Microscopy: Observing Microorganisms

• Observing microorganisms through a microscope involves microscopy and staining techniques.

Microscopy

• Microscopy uses light or electrons to magnify objects, often employing a series of lenses.
• Micrometer (µm) equals 0.000001m or 1x10-6m
• Nanometer (nm) equals 0.000000001m or 1x10-9m
• Angstrom (Å) equals 0.0000000001m or 1x10-10m

General Principles

• Resolution is the ability to distinguish objects that are separate, also known as resolving power.
• Contrast refers to the differences in intensity between two objects or between an object and its background; staining increases contrast, which is important for determining resolution.
• Magnification is the apparent increase in the size of an object, with total magnification achieved through both ocular and objective lenses.
• Example magnification calculation: 10X ocular lens * 40X objective lens = 400X total magnification.

Microscope Types

• Early microscopes, like those used by van Leeuwenhoek, were simple and could magnify up to 300X.
• Brightfield microscopes are inexpensive and easy to use, specimens appear on a bright background, can observe stained specimen and count microbes but cannot see viruses; resolution is ~0.2µm
• Compound light microscopes use a series of lenses; light rays pass through specimen and into one of the objective lenses; oil immersion lenses increase resolution because light does not refract

Fluorescence Microscopy

• Fluorescent substances absorb UV light and emit visible light, increasing resolution and contrast; fluorescent microbes "glow" and can be detected.
• Some cells and molecules naturally fluoresce, while others require staining.
• It enables quick detection or identification of microbes in clinical samples and is used in fluorescent antibody techniques like immunofluorescence & flow cytometry.

Electron Microscopy

• Electron microscopy gives detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms.
• It uses electromagnetic lenses instead of glass and magnifies objects 10,000X to 100,000X with nanometer resolution (0.000000001 m).
• It uses electrons instead of light, which provides the advantage of much shorter wavelength than visible light, resulting in better resolution.

Transmission Electron Microscopy

• Resolution is 2.5nm and magnification is 10,000X – 100,000X

Scanning Electron Microscopy

• It overcomes the sectioning problem in TEM and provides a 3-D view of specimens.
• Resolution is 20nm and magnification: 1000X – 10,000X
• Able to study surface features of cells and viruses.

Fixing & Staining

• Staining increases contrast and resolution by coloring specimens with stains and dyes.
• Sharp images require contrast, and staining changes the refractive index of specimens from that of the medium.
• Preparing specimens for staining requires a thin film of microorganisms, air-drying the sample onto a slide, and attaching it to the surface by heat or chemical fixation.

Staining Classifications

• Staining colorizes microorganisms to emphasize specific structures but is not always desirable, as some organisms cannot be stained, and staining can cause distortion.
• Most stains contain a positive ion (basic dye), useful due to bacteria slight negative charge
• Simple stains use a single basic dye to highlight entire microorganisms for visualization of cell shapes and basic structure, also used to count cells
• A mordant may be added to intensify the stain by increasing affinity or coating a structure to make it easier to see.
• Differential stains employ more than one dye and react differently with different kinds of bacteria, used for differentiation; examples are Gram stain, acid-fast stain and endospore stain
• Special stains color and reveal specific structures of microorganisms and are sometimes used as a diagnostic aid; examples are Negative capsular stain and Flagellar stain

Common Basic Dyes

• Crystal violet
• Methylene blue
• Malachite green
• Safranin

Gram Staining Procedure

1. Slide is flooded with crystal violet for 1 minute, then rinsed with water.
   • Result: All cells are stained purple.
2. Slide is flooded with iodine for 1 minute, then rinsed with water.
   • Result: Iodine acts as a mordant; all cells remain purple.
3. The smear is decolorized by flooding the slide with a solution of ethanol and acetone for 10-30 seconds, then rinsed with water.
   • Result: Gram-positive cells remain purple, but Gram-negative cells are now colorless.
4. Slide is flooded with safranin for 1 minute, then rinsed with water and blotted dry.
   • Result: Gram-positive cells remain purple, Gram-negative cells are pink.

• Gram-positive bacteria stain purple/blue, while Gram-negative bacteria stain red/pink.

Acid-Fast Staining

• Acid-fast stains work for bacteria with "waxy" cell walls such as Mycobacterium tuberculosis, Mycobacterium leprae and also stains some Nocardia sp.
• The procedure after fixation is to apply carbolfuchsin (hot pink dye), apply heat, cool, rinse with H₂O, decolorize using acid-alcohol
  • All bacteria that are not "acid-fast" are decolorized, while Mycobacterium have more cell wall lipids than most bacteria
  • The dye is more soluble in this lipid than in acid-alcohol
• Mycobacterium bovis stains pink (acid-fast), while Streptococcus pyogenes stains blue (non-acid-fast).

Endospore Staining

• Endospores have highly impermeable walls and cannot be stained w/ normal methods
• Various Clostridium sp. and Bacillus sp. produce endospores
• Staining methods after fixation include flooding the slide with malachite green and heating to drive in the stain, which penetrates the endospores with a green stain, followed by counterstaining with safranin, where the remainder of the cell stains red.

Capsule Staining

• Acidic dyes (eosin, nigrosine) are repulsed by the negative charges on the cell surface and therefore do not stain them
• Negatively charged bacterial capsules do not accept most stains; encapsulated bacterial cells appear to have an unstained halo around them, standing out against dark background
• This creates a contrast between the unstained microorganisms and the stained background, making it easier to observe their shape, size, and other structural details.

Flagella Staining

• Thin flagella are invisible using light microscopy
• Mordant (tannic acid) builds up the diameter of the flagella until they are visible when stained with carbolfuchsin.

Practice Questions & Answers

• What is the purpose of a decolorizing agent in the Gram staining process?
  • To differentiate between Gram-positive and Gram-negative bacteria.
• Which of the following is the primary stain used in acid-fast staining to identify acid-fast bacteria?
  • Carbol fuchsin red.
• What is the purpose of negative staining method?
  • To stain the background around the microorganisms.
• In the Gram staining process, what is the role of iodine?
  • To form a complex with crystal violet.
• Name the type of staining used in differential staining method.
  • Gram staining