L.A.T = % IFIAT * ISAT *1

2.3.1.1 Single species experiments

In all single species experiments the bacterium used was Streptococcus sanguis NCTC 10904. Tryptone soya agar plates (Oxoid Ltd., Basingstoke, UK) were inoculated with the type culture and then incubated aerobically at 37°C for 24 hours. Following incubation, individual colonies were transferred from the agar plates to 10 ml tryptone soya broth (Oxoid). This broth was incubated

T h e effect of phosphate-based glasses on the form ation and viability o f oral bacterial biofilms April M iranda Mulligan

aerobically at 37°C for 24 hours. After this time, the resulting culture was added to 100 ml sterile tryptone soya broth. This broth was incubated at 37°C for a further 24 hours. The resulting culture was then Gram stained to check the purity of the culture. There was no sign of contamination so the culture was distributed in 1 ml aliquots into cryovials (Fisher Scientific, Loughborough, UK). These were stored at -70°C.

2.3.1.2 Microcosm experiments (supragingival conditions)

In all microcosm experiments carried out under supragingival conditions, pooled saliva was used. Saliva was collected from 10 healthy individuals, pooled together, mixed and then distributed in 1 ml aliquots into cryovials. These cryovials were then stored at -70°C.

2.3.1.3 Microcosm experiments (subgingival conditions)

In all microcosm experiments carried out under subgingival conditions, pooled subgingival plaque was used. Subgingival plaque was collected from individuals requiring treatment for moderate to advanced Adult Periodontitis from the Department of Periodontology at the Eastman Dental Hospital. Each subgingival plaque sample (containing subgingival plaque from at least two periodontal pockets) was suspended in 1 ml of reduced transfer fluid (RTF) (cryoprotectant) and stored at -70°C until required. When needed, the samples were de-frosted and centrifuged at 1 0 , 0 0 0 rpm in a benchtop centrifuge

(Eppendorf UK) for 10 minutes. The RTF was removed, leaving a pellet at the bottom of the centrifuge tube. The pellets were suspended in 20 ml of pre­ reduced Brain Heart Infusion Broth (Oxoid) with 10% glycerol (Sigma). The

T h e effect o f phosphate-based glasses on the formation and viability of oral bacterial biofilms April M iranda Mulligan

samples were then combined, mixed and distributed in 1 ml aliquots into

cryovials.

RTF was prepared as described by Syed & Loesche (1972) using several stock solutions. Tables 2.7 to 2.10 show the compositions of the stock solutions used (all chemicals were obtained from Sigma). Table 2.11 shows the composition of RTF.

Table 2.7 Composition of stock solution 1 used during the production of RTF Ingredients Quantity per 100 ml distille d w ater

(9) Dibasic potassium phosphate 0 . 6

Table 2.8 Composition of the magnesium sulphate stock solution used during the production of RTF

Ingredients Quantity per 100 ml distille d water (9)

T h e effect of phosphate-based glasses on the form ation and viability of oral bacterial biofilms April M iranda Mulligan

Table 2.9 Composition of stock solution 2 used during the production of RTF Ingredients Quantity per 99 ml distilled water

Potassium chloride 1 2 g

Ammonium sulphate 1 2 g

Monobasic potassium phosphate 0 . 6 g

Magnesium sulphate stock solution 1 ml

Table 2.10 Composition of sodium carbonate stock solution used during the production of RTF

Ingredients Quantity per 10 ml distilled water (g)

Sodium carbonate 0 . 8

Table 2.11 Composition of 100 ml RTF

Ingredients Quantity (ml)

Stock solution 1 7.5

Stock solution 2 7.5

Sodium carbonate stock solution 0.5

Distilled water 80.0

The medium was autoclaved at 120°C for 15 minutes. Once cooled, a solution of the reducing agent dithiothreitol (0.02 g in 5 ml distilled water) (Sigma), which had been filter sterilised using Nalgene 0.2 |im syringe filters (Fisher Scientific),

T h e effect of phosphate-based glasses on the form ation and viability o f oral bacterial biofilms April M iranda Mulligan

2.3.2 NUTRIENT SOURCE 2.3.2.1 Artificial saliva

Artificial saliva, as described by Pratten et al. (1998c), was used as a nutrient source in all single species and microcosm experiments (supragingival conditions). The composition of the artificial saliva used is shown in Table 2.12.

Table 2.12 Composition of artificial saliva

Ingredients _g/L

‘Lab-lemco’ powder ® 1 . 0

Yeast extract ® 2 . 0

Proteose peptone ® 5.0

Mucin Type III 2.5

Sodium chloride ^ 0.35 Calcium chloride ^ 0 . 2 Potassium chloride ^ 0 . 2 ® Supplied by Oxoid ^ Supplied by Sigma ^ Supplied by BDH

The medium was autoclaved at 121°C for 15 minutes. After autoclaving, 1.25ml/L of 40% (w/v) filter sterilised urea (Sigma) was added.

T h e effect o f phosphate-based glasses on the form ation and viability of oral bacterial biofilms April M iranda Mulligan

2.3 2.2 Artificial gingival crevicular fluid

Artificial gingival crevicular fluid (GCF) was used in all microcosm experiments (subgingival conditions). This contained 600 ml/L of RPMI medium (without L- glutamine, without phenol red) (Sigma) and 400 ml/L horse serum (Oxoid). In addition, 10 ml/L of a 0.5 mg/ml stock solution of haemin (Sigma) was added (to give a final concentration of 5 pg/ml in artificial GCF). Also, 10 ml/L of a 0.1 mg/ml stock solution of menadione (Sigma) was added (to give a final concentration of 1 pg/ml in artificial GCF).

The haemin stock solution was prepared by dissolving 50 mg haemin in 1 ml sodium hydroxide (NaOH) (BDH) (1 mole). This was then diluted to 100 ml with sterile distilled water. The solution was filter sterilised. The stock solution was stored at 4°C for a period of one month.

The menadione stock solution was prepared by dissolving 10 mg menadione in 2 ml ethanol (BDH) (95%). This was then diluted to 100 ml with sterile distilled water. The solution was filter sterilised. The stock solution was stored at 4°C for a period of one month.