103 Chapter Notes -Hans Christian Gram, Gram Staining, Crystal Violet

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amanyt3

5 Dec 2022

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NIMS University

Department

Microbiology

Course

103

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The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. In brief, the procedure involves the application of a solution of iodine (potassium iodide) to cells previously stained with crystal violet or gentian violet. This procedure produces "purple colored iodine-dye complexes" in the cytoplasm of bacteria. The cells that are previously stained with crystal violet and iodine are next treated with a decolorizing agent such as 95% ethanol or a mixture of acetone and alcohol. The difference between gram-positive and gram-negative bacteria is in the permeability of the cell wall to these "purple colored iodine-dye complexes" when treated with the decolorizing solvent. While gram-positive bacteria retain purple iodine-dye complexes after the treatment with the decolorizing agent, gram-negative bacteria do not retain complexes when decolorized. To visualize decolorized gram-negative bacteria, a red counter stain such as safranin is used after decolorization treatment.

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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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B. Fill in the table based on the two images shown: Image 1 Image 2 Gram reaction Shape of cells

1. 2. Fill in the following table describing the steps of the gram stain: Step Reagent Purpose of reagent time Color of gram Positive cells after this step Color of gram Negative cells after this step 1 2 3 4 Answer the following questions:

1. Why is the gram stain technique more valuable than the simple stain technique in the diagnostic laboratory? 2. Suppose distilled water was accidentally used instead of alcohol in the Gram stain procedure. What would be the color of gram positive and gram negative bacteria at the end of the procedure? 3. What is the purpose of the mordant?

4. Why do gram positive bacteria retain the primary stain? 5. Suppose you did a gram stain on a sample of bacteria, and you saw bacilli that were gram positive next to bacilli that looked gram negative. What are two ways you could interpret these results?

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1. What would a gram positive bacteria look like if you forgotthe alcohol step during the Gram stain procedure? Why?

2. What would a gram negative bacteria look like if you forgotthe alcohol step during the Gram stain procedure? Why?

3. What would a gram positive bacteria look like if you forgotthe safranin step during the Gram stain procedure? Why?

4. What would a gram negative bacteria look like if you forgotthe safranin step during the Gram stain procedure? Why?

Thank you!!

103 Chapter Notes -Hans Christian Gram, Gram Staining, Crystal Violet

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