Part II: Microscopy
smear examination procedures

Because the human eye cannot see objects with a diameter of less than 0.1mm, individual bacteria can only be detected with a microscope. The function of the magnifying lens system of the microscope is to magnify the objects within the microscopic field to a size which can be detected by the human eye. In addition to magnification, two other factors, contrast and resolution, are of great importance. In order to be perceived through the microscope, an object must possess a certain degree of contrast with its surrounding medium: in order to produce a clear magnified image, the microscope must possess a resolving power sufficient to permit perception of individual objects. The degree of contrast can be greatly increased by staining procedures: treatment with dyes that bind selectively either to the whole cell or to certain cell components. The acid-fast stain is used to obtain information on the composition of the cell walls of tubercle bacilli. The types of microscopy most commonly used for observing acid-fast bacilli are bright-field and fluorescence microscopy. In bright-field microscopy the light passes through the bacilli and the variation in colour due to staining show the form of the organisms. Fluorescence stains are usually organic substances which absorb ultraviolet light and remit part of the energy as light of longer wavelength which can be observed through the eyepiece as fluorescence. When exposed to ultraviolet light, the fluorescent bacilli are perceived as brightly coloured organisms against a dark background.

A binocular microscope (ie. one with two eye pieces) is recommended for the examination of stained smears. If a monocular microscope is used, both eyes should be kept open while looking into the eyepiece, to prevent eye fatigue. If no electricity is available, daylight must be used as light source and the table with the microscope be placed in front of a window.

Components of the microscope
The components of the microscope are illustrated in Figure 3.

Mechanical part
The base or foot of the microscope should be sufficiently heavy to be stable as it supports the stage on which the slide is placed. The slide is kept steady by clips attached to the stage; movements are guided by a micrometric screw with vernier to help relocate a field of examination.

The limb supports the eyepiece holder and the objective holder. In the monocular microscope the two devices are separated by a tube with the distance between the eyepiece and the objective approximately 170mm. In binocular microscopes the tube is replaced by a system of prisms.

In the monocular model, the eyepiece holder is a simple tube, while in the binocular model, the prisms are placed within the eyepiece holder. The two eyepieces can be moved to adjust to the distance between the observer?s pupils; one of the eyepieces moves around its axis to correct any difference in convergence between both eyes.

The objective holder can be rotated as well as replaced, if necessary.

Optical outfit
The mirror, which has two faces, plane and concave, sends parallel rays from the light source to the optical axis of the microscope.

The light source is usually incorporated in the foot of the microscope. The fixed light source keeps a constant distance and alignment. Usually small halogen bulbs of great intensity are used. Tungsten filament lamps can also be employed.

The condenser is a lens that focuses the light on the slide. It is centered by moving the centering screws until the iris is concentric with the opening of the back of the objective. The greater the aperture of the diaphragm the wider the angle of vision. The light opening must be smaller than the opening of the objective used.

Objectives
Objectives are classified according to their numerical aperture (NA) and magnification. The NA describes the properties of the objective lens. Up to a certain limit an increase in the NA of the objective lens increases resolving power.

The passage of light through curved lenses produces the separation of the spectrum in the different wave lengths. This results in coloured bands in the periphery of the lens due to a chromatic aberration. The greater the magnification, the greater the aberration will be. The different colours (wave lengths) do not focus on the same place, producing spherical aberration. These are compensated for by using a combination of lenses. Achromatic objectives are corrected for two colours of the chromatic aberration (yellow and green) and for one of the spherical aberration. Apochromatic objectives do not present chromatic aberrations throughout the whole scale of visible spectrum provided there is an optimal distance between the object and the lens of the objective. They are classified by distance in mm or fractions of mm. They give clearer and crisper images than the achromatic lenses and can be used with higher power oculars. The flat apochromatic lenses have a correction for spherical aberration.

Immersion objectives
Dry objectives are those where the front lens is in contact with no other medium than the air (refraction index nil). The refraction index of glass (as used for microscope slides) is 1.51. As a result, light rays are refracted in passing from the glass to the air; some fall beyond the visual scope of the objectives. With oil-immersion objectives, the oil is used to fill the space between the front lens and the slide. To achieve high magnification, immersion oil is placed between the slide and the immersion objective lens. Unlike air, immersion oil has the same refractive index as glass and consequently improves the resolving power of the lens.

The eyepiece or ocular is composed of two lenses mounted on two ends of a tube. The lower lens is called the collector or field glass; it flattens and clears the real image and therefore completes the action of the objectives. The upper lens enlarges the image and therefore gives a virtual image of the object.

In brief, the objective produces an image of the preparation inside the microscope, and the observer looks at the image through the eyepiece. To form a clear image the lens must focus each point in the slide to give a point in the image.

Use of the microscope

• Check for broken or damaged parts
• Ensure that the light source is well regulated and focused
• Ensure that the lenses, mirrors and other light-conducting surfaces are clean
• Ensure that the condenser is in the upper position with the  diaphragm open
• Adjust the light, mirror, condenser and diagram so that a  strong beam of light is directed towards the objective lens
• Turn the coarse adjustment knob to move the objective lens away from the stage
• Rotate the nosepiece so that a low power objective lens (5x  or 10x) is directly over the condenser
• Place the slide on the stage so that the smear is directly under the objective lens
• Look from the side of the stage to observe the space between the slide and the objective lens. Slowly turn the coarse focus knob to bring the objective  lens close - but not touching - the smear
• Adjust the light intensity so that it is bright but not  uncomfortable when looking into the eyepiece. This may be done by changing the intensity  of the lamp, changing mirror surfaces, using dark filters, adjusting the diaphragm or  adjusting the condenser. Usually, adjusting the diaphragm is sufficient
• While looking into the eyepiece, slowly turn the coarse  focus knob to separate the objective lens and the stage. The smear should come into focus within a few turns
• Turn the fine focus knob until the smear is seen most  clearly
• While looking from the side, turn the nosepiece to select a  higher power lens. Ensure that the lens does not touch the slide. The smear should almost  be in focus. The best focus can be achieved by adjusting the fine focus knob. A light adjustment may also be helpful
• To use the oil immersion lens, turn the nosepiece so that  the lens is over the smear. Put a drop of immersion oil on the smear - do not touch the slide with the oil applicator but allow the drop of oil to fall freely onto the smear
• Lower the 100x immersion lens so that it comes into contact  with the oil. Slowly bring the immersion lens upwards until the image of the smear appears. Adjust the fine focus knob to focus

Examination procedures
To obtain excellence in microscopic examination, a good microscope and a comfortable work area is required. Reading of smears must be systematic and standardised to ensure that a representative area of the smear is examined. To ensure that an area is covered only once, the smear should be examined in an orderly manner and the following procedure is recommended:

• Always examine carbolfuchsin-stained smears with a 100x oil immersion objective
• Examine fluorochrome-stained smears within 24 hours of staining as the fluorescence may  fade with time. Smears that cannot be examined immediately after staining should be kept in the dark, preferably in a refrigerator, for a maximum of 24 hours
• If possible include a known positive slide and a known negative slide with each day?s work. The positive control ensures the staining capability of the solutions and of the staining procedure. The  negative control confirms that acid-fast contaminants are not present in the stains or in  other solutions
• Make a series of systematic sweeps over the length of the smear. After examining a microscopic field, move the slide longitudinally so that the neighbouring field to the right can be examined. Search each field thoroughly
• Examine a minimum of 100 fields before the smear is reported as negative. For a skilled  microscopist this will take approximately five minutes. In a smear of 1.5cm x 1.5cm the  number of microscopic fields in one length of the slide corresponds to around 100. If the smear is moderately or heavily positive fewer fields may be examined and a report of ?positive? may be made even though the entire smear has not been examined
• At the end of examination, take the slide from the microscope stage, check the identification number, and note the result. Dip the slide into xylene to remove the  immersion oil and place it in a box for examined slides
• Before examining the next slide, wipe the immersion lens with a piece of lens tissue
• Unexpected objects may be seen when using the microscope. If these objects move only  when the slide is moved, they may be materials occasionally found in the specimen or  object, precipitated fixatives or stains, contaminants from the stains or contaminants in  the immersion oil
• Artefacts that move only when the eyepiece is rotated are in the eyepiece or on its lenses. Artefacts may also be caused by material on the condenser lenses, mirror, or light source
• Keep all the slides for external quality control, according to established procedures (see I. Organisation and management).
• Analyse results on a weekly and monthly basis for percentage of positive results.  Investigate any sharp differences from the norm

Morphological characteristics of acid-fast bacilli

• Acid-fast bacilli are approximately 1-10Fm long and  typically appear as slender, rod-shaped bacilli, but they also may appear curved or bent
• With carbolfuchsin staining, tubercle bacilli look like fine red rods, slightly curved, more or less granular, isolated, in pairs or in groups,  standing out clearly against the blue background
• Individual bacteria may display heavily stained areas  referred to as ?beads? and areas of alternating stain may produce  a banded appearance
• With fluorochrome staining, tubercle bacilli are rod shaped  and emit a bright yellow fluorescence against a pale yellow (potassium permanganate) or  orange (acridine orange) background
• Some mycobacteria other than M. tuberculosis may  appear pleomorphic, ranging in appearance from long rods to coccoid forms, with more uniform distribution of staining properties
• Organisms other than mycobacteria may demonstrate various  degrees of acid-fastness. Such organisms include Rhodococcus spp., Nocardia spp., Legionella spp., and the cysts of Cryptosporidium and Isospora spp.
• Rapidly growing mycobacteria may vary in their abilities to  retain acid-fast stains

Causes of error in microscopy
Connected with the specimen

• Inadequate specimen quality and/or volume
• Inefficient washing of re-usable containers may leave residual bacilli which give rise  to false positives
• Carelessness in marking the container. Marking should be done on the body of the container and not on its lid

Connected with the preparation of the smear

• Insufficient or poorly lit work surface
• Mixing-up of slides
• Preparing too many slides at once. A maximum of 12 is  recommended
• Utilisation of slides that have been positive. These should  be discarded
• Specimen contamination due to careless use of loops, pipettes or wooden applicators

Connected with the staining

• Using scratched slides on which deposits of stain may look like bacilli
• Using unfiltered fuchsin which may contain crystals
• Carelessness in heating the fuchsin, allowing it to dry and  crystallise on the smear
• Inadequate decolourising of the smear which may leave red  stain on saprophytic bacilli which then appear to be acid-fast

Connected with the microscopic examination

• Failure to check the slides and renumber them should the number be obscured during  staining. This may lead to substitutions
• Failure to clean the immersion lens with lens tissue after each examination, especially  after a smear was found to be positive
• Presence of bacilli in the immersion oil owing to the practice of touching the smear with the neck of the bottle. A dropper bottle should be used and the drop allowed to fall  without any contact between bottle and slide
• Erroneous recording of the results

Troubleshooting guide for microscopy

Consequences of false-positive and false-negative smears
False-positive results

• Patients are started on treatment unnecessarily
• Tuberculosis medications are wasted
• In follow-up examinations the intensive phase of treatment is continued longer than  necessary
• Patients may loose confidence in the National Tuberculosis Programme

False-negative results

• Patients with tuberculosis are not treated, resulting in suffering, spread of  tuberculosis and death
• Intensive phase treatment is not extended for the required duration, resulting in inadequate treatment
• Patients may loose confidence in the National Tuberculosis Programme