W1 TH

Examination of Living Microorganisms

Introduction

The study of living microorganisms is fundamental in microbiology, providing insight into their structure, behavior, and interactions. This section covers essential microscopy techniques, the observation of microbial motility, and the classification of microbes.

Observing Living Microbes

Brightfield Microscopy

Brightfield microscopy is a common method for observing unstained microorganisms. It uses visible light to illuminate specimens, making objects appear dark against a bright background.

• Objects: Dark

• Background: Bright

• Application: Used to observe living microbes in wet mounts and hanging drop slides.

Lab Techniques for Observing Motility

• Wet Mount Technique: Used to distinguish between types of motility, such as Brownian movement and true motility.

Brownian Movement

Definition and Characteristics

Brownian movement refers to the random, erratic motion of particles caused by collisions with molecules in a fluid. This phenomenon was first described by Robert Brown in 1827.

• Random Motion: No specific direction or pattern.

• Dependence on Particle Size: Smaller particles exhibit more pronounced movement.

• Temperature Influence: Higher temperatures increase the movement due to increased molecular collisions.

• Diffusion: Brownian motion contributes to the diffusion of particles from areas of high concentration to low concentration.

Examples of Brownian Movement

• Pollen Grains in Water: Random motion observed under a microscope due to collisions with water molecules.

• Dust Particles in Air: Movement in sunlight beams as air molecules collide with dust.

• Ink in Water: Ink particles spread randomly when a drop of ink is added to water.

Wet Mount Technique

Definition and Purpose

A wet mount is a microscopy technique where a specimen is suspended in a liquid (usually water) on a slide, covered with a coverslip, and viewed under a microscope. It is commonly used to observe living, unstained microorganisms and other biological samples.

• Advantages: Allows examination of living organisms in their natural state and movement.

• Limitations: Limited by contact and longevity of the specimen.

Wet Mount Technique Procedures

• Suspend the specimen in a drop of liquid.

• Use a transfer pipette for sample placement.

• Cover with a coverslip, avoiding air bubbles.

• Adjust the microscope for optimal viewing.

• Use low and high power lenses (10x–40x) for observation.

• Alcohol or Gram's iodine may be used to better visualize motile organisms.

Use of Alcohol or Gram's Iodine

Purpose and Application

• Alcohol: Used for cleaning slides and removing contaminants. It can also be used in staining procedures to decolorize cells.

• Gram's Iodine: Used as a mordant in Gram staining, helping to fix the dye within the cell wall and distinguish between Gram-positive and Gram-negative bacteria.

Microbiomes

Definition and Observation

A microbiome is a community of microbes that compose the biomass of a given environment. Microbiomes include protists, algae, fungi, and bacteria.

• Observation: Larger microbes in microbiomes are easily observed using the hanging drop technique.

• Petroleum Jelly Seal: Used in hanging drop preparations to reduce evaporation of the suspended drop.

Hanging Drop Technique

Procedures

• Obtain a clean hanging drop (depression) slide.

• Apply petroleum jelly to the edges of the coverslip.

• Place a drop of microbial suspension on the coverslip.

• Invert the slide so the drop is suspended in the depression.

• Adjust the microscope as needed for observation.

Why Should the Drop Be Hanging?

• Prevents Compression: Microorganisms move freely without being squashed by a coverslip.

• Reduces Evaporation: Hanging drops maintain the environment needed for longer observation.

• Clear Observation Field: Provides a three-dimensional environment for accurate observation of motility.

Why Not Use Oil Immersion Lens with Hanging Drop?

• Risk of Contact: Oil immersion requires oil application, which can disturb the hanging drop.

• Adequate Magnification: Lower-power objectives (10x–40x) are sufficient for viewing the drop and observing motility.

Advantages of Hanging Drop over Wet Mount

• Better Observation of Motility: Hanging drop allows for more natural movement and longer observation.

• Reduced Evaporation: Petroleum jelly seal helps maintain the drop.

• No Crushing: Organisms are not compressed, preserving their structure and motility.

Why Are Microorganisms Hard to See in Wet Preparations?

• Transparency: Many microorganisms, especially bacteria, are nearly transparent and blend with the background.

• Rapid Movement: Live microorganisms move quickly, making it difficult to keep them in focus.

Bacterial Infections

Overview

Bacterial infections can affect various organ systems, including the respiratory, digestive, urinary, reproductive, skin, and nervous systems. Understanding the diversity and impact of bacterial pathogens is essential in microbiology.

Prokaryotes vs. Eukaryotes

Comparison Table

Feature	Prokaryotes	Eukaryotes
Nucleus	No true nucleus; DNA in nucleoid region	Membrane-bound nucleus containing DNA
Membrane-bound organelles	Absent	Present (mitochondria, ER, Golgi, etc.)
Size	Smaller (0.1–5.0 μm)	Larger (10–100 μm)
DNA	Single, circular DNA	Multiple, linear chromosomes
Ribosomes	70S ribosomes	80S ribosomes
Cell Division	Binary fission	Mitosis and meiosis
Examples	Bacteria, Archaea	Plants, Animals, Fungi, Protists

Microbial Size and Magnification Table

Organism	Approximate Size	Most Likely Magnification	Lab Notes
Viruses	20–300 nm	Not visible	Require electron microscopy
Bacteria	~0.5–5 μm	1000× (oil)	Observe motility, chemical stains
Fungi (Yeast/Mold)	Yeast: ~5–10 μm; Mold: hyphae several μm	400–1000×	Observe budding, chemical stains
Protozoa	~10–50 μm	100–400×	Observe motility, chemical stains
Algae	~10 μm to several mm	40–400×	Larger species visible at low power

Additional info:

• These notes expand on the basic principles of microscopy and microbial observation, providing context for laboratory techniques and microbial classification.

• Tables have been recreated and expanded for clarity and completeness.