DNA Basics
Bacterial Cell Walls
(or Gram Stain “without the mess”)
Bonnie Ownley and Robert Trigiano
Department of Entomology and Plant Pathology
The University of Tennessee, Knoxville
Purpose

To visualize DNA by breaking open bacterial
cells on a slide in an alkaline solution (KOH)
and releasing a thread of DNA that can be lifted
from the slide surface with a toothpick.

To study differences in the composition of
bacterial cell walls because only Gram-negative
bacteria lyse in 3% KOH; Gram-positive
bacteria do not.
Background information

Students need to be familiar with
basic cell structure including cell
walls and the presence of
deoxyribonucleic acid (DNA) in
the cell.
 DNA
is the basis of life because it
contains the genetic code for all
living organisms
 The
genes contained in an
organism’s DNA make up the
blueprint for that organism, and
determine its appearance and all of
its functions

Deoxyribonucleic acid =
DNA

DNA is a very long,
double-stranded, helical
molecule made of many
nucleotide units

Replication of DNA
occurs through the
pairing of nucleotide
bases to form new

Each nucleotide unit is
composed of
a
sugar (deoxyribose)
 a phosphate group
 a nitrogenous base
Molecular structure of DNA base pairs
Pairing of nucleotide bases in DNA
T---A
C---G
G---C
A---T
A---T
T---A
C---G

Adenine (A) always
pairs with thymine
(T)

Cytosine (C) always
pairs with guanine
(G)
Transcription and translation of a
genetic message into a protein
“transcription”
DNA
“translation”
mRNA
protein
[chains of amino acids]
tRNA
+
ribosomes
Diagram of transcription and translation
 Chromosomes
are composed
primarily of long, thin strands of
DNA
 In
eukaryotic organisms, (plants,
animals and fungi) chromosomes
are contained in a membranebound nucleus
 In
prokaryotes, such as bacteria, the
single chromosome is in the
cytoplasm and may be
accompanied by smaller pieces of
circular DNA called plasmids
 The
first step in molecular biology
and genetic engineering is to
isolate DNA from cells
 Because
the genetic code of DNA
is nearly universal, any gene can
potentially be transferred from a
donor organism and function in a
related or unrelated recipient
organism
 Rapid
and simple visualization of
the stringing effect of DNA from
lysed bacterial cells may help
attract the interest of students to
these subjects
Bacterial cell with a single large
chromosome and an extra-circular piece
of DNA known as a plasmid
Agrobacterium tumefaciens, the crown
gall bacterium, a natural genetic engineer
Release of chromosomal and plasmid
(arrows) DNA from an unidentified
bacterium
Bacterial Cell Walls
Gram-stain reaction
Gram-stain reaction

In 1884, Hans Christian Gram found
that bacteria could be divided into two
groups
 cells
that retained crystal violet stain
solution (Gram-positive bacteria)
 cells that did not (Gram-negative)

This differential stain reaction reflects a
difference in cell wall structure

Gram-positive
bacteria have a cell
wall with a very
thick layer of
peptidoglycan

Gram-negative
bacteria have a cell
wall with several
thin layers of
peptidoglycan,
protein and
lipopolysaccharide
G+ and G- cell wall
differences
 The
Gram-stain reaction can
identify bacteria as Gram-positive
or Gram-negative, but messy stains
and expensive microscopes are
needed
 The
KOH test is a faster and
simpler method to determine the
same reaction
 The
KOH method was developed
by a Japanese scientist named Ryu
in 1938.
 When
Gram-negative bacteria are
placed in an alkaline solution (3%
KOH), the cells walls are destroyed,
and the cell contents, including the
DNA, are released.
 The
Gram stain is an important
diagnostic tool in human medicine
because some antibiotics are
effective against only Gramnegative or Gram-positive bacteria.
 Bacteria
also cause plant diseases.
Most plant pathogenic bacteria are
Gram-negative.
Materials and Methods
 Materials
needed for conceptual
demonstration:
 hollow
plastic eggs that open into
two halves
 string or yarn
 chopsticks

Place a long piece of string (DNA)
inside a plastic egg (bacterial cell wall).

Simulate lysis (breaking open) of the
bacterial cell by opening the egg, which
releases the string (DNA).

Picking up the string with a chopstick
is analogous to picking up a string of
viscous DNA with a toothpick.
Prepare students for demonstration
 Use
plastic eggs (to represent the
bacterial cells) containing string (to
represent the DNA)
A
chopstick (toothpick) could be used
to pick up the string (DNA) only
when the egg is broken open, and the
string is released.
For more advanced classes..
 Technique
is useful for
determining the Gram-stain
reaction of bacteria, without the
use of stains.
 The
Gram stain is essential in the
identification and classification of
bacteria.
Materials and Methods

Materials for the KOH test:
 Culture
plates of Gram-negative
(Pseudomonas spp.) and Gram-positive
(Bacillus subtilis) bacteria
 Flat wooden toothpicks
 Glass microscope slides
 Dropper bottle containing 3% (w/v)
potassium hydroxide (KOH)

Transfer bacterial cultures to fresh
media 24-48 hours before conducting
the test

Older cultures may give a Gramvariable reaction

Streak the culture plates generously to
provide sufficient quantities of bacteria
 Pick-up
a mass of bacteria on the
toothpick and transfer it to a glass
slide with 2-3 drops of 3% KOH
 Use
the toothpick to agitate the
bacteria in the liquid with a rapid,
circular motion

Gram-negative bacteria will break
down in the 3% KOH, and the liquid
will become viscous in 15-30 seconds

Continued agitation will increase the
viscosity of the liquid

The DNA released from the lysed cells
of Gram-negative bacteria can be lifted
from the slide surface on the toothpick
when it is drawn up slowly
 No
viscosity will be observed in
the KOH solution with Grampositive bacteria
Stringing DNA
For more ideas/resources

Go to the American Phytopathological
Society website http://www.apsnet.org

Contact info: Bonnie Ownley
 865-974-0219
 bownley@utk.edu

Clark, D.P., and Russell, L.D. 2000. Molecular
Biology: made simple and fun. Cache River
Press, St. Louis, MO [ISBN 1-889899-04-6]