Proteose-peptone

Escherichia coli TOP10 (Invitrogen) and E. coli CA434 [39] (link) were cultured in Luria-Bertani (LB) medium, supplemented with chloramphenicol (25 µg/ml), where appropriate. Routine cultures of C. difficile 630 Δerm[40] (link) and C. difficile R20291 were carried out in BHIS medium (brain heart infusion medium supplemented with 5 mg/ml yeast extract and 0.1% [wt/vol] L-cysteine) [41] (link). C. difficile medium was supplemented with D-cycloserine (250 µg/ml), cefoxitin (8 µg/ml), lincomycin (20 µg/ml), and/or thiamphenicol (15 µg/ml) where appropriate. A defined minimal media [18] (link) was used as uracil-free medium when performing genetic selections. A basic nutritive mannitol broth for growth assays of C. difficile strains were prepared as follows : Proteose peptone no. 2 4% [wt/vol] (BD Diagnostics, USA), sodium phosphate dibasic 0.5%[wt/vol], potassium phosphate monobasic 0.1%[wt/vol], sodium chloride, 0.2% [wt/vol], magnesium sulfate, 0.01% [wt/vol], mannitol, 0.6% [wt/vol] with final pH at +/−7.35. For solid medium, agar was added to a final concentration of 1.0% (wt/vol). Clostridium sporogenes ATCC 15579 was cultivated in TYG media [7] (link). All Clostridium cultures were incubated in an anaerobic workstation at 37°C (Don Whitley, Yorkshire, United Kingdom). Uracil was added at 5 µg/ml, and 5-Fluoroorotic acid (5-FOA) at 2 mg/ml. All reagents, unless noted, were purchased from Sigma-Aldrich.

Bacteria were standardized (1 × 10⁷ cfu/mL) in artificial saliva (AS), which contained the following constituents, as described previously
[11 (link)]. This included porcine stomach mucins (0.25% w/v), sodium chloride (0.35 w/v), potassium chloride (0.02 w/v), calcium chloride dihydrate (0.02 w/v), yeast extract (0.2 w/v), lab lemco powder (0.1 w/v), proteose peptone (0.5 w/v) in ddH₂O (Sigma, Poole, UK). Urea was then added to independently to a final concentration of 0.05% (v/v). To initiate multispecies biofilm development the pioneer species S. mitis biofilm were first formed for 24 h in 5% CO₂ on 13 mm diameter Thermanox™ coverslips within 24 well plates (Corning, NY, USA). The supernatant was then removed and F. nucleatum added, which was incubated anaerobically at 37°C for a further 24 h. The supernatant was removed and P. gingivalis and A. actinomycetemcomitans added to the dual species biofilm, which was incubated anaerobically at 37°C for a further 4 days, replacing the AS daily to produce a mixed four species biofilm (Figure 
1A).

A research team of four individuals visited a poultry processing plant to investigate the sites and diversity of Campylobacter contamination. Three days after this visit, one member of the research team became ill with watery diarrhoea, fever and severe abdominal cramps. The diarrhoea terminated after about six days. Six days after the onset of symptoms a faecal sample was cultured as described below and C. jejuni was isolated. Four separate colonies from the faecal sample were recovered and stored.
The team sampled two flocks (flocks 1 and 2) at the abattoir. The flocks were reared on different farms, and sequentially processed through the processing plant. Caeca were removed from the poultry carcasses during evisceration, and the contents sampled aseptically. Swab samples were also collected from various sites in the abattoir and from up to five poultry carcasses [50] as they progressed through the processing plant. The methods of chicken sampling and culture have been previously described [51] (link). Briefly all samples were directly plated onto blood agar containing selective antibiotics [52] (link) with actidione (100 ug/ml) and cefoperazone (30 mg/ml), incubated microaerobically at 37°C for 2 days, with or without pre-enrichment in Exeter medium [53] (link). Identification was based on microaerobic growth at 42°C, hippurate and indoxylacetate hydrolysis and catalase and oxidase activities. A single colony from each sample was stored in glycerol broth (10% v/v glycerol in 1% w/v proteose peptone) at −80°C for subsequent typing.
All isolates were fla-typed according to the technique of Ayling et al. [23] (link) using the restriction enzymes DdeI and HinfI. Representatives of the human and chicken isolates were also serotyped [54] (link) and molecular typed by pulsed-field gel electrophoresis (PFGE) using the enzymes SmaI and KpnI [55] (link), amplified fragment length polymorphism (AFLP) [56] (link) and multilocus sequence typing (MLST) [57] (link).
Phenotypic characterization of strain M1 (Laboratory designation 99/308) was performed to determine in vitro invasiveness of INT407 cells and CaCO2 cells and to detect the expression of cytolethal distending toxin (CDT) as previously described [58] (link), [59] (link).

The clinical A. castellanii strains 1BU (ATCC PRA-105) and SIN20, both genotype T4, were isolated from two different patients diagnosed with infectious keratitis in 1998 and 2020, respectively. Both strains were maintained and grown axenically in proteose peptone–yeast extract–glucose (PYG) medium, containing 10 g proteose peptone, 10 g yeast extract, 5 g NaCl, 5 g glucose, 0.7 g Na₂HPO₄ and 0.7 g KH₂PO₄ per litre, and maintained at 34 °C.

T. thermophila B2086 (II), CU428 (VII), and CU427 (VI) were obtained from the National Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/, Cornell University, Ithaca, NY). Cells were cultured at 30 °C in a super proteose peptone medium (1% proteose peptone, 0.1% yeast extract, 0.2% glucose, and 0.003% EDTA-Fe). For starvation, log-phase cells were washed with 10 mmol/L Tris–HCl (pH 7.4) and resuspended in 10 mmol/L Tris–HCl (pH 7.4) at 30 °C for 16–24 h. The distinct mating type cells were mixed and initiated sexual development.