M3: Staining

Question 1

Gram Staining overview

Answer 1

• discovered in 1884 by Hans Christian Gram
• differential stain that divides bacteria into 2 categories (Gram-positive and Gram-negative) based on their interactions with crystal violet & iodine dyes
• process: add crystal violet and iodine die, then wash with alcohol, re-dye with Safranin (pink)

Question 2

Gram-positive bacteria

Answer 2

• has thick cell wall with overlapping peptidoglycan layers
• peptidoglycan traps crystal violet & iodine inside cell, doesn’t let it leave when washed with alcohol, keeping it from absorbing Safranin dye
• -> stained cell appears purple

Question 3

Gram-negative bacteria

Answer 3

• thin peptidoglycan layer in cell wall, surrounded by outer membrane of lipopolysaccharides
• thin peptidoglycan layer / membrane doesn’t retain crystal violet & iodine once washed with alcohol, absorbs pink Safranin dye afterwards
• -> stained cell appears pink

Question 4

dyes used for Gram-staining (3)

Answer 4

1. crystal violet
2. iodine
3. safranin

Question 5

disadvantage of Gram-staining

Answer 5

cells must be attached tightly to glass slide so they are not lost during washing –> process of attaching them kills the cells, so can’t examine cell’s motility as part of Gram-stain

Question 6

decolorization wash

Answer 6

final step of Gram-staining: wash sample with alcohol, which removes crystal violet & iodine from gram-negative cells

Question 7

heat fixation

Answer 7

• method of attaching cells to slide

- process: place sample on slide, then pass slide through flame until all liquid evaporates

Question 8

chemical fixation

Answer 8

uses paraformaldehyde, ethanol, or methanol to attach cells to slide

Question 9

differential stain

Answer 9

technique separating specimens into subgroups

Question 10

wet mount

Answer 10

• method of preparing live samples for viewing
• process: prepare small liquid culture containing microorganism, put one drop on slide, cover slide with glass coverslip (protects objective and keeps sample in place)

Question 11

simple staining

Answer 11

• used to quickly observe size/shape/arrangement of cells
• process: use positively charged dye (e.g. methylene blue, crystal violet, safranin, fuchsin) to bind and stain the negatively charged membrane of microorganism

Question 12

negative staining

Answer 12

• stain everything except micro-organism
• process: use negatively charged dye (nigrosin aka india ink) –> repelled by cell membrane
• mildly invasive, may kill micro-org or not
• contraindicated for pathogenic samples

Question 13

acid-fast staining

Answer 13

• aka Ziehl-Neelsen stain
• differential stain, used on bacterial strains with high degree of resistance to decolorization (most commonly mycobacterium tuberculosis)
• process: stain cells red with carbolfuchsin dye, then counterstain with methylene blue –> acid-fast bacteria (what looking for) stay red, others go blue

Question 14

giemsa

Answer 14

• differential stain, often used clinically
• combined with wrights stain to test for bacteria w/in blood smears
• human blood cells = purple, bacterial cells (e.g. malaria) = pink

Question 15

pathogen causing TB

Answer 15

mycobacterium tuberculosis

Question 16

wright’s stain

Answer 16

stain for rbcs