Apr 13, 2026

Halobacteriovorax sp. GFR8 growth and aquarium inoculation protocol

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjexk5gk5/v1
  • 1Georgia Institute of Technology;
  • 2NOAA;
  • 3University of Miami
Lauren Speare
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjexk5gk5/v1
Protocol Citation: Lauren Speare, Stephanie Rosales, Athena Peterson 2026. Halobacteriovorax sp. GFR8 growth and aquarium inoculation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjexk5gk5/v1
Manuscript citation:
Lumpkin et al., In Prep
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
Created: July 28, 2025
Last Modified: April 13, 2026
Protocol  Integer ID: 223463
Keywords: most coral microbiome, low abundance member of most coral microbiome, aquarium inoculation protocol coral, induced coral disease, vibrio fischeri es114 revive halobacteriovorax sp, coral disease, halobacteriovorax sp, microbiome shifts in montastrea cavernosa, obligate marine predator, bacterial strain, new probiotic treatment, microbiome shift, held coral, stony coral, additional optimization for other strain, use in aquaria, other strain, gfr8 prepare 2nd vibrio fischeri es114 culture, tissue loss severity in acropora cervicorni, induced bleaching, strain, aquaria
Funders Acknowledgements:
NOAA Ruth D. Gates Coral Restoration and Innovation Grants
Grant ID: NA25NMFX482C0004-T1-01
Abstract
Corals are facing a myriad of stressors including bacterially-induced disease and few solutions have been developed that can prevent or treat such disease once it establishes in aquaria-held corals. Below we describe a new probiotic treatment for bacterially-induced coral disease that employs a natural, low abundance member of most coral microbiomes. Halobacteriovorax sp. GFR8 can prevent disease progression when applied after the first signs of bacterially-induced bleaching (Speare et al., 2025; Lumpkin et al., In Prep), act prophylactically to reduce tissue loss severity in Acropora cervicornis (Lumpkin et al., In Prep), and prevent microbiome shifts in Montastrea cavernosa (Welsh et al., 2017). The following protocol describes how to grow and prepare this obligate marine predator, Halobacteriovorax sp. GFR8, for use in aquaria held stony corals.
There are nine steps to this protocol that are completed over five days:
  1. Clean and prepare the work area
  2. Quantify culturing needs
  3. Prepare first prey culture of Vibrio fischeri ES114
  4. Revive Halobacteriovorax sp. GFR8
  5. Prepare 2nd Vibrio fischeri ES114 culture
  6. Amplify Halobacteriovorax sp. GFR8
  7. Inspect for clearing and quantify Halobacteriovorax sp. GFR8 abundance
  8. Isolate Halobacteriovorax sp. GFR8
  9. Inoculate aquaria with filtered Halobacteriovorax sp. GFR8 culture
This protocol was optimized for the two bacterial strains employed (Vibrio fischeri ES114 and Halobacteriovorax sp. GFR8) and may require additional optimization for other strains. This protocol was written by Dr. Lauren Speare and Dr. Stephanie Rosales, and validated by Athena Peterson.
Guidelines
  1. This procedure uses bacterial cultures (Vibrio fischeri ES114 & Halobacteriovorax sp. GFR8) and should only be performed in a BSL1 or BSL2 laboratory.
  • Complete your institution’s basic biosafety level 1 or 2 training.
  • Wear a lab coat and safety glasses throughout the procedure
  • Kill all cultures with bleach prior to disposal in appropriate waste containers.
Materials
Materials designated with '*' should be autoclaved prior to use.
Media:
PP25: Instant Ocean salts will precipitate out of solution if autoclaved. Sterilize media by passing through a 0.22um filter.
  • 1 L DI Water
  • 1 g Polypeptone
  • 25 g Instant Ocean Salts
Reusable Materials:
125 mL Erlenmeyer Flasks*
500 mL Erlenmeyer Flasks*
500mL beakers (for autoclave waste / used wooden sticks)
Serological pipette
200 μL pipette
Wooden sticks (sterilized in a beaker)*
Consumable Materials:
10 mL serological pipette tips
200 μL pipette tips (autoclave if tips do not come pre-sterilized)*
0.22 μm filter column
0.45 μm filter column
Foil
Autoclave tape
Labeling tape
10% Bleach
Equipment:
Shaking incubator capable of oscillation at 85 rpm and temperature control (28°C)
-80 °C Freezer
Vacuum pump or line
Autoclave
Strains:
Vibrio fischeri ES114 (prey)
Halobacteriovorax sp. GFR8 (predator)
Experimental Preparation
Clean and prepare the work area
While wearing new gloves, thoroughly spray the work area with 75% Ethanol (or higher %). Wipe with a kim wipe and discard. Repeat this step each time cultures are handled/inspected.
If contamination becomes a problem, perform the following culturing procedure in a hood.
All bacterial cultures must be bleached prior to disposal. Reusable equipment should be bleached, washed with soap and water, and autoclaved after each use.

Quantify culturing needs
Calculate the volume of Halobacteriovorax sp. GFR8 (predator) needed. Final filtered cultures of Halobacteriovorax sp. GFR8 should be added in a culture : tank volume ratio of 1:75. For example, to inoculate a 2.5-gallon tank, 125 mL Halobacteriovorax sp. GFR8 culture should be prepared.
The following procedure assumes inoculation of a single 2.5-gallon tank. To increase the yield of Halobacteriovorax sp. GFR8 (either to account for more tanks or tanks with larger volumes), only adjust the number of flasks used, not the volumes of media within each flask.
Revive Halobacteriovorax sp. GFR8
Day 0: Prepare first prey culture of Vibrio fischeri ES114.
Using a serological pipette, fill two 125 mL flasks with 50 mL PP25 each. One flask will be used to grow Halobacteriovorax sp. GFR8 + prey and the other will be a “prey-only” control (see step 4).
Label each flask with the prey ID (V. fischeri ES114), procedural step, and the date. See example below.

V. fischeri ES114
Step 3
August 1 2025
Inoculate each flask with V. fischeri ES114 from a -80 °C stock. Using a sterile toothpick, collect a glob (the size of ~½ a grain of rice) of frozen V. fischeri ES114 culture and swirl in a flask. Place the used stick into a "dirties" beaker for later autoclaving. Repeat for each flask.
Incubate V. fischeri ES114 cultures shaking at 85 rpm, 28 °C overnight.
Day 1: Revive Halobacteriovorax sp. GFR8.
Add a label to one of the two V. fischeri ES114 125 mL flasks from step 3 with Halobacteriovorax sp. GFR8 and the date. Both flasks should be visibly turbid (see Figure 1). If cultures are not turbid, repeat step 3 or check the viability of the V. fischeri ES114 -80 °C stock.

V. fischeri ES114
+ Halobacteriovorax sp. GFR8
Step 4
August 2 2025

Inoculate the Halobacteriovorax sp. GFR8 flask from a -80 °C stock. Using a sterile toothpick, collect a glob of Halobacteriovorax sp. GFR8 culture and swirl it in the flask. Place the used stick into a "dirties" beaker. DO NOT inoculate the prey, only control.
Incubate both flasks shaking at 85rpm, 28 °C for 48-72 hours.
Day 1: Prepare 2nd V. fischeri ES114 culture.

Using a serological pipette fill two 500 mL flasks with 250 mL PP25 each. Label each flask with the prey ID (V. fischeri ES114), procedural step, and the date.

V. fischeri ES114
Step 5
August 2 2025
Inoculate each flask with V. fischeri ES114 from a -80 °C stock. Using a sterile toothpick, collect a glob of culture and swirl it in the flask. Place the used stick into a "dirties" beaker. Repeat for each flask.
Incubate V. fischeri ES114 cultures shaking at 85 rpm, 28 °C overnight.
Day 2 (or once clearing is visible): Amplify Halobacteriovorax sp. GFR8

Inspect cultures from step 3 for clearing. The control flask (only containing V. fischeri ES114) should be visibly turbid (see Figure 1, left) while the Halobacteriovorax sp. GFR8 flask should show some clearing (see Figure 1, right). DO NOT PROCEED until clearing is visible in the Halobacteriovorax sp. GFR8 flask from step 3 (most likely by day 2 or 24 hours after Halobacteriovorax sp. GFR8 inoculation).




Add a label to one of the two V. fischeri ES114 500 mL flasks from step 5 with Halobacteriovorax sp. GFR8 and the date. Both flasks should be visibly turbid (see Figure 1). If cultures are not turbid, repeat step 5 or check the viability of the V. fischeri ES114 -80 °C stock.

V. fischeri ES114
+ Halobacteriovorax sp. GFR8
Step 6
August 3 2025

Using a serological pipette, transfer 10 mL of cleared Halobacteriovorax sp. GFR8 culture into the labeled Halobacteriovorax sp. GFR8 flask. Discard pipette. DO NOT inoculate V. fischeri ES114 only control.
Incubate Halobacteriovorax sp. GFR8 cultures and control cultures (V. fischeri ES114 alone) shaking at 85 rpm, 28 °C for 24 hours.
Discard cultures from Steps 3 & 4 by bleaching culture, washing with soap, and autoclaving.
Inoculate Halobacteriovorax sp. GFR8
Day 3: Isolate Halobacteriovorax sp. GFR8
Inspect Halobacteriovorax sp. GFR8 for clearing. DO NOT PROCEED until clearing is visible in the freshly passaged cultures (likely by day 3).
Separate V. fischeri ES114 from Halobacteriovorax sp. GFR8 cultures via filtration. Pass culture through a 0.45 μm filter. A vacuum pump with filter columns rather than syringe filters is highly recommended to speed up the process. Liquid that has passed through the filter should be clear and contain only Halobacteriovorax sp. GFR8 cells.
Cultures from steps 5 and 6 can be discarded (bleached, washed, and autoclaved) after filtrate has been collected.
Day 3: Inoculate aquaria with filtered Halobacteriovorax sp. GFR8 culture
Pause water flow in aquaria and pause new water input. Note the time
Using sterile beakers (or another sterilized vessel), remove 10% of tank volume (i.e., remove 1 L from a 2.5-gallon tank) and discard.
Pour filtered Halobacteriovorax sp. GFR8 culture into the aquarium at a final ratio of 1:75. (i.e., add 125 mL Halobacteriovorax sp. GFR8 culture for a 2.5-gallon tank).
Keep inflow water paused for ~3 hours.
Acknowledgements
We would like to thank the Lirman Lab (University of Miami) for providing corals with which to test and develop this protocol and a NOAA Ruth D. Gates Coral Restoration Innovation Grant for funding this work.