Microscopy Techniques for Observing Living Microorganisms

Examination of Living Microorganisms

Introduction to Observing Living Microbes

Microscopy is a fundamental technique in microbiology for examining the structure, motility, and behavior of living microorganisms. Two common methods for observing live microbes are the wet mount and hanging drop techniques, each with specific advantages and limitations.

Microscopy Techniques

Brightfield Microscopy

• Brightfield microscopy is a standard light microscopy technique where the specimen appears dark against a bright background.

• It is commonly used to observe unstained microorganisms in wet mounts and hanging drop slides.

• Provides basic visualization but may lack contrast for transparent organisms.

Wet Mount Technique

The wet mount technique involves suspending a specimen in a liquid on a microscope slide, covering it with a coverslip, and observing it under a microscope. This method is simple and allows for the examination of living, unstained microorganisms in their natural state.

• Advantages: Quick preparation, observation of live movement, and natural morphology.

• Limitations: Low contrast, rapid evaporation, and potential compression of organisms by the coverslip.

Wet Mount Procedure

• Stir or shake the infusion to suspend organisms.

• Transfer a drop to a slide using a Pasteur pipette or inoculating loop.

• Carefully place a coverslip over the drop and gently press to spread the liquid.

• Observe under low and high power objectives, adjusting light as needed.

• For motility studies, alcohol or Gram’s iodine may be used to reduce movement.

Hanging Drop Technique

The hanging drop method is used to observe live microorganisms in a three-dimensional, hydrated environment, minimizing evaporation and preventing compression. This technique is especially useful for studying microbial motility.

• Advantages: Preserves natural motility, reduces evaporation, and prevents crushing of delicate organisms.

• Limitations: More complex preparation, not suitable for oil immersion objectives.

Hanging Drop Procedure

• Apply a ring of petroleum jelly around the concavity of a depression slide.

• Place a loopful of microbial suspension on a coverslip.

• Invert the depression slide over the coverslip so the drop is suspended in the well.

• Examine under low or medium power objectives.

Comparison: Wet Mount vs. Hanging Drop

• Wet Mount: Easier and faster to prepare, but organisms may be compressed and dry out quickly.

• Hanging Drop: Maintains hydration, prevents compression, and allows for extended observation of motility.

Motility in Microorganisms

Brownian Movement vs. True Motility

• Brownian Movement: Random, erratic motion of particles due to collisions with molecules in a fluid. Not a form of true motility.

• True Motility: Directed movement powered by cellular structures such as flagella or cilia.

Examples of Brownian Movement

• Pollen grains in water

• Dust particles in air

• Ink particles dispersing in water

Staining and Chemical Effects

Use of Alcohol and Gram’s Iodine

• Alcohol: Disrupts cell membranes, leading to cell death or reduced motility.

• Gram’s Iodine: Denatures proteins and enzymes, inactivating flagella and disrupting cellular function.

Microbiomes

Definition and Composition

A microbiome is a community of microorganisms, including protozoa, algae, fungi, and bacteria, that inhabit a particular environment. Larger members of the microbiome can be observed using the hanging drop technique.

Microscopy and Microbial Size

Size Ranges and Magnification

Organism	Approximate Size	Brightfield Magnification	Lab Notes
Viruses	~20–300 nm	Not visible	Require electron microscopy
Bacteria	~0.5–5 µm	1000× (oil immersion)	Usually require staining (Gram stain)
Fungi (Yeast)	~3–10 µm	400×–1000×	Often show budding
Fungi (Molds/Hyphae)	Width ~5–10 µm	100×–400×	Branching structures easily seen
Protozoa	~10–100 µm	100×–400×	Often motile; may be unstained
Algae	~10 µm to several mm	40×–400×	Larger species visible at low power

Prokaryotes vs. Eukaryotes

Key Differences

Feature	Prokaryotes	Eukaryotes
Nucleus	No true nucleus; DNA in nucleoid region	Membrane-bound nucleus
Organelles	No membrane-bound organelles	Contains membrane-bound organelles
Size	0.1 to 5.0 µm	10 to 100 µm
Genetic Material	Single, circular DNA; may have plasmids	Multiple, linear chromosomes; no plasmids (typically)
Ribosomes	70S (smaller)	80S (larger)
Cell Division	Binary fission	Mitosis and meiosis
Examples	Bacteria, Archaea	Plants, Animals, Fungi, Protists

Bacterial Infections

Common Sites and Causative Agents

Bacterial infections can affect various body systems, with specific organisms commonly associated with each site.

Site	Common Pathogens
Skin Infections	Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa
Sexually Transmitted Diseases	Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Ureaplasma urealyticum, Haemophilus ducreyi
Urinary Tract Infections	Escherichia coli, Other Enterobacteriaceae, Staphylococcus saprophyticus, Pseudomonas aeruginosa

Frequently Asked Questions

Why should the drop be hanging in the hanging drop method?

• Prevents compression of organisms, allowing for natural movement.

• Minimizes evaporation, maintaining hydration for longer observation.

• Provides a clear, three-dimensional observation field.

Why is oil immersion not used with the hanging drop procedure?

• Oil immersion could disrupt or contaminate the suspended drop, causing collapse or altered behavior.

• Lower-power objectives (10x or 40x) are sufficient for observing motility in the hanging drop method.

Why are microorganisms hard to see in wet preparations?

• Many microorganisms are nearly transparent and blend into the liquid background.

• Rapid movement of live organisms makes them difficult to focus and observe in detail.