Aug 28, 2020
Fermentor Growth of Streptococcus sanguinis
- 1Philips Institute for Oral Health Research, Virginia Commonwealth University
Protocol Citation: Tanya Puccio, Todd Kitten 2020. Fermentor Growth of Streptococcus sanguinis. protocols.io https://dx.doi.org/10.17504/protocols.io.bkayksfw
Manuscript citation:
Puccio, T., Kunka, K.S., Zhu, B., Xu, P., and Kitten, T. (2020). Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogen Streptococcus sanguinis. bioRxiv. doi: 10.1101/2020.08.06.240218
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 25, 2020
Last Modified: August 28, 2020
Protocol Integer ID: 41016
Keywords: Fermentor, biostat, Streptococcus, sanguinis, manganese depletion,
Abstract
Streptococcus sanguinis 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 S. sanguinis, 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.
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Guidelines
These intructions are specific to a Sartorius Stedim Biostat® B with a 1.5 L capacity UniVessel® Glass + BioPAT® Fundalux, DO probe, and pH probe. Adjustments may be required if using different equipment.
Materials
MATERIALS
RNeasy Mini KitQiagenCatalog #74104
RNase-Free DNase SetQiagenCatalog #79254
Bacto™ Brain Heart InfusionThermo FisherCatalog #237300
Antifoam 204SigmaCatalog #A8311
Potassium HydroxideFisher ScientificCatalog #1310-58-3
Sodium HydroxideFisher ScientificCatalog #1310-73-2
Hydrochloric AcidFisher ScientificCatalog #7647-01-0
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
RNAprotect Bacteria ReagentQiagenCatalog #76506
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
Needleless Injection Site Swabbable Female Luer Lock to Barb ConnectorQosinaCatalog #80210
STEP MATERIALS
RNAprotect Bacteria ReagentQiagenCatalog #76506
RNeasy® Mini KitQiagenCatalog #74104
RNase-Free DNase SetQiagenCatalog #79254
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
Protocol materials
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
RNeasy Mini KitQiagenCatalog #74104
Needleless Injection Site Swabbable Female Luer Lock to Barb ConnectorQosinaCatalog #80210
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
Antifoam 204Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8311
Sodium HydroxideFisher ScientificCatalog #1310-73-2
RNAprotect Bacteria ReagentQiagenCatalog #76506
RNase-Free DNase SetQiagenCatalog #79254
Hydrochloric AcidFisher ScientificCatalog #7647-01-0
RNAprotect Bacteria ReagentQiagenCatalog #76506
RNeasy® Mini KitQiagenCatalog #74104
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
Potassium HydroxideFisher ScientificCatalog #1310-58-3
RNase-Free DNase SetQiagenCatalog #79254
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
Bacto™ Brain Heart InfusionThermo FisherCatalog #237300
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
RNAprotect Bacteria ReagentQiagenCatalog #76506
RNase-Free DNase SetQiagenCatalog #79254
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
RNeasy® Mini KitQiagenCatalog #74104
Before start
- The afternoon before the run, start a 40 mL pre-culture at 37 °C and appropriate oxygen concentration with antibiotics (if appropriate).
- Make5 L of BHI with 25 Parts per Million (PPM) antifoam. Autoclave 02:00:00 .
- Set up fermentor according to manufacturer's specfications/user's needs.
When running an experiment for the first time, test to see how long it takes for the media to flow from the carboy to the vessel at the chosen flow rate (will vary depending on the length of the tubing used).
Inoculation
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 7.4 800 mL
Equipment
Biostat® B
NAME
Benchtop Bioreactor
TYPE
Sartorius
BRAND
N/A
SKU
Equipment
UniVessel® Glass 1.5 L capacity
NAME
Vessel
TYPE
Sartorius Stedim
BRAND
N/A
SKU
LINK
Equipment
BioPAT® Fundalux
NAME
Optical density probe
TYPE
BioPAT
BRAND
N/A
SKU
Equipment
EasyFerm Plus PHI VP 120 Pt100
NAME
pH probe
TYPE
Hamilton
BRAND
238633-1111
SKU
Equipment
InPro6800/12/160/4112111
NAME
Dissolved oxygen probe
TYPE
Mettler Toledo
BRAND
5230580
SKU
Remove 15 mL media from vessel to incubate 37 °C as a check for contamination.
Remove 500 µL media and store at -80 °C for metabolomics analysis.
Take OD600 reading using 1 mL of 40-mL overnight pre-culture.
Equipment
Genesys 150
NAME
UV-VIS Spectrophotometer
TYPE
Thomas Scientific
BRAND
1160V96
SKU
Centrifuge remaining volume 39 mL of overnight culture.
4303 x g, 4°C, 00:10:00
Decant supernatant and resuspend in 15 mL BHI.
Inoculate into vessel using 20-mL syringe.
Ramping up air flow
Ramping up air flow
As the absorbance units (AU) increase, gradually ramp up the air flow.
When AU reaches 0.10, turn off the DO control and set air flow to 0.03 lpm.
When AU reached 0.30, set air flow to 0.20 lpm.
Note
If the experiment is meant to be low oxygen, skip this step.
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%.
Note
For our fermentor, 17% ~ 700 mL h-1
Note
If the experiment is meant to be low oxygen, do not increase the air flow.
Expected result
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
Experimental Conditions
Before proceeding with sample collection, allow culture to adjust to media flow for 01:00:00 .
At 1 h post-media flow (T-20), remove sample 40 mL from vessel using a syringe (usually 60 mL capacity) using the sampling port.
At the predetermined time, add EDTA to 100 micromolar (µM) to carboy in 5 mL BHI using a syringe into the inoculation port.
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
Note
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.
At T0 (20 min after first sample), add EDTA to 100 micromolar (µM) to vessel in 5 mL BHI using a syringe into the inoculation port.
EDTA 0.5 M pH 8.0InvitrogenCatalog #AM9261
Collect the remaining samples at T10, T25, and T50.
Sample Collection Protocols
Sample Collection Protocols
For metabolomics samples, aliquot 30 mL of cell culture into conical tube.
Swirl immediately in a dry ice/ethanol bath for 00:01:00 .
Centrifuge sample immediately.
4303 x g, 4°C, 00:05:00
Aliquot 500 µL of supernatant media and store at -80 °C .
Remaining volume can be decanted or stored at -20 °C for hydrogen peroxide quantification or other analysis.
Store cell pellet at -80 °C until ready for analysis.
For RNA samples, set up tubes for each sample containing 4 mL RNAprotect.
RNAprotect Bacteria ReagentQiagenCatalog #76506
Add 2 mL of cell culture to RNAprotect tubes. Vortex for 00:00:05 .
Incubate at Room temperature for at least 00:05:00 but less than 03:00:00 .
Centrifuge cell culture in RNAprotect 4303 x g, 4°C, 00:10:00 .
Store at -80 °C until ready to isolate RNA.
Isolate RNA and remove DNA.
RNeasy® Mini KitQiagenCatalog #74104
RNase-Free DNase SetQiagenCatalog #79254
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
Store RNA at -80 °C until ready for analysis.