Protocol

Fluorescence In Situ Hybridization for the Identification of Environmental Microbes

Department of Environmental Microbiology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany

Corresponding author (annelie.pernthaler{at}ufz.de).

INTRODUCTION

This protocol presents methods for the phylogenetic identification of microorganisms in environmental samples (e.g., water and sediments) by means of fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, followed by signal amplification with catalyzed reporter deposition (CARD). In this procedure, FISH probes are conjugated with the enzyme horseradish peroxidase (HRP). After hybridization, the subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities on target cells than after FISH with probes labeled directly with fluorochromes. This protocol describes the custom labeling of tyramides with different fluorochromes, as well as sample preparation and cell permeabilization strategies for various microbial cell wall types. A protocol for sequential multicolor CARD-FISH for the simultaneous detection of different phylogenetic groups is also presented.

RELATED INFORMATION

The cell wall compositions of bacteria and archaea from different environments can vary greatly, necessitating different permeabilization and fixation strategies for each cell type. Protocols have been developed for CARD-FISH of marine planktonic archaea and bacteria (Pernthaler et al. 2002; Teira et al. 2004), for marine benthic bacteria (Ishii et al. 2004) and for freshwater samples dominated by Actinobacteria (Sekar et al. 2003). An example of bacterioplankton labeled using this technique is presented in Figure 1 .

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Figure 1. North Sea bacterioplankton after CARD-FISH. (Blue) DAPI staining; (red) probe targeting Roseobacter. The figure width is ~100 µm.

MATERIALS

Reagents

Store all succinimidyl esters protected from light at –20°C. Use for tyramine labeling within days after purchase.

Achromopeptidase solution

Use for CARD-FISH staining of Gram-positive freshwater Actinobacteria (see Step 30.iii.)

Agarose, low melting point (0.1% [w/v], prepared in H₂O)

Alexa Fluor succinimidyl ester 350 (see Step 1)

Alternative dyes include:

Alexa Fluor succinimidyl ester 488

Alexa Fluor succinimidyl ester 546

Alexa Fluor succinimidyl ester 633

5-(and 6-) Carboxyfluorescein succinimidyl ester (CF)

CARD-FISH amplification buffer

CARD-FISH hybridization buffer

Citifluor mountant media (or other low-fluorescence glycerol mountant containing an antibleaching agent)

Store protected from light at 4°C. If desired, mix DAPI directly in the mounting medium to a final concentration of 1 µg/mL and add 10% v/v 1X PBS; prepare fresh each month.

DAPI (4',6-diamidine-2'-phenylindole dihydrochloride; 1 µg/mL)

Store protected from light at -20°C.

DMF (dimethylformamide, anhydrous)

Ethanol (50% [v/v], prepared in 1X PBS) (for sediment samples only)

Ethanol (50%, 80%, 96% [v/v] in H₂O, and 100%)

Formaldehyde (37% [w/v])

H₂O₂ (30% [v/v] stock)

Store at 4°C.

HCl (10 mM) (optional; see Step 45)

IPBA (p-iodophenylboronic acid) (optional; see Step 6) (Aldrich 471933)

IPBA enhances the CARD-FISH signal. Protect from light.

Lysozyme (10 mg/mL in CARD-FISH proteinase buffer)

Prepare fresh immediately before use.

Oligonucleotide probes, HRP-labeled (Biomers, Germany)

Store 50-µL aliquots at -20°C. Thaw only once; store thawed aliquots for up to 6 mo at 4°C.

Use a nonsense probe (e.g., NON338) as a negative control.

Phosphate-buffered saline (PBS) (pH 7.6)

Proteinase K (0.1-10 µg/mL in CARD-FISH proteinase buffer)

Prepare fresh immediately before use.

Use for microbes not amenable to other permeabilization strategies. The optimal working concentration should be determined empirically for each sample (see Step 30.iv).

SDS (sodium dodecyl sulfate; 20% [w/v])

Tyramine HCl stock

Prepare immediately before use.

Equipment

Centrifuge, clinical (for sediment samples only)

Chamber, humidified

Desiccator

Filter paper, blotting (e.g., Whatman)

Filtration apparatus (for aqueous samples only)

Forceps

Gloves, powder-free

Hybridization oven, equipped with internal rotation shaker, variable temperature (37°C -46°C)

Laboratory tissues (optional; see Step 19)

Membrane filters, white polycarbonate, type GTTP, 0.2-µm pore size, 47-mm diameter (for aqueous samples only)

Micropipettors, 10-, 100-, and 1000-µL, with corresponding tips

Microscope, epifluorescence

Microscope slides, glass (for aqueous samples only)

Microscope slides, diagnostic, epoxy-printed, 10-well (for sediment samples only)

Microwave oven

Parafilm (for aqueous samples only)

Pencil, lead (for aqueous samples only)

Petri dishes, plastic (for aqueous samples only)

Rotary shaker

Sonicator probe (for sediment samples only)

Spectrophotometer

Tubes, 50-mL

Vacuum or freeze dryer (see Step 5)

Vials, 0.5- or 2.0-mL

Waterbath preset to 55°C (for sediment samples only)

METHOD

In addition to procedures for preparation of fluorescently labeled substrates (Steps 1-6), probe assessment (Steps 7-9), and CARD-FISH (Steps 28-44 and 45-48 [multicolor]), separate protocols are described for the processing of aqueous and sedimentary samples (Steps 10-19 and 20-27, respectively).

Tyramide Labeling

Custom labeling large batches of tyramides is fast and straightforward and should be started at least 2 d before CARD-FISH is to be performed. Because succinimidyl esters can hydrolyze rapidly, all reagents must be anhydrous, and the active dye and tyramine HCl stocks must be prepared just before use. Fluorescently labeled tyramides can also be purchased, but are considerably more expensive.

1. Prepare the dye stock(s) of choice:

Stock Amount to add Succinimidyl ester DMF Alexa350 5 mg 500 µL Alexa488 1 mg 100 µL Alexa546 1 mg 100 µL Alexa633 1 mg 100 µL CF 100 mg 10 mL

2. Add the dye stock of choice (from Step 1) in a 1.1-fold molar excess to the tyramine HCl stock solution:

Active dye Amount to add Dye stock Tyramine HCl stock Alexa350 500 µL 193 µL Alexa488 100 µL 25.2 µL Alexa546 100 µL 14.7 µL Alexa633 100 µL 13.1 µL CF 10 mL 3.3 mL

3. Incubate in the dark for 6-12 h at room temperature.

4. Dilute the reaction mixture with 100% ethanol to a final concentration of 1 mg of active dye/mL.

5. Dispense into 20-µL aliquots. Lyophilize in a freeze-dryer or under vacuum at room temperature.
Desiccated tyramides are stable for years at -20°C.

6. Reconstitute tyramides in 20 µL of DMF containing 20 mg/mL of IPBA (Alexa₄₈₈, Alexa₅₄₆, Alexa₆₃₃, or CF) or in sterile H₂O (Alexa₃₅₀).
Store tyramides in DMF at -20°C. Store tyramides in H₂O at 4°C.

Quality Check of Probes

Purified oligonucleotide probes frequently are delivered lyophilized. Before use, test the probes to ensure that the actual concentration corresponds to that claimed by the manufacturer.

7. Reconstitute lyophilized probes in 50 µL of H₂O. Dilute 1 µL of this stock 1:100 with H₂O.

8. Assess the probe quality:

To assess probe concentration

Both HRP and the probe contribute to the absorption maximum at 260 nm. The measured concentration therefore must be decreased by a correction factor of 0.276.

i. Measure the absorbance of the diluted stock at 260 nm and 404 nm (i.e., the absorbance maximum of HRP).

ii. Calculate the concentration of the single-stranded DNA oligonucleotide present in the sample:

OD_{260 adj} = OD₂₆₀ - (OD₄₀₄ x 0.276)

DNA concentration (ng/µL) = OD_{260 adj} x 20

To assess labeling efficiency

iii. Determine the OD₂₆₀/OD₄₀₄ ratio.
Presuming optimal labeling, the peak ratio (OD₂₆₀/OD₄₀₄) should be ~3.

9. Prepare working solutions of the probe at a concentration of 50 ng DNA/microliter. Store in 50- to 100-µL aliquots at -20°C.
Repeated freeze-thawing damages the peroxidase. Once thawed, HRP-labeled probes should be stored at 4°C and must not be refrozen. Probe working solutions stored at 4°C can be used for up to 6 mo.

Fixation and Preparation of Aqueous Samples

To prevent cell loss during permeabilization, the cells must be attached to polycarbonate filters (e.g., with agarose). Cell densities should be checked before and after pretreatment and hybridization.

10. Add formaldehyde to the sample to a final concentration of 2% (v/v). Fix for 1 h at room temperature (or for up to 24 h at 4°C).
This works well for marine plankton and benthos.

11. Filter the samples gently (i.e., at a pressure of ~5 mm Hg) onto white polycarbonate membrane filters.

12. Wash twice by filtration with 5-10 mL of particle-free H₂O.

13. Air-dry the filters.
Dried filters can be stored at -20°C for several months to years.

14. Boil the 0.1% agarose in H₂O completely in a microwave oven.

15. Fill a Petri dish with agarose. Allow the agarose to cool to 35°C-40°C.

16. Dip both sides of polycarbonate filters (from Step 13) in the agarose. Place the filters with the cells facing down onto a clean glass plate coated with Parafilm.

17. Allow the filters to dry for ~10-30 min at 20°C-40°C.

18. Soak the filters in 80%-96% ethanol for 1 min. Carefully peel the filters off the glass plate.

19. Air-dry the filters on a laboratory tissue or filter paper.

Fixation and Preparation of Sediment Samples

20. Add formaldehyde to the sample to a final concentration of 2% (v/v). Fix for 1 h at room temperature (or for up to 24 h at 4°C).

21. Centrifuge the samples at 16,000g for 10 min. Resuspend in 1X PBS. Repeat twice (i.e., three washes total in PBS).
Samples can be stored in 50% ethanol in PBS at -20°C until further processing.

22. To detach cells from sediment particles, mix 100 µL of sediment with 900 µL of 1X PBS.

23. Sonicate with a sonication probe at minimum power for 20 sec.
Adjust sonication conditions depending on the sediment/soil type used.

24. Mix the sonicated sample with 0.1% agarose in PBS containing 0.001% SDS. Incubate at 55°C for 3-5 min.

25. Pipet 10 µL of the sample suspension into the wells of the diagnostic slides. Allow to dry at room temperature.

26. Dehydrate the slides in ethanol:

i. Incubate in 50% ethanol for 5 min.

ii. Incubate in 80% ethanol for 1 min.

iii. Incubate in 96% ethanol for 1 min.

27. Dry the slides at room temperature.

Inactivation of Endogenous Peroxidases

Prokaryotes sometimes contain enzymes with pseudoperoxidase activity. If these enzymes are not inactivated, they can react with the fluorescently labeled tyramides and produce a nonspecific hybridization signal.

28. Incubate the filter sections (from Step 19) or slides (from Step 27) in 50 mL of 0.1% H₂O₂ for 1-2 min at room temperature.
The precise length of incubation should be determined empirically for each sample.

29. Wash the samples with 50 mL of H₂O twice (1 min each wash) at room temperature.
Samples can be permeabilized immediately or air-dried and stored at 4°C.

Permeabilization

To facilitate penetration of HRP-conjugated probes, cell walls often need to be permeabilized with at least one of the following enzymes. There is a trade-off between permeability and cell integrity that must be taken into account to optimize results for individual sample types. Prepare the enzyme solutions fresh immediately before use.

30. Permeabilize cell walls as required for the sample of interest:
After permeabilization, samples can be hybridized immediately. Alternatively, dehydrate the filters with 50 mL of 50% ethanol, air-dry, and store long-term at -20°C.

To permeabilize with lysozyme

i. Incubate the samples in 10 mg/mL lysozyme solution for 60 min at 37°C.

ii. Wash the samples with 50 mL of H₂O twice (1 min each wash) at room temperature.

To permeabilize with achromopeptidase

For CARD-FISH staining of Gram-positive freshwater Actinobacteria, cell walls need to be permeabilized with lysozyme, followed by a digestion with achromopeptidase.

iii. Incubate samples (from Step 30.ii) in the achromopeptidase solution for 30 min at 37°C.

To permeabilize with proteinase K

For other microbes not amenable to other permeabilization strategies, treatment with proteinase K is the best alternative. However, cell lysis can occur at high enzyme concentrations or after prolonged incubation with proteinase K. Also, the fixation conditions (i.e., duration, formaldehyde concentration) as well as the cell wall composition of the target organisms can influence the degree of permeabilization. Therefore, the appropriate concentration of proteinase K and the optimal incubation period should be modified for particular samples and target cells.

iv. Incubate the samples in 0.1-10 µg/mL proteinase K for 5-30 min at 37°C.

v. Wash the samples three times in 50 mL of H₂O, 1 min each wash.

Hybridization

The CARD-FISH hybridization and amplification buffers need to be prepared at least half a day in advance.

31. Dilute the HRP-conjugated oligonucleotide probe (from Step 9) 1:300 with CARD-FISH hybridization buffer.
Hybridize some samples with a nonsense probe (e.g., NON338) as a negative control to ensure that the observed CARD-FISH signals are specific.

32. Add the probe hybridization mix to the samples:

For filter-mounted samples

i. Cut filters in sections.
About 16 sections can be obtained from a filter with a diameter of 47 mm.

ii. Label sections with a lead pencil.
Avoid using other markers; they might contain fluorescent compounds.

iii. Place filter sections in reaction vials (0.5-2 mL, depending on the size of sections). Pipette the probe hybridization mix onto the sections.
At least two-thirds of the total volume of the reaction vial should be filled with buffer.

iv. Hybridize on a rotary shaker (~10 rpm) for 2-15 h at 46°C.
Do not let the filter sections dry out during hybridization; this can reduce the activity of the HRP.

For slide-mounted samples

v. Pipette 10-µL aliquots of the probe hybridization mix onto the samples in each well.

vi. Hybridize in a humidified chamber for 2-15 h at 46°C.

Catalyzed Reporter Deposition (CARD)

Most FISH protocols include a stringent washing step after hybridization; this is omitted here because CARD-FISH works with a probe concentration 30-fold lower than FISH using fluorochrome-labeled probes. Also, the washing step after CARD-FISH does not differ dramatically from the stringent washing step after FISH with fluorochrome-labeled probes.

33. Remove the filter sections from the hybridization buffer (or aspirate the hybridization mix from the slide wells). Incubate samples in 50 mL of 1X PBS for 15 min at room temperature.

34. Dilute 5 µL of 30% H₂O₂ in 1 mL of 1X PBS.

35. Mix 1 mL of CARD-FISH amplification buffer with 10 µL of the diluted H₂O₂ solution and 1-5 µL of fluorescently labeled tyramide (from Step 6).

36. Place filter sections in fresh reaction vials. Pipette the CARD-FISH amplification buffer/tyramide solution into the vials (or cover the slides with the solution).

37. Incubate in the dark for 10-15 min at 37°C-46°C.
The FISH signal intensity increases with temperature.

38. Wash the samples in the dark in 50 mL of 1X PBS for 5-15 min at room temperature.

39. Wash the samples with 50 mL of H₂O twice (1 min each wash) at room temperature.

40. Wash the samples in 50 mL of 50% ethanol for 1 min at room temperature. Allow to air-dry.
If labeling with more than one probe, proceed to Step 45.

41. Stain the samples with 1 µg/mL DAPI for 5-10 min at room temperature. Wash with H₂O for 1 min.

42. Wash with 100% ethanol for 1 min at room temperature. Allow the sections to air-dry.

43. Mount filters on slides using Citifluor or another low-fluorescence glycerol mountant containing an antibleaching agent.
Stained preparations can be stored at -20°C until further processing.

44. Examine using an epifluorescence microscope.
See Troubleshooting.

Multicolor CARD-FISH

To localize two or more target species simultaneously (e.g., to clarify the distribution of microorganisms within a dense community, such as in the case of biofilms or symbioses), the hybridization and cytochemical detection of different targets must be performed sequentially. Select appropriate fluorochromes for multicolor FISH; Alexa dyes are the fluorochromes of choice for this purpose.

45. Inactivate the HRP from the first hybridization by incubating the samples (from Step 40) in 0.1% H₂O₂ for 1-2 min at room temperature.

46. Wash the samples twice with H₂O, 1 min each wash.

47. Hybridize with a second HRP-conjugated oligonucleotide probe as described in Steps 31 and 32.

48. Visualize the second probe using a different fluorescently labeled tyramide reporter as described in Steps 33-44.
See Troubleshooting.

TROUBLESHOOTING

Problem: There is high background fluorescence.

[Steps 44 or 48]

Solution: Consider the following:

1. The tyramide concentration could be too high. Either decrease the tyramide concentration or increase the concentration of the blocking reagent.

2. If the background is covered with tiny fluorescent dots, the amount of probe might be too high. Check the probe concentration; 0.2 ng/µL is sufficient.

3. Increase the duration and/or number of washes with fresh H₂O after CARD.

Problem: There is low FISH signal intensity.

[Steps 44 or 48]

Solution: Consider the following:

1. If the ribosomal content of the target cells is low, increase the tyramide concentration or the temperature during the signal amplification step (up to 46°C). Prolonging the hybridization time (up to 15 h) can also help.

2. Verify the activity of the HRP-conjugated probes using a positive control; probes should be thawed only once and should not be stored for longer than 6 mo at 4°C.

3. Estimate the amount of unlabeled oligonucleotide spectrophotometrically to ensure that HRP is present in sufficient amount.

4. It is possible that the probe is not able to penetrate the cell wall. If this is the case, modify the permeabilization protocol.

5. Verify all reagents, particularly the concentration and reactivity of the tyramide, the pH of the PBS (~7.6), and the concentration and age of the H₂O₂; the working peroxide solution should be prepared fresh from the stock immediately before use.

REFERENCES

Ishii K, Mußmann M, MacGregor BJ, Amann R. 2004. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol Ecol 50: 203–212.[Medline]

Pernthaler A, Pernthaler J, Amann R. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol 68: 3094–3101.[Abstract/Free Full Text]

Sekar R, Pernthaler A, Pernthaler J, Warnecke F, Posch T, Amann R. 2003. An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization. Appl Environ Microbiol 69: 2928–2935.[Abstract/Free Full Text]

Teira E, Reinthaler T, Pernthaler A, Pernthaler J, Herndl GJ. 2004. Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by bacteria and archaea in the deep ocean. Appl Environ Microbiol 70: 4411–4414.[Abstract/Free Full Text]