Download DNA the Easy Way

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
 [email protected]

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]