Chapter 5: Microscopy

Question 1

List the general principles of microscopy.

Answer 1

Wavelength of radiation 𝛌

Magnification:

Resolution

Contrast

Question 2

Explain how the use of oil affects microscopy.

Answer 2

When a high-power microscope objective is used with air as the medium between the lens and the specimen, light refracts and scatters, limiting resolution and clarity. Immersion oil is used to fill this gap, dramatically improving the image quality by minimizing light refraction and increasing the microscope’s numerical aperture.

Question 3

Distinguish between different types of light microscopy and discuss the advantages of each.

Answer 3

Bright-field + Staining:
Specimen appears dark and background appears light

Dark-field:
Specimen appears light and background appears dark. Condenser is thicker/there are two lenses. Does not require specimens to be stained – good for live imaging.

Phase:
Also good for live imaging, internal structures of microbes. Differs from dark field in that both direct and scattered light make it up to
the eye piece. Light sent through the specimen in different phases.

-Phase Contrast: Great for looking at small/fine details on exterior of cells

-Differential Interference Contrast: 3-D experience.

Fluorescent: Use smaller wavelength (UV) radiation for imaging. Increases resolution, so it’s great for visualizing small structures. Some bacteria naturally fluoresce, and others need the help of fluorescent dyes that label them – fluorophores.

Immunofluorescent:
Similar in practice to fluorescence microscopy, except now add in extra
specificity. Antibodies bind to very specific sites/proteins in or on cells.

Confocal: Microscopy that can capture a 3-D image of a sample by taking pictures at multiple depths/focal planes through the sample and compiling a “z-stack.” Called optical sectioning. The process of creating a three dimensional reconstruction of a specimen
from images captured at different focal planes. Not great for live imaging, since lasers can be toxic.

Question 4

Discuss the similarities and differences between light microscopy, confocal microscopy and electron microscopy.

Answer 4

Differ in their illumination source, resolution, and imaging capabilities.

Light microscopes use visible light to create images, allowing for the observation of living samples at relatively low resolutions.

Confocal microscopes are a specialized type of light microscope that uses a pinhole to block out-of-focus light, improving resolution and enabling optical sectioning of thick specimens.

Electron microscopes use a beam of electrons, achieving much higher resolution and magnification but requiring samples to be dead and imaged in a vacuum.

Question 5

Distinguish between simple and differential stains. List and describe several types.

Answer 5

Simple stains use a single dye to stain all cells and are used to determine cell morphology, shape, and arrangement, while differential stains use multiple dyes to distinguish between different types of cells or structures based on their chemical or structural properties.

Simple stains: methylene blue, safranin, crystal violet

Differential stains: Gram stain, acid fast stain, endospore, etc.

Question 6

Explain the importance of staining in microscopy and how dyes function.

Answer 6

Importance of Staining:
-Enhanced Visibility
-Structural Detail: Dyes can selectively bind to specific cellular components, allowing for the detailed observation of structures like the nucleus, cell wall, or flagella
-Cell Differentiation
-Identifying Microorganisms: Staining is used to identify specific bacteria or other pathogens

How Dyes Function:
-Selective Binding
-Electrostatic Attraction: Many dyes are ionic.
-Basic (Cationic) Dyes: These positively charged dyes, like methylene blue, are attracted to the negatively charged components within cells, such as nucleic acids and bacterial cell walls, coloring the cell.
-Acidic (Anionic) Dyes: These negatively charged dyes are attracted to positively charged components or stain the background rather than the cell, as seen in negative staining with India ink.
-Selective Uptake: Dyes may be taken up by certain structures and not others, leading to differential coloration.

Question 7

Explain the steps of the Gram stain procedure.

Answer 7

● Crystal violet: primary stain- To increase contrast, sticks inside peptidoglycan.

● Gram’s iodine: mordant- Helps keep the color in (but not too well)

● Alcohol/acetone: decolorizer- Takes the color out of the Gram negative.

● Safranin: counterstain- Stains the Gram negative pink.

Question 8

Brightfield Staining

Answer 8

● Chromophore- The colored portion of a dye

● Basic dye- A salt containing a cationic chromophore, which bonds to negatively charged molecules. Most common since external cell structures are usually (-) charge.

● Acidic dye- A salt containing an anionic chromophore, which bond to
positively charged molecules.

● Can simply stain bacteria to increase _________ between organisms and bright background.

● Can dye bacteria/cells differentially based on certain features (cell wall
thickness, capsule/mycolic acid presence).
○ Differentiate between types of bacteria

Question 9

Acid Fast

Answer 9

● To differentially stain bacterial cells with and without mycolic acid and associated lipids in their cell walls.
(Mycobacterium species)

● Ziehl-Neelsen protocol uses carbolfuchsin and penetrating heat (steam) to stain acid-fast bacteria
● Decolorize with acid-alcohol
● Counterstain with methylene blue

acid-fast- red
non-blue

Question 10

Endospore

Answer 10

● To visualize bacterial endospores and differentiate from non sporulating bacteria
● Hard for other stains to penetrate endospores without heating
● Tells about conditions of bacteria–why do endospores form?
● Use penetrating heat (as with acid-fast) to get malachite green into spore coat
● Counterstain rest of cell(s) with safranin

Question 11

Capsule

Answer 11

● To visualize bacteria with capsules
● Hard for other stains to penetrate
capsule layers
● Capsules denature at high
temperatures, so be careful in the lab!

Maneval’s Procedure:
○ Congo red is actually the counterstain
○ Maneval’s solution:
■ Acetic acid lowers pH → Congo
Red turns blue (pH indicator)
■ Mordant for capsule
■ Acid fuchsin stains bacterial cell
● penetrates capsule but
doesn’t stain it

Capsule is colorless!

Question 12

Transmission Electron Microscopy

Answer 12

● Electrons pass through sample, knocking other electrons off negative stain, giving a negative image of the sample
● Better resolution and ability to image smaller structures
● Energy intensive process, samples must be sliced very thin, no live imaging

Question 13

Scanning Electron Microscopy

Answer 13

● Electrons bounce off the surface of structures
● Amazing images of external cell structures, but can’t see all the way
through the cells
● Can visualize structures 1 nm - 1 m, resolution <1 nm𝜇
● 3 D images

Question 14

Atomic Force Microscopy

Answer 14

● Uses a scanning probe that travels
over the surface of a sample and a
laser to obtain high resolution
images of sample topography
● Atomic scale resolution, 1 billion x
magnification!!!