The overall goal of this procedure is to observe an measure the binding of bacteria to plant surfaces. This method can answer important questions in both plant pathology and in food safety. In particular, it can answer questions about how bacteria bind to plant surfaces and how they can be removed.
The main advantage of this technique is that it's easy, quick, and inexpensive. To prepare seedlings grown in water, place a small number of seeds in a 30, 50, 100, or 150 milliliter glass beaker. Here tomato seeds are used.
Cover the seeds with 80%ethanol and swirl briefly. Then let the seeds soak for one minute. Pour off the ethanol and submerge the seeds in a solution of 50%commercial bleach and 0.1%Triton X-100 and tap water.
Allow the seeds to soak for 20 minutes or longer if the seeds are large, like bean seeds. Pour off the bleach mixture and wash the seeds three times with sterile water, letting the seeds soak in the water for one minute for each wash. Pour the seeds and a small amount of water into a sterile petri dish and incubate the seeds in the dark until the seedlings reach the desired size, between one centimeter and 10 centimeters.
To prepare seeds or seedlings grown in sand, after sterilizing the seeds and planting materials inoculate the seeds or seedlings with bacteria according to the text protocol. Using a sterile glass rod, make a shallow hole in the sand just deep enough to place the seed below the surface. Place the seed in the hole and cover it with a thin layer of sand.
Then use sealing film to seal the top of the container to prevent loss of water and entry of additional microbes. Grow the plants in the lab under a light or in the greenhouse at an appropriate temperature and day length for the species and variety of plant. Plant the seedlings in the sand.
Use a sterile glass rod to make a hole. Then with a sterile stainless steel crochet hook guide the root carefully into the hole and use sand to fill the sides of the hole. After incubating plants with bacteria according to the text protocol, prepare a sample for microscopy by placing it in a drop of water or incubation medium on a microscope slide and directly observe it.
If there are no visible free bacteria, there may have been bacterial death or bacterial binding to the container in which the incubation was carried out. Wash the sample in water or incubation medium by placing it in a vial of liquid and gently inverting the vial. Then place the sample on the microscope slide in fresh liquid for observation.
To mount the sample use an ordinary cover slip. If the sample is thick and makes a bulge add the liquid and sample into the well of a press apply cover slip that has a ring of rubber or plastic around the edge. Place the slide on top and press down gently to seal the cover slip to the slide.
Invert the slide to examine the sample. Alternatively, use an algae counting slide and cover slip in a similar manner and view at no more than 20 times magnification. To determine the number of viable bacteria found to plants grown in sand, begin by removing the sealing material from the top and bottom of the container.
Place the container over a piece of sterile paper and gently knock the container against the surface to loosen the sand before gently lifting the sand with the plant from the container. When the cylinder of sand and the plant are free on the paper, split the sand down the middle to reveal the plant root. If desired, take samples of the sand from near the edge of the container as well as near the root.
This may be useful to determine the spread, accumulation, and growth of bacteria. Pick up the root and remove the sand and bacteria that are loosely adhered by dipping the root in a measured volume of water or buffer and gently shaking. Determine the viable cell count of the bacteria in the resulting suspension by plating it on suitable mediums such as luria agar.
This represents the number of bacteria loosely associated with the root. Finally, remove the tightly bound bacteria by sonication and determine their numbers. This graph shows the effect of two different cellulose minus mutations on the ability of the bacteria to colonize tomato roots.
Although the standard deviations of some measurements were as high as 0.9 log base of 10, the reduction in binding of the cellulose minus mutants is clearly evident. In this experiment the binding to alfalfa sprouts of wild type E.Coli 0157:H7 in mutants unable to make various exopolysaccharides was measured. In both strains, the production of poly-beta one six glucuronic acid, or PGA, appeared to make the largest contribution to the binding of pathogenic E.coli to plant surfaces.
Colanic acid also played a significant role in binding. Here two strains of bacteria that are unable to bind to plant surfaces were engineered to produce PGA to determine if it is sufficient to cause bacterial binding to tomato roots. In the case of A.tumefaciens A1045, PGA caused the bacteria to bind individually to the root surface.
On the other hand, S.meliloti bound in large clusters, in which only a few of the bacteria were directly attached to the root, and the majority of the bacteria were attached to other bacteria. Once mastered, this technique can be done in less than one hour for initial setup, plus incubation time, and between 10 minutes and one hour scoring time, depending on the sample. While attempting this procedure it's important to remember that bacteria can stick to almost any surface.
After watching this video you should have a good understanding of how to observe binding of bacteria to plant surfaces. Determine where on the plant the bacteria bind and determine the number of bacteria bound. Don't forget that if you carry out these procedures with human pathogens the procedure can be extremely hazardous and precautions such as working in a containment hood and wearing gloves should always be taken.
