Lecture 2 - observing microbes

Question 1

Differential stains

Answer 1

Stain particular types of cells

Question 2

Mechanism of Gram stain

Answer 2

Differentiation between bacteria based on cell wall structure
- Gram positive: retain crystal violet due to thick peptidoglycan cell wall
- Gram negative: readily lose violet stain when washed with alcohol

Question 3

Process of Gram staining with crystal violet

Answer 3

1. Add crystal violet
2. Iodide complexes with crystal violet and reduces its mobility (resists de-staining)
3. Decolorizer (ex: alcohol) releases loosely trapped stain

Question 4

Why does Gram positive bacteria retain crystal violet better than Gram negative?

Answer 4

10-15x thicker peptidoglycan cell wall. Gram negative bacteria usually have 1-2 layers of peptidoglycan and an outer membrane made of some other material.

Question 5

What does acid-fast stain use and stain?

Answer 5

Uses carbol-fuchsin to stain Mycobacterium species (ex: Mycobacterium tubercuosis). Resistant to decolorization with an acid.

Question 6

What does spore-stain use and stain?

Answer 6

Uses malachite green to detect endospores of Bacillus and Clostridium species (ex: tetanus, botulism)

Question 7

What does a negative stain do?

Answer 7

Adds contrast to media to make cells and other structures (ex: capsules) more visible.

Question 8

How does fluorescence microscopy work?

Answer 8

Incident light is absorbed by a fluorophore and re-emitted at a longer wavelength.

Question 9

What is the advantage of fluorescence microscopy?

Answer 9

Can be used to specifically label certain things; helps determine intracellular localization. Can watch cellular processes happen.

Question 10

Difference between using fluorescently labeled antibodies and using fluorescent proteins?

Answer 10

Use of fluorescent antibodies requires you to fix and permeabilize the cell. Use of fluorescent proteins makes use of gene editing; can attach a sequence for a fluorescent protein to the protein of interest’s DNA sequence –> make a tagged protein.

Question 11

Examples of fluorescent molecules?

Answer 11

FITC, acridine orange (AO), and DAPI.

Question 12

How do acridine orange (AO) and DAPI work?

Answer 12

Only fluorescent when they are bound to nucleic acids. Require a hydrophobic environment to fluoresce, so they only do so when stacked between layers of nucleic acids.

Question 13

Example of fluorescent protein?

Answer 13

GFP

Question 14

Examples of observations made with fluorescence microscopy

Answer 14

1. Monitoring the actions of one bacterial species delivering DNAse to another species –> killing of the receiving species via DNA degeneration (seen with DAPI)
2. Observing ring formation of FtsZ for cytokinesis.

Question 15

Wavelength and resolution of electron microscopy

Answer 15

1.2 nm wavelength, 1 nm (10^-9 m) resolution

Question 16

How does electron microscopy work? Contrast with fluorescent or bright field microscopy

Answer 16

1. Heavy metal “stains” like electron-dense uranyl acetate are used to increase contrast
2. Electron beam is focused by magnetic fields instead of glass lenses

Question 17

Transmission electron microscopy (TEM) vs scanning electron microscopy (SEM)

Answer 17

TEM: electrons pass through specimen to reveal internal structures, higher resolution
SEM: electrons scan specimen surface that is coated in an electrically conductive material, reveals external features in 3D

Question 18

Issues with electron microscopy

Answer 18

1. Things may look thicker due to coating with heavy metals
2. Distortion of cell features can be caused by ice formation (e- microscopy requires cold temps) or cell fixation process

Question 19

Electron cryotomography

Answer 19

Attempts to mitigate distortion from ice crystal formation by flash-freezing cell with liquid ethane. Traps liquid water in a native-like state to minimize distortion