Unit 3: Microscopic examination of different groups of microorganisms

I. Orientation — Principles of microbial microscopy

Microscopic examination uses magnification, resolution, contrast, and staining to reveal the size, shape, arrangement, and internal or surface features of microorganisms. Bacteria are generally observed with a compound light microscope, whereas yeast and molds can often be examined as larger, multicellular or unicellular structures under lower magnification.

A. To microscopically examine bacteria, yeast and mold (fungi)

The central principle is to prepare a representative specimen, produce sufficient contrast, select appropriate magnification, and interpret visible structures without confusing artifacts with microorganisms.

• Magnification: Total magnification is calculated as:

  Total magnification = ocular-lens magnification × objective-lens magnification

A 10× eyepiece with a 100× oil-immersion objective gives 1000× total magnification.

• Resolution: Resolution is the ability to distinguish two close points as separate. It depends more on numerical aperture and wavelength than on magnification alone; excessive magnification without improved resolution produces an enlarged but blurred image.

• Contrast: Contrast is the difference in brightness between the specimen and background. Unstained cells have low contrast, so staining, reduced illumination, or phase-contrast microscopy may be used.

• Specimen preparation: A smear must be thin, evenly distributed, air-dried, and fixed when a permanent stained preparation is required. Thick smears obscure individual cells and produce uneven staining.

• Microscope sequence: Objectives are normally changed from scanning or low power to high dry and then oil immersion. The coarse adjustment is used only with low power; fine adjustment is used at high power.

• Safety and accuracy: Treat all unknown cultures as potentially hazardous. Wear laboratory protection, avoid aerosol generation, disinfect work surfaces, and never return contaminated immersion oil or disposable materials to clean areas.

II. Bacteria — Examination of small prokaryotic cells

Bacteria are unicellular prokaryotes that lack a membrane-bound nucleus. Their small size, commonly about 0.5–5 µm, makes careful smear preparation and high-resolution observation essential.

A. Purpose and preparation of bacterial microscopy

The purpose of bacterial microscopy is to determine cellular morphology, arrangement, staining reaction, motility when applicable, and the presence of structures such as endospores or capsules.

• Smear preparation: Place a small drop of sterile water on a clean slide when using a solid culture, mix in a minute amount of growth, and spread it into a thin film. A loopful of broth culture can usually be spread directly.

• Air-drying: Allow the smear to dry completely before heat fixation. Heating a wet smear can boil cells, distort their shape, and create misleading clumps.

• Heat fixation: Pass the dry slide briefly through a flame several times, or use an approved fixation method. Fixation attaches cells to the glass and kills many organisms, but excessive heat causes shrinkage and poor morphology.

• Oil immersion: Place immersion oil over the stained smear and use the 100× objective. Oil reduces refraction between the slide and objective, allowing the lens to collect more light and improve resolution.

B. Bacterial staining and morphology

Staining increases contrast and may classify bacteria according to cell-wall properties or demonstrate specific structures.

• Simple stain: A basic dye such as methylene blue, crystal violet, or safranin colors cells uniformly. It reveals shape and arrangement but does not reliably distinguish bacterial groups.

  • Cocci: Spherical cells, such as individual cocci, diplococci, chains, or clusters.
  • Bacilli: Rod-shaped cells, occurring singly, in pairs, or in chains.
  • Vibrios and spirals: Curved rods