Sample preparation for microscopy

Microscopes are the instruments which enable us the observation of microscopic objects such as microorganisms, bacteria, viruses, fungi and protozoa. They are used for studying cellular organization of plant and animal tissues, cell division or mitosis-meiotic processes. Microscopes are employed for detection of pathological parameters such as presence of parasites in blood, morphological features of white and red blood cells. Importance of microscopical analyses in forensic and diagnostics is well known. In addition to these biological applications, microscopes have potential usefulness in sciences like nanotechnology, physics, chemistry, environment and metallurgy. Magnifying power of microscope is either derived from light or electrons. Depending upon the source of magnification, 2 types of microscopes: light (optical) and electron, are used by microbiologists. Both dead and living cells can be observed but needs to be prepared by specific microscopic techniques for observation under microscope. Let's see different types and modifications of specimen preparation techniques for microscopy.

1. Light microscopy preparation:
Various types of optical microscopes like bright field, dark field, phase contrast and fluorescence are employed for different purposes. Depending upon the type of microscope and application specimen or object is prepared for observation.

Bright field microscope- In this type, microscopic field appears bright against dark sample. Sample absorbs light and appears dark. This is essential for observation of microbial cells especially bacteria as they don't absorb much light. Increase in light absorption capacity of microbe is made possible by staining them with appropriate dyes. Three types of dyes, acidic (eosin, acid fuchsin), basic (crystal violet, methylene blue) and neutral (combined acidic and basic dyes) are used in the staining. Dye application is followed by treatment with mordant solution (iodine, tannic acid) which forms insoluble complex with dye and deposit it in cell membrane or cell wall. Sample smears are fixed by heat or formalin solution to prevent washing away by dye or water. Stained smears can also be permanently preserved by treating them with Canada balsam solution. Immersion oils are also used to increase resolving power and to decrease working distance of objective lens. Stained preparations are required as live bacteria don't reveal much structural details. The only drawback of staining is that samples cannot be observed in living state. Staining the cells also provides greater contrast and color differentiation required for observation. Different types of stains and staining procedures are available for microorganisms. Special staining techniques are employed to demonstrate external morphological features such as shape, cell arrangement, cell wall, flagella, pili, capsule and internal structures like endospores, cytoplasmic granules and nucleus of microbial cell. Coloring or staining involves steps like drying and fixing the smear followed by sequential treatment with dye or dye mixture, mordant, decolorizer and the counter stain. Simple stains such as methylene blue impart their color directly to the cell. Bacteria are generally stained with basic dyes as they have acidic cytoplasm. Some bacterial features such as capsules don't take up the dye, therefore they are negatively stained. For this, bacterial suspension is mixed with dye nigrosin or India ink, smeared and dried. Under microscope, cells appear as unstained bright bodies against dark colored background. Some bacteria (spirochetes) are not stained by simple stains, can be observed by negative staining. Very fragile structures like flagella are stained by dye impregnation. Since they are very thin, are treated with dye solution so as to allow deposition of dye on the structure. This renders staining as well as increase in thickness of that structure. Differential staining procedures like Gram reaction and acid fast staining impart different coloration to different bacteria or their structural components. Gram stain differentiates bacteria into 2 broad groups: Gram positive and Gram negative depending upon their difference in cell wall composition. Gram staining is very essential technique in identification and classification of bacteria. It is also an ideal example of staining method of bacteria and therefore felt mandatory to describe in this article. It was invented by the scientist Christian Gram in 1884. It involves 4 steps: First step is primary staining of smear with crystal violet solution. In second step, iodine solution is applied as mordant to fix primary stain into cell wall. Primary stain is decolorized with alcohol or acetone. Fourth step is counter staining by neutral red or safranine. Each step is carried out with definite time followed by intermittent washing steps in distilled water. Gram negative bacteria take the color of counter stain while Gram positive bacteria retain the violet color of crystal violet. Similarly like bacteria, yeasts, fungi, actinomycetes, algae, protozoa and special group of bacteria such as rickettsias are stained for microscopic studies.

Dark field microscope- In this microscopy, specimen appears bright against dark background and this is achieved by condenser of microscope. Dark field microscopy is used for observation of unstained samples. Specimen in their living state can be observed. For this, wet mount and hanging drop preparations are done. Microbial cell suspension is placed (as drop and not smear) on grease free cavity slide. Coverslip is placed and sealed with wax to avoid evaporation of suspension drop. Although, morphological characters are not revealed but individual cell movement like motility can be seen.

Phase contrast microscope- It is used to observe unstained cells and their cytological differences. Its working is based on principle that altered phase relations by illumination are induced by otherwise invisible elements in cytoplasm. Microscope is provided with phase contrast objective and special condenser. This enables to distinguish unstained structures within cell which differ only slightly in their refractive indices. Since no staining is required, chemical and morphological changes do not occur; living or static microbial cells can be observed.

Fluorescence microscopy- The working of florescence microscope is based on phenomenon of fluorescence. When a dye absorbs light of certain wavelength, also emits light of less energy and longer wavelength. In practice, microbes are stained with fluorescent dyes such as fluorescein or rhodamine. Routinely employed staining is fluorescent antibody technique (FAT), also known as direct immunofluorescence method. In this, fluorescent dye is combined with microbial cell antibodies; such labeled antibodies are mixed with microbial suspension and observed under fluorescence microscope. FAT has been very helpful in diagnostic pathology for identification of pathogens like Mycobacterium tuberculosis, Vibrio cholera, Neisseria meningitidis, Candida albicans, fecal coliforms and streptococci, pseudomonads, Polio and Rabies viruses.

2. Electron microscopy preparation: Electron microscopic techniques are considerably different from light microscopy. They give higher resolution and magnification using extremely narrow wavelength electron beam in presence of magnetic field. Transmission and scanning electron microscope have variable applications and sample preparation techniques. Some of them are described here.

Scanning electron microscope- Specimen is subjected to surface scanning by electron beam. Depending upon shape and chemical composition, irradiated specimen emits secondary electrons. It reveals surface topography and 3-D structure of the specimen.

Transmission electron microscope (TEM) - Specimens should be ultrathin to be observed by TEM. Even bacterial cells are very thick to observe intracellular details. Ultrathin sectioning is done by special technique. Bacterial cells are embedded in plastic block and cut into ultrathin slices (60nm). Sections are then stained by uranium or lanthanium salts. Since cells are sliced at different angles, every detail about structural organization is obtained. Unstained ultrathin sections can be observed by freeze fracturing. Here specimen is not stained but carbon replicas are prepared to reveal extracellular details. In another technique, specimen is dried on special copper grid and placed in vacuum. Atoms of heavy metal platinum are projected and deposited from angled highly heated filament on specimen. This is called shadow casting. TEM observation of shadowed image reveals details about shape of specimen. Negative staining similar to light microscopy but electron dense phosphotungstic acid is used as stain. It is used to observe fine details of viruses, flagella and pili.

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