BMSC 200 Lecture Notes - Lecture 5: Gram Staining, Teichoic Acid, Gram-Negative Bacteria

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roseeel181

2 Feb 2017

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Species of bacteria separated into two groups based on the gram stain. Gram postives and gram negatives have different cell wall structures: gram negative cell wall. Two layers: lps and peptidoglycan: gram positive cell wall. The gram stain is widely used in microbiology: gram positive bacteria appear purple and gram negative appear red. Procedure: flood the heat fixed smear with crystal, add iodine, decolorize with alcohol, counterstain with safranin. Result: all cells purple, all cells remain purple, gram positive cells are purple gram negative are colourless, gram positive purple and gram negative are red. Gram positive cell walls: contain 90% peptidoglycan, common to have teichoic acids (acidic substances) embedded in their cell wall. Lipoteichoic acids: teichoic acids covalently bound to membrane lipids. Note: some prokaryotes lack cell walls: mycoplasms. Lipopolysaccharide found only on the outer surface of the outer membrane of gram negative bacteria. Lipid a component = endotoxin released when gram negative cells are lysed.

Related Questions

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).

yellowspider532

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

Question 1

What is the last step of successfully preparing a bacterial smear for a simple stain?

		Spread cells with inoculating loop
		Label the slides
		Heat fixation
		Air dry the smear

Question 2

When looking at your smear under the microscope, all you see is darkness (despite shining light through your slide)- the best explanation is:

		all your bacteria are dead
		your smear was too thin
		forgot to add the bacteria
		your smear was too thick

Question 3

If a microbe can ferment lactose and its fermentation products include lactic acid and CO2, then the results for this assay should be:

		the broth will appear yellow and there will be a gas bubble in the Durham tube
		the broth will appear red and there will be no gas bubble in the Durham tube
		the broth will appear yellow and there will be no gas bubble in the Durham tube
		the broth will appear red and there will be a gas bubble in the Durham tube

True/Flase

Question 1

If the result for this carbohydrate test is negative, then the organism had NO growth in the tube.

Question 2

The biochemical basis of the Gram stain is the thickness of peptidoglycan layers surrounding cells. Therefore a Gram positive cell with a thin layer of peptidoglycan should stain as Gram negative.

Question 3

If the iodine is not applied during the Gram stain, then gram positive cells will likely stain pink

Question 4

A simple stain allows you to see the cell shape and arrangement.

Question 5

If there were no limit to magnification, a light microscope could enlarge microbes infinitely.

Question 6

When doing a bacteria smear from a broth medium you should first place one to two loopfuls of water on the center of the slide?

BMSC 200 Lecture Notes - Lecture 5: Gram Staining, Teichoic Acid, Gram-Negative Bacteria

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