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DNA Basics Bacterial Cell Walls (or Gram Stain “without the mess”)

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.

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DNA Basics Bacterial Cell Walls (or Gram Stain “without the mess”)

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  1. DNA BasicsBacterial Cell Walls(or Gram Stain “without the mess”) Bonnie Ownley and Robert Trigiano Department of Entomology and Plant Pathology The University of Tennessee, Knoxville

  2. 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.

  3. Background information • Students need to be familiar with basic cell structure including cell walls and the presence of deoxyribonucleic acid (DNA) in the cell.

  4. 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

  5. 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

  6. Each nucleotide unit is composed of • a sugar (deoxyribose) • a phosphate group • a nitrogenous base

  7. Molecular structure of DNA base pairs

  8. T - - - A C - - - G G - - - C A - - - T A - - - T T - - - A C - - - G Pairing of nucleotide bases in DNA • Adenine (A) always pairs with thymine (T) • Cytosine (C) always pairs with guanine (G)

  9. Transcription and translation of a genetic message into a protein “transcription” “translation” DNA mRNA protein [chains of amino acids] tRNA + ribosomes

  10. Diagram of transcription and translation

  11. Chromosomes are composed primarily of long, thin strands of DNA • In eukaryotic organisms, (plants, animals and fungi) chromosomes are contained in a membrane-bound nucleus

  12. In prokaryotes, such as bacteria, the single chromosome is in the cytoplasm and may be accompanied by smaller pieces of circular DNA called plasmids

  13. 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

  14. Rapid and simple visualization of the stringing effect of DNA from lysed bacterial cells may help attract the interest of students to these subjects

  15. 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

  16. Release of chromosomal and plasmid (arrows) DNA from an unidentified bacterium

  17. Bacterial Cell Walls Gram-stain reaction

  18. 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

  19. G+ and G- cell wall differences • 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

  20. 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

  21. 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.

  22. The Gram stain is an important diagnostic tool in human medicine because some antibiotics are effective against only Gram-negative or Gram-positive bacteria. • Bacteria also cause plant diseases. Most plant pathogenic bacteria are Gram-negative.

  23. Materials and Methods • Materials needed for conceptual demonstration: • hollow plastic eggs that open into two halves • string or yarn • chopsticks

  24. 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.

  25. 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.

  26. 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.

  27. 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)

  28. Transfer bacterial cultures to fresh media 24-48 hours before conducting the test • Older cultures may give a Gram-variable reaction • Streak the culture plates generously to provide sufficient quantities of bacteria

  29. 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

  30. 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

  31. No viscosity will be observed in the KOH solution with Gram-positive bacteria

  32. Stringing DNA

  33. 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]

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