Intro to Bacterial infections

Question 1

What are the 4 steps to a gram stain?

Answer 1

Pour on crystal violet stain (blue dye) and wait 60 sec

Wash with water and flood with iodine solution. Wait 60 sec.

Wash with water and decolorize with 95% alcohol

Counterstain with Safranin (red dye). Wait 30 sec and wash with water.

Question 2

What color do Gram Positive bacterial stain? Why?

Answer 2

BLUE, holds onto crystal violet stain

B/c there is a thick peptidoglycan layer that traps the large dye molecule

Question 3

What color do Gram Negative bacteria stain? Why?

Answer 3

RED, holds onto Safranin

B/c crystal violet is washed out when alcohol is added b/c the outer lipid containing cell membrane is dissolved by alcohol

Question 4

Describe the differences in cell membrane b/w Gram (-) and (+) bacteria. (Don’t forget the antigenic determinants!)

Answer 4

Gram (+):
2 Layers:
-Inner cytoplasmic membrane
-Outer thick peptidoglycan layer with extensive crosslinking of AA side chains

Low lipid content
Vulnerable to lysozyme and PCN attack
Imp polysaccharide: Teichoic acid (imp for antigenic identification)

Gram (-): 
3 layers:
	-Inner cytoplasmic layer
	-Thin peptidoglycan layer with simple cross linking
	-Outer membrane w/ LPS 

High Lipid content 
Endotoxin (LPS) - Lipid A = antigenic determinant 
Periplasmic space
Porin channels
Resistant to lysozyme and PCN attack 
Imp lipoprotein = murein lipoprotein

Question 5

Why are Gram (+) bacterial vulnerable to PCN but not Gram(-)?

Answer 5

Gram (+) bacteria’s thick peptidoglycan layer doesn’t block diffusion of low molecular weight compounds so PCN can go through and attach to teichoic acid

Gram (-) lipopolysaccharide containing outer cell membrane blocks PCN passage

Question 6

What are the 3 major groups of bacteria?

Answer 6

Cocci = spherical
Bacilli = rods (Coccobacilli = short rods)
Spiral

Rest are pleomorphic = lacking distinct shape