Putthoff Lecture

Question 1

LO 1:

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

What does the Gram stan do overall

Answer 1

Gram stain separates bacteria into two groups: Gram+ and Gram-.

Gram stain allows clinician to determine round shape or rod shape

Question 2

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

How to prepare material on slide

Answer 2

smear it, then heat it to fix the bacterias on the slide

Question 3

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

4 steps of gram stain: step 1

Answer 3

pour on crystal violet stain (blue dye), then wait 60s

Question 4

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

4 steps: step 2

Answer 4

wash off with water, flood with iodine solution.

wait 60s.

Question 5

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

4 steps: step 3

Answer 5

wash off with water and then “decolorize” with 95% alcohol

Question 6

Specifically delineate/describe steps+reagents utilized in the gram strain and its use/ability to subdivide + identify bacteria

4 steps: step 4

Answer 6

Counter stain with safranin (red dye)

wait 30 sec

Question 7

cells that absorb crystal violet and hold it =

Answer 7

appear blue = gram positive

possess peptidoglycan layer/cell wall

Question 8

cells that absorb safranin instead =

Answer 8

appear red = gram negative

possesses peptidoglycan layer/cell wall (as well as gram + cells: both do)

Question 9

Gram positive cell wall

thickness, cross linking of amino acid side chains

Answer 9

Thick, extensive cross linking

Question 10

Gram negative cell wall

thickness, cross linking of amino acid side chains

Answer 10

thin, simple cross linking pattern

Question 11

Gram positive cell wall

cell wall constitution

Answer 11

cross linked peptidoglycan, teichoic acid, polysaccharides, other proteins

inner surface of cell wall touches cytoplasm

teichoic acid is important for identification

no cholesterol in membrane or sterols

Question 12

Gram negative cell wall constitution

cell wall constitution

Answer 12

gas three layers (two cell membranes btw):

Outer membrane (has LPS)
Peptidoglycan 
Cytoplasmic membrane (no cholesterol or sterols but contains embedded proteins)

outer cytoplasmic membrane

Question 13

Periplasmic space

Answer 13

between G- inner cell membrane and peptidoglycan layer

contains gel with proteins/enzymes

Question 14

G- PEPTIDOGLYCAN LAYER

Answer 14

peptidoglycan layer contains NO teichoic acid but has Muerin lipoprotein, which extends up to the outer cell membrane

Question 15

Meurin lipoprotein

Answer 15

helical protein

extends from inner peptidolgycan wall to attach to cytoplasmic outer cell wall

Question 16

LPS

components

Answer 16

composed of 3 covalently linked components

1) outer carb chain 1-50 oligosaccharides that extend into the surrounding area: O-specific side chain (outer)
2) water soluble “center” is the CORE POLYSACCHARIDE
3) INTERIOR = LIPID A, a disaccharide with multiple fatty acid tails reaching into the membrane.

LIPID A is the “toxic” part: it is called an endotoxin

Question 17

LIPID A

Answer 17

a disaccharide with multiple fatty acid chains

responsible for LPS’s toxicity

Question 18

Gram + cells versus Gram - cells

Answer 18

Gram +

3 layer, outer membrane with LPS 
high lipid content 
endotoxin (LPS)
periplasmic space
porin 
resistant to lysozyme 
resistant to penicillin 

Gram -

2 layers, peptidoglycan 40-60+ of outer cell layer 
low lipid content
no endotoxin
no periplasmic space
no porin
vulnerable to lysozyme and penicillin

Question 19

clinical significance of LPS/NON LPS

Answer 19

LPS layer blocks diffusion of low molecular weight compounds like penicillin that can destroy the peptidoglycan cell wall.