Microscopic tests: Image factors

• Microbiological tests are performed microscopically
• The microscope is an essential tool for microbiological studies

Factors Affecting Microscopic Image

• A clear microscopic image through a microscope depends on three key factors: increase, contrast, and resolution

Increase

• Increase is the most important function of a microscope
• It is the process of making an image larger using a convex lens
• This lens redirects parallel light rays to converge at a "focal point", creating an enlarged and inverted image of the object

Contrast

• Contrast is crucial for improving the visual perception of an image
• Light that reaches an object can either be absorbed or transmitted
• Microscopes often work by transmitting light through an object
• Contrast refers to the differences in light intensity between the object and its surrounding medium
• It is necessary to distinguish an object

Enhancing Contrast

• Contrast can be artificially enhanced by adding stains with chemical compounds that affect cells
• Specialized microscopes, like "phase contrast" microscopes, can also be used

Resolution

• Resolution depends on the lens system of the microscope
• The resolving power is the measure of the minimum distance between two distinct points that can be distinguished as separate entities
• Aim for the smallest "resolving power" figure possible for clearer resolution

Factors Affecting Resolving Power

• Resolving power depends on the wavelength of light used, the size of the first lens for magnification, and the index of refraction of the material

Refraction Index

• The refraction index determines how much the path of light deviates when it passes from one medium to another

Light Wavelength

• Shorter wavelengths of light in illumination provide greater resolving power

The Resolving Power Metric

• It is the shortest distance between two points that can still be distinguished as separate

Types of Microscopes

Optical Microscope

• It relies on optical lenses, using light or photons
• Microscopes in this category can be referred to as light or clear field microscopes

Parts of a Microscope

• Ocular: the lens near the eye that captures and enlarges the image formed in the objectives
• Revolver: a rotating mechanism used to switch between different objective lenses
• Macrometer screw: moves the platform quickly and coarsely up and down
• Micrometer screw: moves the platform up and down more precisely
• Objective: the lens positioned near the sample
• Platen: the platform where the sample is placed, allowing light to pass through
• Focus: directs luminous rays towards the condenser

Compound Optical Microscope

• The compound optical microscope is formed with more than one lens
• Ocular magnification typically ranges from 5x to 20x
• Objectives come in two types: dry objectives and immersion objectives

Dry Objectives

• Dry objectives are used without placing any substance between the lens and the sample
• Common magnification levels include 4x, 10x, 20x, 40x, and 60x

Immersion Objectives

• Immersion objectives require placing a drop of cedar oil or immersion oil between the objective and the preparation
• 100x is the magnification level

Ultraviolet Light Microscope

• They employs lenses made of quartz or fluorite materials, as conventional glass blocks ultraviolet light
• In this type of microscope, the sample is not viewed directly through the eyepieces, as UV light can be harmful to the eyes
• The image is transmitted to a sensor and observed through a screen

Dark Field Microscope

• It utilizes a very weak light to illuminate the sample
• Its condenser is specifically designed to deflect light from the center of the field of view
• This results in highlighting the outline of microbes against a dark background

Phase Contrast Microscope

• It allows the observation of unstained cells and is useful for living cells

Electron Microscope

• It uses electrons to visualize specimens at a much smaller scale
• It operates with a beam of electrons generated by an electron gun, accelerated by high voltage, and focused by magnetic lenses, resulting in black and white vision

Electron Microscope Types

• Transmission Electron Microscope (TEM)
• Scanning Electron Microscope (SEM)

Transmission Electron Microscope Function

• Transmission electron passes through the sample

Scanning Electron Microscope Function

• Scans the surface of the cell to produce high-resolution 3D images

Sample Preparation

• The sample is coated with a thin layer of carbon or metal, such as gold, to make it conductive

Fresh Preparations

• Most microorganisms require a special preparation to be seen at the microscope due to their low contrast

Fresh Preparation Techniques

• Fresh simple: places a drop of the sample on a slide, covers with a coverslip
• Hanging drop: seals the four sides of the coverslip, places a drop of the sample, inverts the slide, placing a special slide and covers the drop

Stains

• Bacteria are normally transparent that cannot be seen in fresh state
• Staining is the procedure for visualization through coloring
• Dyes are used to stain
• They are chemical compounds that increase the contrast
• With staining the bacteria can be observed easier

Stain effectiveness

• Most of the dyes will stain if the microorganisms are dead and attached to the slide

Types of Fixation Techniques

• Fixation aims to preserve cells by killing them and permanently adhering cells to the slide
• Heat Fixation
• Chemical Fixation

Heat Fixation

• Distributes a thin film of samples on the slide
• Then, dries and quickly passes the slide through a flame to kill the specimen and fix the particles to the slide

Chemical Fixation

• Applies different chemicals to kiill the organisms and fix them to the slide
• The following chemicals are sued: Methanol, Formaldehyde, Acetone, Glutaraldehyde, Ethanol, and Picric acid

Dyes

• The added dye stains
• Washes away the rest with excess water

Used Dyes

• Methylene blue , Basic Fuchsin, Crystal violet, Safranin, Acid Fuchsin, and Eosin.

Types of Stains

• Simple Stains
• Differential Stains
• Specific Stains

Simple Stains

• It applies a single dye to increase the contrast
• All structures in the smear are stained with the same color and appear uniform
• Simple staining can be used to determine basic cell shape and cell arrangement but cannot differentiate between different bacterial types
• It applies the smear; fixed and it applies a stain until absorbed
• Wash the excessive of dye and let it dry

Differential Stains

• Applies a secondary dye with a color that contrasts the primary
• It is uses in the identification of microorganisms
• It has two steps
• Primary stain applies color to the sample
• Counterstain contrasting color the cells not reacting with the primary dye

Specific Stains

• It stains particular structures for microorganisms
• Endospores, Flagellas, and capsules