Insect Preservation Methods Allow Quantification of Dispersing Microorganisms by Scanning Electron Microscopy

Abstract Insects may act as dispersal vectors for microbes where microbes can temporarily adhere to insect exoskeletons. Microbes carried by honey bees (Apis mellifera) may experience a range of dispersal outcomes, from successful colonisation of a new habitat to predation by grooming bees, partly depending on their location on the bee exoskeleton and the carried population size. I tested four methods for handling collected bees, and examined the bees in a scanning electron microscope, imaging and counting microbial cells attached to tarsal claws. Freeze-dried bees carried more microbial cells than bees that were pinned and air-dried, preserved in 96% ethanol then dried by CO2 critical-point drying, or bees washed with potassium phosphate then preserved in 96% ethanol, but some microbes were found on bees from every treatment. The similarity in microbial passengers found between air-dried and ethanol-preserved bees suggests that examination by electron microscopy could be used to address questions regarding microbial dispersal by pollinators already present in collections associated with other pollination research.

The use of HMDS (hexamethyldisilazane) to Replace Critical Point Drying (CPD) in the Preparation of Tardigrades for SEM (Scanning Electron Microscope) Imaging

Transactions of the Kansas Academy of Science ◽

10.1660/062.112.0407 ◽

2009 ◽

Vol 112 (3-4) ◽

pp. 198-200 ◽

Cited By ~ 19

Author(s):

Shawn Shively ◽

William R. Miller

Keyword(s):

Electron Microscope ◽

Scanning Electron Microscope ◽

Critical Point ◽

Critical Point Drying ◽

Microscope Imaging ◽

Electron Microscope Imaging ◽

The Critical Point Drying Method Applied to Scanning Electron Microscopy of L-929 Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068382 ◽

1970 ◽

Vol 28 ◽

pp. 278-279

Author(s):

Charles TurnbiLL ◽

Delbert E. Philpott

Keyword(s):

Electron Microscopy ◽

Carbon Dioxide ◽

Surface Tension ◽

Critical Point ◽

Freeze Drying ◽

Attractive Alternative ◽

Crystal Formation ◽

Critical Point Drying ◽

Time Required ◽

The advent of the scanning electron microscope (SCEM) has renewed interest in preparing specimens by avoiding the forces of surface tension. The present method of freeze drying by Boyde and Barger (1969) and Small and Marszalek (1969) does prevent surface tension but ice crystal formation and time required for pumping out the specimen to dryness has discouraged us. We believe an attractive alternative to freeze drying is the critical point method originated by Anderson (1951; for electron microscopy. He avoided surface tension effects during drying by first exchanging the specimen water with alcohol, amy L acetate and then with carbon dioxide. He then selected a specific temperature (36.5°C) and pressure (72 Atm.) at which carbon dioxide would pass from the liquid to the gaseous phase without the effect of surface tension This combination of temperature and, pressure is known as the "critical point" of the Liquid.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066097 ◽

1971 ◽

Vol 29 ◽

pp. 410-411 ◽

Cited By ~ 1

Author(s):

Jane A. Westfall ◽

S. Yamataka ◽

Paul D. Enos

Keyword(s):

Electron Microscopy ◽

Osmium Tetroxide ◽

External Surface ◽

Three Dimensional ◽

Surface Structures ◽

Transmission Microscopy ◽

Transmission Electron ◽

Freeze Dried ◽

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Preservation of Plant Cell Surfaces For Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072800 ◽

1973 ◽

Vol 31 ◽

pp. 472-473

Author(s):

Linda M. Sicko ◽

Thomas E. Jensen

Keyword(s):

Electron Microscopy ◽

Critical Point ◽

Filter Paper ◽

Freeze Drying ◽

Cell Surfaces ◽

Air Drying ◽

Popular Method ◽

Critical Point Drying ◽

The use of critical point drying is rapidly becoming a popular method of preparing biological samples for scanning electron microscopy. The procedure is rapid, and produces consistent results with a variety of samples. The preservation of surface details is much greater than that of air drying, and the procedure is less complicated than that of freeze drying. This paper will present results comparing conventional air-drying of plant specimens to critical point drying, both of fixed and unfixed material. The preservation of delicate structures which are easily damaged in processing and the use of filter paper as a vehicle for drying will be discussed.

Scanning Electron Microscopy-cuticular Lesions on the Roundworm Ascaris Suum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010006756x ◽

1970 ◽

Vol 28 ◽

pp. 114-115

Author(s):

P. A. Madden ◽

W. R. Anderson

Keyword(s):

Electron Microscopy ◽

Freeze Drying ◽

Room Temperature ◽

Secondary Electron ◽

Distilled Water ◽

Freeze Dried ◽

Silver Paint ◽

To Come ◽

The intestinal roundworm of swine is pinkish in color and about the diameter of a lead pencil. Adult worms, taken from parasitized swine, frequently were observed with macroscopic lesions on their cuticule. Those possessing such lesions were rinsed in distilled water, and cylindrical segments of the affected areas were removed. Some of the segments were fixed in buffered formalin before freeze-drying; others were freeze-dried immediately. Initially, specimens were quenched in liquid freon followed by immersion in liquid nitrogen. They were then placed in ampuoles in a freezer at −45C and sublimated by vacuum until dry. After the specimens appeared dry, the freezer was allowed to come to room temperature slowly while the vacuum was maintained. The dried specimens were attached to metal pegs with conductive silver paint and placed in a vacuum evaporator on a rotating tilting stage. They were then coated by evaporating an alloy of 20% palladium and 80% gold to a thickness of approximately 300 A°. The specimens were examined by secondary electron emmission in a scanning electron microscope.

Development of the Foramen Secundum in the Chick as Observed by Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100091044 ◽

1976 ◽

Vol 34 ◽

pp. 212-213

Author(s):

M.J.C. Hendrix ◽

D.E. Morse

Keyword(s):

Electron Microscopy ◽

Procaine Hydrochloride ◽

Cardiac Anomaly ◽

Chick Embryos ◽

Congenital Cardiac Anomaly ◽

Septal Defects ◽

Critical Point Drying ◽

Atrial septal defects are considered the most common congenital cardiac anomaly occurring in humans. In studying the normal sequential development of the atrial septum, chick embryos of the White Leghorn strain were prepared for scanning electron microscopy and the results were then extrapolated to the human heart. One-hundred-eighty chick embryos from 2 to 21 days of age were removed from their shells and immersed in cold cacodylate-buffered aldehyde fixative . Twenty-four embryos through the first week post-hatching were perfused in vivo using cold cacodylate-buffered aldehyde fixative with procaine hydrochloride. The hearts were immediately dissected free and remained in the fixative a minimum of 2 hours. In most cases, the lateral atrial walls were removed during this period. The tissues were then dehydrated using a series of ascending grades of ethanol; final dehydration of the tissues was achieved via the critical point drying method followed by sputter-coating with goldpalladium.

The time course of calcium deposition in shells of the barnacle (Balanus amphititre amphititre) during cyprid-juvenile metamorphosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168621 ◽

1994 ◽

Vol 52 ◽

pp. 178-179

Author(s):

Nancy R. Wallace ◽

Craig C. Freudenrich ◽

Karl Wilbur ◽

Peter Ingram ◽

Ann LeFurgey

Keyword(s):

Electron Microscopy ◽

Time Course ◽

Vertical Plane ◽

Mantle Cavity ◽

Qualitative Assessment ◽

Calcium Deposition ◽

Free Swimming ◽

Freeze Dried ◽

The morphology of balanomorph barnacles during metamorphosis from the cyprid larval stage to the juvenile has been examined by light microscopy and scanning electron microscopy (SEM). The free-swimming cyprid attaches to a substrate, rotates 90° in the vertical plane, molts, and assumes the adult shape. The resulting metamorph is clad in soft cuticle and has an adult-like appearance with a mantle cavity, thorax with cirri, and incipient shell plates. At some time during the development from cyprid to juvenile, the barnacle begins to mineralize its shell, but it is not known whether calcification occurs before, during, or after ecdysis. To examine this issue, electron probe x-ray microanalysis (EPXMA) was used to detect calcium in cyprids and juveniles at various times during metamorphosis.Laboratory-raised, free-swimming cyprid larvae were allowed to settle on plastic coverslips in culture dishes of seawater. The cyprids were observed with a dissecting microscope, cryopreserved in liquid nitrogen-cooled liquid propane at various times (0-24 h) during metamorphosis, freeze dried, rotary carbon-coated, and examined with scanning electron microscopy (SEM). EPXMA dot maps were obtained in parallel for qualitative assessment of calcium and other elements in the carapace, wall, and opercular plates.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155657 ◽

1989 ◽

Vol 47 ◽

pp. 736-737

Author(s):

T. Inoué ◽

H. Koike

Keyword(s):

Electron Microscopy ◽

Low Temperature ◽

Liquid Nitrogen ◽

Specimen Preparation ◽

Chemical Fixation ◽

Brown Adipose ◽

Critical Point Drying ◽

Temperature Scanning ◽

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100145820 ◽

1986 ◽

Vol 44 ◽

pp. 902-903

Author(s):

Alan Beckett

Keyword(s):

Electron Microscopy ◽

Low Temperature ◽

Conformational Changes ◽

Chemical Vapour ◽

List Type ◽

Dimensional Changes ◽

Critical Point Drying ◽

Temperature Scanning ◽

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

Facile preparation of nanofiller-paper using mixed office paper without deinking

TAPPI Journal ◽

10.32964/tj14.3.167 ◽

2015 ◽

Vol 14 (3) ◽

pp. 167-174 ◽

Cited By ~ 8

Author(s):

QIANQIAN WANG ◽

J.Y. ZHU

Keyword(s):

Cellulose Nanofibrils ◽

Ash Content ◽

Raw Material ◽

Mechanical Grinding ◽

Microscope Imaging ◽

Facile Preparation ◽

Electron Microscope Imaging ◽

Thermal Stability Of ◽

Scanning Electron ◽

Office Paper

Mixed office paper (MOP) pulp without deinking with an ash content of 18.1 ± 1.5% was used as raw material to produce nanofiller-paper. The MOP pulp with filler was mechanically fibrillated using a laboratory stone grinder. Scanning electron microscope imaging revealed that the ground filler particles were wrapped by cellulose nanofibrils (CNFs), which substantially improved the incorporation of filler into the CNF matrix. Sheets made of this CNF matrix were densified due to improved bonding. Specific tensile strength and modulus of the nanofiller-paper with 60-min grinding reached 48.4 kN·m/kg and 8.1 MN·m/kg, respectively, approximately 250% and 200% of the respective values of the paper made of unground MOP pulp. Mechanical grinding duration did not affect the thermal stability of the nanofiller-paper.