3-7

Question 1

what is a primary stain?

Answer 1

first stain used in a differential staining technique

stains all cells the same color, regardless if +/-
crystal violet

Question 2

what is a mordant?

Answer 2

enhances the staining of the primary stain

forms an insoluble CV (crystal violet) iodine complex

Question 3

what are the steps in Gram stain?

Answer 3

1. primary stain crystal violet
2. mordant Gram’s iodine
3. decolorization 95% alcohol/ethanol
4. counterstain safranin

Question 4

what happens during decolorization?

Answer 4

removes primary stain from certain bacteria

alcohol/ethanol removes primary stain from negatively charged bacteria (will be colorless)

+ purple

Question 5

what happens during the counterstain/ secondary stain?

Answer 5

stains decolorized MOs pink

safranin

Question 6

what is the backbone of G+ bacteria?

Answer 6

matrix of peptidoglycan connected by 5 glycine interbridges

Question 7

in G+ how are sheets of peptidoglycan connected?

Answer 7

wall teichoic acids

and anchored into plasma membrane via lipotheichoic acid

Question 8

describe cell wall of gram - bacteria?

Answer 8

outer membrane composed of lipids, lipopolysaccharides, phospholipids, and proteins

very little peptidoglycan

no teichoic acid

Question 9

during Gram Staining, when decolorizing, what happens to G+ bacterial cell wall?

Answer 9

cell wall shrinks and limits the removal of crystal violet

Question 10

during Gram staining, when decolorizing, what happens to G- cell wall?

Answer 10

outer membrane has holes form and ethanol washes crystal violet out since there is no matrix of peptidoglycans to retain the stain

Question 11

what are some problems that can occur with Gram Stains?

Answer 11

1. over decolorization (convert G+ into being seen as G-)
2. Age of culture (cultures over 24 hours of age will result in decolorization)
3. Gram variability (some bacteria will show both G+ and G- regardless of age or decolorization skills (both pink and purple))
4. Too thick of smear (see both G+&G- color results)
5. too thin of smear (result in decolorization of G+ bacteria)
6. under-decolorize (produce purple gram negative cells)
7. staining solution not adequately filtered or is old

Question 12

results of gram staining?

Answer 12

Staphylococcus aureus: +, purple
streptococcus

Escherichia coli: -, pink
pseudomonas
klebsiella