GeneFISH – combined detection of genes and rRNA in microorganisms

A. Sample preparation (fixation, permeabilization and peroxidase inactivation)

 

 

When you are using this protocol, please cite:

Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ. Microbiol. 12: 3057-3073.

 

 

Introduction to geneFISH           

A. Sample preparation

A1. Sample fixation

A2. Sample immobilization

A3. Embeding in agaroze

A4. Permeabilization

A5. Inactivation of endogeous peroxidases

B. gene detection

B1. gene probes

B1.1. probe design

B1.2 probe synthesis

B1.3 determination of hybridization conditions for hybridization and washing

B2. gene hybridization

B3. antibody binding

B4. gene CARD and inactivation of HRPs from the gene detection step

C. rRNA detection

C1. rRNA hybridization

C2. rRNA CARD

D. Mounting for microscopy and counterstaining

E. Microscopy

F. List of materials

A. Sample preparation (fixation, permeabilization and peroxidase inactivation)

            Several steps are necessary before rRNA or gene hybridization. Fixation and permeabilization are needed to conserve cell morphology and allow high molecular weight molecules (polynucleotides, HRP-conjugated antibodies, HRP-conjugated oligonucleotide probes) to freely diffuse into the cells. Inactivation of peroxidases (endogenous or introduced in the rRNA / gene detection steps) is critical for avoiding false positive signals and has to be tested thoroughly before performing geneFISH. There are no standardized protocols for these treatments. They differ with the sample type and with the microorganisms targeted. For example, we used different permeabilization and peroxidase inactivation procedures for E. coli clones than for Crenarchaea cells in the seawater samples. Also, we noticed that the inactivation of HRP introduced with the rRNA probes required a harsher treatment than the inactivation of endogenous peroxidases. Different protocols for fixation, permeabilization and peroxidase inactivation have been previously described and discussed (Pernthaler et al., 2002; Pernthaler and Amann, 2004; Pernthaler et al., 2004; Pavlekovic et al., 2009).

 

A1. Sample fixation

            The fixation procedure depends from sample to sample. Generally, the samples for geneFISH can be fixed in 1% paraformaldehyde, however, depending on the sample, other fixations can be tested. For more details about fixation, please check the fixation protocol on the Max Planck Institute for Marine Microbiology FISH page.

A2. Sample immobilization

            Depending on their type, the samples for geneFISH can be mobilized either on membrane filters (cell suspensions from pure cultures, enrichments, water columns or sediment slurries) or on glass slides (tissue sections).

Procedure

Cell susspensions

• x µl of PFA fixed cell suspension are resuspended in 1X PBS up to a final volume of 10 ml, vortexed and filtered at -200 mBar on 25 mm filters, 0.2 µm, Cyclopore (Cat. no. 7060-2502 from Whatman)
• 10 ml miliQW wash, in the filtration tower
• air dry
• -20°C store,

Make sure the filtration towers are clean (if necessary, scrub them with detergent, but then rinse plenty).

A3. Embedding in agarose

            When working with filters, an agarose embedding step can be used to avoid cell loss. On the other hand, agarose could promote increased background in the gene detection channel, so, if possible, this step should be avoided. Because of the high temperatures used during the protocol, the low melting point agarose should not be used.

Procedure

• 0.1% LE agarose (Ambion, 50 mg agarose in 50 ml miliIQW – first 25 ml to boil, then add up to 50 ml)
• Add droplets of 25 µl on a Petri Plate
• Place filters face down in one droplet of agarose
• 20 min at 35°C dry
• + 80% ethanol, peel filters off
• Air dry

A4. Permeabilization

            As with CARD-FISH, the permeabilization depends on the sample type. For bacteria lysozyme can be used, while for crenarchaea, HCl or proteinase K should be used. For more details, see the CARD FISH protocol on the Max Planck Institute for Marine Microbiology FISH page. It is better to avoid too harsh permeabilization, otherwise the cells will be lyzed.

Procedure

• Prepare a 0.5 mg/ml Lysozyme solution
• Place filters face up in Petri Dish, on ice
• Cover each filter with 1 ml Lysozyme sol,
• Incubate for 1 h, on ice
• Quick wash in miliQW

Table 1: Lysozyme buffer

A5. Inactivation of endogenous peroxidases

            The geneFISH protocol uses horseradish peroxidase (HRP) for detection of both the gene and the rRNA. Some of the microorganisms can have their own peroxidases, which, if not inactivated, can give false positive signals. For inactivation of peroxidases different chemicals can be used: HCl (0.01 – 0.1 M), methanol, H₂O₂ in water or in methanol. It might be necessary to optimize this step for difficult samples. For more details, see the CARD FISH protocol on the Max Planck Institute for Marine Microbiology FISH page.

Procedure (inactivation with HCl)

• Transfer filters in ~ 50ml of 0.05 M HCl (50 ml miliQW + 250 µl 37% HCl)
• 10 min at RT
• 2 min in 1xPBS
• 5 min in 1xPBS
• 1 min miliQW
• 96% ethanol
• Air dry

Filters can be stored at -20°C at this point, and the protocol continued at a later date.

References:

Pavlekovic, M., Schmid, M.C., Schmider-Poignee, N., Spring, S., Pilhofer, M., Gaul, T., Fiandaca, M., Löffler, F.E., Jetten, M., Schleifer, K.-H., and Lee, N.M. (2009) Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment. J Microbiol Methods 78: 119-126.

Pernthaler, A., and Amann, R. (2004) Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl Environ Microbiol 70: 5426-5433.

Pernthaler, A., Pernthaler, J., and Amann, R. (2002) Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68: 3094–3101.

Pernthaler, A., Pernthaler, J., and Amann, R. (2004) Sensitive multi-color fluorescence in situ hybridization for the identification of environmental microorganisms. In Molecular Microbial Ecology Manual. Kowalchuk, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D., and van Elsas, J.D. (eds): Springer Netherlands.