Development and optimisation of a bacteriological liquid growth medium

The development of a single liquid growth medium that could be used to culture any viable bacteria present in CAP and LITA tissue was investigated. While the ultimate aim was to isolate and identify any viable bacteria species present in atherosclerotic plaque tissue, particular emphasis was placed on developing a liquid medium with the ability to support the growth of RCB. Thus, P. gingivalis 11834/W50, T. forsythia & T. denticola were employed as test species to assess media development at every stage in the process. Firstly, media known to support the individual growth of each RCB test species (BHI, TSB and TYGVS) were assessed to establish whether each medium could support the growth of all or more than one RCB species. Testing how readily each RCB species was supported in each alternative medium provided a foundation “base medium” from which the most efficacious medium could be further developed to include the growth of all RCB test species/strains. Therefore, media development was performed in two distinct stages.

Stage 1. Test the efficacy of BHI, TSB and TYGVS media (appendices A, B & C) to support the growth of all or more than one RCB species and establish a “base medium” that may be further developed.

Stage 2. Further development of the “base medium” through a stepwise supplementation of specific growth reagents known to support the growth of any RCB species not satisfied in stage 1 assessment.

2.3.2.1 Stage 1 - Establishment of a base medium that supports the growth of RCB

P. gingivalis is typically grown in BHI broth (appendix A), while T. forsythia and T. denticola are commonly grown in TS broth (appendix B) and TYGVS (appendix C),

respectively. To determine which media was most supportive, each medium was inoculated individually with a single RCB species per medium replicate for the growth of pure cultures only (table 2.01). Prior to inoculation, bacteria numbers present in a starter culture (OD600 0.4 – 0.6) were counted as described in section 2.3.1.1. Twenty milliliters

of pre-reduced, pre-warmed media were each inoculated with ~1 x 105 bacteria. Cultures were incubated anaerobically for 8 days within a previously described anaerobic system (section 2.3.1). Cultures were repeated by performing three technical replicates for each

69

RCB species and alternative medium combination (table 2.01). Liquid culture turbidity was assessed by daily measurement of OD600 values, as previously detailed (section

2.3.1.1). Finally, the recorded OD600 values were used to plot line graphs to show standard

growth curves of each test species in each of the liquid media.

The most supportive “base medium” was selected based on three general criteria:-

1. The number of RCB species/strain supported by the medium.

2. The consistency of growth in the medium based on three technical replicates. 3. Final yield of each bacterium in each alternative medium, taken as a mean of three

technical replicate OD600 values.

P. gingivalis

Media W50 11834 T. forsythia T. denticola

Alternative

TSB TSB BHI BHI

TYGVS TYGVS TYGVS TSB

Typical BHI BHI TSB TYGVS

Table 2.01: Alternative growth media used in stage 1 to culture test bacteria species/strains. Each

“alternative medium” was used to culture each bacteria species in technical replicates of three. Bottom row shows the “typical media” that have previously been shown support the growth of each test species/strain (Abaibou et al. 2001; Roy et al. 2010).

2.3.2.2 Stage 2 - Supplementation of a base medium with specific growth reagents

The second stage of medium development involved building upon the “base medium” established in stage 1, (TSB; appendix A). Since T. denticola failed to grow within the stage 1-established medium, particular emphasis was placed on incorporating growth reagent supplements known to support the growth of T. denticola (Fenno 2005). Such reagents were selected by considering several complex broth media formulations in common use for growth of T. denticola (Wyss 1992; J. Fenno 2005). These media share common features including sources of trace elements, peptides, amino acids, and trace nutrients, as well as reducing agent(s), volatile fatty acids (VFA), and heat-inactivated animal sera, typically rabbit.

70

All selected growth reagents except volatile fatty acid (VFA) solution were tested as single compounds. VFA solution comprised seven VFA’s and was tested in media as an overall percentage of the final medium volume (table 2.02). Briefly, a stock solution of VFA was prepared comprising, 1.42 % glacial acetic acid (v/v), 0.5% propionic acid (v/v), 0.34% butyric acid (v/v), 0.084% valeric acid (v/v), 0.084% isobutyric acid (v/v), 0.084% isovaleric acid (v/v) and 0.084% methylbutyric acid (v/v). Table 2.02 details the final volume (µl/ml, v/v) of each acid when used at 4, 6, 8 and 10% of the final medium volume.

For the purpose of testing the impact on bacterial growth in the presence of different growth reagents, base media were prepared, each containing an increasing concentration of a single growth reagent supplement, which were compared to bacterial growth in a negative control media (without tested reagent) as detailed in table 2.03. Each varying test medium was inoculated with only pure cultures for separate growth of one RCB species or strain per medium at a density of ~1 x 105 bacteria cells and incubated for 8 days in a previously described anaerobic environment (section 2.3.1).

The impact of each reagent concentration on bacterial growth was assessed daily by measuring OD600 values, as detailed earlier in section 2.3.1.1. When maximum OD600

values were observed, approximately 108 – 120 h post inoculation, bacterial numbers were considered to be at their greatest density i.e. during late-log/early-plateau growth phase. These maximum OD600 values were compared between test reagent media and negative

control media. A reagent concentration was considered to be optimum when the lowest possible reagent concentration provided the greatest increase in bacterial growth compared to control medium. When an optimum concentration was established for one reagent, that reagent was incorporated as part of the complete growth medium prior to the next round of reagent testing. Table 2.04 lists the reagents in order of assessment and shows the progressive development of each supplemented medium as the optimum concentration of each growth reagent was established.

71

Final volume of VFA in 1 L medium (v/v) based on percentage used Volatile fatty acids 4% mM 6% mM 8% mM 10% mM Glacial acetic acid (µl/ml) 0.57 9.92 0.85 14.9 1.14 19.8 1.42 24.8 Propionic acid (nl/ml) 200 2.67 300 4.00 400 5.33 500 6.67 Butyric acid (nl/ml) 104 1.47 156 2.21 208 2.94 340 3.68 Valeric acid (nl/ml) 33.6 0.31 50.4 0.46 67.2 0.61 84 0.76 Isobutyric acid (nl/ml) 33.6 0.37 50.4 0.55 67.2 0.74 84 0.92 Iisovaleric acid (nl/ml) 33.6 0.30 50.4 0.46 67.2 0.61 84 0.76 Methylbutyric acid (nl/ml) 33.6 0.31 50.4 0.46 67.2 0.62 84 0.77

Table 2.02: Individual VFA concentrations used as part of a solution to test bacteria growth in culture

medium. Stock solutions used for media containing 7 VFAs. Table shows the volume and molarity of each VFA in 100 ml medium (v/v) based on the percentage of the VFA stock solution in the final media volume.

Concentration gradient Media supplements #1 #2 #3 #4 #5 (NH4)2SO4 (mg/ml) NC 0.25 0.5 0.75 1 K2HPO4 (mg/ml) NC 1 1.25 1.5 1.75 NaCl2 (mg/ml) NC 0.5 0.75 1 1.25 VFA solution (%) NC 4 6 8 10 Thiamine pyrophosphate (µg/ml) NC 12.5 13 13.5 14 Sodium pyruvate (µg/ml) NC 275 300 325 350 Rabbit serum (%) NC 5 10 15 -

Table 2.03: Media supplements selected for stage 2 of bacterial growth medium development. Media

supplements are listed in the left-hand column in order of assessment. A concentration gradient of each reagent was tested to establish the optimum concentration of each reagent required for bacterial growth (columns #2 – #5). Bacterial growth in media containing each reagent concentration was compared to bacterial growth in a negative control medium (NC; colum #1). Negative control medium comprised optimum concentrations of all previously tested reagents, minus the reagent being tested or subsequently untested reagents.

Finally, data were presented by plotting bar graphs of OD600 values for each test species

cultured in media containing concentration gradients of growth reagents. Statistical testing was conducted by performing ANOVA, Levene’s test and Student’s t-test and the significance of each test result was indicated on bar graphs as one, two or three asterisks dependent on test result probabilities of ≤0.05, ≤0.01 or ≤0.001, respectively (section 2.7.1). Moreover, to present data for the growth of RCB species in media containing only established optimum reagent concentrations, OD600 values were plotted as line graphs to

72

Stage 2 medium reagents

A B C D E F G M edi um c om pos it ion

(NH4)2SO4 (NH4)2SO4 (NH4)2SO4 (NH4)2SO4 (NH4)2SO4 (NH4)2SO4 (NH4)2SO4

- K2HPO4 K2HPO4 K2HPO4 K2HPO4 K2HPO4 K2HPO4

- - NaCl2 NaCl2 NaCl2 NaCl2 NaCl2

- - - VFA VFA VFA VFA

- - - - TPP TPP TPP

- - - SP SP

- - - RS

Table 2.04: Progressive development of bacterial growth medium. Lists of each individual growth reagent supplement in order of assessment. Each column shows the progressive development of the medium through the incorporation individual reagents (A – G). Reagents were tested individually as a concentration gradient by measuring their impact on bacterial growth to identify the optimum medium concentration required to support the growth of each red complex bacteria species. As an optimum concentration was established for one reagent, that reagent was then incorporated as part of the complete growth medium prior to the next round of reagent testing. (NH4)2SO4 (Ammonium Sulphate), K2HPO4 (Potassium Phosphate), NaCl

(Sodium Chloride), VFA (volatile fatty acid), TPP (thiamine pyrophosphate), SP (sodium pyruvate) and RS (rabbit serum).