Materials needed for conceptual demonstration:
- plastic eggs, the hollow kind that open into two halves
- string or yarn
Stuff a long piece of string or yarn (DNA) into one half of a plastic egg (bacterial cell wall) and then close the egg with its other half to make a “bacterium”. Give this stuffed egg and a chopstick to a student. The student then simulates lysis (breaking open) of the bacterial cell by opening the egg, which releases the string or yarn (DNA). Picking up the string or yarn with a chopstick is analogous to picking up a string of viscous DNA with a toothpick in the test described below.
Materials needed for the KOH test are:
- Culture plates of Gram-negative and Gram-positive bacterial isolates
(only Gram-negative bacteria are needed to visual the
DNA stringing, e. g. Pseudomonas syringae, Erwinia
herbicola, Erwinia carotovora, Escherichia coli)
- flat wooden toothpicks
- glass microscope slides
- dropper bottle containing 3% (w/v) potassium hydroxide (KOH)
Bacterial cultures should be transferred to fresh media about 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 for class use.
A mass of bacteria is picked up on the toothpick and transferred 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 lyse (break down) in the 3% KOH, and the liquid will become viscous in 15-30 seconds. Continued agitation should 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 (FIGURE 6). No viscosity will be observed in the KOH solution containing Gram-positive bacteria. Students are generally amazed at the quantity of DNA that is visible. Some like to compete to see who can draw up the longest string of DNA on their toothpick.
Figure 6. (A) The cell walls of Gram-negative bacteria are lysed by 3% KOH. Students can quickly begin stringing DNA from the lysed cells with a toothpick. (B) Close-up. (Courtesy G.L. Schumann)
Design your own experiment:
We do not recommend isolating bacteria from humans, e. g. hands, saliva, blood, etc., due to the potential risk of obtaining bacteria capable of causing disease (pathogenic) in humans.
Ideas for advanced classes…
1) Students could isolate their own cultures from soil for testing. Add a tiny pinch of soil to a test tube of sterile water, mix to suspend the soil particles, and make a series of ten-fold dilutions (100to 10-4) of this suspension in water. Then spread an aliquot of each dilution onto a bacteriological medium; use a separate plate of medium for each dilution of the soil suspension. After allowing time for the bacteria to grow, select the dilution plate(s) that contain individual bacterial colonies and test these colonies to determine if they are Gram-negative or Gram-positive using the KOH test. Compare different kinds of soil, e.g. sandy, clay, loam, garden, forest, etc. Dilution plating allows you to estimate the total bacterial population in your soil samples by counting the number of bacteria on the dilution plate which gave individual colonies and calculating to determine the total number of bacteria present in the initial soil suspension. By keeping count of the number of Gram-positive and Gram-negative colonies as you test the colonies on the same dilution plate, you will then be able to calculate the respective frequencies of the Gram-negative and Gram-positive bacteria in the total bacterial population. In this way you can compare different kinds of soils.
2) Students could isolate bacteria from leaves for testing. Press the surface of a leaf to the surface of a bacteriological medium contained in a petri dish. When the bacterial colonies grow, test them to determine if they are Gram-positive or Gram-negative using the KOH technique. Compare colonies isolated from the upper and lower surfaces of the leaf, leaves from different plant species, or leaves from indoor and outdoor plants. Are bacteria found on plant surfaces potential plant pathogens? Many bacteria found on plant surfaces are used for biological control of plant diseases. How might scientists determine if bacteria from a plant surface were pathogens or beneficial to the plant?