A procedure for handling microorganisms during preparation for electron microscopy

Abstract

There are a number of procedures for preparing bacteria and other small organisms for electron microscopy using various types of elaborate filter systems such as millipore or nucleopore filters (Watson et a1.,1980; Lloyd et a1.,1985).In some cases fixation and dehydration are performed in small vials or well slides, using a Pasteur pipette to withdraw solutions (Poirier et al., 1979); in other cases the material is centrifuged (Klainer and Betsch, 1970;Lloyd et al., 1985;Jones et al., 1986) before each change. This constant manipulation often results in broken cells, loss of cilia or flagella, and general damage to the tissue.In our laboratory we have used a relatively simple filter method for processing bacteria from colonies grown on agar in Petri dishes. Bacteria were grown on TrypSoy agar plates for 24 hours at 35 degrees centigrade. To each plate were added 7.0 ml. of 3.5% glutaraldehyde in 0.1M Na caccdylate buffer, pH 7.4.After 1 hour the fixative was remved by aspiration and the agar surface was rinsed with cacodylate buffer 3 times (5 min. each). The material was then postfixed in 1% osmium tetroxide under the fume hood for fifteen minutes. The plates were again rinsed 3 times with the cacodylate buffer. The surface growth from each plate was carefully scraped into a small cup made of lens paper ( fig. 1) and the cup was immersed in a glass vial containing 50% acetone. The cup was made by cutting a 7.5 cm. square of lens paper. The square was then folded diagonally in half. With the large angle of the resulting triangle at the bottom, each side with the acute angle was folded to the middle of the opposite side. angle were separated and folded down. The flaps that were folded down were then inserted between the trm layers of lens paper. Using a pair of forceps the lens paper cup was passed through a graded series of acetone to absolute acetone. After dehydration the lens paper cup together with the tissue was embedded in araldite. The size of the lens paper cup used for processing tissue for transmission electron microscopy would depend on the size of the embedding mold.In processing tissue for scanning electron microscopy, after dehydration, the top t m centimeters of the cup were remved and the cup was closed by folding down the top to prevent loss of cells during critical point drying. The cup was then transferred to a Polaron critical point drier and dried with carbon dioxide. Follawing critical point drying the lens paper cup was opened and an aluminum stub coated with silver paint, carbon paint, or double stick tape, was irranediately inverted onto the dried bacterial cells. A l l three conductive adhesives worked equally well.The cells were gold-palladium coated in an IS1 sputter coater and examined with an Hitachi S-510 scanning

Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy

“…Several techniques have been developed for preparing sperm (Baccetti and Burrini, 1973;Gould, 1973;Schneider, 1973; Van der Horst and Cross, 19781, bacteria, and other small organisms for scanning electron microscopy (SEM) (Griffin et al, 1987;Klainer and Betch, 1970;Poirier et al, 1979). Most techniques for mammalian sperm involve repeated washing to dilute/remove the viscous and granular secretions in the ejaculate, particularly those originating from the prostate.…”

Rapid Communication. A Micro‐sample critical point drying device for small SEM and TEM specimens

“…Microstik [Ted Pella, Inc.]), (Whitcomb, 1985) charge-binding substrates like poly-L-lysine, (Mazia et al, 1975) various plastic substrates including Formvar, or sample entrapment devices like Millipore, Nucleopore and paper filters (Griffin et al, 1987). These methods have the advantage of ease in handling; however, each substrate deforms the specimen to some degree in order to bind it effectively.…”

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Accomplishing effective critical point drying (CPD) on small samples for SEM and TEM is typically accomplished by attaching the sample to some form of substrate using adhesives like "grid glue" (ethylene dichloride-extracted cellophane tape adhesive) or other proprietary formulas (e.g. Microstik [Ted Pella, Inc.]), (Whitcomb, 1985) charge-binding substrates like poly-L-lysine, (Mazia et al, 1975) various plastic substrates including Formvar, or sample entrapment devices like Millipore, Nucleopore and paper filters (Griffin et al, 1987). These methods have the advantage of ease in handling; however, each substrate deforms the specimen to some degree in order to bind it effectively.

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