Lesson Plan: DNA the Easy Way (and “Gram Stain” Without the Mess)

Learning objectives:

1. To visualize DNA from cells.
   
   
2. For advanced classes, to understand the basis and importance of the Gram-stain reaction and to perform the KOH test equivalent.

Exercise description:

Bacterial cells are placed in 3% (w/v) potassium hydroxide (KOH) solution. This alkaline solution lyses (breaks down) the cell walls of Gram-negative bacteria, releasing the contents of the cells. Agitation of this solution with a toothpick creates a viscous liquid. The increase in viscosity is due to the release of the DNA, which can then be drawn up in a string on the toothpick. Gram-positive bacteria will not lyse in 3% KOH, so this demonstration can be used to determine Gram-stain reaction for bacterial identification.

Study questions:

1. What is the basic structure of DNA?
2. Why is the genetic code described as “nearly universal”?
3. How are genes expressed?
4. Why is the Gram-stain reaction so important in bacterial taxonomy?
5. What physical properties of bacterial cells cause the difference in Gram-staining reaction?
6. Which genera of plant pathogenic bacteria are Gram-positive, and which Gram-negative?

Answers to Study Questions:

1. What is the basic structure of DNA?
   DNA is a double helix composed of nucleotide units. Each unit consists of a sugar, a phosphate group, and a nitrogenous base (adenine, guanine, cytosine, or thymine). The nitrogenous bases pair (A with T and C with G) to form the “rungs” of the double helix ladder.
   
2. Why is the genetic code described as “nearly universal”?
   The same genetic code is used by all living organisms: plants, animals, fungi, bacteria, even viruses. It is this “nearly universal” genetic code that makes genetic engineering- the transfer of genes from one species to another- possible.
   
3. How are genes expressed?
   Genes in the DNA are transcribed into messenger RNA which carries the genetic message to the cytoplasm where it is translated into chains of amino acids. The amino acid chains form proteins, many of which are enzymes. Enzymes are the biological catalysts that control cell function.
   
4. Why is the Gram-stain reaction so important in bacterial taxonomy?
   Hans Christian Gram was originally looking for a stain for all bacteria to make them more easily visible in a microscope field. In the process of looking for a universal stain, he stumbled upon a fundamental difference between two distantly related groups of bacteria - those that retained the stain (Gram-positive ) and those that did not (Gram-negative). Thus, today the Gram stain is one of the first steps in identifying an unknown bacterial culture. Gram reaction is important in medicine because some antibiotics are effective against only Gram-negative bacteria (e.g. erythromycin) and some against only Gram-positive ones (e.g. penicillin, actinomycin).
   
5. What physical properties of bacterial cells cause the difference in Gram-staining reaction?
   Gram-positive bacteria have a cell wall composed of a single macromolecule of peptidoglycan (about 15-80 nanometers thick). Gram-negative bacteria have a thin peptidoglucan layer surrounded by a lipopolysaccharide-containing bilayer. These differences in cell wall structure cause the difference in the Gram-stain reaction.
   
6. Which genera of plant-pathogenic bacteria are Gram-positive, and which Gram-negative?
   Clavibacter (previously known as Corynebacterium), Bacillus, and Streptomyces species are Gram-positive. The other common genera (Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Xanthomonas) are all Gram-negative.