Differentiating Organisms Using The Gram Stain

Contents

TOC o “1-3” h z u HYPERLINK l “_Toc380071602” Introduction PAGEREF _Toc380071602 h 1

HYPERLINK l “_Toc380071603” Background information PAGEREF _Toc380071603 h 1

HYPERLINK l “_Toc380071604” Procedure PAGEREF _Toc380071604 h 3

HYPERLINK l “_Toc380071605” Results PAGEREF _Toc380071605 h 3

HYPERLINK l “_Toc380071606” Explanation PAGEREF _Toc380071606 h 3

IntroductionThe importance of bacteria in an ecosystem cannot be gainsaid. There has been an increase in the number of studies that focus on the various aspects of bacteria in the recent times. These are mainly aimed at gaining an understanding on the inner workings of bacteria so as to enhance the manufacture of antibiotics, disinfectants and contribute to the overall health and well being of human beings. It goes without saying that there are variations in the nature of bacteria, with some being useful while others amount to disease-causing pathogens. This underlines the importance of bacteria differentiation, which has become a fundamental pillar in biology and biological lessons. Various techniques of differentiating organisms have been devised, one of which is the Gram Stain.

AIM of this Study: To determine the effectiveness of the Gram Stain in differentiating organisms.

Hypothesis: It is possible to differentiate the nature of bacteria through Gram Stain technique.

Background informationGram staining refers to a common technique that is used in differentiating two, enormous categories of bacteria on the basis of the varied components of their cell wall. The procedure colors the cells of bacteria as red or violet thereby distinguishing between Gram Negative and Gram-positive groups of bacteria. This technique was developed in the 19th century by a Danish bacteriologist (Mohan, 2009. pp. 31).

The technique involves the application of varied dyes that result in some of the bacteria staining pink while others stain purple. The bacteria that stain purple are viewed as Gram-Positive while those that stain pink are considered gram negative.

The rigid structure pertaining to the peptidoglycan gives bacteria their shape. It also protects the prokaryotes from the environment, while also surrounding that plasma membrane. Peptidoglycan refers to an enormous polymer of interlocking chains pertaining to identical monomers that are connected using inter-peptide bridges. Bacteria cells are extremely similar from the peptidoglycan moving inwards (Mohan, 2009. pp. 31). However, the bacterial world splits into two key classes moving inwards. These are Gram Positive Cells and Gram-negative cells. Peptidoglycan, in Gram-positive bacterial cells, makes up almost 90% of the compact and thick cell wall that is the outermost cell wall structure pertaining to Gram+ cells. Gram-negative cell walls are less compact, thinner and more chemically complex, with peptidoglycan making up between 5% and 20% of the cell walls (Mohan, 2009. pp. 31). It is also worth noting that the peptidoglycan does not make up the outermost layer, rather it lies between the outer membrane and the plasma membrane. The outer-membrane is more or less similar to the plasma membrane, but comes off as less permeable and is made up of lipopolysaccharides, which are harmful substances that are classified as endotoxin. These variations in the composition are the basis for the Gram stain protocol.

ProcedureBacteria smear is heat-fixed to the microscope slide then dried using heat. This heat kills bacteria while attaching the sample to the slid so as to prevent the ease of washing away the sample.

Crystal violet stain is applied to the heat fixed bacteria smear. The slide is them rinsed with water after a minute. (Mohan, 2009. pp. 33) Iodine is applied to the slide and then rinsed with water after a minute.

Acetone alcohol (decolorizer) is applied to the slide. The slide is then rinsed with water after about 10n seconds.

Secondary stain (safranin) is applied on the slide, then rinsed off the slide after a minute.

The slide is then blotted dry, making it ready for viewing under oil immersion using bright-field compound microscope.

ResultsOn examining the blot under oil immersion using bright-field compound microscope, there is a clear distinction of the Gram-positive and Gram negative Bacteria. The Gram-positive bacteria have a blue appearance while the Gram-negative bacteria take on a pinkish red appearance.

ExplanationThe results are attributed to variations in the thickness of the peptidoglycan layer of the cell membrane for the gram-negative and Gram-positive bacteria. The Gram-positive bacteria have a significantly thicker peptidoglycan layer, in which case it retains the crystal violet stain in the process of decolorisation. The Gram-negative bacteria, on the other hand, lose their crystal violet stain, instead getting stained by safranin during the final process of staining. The inability of the Gram-negative bacteria to retain the crystal violet in the process of decolorising is attributed to their thin, peptidoglycan walls (Mohan, 2009. pp. 34).

The blue-violet appearance that the Gram-positive cells incorporate results from the crystal violet, which is the primary stain. The crystal violet binds or adheres to the iodine mordant thereby causing the enormous molecule to be trapped between the layers peptidoglycan pertaining to the Gram-positive cells (Mohan, 2009. pp. 34). The decolorisation does not eliminate the crystal violet stain that is trapped in the Gram-positive cell wall. However, it does eliminate the stain that is trapped in the thin layer of the Gram-negative bacteria’s peptidoglycan. This marks the fundamental pillar of the Gram stain technique. The secondary stain (counterstain) of safranin imparts the pink color on the colorless gram-bacteria. However, it does not modify the color of the Gram-positive cells. This proves that it is possible to differentiate the groups of bacteria by the composition of their cell wall using Gram stain technique (Mohan, 2009. pp. 34).

References

Mohan, S. K. (2009). Gram stain: looking beyond bacteria to find fungi in gram stained smear. a laboratory guide for. [S.l.], Authorhouse.