Protocol

Author

Michelle Watt

Additional initiating authors

Kerry Vinall and Xavier Sirault

Overview

The term “rhizosphere” was first proposed Lorenz Hiltner in 1904 to describe the soil influenced by the root, through exudates that modify bacterial numbers and types (see Hartmann et al., 2007 for a fascinating description of Hiltner’s research and life). Since then, researchers have been quantifying, localising and trying to understand the function of these bacteria and their influence on plant growth. This protocols is to directly see and quantify rhizosphere bacteria on the root.

Bacteria on roots can be seen and counted directly by staining or labelling with a DNA probe with a dye and viewing with a microscope. Some stains such as DAPI intercalate with little specificity into the walls and nucleic acids of likely all bacteria, are suitable for total counts of bacteria. Specific nucleic acid probes of a sequence of about 15 to 20 bases can be purchased with a conjugated fluorescent dye, and are hybridised to ribosomal RNA within the bacterial cells in a process known as FISH (fluorescence in situ hybridisation). These probes are designed from 16S ribosomal DNA sequences that can be specific to the species level of bacteria, and thus can be used to localise, quantify and identify types of bacteria

Background

The protocol included below outlines a process for using fluorescence in situ hybridisation (FISH) to quantify and identify bacteria in the rhizosphere:

1. a quick FISH protocol guide for roots in suspension (using confocal microscopy).
   This is a quick protocol guide, assuming:
   • All solutions and slides prepared previously
   • A formamide concentration of 30%
2. a more detailed protocol: Counting bacteria by Fluorescence Microscopy (for rhizosphere washed samples)

Materials/Equipment

• Coated ordinary slides (check there are enough)
• 200ml beaker filled with ice
• P1000, P200, P20 and P2 pipettes and tips.
• Sterile Eppendorfs
• 1 2ml screw top tube (nunc tube) for hyb (hybridisation) solution
• 22x22mm cover slips
• 100% EtOH
• Solutions: formamide, probe, 15ml sterile filtered MQH₂O, sterile filtered Tris-HCl pH 7.2, sterile filtered 5M NaCl, filtered 10% SDS.
• Confocal microscope
• Distilled water
• 0.2 μm Nucleopore filter
• UTHSCSA Image Tool (The University of Texas Health Science Centre at San Antonio; available online as shareware).

Units, terms, definitions

Fluorescence in situ hybridisation FISH

Hyb oven

SDS

Procedure

Quick FISH protocol guide for roots in suspension (using confocal microscopy)

This protocol assumes:

• All solutions and slides prepared previously
• A formamide concentration of 30%

Preparation

1. Set hyb oven to 46^∘C.
2. Set water bath to 48^∘C

Hybridisation

Formamide is Toxic and readily absorbed through the skin. Use gloves and dispose of in an appropriate container.

1. Cut pieces to desired length under 50% ethanol; on dental wax sheet
2. Put through EtOH series 50%, 75%, 100%, 3min in each.
3. Create Hyb solution (for 2ml)
   In this order:
   • 360μl 5M NaCl
   • 40μl 1M Tris-HCl
   • 600μl formamide
   • 998μl milli-Q water
   • 2 l of 10% SDS (add to lid)
4. Prepare 96μl hyb buffer (make 2ml and aliquot) and 3μl of each required probe in an eppendorf
5. Add root piece to hyb and probe solution
6. Put at 46^∘C 1-2 hours

Post-hybridisation

For root pieces

1. Remove root from solution with forceps, blot on Kimwipe
2. Place on slide (preferably cavity slide) mounted in Citifluor Gloves should be used with citifluor at the manufacturer’s recommendation; they do not provide particularly detailed MSDS information.
3. Observe on confocal

On the confocal

• Use a x63 water immersion lens
• Use settings appropriate to the probe.
  For example:
  • Objective: 63x water immersion
  • Beam expansion: 6
  • Pinhole: 1.00 Airy (AE)
  • Gain PMT of the wavelength in use: 800
  • Format: 1024*1024
  • Scan speed: 400hz
  • Laser level %: 50
• Bacterial counts were performed in UTHSCSA Image Tool. See Watt et al., 2006 for details of quantifying clustering and distances between cells on the root, and their relationship to features on the root.

Literature references

Fluorescence in situ hybridization (FISH)

Watt, M., Hugenholtz, P., White, R., and Vinall, K. (2006) Numbers and locations of native bacteria on Field-grown wheat roots quantified by fluorescence in situ hybridization (FISH), Environmental Microbiology 8 (5), 871-884. doi:10.1111/j.1462-2920.2005.00973.x

General reference for the Rhizosphere

Hartmann A., Rothballer M., Schmid M. (2008) Lorenz Hiltner, a pioneer in rhizosphere microbial ecology and soil bacteriology research. Plant and Soil 312:7-14.

Health, safety & hazardous waste disposal considerations

• Formamide is Toxic and readily absorbed through the skin. Use gloves and dispose of in an appropriate container.
• Gloves should be used with citifluor at the manufacturer’s recommendation; they do not provide particularly detailed MSDS information.