TITLE: Fermentor Growth of Streptococcus sanguinis
AUTHORS: Tanya Puccio, Todd Kitten

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Streptococcus sanguinis</span><span> is a lactic acid-forming bacterium that can be cultured in both aerobic and anaerobic conditions. It is a primary colonizer of the oral cavity but can also cause a heart disease called infective endocarditis. Our main objective for this protocol was to grow large, controlled cultures before and after manganese depletion for various analyses. In order to obtain large scale, reproducible growth of </span><span style = "font-style:italic;">S. sanguinis, </span><span>a biostat was used to maintain controlled conditions. After optimization, it was determined that traditional chemostat conditions (Burne and Chen, 1998) were not appropriate for these experiments. Thus, we decided on this modified protocol. Using this method, we were able to identify a concentration of the non-specific metal chelator, EDTA, that would lead to a decreased growth rate in a manganese transporter mutant but not in the wild-type strain.</span></div></div>

[STEPS]
?. [Inoculation]
Set up fermentor to experiment specifications: pO2 set to 5% with 0.03 lpm max air flow; nitrogen set at 0.08 lpm; stirrer set at 250 rpm;
37 °C
800 mL
?. [Inoculation]
Remove   media from vessel to incubate  as a check for contamination.
15 mL
37 °C
?. [Inoculation]
Centrifuge remaining volume   of overnight culture.
39 mL
Centrifuge: 4303 34, 10 min, 4 10
?. [Inoculation]
Resuspend in 15 mL BHI.
?. [Inoculation]
Take OD600 reading using  of 40-mL overnight pre-culture.
1 mL
?. [Inoculation]
Decant supernatant and resuspend in  BHI.
15 mL
?. [Inoculation]
Inoculate into vessel using 20-mL syringe.
?. [Ramping up air flow]
As the absorbance units (AU) increase, gradually ramp up the air flow.
?. [Ramping up air flow]
When AU reaches 0.10, turn off the DO control and set air flow to 0.03 lpm.
?. [Ramping up air flow]
When AU reached 0.30, set air flow to 0.20 lpm.
If the experiment is meant to be low oxygen, skip this step.
?. [Ramping up air flow]
When AU peaks and begins to drop, set air flow to 0.50 lpm. Turn on media pumps: input at 17% and output at 34%.
For our fermentor, 17% ~ 700 mL h-1
If the experiment is meant to be low oxygen, do not increase the air flow.
SK36 (wild type) growth should not be drastically affected by the addition of EDTA. For the primary Mn transporter mutant (ΔssaACB), the OD will increase initially and then should start to drop in OD (by 0.01 AU) ~38 min after EDTA addition.
?. [Experimental Conditions]
Before proceeding with sample collection, allow culture to adjust to media flow for .
?. [Experimental Conditions]
At 1 h post-media flow (T-20), remove sample  from vessel using a syringe (usually 60 mL capacity) using the sampling port.
40 mL
?. [Experimental Conditions]
At the predetermined time, add EDTA to   to carboy in  BHI using a syringe into the inoculation port.
5 mL
Using the flow time calculated previously (see Before Starting), determine how long it will take for media to flow from carboy to vessel and subtract from 20 min. For example, if it takes 4 min to flow from carboy to vessel, add EDTA 16 min after the first sample was removed.
?. [Experimental Conditions]
At T0 (20 min after first sample), add EDTA to  to vessel in  BHI using a syringe into the inoculation port.
5 mL
?. [Experimental Conditions]
Collect the remaining samples at T10, T25, and T50.
?. [Sample Collection Protocols]
For metabolomics samples, aliquot  of cell culture into conical tube.
30 mL
?. [Sample Collection Protocols]
Swirl immediately in a dry ice/ethanol bath for .
?. [Sample Collection Protocols]
Centrifuge sample immediately.
Centrifuge: 4303 34, 5 min, 4 10
?. [Inoculation]
Remove  media and store at  for metabolomics analysis.
500 µl
-80 °C
?. [Sample Collection Protocols]
Aliquot  of supernatant media and store at  .Remaining volume can be decanted or stored at  for hydrogen peroxide quantification or other analysis.
500 µl
-80 °C
-20 °C
?. [Sample Collection Protocols]
Store cell pellet at  until ready for analysis.
-80 °C
?. [Sample Collection Protocols]
For RNA samples, set up tubes for each sample containing   RNAprotect.
4 mL
?. [Sample Collection Protocols]
Add  of cell culture to RNAprotect tubes. Vortex for .
2 mL
?. [Sample Collection Protocols]
Incubate at  for at least  but less than .
0 Room temperature
?. [Sample Collection Protocols]
Centrifuge cell culture in RNAprotect  .
Centrifuge: 4303 34, 10 min, 4 10
?. [Sample Collection Protocols]
Isolate RNA and remove DNA.
?. [Sample Collection Protocols]
Store at  until ready to isolate RNA.
-80 °C
?. [Sample Collection Protocols]
Store RNA at  until ready for analysis.
-80 °C