepared specimens for sem and confocal reflected light microscopy

1988 ◽  
Vol 46 ◽  
pp. 98-99
Author(s):  
R. E. Crang ◽  
B. Thompson
Keyword(s):  
Light Microscopy ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Scanning Microscope ◽  
Minimum Essential Medium ◽  
Culture Cells ◽  
Hindlimb Muscles ◽  
Tissue Culture Cells ◽  
Small Depth ◽  
Reflected Light

Biological specimens prepared for SEM are difficult to observe in detail by means of light microscopy. Conventional epi-illuminescence produces relatively poor images with reflected light due to poor contrast and multiple scattering of the light (1). However, we have found that mammalian tissue culture cells prepared for SEM by means of the co-polymerized glutaraldehyde-carbohydrazide (GACH) technique (2), can be alternately observed by means of SEM and reflected light microscopy if the light optical instrument is a confocal tandem scanning microscope (TSM). The images produced with TSM show resolution comparable to that of the SEM at magnifications ≤ 1,000 X. Furthermore, due to the extremely small depth of focus containing high contrast and brightness, optical sections can be produced which bear detail similar to that in conventional TEM observations.Fibroblasts and myotobes, from newborn rat hindlimb muscles were grown in Eagle's minimum essential medium with 10% fetal calf serum on collagen- coated glass cover slips for a period of 7 d (3).

Electron Microscopic Examination of DNA in an Alkaline Sucrose Gradient

1975 ◽  
Vol 33 ◽  
pp. 612-613
Author(s):  
W. D. Garner ◽  
R. E. Nordquist ◽  
R. H. Bottomley ◽  
J. M. Hawrylko
Keyword(s):  
Mammalian Cells ◽  
Sucrose Gradient ◽  
Electron Microscopic Examination ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Ultrastructural Characteristics ◽  
Rat Hepatoma ◽  
Hepatoma Tissue

The technique of lysing cells on top of an alkaline sucrose gradient prior to sedimentation was first designed to examine single stranded DNA from protoplasts. The lysis of mammalian cells and sedimentation of the released DNA has not been examined thoroughly by EM. The present work describes the ultrastructural characteristics of DNA species found after whole cell lysing and sedimentation of the DNA through an alkaline sucrose gradient.The DNA of H4-II-E rat hepatoma tissue culture cells was prelabelled with H3TdR (luci/ml) for 18 to 24 hours prior to lysis. Cells were layered into a lysing layer (0.1 M EDTA and 0.5 N NaOH, pH 13.25) on top of a 5-202or 5-25% alkaline sucrose gradient (0.01 M EDTA, 0.1 N NaOH and 0.9 M NaCl). After centrifugation the gradients were fractionated from the bottom. The fractions containing the majority of H3TdR were applied directly on grids at pH 8.5 or 12. Samples were stained with uranyl acetate.

scholarly journals Decreased protein-synthetic activity is an early consequence of spermidine depletion in rat hepatoma tissue-culture cells

1984 ◽  
Vol 217 (3) ◽  
pp. 731-741 ◽  
Author(s):  
B B Rudkin ◽  
P S Mamont ◽  
N Seiler
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Synthetic Activity ◽  
Cell Culture Medium ◽  
Irreversible Inhibitor ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Htc Cells ◽  
Rat Hepatoma ◽  
Hepatoma Tissue

Hepatoma tissue-culture (HTC) cells were exposed to DL-alpha-difluoromethylornithine (DFMeOrn), a specific irreversible inhibitor of ornithine decarboxylase. Concomitantly with the decrease in spermidine, a decrease in the amount of ribosomes in polyribosomes was observed. Spermine concentrations remained essentially comparable with those in cells not exposed to this inhibitor. Exposure of putrescine- and spermidine-depleted HTC cells to spermidine or spermine rapidly reversed the effect of DFMeOrn on polyribosome profiles, whereas addition of putrescine to the cell culture medium had an effect only after its transformation into spermidine and spermine. The results show that the perturbation of polyribosome formation in DFMeOrn-treated HTC cells is due to spermidine deficiency and that a normal polyamine complement is required for optimal protein-synthetic activity in these cells. The results also indicate that protein synthesis is perturbed before DNA synthesis during depletion of putrescine and spermidine in HTC cells.

Vaccinia-lassa recombinant produces lassa-like inclusions

1989 ◽  
Vol 47 ◽  
pp. 1036-1037
Author(s):  
C. S. Goldsmith ◽  
H. G. Morrison ◽  
D. D. Auperin ◽  
S. G. Whitfield ◽  
E. L. Palmer
Keyword(s):  
Tissue Culture ◽  
Lassa Fever ◽  
Uranyl Acetate ◽  
Envelope Glycoproteins ◽  
Effective Vaccine ◽  
Lassa Virus ◽  
Recombinant Vaccinia ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Vaccinia Viruses

Lassa virus, a member of the Arenaviridae, is the etiologic agent of Lassa fever. The virus is endemic in widespread areas of West Africa, particularly Nigeria, Liberia, and Sierra Leone. Rough estimates indicate that there are upwards of 300,000 human infections annually, resulting in approximately 5000 fatalities. A safe and effective vaccine for Lassa fever is not yet available. Recent studies in our laboratories have focused on the development of recombinant vaccinia viruses that express the nucleoprotein and envelope glycoproteins of Lassa virus as potential vaccine candidates. Tissue culture cells infected with various recombinant viruses were examined by thin-section TEH for any morphological alterations.Tissue culture cells were infected with wild-type Lassa virus or with recombinant vaccinia viruses expressing either the Lassa virus nucleoprotein (NP). the envelope glycoproteins (GPI,GP2), or NP, GPI, and GP2 in combination. Cells were fixed in 2.5% glutaraldehyde in 0.2M cacodylate buffer, postfixed in buffered 1% osmium tetroxide, and en bloc stained with 4% uranyl acetate. The samples were dehydrated and embedded in Polysciences Epon-substitute and Araldite. Sections were stained with uranyl acetate and lead citrate.

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