Microscopy

Question 1

Lenses and the Bending of Light

Answer 1

• Light is refracted (bent) when passing from one medium to another.
• Refractive index is a measure of how greatly a substance slows the velocity of light.
• Direction and magnitude of bending is
  determined by the refractive indices of the
  two media forming the interface.

Question 2

Lenses

Answer 2

Lenses focus light rays at a specific place called the focal point (F).
* Distance between centre of lens and focal point is the focal length (f).
* Strength of lens related to focal length:
– short focal length more magnification

Question 3

The Light Microscope

Answer 3

Are compound microscopes, because image
is formed by action of 2 lenses

Question 4

The Bright-Field Microscope

Answer 4

Produces a dark image against a brighter
background.
* Has several objective lenses.
Parfocal microscopes remain in focus when
objectives are changed.
* Total magnification: a product of the
magnifications of the ocular lenses and the
objective lenses.

Question 5

Microscope Resolution

Answer 5

Resolution is the ability of a lens to separate
(distinguish) small objects that are close
together.
* Wavelength of light used is major factor in
resolution: shorter wavelength  greater resolution.

Question 6

Replacing oil with air

Answer 6

Replacing air with oil means that many light rays which did not enter the objective lens due to reflection and refraction at the surface of the lens will now enter the lens, increasing the numerical aperture and the resolution of the lens.

Question 7

Good objective lenses

Answer 7

Objective lenses with great resolving power have large numerical apertures and short working distances.

Question 8

PREPARATION AND STAINING OF SPECIMENS

Answer 8

Increases visibility of specimen (increases
contrast with background).
* Staining can be used to accentuate specific
morphological features.
* Preserves specimens.

Question 9

Fixation

Answer 9

Preserves internal and external structures and fixes them in position.
* Organisms usually killed and firmly attached to microscope slide during fixation.
– Heat fixation – routine use with bacteria and archaea.
* preserves overall morphology but not internal structures.
– Chemical fixation – used with larger, more delicate organisms.
* protects fine cellular substructure and morphology

Question 10

Dyes and Simple Staining

Answer 10

• Dyes
  – Make internal and external structures of
  cell more visible by increasing contrast with
  background.
  – Have two common features:
• Chromophore groups:
  – Chemical groups with conjugated double bonds.
  – Give dye its color.
• Ability to bind cells.

Question 11

Dyes and Simple Staining (continued)

Answer 11

• Ionizable dyes have charged groups:
  – Basic dyes have positive charges.
  – Acidic dyes have negative charges.
• Simple stains:
  – A single stain is used.
  – Used to determine size, shape, and arrangement of bacteria

Question 12

Differential Staining

Answer 12

Divides microorganisms into groups based on their staining properties:
– e.g. Gram stain.
– e.g. acid-fast stain (e.g. for Mycobacterium).
* Differential stain used to detect presence or absence of structures (e.g. endospores, flagella, capsules).

Question 13

Mechanism of Gram Stain Reaction

Answer 13

Result of Gram stain is due to differences in the nature of cell wall between Gram positives and
Gram negatives.
* Shrinkage of the pores of thick peptidoglycan layer of Gram-positive cells prevents loss of crystal violet during decolourisation step.
* Thinner peptidoglycan layer and larger pores of Gram-negative bacteria does not prevent loss of crystal violet.

Question 14

Bacteria and Archaea reproduction

Answer 14

Haploid only, asexual reproduction by
binary fission, budding or filamentous
growth.
– All cells must replicate the genome and
segregate the daughter genomes prior to
division.

Question 15

THE BACTERIAL CELL CYCLE

Answer 15

The cell cycle is a sequence of events from
the formation of the two new cells through to the next cell division; most bacteria divide by binary fission.
* Two pathways function during cycle:
– DNA replication and partition.
– Cytokinesis.

Question 16

CHROMOSOME REPLICATION AND
PARTITIONING

Answer 16

Most bacterial chromosomes are circular.
* Single origin of replication – site at which replication begins.
* Terminus – site at which replication is terminated,
located opposite of the origin.
* Replisome – group of proteins needed for DNA synthesis.
* DNA replication proceeds in both directions from the
origin.