MMI133 Lecture Notes - Lecture 2: Gram Staining, Mycolic Acid, Gram-Negative Bacteria

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18 Jun 2018

School

University of Alberta

Department

Medical Microbiology and Immunology

Course

MMI133

Professor

Judy Gnarpe

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A. Differentiation of Bacterial Organisms

Simple Stains - used to check only if bacteria is present (1 stain)

Differential Stain - to tell if more than one type of bacteria is present

Gram Staining - diagnostic stain test to identify disease causing bacteria based on wall structure.

The Gram Staining Process

1. Fix Specimen onto the slide, this is done by adding methanol to the slide or passing it through a

flame.

2. Add Crystal Violet to the slide to penetrate cells, this is the primary stain

3. Add Iodine onto slide, washing away excess violet stain after 1 minute. It is a mordant that helps

the stain stay in cells, the molecule formed by mixing and iodine in cell prevents stain from

leaving the cell wall containing peptidoglycan mesh (done by a larger molecule created).

4. Apply decolorizer (acetone alcohol), to rinse away remaining purple and brown colour after 1

minute. The alcohol dehydrates and shrinks the peptidoglycan mesh, becoming dense. This is the

differentiation step, as negatives do not have this wall.

5. To enable visualization, add safranin (pink dye) to slide to counterstain the Gram negative

bacteria, not affecting the gram positive. Dry slide

Gram positive

● Purple/blue stain

● Thick layer made of peptidoglycan (like a mesh)

● Contains teichoic acid

● No lipopolysaccharide (LOS)

● No outer membrane made of periplasm

● Generally more susceptible to penicillin

Gram negative

● Pink stain

● Thin layer of peptidoglycan

● No teichoic acid

● Contains endotoxin (LOS) with “O” antigen

● Generally less susceptible to penicillin

● Outer membrane with periplasm

Bacteria not affected by the Gram Stain - Mycobacterium and Mycoplasma

● Mycolic acid prevents gram stain

● Mycoplasma contains no cell wall to hold in stain

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Acid Stain Fast/Ziehl-Neelsen Stain is another, but rarer option

● Used to detect tuberculosis and few other organisms with high lipid/wax content cell wall

(Mycobacterium)

● Also can detect a parasite known as cryptosporidium

● Uses carbol fuchsin, heat and acid alcohol

B. Bacterial Morphology

Spirillum - corkscrew shaped

Bacillus - rod shaped

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Coccus - spherical

Prokaryotic Cell

Fimbriae/Pili - transfer genes (sex pilus) & attach to cell tissue, adhere to mucosal membranes. The

transfer is known as conjugation

Capsule or Glycocalyx - polymer of polysaccharide or polypeptide which provides a sugar coating

Axial Filament - internal flagellum used for mobility by spirochaetes

Nucleoid - Contains 1 chromosome (versus 46 in humans) attached to the plasma membrane containing

genetic information

Ribosomes - site of protein synthesis, 2 subunits composed of protein and mRNA

Inclusions - reserves for nutrient deposits in membrane bounded vacuoles

Plasmids - a small circular piece of DNA in some bacteria that code for a restricted number of proteins

Bacterial Origins in Eukaryotic Cells

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Document Summary

Simple stains - used to check only if bacteria is present (1 stain) Differential stain - to tell if more than one type of bacteria is present. Gram staining - diagnostic stain test to identify disease causing bacteria based on wall structure. The alcohol dehydrates and shrinks the peptidoglycan mesh, becoming dense. This is the differentiation step, as negatives do not have this wall: to enable visualization, add safranin (pink dye) to slide to counterstain the gram negative bacteria, not affecting the gram positive. Thick layer made of peptidoglycan (like a mesh) Bacteria not affected by the gram stain - mycobacterium and mycoplasma. Mycoplasma contains no cell wall to hold in stain (mycobacterium) Also can detect a parasite known as cryptosporidium. Uses carbol fuchsin, heat and acid alcohol. Acid stain fast/ziehl-neelsen stain is another, but rarer option. Used to detect tuberculosis and few other organisms with high lipid/wax content cell wall: bacterial morphology.

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

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MMI133 Lecture Notes - Lecture 2: Gram Staining, Mycolic Acid, Gram-Negative Bacteria

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