High-pressure freezing and freeze substitution fixation of the plant pathogenic fungus Exobasidium vaccinii

1996 ◽  
Vol 54 ◽  
pp. 78-79
Author(s):  
Elizabeth A. Richardson ◽  
Charles W. Mims
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Plant Pathogens ◽  
Pathogenic Fungus ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Large Samples ◽  
Reproductive Structures ◽  
Transmission Electron ◽  
Fungal Genus

Members of the small fungal genus Exobasidium are all plant pathogens. Most species are noted for their ability to produce large fleshy galls on leaves and flower parts. They produce their hyphae and specialized absorbing structures known as haustoria inside these galls and their sexual reproductive structures (basidia) on gall surfaces. The objective of this study was to examine the feasibility of using high pressure freezing (HPF) followed by freeze-substitution (FS) fixation to prepare Exobasidium induced galls on Rhododendron sp. for study with transmission electron microscopy (TEM). HPF fixation followed by FS fixation is considered to be the best method for preserving large samples of plant tissue for TEM. This approach has shown promise for the examination of host-pathogen relationships in fungal diseases of plants.

Ultrastructure of the host-parasite interaction in leaves of Duchesnea indica infected by the rust fungus Frommeëla mexicana var. indicae as revealed by high pressure freezing

2001 ◽  
Vol 79 (1) ◽  
pp. 49-57 ◽  
Author(s):  
C W Mims ◽  
C Rodriguez-Lother ◽  
E A Richardson
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Pressure ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Transfer Cells ◽  
Host Cells ◽  
High Pressure Freezing ◽  
Transmission Electron ◽  
Duchesnea Indica

A combination of scanning and transmission electron microscopy was used to examine the host-pathogen relationship in leaves of Duchesnea indica (Andrz) Focke infected by the rust fungus Frommeëla mexicana var. indicae McCain & Hennen. Samples for transmission electron microscopy were prepared using high pressure freezing followed by freeze substitution. This protocol provided excellent preservation of both host cells and fungal haustoria. Each haustorium of F. mexicana var. indicae possessed a long slender neck with a neck band and an expanded body that contained two nuclei positioned close together. The haustorial body was lobed and sometimes even branched but lacked septa. Details of the extrahaustorial membrane that separated each haustorium from the cytoplasm of its host cell were particularly well preserved. Extensive labyrinth cell wall ingrowths developed around haustorial necks, as well as elsewhere, in infected cells. These ingrowths appeared to be identical to those present in plant transfer cells. Transfer cells are thought to be involved in intensive solute transfer over short distances. This appears to be the first report of the development of transfer cells in response to infection by a plant pathogenic fungus.Key words: haustoria, transfer cells, freeze substitution, electron microscopy.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

1990 ◽  
Vol 48 (3) ◽  
pp. 692-693
Author(s):  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Pathogenic Fungus ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Ice Crystals ◽  
High Pressure Freezing ◽  
Thin Walled Structures ◽  
Cytological Changes ◽  
Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 690-691
Author(s):  
G. J. Celio ◽  
E. A. Richardson ◽  
C. W. Mims
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Euphorbia Pulcherrima ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Transmission Electron ◽  
Dextran Solution ◽  
Leaf Disks ◽  
Diamond Knife

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

Morphology of the lateral fields of the infective juvenile of the entomopathogenic family Steinernematidae (Nematoda) using high pressure freezing (HPF)

Nematology ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 69-74
Author(s):  
Zdeněk Mráček ◽  
Jiří Nermut’ ◽  
Martina Tesařová ◽  
Vladimír Půža
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Pressure ◽  
Infective Juvenile ◽  
Field Pattern ◽  
Taxonomic Character ◽  
High Pressure Freezing ◽  
Lateral Field ◽  
Transmission Electron ◽  
Scanning Electron

Summary The lateral field pattern of infective juveniles of the nematode family Steinernematidae is an important taxonomic character. Scanning electron microscopy (SEM) shows the number of ridges and lines or incisures clearly, but does not provide other details. In the present study, ten species from six clades of Steinernematidae have been studied for their lateral field morphology using SEM and high pressure freezing (HPF) with transmission electron microscopy (TEM). Both methods indicated the same number of ridges and lines, although HPF/TEM resulted in a more detailed morphology with differences between the species. The tips of the ridges are either finely rounded or pointed and the lines are V-shaped or have a broadened bottom. These characters represent an additional pattern that may be characteristic for some species within the phylogenetic clades. Further studies of the lateral field morphology of other species is needed to ascertain whether each pattern is clade specific and phylogenetically valuable.

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