Leaf Bacteria

Are There Guard-Dog Bacteria in Leaves?

Another potential for "Probiotics"?

(Sneak preview! This work will tie in with many others spanning both the animal and plant kingdoms. It is part of a new branch of immunology that young scientists almost alone are exploring. VERY EXCITING!)

It is known that all the membranes at the openings of our bodies are coated with a layer of very special bacteria. Imagine two groups known as the lactic acid bacteria, and the propionic acid bacteria. Only a few species within each group are the NORMAL residents of our membranes. This small collection of species are often normally found in high numbers in milk and cheeses. Hence, we shall refer to them as the DAIRY BACTERIA. While other groups are exploring the functions of these bacteria in milk and on animal membranes, you will be the group to make the huge immunological jump over to a new kingdom! Up to now, immunology has been thought to be only in realm of animals. Well-, you now have the opportunity to move it farther afield - MUCH farther afield. Indeed, into the field!

Keep in mind that it has been for many decades that the leaves of most plants are closely associated with these "dairy bacteria." Of course, what was thought was that the bacteria coated the leaves. Thus, when you are asked the following question, what should pop immediately to mind?

THE QUESTION. Inside leaves is a spongy layer of odd-shaped cells that are tender, moist and surrounded by air that comes into the leaves through pores called stomata, which are mostly in the undersides of the leaves. (Click the image for a line-drawing you may use in any reports you are writing.) How is it that these delicate moist cells do not get infected by any of the environmental bacteria and fungi that float in through the stomata? Are these tender cells being protected by guard-dog bacteria such as is putatively happening on the tender membranes at the openings of your body?

How can you find out? How can you look for bacteria inside of a thin leaf? How do you bypass the bacteria coating the outside of the leaf? How do you get at the bacteria in that thin leaf?

(All black numbers in the following procedures refer to pages in Pierce and Leboffe's Exercises for the Microbiology Laboratory, and all red numbers to the companion Leboffe and Pierce's A Photographic Atlas for the Microbiology Laboratory. Both are published by Morton Publishing Company.)

We first consider the thin-ness problem. Easy! Get a thick leaf!
Sterilize the outside of the leaf by dipping it in 1/30 household bleach for a minute, and then rinsing it several times in autoclaved water that has been allowed to cool.
Holding both the base and tip, bend the leaf so that it cracks open exposing its inside layers (5).
Rub a sterile swab onto the newly exposed layers, and then rub the swab (11) onto petri plates containing various types of agar (nutrient, MacConkey (61, 14), tomato-juice, phenylethanol (57, 16), and other selective agars).
Incubate the plates at room temperature (since that is the temperature that the leaf originally was).
Which types of colonies (1) were the most common (don't just look at big colonies, little colonies are equally important. Remember that each colony, whether large or small, grew up from a single bacterium.
Make "streak plates" (52, 9) for each of the different types of colonies found. Allow the streak plates to grow and give colonies.
Transfer some of the colonies to separate sections of another plate that will act as your "stock plate." Once those grow up, then make a microscopic examination of each type of bacterium (27-49, 21-36)
Further identification can be done by testing for growth on other media (63-98, 9-20, 37-82).

But are they guard dogs with teeth?

You must now devise a test to see if these bacteria are able to kill other bacteria. You will have to mix suspensions of these bacteria with a second type of bacteria to see if the second type is killed off. See if you can devise this experiment.

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