Ch. Three

Question 1

Compound Light Microscope

Answer 1

• ocular lens: remagnifies the image formed by the objective lens
• objective lenses: primary lenses that magnify the specimen
• condenser: focuses light through specimen
• illuminator: light source

Question 2

Simple Stains

Answer 2

• use of a single basic dye
• mordant may be used to hold stain or coat the specimen to enlarge it
• purpose to highlight entire microorganism to see shape

Question 3

Differential Stains

Answer 3

• used to distinguish between bacteria

- gram and acid-fast stain

Question 4

Gram Stain

Answer 4

• classifies bacteria into gram-positive or gram-negative
• multi-step procedure:
  1. primary stain: heat-fixed smear covered in crystal violet; imparts its color to all cells
  2. mordant: purple dye is washed off, and smear is covered with iodine which intensifies the dark purple color
  3. decolorizing agent: washed with alcohol-acetone solution which removes the purple from the cells of some species
  4. Counterstain: alcohol is rinsed off, slide is then stained with safranin (basic red dye), then washed again and examined
• purple dye and iodine combine in cytoplasm and color it, bacteria that retain color after alcohol is gram-positive and bacteria that recolor are gram-negative
• because gram positive keep purple color they are not affect by safranin

Question 5

Acid-Fast Stain

Answer 5

• it can be used to detect tuberculosis and leprosy causing organisms of the Mycobacterium species
  1. red dye, carbolfuchsin is applied to a fixed smear and then heated (enhances penetration and rentention of dye)
  2. cooled and washed; smear is treated with acid-alcohol, a decolorizer, which removes red stain from bacteria that are not acid-fast
  3. acid-fast microorganisms retain color because carbofuchsin is more soluble in the cell wall lipids than in acid-alcohol
  4. in non-acid-fast bacteria, whose cell walls lack lipid components, carbofuchsin is removed leaving cells colorless
  5. smear is stained with methylene blue, counterstain, and non-acid-fast cells appear blue

Question 6

Endospore Stain

Answer 6

• endospore is resistant, dormant structure to protect bacterium from adverse environmental conditions
• schaeffer-fulton endospore stain:
  1. primary stain: malachite green, usually with heat to penetrate endospore wall
  2. decolorize cells: water to remove malachite green from all except endospores
  3. counterstain: safranin is applied to all parts of cell except endospores
• endospores appear green within pink or red cells

Question 7

Flagella Staining

Answer 7

• carbolfuchsin + potassium alum solution

- use of Mordant too build up diameters of flagella until visible

Question 8

Capsule Stain

Answer 8

• capsule materials are soluble in water and may be dislodged or removed during rigorous washing
  1. mix bacteria in a solution containing a fine colloidal suspension of colored particles (india ink or nigrosin) to provide a contrasting background
  2. then stain the bacteria with a simple stain, such as safranin
  3. because of chemical composition, capsules do not accept most biological dyes and thus appear as halos surrounding each stained bacterial cell