Lecture 5

Question 1

What is Gram positive bacteria and what color is it?

Answer 1

Retains 1st dye–>PURPLE

Question 2

What is Gram negative bacteria and what color is it?

Answer 2

Does not retain 1st dye–>PINK

Question 3

What are the 4 steps of Gram stain?

Answer 3

1. Crystal violet
2. Iodine treatment
3. Alcohol/acetone
4. Safranin

Question 4

How can we make sure that bacteria won’t be washed off from the slide?

Answer 4

1. Heat fixate the slide/cells–>cells get stuck to the slide
2. Use methanol

Question 5

What does the diference in Gram stain color reflect?

Answer 5

Differences in bacteria cell wall

Question 6

What is the structure of Gram positive cells?

Answer 6

-THICK peptidoglycan layer
-1 inner membrane

Question 7

What is the striucture of Gram negative cells?

Answer 7

-THIN peptidoglycan layer
-2 membranes layers–>inner & outer

Question 8

True or false: Gram positive cells have an outer membrane

Answer 8

False
-only 1 membrane–>inner

Question 9

True or false: Gram negative cells have a THICK layer of peptidoglycan

Answer 9

False
-thin layer

Question 10

What makes the cell wall negatively charged in Gram (+) bacteria?

Answer 10

Teichoic Acid

Question 11

Why can crystal violet bind to Gram (+) bacteria

Answer 11

Teichoic acid makes the wall negative, crystal violet has a (+) charge so it bonds

Question 12

What is the outer membrane of Gram (-) made of

Answer 12

-LPS
-lipoproteins
-phospholipids

Question 13

What is LPS?

Answer 13

Gives negative charge to cell wall to Gram (-) bacteria, does NOT use techoic acid

Question 14

Why is iodine used in Gram stain?

Answer 14

Makes a complex w/ crystal violet
-size of crystal violet becomes larger
-acts as a primer

Question 15

Why do Gram (+) cells remain blue?

Answer 15

After iodine is used the size of crystal violet becomes larger
-becomes larger than pores in cell wall
-so blue stain remains (cannot leave pores)

Question 16

Why are Gram (-) cells colorless after decolorizing step (alcohol)?

Answer 16

The pores of cell wall become dehydrated from alcohol
-The outer membrane becomes impaired & the dye can leave

Question 17

Why is Gram (+) purple?

Answer 17

Blue (crystal violet) mixed with second stain step (safranin) makes purple

Question 18

True or false: Gram (+) can stay in the stationary phase for a long time

Answer 18

False
-cannot stay on the stationary phase for too long or it loses cell wall characteristics
-Cannot retain dye efficiently
-can form endospores

Question 19

What is mycolic acid layer?

Answer 19

Waxy layer
-makes it difficult for drugs to enter cell
-found in Mycobacterium tuberculosis

Question 20

Is Mycobacterium Gram (+) or (-)?

Answer 20

Neither
-They are acid-fast so it needs a stain that is differential stain. crystal violet/safranin cannot enter

Question 21

What color is bacteria with and without mycolic acid?

Answer 21

Without–>blue
With–>hot pink

Question 22

What is the purpose of SDS?

Answer 22

Breaks down fatty acids & lipids in membrane

Question 23

In DNA extraction lab what does the supernatant contain and what does the pellet contain after centrifuge

Answer 23

-The supernatant contains the DNA
-The pellet contains non-DNA and cell debris

Question 24

In DNA extraction lab what is the purpose of the ethanol step?

Answer 24

Washing step
-removes anything that is not pure DNA