Specimen and Slide Preparation

Specimen Collection

The first step is specimen collection, where a sample of the microorganism to be studied is obtained from a source.
Methods of collection can vary based on the source, such as swabbing, scraping, aspiration, or biopsies.
It is crucial to ensure that the specimen is uncontaminated and represents the microorganism or microbial community accurately.

Sample Storage and Transport

After collection, specimens need to be properly stored and transported to prevent any changes in the microbial population.
The sample is often stored in sterile containers or transport media.
Ideally, specimens should be processed as soon as possible, within 2 hours of collection.

Staining

Staining is a crucial process in slide preparation where a dye is used to increase the contrast of the microorganisms, making them easier to see under a microscope.
Most commonly, the Gram stain is employed, which differentiates bacteria into Gram-positive (which retain the violet crystal stain) and Gram-negative (which do not retain the violet stain).
Further staining methods, such as Ziehl-Neelsen stain or Acid-fast stain, are used for specific types of bacteria.

Slide Preparation

There are two main types of slide preparation: wet mount and fixed smear.
Wet mounts are quick and easy to make, making them ideal for viewing live microorganisms and determining motility, but they dry out quickly and cannot be stained.
A fixed smear involves heat-fixing the specimen to the slide, which kills the cells but adheres them to the slide, allowing for staining and longer-term study.

Microscopic Examination

Once the slide is prepared and stained, it can be examined under a microscope.
Microscope binoculars should be properly adjusted to the user’s inter-pupillary distance, and the viewer should focus on the slide using the 10x objective, switching to the oil immersion lens (100x) for detailed viewing.
Cells can be measured, and the morphological characteristics, such as cell shape and arrangements, can be systematically observed and recorded.