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In the production of soft rot, Erwinia carotovora produces pectinase, an extracellular enzyme which breaks down pectin, a polysaccharide that cements plant cells together. The pectinase produced by E. carotovora affects the storage tissues of a wide variety of plants with resultant economic loss. This organism is a member of the Family Enterobacteriaceae and possesses the characteristics tabulated in Section 10-3.

Period 1

Materials

Note: For this and the following periods, Materials that are to be used by the entire class are not listed quantitatively. Otherwise, the amounts of materials needed are listed for each person.

Slices of a potato suspected to be suffering from soft rot

1 plate of MacConkey Agar

1. Locate the area of soft rot on the potato slice. Touch a sterile loop to the edge of the area and streak the plate of MacConkey Agar for isolated colonies.
2. Incubate at 30°C for 1-2 days.

Period 2

Materials

Healthy potatoes previously immersed in 1% hypochlorite (appropriately-diluted Clorox) and presently in a 70% ethanol bath.

Potato knives

1 sterile petri dish with 1-2 sheets of filter paper

1 tube of sterile water (5 ml)

1 culture of Escherichia coli

picture of a MAC plate with E. carotovora.

1. For your group of 3-4 people, obtain a potato from the ethanol bath; handle only at the ends. Place on a fresh paper towel.
2. From the middle of the potato, aseptically cut 1/2-inch slices (1 per person). Place your slice into the sterile petri dish. Moisten the paper in the dish with the sterile water.
3. On the surface of the slice, make two parallel, 1-inch-long cuts about 2 inches apart, each about 1/4 inch deep.
4. Examine your Mac plate, E. carotovora appears as deep red, small colonies. The red color is caused by the fermentation of lactose, a resulting drop in pH, and an accumulation of the red dye, neutral red. Make careful note of the colony appearance. (Would this appearance automatically cause the organism to be termed a coliform?)
5. Inoculate one cut with a suspected E. carotovora colony from the plate inoculated in the first period.
6. Inoculate the other cut with a loopful of the E. coli culture.
7. Incubate the potato plate at 30°C for 2-3 days.

Period 3

Materials

1 plate of MacConkey Agar

picture of rotten potato

1. Observe the potato slice for evidence of soft rot. Describe the symptoms. Are they similar to what you saw in the original infection?
2. Touch a sterile loop to the edge of the soft rot and streak the MacConkey Agar plate for isolated colonies.
3. Incubate at 30°C for 1-2 days.

Period 4

Picture of second MAC plate.

1. Observe the colonies on the MacConkey Agar plate. Do the colonies appear to be the same type observed previously?
2. How do the foregoing procedures and observations fit in with Koch's Postulates? Assign each task you have done in this protocol to a postulate.

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