This article describes a method for observing the interaction between plant root hairs and pathogenic bacteria using microscopy. The protocol includes sample preparation, washing techniques, and microscopy tips to enhance visualization.
Take root samples from plant seedlings grown in sand containing pathogenic bacteria.
Mount the sample using water and a coverslip.
Observe under a phase-contrast microscope to enhance contrast and visualize the root hair and bacteria without staining.
Place the sample in a vial of water.
Invert the vial to wash the sample and remove free and loosely attached bacteria.
Mount the washed sample.
If the sample is thick, use a press-apply coverslip with an elevated adhesive edge to prevent sample damage.
Place some water and the sample inside the coverslip and press the slide on top to seal it.
Invert the slide and view under the microscope.
Count the number of bacteria attached to the root hair.
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.
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