Visualization, adhesiveness, and cytochemistry of the extracellular matrix produced by urediniospore germ tubes of Puccinia sorghi

1991 ◽  
Vol 69 (9) ◽  
pp. 2044-2054 ◽  
Author(s):  
Rajendra Chaubal ◽  
V. A. Wilmot ◽  
Willard K. Wynn
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Rust Fungus ◽  
Cetylpyridinium Chloride ◽  
Freeze Substitution ◽  
Ruthenium Red ◽  
Puccinia Sorghi ◽  
Temperature Scanning ◽  
Germ Tubes ◽  
Scanning Electron

Adherence of germinating urediniospores of the common maize rust fungus (Puccinia sorghi Schw.) to substrata was studied by ultrastructural and cytochemical examination of extracellular matrix produced by germ tubes in conjunction with measurements of adhesion to plastic and glass surfaces. Copious amounts of extracellular matrix on germ tubes could consistently be visualized by scanning and transmission electron microscopy only when (i) a cationic detergent (cetylpyridinium chloride, polydiallyldimethylammonium chloride) or a cationic stain (ruthenium red, alcian blue, cuprolinic blue) was added to the fixation solutions, (ii) germ tubes were fixed by rapid-freezing and freeze-substitution and observed with a scanning electron microscope, or when (iii) germ tubes were observed in a frozen-hydrated state by low-temperature scanning electron microscopy. Incubation of germinated spores with dilute alkalies (NaOH, KOH), pronase E (nonspecific protease), and laminarinase (β-1,3 (1,3; 1,4-glucanase) removed the extracellular matrix and detached germ tubes from surfaces. Treatments with water, dilute acids, ionic and neutral detergents, organic solvents, hydrocarbons, and several polysaccharide-degrading enzymes did not remove the extracellular matrix and also did not detach germ tubes. These results, together with staining patterns obtained with lectins and other polysaccharide-specific reagents, indicate that the extracellular matrix is composed mainly of glycoproteins rich in acidic amino acids and β-1,3-glucan polymers, and that it is probably responsible for the adhesion of the rust germ tubes to the host leaf surfaces. Key words: Puccinia sorghi, germ tube adhesion, extracellular matrix, cytochemistry.

The surface of Erysiphe graminis and the production of extracellular material at the fungus – host interface during germling and colony development

1995 ◽  
Vol 73 (2) ◽  
pp. 272-287 ◽  
Author(s):  
T. L. W. Carver ◽  
B. J. Thomas ◽  
S. M. Ingerson-Morris
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Temperature ◽  
Erysiphe Graminis ◽  
Colony Development ◽  
Extracellular Material ◽  
Fungal Enzymes ◽  
Temperature Scanning ◽  
Germ Tubes ◽  
Scanning Electron

Low temperature scanning electron microscopy was used to study the development of Erysiphe graminis DC f.sp. avenae Marchal from germination through infection to sporulation of the fungal colony. To clarify resolution of the fungus–host interface and facilitate interpretation of fungal surface structures, epicuticular waxes were removed from host leaves before inoculation. Whereas conidia were covered in spinelike protrusions or globular bodies, young germ tubes, appressoria, and hyphae were initially smooth walled, but by 15 h after inoculation, wartlike bodies, resembling globular bodies on conidia, were present on first appressorial lobes; these increased in number, eventually covering the appressorium surface and appearing on contiguous hyphae. Wartlike bodies also appeared at junctions of hyphal branches, on hyphal appressoria, and on conidiophore basal cells. Their function, if any, is unknown. The meristematic zone, at the apex of the conidiophore basal cell, remained smooth walled, but globular bodies appeared on the wall of young conidia as soon as the limiting septum had formed. Observations with the fungus in situ revealed the presence of amorphous extracellular material around primary germ tubes and appressorial lobes. Extracellular material was also present beneath appressorial germ tubes and hyphae but it was hidden unless the fungus was displaced. It could not be seen beneath conidia. The extracellular material appeared to be adhesive, sticking the fungus firmly to the host surface. Removal of the fungus showed that the extracellular material was deposited close to the tip of developing germ tubes and hyphae. It was particularly thick around primary germ tubes and appressorial lobes, and a discrete ring of extracellular material was often visible around penetration pores (holes in the leaf surface seen beneath primary germ tubes and appressorial lobes). In addition to its adhesive properties, the extracellular material may act as a matrix in which fungal enzymes are sited and focused for attack on the host. Key words: Erysiphe graminis, low temperature scanning electron microscopy, extracellular material, fungal adhesion, fungal surface morphology.

Ultrastructural preservation of biofilms formed by non-typeable Hemophilus influenzae

Biofilms ◽  
2004 ◽  
Vol 1 (3) ◽  
pp. 165-182 ◽  
Author(s):  
P. Webster ◽  
S. Wu ◽  
S. Webster ◽  
K. A. Rich ◽  
K. McDonald
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Freeze Substitution ◽  
Ruthenium Red ◽  
Chemical Fixation ◽  
Chronic Infections ◽  
Liquid Propane ◽  
Hemophilus Influenzae ◽  
Critical Point Drying ◽  
Bacterial Ultrastructure

There is growing evidence to suggest that non-typeable Hemophilus influenzae (NTHi), an important cause of otitis media in children, is able to grow as a biofilm in the middle ear. This observation may help to explain bacterial persistence in chronic infections. We evaluate the usefulness of rapid freezing and freeze substitution as a means of preparing biofilms for ultrastructural examination by comparing the morphology of cryofixed specimens with the morphology produced using more conventional chemical fixation and dehydration methods. Chemical fixation and dehydration methods produced substantial ultrastructural damage to individual NTHi in the biofilm and loss of extracellular matrix, even in the presence of ruthenium red. In comparison, cryofixed and freeze-substituted NTHi biofilms showed significantly improved preservation of bacterial ultrastructure and biofilm organization. The intracellular contents of NTHi prepared using the cryomethods showed little evidence of aggregation, and bacteria within the biofilm were closely packed and surrounded by an abundant extracellular matrix. Although high-pressure freezing of NTHi biofilms followed by freeze substitution was highly effective for preserving ultrastructure when examined by transmission electron microscopy, immersion in liquid propane offered an alternative, “less technical”, freezing method. Immersion in liquid propane followed by freeze substitution and critical point drying was most effective for preserving ultrastructural details in specimens examined by scanning electron microscopy.

The pattern of colony development and the formation of the uredinium of Uromyces viciae-fabae on Vicia faba

1987 ◽  
Vol 65 (10) ◽  
pp. 1998-2006 ◽  
Author(s):  
A. Beckett ◽  
A. M. Woods
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Extracellular Matrix ◽  
Low Temperature ◽  
Host Cells ◽  
Colony Development ◽  
Colloidal Iron ◽  
Thin Sections ◽  
Temperature Scanning ◽  
Scanning Electron

Low-temperature scanning electron microscopy of frozen-hydrated specimens and transmission electron microscopy of cytochemically stained thin sections and enzymically digested samples have been used to study the pattern of colony development from the initial infection in the substomatal chamber of the leaf to the production of urediniospores within uredinia. Rapid immobilization of leaf samples by freezing enables them to be fractured, revealing information on internal structural relationships between intercellular hyphae and host cells. Extracellular matrix substances, which are well preserved by low-temperature scanning electron microscopy, play an important role in the cohesion of hyphae during the early stages of uredinium formation. Cytochemical staining indicates the presence of two such matrix materials: (i) colloidal iron-negative matrix surrounding intercellular hyphae, forming a pad between adjacent hyphal cells and (ii) a colloidal iron-positive matrix between hyphal and host cell walls. A positive reaction with colloidal iron indicates acidic mucopolysaccharides. Enzymic digestion with pronase and the staining reaction with silicotungstic acid – chromic acid suggests that a glycoprotein is present in the matrix surrounding intercellular hyphae. Possible roles for the extracellular matrix, including hyphal cohesion, cell–cell communication, and protection against desiccation, are discussed.

Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 736-737
Author(s):  
T. Inoué ◽  
H. Koike
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Temperature ◽  
Liquid Nitrogen ◽  
Specimen Preparation ◽  
Chemical Fixation ◽  
Brown Adipose ◽  
Critical Point Drying ◽  
Temperature Scanning ◽  
Scanning Electron

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.

Criteria for the evaluation of low-temperature Scanning Electron Microscopy

1986 ◽  
Vol 44 ◽  
pp. 902-903
Author(s):  
Alan Beckett
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Low Temperature ◽  
Conformational Changes ◽  
Chemical Vapour ◽  
List Type ◽  
Dimensional Changes ◽  
Critical Point Drying ◽  
Temperature Scanning ◽  
Scanning Electron

Low temperature scanning electron microscopy (LTSEM) has been evaluated with special reference to its application to the study of morphology and development in microorganisms. A number of criteria have been considered and have proved valuable in assessing the standard of results achieved. To further aid our understanding of these results, it has been necessary to compare those obtained by LTSEM with those from more conventional preparatory procedures such as 1) chemical fixation, dehydration and critical point-drying; 2) freeze-drying with or without chemical vapour fixation before hand.The criteria used for assessing LTSEM for the above purposes are as follows: 1)Specimen immobilization and stabilization2)General preservation of external morphology3)General preservation of internal morphology4)Exposure to solvents5)Overall dimensional changes6)Cell surface texture7)Differential conformational changes8)Etching frozen-hydrated material9)Beam damage10)Specimen resolution11)Specimen life

Neutrophil Derived Cathepsin G Induces Potentially Thrombogenic Changes in Human Endothelial Cells: a Scanning Electron Microscopy Study in Static and Dynamic Conditions

1994 ◽  
Vol 72 (01) ◽  
pp. 140-145 ◽  
Author(s):  
Valeri Kolpakov ◽  
Maria Cristina D'Adamo ◽  
Lorena Salvatore ◽  
Concetta Amore ◽  
Alexander Mironov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Thrombus Formation ◽  
Cathepsin G ◽  
Arterial Segment ◽  
Dynamic Conditions ◽  
Activated Neutrophils ◽  
Scanning Electron

SummaryActivated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer.We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions.Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment.Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an “in vivo” model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential.This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.

scholarly journals Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 51
Author(s):  
Michela Relucenti ◽  
Giuseppe Familiari ◽  
Orlando Donfrancesco ◽  
Maurizio Taurino ◽  
Xiaobo Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Ion Beam ◽  
Ruthenium Red ◽  
Variable Pressure ◽  
Sem Images ◽  
Advantages And Disadvantages ◽  
Pros And Cons ◽  
Microscopy Techniques ◽  
Scanning Electron

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

Sign in / Sign up

Export Citation Format

Share Document