BISC 303 Chapter Notes - Chapter 3: Gram Staining, Crystal Violet, Gram-Negative Bacteria

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18 Jun 2018

School

Simon Fraser University

Department

Department of Humanities Biological Sciences

Course

BISC 303

Professor

Julian Guttman

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Mechanism of Gram

Staining

•The difference between typical Gram-positive bacteria and

typical Gram-negative bacteria is due to the physical

nature of their cell walls. If the cell wall is removed, typical

Gram positive bacteria stain Gram negative. Furthermore,

bacteria that never make cell walls, such as the

mycoplasmas, also stain Gram negative. 1... Differential

staining (section 2.3)

•During the Gram -staining procedure, bacteria are first

stained with crystal violet, a dye with a positive charge

that is attracted to the bacterial cell's net negative charge,

and next treated with iodine.

•Iodine is a mordant that interacts with the crystal violet,

forming an insoluble complex and thus promoting dye

retention.

•When bacteria are treated with ethanol in the

decolorization step, the alcohol is thought to shrink the

pores of the thick peptidoglycan found in the cell walls of

typical Gram-positive bacteria, causing the peptidoglycan

to act as a permeability barrier that prevents loss of crystal

violet.

•Thus the dyeiodine complex is retained during the

decolorization step and the bacteria remain purple, even

after the addition of a second dye, required by the

procedure.

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Related Questions

1.This week, you will be conducting Gram stains of bacteria.Which of the following statements are true? (Mark all thatapply)

		Gram-negative bacteria do not stain purple because they do nottake-up crystal violet or iodine
		The peptidoglycan in the walls of Gram-positive bacteria bindsto the crystal violet stain more easily than walls of Gram-negativebacteria do
		Gram-positive bacteria have cell walls with a thickerpeptidoglycan layer than walls of Gram-negative bacteria
		Gram-negative bacteria do not stain purple, because their cellwall structure allows purple-colored iodine-dye complexes to washout of the cells more easily than the cell wall structure ofGram-positive bacteria does
		Gram-negative bacteria have an outer membrane (outside thepeptidoglycan layer in their wall) that Gram-positive bacterialack

What evidence is used to inform our understanding ofrelationships among species? (mark all that apply)

		Biochemical similarities between existing species
		Fossils
		Morphological similarities between existing species (presence ofhair, backbones, etc.)
		DNA sequences and molecular clocks

For each of the groups of organisms below, indicate whether theybelong in (A.) Archaea, (B.) Bacteria, or (C.) Eukarya

- A. B. C.	Methanogens (prokaryotes that produce methane)
- A. B. C.	Gram-positive bacteria
- A. B. C.	Fungi
- A. B. C.	Animals
- A. B. C.	Cyanobacteria
- A. B. C.	Green algae
- A. B. C.	Red algae
- A. B. C.	Amoebas
- A. B. C.	Thermophiles (prokaryotes of hydrothermal vents)
- A. B. C.	Plants
- A. B. C.	Free-living relatives of mitochondria
- A. B. C.	Single-celled organisms with a circular strand of DNA, without anuclear envelope and without peptidoglycan in their cell walls

Select all true statements about the relative diversity ofdifferent groups of organisms from among the followingstatements:

		There are more species of plants on Earth than species ofmolluscs
		There are more plant species on Earth than mammal species
		There are more species of plants on Earth than species offungi
		There are more species of fungi on Earth than mammal species
		There are more species of plants on Earth than species ofarthropods
		There are more flatworm species on Earth than mammal species

beigegerbil183

A 47 year old female patient presents herself to you with symptoms of pneumonia. All indications are that the patient is suffering from an infection of Streptococcus pneumoniae, a Gram-positive bacterium. Since the patient is not allergic to penicillin, you prescribe a dose of amoxicillin, a semi-synthetic penicillin. This drug works by blocking the peptide cross-links, between the muramic acid subunits, of peptidoglycan chains. This leads to a destabilization of the bacterial cell, leading to its rupture.

However, after the patient finished her course of amoxicillin, no improvement in symptoms were reported. Perplexed, you decide to culture and examine the bacteria infecting her lungs. The test you order is a Gram stain test, which comes back as Gram-negative. You feel like a failure as a health care provider due to your misdiagnosis of this bacterium.

The same day, you put the patient on an aminoglycoside class antibiotic. This antibiotic is Gram-negative specific and works by shutting down ribosomes. You are dismayed and confused when this treatment also fails.

To get a full read on the disease, you have a barcoding DNA test done on the causative bacterium. The test comes back and it clearly shows that the bacterium is within the group of Gram-positive bacteria. With the results of this test, you come up with the theory that the patient is infected with a strain of Drug Resistant Streptococcus pneumoniae (DRSP), resistant to amoxicillin. You decide to treat the DRSP by administering intravenous vancomycin, which is indicated for the treatment of serious, life-threatening infections by Gram-positive bacteria. This treatment also fails.

In the end, you figure out that you were wrong about everything, and that there was a simple solution to the treatment of the patient.

Now knowing that the patient was infected with Mycoplasma pneumoniae, what is the likely explanation for the false Gram-negative test?

Before you answer this question you may want to know how Gram staining works. Briefly, the Gram stain is a differential stain meaning that you are using two dyes that stain different structures. Based on the structural differences of the bacteria, the dyes will interact differently with them, producing different results (colors). The Gram stain (Crystal Violet together with Gram's iodine) stains thick layers of peptidoglycan purple and thin layers of peptidoglycan very, very lightly purple. The second stain (or counterstain) is safranin. Safranin is a dye which interacts with and binds to lipid bilayers. Yes, safranin also stains the cytoplasmic membrane of Gram-positive bacteria, but the deep purple color of the Gram stain makes it impossible to see.

A.	The Gram stain was able to penetrate the cell making it turn pink
	B.	The counterstain stained the cytoplasmic membrane, making it look like a Gram-negative bacterium
	C.	The counterstain stained the mycolic acid in the cellular envelope, thus making it look like a Gram-negative bacterium
	D.	The Gram stain lightly stained the present peptidoglycan (making it look pink rather than red).

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BISC 303 Chapter Notes - Chapter 3: Gram Staining, Crystal Violet, Gram-Negative Bacteria

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