Criterios básicos para una movilidad ciclista ideal

2.2.2.1 Anaerobic glycerol solution

Composition of the anaerobic glycerol solution and order of component addition are given in Table 2-3. The mixture was brought to the boil by heating in a microwave oven and allowed to cool under the continuous flow of CO2. Once cool 0.125g L.cysteine·HCl·H2O was added per 500 mL and the glycerol solution transferred to serum bottles while being flushed with CO2. The bottles were sealed with butyl rubber bungs and sealed with crimp caps before being autoclaved.

Table 2-3. Anaerobic glycerol solution components.

Component Volume or weighta

Salt solution A 85 mL Glycerol 200 mL dH2O 130 mL Salt solution 2B 85 mL Resazurin solution 0.1% (w/v) 50 μL NaHCO3 2.5 g

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2.2.2.2 Rumen fluid collection for preparation of media

Samples of the whole rumen contents are collected from two hay-fed fistulated cows. Feed was withheld from the animals after 4 pm, and rumen contents collected at 9 -10 am the following morning. Rumen contents were filtered through a single layer of cotton cheesecloth with a mesh size of approx. 1 mm (Stockinette; Cirtex Industries Ltd., Thames, New Zealand) into a collection vessel and then fine particulate material was removed by centrifugation at 10,000 × g for 20 min. The resulting supernatant

(rumen fluid) was frozen at –20 °C. Before use, the rumen fluid was thawed and centrifuged a second time at 10,000 × g for 20 min.

2.2.2.3 Clarified rumen fluid for preparation of media

Thawed rumen fluid collected as described in previous section (2.2.2.2) was heated in a microwave until the point of boiling and then allowed to cool under a continuous flow of oxygen-free nitrogen gas for at least 10 min. The anoxic rumen fluid was then sealed in a nitrogen flushed serum bottle with a butyl rubber stopper and autoclaved. Once rumen fluid equilibrated to room temperature, 0.08 M MgCl2·6H2O (1.63g per 100 ml) and 0.08 M CaCl2·2H2O (1.18g per 100 ml) was added, forming a heavy precipitate. The rumen fluid was then centrifuged at 25,500 × g at 4 °C for 20

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2.2.2.4 GenRFV – General Rumen Fluid-Vitamin mix

The GenRFV (Table 2-4) was prepared by adding the listed components to 100 mL of clarified rumen fluid and dissolved by heating until boiling in a microwave oven. The resulting mixture was made anoxic by being allowed to cool under a continuous flow of oxygen free nitrogen gas for 15 min. The mixture was passed through a filter (0.2 μm pore diameter) into a sterile nitrogen flushed serum vial sealed with a butyl rubber stopper. Finally, 2 mL of vitamin 10 concentrate solution (Table 2-7; 2.2.1.8) per 100 mL of mixture was added anoxically, using a sterile syringe and needle. The final growth media containing GenRFV were prepared by adding 0.5 mL of GenRFV to 9.5 mL of medium; this results in final medium concentrations: 5% (v/v) rumen fluid, 0.5 mM of cellobiose, 1 mM each of glucose, xylose, and arabinose, 5 mM lactate, 4 mM each of Ca2+ and Mg2+, and 1 g each of casamino acids, Bacto-peptone, and yeast extract per L.

Table 2-4. GenRFV solution components.

Component Volume or weight

D-glucose (Labserv, Auckland, NZ) 0.36 g

D-cellobiose (Sigma-aldrich, Steinheim, Germany) 0.34 g D-xylose (Sigma-aldrich, Steinheim, Germany) 0.30 g L-arabinose (Sigma-aldrich, Steinheim, Germany) 0.30 g Na L-lactate syrup ( Applichem, Damstalt, Germany) 0.88 ml

Casamino acids (BD, Claix, France) 2 g

Bacto-peptone (BD, Claix, France) 2 g

Yeast extract (BD, Claix, France) 2 g

Rumen fluid 100 ml

Vitamin 10 concentrate solutiona 2 ml

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2.2.2.5 2xS GenRFV - 2× Sugars General Rumen Fluid - Vitamin mix

2xS GenRFV was made by the same procedure as was GenRFV; except that the 2xS GenRFV contained two-fold as high concentration of D-glucose, D-cellobiose, D- xylose and L-arabinose (Table 2-5). The final growth media containing 2xS GenRFV were prepared by adding 0.5 mL of 2xS GenRFV to 9.5 mL of medium; this results in final medium concentrations: 5% (v/v) rumen fluid, 1.0 mM of cellobiose, 2 mM each of glucose, xylose, and arabinose, 5 mM lactate, 4 mM each of Ca2+ and Mg2+, and 1 g each of casamino acids, Bacto-peptone, and yeast extract per litre.

Table 2-5. 2xS GenRFV solution components.

Component Volume or weight

D-glucose 0.72 g D-cellobiose 0.68 g D-xylose 0.60 g L-arabinose 0.60 g Na L-lactate syrup 0.88 ml Casamino acids 2 g Bacto-peptone 2 g Yeast extract 2 g Rumen fluid 100 ml

Vitamin 10 concentrate solutiona 2 ml

a_{Added to prepared anoxic mixture of all other components listed above}

2.2.2.6 GenV mix

GenV mix was modified from GenRFV mix with the clarified rumen fluid replaced with distilled water.

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2.2.2.7 2xS GenV mix

2xS GenV mix was modified from 2xS GenRFV mix with the clarified rumen fluid replaced with distilled water.

2.2.2.8 Titanium (III) NTA solution

Titanium (III) NTA (nitrilotriacetic acid) solution was made as previously described by Moench et al.(1983). 1 L of distilled water was brought to the boil and

allowed to cool under continuous flow of oxygen free nitrogen gas. 9.6 g of nitrilotriacetic acid (Sigma, Sreinheim, Germany) was added to 300 mL of the anaerobic water while under continuous nitrogen gas flow. The pH was adjusted to 9.0 with NaOH. To this mixture, 9.6 mL of 20% TiCl3 solution (BDH, VWR International Ltd Poole, UK), kept under N2, was slowly added, while maintaining the pH above 2.0 with Na2CO3 to avoid unwanted precipitates forming. The final pH was adjusted to 7.0 with anaerobic Na2CO3 solution and made up to the final volume of 500 mL with anaerobic water, before being transferred to a sealed N2-flushed sterile serum vial through a sterile filter (0.22 μm pore size; Milipore) using a sterile syringe and needle. This results in final concentration of 25 mM Ti III and 100 mM NTA.

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