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 genes contained in an organism’s DNA make up the blueprint for that organism, and determine its appearance and all of its functions
The
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 strands
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”
“translation”
DNA
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 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
Because
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
that retained crystal violet stain solution (Gram-positive bacteria) cells that did not (Gram-negative)
cells
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 KOH test is a faster and simpler method to determine the same reaction
The
The
KOH method was developed by a Japanese scientist named Ryu in 1938. 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.
When
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:
plastic eggs that open into two halves string or yarn chopsticks
hollow
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)
chopstick (toothpick) could be used to pick up the string (DNA) only when the egg is broken open, and the string is released.
A
For more advanced classes..
Technique
is useful for determining the Gram-stain reaction of bacteria, without the use of stains. Gram stain is essential in the identification and classification of bacteria.
The
Materials and Methods
Materials for the KOH test:
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)
Culture
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 the toothpick to agitate the bacteria in the liquid with a rapid, circular motion
Use
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 1889899-04-6]