Fluorescence in situ hybridisation (FISH)

2.11.1 Rumen samples

The sheep used were part of a long-term experiment to identify and breed low-CH4

emitting sheep (Pinares-Patiño et al., 2013b). They were fed lucerne pellets twice daily and rumen samples were collected by stomach tubing 2 h after the morning feeding.

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2.11.2 Rumen fluid fixation and preservation for FISH

Rumen samples were kept on ice immediately after collection and transported to the laboratory. The samples were fixed in two different ways: one in 4% paraformaldehyde (PFA) (w/v) solution:sample (1:3), and second in absolute ethanol:sample (1:1). PFA- fixed samples were used in FISH experiments to identify Quinella in rumen samples by microscopy, while ethanol-fixed samples were used for FISH combined with single cell sorting of Quinella. Post sampling processing steps for both types of fixation were the same except that the ethanol-fixed samples were processed immediately on arrival into the laboratory and PFA-fixed samples were kept at 4 °Cfor 2 h. Samples were centrifuged for 8,000 g for 5 min. Supernatants were discarded and the pellets were washed twice in PBS buffer by repeating the centrifugation step. Finally, pellets were diluted in 750 μl of PBS and pure ethanol (1:1) and stored at 20 °C.

2.11.3 FISH probe design

Sequences from the SILVA database (refined by Henderson et al. (2017) in combination with 16S rRNA clone library sequences of Quinella were used as reference sequences to design FISH probes. Initially the ARB software Probe Design Tool was used to find Quinella specific probes but this was unsuccessful. Then, 16S rRNA gene sequences from

Quinella were inspected manually in MEGA 7 (Kumar et al., 2016) for signature

conserved 18-20 bp length sequences. Shortlisted probe sequences were tested using the ARB Probe Match Tool to confirm specificity. Specific probes with exact matches to Quinella sequences were synthesised by IDT (Custom Science, Auckland, New Zealand) with three different 5' fluorochromes (Cy3, Cy5 and Alexa488). Details are given in Table 5.1. A bacterial-specific domain-level probe EUB338 (5´ GCTGCCTCCCGTAGGAGT; target site E. coli position. 338-355), and a nonsense probe nonEUB338 (the reverse and complement of EUB338) were used as positive and negative controls respectively. Probes were aliquoted (2.5 μg) in 200 μl microcentrifuge tubes and stored at 20 °C. Before use, probes were resuspended in 50 μL of sterile nuclease-free water to make a working concentration of 50 ng/μL.

2.11.4 FISH probe testing

Newly designed probes were tested using PFA-fixed rumen fluid samples. Probe stringencies (conditions at which probes specifically hybridised only to Quinella-like

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cells) were optimised by varying formamide concentration, NaCl concentration and hybridisation temperature. The rest of the procedure was as described by Hugenholtz et al. (2001) with slight modifications. Briefly, 3-5 μL (depending upon cell concentration) samples of fixed cells were applied on 10-well FISH slides and air dried for at least 3 h or overnight. Slides were then dehydrated using a series of ethanol washes starting at 50%, 80% and finally 100% ethanol for 3 min each and finally air dried. A hybridisation oven was pre-warmed to the desired hybridisation temperature, e.g. 46 °C, 50 °C or 52 °C. Hybridisation buffers (8 μL; see Table 2.2) with various formamide and NaCl concentrations were applied to different slides in duplicate. Probes (1 μL/well of 50 ng/μL) were applied either singly or in combination to different wells and mixed gently with a micropipette tip without touching the well surface. Each slide was then transferred carefully into a 50 mL Falcon tube containing moistened paper towels, capped firmly and placed horizontally in the hybridisation oven for 2 h. After hybridisation, slides were well rinsed with the same pre-warmed wash buffer (Table 2.2) to remove unhybridised probes. Slides were then transferred into Falcon tubes containing wash buffer (Table 2.2) and placed in a waterbath (at a temperature 2 °C higher than the hybridisation temperature) for 15 min. Slides were then rinsed briefly with ice-cold distilled water and dried immediately either with compressed air or in a 39 °C oven. Antifade mounting solution (VECTASHIELD; Vector Laboratories Ltd., Burlingame, CA, USA) was applied carefully on the slides and covered with a coverslip without trapping air bubbles in the wells. Slides were then visualised by epifluorescence microscopy using the appropriate filters (for each probe’s fluorochrome).

2.11.5 Clone FISH

Clone FISH (Schramm et al., 2002) was performed to test the newly-designed FISH probes. 16S rRNA genes clones (i.e. E. coli containing pCR2.1 plasmid with a Quinella 16S rRNA gene insert) with exact sequence matches were used as positive controls whereas cloned sequences with one or more mis-matches were used as negative controls. These were clones generated as described in section 2.7.6. First, the inserts and vectors were sequenced to ensure they had the correct sequence orientation (5'-3') using primer 514R that targets a region flanking the cloning site of the vector (Table 2.1), so that the RNA generated contained the correct probe binding sites. Clones with the wrong orientation (5'-3') were sub-cloned to get the correct orientation. Briefly, plasmids were extracted from Quinella clones (i.e., E. coli containing pCR2.1 plasmid with Quinella

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16S rRNA gene inserts) and 16S rRNA gene sequences were amplified using 27f and 1492r primers (Table 2.1). Amplified sequences were then ligated into the plasmid (pCR2.1) and transformed in E. coli JM109 (DE3). Clones were then screened again by sequencing for examples with the desired orientation. Clones with the correct orientation were then grown in LB medium containing ampicillin, until they achieved an optical density of 0.4 (600 nm), then 100 μl of 100 mM IPTG were added to 10 ml of grown cells and incubated at 37 °C for 1 h. 50 μl (34 mg/ml) of chloramphenicol was then added to the cells which were further incubated for 3 h at 37 °C. This process was used to increase the copy number of transcribed 16S rRNA to increase the FISH probe targets so as to enhance hybridisation signal intensity (Schramm et al., 2002). Methods described in section 2.11.4 were followed to determine conditions for probe stringency.

2.11.6 Liquid FISH

A liquid FISH method was developed using concentrated Quinella samples (section 2.12.3) and probe Quin1231. FISH probe stringency was tested using the protocol described in section 2.11.4 with some changes. Here all steps were conducted in 1.5 mL microfuge tubes. First, 800 μL of TE buffer (10 mM Tris and 1 mM EDTA, pH 7.5 with HCl) was added to 200-500 μL (depending on cell density) of ethanol-fixed concentrated

Quinella cells and centrifuged at 8,000 g for 5 min at 20 °C. The supernatant was

discarded and the pellet was washed twice with 1 mL TE buffer by re-suspending it and then centrifuging it at 8,000 g for 5 min at 20 °C and discarding the supernatant. The pellet was then re-suspended in 1 mL of 0.22 μm-filtered TE-lysozyme (1 mg/mL) solution and incubated for 10 min at room temperature. The mixture was centrifuged at 8,000 g for 3 min at 4 °C and the supernatant was removed. The pellet was then washed again by adding 1 mL of PBS and centrifuging at 8,000 g for 3 min at 4 °C and discarding the supernatant. The pellet was then re-suspended in 500 μL of hybridisation buffer by gentle mixing. 70 μL aliquots of this homogenised suspension were transferred to 1.5 mL plastic tubes and 7 μL of the desired probe (50 ng/μL) was added into each tube. Hybridisation was carried out at the desired temperature for 2 h. Remaining hybridisation buffer was kept at the same temperature whereas the wash buffer at 2 °C warmer. After hybridisation, 150 μL of the pre-warmed hybridisation buffer was added to each tube and centrifuged at 8,000 g for 3 mins at 20 °C. The supernatant was removed and 200 μl of the pre-warmed wash buffer was added. The pellet was re-suspended by pipetting and then incubated at the wash buffer temperature for 20 min. Wash buffer was then removed

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by centrifuging at 8,000 g for 3 min at 20 °C. Finally, the pellet was re-suspended in 100 μL of PBS and hybridisation with the probe was checked by epifluorescence microscopy using various filters (appropriate for each probe’s fluorochrome). A hybridisation temperature of 46 °C and a wash temperature of 48 °C was found to result in optimal

Quinella-specific probe hybridisation to large Quinella-like cells, without binding to

other cell types.

Table 2.2 Hybridisation and washing buffer preparation for FISH.

Formamide concentration (v/v, %)

0 20 40 60 80

Hybridisation buffer Volume (μL)

5 M NaCl 360 360 360 360 360

1 M Tris-HCl, pH 8.0 40 40 40 40 40

100% formamide 0 400 800 1200 1598

Sterile Milli-Q water 1598 1198 798 398 0

10% (w/v) SDS 2 2 2 2 2

Total volume 2000 2000 2000 2000 2000

Wash buffer Volume (μL)

5 M NaCl 9000 2150 460 40 0

1 M Tris-HCl, pH 8.0 1000 1000 1000 1000 1000

0.5 M EDTA 0 500 500 500 175

Sterile Milli-Q water 39950 46300 47990 48410 48775

10% (w/v) SDS 50 50 50 50 50

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