Microscopy-Based Evaluation of Bacterial Attachment to Plant Root Surfaces

Abstract

Source: Matthysse, A. G. Adherence of Bacteria to Plant Surfaces Measured in the Laboratory. J. Vis. Exp. (2018).

This video demonstrates the evaluation of bacterial attachment to plant roots using phase-contrast microscopy. Root samples from seedlings incubated with bacteria are first mounted to check for free bacteria. The samples are then washed to remove loosely attached bacteria and remounted using a suitable coverslip to prevent damage. Finally, the number of bacteria attached to the root hairs is counted under the microscope.

Protocol

1. Inoculation of the Bacteria

1. Determine the number of bacteria to be inoculated with reference to the measurement to be used to determine bacterial adhesion and the length of the incubation time.
2. For microscopic studies involving incubation times less than 1 day, inoculate relatively large numbers of bacteria. Add an amount of culture to reach a final bacterial concentration of more than 10⁶ bacteria per mL. For longer incubation times, decrease the bacterial inoculum size.
3. For studies in which bacterial adhesion will be measured by viable cell counts, add an amount of culture to reach a final bacterial concentration of 10³

Results

Figure 1: Steps in the preparation of a sample for determining the number of bound bacteria. Agrobacterium tumefaciens binding to tomato root hairs (A, B, and C) and to nylon threads (D, E, and F). In samples mounted in water without.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Light microscope	any	N/A	phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.
Seeds	any	N/A	make a note of the seed lot number and the cultivar
Bleach	any	N/A
Bath sonicator	any scientific supply company	N/A
Triton X-100	Sigma-Aldrich	T9284
Nutrient agar	Difco	2001-01-08
Soytone	Difco	24360
Sand	sea sand Fisher Scientific	S25
Sand	Sigma-Aldrich	S9887
Containers	Stuewe & Sons, Inc.	Ray-Leach cone-tainers	many different sizes are available to suit the type of plant you wish to grow
Parafilm	any scientific supply company	N/A
MS salts	Sigma-Aldrich	M5524
Sedgwick-rafter counting cell	Hauser Scientific	HS3800
Probe-clip press-seal incubatin chamber	Sigma-Aldrich	Z359483
Rifampicin	Sigma-Aldrich	R3501