Week 1 Lecture 3 Microscope Modalities

Front 1

Identify which type of microscope you would use to best visualise the following:
a. stained bacterial smear,
b. motile unstained bacteria,
c. stained tissue to identify intracellular microbes,
d. sample pre-stained with fluorescent dye,
e. intracellular detail smaller than 1 m, and
f. unstained cells with intracellular detail appearing in colour.

Back 1

a. Bright field
b. Phase contrast
c. Bright field
d. ??
e. Dark field
f. ??

Front 2

State the major uses of dark field microscopy, how microbes appear when viewed using this technique, and name three different microbial genera that would be best viewed using this type of approach.

Back 2

It uses indirect visible light to observe the specimen that appears as light/dark against dark background. Provides differences in contrast hence specimens don’t require staining. Works well with thicker specimens.
Treponema pallidum in lesions,
Borreliae in blood,
Leptospires in urine

Front 3

Define the following:
a. mordant,
b. simple stain,
c. differential stain,
d. acid fast stain
e. flagella stain.

Back 3

a. mordant,
A mordant is a chemical that helps a stain adhere to cells. Eg Iodine
b. simple stain,
Simple Staining refers to a single basic dye used to highlight a microorganisms shape and arrangement.
c. differential stain,
React differently with different organisms or cell components.
This is used to differentiate organisms that have different staining properties
d. acid fast stain
AKA Ziehl-Neelsen stain. Primary stain: carbolfuchsin, heated (dissolves in a waxy layer of the cell
wall). Decolouriser: acid alcohol. Counterstain: methylene blue (background)
e. flagella stain.
Mordant is used to build up the flagella using carbolfuchsin. Since staining flagella can be complicated, other methods can be used to determine motility

Front 4

Identify 3 decolourizing agents.

Back 4

Alcohol-acetone
acetone,
alcohol,
iodine-acetone

Front 5

Briefly describe the steps and chemicals required for performing the Gram stain. State the purpose of each component and identify in what circumstances you would use the Gram stain.

Back 5

1. Primary stain: crystal violet. This binds to (i.e. colours) all cells.
2. Add mordant: iodine. A mordant is a chemical that helps a stain adhere to cells.
3. Decolourising: alcohol-acetone. This decolourises some cells but not others. As a result of crystal violet stain, Gram positive cells appear purple (dark), but Gram negative cells appear colourless (due to cell wall structure).
4. Counterstain: safranin or carbolfuchsin, which stains all cells. So now: Gram positive cells appear purple (dark), and Gram negative cells appear pink (light).

Front 6

Explain why a Gram positive organism may appear Gram negative in a specimen.

Back 6

over-decolourization of the specimen,
smear prepared from old culture/sample,
iodine solution is expired (turns yellow instead of brown),
there is cell wall damage in the specimen (e.g. due to excessive heat fixation, antibiotic exposure of the specimen, or due the actions of autolytic enzymes)

Front 7

Name the stains that you would use to identify the following:
a. spores,
b. peptidoglycan cell wall,
c. capsule
d. flagella,
e. waxy cell wall.

Back 7

Capsules are demonstrated by negative staining,(wet-mount India Ink preparations)

Front 8

Describe why Ink is a good stain for some fungal pathogens.

Back 8

??

Front 9

Name the major factor that limits imagining in fluorescence microscopes that can detect multiple emission spectra.

Back 9

??

Front 10

Define Stokes Shift.

Back 10

Stokes Shift (def) is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

Front 11

Differentiate images acquired using light, phase contrast, dark field, fluorescence and electron microscopes.

Back 11

???