Chapter Capsule

A. Microbiology as a science is very dependent on a number of specialized laboratory techniques. Laboratory steps routinely employed in microbiology are inoculation, incubation, isolation, inspection, and identification.

B. To prepare a culture (visible growth specimen), a medium (nutrient substrate) is inoculated (implanted, seeded) with a sample.

C. Methods of isolation. Cells are spread over a large area (streak plate and spread plate) or are diluted in a large volume (pour plate) so that individual cells are completely separated and can grow into colonies.

D. The cultures are incubated, subcultured, observed macroscopically and microscopically, and identified by means of morphological, physiological, genetic, and serological methods.

E. A pure culture consists of a single type of microbe; a mixed culture deliberately contains more than one type; and a contaminated culture is tainted with some intruding microbe.

A. Physical Subgroups

They can be liquid (broth, milk), semisolid, or solid, depending upon the absence or the quantity of solidifying agent (usually agar and gelatin).

B. Chemical Subgroups

A synthetic medium is any preparation that is chemically defined, but a medium containing a poorly identified component is nonsynthetic.

C. Functional Subgroups

1. A general-purpose medium is used to grow a wide assortment of microbial types.

2. An enriched medium is supplemented with blood or tissue infusion to culture fastidious species.

3. A selective medium permits preferential growth of certain organisms in a mixture and inhibits others. Highly selective media are used expressly to favor the growth of one organism over another.

4. A differential medium distinguishes among different microbes by bringing out their variations in a particular reaction.

5. A reducing medium limits oxygen availability and is useful for cultivating anaerobes.

6. The ability to utilize sugars can be determined with carbohydrate fermentation media.

7. To convey fragile microbes, special transport media are needed to stabilize viability.

8. Assay media are used to evaluate the effectiveness of antimicrobial agents.

9. Environmental and industrial surveillance routinely call for enumeration media to determine the number of microbes in food, drinking water, soil, sewage, and other sources.

10. In certain instances, microorganisms have to be grown in animals and bird embryos.

A. Optical, or light, microscopy depends upon lenses that refract light rays, drawing them to a focus to produce a magnified image.

B. A simple microscope consists of a single magnifying lens, whereas a compound microscope relies on two lenses: the ocular lens and the objective lens. The objective lens is responsible for the real image, and the ocular lens forms the virtual image.

C. The total power of magnification is calculated from the product of the ocular and objective magnifying powers.

D. Resolution, or the resolving power, is a measure of a microscope’s capacity to make clear images of very small objects. Resolution is improved with shorter wavelengths of illumination and with a higher numerical aperture of the lens.

E. Modifications in the lighting or the lens give rise to the bright-field, dark-field, phase-contrast, interference, and fluorescence microscopes.

F. Magnification and resolution in the electron microscope obey the same laws as in optical microscopes. Fundamental substitutions include electron emission for light, electromagnets for focusing the lens, and the proper apparatus to provide the necessary voltage and vacuum. Compared with optical microscopes and imagery, the transmission electron microscope (TEM) is analogous to the bright-field microscope, and the scanning electron microscope (SEM) corresponds to the dark-field microscope.

A. Specimen preparation of optical microscopy is governed by the condition of the specimen, the purpose of the inspection, and the type of microscope being used.

B. Wet mounts and hanging drop mounts permit examination of the characteristics of live cells such as motility, shape, and arrangement.

C. Stained, fixed mounts are made by drying and heating a thin film of specimens called a smear.

D. To provide more detailed information on cells, a smear is stained with one or more dyes.

E. A basic (cationic) dye carries a positive charge, and an acidic (anionic) dye bears a negative charge, thus they selectively bind to oppositely charged surfaces. The surfaces of microbes are negatively charged and attract basic dyes. This is the basis of positive staining. In negative staining, the microbe repels the dye and it stains the background.

F. Applications of Staining

1. A simple stain uses just one dye, such as methylene blue, malachite green, crystal violet, basic fuchsin, or safranin. It is useful in determining cell morphology.

2. A differential stain requires a primary dye and a contrasting counterstain; consequently, the procedure is more involved. Classic examples of differential stains are the Gram stain, acid-fast stain, and endospore stain.

3. Differential stains have practical diagnostic significance. The ability of the Gram stain to distinguish gram-positive bacteria and gram-negative bacteria is useful in identification and choosing drug therapy. The acid-fast stain is useful in diagnosing tuberculosis and leprosy. The spore stain is helpful in anthrax, botulism, and tetanus diagnosis.

4. Special stains are designed to bring out distinctive characteristics, as with the spore stain, capsule stain, and flagellar stain. Finding internal granules by means of a simple stain is valuable in diphtheria diagnosis.