Cell Microscopy, Staining & Bacterial Smear Techniques

These flashcards cover key points from the lecture on ribosome transport, fluorescence and electron microscopy, staining theory, bacterial smear preparation, and the Gram stain’s diagnostic value.

Why are most ribosomes located near the nucleus?

Because the nucleus produces mRNA instructions and ribosomes translate them into proteins, so proximity speeds protein synthesis.

What cellular structures guide ribosomes to membranes for membrane-protein synthesis?

Microtubules serve as tracks for intracellular transport of ribosomes to the membrane.

What is the main advantage of fluorescence microscopy over other light-based methods?

It allows highly specific, non-toxic staining of individual molecules in living cells for real-time observation.

What physical property limits conventional light microscopy resolution?

The relatively long wavelength of visible light.

Why are electrons used in electron microscopy instead of high-energy photons like γ-rays?

Electrons have a much shorter wavelength without destroying the sample, whereas γ-rays would obliterate it.

Approximately how much magnification can a transmission electron microscope (TEM) achieve?

About 100,000× magnification.

List two major drawbacks of electron microscopes.

Very high cost/large size and complex, hazardous specimen preparation (e.g., metal or radioactive coatings).

What is the key difference between TEM and scanning electron microscopy (SEM)?

TEM shows internal ultrastructure; SEM provides surface (topographical) images only.

What sample-preparation method is commonly used to slice biological material thin enough for TEM?

Cryo-fracture (freeze-fracturization) followed by metal coating.

Why do biologists stain specimens before light microscopy?

To increase contrast between the specimen and its background, making details visible.

What ion charge does an acidic dye’s chromophore carry when dissolved?

A negative charge.

Which charge predominates on the exterior of eukaryotic cell membranes, and what type of dye best stains them?

A slight positive charge; therefore, an acidic (negatively charged) dye binds best.

Bacterial surfaces are slightly negative. Which class of stain is therefore preferred?

Basic (positively charged) stains.

Give two common basic stains used in microbiology.

Crystal violet and methylene blue (safranin is another example).

Which pink acidic dye is the most widely used tissue stain in biology?

Eosin.

What information does a simple stain provide, and why is it often skipped in microbiology labs?

It gives morphology and arrangement, but Gram staining provides the same plus cell-wall information, making simple stains redundant.

Define a differential stain.

A staining procedure that uses two dyes but only one is retained, allowing differentiation between cell types.

What is a structural stain intended to reveal?

A specific cellular structure such as a flagellum, capsule, slime layer, or endospore.

Why can’t you add dyes to a traditional wet mount?

The dye would diffuse through the liquid droplet and uniformly color everything, eliminating contrast.

State one advantage and one limitation of wet mounts.

Advantage: observe live motility/behavior; Limitation: low magnification and no staining possible.

What are the three primary purposes of heat fixation during a smear preparation?

Kill the cells, adhere them firmly to the slide, and permeabilize cell walls to enhance stain uptake.

Describe the recommended heat-fixing procedure for a bacterial smear.

Pass the dried slide quickly through the Bunsen burner flame three times, ~1 second each pass.

How does a Gram-positive cell wall differ from a Gram-negative one?

Gram-positive has a thick multilayer peptidoglycan wall; Gram-negative has a thin peptidoglycan layer plus an outer LPS membrane.

Why are Gram-positive bacteria generally more susceptible to penicillin?

Penicillin targets peptidoglycan synthesis, and Gram-positives rely on a thick peptidoglycan wall for integrity.

What chemical acts as the decolorizer in a Gram stain?

Acetone-alcohol (similar to nail-polish remover).