Sesión para el desarrollo de las estrategias de memoria

Anaerobic glycerol solution (40% v/v)

Anaerobic glycerol solution is composed of 4.4 M glycerol (BDH, VWR International Ltd, Poole, UK) in water with the components of Table 2.2 added in the order listed. The solution was mixed, boiled for 5 min, cooled to room temperature under

a continuous flow of O2-free CO2. Once cooled, L-cysteine-HCl (0.125 g) was added per

500 mL of the glycerol solution and transferred to serum bottles in 70 mL aliquots while

being flushed with CO2. The bottles were sealed with butyl rubber bungs and sealed with

aluminium crimp caps before being autoclaved at 121 ºC for 20 min. Table 2.2. Anaerobic glycerol solution.

Component Volume or weight

dH2O 130 mL

Salt solution A 85 mL

Glycerol 200 mL

Salt solution 2B 85 mL

Resazurin solution (1% w/v) 2 drops

NaHCO3 2.5 g

L-cysteine-HCl 0.125 g

DNA-free water

Ultrapure water was collected from a Milli-Q Integral Water Purification System (EMD Millipore/Merck Millipore, Darmstadt, Germany) and sterilised by autoclaving, followed by microfiltration. The water was then irradiated with UV light (254 nm, 6 W) for 8 h.

Diethylpyrocarbonate (DEPC)-treated H2O

DEPC (0.1% v/v, Sigma-Aldrich, St. Louis, MO, USA) was added to dH2O and

33 Table 2.1. Bacterial strains used in this thesis.

Species Strain Accession number * Source/Reference

Butyrivibrio fibrisolvens AB2020 NA Noel (2013) Butyrivibrio fibrisolvens D1T DSM 3071 Bryant and Small (1956a) Butyrivibrio fibrisolvens FE2007 NA Noel (2013) Butyrivibrio fibrisolvens MD2001 NA Noel (2013) Butyrivibrio fibrisolvens ND3005 NA Noel (2013) Butyrivibrio fibrisolvens WTE3004 NA Noel (2013) Butyrivibrio fibrisolvens YRB2005 NA Noel (2013) Butyrivibrio hungatei JK615T DSM 14810 Kopecny et al. (2003) Butyrivibrio hungatei MB2003 NA Palevich (2011) Butyrivibrio hungatei NK4A153 NA Noel (2013) Butyrivibrio proteoclasticus B316T DSM 14932 Attwood et al. (1996) Butyrivibrio proteoclasticus FD2007 NA Noel (2013) Butyrivibrio proteoclasticus P6B7 NA Noel (2013)

Butyrivibrio sp. AC2005 NA Noel (2013)

Butyrivibrio sp. AD3002 NA Noel (2013)

Butyrivibrio sp. AE2005 NA Noel (2013)

Butyrivibrio sp. AE2015 NA Noel (2013)

Butyrivibrio sp. AE2032 NA Noel (2013)

Butyrivibrio sp. AE3003 NA Noel (2013)

Butyrivibrio sp. AE3004 NA Noel (2013)

Butyrivibrio sp. AE3006 NA Noel (2013)

Butyrivibrio sp. AE3009 NA Noel (2013)

Butyrivibrio sp. FC2001 NA Noel (2013) Butyrivibrio sp. FCS006 NA Noel (2013) Butyrivibrio sp. FCS014 NA Noel (2013) Butyrivibrio sp. LB2008 NA Noel (2013) Butyrivibrio sp. LC3010 NA Noel (2013) Butyrivibrio sp. MB2005 NA Noel (2013) Butyrivibrio sp. MC2013 NA Noel (2013) Butyrivibrio sp. MC2021 NA Noel (2013) Butyrivibrio sp. NC2002 NA Noel (2013) Butyrivibrio sp. NC2007 NA Noel (2013) Butyrivibrio sp. NC3005 NA Noel (2013) Butyrivibrio sp. VCB2001 NA Noel (2013) Butyrivibrio sp. VCB2006 NA Noel (2013) Butyrivibrio sp. VCD2006 NA Noel (2013) Butyrivibrio sp. WCD2001 NA Noel (2013) Butyrivibrio sp. WCD3002 NA Noel (2013)

Butyrivibrio sp. WCE2006 NA Noel (2013)

Butyrivibrio sp. XBB1001 NA Noel (2013)

Butyrivibrio sp. XPD2002 NA Noel (2013)

Butyrivibrio sp. XPD2006 NA Noel (2013)

Escherichia coli DH5α DSM 6897 Scheutz and Strockbine (2005) Prevotella ruminicola 23 NA Avguštin et al. (1994) Pseudobutyrivibrio ruminis AD2017 NA Noel (2013) Pseudobutyrivibrio ruminis CF1b NA Noel (2013) Pseudobutyrivibrio ruminis HUN009 NA Noel (2013) Pseudobutyrivibrio sp. LB2011 NA Noel (2013) Pseudobutyrivibrio sp. MD2005 NA Noel (2013) Pseudobutyrivibrio xylanivorans MA3014 NA Palevich (2011) Pseudobutyrivibrio xylanivorans Mz5T DSM 14809 Kopecny et al. (2003) Streptococcus bovis 2B ATCC 33317 Iverson and Millis (1976)

* DSM: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany. NA, not associated with an accession number. T_{, indicates type strain.}

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dNTP solution

Deoxynucleotide triphosphates (dNTPs), dATP, dCTP, dGTP, and dTTP were supplied separately at 100 mM (Thermo Fisher Scientific Inc., Waltham, MA, USA),

mixed in equimolar concentrations and diluted in dH2O to make a 20 mM total dNTP

stock solution. Stocks were stored at -20 ºC until required.

EC buffer

Ethylene diamine tetraacetic acid (EDTA) (100 mM), NaCl (1 M), N-

Laurylsarcosine (35 mM, Sigma-Aldrich, St. Louis, MO, USA) and Trizma base (6 mM,

Thermo Fisher Scientific Inc., Waltham, MA, USA) were prepared in dH2O, adjusted to

pH 7.6 with 1 M NaOH, then autoclaved at 121 ºC for 20 min.

EDTA-Sarkosyl solution

EDTA (0.5 M) and N-Laurylsarcosine (35 mM) were prepared in dH2O and pH

adjusted to pH 8.0 with 1 M NaOH, then autoclaved at 121 ºC for 20 min.

Mineral solution I (Salt solution 2B)

KH2PO4 (45 mM) and KH2PO4.H2O (60 mM), all supplied by VWR International

Ltd, Poole, UK, were dissolved in dH2O and autoclaved at 121 ºC for 20 min.

Mineral solution II

This solution contained (NH4)2SO4 (45 mM), CaCl2.2H2O (10 mM), MgSO4.7H2O

(10 mM), NaCl (200 mM) and KH2PO4 (45 mM). All ingredients (supplied by VWR

International Ltd, Poole, UK) were dissolved in dH2O and autoclaved at 121 ºC for 20

min.

Modified Karnovsky’s fixative

Paraformaldehyde powder (VWR International Ltd, Poole, UK) was dissolved in

100 mL dH2O and heated to 60 ºC with the drop wise addition of 1 M NaOH until the

solution became transparent. Once cooled, Buffer Salts (2.51 g Na2HPO4.12H2O and 0.41

g KH2PO4) and Glutaraldehyde Solution (12 mL of 25% v/v glutaraldehyde in dH2O)

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Orange G loading dye (6× stock solution)

The Orange G loading dye contained 20 mM Tris-HCl (pH 7.6), 0.4% (w/v) 1- phenylazo-2-naphthol-6, 8-disulfonic acid disodium salt (Orange G sodium salt), 30% (v/v) glycerol, 60 mM EDTA (pH 8.0) and 0.1% (w/v) sodium dodecyl sulfate (SDS). Dye aliquots were stored short-term at 4 ºC or long-term at -20 ºC.

Phosphate-buffered saline (PBS) solution (10× stock solution)

To make 10× concentrated stock solution, CaCl2 (0.01 g) and MgCl2 (0.01 g) were

dissolved in 100 mL dH2O and added to a solution containing NaCl (8.00 g), KCl (0.20

g), Na2HPO4 (1.44 g) and KH2PO4 (0.24 g) dissolved in 1.0 L dH2O (Sambrook et al.,

1989). The combined solution was adjusted to pH 7.4, mixed well and autoclaved at 121 ºC for 20 min (Neinhuis and Edelmann, 1996).

Paraformaldehyde (PFA) solution (4% w/v)

PFA powder (12 g, VWR International Ltd, Poole, UK) was dissolved in 195 mL

dH2O, heated to 60 ºC with the drop-wise addition of 1 M NaOH until the solution became

transparent (Neinhuis and Edelmann, 1996). A volume of 99 mL of 3× PBS solution was added and once cooled, the pH was adjusted to 7.2, it was then filter sterilised and stored at -20 ºC.

Saline-EDTA solution

Saline-EDTA solution was prepared by adding EDTA powder (VWR International

Ltd, Poole, UK) to a concentration of 10 mM, and NaCl to 150 mM to dH2O. The pH was

adjusted to 8.0 and solution was autoclaved at 121 ºC for 20 min.

Salt solution A

To make Salt solution A, NaCl (6.0 g), (NH4)2SO4 (1.5 g), KH2PO4 (3.0 g),

CaCl2.H2O (0.79 g) and MgSO4.7H2O (1.2 g) were dissolved in 1.0 L of dH2O.

Salt solution 2B

To make salt solution B, either K2HPO4 (6.0 g) or K2HPO4.H2O (7.9 g) were added

36

SDS solution (20% w/v)

SDS (200 g) was dissolved in dH2O, pH adjusted to 7.2 and volume adjusted to 1.0

L, followed by filter sterilization into a 1.0 L sterile Schott bottle.

Substrate solutions

Stock solutions (20% w/v) were made for 32 sugar substrates. The appropriate

amount of sugars were dissolved in 20 mL of dH2O in N2-filled 100 mL Schott bottles

and autoclaved at 121 ºC for 20 min. For insoluble polysaccharide substrates, 4 g of each

substrate was added to 20 mL of dH2O in N2-filled 100 mL Schott bottles, mixed by

heating with stirring and autoclaved at 121 ºC for 20 min. The D (+) enantiomers of all

substrates were used and were as follows: amygdalin, arabinose, cellobiose, cellulose, dextrin, esculin, fructose, galactose, glucose, glycerol, glycogen, myo-inositol, inulin, lactose, maltose, mannitol, mannose, melezitose, melibiose, pectin, raffinose, rhamnose, ribose, rutin, salicin, sorbitol, starch, sucrose, trehalose, xylan, xylitol and xylose. The solutions were stored in the dark at room temperature until use (Leedle and Hespell, 1980).

Tris-acetate-EDTA (TAE) buffer (50× stock solution)

A solution containing glacial acetic acid (950 mM, VWR International Ltd, Poole, UK), EDTA (50 mM) and Trizma base (2 M, Thermo Fisher Scientific Inc., Waltham,

MA, USA) was prepared in dH2O, pH adjusted to 8.0 with 1 M NaOH and autoclaved at

121 ºC for 20 min. A working solution (1×) was made by diluting the stock solution 1:50

in dH2O.

Tris-borate-EDTA (TBE) buffer (5× stock solution)

A solution containing boric acid (445 mM, VWR International Ltd, Poole, UK),

EDTA (10 mM) and Trizma base (445 mM) was prepared in dH2O and autoclaved at 121

ºC for 20 min. A working solution (0.5×) was made by diluting the stock solution 1:10 in

dH2O.

Tris-EDTA (TE) buffers

Three types of TE buffer were used: TE buffer 10/0.1 (10 mM Trizma base and 0.1 mM EDTA), TE buffer 10/1 (10 mM Trizma base and 1 mM EDTA) and TE buffer

37

10/100 (10 mM Trizma base and 0.1 M EDTA). The buffer components were dissolved

in dH2O, pH adjusted to 8.0 and autoclaved at 121 ºC for 20 min.

Tris-EDTA-sucrose (TES) buffer

EDTA (1 mM), sucrose (250 mM) and Trizma base (10 mM) were dissolved in

dH2O, pH adjusted to 7.5 and autoclaved at 121 ºC for 20 min.

Volatile fatty acid (VFA) solution

Butyric acid (114 mM), iso-butyric acid (28 mM) , iso-valeric acid (24 mM), n-

valeric acid (24 mM) (all supplied by Sigma-Aldrich, St. Louis, MO, USA), D, L-2-methyl

butyric acid (24 mM), propionic acid (202 mM) (both supplied by Merck, Darmstadt,

Germany) and acetic acid (710 mM) were prepared in dH2O, pH adjusted to 7.5 with 1 M

NaOH and the mixture was stored at -20 ºC (Attwood et al., 1998).

Wash solution for Pulse-Field Gel-Electrophoresis

NaCl (2 M) and Trizma base (20 mM) were dissolved in dH2O, pH adjusted to 7.6

with 6 M HCl and autoclaved at 121 ºC for 20 min.