Microscopy and staining of bacteria

Bacterial cells contain approximately 80% water by weight and this accounts for their very low refractility, i.e. they are transparent when viewed under ordinary transmitted light.

Consequently, in order to visualize
bacteria under the microscope, the cells must be killed and stained with some compound that scatters the light or, if live preparations are required, special adaptations must be made to the microscope.

Such adaptations are found in phase-contrast, dark-ground and differential-interference contrast microscopy.

The microscopic examination of fixed and stained preparations is a routine procedure in most laboratories, but it must be appreciated that not only are the cells dead but they may also have been altered morphologically by the often quite drastic staining process.

The majority of stains used routinely are
basic dyes, i.e. the chromophore has a positive charge and this readily combines with the abundant negative
charges present both in the cytoplasm in the form of nucleic acids and on the cell surface.

These dyes remain firmly adhered even after the cells have been washed with water.

This type of staining is called
simple staining, and all bacteria and other biological material are stained the same colour.

Differential staining is a much more useful process as different organisms or even different parts of the same cell can be stained distinctive colours.

To prepare a film ready for staining, the glass microscope slide must be carefully cleaned to remove all traces of grease and dust.

If the culture of bacteria is in liquid form, then a loopful of suspension is
transferred directly to the slide.

Bacteria from solid surfaces require suspension with a small drop of water
on the slide to give a faintly turbid film.

A common fault with inexperienced workers is to make the film too thick. The films must then be allowed to dry in

When thoroughly dry, the film is fixed by passing the back of the slide through a small Bunsen flame until the area is just too hot to touch on the palm of the hand.

The bacteria are killed by this procedure
and are also stuck onto the slide. Fixing also makes the bacteria more permeable to the stain and inhibits lysis.

Chemical fixation is commonly carried out using formalin or methyl alcohol; this causes less damage to the specimen but tends to be used principally for
blood films and tissue sections.

Differential stains

A large number of differential stains have been developed, and the reader is referred to the bibliography for more details.

Only a few of those available will be discussed here.

Gram stain. By far the most important in terms of use and application is the Gram stain, developed by Christian Gram in 1884 and subsequently modified.

The fixed film of bacteria is flooded initially with a solution of methyl violet.

This is followed by a solution of Gram’s iodine, which is an iodine– potassium iodide complex acting as a mordant, fixing the dye firmly in certain bacteria and allowing easy removal in others.

Decolourization is achieved with either alcohol or acetone or mixtures of the two.

After treatment, some bacteria retain the stain and appear dark purple and these are called Gram positive.

Others do not retain the stain and appear colourless (Gram negative). The colourless cells may be stained with a counterstain of contrasting colour, such as 0.5% safranin, which is red.

This method, although extremely useful, must be used with caution as the Gram reaction may vary with the age of the cells and the technique of the operator.

For this reason, known Gram-positive and Gram-negative controls should be stained alongside the specimen of interest.

Ziehl–Neelsen acid-fast stain.

The bacterium responsible for the disease tuberculosis (Mycobacterium tuberculosis) contains within its cell wall a high proportion of lipids, fatty acids and alcohols, which render it resistant to normal staining procedures.

The inclusion of phenol in the dye solution, together with the application of heat, enables the dye (basic fuchsin) to penetrate the cell and, once attached, to
resist vigorous decolourization by strong acids, e.g. 20% sulphuric acid.

These organisms are therefore
called acid fast. Any unstained material can be counterstained with a contrasting colour, e.g. methylene blue.

Fluorescence microscopy

Certain materials when irradiated by short-wave radiation (e.g. UV light) become excited and emit visible light of a longer wavelength.

This phenomenon is termed fluorescence and will persist only for as long as the material is irradiated.

A number of dyes have been shown to fluoresce and are useful in that they tend to be specific to various tissues, which can then be demonstrated by UV irradiation and subsequent fluorescence of the attached fluorochrome.

Coupling antibodies to the fluorochromes can enhance specificity, and this technique has found wide application in microbiology.

As with the staining procedures described earlier, this technique can only
be applied to dead cells.

The three following techniques have been developed for the examination of living organisms.

Dark-ground microscopy

The usual function of the microscope condenser is to concentrate as much light as possible through the specimen and into the objective lens.

The dark-ground condenser performs the opposite task, producing a hollow cone of light that comes to a focus on the specimen.

The rays of light in the cone are at an oblique angle, such that after passing across the specimen, they continue without meeting the front lens of the objective, resulting in a dark background.

Any objects present at the point of focus scatter the light, which then enters the objective and shows up as a bright image against the dark background.

Specimen preparation is critical, as very dilute bacterial suspensions are required, preferably with all the objects in the same plane of focus.

Air bubbles must be absent from both the film and the immersion oil, if used.

Dust and grease also scatter light and
destroy the uniformly black background required for this technique.

With this technique it is not possible
to see any real detail but it is useful to study motility.

Phase-contrast microscopy

This technique allows us to see transparent objects well contrasted from the background in clear detail and is the most widely used image-enhancement method in microbiology.

In essence, an annulus of light is produced by the condenser of the microscope and focused on the back focal plane of the objective, where a phase plate, comprising a glass disc containing an annular depression, is situated.

The direct rays of the light source annulus pass through the annular groove and any diffracted rays pass through the remainder of the disc.

Passage of the diffracted light through this thicker glass layer results in retardation of the light.

This alters its phase relationship to the direct rays and increases contrast.

Differential-interference contrast microscopy

This method uses polarized light and has other applications outside the scope of this chapter, such as detecting surface irregularities in opaque specimens.

It offers some advantages over phase-contrast microscopy, notably the elimination of haloes around the object edges, and enables extremely detailed
observation of specimens.

It does, however, tend to be more difficult to set up.

Electron microscopy

The highest magnification available using a light microscope is approximately ×1500.

This limitation is imposed not by the design of the microscope itself,
as much higher magnifications are possible, but by the wavelength of light.

An object can only be seen if it causes a ray of light to deflect. If a particle is
very small, then no deflection is produced and the object is not seen.

Visible light has a wavelength
between 0.3 µm and 0.8 µm, and objects less than 0.3 µm will not be clearly resolved, i.e. even if the magnification were increased no more detail would
be seen.

In order to increase the resolution it is
necessary to use light of a shorter wavelength, such as UV light. This has been done and resulted in some
useful applications but generally, for the purposes of increased definition, electrons are used and they can be thought of as behaving like very short wavelength light.

Transmission electron microscopy requires the preparation of ultrathin (50 nm to 60 nm) sections of material mounted on grids for support.

Because of the severe conditions applied to the specimen during preparation, and the likelihood of artefacts, care must be taken in the interpretation of information from electron micrographs.

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