Lyophilization of Bdellovibrio bacteriovorus 109J for Long‐Term Storage

Mélanie J. Boileau1, Rinosh Mani2, Kenneth D. Clinkenbeard3

1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, 2 Diagnostic Center for Population and Animal Health, College of Veterinary Medicine, Michigan State University, Lansing, Michigan, 3 Veterinary Pathobiology and Graduate College, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 7B.3
DOI:  10.1002/cpmc.29
Online Posting Date:  May, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Bdellovibrio bacteriovorus 109J is a Gram‐negative predatory bacterium with obligate host dependency on other Gram‐negative bacteria. This bacteriolytic predator collides with, enters, and establishes growth within the prey (host) periplasm, eventually lysing the prey cell wall to release fresh, motile B. bacteriovorus progeny. Laboratory maintenance of B. bacteriovorus has been previously described by other investigators. The protocols included in this unit deal with the technique required to lyophilize or freeze dry host‐dependent B. bacteriovorus. This is an alternative means to frozen glycerol stocks for the long‐term storage of B. bacteriovorus. It includes the cultivation process and methods to lyophilize B. bacteriovorus as well as recommended storage conditions. In addition, this unit provides insight on the formulation's shelf‐life including the time to active culture after reviving lyophilized stocks of B. bacteriovorus following short‐, medium‐, and long‐term storage. © 2017 by John Wiley & Sons, Inc.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Culturing and Preparing Bdellovibrio bacteriovorus for Lyophilization
  • Alternate Protocol 1: Culturing Bdellovibrio bacteriovorus Using Moraxella Bovis as Prey
  • Basic Protocol 2: Lyophilizing Bdellovibrio bacteriovorus for Long‐Term Storage
  • Basic Protocol 3: Reviving Bdellovibrio bacteriovorus from Lyophylized Stocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Culturing and Preparing Bdellovibrio bacteriovorus for Lyophilization

  Materials
  • Prey cells (e.g., Escherichia coli strain 012207 or alternatively, E. coli K12, see Lambert & Sockett, ; unit 7.2)
  • ≤7 day old active B. bacteriovorus stock culture (from previous prey lysate)
  • 5% brain heart infusion agar with 5% sheep blood (BAP; Hardy Diagnostics, cat. no. A10; see also appendix 2C)
  • Dilute nutrient broth (DNB) medium (see recipe)
  • Peptone yeast extract (PYE) medium (see recipe)
  • Microbial freeze drying buffer, filter sterilized (OPS Diagnostics, cat. no. MFDB 500‐06)
  • 50‐ml conical centrifuge tubes, sterile
  • 75‐cm2 rectangular flask with vented caps or equivalent (i.e., 250‐ml conical flask), sterile
  • Serological pipets, individually wrapped (suggested sizes: 5, 10, and 25 ml), sterile
  • Cotton swab, individually wrapped, sterile
  • Pipet tips (1000 µl) with filter, sterile
  • 10‐µl inoculation loop, individually wrapped, sterile
  • Microscope glass slides and coverslips
  • 37°C static incubator
  • 30°C shaking incubator (target speed: 180 rpm)
  • Serological pipet dispenser
  • Serial pipet (1000 µl capacity)
  • Vortex mixer
  • Centrifuge and rotor (capable of handling 50‐ and 250‐ml centrifuge tubes and speeds up to 10,000 × g)
  • Spectrophotometer with cuvettes
  • Phase contrast microscope with a 100× oil immersion lens (Olympus BX41, Hitschfel Instruments)

Alternate Protocol 1: Culturing Bdellovibrio bacteriovorus Using Moraxella Bovis as Prey

  Additional Materials (also see protocol 1)
  • Prey cells (e.g., non‐hemolytic Moraxella bovis 12040577, or alternatively β‐hemolytic M. bovis Epp63‐300)
  • ≤7 day old active B. bacteriovorus stock culture (from previous E. coli prey lysate)
  • 3‐ml plastic syringes, luer lock, individually wrapped, sterile
  • Acrodisc syringe filters, 0.8‐ and 0.45‐µm pore size, individually wrapped, sterile (Pall Corporation)
  • Low form graduated glass beakers with spout, sterilized (suggested size: 10 and 25 ml)

Basic Protocol 2: Lyophilizing Bdellovibrio bacteriovorus for Long‐Term Storage

  Materials
  • B. bacteriovorus cell suspension (from step 6, protocol 1)
  • Microbial freeze drying buffer, sterile filtered (OPS Diagnostics, cat. no. MFDB 500‐06)
  • Liquid nitrogen
  • Freeze dry glass chamber (600 ml), clear, autoclavable with fitted lid and adaptor (Labconco, cat. no. 7540800)
  • Freeze dry glass serum vials (10 ml), clear, autoclavable (OPS Diagnostics, cat. no. LBVC 10‐400‐11)
  • Kimwipes KIMTECH Science delicate task wipers, sterilized (Kimberly‐Clark Professional, cat. no. 05511)
  • Trash bag twist ties
  • Crucible tongs (to hold freeze drying vials in liquid nitrogen)
  • Styrofoam container with lid (to hold liquid nitrogen)
  • FreeZone lyophilizer, 4.5 liters console freeze dry system with PTFE‐coated collector (Labconco, cat. no. 7751021)
  • 13‐mm split rubber stoppers, autoclavable (OPS Diagnostics, cat. no. RSSB 13‐500‐13)
  • Thumb forceps, sterile
  • 13‐mm aluminum tear‐off seal (OPS Diagnostics, cat. no. TNAS 13‐500‐15)
  • 13‐mm crimper (OPS Diagnostics, cat. no. KIHC 13‐17)
  • Refrigerator (4°C)

Basic Protocol 3: Reviving Bdellovibrio bacteriovorus from Lyophylized Stocks

  Materials
  • Prey cells, e.g., E. coli strain 012207 or alternatively, E. coli K12 (Lambert & Sockett, )
  • ≤1 year old lyophilized B. bacteriovorus (grown on E. coli; stored at 4°C in a vial, sealed and light protected)
  • 5% brain heart infusion agar with 5% sheep blood (BAP; Hardy Diagnostics, cat. no. A10; see also appendix 2C)
  • Dilute nutrient broth (DNB) medium (see recipe)
  • Peptone yeast extract (PYE) medium (see recipe)
  • Deionized distilled water, sterile
  • 50‐ml conical centrifuge tubes, sterile
  • 75‐cm2 rectangular flask with vented caps or equivalent (i.e., 250‐ml conical flask), sterile
  • Serological pipets, individually wrapped (suggested sizes: 5, 10, and 25 ml), sterile
  • Cotton swab, individually wrapped, sterile
  • Pipet tips (1000 µl) with filter, sterile
  • 10‐µl inoculation loop, individually wrapped, sterile
  • Microscope glass slides and coverslips
  • 37°C static incubator
  • 30°C shaking incubator (target speed: 180 rpm)
  • Serological pipet dispenser
  • Serial pipet (1000 µl capacity)
  • Vortex mixer
  • Centrifuge and rotor (capable of handling 50‐ and 250‐ml centrifuge tubes and speeds up to 10,000 × g)
  • Spectrophotometer with cuvettes
  • Phase contrast microscope with a 100× oil immersion lens (Olympus BX41, Hitschfel Instruments)
  • Refrigerator (4°C)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

  Boileau, M. J., Clinkenbeard, K. D., & Iandolo, J. J. (2011). Assessment of Bdellovibrio bacteriovorus 109J killing of Moraxella bovis in an in vitro model of infectious bovine keratoconjunctivitis. Canadian Journal of Veterinary Research, 75, 285–291.
  Boileau, M. J., Mani, R., Breshears, M. A., Gilmour, M., Taylor, J. D., & Clinkenbeard, K. D. (2016). Efficacy of Bdellovibrio bacteriovorus 109J for the treatment of dairy calves with experimentally induced infectious bovine keratoconjunctivitis. American Journal of Veterinary Research, 77, 1017–1028. doi: 10.2460/ajvr.77.9.1017
  Lambert, C., & Sockett, R. E. (2008). Laboratory maintenance of Bdellovibrio. Current Protocols in Microbiology, 9, 7B.2.1–7B.2.3. doi: 10.1002/9780471729259.mc07b02s9
  Martin, M. O. (2002). Predatory prokaryotes: An emerging research opportunity. Journal of Molecular Microbiology and Biotechnology, 4, 467–477.
  Rogosky, A. M., Moak, P. L., & Emmert, E. A. (2006). Differential predation by Bdellovibrio bacteriovorus 109J. Current Microbiology, 52, 81–85. doi: 10.1007/s00284‐005‐0038‐6
  Ruby, E. G. (1991). The genus Bdellovibrio. In A. Balows, H. G. Truper, M. Dworking, W. Harder, & K. H. Schleifer (Eds.), The prokaryotes, 2nd ed. (pp. 3400–3415). New York, NY: Springer‐Verlag.
  Seidler, R. J., & Starr, M. P. (1969). Factors affecting the growth of Bdellovibrio bacteriovorus developing within E. coli. Journal of Bacteriology, 97, 912–923.
  Stolp, H. (1973). The Bdellovibrios: Bacterial parasites of bacteria. Annual Review of Phytopathology, 11, 53–76. doi: 10.1146/annurev.py.11.090173.000413
  Stolp, H., & Starr, M. P. (1963). Bdellovibrio bacteriovorus gen. et sp. n., a predatory, ectoparasitic, and bacteriolytic microorganism. Antonie Van Leeuwenhoek, 29, 217–248. doi: 10.1007/BF02046064
  Tudor, J. J., McCann, M. P., & Acrich, I. A. (1990). A new model for the penetration of prey cells by bdellovibrios. Journal of Bacteriology, 172, 2421–2426. doi: 10.1128/jb.172.5.2421‐2426.1990
  Varon, M., & Shilo, M. (1980). Ecology of aquatic bdellovibrios. In M. R. Droop & H. W. Jannesch (Eds.), Advances in aquatic microbiology, Vol. 2 (pp. 1–41). London, UK: Academic Press.
Key References
  Dwidar, M., Kalanjana Monnappa, A., & Mitchell, R. J. (2012). The dual probiotic and antibiotic nature of Bdellovibrio bacteriovorus. BMB Reports, 45, 71–78. doi: 10.5483/BMBRep.2012.45.2.71
  Review of potential applications of Bdellovibrio.
  Lambert, C., & Sockett, R. E. (2008). See above.
  Describes laboratory maintenance of host‐dependent and host‐independent Bdellovibrio.
  Martin, M. O. (2002). Predatory prokaryotes: An emerging research opportunity. Journal of Molecular Microbiology and Biotechnology, 4, 467–477.
  Review of widespread potential applications of Bdellovibrio.
  Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation of micro‐organisms by drying: A review. Journal of Microbiological Methods, 66, 183–193. doi: 10.1016/j.mimet.2006.02.017
  Overview of freeze drying of microorganisms.
  Hubakek, Z. (2003). Protectants used in the cryopreservation of microorganisms. Cryobiology, 46, 205–229. doi: 10.1016/S0011‐2240(03)00046‐4
  General review of protectants used for lyophilization and long‐term storage of various microorganisms.
Internet Resources
  http://opsdiagnostics.com/notes/ranpri/bacteria_lyophilization_overview.htm
  Describes basic principles and tips for freeze drying microorganisms.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library