Basic Methods for Examination of Microbial Morphology

Morphological traits of bacterial cells (their sizes, shapes, details of inner structure, etc.) are studied by versatile methods of microscopy.

The efficacy of any microscopical technique is actually determined by its resolving power (resolution of microscope).

Resolving power is the minimal distance that yet allows to distinguish 2 objects of microscopy as 2 separate images in the field of view.

Resolving power of optical microscopes, where the visible light is collected, is described by the classical formula of E. Abbe:

d = λ / 2n*sinα,

where d is the resolving power of microscope, λ designates the wavelength of light, collected by microscope objective lens, sinα is the angular aperture of objective lens, n is the refractory index of immersion medium for objective lens.

According to that ratio, the maximum resolving power of conventional optical (light) microscope should be around 200 nm. It is approximately equal to the half of light wavelength that is used for microscopy. In case of violet component of visible light with the shortest wavelength of 400 nm it brings resolution to its theoretical value of about 200 nm.

Below this distance, the diffraction of visible light on the examined objects limits the further increase of resolving power.

Standard bright field microscopes used in bacteriology are usually equipped with 100-power objective lenses and 10 power eyepieces. Therefore, their total magnification is equal to 1000 times.

Current advanced optical systems with immersion objective lenses of power 150 give efficient resolution near 150 nm. The sizes of microorganisms can be determined by various measuring devices placed inside the eyepieces, e.g. ocular micrometer or reticle, containing scale or measuring grids.

The diagnostic value of bright field microscopy in bacteriology is improved with a great number of staining methods.

Various organic dyes (acid, basic, or neutral) can be employed here. They bind to certain microbial structures by physical and chemical interactions (ionic, hydrophobic, covalent and others).

Simple stain technique involves a single dye (methyl violet, methylene blue, fuchsin, etc.).

Differential stain presumes the use of two and more dyes. They stain distinct parts of bacterial cells (tinctorial properties of bacteria) thus fostering microbial discrimination.

Among these methods are Gram stain, Neisser’s volutin granules stain, Gin’s capsule stain, Ziehl-Neelsen acid-fast bacilli stain and many others.

Gram stain discriminates the differences in structure of bacterial cell wall. The method includes several steps: staining of flame-fixed slide with crystal violet (methyl violet, or gentian violet); treatment with Lugol’s iodine solution; ethanol decolorization; fuchsin counterstain.

Due to large amounts of negatively charged cell wall peptidoglycans and nucleoproteins gram-positive bacteria stain violet. They retain the first basic dye (crystal violet) in complex with iodine within their thick cell wall. Gram-negative bacteria stain pink as crystal violet is washed out by ethanol from the thin cell wall that is few of peptidoglycan, and bacterial bodies are counterstained with fuchsin.

Ziehl-Neelsen stain is used for special staining of acid-fast bacteria that carry exuberant amounts of lipids. Neutral lipids poorly absorb aniline dyes. Great lipid contents (mycolic acids, waxes) is typical for acid-resistant mycobacteria (M. tuberculosis, M. leprae), and actinomycetes.

The slide is exposed to Ziehl carbol fuchsin and steamed upon burner until vapor appearance. Then it is treated with sulfuric acid and counterstained with methylene blue solution.

Due to acid resistance and poor permeability for dyes, only acid-fast bacteria retain the primary red color whereas all other bacteria stain blue.

Romanowsky-Giemsa stain is the universal differential technique that is widely applied for discrimination of many bacteria (e.g., spiral-shaped borreliae, treponemas, or leptospira) as well as for protozoans, and mammalian cells (e.g., for blood cell count). It uses Romanowsky-Giemsa’s complex stain (mixture of azure, eosin, and methylene blue dyes).

Following this method, the examined bacteria stain violet-purple, protozoan nuclei red-violet, their cytoplasm – blue; mammalian cell nuclei – red, their cytoplasm – blue.

The results of other differential stains (e.g., Ozheshko spore stain, Neisser’s stain for volutin granules, or Gin’s stain for capsule) provide the identification of these optional microbial structures among various groups of bacteria (see above for details).

Standard staining methods usually operate with fixed inactivated specimens that don’t allow to observe the functional activity of bacteria. This is possible only by studying live bacterial cells within native (not fixed) specimens.

For examination of semi-transparent bacterial cells without staining, dark-field microscopy is broadly used. It is performed on the base of conventional bright field microscope supplied with special condenser. Dark-field condenser blocks the rays that directly come to the aperture of objective lens along optical axis of microscope. Other light rays, mirrored by condenser, pass through focal plane of the slide at large angles, thus missing objective aperture as well. As the result, the field of view becomes dark. When semi-transparent small objects (e.g., native bacterial cells) are placed in focal plane, the oblique rays will be reflected into objective lens by microbial bodies making bacteria visible. For instance, dark-field microscopy can easily determine motile thin and long bacteria, such us numerous species of spirochetes (treponemas, leptospirae, and borreliae) that are in width less than 0.2 μm.

Another powerful and reliable method for visualization of living bacterial cells is phase contrast microscopy. This technique exploits bright field optical microscopes equipped with special phase contrast devices. This device is based on the principle of light phase shift when the light passes through media with unequal refractive indices – bacterial cells and aqueous microbial surroundings, cellular cytoplasm and more dense nucleoid or nucleus. Phase contrast device transforms the shift of phase of light into differencies in light intensity.

Phase contrast microscopy permits direct observations of complex bacterial processes, such as growth and reproduction. Also it makes possible the study of internal structures of bacterial cells.

Another highly efficient method for observation of living unstained microbial objects was devised on the base of polarization contrast principle. This method of differential interference contrast (or DIC) detects the alterations of polarized light beams when they pass through non-homogenous transparent structures. Microscopy with DIC creates optical images with 3D-like reliefs. Also it allows optical sectioning of thick non-stained specimens.

High perspectives are related with luminescent microscopy. This technique uses the vast number of luminescent dyes for micobial stain (e.g., fluorescein, acridine orange, auramine, rhodamine, ethydium bromide, SYBR Green, the dyes of Alexa Fluor family and many others). Such dyes easily stain either fixed or vital (native) specimens of bacterial or mammal cells allowing the study of bacterial physiology and pathology.

The method exploits luminescent microscope with UV-source of light for excitation of dye fluorescence. After excitation, the dye begins to emit fluorescence of longer wavelength. The method provides differential staining of various microbial structures (nucleoid, volutin granules, spores and others).

Current striking advances of luminescent microscopy primarily ensue from the employment of laser as the source of illuminating light. Laser beams are easily controlled and can be focussed within minimal volume. The advantages of this technology were realized in the method of laser scanning confocal microscopy (LSCM).

The microscope for LSCM uses lasers of various wavelengthes for excitation of object fluorescence. Every moment of time the laser stimulates emission of fluorescence in certain point of stained specimen. The emitted light is collected by objective lens and then passes to the fluorescence detector. In the center of optical path before the detector, a small pinhole (or confocal diaphragm) is situated. It allows to pass further only the light generated directly in point of laser excitation within focal plane. This greatly reduce the size of analyzed specimen point and brings LSCM resolution closer to theoretical limit.

Laser beam of LSC microscope rapidly scans one horyzontal plane of specimen and creates its computer optical image. Then it moves along the vertical axis and repeats the operation. Computer analysis of accumulated images (image stack) generates real 3D reconstruction of microscopical objects. Unlike any other method, LSCM provides real-time 3D-scanning of large living microbial communities like biofilms. For instance, it enables to trace bacterial behaviour within biofilms including their complex interactions with antibiotics and other biocides.

Current developments of laser flourescent microscopical technologies open new horizons in all fields of modern cytology and microbiology. The most advanced novel methods created opportunities to overcome resolving power limitations that are essential for standard optical microscopes.

As an example, stimulated emission-depletion fluorescent microscopy (STED-microscopy) has seriously higher resolution equal to ~60 nm. Similarly, high efficacy is characteristic for multiphoton fluorescent microscopy. All these methods pertain to superresolution light microscopy.

Laser confocal microscopy with technology of fluorescence resonance energy transfer (or FRET) makes possible to analyse direct interactions of molecules within living cells (e.g., toxins and their receptors), calculating distances between active reactants (“nanometric ruler” function).

Nevertheless, the profound study of intimate details of microbial structure on molecular and atomic levels requires devices and facilities, operating on principles other than optical microscopy.

Classical technology that demonstrates superior resolving power in comparison with all other methods is electron microscopy.

The extremely high resolution of electron microscope ensues from the situation that the wavelength of electrons is substantially shorter than the wavelength of photons of visible light.

There are two basic kinds of electron microscopes: the transmission electron microscope (ТЕМ), and the scanning electron microscope (SEM).

TEM electron microscopy uses the beam of electrons expelled from an electron gun under the high voltage. The electron beams are oriented and focused by the electromagnetic condenser lenses onto a specially prepared thin specimen.

After differential scattering of emitted electrons on atomic and molecular groups of the specimen, some quota of electrons passes across the specimen, depending on its local densities. Such electrons are collected and focused by electromagnetic lens of an objective. This creates the image of the specimen that is further processed with the projector electromagnetic lenses for additional magnification. The visualization of an image can be performed with fluorescent screen that emits the light under electron strikes. ТЕМ efficient resolution operates in the range of 0,1-1 nm (at nanometer or angstrem scales). As the result, even small viruses with their diameters of 30-50 nm can be easily detected and characterized.

SEM electron microscopy gains three-dimensional images of microscopical objects. However, this technique demonstrates generally lower resolution than transmission electron microscopy.

Possibility of 3D-imaging arises from highly precise focusing of electrons within a smallest point on specimen surface at time of scanning. The electrons, passing through the surface of investigated objects, generate various forms of secondary radiation (e.g., secondary electrons) that can be registered, amplified and analyzed with subsequent computer reconstruction of 3D object image.

Despite superior resolution of electron microscopy, this technology has essential limitations, as it operates only with artificially modified fixed objects.

Atomic force microscopy (AFM) allows to perform real-time study of live microorganisms at high resolution that might be comparable with scanning electron microscopy.

Unlike all previosly mentioned techniques, AFM doesn’t use any kind of radiation (e.g., light or electron beam) to create object image.

AFM estimates the minimal physical forces that arise from AFM-scanning of micoscopical objects on ultra-low distances. These forces act between the surface of studied object and the tip of extremely sensitive sensor of atomic force microscope (termed as cantilever).

Laser beams control the position of the sensor (cantilever), when it scans the surface of the specimen. At the end of scanning the detailed 3D-image of the shapes of microscopical objects is generated with resolution near to molecular level.

AFM can study live bacterial cells and viruses in conditions, closer to their natural surroundings. It makes possible to visualize the essential long-term events of microbial life cycle – bacterial division, spore formation, viral entry and reproduction and others.

In addition, AFM provides exclusive data about the state of bacterial external structures – cell wall architecture, cytoplasmic membrane viscosity and fluidity, organization of bacterial flagella and pili, binding activities of adhesins, clustering of surface proteins, etc.

Future perspectives are related with the combination of advantages of atomic force microscopy and advanced methods of fluorescent light imaging.