Microbiology Chapter: Gram Staining and Techniques

Microbiology and Parasitology (Laboratory) - BIOL 014

• This course is offered at the Polytechnic University of the Philippines, College of Science, Department of Biology.
• The material covered includes Laboratory Discussion 2, Microscopy and Slide Preparations.
• The document details the use of microscopes, including their principles, types, and components (e.g., ocular lenses, objective lenses).
• Microscopes allow the study of living organisms and species not visible to the naked eye.
• The first microscope employed a single lens (Sir Antonie van Leeuwenhoek), achieving 300X magnification.
• Double-lens microscopes arrived later, in the late 16th century.

Microscopy: Principles behind Microscopes

• Microscopes function by magnifying the structure of living organisms to study them.
• Light path, magnification, and intermediate image (inverted from that of the specimen) are critical components of the microscope.
• Ocular lenses (eyepieces): Magnify the intermediate image.
• Objective lenses magnify the specimen.
• Stage holds the specimen.
• Condenser focuses light on the specimen.
• Focusing knobs regulate focus.
• Light source illuminates the specimen.
• Magnification is crucial in visualizing minute specimens (e.g., 100x, 400x, 1000x).

Different Types of Microscopes and Uses

• Light microscopes use natural or artificial light.
• Bright-field microscopes, dark-field microscopes, phase-contrast microscopes, and fluorescence microscopes are examples of light microscopes.
• Electron microscopes use electrons.
  • Transmission electron microscopes and scanning electron microscopes are examples.
• Each type has specific applications in studying specimens.

Bright-Field Microscope

• This type produces a dark image on a brighter background.
• It usually has multiple objective lenses.
• It can be simple or compound.

Dark-Field Microscope

• It creates a bright image of the specimen against a dark background.
• This highlights the specimen and is effective for observing unstained living tissue.

Phase-Contrast Microscope

• This microscope generates a brighter image against a dark background due to the out-of-phase rays.
• It allows observation of intracellular components.

Fluorescence Microscope

• UV, violet, or blue light is used to illuminate specimens stained with fluorochromes.
• The specimen emits a bright image of the object.

Electron Microscope

• Using high-energy electrons, specimens are observed on a very fine scale.
• Transmission and scanning electron microscopes.

Micrometry

• Micrometry is used to measure specimen dimensions under a microscope.
• Two types of micrometers are used: stage micrometers (calibrated) and ocular micrometers (non-calibrated).
• Calibration constants are necessary to convert ocular micrometer measurements to real world units (e.g., µm).
• To calibrate an ocular micrometer, the number of ocular units is compared with the corresponding measurement on the stage micrometer.

Slide Preparations: Wet Mount, Hanging Drop, and Staining

• Wet mounts and hanging drop methods preserve the natural size and shape of specimens, ideal for observing activities like motility and binary fission.
• Gram staining: a differential staining procedure that distinguishes between Gram-positive and Gram-negative bacteria based on cell wall composition.
• Gram-positive bacteria have a thick peptidoglycan layer.
• Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane.
• The staining process utilizes specific reagents like crystal violet, iodine, ethanol, and safranin.
• Different slide preparation procedures (wet mount, hanging drop, gram staining) support the different types of microscope observation.

Microbial Cell Morphology

• Morphology refers to cell shape in microbiology.
• Bacterial shapes include coccus, rod, spirillum, spirochete, and filamentous bacteria.
• Fungi include nonseptate and septate hyphae, sporangiospores, conidia, and various spore forms, conidia, and mycelium types.
• Different flagellar arrangements exist.

Laboratory Activity 1: Microscopy

• This activity encompasses various microscopy techniques (e.g., letter "e" mounting, wet mount, hanging drop, micrometry, Gram staining).
• Each technique has specific materials and procedures to follow.
• Expected outputs from the exercises include microscopic images of various prepared slides and specimen measurements.