Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Summary

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Abstract

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Introduction

A scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate an image from secondary electrons that shows the morphology and topography of a sample¹. SEM can be used to directly determine the physical size of a sample, the surface structure, and the three-dimensional shape, and offers greater resolution and larger depth of field compared to light microscopy. Another form of electron microscopy (EM), transmission electron microscopy (TEM) uses focused electrons that pass through the sample, generating images with fine details of internal structure. While TEM has higher resolution than light or SEM and can be used....

Protocol

1. Preparation and Fixation

1. Prepare cyanobacteria.
   1. Grow unialgal cultures in F/2 media^24,25 at a temperature of 28 °C on a 14:10 h light dark cycle. Transfer sufficient culture to a 1.5 mL microcentrifuge tube such that after allowing 15 min to settle, the bacterial pellet is approximately 0.05 mL in size. Remove the media and replace with 1.5 mL of fixative (1.25% glutaraldehyde, 0.1 M phosphate buffer pH 7.0), gently invert several times, and incubate overnight at 4 °C.
   2. Transfer the fixed cells with a glass pipette into a 10 mL filtration rig (

Results

Cyanobacteria are a prokaryotic group of organisms that are critical to the global carbon, oxygen, and nitrogen cycles^28,29. Of the estimated 6000 species of cyanobacteria³⁰, most have a mucilaginous sheath that cover and connect the cells together and to other structures³¹, which along with the shape, can be resolved microscopically³². Cell size, shape, and p.......

Discussion

Here we described a protocol using SEM to obtain detailed information about external morphological characteristics of three types of organisms that others can apply to examine features of many types of organisms or tissues. Within each step of the protocol, there are potential points of error that may arise and are discussed in detail below.

While the volumes for fixative and washes given here are specific, in general the fixative and washes should be 5-10x the volume of the specimen. All fixa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
gold–palladium	Ted Pella, Inc.	91212
Silver conductive adhesive 503	Electron Microscopy Sciences	12686-15
Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 8 μm, polycarbonate	Sigma	WHA110614
Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.8 μm, polycarbonate, black	Sigma	WHA110659
Whatman Nuclepore Track-Etched Membranes; diam. 25 mm, pore size 0.2 μm, polycarbonate	Sigma	WHA110606
aluminum weighing dish	Fisher Scientific	08-732-100
aluminum mounting stubs (12 mm)	Electron Microscopy Sciences	75210
aluminum mounting stubs (25 mm)	Electron Microscopy Sciences	75186
Adhesive tabs	Electron Microscopy Sciences	76760
conductive carbon adhesive tabs	Electron Microscopy Sciences	77825
F/2 media	Culture Collection of Algae at the University of Texas at Austin	https://utex.org/products/f_2-medium	No Catalog number given - see link
AF6 media	Bigelow - National Center for Marin Algae and Microbiota	https://ncma.bigelow.org/media/wysiwyg/Algal_recipes/NCMA_algal_medium_AF6_1.pdf	No Catalog number given - see link
Soil water medium	Carolina Biological Supply Company	153785
Polyethylene glycol tert-octylphenyl ether (Triton X-100)	VWR	97062-208
Hummer 6.2 Sputter Coater	Anatech USA	http://www.anatechusa.com/hummer-sputter-systems/4	No Catalog number given - see link
Hitachi 3400N-II SEM	Hitachi	https://www.hitachi-hightech.com/us/product_list/?ld=sms2&md=sms2-1&sd=sms2-1 -2&gclid=EAIaIQobChMIpq_jtJfj3A IVS7jACh2mdgkPEAAYASAAEgKA nfD_BwE	The company doesn't appear to sell this model any longer