TITLE: Dilution-to-Extinction Experiment Protocol
AUTHORS: Shelby J Barnes, Michael Henson, Celeste Lanclos, Jordan Coelho, Cameron Thrash

[DESCRIPTION]
Dilution-to-Extinction Experiment Protocol

[STEPS]
SECTION: Collection and Filtration
2. Culturing Hardware Preparation 
SECTION: Inoculum Sample Enumeration
5. Sample Preparation 


SECTION: Medium Inoculation
8. 
All steps except incubation are performed inside a biosafety cabinet or laminar flow hood. 
SECTION: Collection and Filtration
3. Collect samples at any source of interest with sterile, acid-washed carboys appropriate to the required volumes, taking as many precautions as practical to avoid contamination. 

SECTION: Collection and Filtration
4. Filter sample(s) prior to enumeration using a (2.7um) Whatman GF/D glass fiber filter to exclude large aggregates/particles and increase the proportion of planktonic cells in the seawater. 



SECTION: Inoculum Sample Enumeration
6. Run Settings and Worklist Setup
SECTION: Inoculum Sample Enumeration
5.1. Enumerate DTE inoculum sample prior to dilution with artificial medium and PTFE plate inoculation. 

Total cells μL-1 needs to be between 1,000 and 5*10^6 for a given sample to stay within the accurate counting range of the BDAccuri. If samples are higher, make appropriate dilutions in sterile medium. 
SECTION: Inoculum Sample Enumeration
5.4. Transfer 400 μL of each of the desired controls into a 0.5 mL microcentrifuge tube and add 198uL of each to 96-well plate. Typically, controls consist of stained and unstained medium and stained and unstained E. coli. 
SECTION: Inoculum Sample Enumeration
5.5. Stain samples and one control with 1x  final concentration SYBR Green (2 μL 100x SYBR Green added to 198 μL sample). 

SECTION: Inoculum Sample Enumeration
6.1. The following is used with BD Accuri C6 Plus Software  for the BD Accuri C6 Plus Flow Cytometer systems from BD Biosciences (San Jose, California, United States). 
SECTION: Inoculum Sample Enumeration
6.4. Plots. Forward scatter (log scale) vs. green fluorescence (log scale), side scatter (log scale) vs. green fluorescence (log scale), and green fluorescence (log scale) vs. yellow fluorescence (log scale)  are used to evaluate the counts. 



SECTION: Inoculum Sample Enumeration
7. Analysis and Gating
SECTION: Inoculum Sample Enumeration
7.1. Cell concentration is calculated using a gated region, which is selected based on the side scatter vs. green fluorescence plot. Forward scatter vs. green fluorescence can also be used but may reduce resolution depending on the sample.



SECTION: Inoculum Sample Enumeration
7.2. Initially, a global gate is applied to plots using the stained artificial medium such that the gate excludes the stained medium signal. 
SECTION: Inoculum Sample Enumeration
7.3. However, especially in the case of isolates, but sometimes with whole seawater, individual samples must have the gate customized to account for variations in signal. In practice, this will be empirically defined, as different media will give different amounts of background noise. 




SECTION: Medium Preparation
1. Defined Medium 


SECTION: Medium Preparation
1.1. Can be prepared in advance according to SJB Artificial Media Protocol dx.doi.org/10.17504/protocols.io.rm7vzy615lx1/v1 or using medium of choice.
SECTION: Inoculated Medium Distribution and Incubation
9. Distribute with a P1200 multichannel pipette or the Eppendorf epMotion 5075 automated liquid handling system.

SECTION: Inoculum Sample Enumeration
5.6. Incubate samples for 30 minutes in the dark. 
SECTION: Large Format Enumeration
12. Carried out in the same manner as enumeration for medium inoculation with the following adaptations for high-throughput counting with 96-well plates.
SECTION: Large Format Enumeration
12.2. In a biosafety cabinet or laminar flow hood, using a multichannel pipette, transfer 198 μL from the deep 96-well incubation plates to 96-well round-bottom counting plates. 



SECTION: Large Format Enumeration
12.1. Aliquoting samples for enumeration can be done with the Eppendorf epMotion 5075 automated liquid handling system or P200 multichannel pipette.
SECTION: Collection and Filtration
2.1. All culturing hardware (carboys, flasks, glassware, PTFE plates, sealing mats, stir bars) are washed first with dishwasher or lab-grade detergent; rinsed with hot tap water, cold tap water, and then DI water; and let dry.
SECTION: Collection and Filtration
2.2. They are then soaked in a 10% HCl acid bath overnight, rinsed six times with DI water and MilliQ water, lids applied or wrapped in foil, and autoclaved prior to storage or use. 
SECTION: Collection and Filtration
3.1. Collectors should wear gloves.
SECTION: Collection and Filtration
3.2. Samples should be transported back to the laboratory with the shortest delay possible to reduce potential bottle effects that can alter the community prior to inoculation. 
SECTION: Inoculum Sample Enumeration
5.3. Transfer 198 μL of each sample, in triplicate, into a round- bottom 96-well plate. 
SECTION: Inoculum Sample Enumeration
5.2. Add two artificial medium controls to count (one stained and one unstained).

Controls. Unstained controls (artificial medium) are used to set a green fluorescence threshold for exclusion of autofluorescence and system noise, respectively. Stained controls ( one stained and one unstained artificial medium sample) are used for gating results around standard noise of the artificial media and identify the potential area for positive cell growth. Control samples are counted along with the experimental samples. 

SECTION: Inoculum Sample Enumeration
6.3. Worklist need to be tailored by application and stain used.  General run settings (w/SYBR Green): "Run with Limits"; volume: 10uL;  flow rate: medium; Thresholds - FSC or SSC: 10,  FL1 or FITC: 100 (thresholds may vary depending on media type and culture signature).


SECTION: Inoculum Sample Enumeration
6.2. Adjust Thresholds. Run control samples in manual collect tab using no thresholds and the general run settings: "Run with Limits" , volume: 10uL , flow rate: medium. The green fluorescence (FL1 or FITC) and forward scatter (FSC)/ side scatter (SSC) thresholds are set to exclude noise based on the unstained controls (Fig. 2). These settings are used for counting the samples in the auto collect tab. 
SECTION: Inoculum Sample Enumeration
6.5. SIP Settings

SIP Rinse: 1 cycle per sample
SIP Clean after completed run
SECTION: Medium Inoculation
8.1. Based on enumeration (Sect. above), cells are diluted in medium such that they can be inoculated into deep 96-well plates using minimal volume. The average number of estimated cells per well should be 1–3 after inoculation. 
SECTION: Medium Inoculation
8.2. Example. With a medium plus inoculum final volume of 900mL, an inoculum cell density of 5*103  cells/mL and final concentration of 1-3 cells/1.7 mL after distribution ; the inoculation volume should be approximately 0.1062 mL or 106 µL.

  
  
SECTION: Inoculated Medium Distribution and Incubation
10. Manual Distribution

Inoculated medium is distributed into sterile, acid-washed deep 96-well plates (2.1 mL PTFE) at 1.7 mL per well in a biosafety cabinet using a P100-1200 µL multichannel  pipette. 
SECTION: Inoculated Medium Distribution and Incubation
11. Automated Distribution

Eppendorf epMotion 5075 can be set up following the manufacturer’s instructions.
SECTION: Inoculated Medium Distribution and Incubation
11.1. Distribution and incubation protocol is consistent between distribution types.
SECTION: Inoculated Medium Distribution and Incubation
10.1. Swirl inoculated medium thoroughly and  aliquot into sterile liquid reservoir for ease of distribution. 
SECTION: Inoculated Medium Distribution and Incubation
10.2. Top full plates with sterile silicon lids. Incubate plates at room temperature (~25C°) for 2 - 4 weeks. 
SECTION: Transferring Plate Isolates and Culture Maintenance
13. Wells with cell densities of 10^4 and higher are considered positive and should be transferred.
SECTION: Transferring Plate Isolates and Culture Maintenance
13.1. The four corners of each plate containing a negative control should have very little signature apart from the media signal and a cell density of 10^3 or less. If a biotic signature is seen in a negative control, consider the plate contaminated and should not be used. 
SECTION: Transferring Plate Isolates and Culture Maintenance
14. Transfer 200uL of each positive well to a 250mL flask filled with 50mL of medium of choice. 
- transferring inoculum and culture volume can vary with preferred starting culture cell density.
SECTION: 16S rRNA PCR Preparation and Purification
19. Once DNA is extracted, prepare the PCR master mix for the number of samples

Volume of gDNA will vary depending on concentration.
Can use taq Polymerase kit (Invitrogen, OneTaq, etc) and dNTP's.
All PCR's require a positive and negative control to verify the run as successful. The positive control consists of gDNA of an already verified bacterial strain and the negative control is a sample with the gDNA replaced with molecular H2O. 

SECTION: 16S rRNA PCR Preparation and Purification
19.1. To each PCR tube add(50uL total volume):
Master Mix: 15uL 
gDNA: 3uL 
H20: 32uL
SECTION: 16S rRNA PCR Preparation and Purification
19.2. Run PCR's using the following Thermocycler conditions:

Lid Temp: 105°C
Sample Volume: 50uL
1. 94°C, 3:00 mins
2. 94°C, 0:30 mins
3. 50.8°C, 0:30 mins
4. 72°C, 2:00
5. GoTo step 2, 34x
6. 72°C, 10:00
7. 4°C, infinite hold
SECTION: 16S rRNA PCR Preparation and Purification
20. Store PCR products in 4°C or continue to gel and purification. 
SECTION: 16S rRNA PCR Preparation and Purification
22. Purify PCR product with either GeneJet PCR purification kit or Ampure XP beads.
SECTION: 16S rRNA PCR Preparation and Purification
22.1. Follow GeneJet PCR purification kit protocol.
OR
Follow Ampure XP bead protocol (lab protocol).

SECTION: Sanger Sequencing Preparation
23. Quantify purified PCR product using Qubit assay (following kit instructions) and set up sequencing samples as follows:
SECTION: Sanger Sequencing Preparation
23.1. For each PCR product there will be two sequencing samples made (one for 16S rRNA 27F Forward and one for 1492R Reverse sequences).
SECTION: Sanger Sequencing Preparation
23.2. Sequencing Sample Setup
(follow sequencing service guidelines)
SECTION: Sanger Sequencing Preparation
23.3. Once samples are set up, submit sequencing request through a sequencing facility (Genewiz, Laragen, etc). 
SECTION: 16S rRNA PCR Preparation and Purification
21. Run a 1.5% agarose gel to verify success of PCR amplification at the correct band length (usually 1500 bp).

Gel reagents in Materials section of protocol.
SECTION: 16S rRNA PCR Preparation and Purification
21.1. 
Set up gel casting tray in stabilizer and use leveler.
Add a 50mL solution of 1.5% agarose and 1x TAE Buffer to the designated gel pyrex bottle.
Microwave in 30 second intervals until all  agarose is dissolved (swirl intermittently).
Add 30ul of 100x SYBR Green to molten agarose solution, swirl and pore into gel mold. 
Remove any bubbles with a pipette tip by either sucking up bubble or moving it to the side. 
Place gel mold in a dark place(SYBR Green is light sensitive) for at least an hour for gel to harden. 
SECTION: Transferring Plate Isolates and Culture Maintenance
14.1. Prepare medium before day of transferring.
SECTION: Identification through 16S rRNA PCR Amplification
15. Either extracted genomic DNA or cells from maintained cultures can be used as the template for the PCR section below. 
SECTION: Identification through 16S rRNA PCR Amplification
16. Culture Filtering 
SECTION: Identification through 16S rRNA PCR Amplification
16.1. Transfer cultures prior to filtering. When cultures are at late log phase create a new transfer group using 200uL of culture into 50mL of fresh medium. (volumes can vary depending on preference)
SECTION: Identification through 16S rRNA PCR Amplification
17. Once transferred, filter 40-45mL of late log phase culture using a 0.1-0.2 Supor PES filter. Filter either by hand or with vacuum filtration. 
SECTION: Identification through 16S rRNA PCR Amplification
17.1. Store filters with cells in the -20°C until ready for DNA extraction.
SECTION: Identification through 16S rRNA PCR Amplification
18. DNA Extraction
SECTION: Identification through 16S rRNA PCR Amplification
18.1. Extract DNA from filtered cells using the GenElute™ Bacterial Genomic DNA Kit or Phenol Chloroform Extraction Protocol dx.doi.org/10.17504/protocols.io.b5iiq4ce
Quantify the gDNA concentration after extraction using the Qubit Fluorometer.
SECTION: 16S rRNA Direct PCR Preparation and Purification
16. Using GoTaq Master Mix G2 reagents set up PCR run as follows.

All PCR's require a positive and negative control to verify the run as successful. The positive control consists of gDNA of an already verified bacterial strain and the negative control is a sample with the gDNA replaced with molecular H2O. 

SECTION: 16S rRNA Direct PCR Preparation and Purification
16.1. To each PCR tube add (50uL total volume):
Master Mix: 25uL 
27F Forward Primer: 2uL
1492R Reverse Primer: 2uL
Culture cells: 1uL 
Molecular H20: 20uL
Minimum cells required in the 1uL of culture template is approximately 1*10^6 cells/mL.
SECTION: 16S rRNA Direct PCR Preparation and Purification
16.2. Run PCR's using the following thermal cycler conditions:

Lid Temperature:105°C
Sample Volume: 50uL
1. 98°C, 10:00 mins
2.94°C, 1:00 mins
3. 50°C, 1:00 mins
4. 72°C, 0:45 mins
5. GoTo step 2, 34x
6. 72°C, 5:00 mins
7. 4°C, infinite hold
SECTION: 16S rRNA Direct PCR Preparation and Purification
16.3. Store PCR products in 4°C or continue to gel and purification. 
SECTION: 16S rRNA Direct PCR Preparation and Purification
17. Run a 1.5% agarose gel to verify success of PCR amplification at the correct band length (usually 1500 bp).
SECTION: 16S rRNA Direct PCR Preparation and Purification
17.1. Gel reagents in Materials section of protocol.

Set up gel casting tray in stabilizer and use leveler.
Add a 50mL solution of 1.5% agarose and 1x TAE Buffer to the designated gel pyrex bottle.
Microwave in 30 second intervals until all  agarose is dissolved (swirl intermittently).
Add 30ul of 100x SYBR Green to molten agarose solution, swirl and pore into gel mold. 
Remove any bubbles with a pipette tip by either sucking up bubble or moving it to the side. 
Place gel mold in a dark place(SYBR Green is light sensitive) for at least an hour for gel to harden. 
SECTION: 16S rRNA Direct PCR Preparation and Purification
18. Purify PCR product with Ampure XP beads.
SECTION: 16S rRNA Direct PCR Preparation and Purification
18.1. Follow Ampure XP bead protocol (lab protocol).
SECTION: Identification through 16S rRNA PCR Amplification
18.2. Follow GenElute kit protocol.
SECTION: Sanger Sequencing Preparation
19. Quantify purified PCR product using Qubit assay (following kit instructions) and set up sequencing samples as follows:
SECTION: Sanger Sequencing Preparation
19.1. For each PCR product there will be two sequencing samples made (one for 16S rRNA 27F Forward and one for 1492R Reverse sequences).
SECTION: Sanger Sequencing Preparation
19.2. Sequencing Sample Setup
(follow sequencing service guidelines)
SECTION: Sanger Sequencing Preparation
19.3. Once samples are set up, submit sequencing request through a sequencing facility (Genewiz, Laragen, etc).