This document provides instructions for performing a Gram staining experiment to classify bacteria as either Gram-positive or Gram-negative. It first explains the theory behind Gram staining, describing the four steps of the procedure and how it is used to differentiate bacteria based on differences in their cell wall structure. Diagrams of typical Gram-positive and Gram-negative cell walls are included. The document then lists the specific reagents, apparatus, and step-by-step method for performing the experiment. References for further information are provided at the end.
This document provides instructions for performing a Gram staining experiment to classify bacteria as either Gram-positive or Gram-negative. It first explains the theory behind Gram staining, describing the four steps of the procedure and how it is used to differentiate bacteria based on differences in their cell wall structure. Diagrams of typical Gram-positive and Gram-negative cell walls are included. The document then lists the specific reagents, apparatus, and step-by-step method for performing the experiment. References for further information are provided at the end.
This document provides instructions for performing a Gram staining experiment to classify bacteria as either Gram-positive or Gram-negative. It first explains the theory behind Gram staining, describing the four steps of the procedure and how it is used to differentiate bacteria based on differences in their cell wall structure. Diagrams of typical Gram-positive and Gram-negative cell walls are included. The document then lists the specific reagents, apparatus, and step-by-step method for performing the experiment. References for further information are provided at the end.
This document provides instructions for performing a Gram staining experiment to classify bacteria as either Gram-positive or Gram-negative. It first explains the theory behind Gram staining, describing the four steps of the procedure and how it is used to differentiate bacteria based on differences in their cell wall structure. Diagrams of typical Gram-positive and Gram-negative cell walls are included. The document then lists the specific reagents, apparatus, and step-by-step method for performing the experiment. References for further information are provided at the end.
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Chemical Engineering Laboratory 1
Pre-lab
Experimental number : 7th experiment
(Gram Staining of Bacteria) Name : Student number : [Experiment 7] Gram staining of Bacteria
1. Objective
Gram staining is very useful technique to classify the sort of bacteria. In this
experiment, we will learn about the gram staining’s mechanism and characteristic of
gram-positive bacteria and gram-negative bacteria.
2. Theory
1) Gram staining
In 1884, Hans Christian Joachim Gram, the Danish scientist, find the staining
method to classify the bacteria by two group. Staining is adding color on
microspecies such as cell and bacteria to identify it easily. The procedures of Gram
staining are as follows.
1. Dye the bacteria with crystal violet.
Crystal violet dissolves to C V +¿ ¿ and C l −¿ ¿ ion. C V +¿ ¿ ion can penetrate the cell’s
membrane and it dyes the cell color to purple. In this process, bacteria dyed to
purple color regardless of bacteria type.
2. Add Gram iodine solution on bacteria.
+¿ ¿ In this procedure, iodine reacts with C V ion and it forms CV-I (crystal violet and
iodine) complex. Also, this procedure strengthens the bond with crystal violet and cell
structure.
3. Add 95% ethanol on bacteria.
Ethanol dissolves the CV-I complex. In this procedure, the color difference occurs
between bacterias. The bacteria which have thick cell wall, CV-I complex is stucked on it and it does not dissolve. So this bacteria remains purple color and we call this
bacteria as gram-positive bacteria. On the other hand, CV-I complex in the bacteria
which have thin cell wall dissolved by ethanol. This bacteria lose their purple color
and become colorless.
4. Counterstain the bacteria with red pigment such as safranin
Safranin dyes the cell with red color. In contrast to gram-positive bacteria, gram-
negative bacteria react with safranin and change their color to red. In some cases,
little amount of remaining CV-I causes the color change to pink. Instead, gram-
positive bacteria remain purple color.
2) Gram-negative bacteria and Gram-positive bacteria
Every bacterias contains peptidoglycan on cell wall. Peptidoglycan is consisted of
two subunits : N-acetyl glucosamine and N-acetyl muramic acid. In the case of gram-
positive bacteria, this peptidoglycan layers crosslink with each other and it shapes
mesh-like thick cell walls. Because of this structure, CV-I complex can’t get out of cell
wall. Also, the compound called Lipoteichoic acid consists the gram-positive
bacteria’s cell wall with peptidoglycan. Gram-positive bacteria have high sensitivity to
pigment and their toxin is removed easily by heating. Also, their toxin can be used for
antibody after treating formalin on toxin.
Gram-negative bacteria have thin peptidoglycan layer, so CV-I complex easily get
out and dissolved by ethanol. Generally, it has high resistance to pigment and
surfactant. It doesn’t need special things for metabolism and their toxin are hard to
get rid of it by using heat.
[Figure 1] Cell structures of gram-positive (left) and negative (right) bacteria
3. Answers to the textbook question
Because we don’t experiment yet, so we can’t decide E. coli and Bacillus are
whether gram-negative and gram-positive. But we already know the color changes
from each procedure, and we can decide which bacteria is gram-negative or positive
