Assessing Root Colonization of Tomato Seedlings by a Pathogenic Bacterium Using Viable Cell Counts

Samenvatting

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

This video demonstrates a protocol for measuring root colonization of tomato seedlings by wild-type and cellulose-deficient pathogenic bacteria. Viable counts of tightly adhering bacteria show greater adhesion and microcolony formation by the wild-type strain, highlighting cellulose’s role in stable root colonization.

Protocol

1. Measurement of adhesion using viable cell counts

1. Determine the number of viable bacteria bound to plants grown in sand
   1. Remove the plant from the container and sand. To remove plants from containers, first remove sealing material on the top and bottom of the container. Place the container over a piece of sterile paper and gently remove the entire sand or soil and plant as one large tapered cylinder by gently knocking the container against a surface to loosen the material.

1. If necessary, use a spatula or rod to loosen the material around the edges going in through the bottom of the container.

2. When the cylinder of sand or soil containing the plant is free on the paper, split it 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 (and accumulation and growth) of the bacteria.
3. Measure the length of the root. Pick up the root and remove the sand and bacteria adhering loosely to the root (the rhizosphere material) by dipping the root in a measured volume of water or buffer and gently shaking it. Determine the viable cell count of the bacteria in the resulting suspension by plating on a suitable medium such as Luria agar. This represents the number of bacteria loosely associated with the root.
4. Remove the tightly bound bacteria by sonication and determine their numbers.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Seeds	Any	N/A	Make a note of the seed lot number and the cultivar
Sand	Sea sand Fisher Scientific	S25
Sand	Sigma-Aldrich	S9887
Conetainers	Stuewe & Sons, Inc.	Ray-Leach cone-tainers	Many different sizes are available to suit the type of plant you wish to grow
Bath sonicator	Any scientific supply company	N/A
Parafilm	Any scientific supply company	N/A