Vitreous Carbon: A New Material for Making Microtome Knives

The quality of sections obtained by microtomy depends to a large extent on the quality and characteristics of the microtome knife itself. Despite the need for improved microtomy techniques, there have been few significant developments since the introduction of glass and diamond knives in the 1950's. The manufacture of microtome knives from vitreous carbon provides new possibilities for developing both improved methods and improved equipment for specimen sectioning. Vitreous carbon has unique physical properties that lend themselves to the generation of precision cutting edges. Such an edge can be obtained either by breaking a piece of vitreous carbon or by using lapidary techniques. The resultant edge seems well adapted to both thick and thin sectioning. The introduction of vitreous carbon as a sectioning tool offers a significant alternative to metal, glass and diamond knives.     

Adaptation of a sliding microtome for sectioning of material embedded in epoxy resins]

The employment of a sliding microtome of sectioning plastic embedded material with glass knives is described. Using a new knife holder and a modificated device for fixing plastic blocks succeeded in cutting sections 1--10 micron thick of relatively large pieces of tissue.     

Precision Sectioning of Wood

In discussing the theories and mechanism of sectioning, it is pointed out that virtually no concrete knowledge exists on the subject. The function of different mechanisms and their role in cutting different types of tissue, and the value of microtome knives versus safety razor blades is discussed. Razor blades failed to cut precise sections; a technic for sharpening a knife to give precise sections is outlined, pointing out errors to avoid in sharpening and sectioning. Various types of knife edges are illustrated by photomicrographs. The procedure of sharpening and sectioning technics for critical results is explained. The mechanism of cutting in wood appeared to be of the crushing and tearing type, indicating the necessity of final polishing in sharpening.     

Improved Methods for Molding, Facing and Sectioning Glycol Methacrylate Embedded Tissue Blocks

Improvements in glycol methacrylate embedding, block facing, trimming, and sectioning are described. The improvements are derived from a novel molding system, a multipurpose instrument for rapid block facing, trimming and examination, and a device for removing unwanted sections from the microtome knife while sectioning is in progress. Together, these methods facilitate specimen preparation and result in a significant reduction of the time required to prepare high resolution, very thin sections for light microscopy.     

Contributions to Semithin Sextioning on A Conventional Rotary Microtome

Procedures for obtaining sections 1 μ thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen bolder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections I micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

Contributions to semithin sectioning on a conventional rotary microtome.

Procedures for obtaining sections 1 micrometer thick on a conventional rotary microtome are described. Hydrophilic resin blocks with adequate hardness and elasticity for semithin sectioning are made by addition of divinylbenzene and methylmethacrylate to a commercial embedding kit. The blocks are pinched between two simple adapters and mounted in the specimen holder of a microtome. A glass knife of the Ralph type with an effective blade length of 25 mm is made from a glass slide and attached to a metal bar with paraffin. The low cost assembly is set in the steel knife holder of a conventional rotary microtome. Sections 1 micron in thickness can be cut from the resin embedded blocks. Staining with the usual staining solutions may be weak due to the thinness of the sections, but the fine resolution and low distortion achieved are compensating gains.     

Glass Knife Adapter for Cutting Epoxy Sections on a Conventional Rotary Microtome

Electron microscopy-style fixation followed by epoxy plastic embedding is often now the method of choice for preparing tissue even for light microscopy; I have found it excellent for fluorescence, autoradiographic and conventional histology (Shaw 1972, 1977). Sections more than about five microns thick can be cut on a really sharp steel knife if the plastic is reasonably soft (Stretton and Kravitz 1973, Shaw 1972), but this is much easier and knife marks are reduced if extra-wide glass knives are used on a special-purpose intermediate microtome like the Sorvall JB-4. Recent budgetary restrictions made us defer purchase of such a microtome, and some alternative had to be devised. I report here a simple but rugged adapter for glass knives which replaces the steel knife in a conventional Leitz rotary microtome and allows thin plastic sections to be cut as easily as with a more sophisticated cutter. It could be adapted for any rotary microtome, and can be readily constructed in most machine shops for negligible cost.     

Improved Paraffin Sectioning with Etched Microtome Knife Edges

Carbon steel microtome knives etched with 0.1 N HNO₃ for 2 min display a very sharp cutting edge. Over a period of 3 yr no damage to the steel has been detected. The effect on paraffin sectioning was observed by comparing acid-treated knives with nonetched but well-sharpened ones. Sections of whole eyes cut with an etched blade showed approximately 15% less compression of the parffin matrix than those sectioned with an untreated knife. Tissues selected from routine autopsy material presented approximately 9% reduction in compression. As a result, excellent ribbons of sections could be cut from 5-7 μ and floated on water at 42—46 C with a minimum of folds or distortion. Etching improved sectioning when knife edges having bevel angles in a range of 31-39° were used, and also when the bevel was decreased to 20°, but the 20° edge gave impractically short service.     

Serial sectioning techniques for a modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

A Motor-Controlled Device for Slow-Motion Cutting with the Ultramicrotome

The preparation of ultrathin sections of high quality with a Porter-Blum microtome requires that the drive wheel be rotated at a slow, uniform rate during actual cutting. A mechanical device to control the motion of the microtome drive wheel consists of a synchronous motor connected to a drive shaft (4 rev/min), which can be intermittently coupled to the rim of the microtome wheel by a friction drive of 1/2-inch diameter. The microtome drive wheel is rotated manually at a rapid rate until the specimen is about to come into contact with the knife; then the mechanical drive is engaged for slow movement during cutting. When the section has been cut, the drive is disengaged by a lever operated with the left hand; after which, the microtome wheel is turned quickly back to the cutting position with the right hand.     

A Motor-Controlled Device for Slow-Motion Cutting with the Ultramicrotome

The preparation of ultrathin sections of high quality with a Porter-Blum microtome requires that the drive wheel be rotated at a slow, uniform rate during actual cutting. A mechanical device to control the motion of the microtome drive wheel consists of a synchronous motor connected to a drive shaft (4 rev/min), which can be intermittently coupled to the rim of the microtome wheel by a friction drive of 1/2-inch diameter. The microtome drive wheel is rotated manually at a rapid rate until the specimen is about to come into contact with the knife; then the mechanical drive is engaged for slow movement during cutting. When the section has been cut, the drive is disengaged by a lever operated with the left hand; after which, the microtome wheel is turned quickly back to the cutting position with the right hand.     

A Ralph Knife Holder Assembly for Use with the Sorvall “Porter-Blum” MT-1 Ultramicrotome

The superiority of plastic embedding for the production of high quality sections for light microscopy is well known, but the use of conventional glass knives with a cutting edge of approximately 4 mm has severely restricted the size of specimens in the past. Ralph knives provide a much longer cutting edge and adapters are available for certain models of microtomes and ultramicrotomes. A modified knife holder for use with the Sorvall “Porter Blum” MT-2 microtome was described by Gorycki and Sohm (1979); however, this is not suitable for the MT-1 model. We have therefore designed and made an adapter which enables Ralph knives to be used with this instrument. The design allows approximately 18 mm of cutting edge to be used on each knife, allowing larger specimens to be sectioned than with a conventional glass knife and reducing the frequency with which the knife needs to be changed when working with smaller blocks.     

Serial Sectioning Techniques for A Modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing; serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

Interferometric analysis of intrasection and intersection thickness variability associated with cryostat microtomy

Summary Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10–20 m. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.     

A Method for the Preparation of Undecalcified Bone Sections for Light Microscopy and Microradiography

A method which gives good quality 1-2 μm thick sections of undecaldfied cancellous and thin cortical bones for light miuoscopy is described. Formalin fixed material is dehydrated in graded acetones and embedded in a modiEed formula of Spurr's low viscosity embedding medium. After a 16 hour polymerisation period at 60 C, sections are cut at 1-2 μm thickness on a Porter-Blum JB4A rotary microtome Using glass knives. Sections are attached to clean glass slides with heat, the resin degraded in bromine vapour and removed in acetone. This allows comparative ease of staining. The technique is rapid, does not interfere with tetracycline fluorescence and the same specimens can be used to prepare thick sections for microradiography.     

Improved Trough for Ultramicrotomy

Troughs made of metal or tape have been used to provide liquid reservoirs for ultrathin sectioning with glass knives (Hayat 1970). However, these types of trough have certain limitations. the metal trough is difficult to use and its paraffin seal is easily broken, causing leaks. Hayat(1970) has even suggested that fumes from heating this paraffin may be carcinogenic. the tape trough is easier to seal but deforms easily while sections are being collected and does not always provide an even surface for the observation of interference colors. This report describes a method of making inexpensive plastic troughs that can provide a reservoir comparable to those used with diamond knives.     

A Microtome Drive That Gives Either A Fast Cutting Stroke Or A Slow Stroke with Fast Return

The construction comprises a 0.3 hp electric motor, a drive mechanism which transforms the speed of the motor to the two actual cutting speeds, and a clutch mechanism which transmits the slow or the fast movement to the microtome. When the slow cutting speed is used the cam (on the microtome flywheel) and lever mechanism actuates the drive to give a quick return to the cutting position after a slow cutting act. This device, which needs little supervision, has given excellent serial sections, uniform both individually and with respect to others in the ribbon, with all the material on which it has been tested.     

The Use of Ethylenediamine in Softening Hard Plant Structures for Paraffin Sectioning

Ethylenediamine has been used as an agent for softening very hard woods prior to sectioning on a sliding microtome. The use of ethylenediamine is recommended for two additional uses: for preparing 1) soft woods in which wide, thin-walled tracheids or vessels tend to collapse during sliding microtome sectioning and 2) plant tissues with sclerenchyma mixed with soft-walled cells (bark, leaves, fruits, etc.) which frequently fail to section well. After softening in ethylenediamine, material is washed, infiltrated, and embedded in paraffin. Preliminary sections are made with a rotary microtome, just exposing the cut surface of the material; this exposed surface is soaked overnight in water. Sectioning is then continued. Sections produced in this fashion are considerably improved. The wood and pith of Podocarpus ustus, a parasitic conifer from New Caledonia, is used as an object to demonstrate improvements in sectioning by the ethylenediamine-paraffin method. Thinner sections with minimal tearing, cell collapse, and unevenness are produced. Sections can be handled easily and stained more effectively than unmounted sections. Variations in timing and in treatment are recommended to suit different materials. Ethylenediamine, used with reasonable caution, is much less hazardous than hydrofluoric acid and is more effective in softening plant material. The ethylenediamine method may be used routinely on any material difficult to section because of hardness.     

A SIMPLE MODIFICATION OF THE SPENCER ROTARY MICROTOME FOR CHUCK WITHDRAWAL ON THE UPSTROKE

A simple and accurate modification of the Spencer Model 820 rotary microtome is described in which the specimen is caused to withdraw from the knife on the upstroke. The modification, when used with glass knives, allows methacrylate-embedded tissues to be easily sectioned in the 1 to 5 β range. Interconvertibility from normal to modified operation is accomplished by simply turning a cam into one of two positions.