Materials and Reagents

Breathable sealing membrane (Breathe-Easy^TM sealing membrane, Diversified Biotech, catalog number: Z380059 )
Centrifuge tubes, 15 ml (Sarstedt, catalog number: 62.554.502 )
Centrifuge tubes, 50 ml (Sarstedt, catalog number: 62.547.254 )
Clear 96-well plates (BRAND, catalog number: 781662 )
Deep 96-well plates, 2.2 ml wells (BRAND, catalog number: 701354 )
Microcentrifuge tubes, 1.5 ml (VWR, catalog number: 20170-038 )
Parafilm M (Bemis, catalog number: HS234526A )
Petri dishes (Sarstedt, catalog number: 82.1472.001 )
Bdellovibrio bacteriovorus HD100 (source: Sockett RE, Queen's Medical Centre Nottingham)
Escherichia coli BW25113 (source: laboratory collection)
Agar (AppliChem, catalog number: A0949 )
Anhydrous sodium acetate (Roth, catalog number: 6773 )
CaCl₂·2H₂O (Emsure, Merk, catalog number: 102382 )
HEPES free acid (Roth, catalog number: 9105 )
MgSO₄·7H₂O (Roth, catalog number: P027 )
Peptone (BD, Bacto^TM, catalog number: 211830 )
Tryptone (BD, Bacto^TM, catalog number: 211699 )
Yeast extract (BD, Bacto^TM, catalog number: 212720 )
NaCl (Applichem, catalog number: A4661.5000 )
CaCl₂ stock solution (see Recipes)
MgSO₄ stock solution (see Recipes)
YT broth (see Recipes)
YPSC broth (see Recipes)
Ca/Mg/HEPES buffer (see Recipes)
Bottom agar (see Recipes)
Top agar (see Recipes)
LB agar (see Recipes)

Equipment

Benchtop centrifuge (from any qualified supplier)
Bottles, 250 ml (VWR, catalog number: 215-1593 )
Bottles, 500 ml (VWR, catalog number: 215-1595 )
Bunsen burner (from any qualified supplier)
Cuvettes (Sarstedt, catalog number: 67.742 )
Flasks, 250 ml (VWR, catalog number: 214-0423 )
Freezer, -80 °C (from any qualified supplier)
Plate incubators (from any qualified supplier)
Laminar flow hood (from any qualified supplier)
Inoculation loop (from any qualified supplier)
Microcentrifuge (Thermo Fisher Scientific, catalog number: 75002471 )
Multichannel pipette (8x) 100-1,200 μl (Gilson, catalog number: FA10039 )
Multichannel pipette (8x) 30-300 μl (Sartorius, catalog number: LH-729140 )
Phase contrast microscope, magnification equal or above 1,000x (from any qualified supplier)
Pipette 0.5-10 μl (Eppendorf, catalog number: 3120000020)
Pipette 10-100 μl (Eppendorf, catalog number: 3120000046)
Pipette 100-1,000 μl (Eppendorf, catalog number: 3120000062)
Plate reader (TECAN, model: Infinite M200 PRO)
Plaque visualization chamber ( Marine et al., 2020 : Figure S9)
Refrigerator, 4 °C (from any qualified supplier)
Shakers (Infors HT, Multitron Standard)
Spectrophotometer (Eppendorf, Biospectrometer basic, catalog number: 6135000017)
Water bath (from any qualified supplier)
Microwave (from any qualified supplier)

Software

Prism 5 (GraphPad, https://www.graphpad.com/scientific-software/prism/)
However, any other statistics software can be used by setting the modified Gompertz equation (Lambert and Pearson, 2000).
WPS Office (Kingsoft, https://www.wps.com/)

Procedure

This protocol details every step of antimicrobial testing on B. bacteriovorus from culturing cells to setting up MIC experiments and determining the MIC point. A general outline of the workflow is summarized below:

Revival of B. bacteriovorus from glycerol stocks
Growth of B. bacteriovorus in liquid culture
Antimicrobial susceptibility testing
Enumeration on double layer agar plates
Antimicrobial susceptibility testing of prey bacteria

Note: All steps that require working in a laminar flow under sterile conditions are indicated with the symbol "*".

Revival of B. bacteriovorus from glycerol stocks
Day 1:
1. Open the glycerol stock tube containing E. coli and scrape the top of the frozen culture with a sterile inoculation loop*.
2. Streak the E. coli cells onto a section at the border of a LB 1% agar plate, by gently touching the LB agar with the inoculation loop containing the E. coli cells and streaking the loop back and forth. To produce single colonies, sterilize the loop and gently drag the bacteria across the surface of the agar from the first section to another section of the plate by streaking through the first section. Repeat this step one or two more times to spread the bacteria from the second or third section to a new section of the plate* ( Slonczewski et al., 2020 : Figure 4.11, p. 131).
3. Incubate plates upside down overnight in an incubator at 37 °C. These plates can be stored at 4 °C for 2 weeks.
  Day 2:
4. Pour 5 ml YT broth inside a 50 ml tube. Inoculate the broth with 4 separately picked E. coli colonies using an inoculation loop* and incubate the culture at 37 °C with shaking at ~170 rpm overnight. This step has to be done late in the afternoon, so the E. coli cells do not overgrow.
  Day 3:
5. Unscrew the lid of bottles containing bottom and top agar and melt the agar in a microwave (500 ml of bottom agar allow to cast around 20-25 plates; the amount of top agar required for 25 plates is 125 ml). Melting the agar may require several minutes, depending on the power setting and the amount of agar to be melted. Make sure that the cap of the bottle is partly unscrewed as to allow for boiling vapor to escape. We suggest to use a minimal power setting and to stop the microwave as soon as the liquified agar begins to boil. Take out the bottle and gently mix it by swirling (careful, when agitated the liquid agar can boil). Repeat heating in the microwave and mixing until all the agar is melted and there are no lumps visible. Then, briefly heat the agar one last time at a higher power setting to melt all remaining lumps not visible by eye.
6. Subsequently, place the bottles in a 55 °C water bath until they reach this temperature (Note 1).
7. Supplement both bottom and top agar with CaCl₂ and MgSO₄ to a final concentration of 2 mM and 1 mM, respectively*.
8. Pour a layer (20-25 ml) of bottom agar in a Petri dish (diameter of 10 cm) and distribute it equally by gently swirling the Petri dish*.
9. Centrifuge 5 ml of an E. coli overnight culture and resuspend it in the exhausted broth of culture (which was used to grow cells) to a final OD₆₀₀ of ~10*.
  Note: The relationship between OD₆₀₀ and cell biomass approximates linearity only at low absorbance values (usually up to 0.3-0.4, depending on the instrument and cuvettes). Therefore, serial dilutions (e.g., 1:2) of the E. coli overnight culture should be prepared for measuring the OD₆₀₀. The construction of a graph of absorbance against dilution factors allows to determine the linear range of values. Linear regression can be used to calculate the OD₆₀₀ value of the undiluted overnight culture.
10. Add 500 μl of the concentrated overnight E. coli culture and 5 ml of the top agar in a 10 ml test tube*.
11. After capping the tube with a sterile lid, gently mix by inversion of the tube for 2 times (avoiding the formation of bubbles)*.
12. Quickly pour the agar onto the Petri dish containing the solidified bottom agar and distribute it evenly by gently swirling the Petri dish*. Leave the dish under the laminar flow hood for the agar to solidify with the lid semi-open.
13. Pipet 50 μl of a thawed B. bacteriovorus glycerol stock (containing 20% (v/v) glycerol) in the center of the double layer agar plate (i.e., a 2-layer plate consisting of bottom and top agar) with E. coli cells within the top agar*.
14. Leave this plate under the laminar flow hood with the lid semi-open to dry the B. bacteriovorus suspension*.
15. After the B. bacteriovorus suspension has dried (after approx. 30 min), seal the plate-lid and plate with parafilm to prevent it from drying out.
16. Incubate the plate for 2 to 3 days at 29 °C until a clear zone (plaque) on the agar is visible (due to E. coli lysis) at the center of the plate where the B. bacteriovorus suspension has been applied (Figure 1).
  Note: Ca/Mg/HEPES double layer agar plates can be stored at room temperature for 2 weeks without significantly affecting B. bacteriovorus viability (Lambert et al., 2008).
  
  Figure 1. B. bacteriovorus plaque on a double-layer agar plate. B. bacteriovorus revived from a glycerol stock form a large plaque on a double-layer plate (containing E. coli in the top agar) at the point of application. Some of the agar has been taken with a pipette from the center of the plaque (holes in the agar).
Grow B. bacteriovorus in liquid culture
Note: B. bacteriovorus predation efficiency is higher in presence of slow growing or stationary phase bacteria preys. Broth of cultures such as DNB (diluted nutrient broth), YT (yeast extract, trypton) and YPSC (yeast extract, peptone, sodium, calcium) have a low concentration of nutrients and result in slow bacterial growth rates and were originally used for the concomitant culturing of B. bacteriovorus and its preys (inoculation of fresh broth with both predatory and prey bacteria, at the same time). Although, most research groups grow B. bacteriovorus on stationary phase prey bacteria from overnight cultures, they still use DNB or YT broth (YPSC is used mostly for the concomitant growth in double layer agar plates). A critical point that affects predation efficiency (and therefore predatory bacteria growth) is the Ca²⁺ and Mg²⁺ concentration and pH of the buffer/broth of culture. Typically, B. bacteriovorus is cultivated at a pH of 7.2 to 7.6 and at a concentration of 2-3 mM Ca²⁺ (CaCl₂) and 1-3 mM Mg²⁺ (either MgSO₄ or MgCl₂). Other broth components, such as peptone and yeast extract, seem to increase B. bacteriovorus viability and durability.
Day 1:
1. Streak out E. coli prey cells on a LB 1% agar plate to get single colonies* and incubate overnight at 37 °C.
  Day 2:
2. Pour 2 ml YT broth in a 15 ml tube. Inoculate this broth with 4 separately picked E. coli colonies using an inoculation loop* and incubate at 37 °C under shaking conditions (~170 rpm) overnight.
  Day 3:
3. Excise a portion of top layer agar from the B. bacteriovorus plaque area (the top agar has a soft gel-like consistency and can be easily taken up using a 1,000 μl pipet set to ≥ 250 μl) and transfer the cells by pipetting up and down into the tube containing the overnight prey culture*. Incubate 1 to 2 days at 29 °C under shaking conditions (~170 rpm) until the culture is clear (lysed).
4. Pour 5 ml YPSC broth in a 50 ml tube. Inoculate this broth with 4 separately picked E. coli colonies using an inoculation* loop and incubate at 37 °C with shaking at ~170 rpm overnight.
  Day 4:
5. When the B. bacteriovorus culture appears clear, check under a phase contrast microscope for the presence of B. bacteriovorus and lysis of prey cells.
  Note: For this purpose, 5 μl of the culture can be used for examination at the microscope. B. bacteriovorus cells can be distinguished from other bacteria by their smaller size and high motility, while bdelloplasts (B. bacteriovorus growing inside preys) are round shaped.
6. Add 100 μl of the B. bacteriovorus culture to the E. coli overnight culture* and incubate for 24 h at 29 °C under shaking conditions (~170 rpm).
7. Pour 5 ml YPSC broth in a 50 ml tube. Inoculate this broth with 4 separately picked E. coli colonies using an inoculation loop* and incubate at 37 °C with shaking at ~170 rpm overnight.
  Day 5:
8. Check the B. bacteriovorus culture under a phase contrast microscope for the presence of B. bacteriovorus and lysis of prey cells.
9. Add 100 μl of the B. bacteriovorus culture (from Day 4) to the E. coli overnight culture* and incubate for 24 h at 29 °C under shaking conditions (~170 rpm).
10. Inoculate the required volume of YPSC with 4 separately picked E. coli colonies using an inoculation loop* and incubate at 37 °C with shaking at ~170 rpm overnight. When preparing this culture, consider the volume necessary to obtain around 2 ml per antimicrobial to be tested (i.e., 2 ml of culture for each master plate).
  Day 6:
11. Check the B. bacteriovorus culture under a phase contrast microscope for the presence of B. bacteriovorus and lysis of prey cells.
12. Add 1 ml of B. bacteriovorus culture (from Day 5) to the flask containing the E. coli overnight culture* and incubate for 24 h at 29 °C under shaking conditions (~170 rpm).
13. Inoculate the required volume of YPSC broth with 4 separately picked E. coli colonies using an inoculation loop* and incubate at 37 °C with shaking at ~170 rpm overnight. Calculate the required volume as follows:
  
  volume E. coli culture = number of master plates * 10 ml + 10 ml
  e.g., for 3 plates a volume of E. coli culture equals to 3 * 10 ml + 10 ml = 40 ml is required

Antimicrobial susceptibility testing (Figure 2, left panel)
Day 7:
1. Prepare antimicrobial two-fold serial dilutions along the rows of clear 96-well plates in 100 μl Ca/Mg/HEPES buffer including controls without antimicrobial (one antimicrobial per plate)*.
  Note: Ideally, the MIC point should fall in the middle of the antimicrobial concentration range. As a starting point, the researcher can use the known MIC values for other bacteria species. If the MIC at a specific initial PPR falls outside the range of values or too close to one end, the test should be repeated with the adjusted concentration range.
2. Check the B. bacteriovorus culture under a phase contrast microscope for the presence of B. bacteriovorus and lysis of prey cells.
3. Dilute the B. bacteriovorus culture (from Day 6) by a factor of 25 in Ca/Mg/HEPES buffer (this dilution is necessary to ensure an initial PPR in the assay within the range of 0.2-0.005)*.
4. Divide this B. bacteriovorus dilution in two separate 50 ml tubes*. One part of this dilution will be inactivated and used as a diluent for preparing the serial dilutions. The other part with active B. bacteriovorus will be used for preparing the serial dilutions and for enumeration. Calculate the required volumes as follows:
  
  Volume active bacteria dilution = number of master plates * 4 ml + 10 ml
  
  Volume inactivated bacteria dilution = number of master plates * 13 ml + 10 ml
5. Inactivate one part of the B. bacteriovorus dilution by incubation in a water bath at 100 °C for 5 min.
6. Prepare B. bacteriovorus two-fold serial dilutions (from Step C3) in a sterile deep 96-well plate along the column line, including controls (no viable B. bacteriovorus present)*.
7. Transfer 50 μl from each well of this plate (Step C6) to the corresponding wells of master plates (Step C1, plates with the antimicrobial dilution series)*.
8. Add 50 μl of the E. coli overnight culture to each plate well*.
9. Measure the blank at a wavelength of 600 nm in a plate reader.
10. Seal master plates with breathable film* and incubate for 24 h at 29 °C under shaking conditions (300 rpm, 3 mm diameter orbit (or throw)).
11. In a spectrophotometer, measure the absorbance at 600 nm of the E. coli overnight culture (measurements have to be performed at different dilutions in fresh broth to be in the linear range of values).
  Day 8:
12. Measure the absorbance of the master plate(s) (from Day 7) at a wavelength of 600 nm in a plate reader.

Enumeration on double layer agar plates
Note: Plating of predatory bacteria for enumeration has to be performed on the same day of the antimicrobial test as cell viability decrease over time and may result in a lower estimate.
Plating on double layer agar plates (or plaque assay) is the most used method to enumerate, separate or identify bacteriophages and predatory bacteria in a sample. Both predatory bacteria and phages lysate prey/host bacteria and form plaques on agar plates. Double layer plates are cast in two steps as a thick layer of agar at the bottom (i.e., bottom agar) and a thin layer on the top (i.e., top agar) containing prey and predatory bacteria. The bottom agar helps to maintain the top agar moist and soft, buffers against pH changes and provides nutrients to prey bacteria. The top agar has a low agar content (to increase the efficiency of plaque formation) and is poured as a thin layer so that plaques form on the surface of the agar (thus increasing visibility) and to prevent plaques from forming on top of each other.
Day 7:
1. Unscrew the lid of a top agar bottle and melt the agar in a microwave.
2. Incubate the bottle in a water bath at 55 °C until it reaches this temperature (Note 1).
3. Supplement the top agar with CaCl₂ and MgSO₄ to a final concentration of 2 mM and 1 mM, respectively*.
4. Centrifuge 5 ml of an E. coli overnight culture at 5,000 x g for 6 min and resuspend it in the exhausted broth of culture (which was used to grow cells) to a final OD₆₀₀ of ~10*.
5. Prepare ten-fold serial dilutions in Ca/Mg/HEPES buffer of the 25x diluted B. bacteriovorus culture (from Step C3)*.
  Note: Place eight 1.5 ml centrifuge tubes in a rack and label them from -1 to -8. Pipet 900 μl of Ca/Mg/HEPES in each tube, then add 100 μl of the B. bacteriovorus 25x diluted culture to the first tube (10^-1 dilution). Replace the tip of the pipet with a new one, mix the content of the first tube and transfer 100 μl of this dilution to the second tube (10^-2 dilution). Repeat the latter step to ten-fold dilute the 10^-2 dilution and so forth for all remaining tubes.
  Typically, 10^-4 to 10^-6 dilutions of a B. bacteriovorus culture result in the formation of single plaques upon plating (Lambert et al., 2008). Duplicate plates are not needed.
6. To a test tube, add 100 μl of a serial dilution, and 500 μl of the concentrated E. coli overnight culture as well as 5 ml of the top agar*.
7. After capping the tube with a sterile lid, gently mix by inversion of the tube for 2 times (avoiding the formation of bubbles)*.
8. Quickly pour the agar onto the Petri dish containing the bottom agar and distribute it evenly by gently swirling the Petri dish*. Leave the dish under the laminar flow hood for the agar to solidify with the lid semi-open.
9. Seal the plate-lid and plate with parafilm to prevent it from drying out*.
10. Incubate for 3 to 4 days at 29 °C until the formation of clear plaques in the top agar (due to E. coli lysis).
11. Estimate the number of B. bacteriovorus cells in the initial inoculation culture by counting plaque forming units (PFU; the equivalent of colony forming units for non-predatory bacteria. The growth of a single B. bacteriovorus cell generates a single plaque).
12. Optional: pictures of plaques can be acquired by the aid of a plaque visualization chamber (Marine et al., 2020: Figure S9).

Antimicrobial susceptibility testing of prey bacteria (Figure 3, left panel)
Day 1:
1. Inoculate YPSC broth with 4 separately picked E. coli colonies using an inoculation loop* and incubate at 37 °C with shaking at ~170 rpm overnight.
  Day 2:
2. Prepare two-fold serial antimicrobial dilutions in 100 μl Ca/Mg/HEPES along the rows of deep 96-well plates including controls without antimicrobial (2 antimicrobials per plate can be tested as triplicates)*.
3. Centrifuge the E. coli overnight culture at 5,000 x g for 6 min, discard the supernatant and re-suspend in Ca/Mg/HEPES buffer (wash step)*.
4. Centrifuge it at 5,000 x g for 6 min and re-suspend in Ca/Mg/HEPES buffer to a final OD₆₀₀ of 0.024*.
5. Add 50 μl of this E. coli suspension and 50 μl of fresh YPSC broth to the wells of master plates (plates with the antimicrobial dilution series)*.
6. Measure the blank at a wavelength of 600 nm in a plate reader.
7. Seal plates with breathable film* and incubate for 24 h at 29 °C under shaking conditions (300 rpm, 3 mm throw).
  Day 3:
8. Measure the absorbance of the master plate(s) (from Day 2) at a wavelength of 600 nm in a plate reader.

Data analysis

Data analysis of B. bacteriovorus antimicrobial test results (Figure 2, right panel)
Note: B. bacteriovorus antimicrobial test should be repeated at least three times for each antimicrobial. Only process the data from three experiments (plates) in which both B. bacteriovorus and E. coli show growth in the wells without antibiotic (control). An antibiotic of known efficacy (e.g., trimethoprim or aztreonam; Marine et al., 2020) can be tested to verify the correctness of the procedure.
1. Estimate from B. bacteriovorus enumerations and E. coli absorbance readings the initial PPRs, by dividing the number of B. bacteriovorus to the number of E. coli cells.
2. Subtract the blanks from the corresponding absorbance values (including controls).
3. Subtract the resulting values from control values of E. coli without B. bacteriovorus at the same antimicrobial concentrations.
4. Convert these absorbance Δ values to growth percentage values.
5. Plot these data in a graph against the logarithm of antimicrobial concentrations.
6. Download the GraphPad file for the Gompertz modified equation from the webpage.
7. Open the file and fit B. bacteriovorus growth inhibition curves to the Gompertz modified equation (Lambert and Pearson, 2000). Apparent MIC values are calculated by the program as the intersection of the line tangential to the lower asymptote with the equation of the line tangential to the inflexion point: MIC = 10^{(M + 1/A)}; where A is the distance between the upper and lower asymptote and M is the logarithmic concentration at the inflexion point (Lambert and Pearson, 2000).
8. For each antimicrobial, plot apparent MIC values against the logarithm of initial PPRs.
9. Determine the reference MIC value at a PPR of 0.02 by linear regression (within the linear range of values).
  
  Figure 2. MIC determination of antimicrobials for B. bacteriovorus. Antimicrobial sensitivity test scheme (left panel). The calculation of the MIC of Aztreonam at the initial PPR of 0.024 is shown as an example (right panel). Alphanumeric characters enclosed by circles indicate the corresponding steps in the text.

Data analysis of E. coli antimicrobial test results (Figure 3, right panel)
Note: E. coli antimicrobial test should be repeated at least three times for each antimicrobial. Only process the data from three experiments (plates) in which E. coli shows growth in the wells without antibiotic (control). An antibiotic of known efficacy (e.g., aztreonam; Marine et al., 2020) can be tested to verify the correctness of the procedure.
1. Subtract the blanks from the corresponding absorbance values (including controls).
2. Convert these absorbance values to growth percentage values.
3. Plot these data in a graph against the logarithm of antimicrobial concentrations.
4. Fit E. coli growth inhibition curves to the Gompertz modified equation (Lambert and Pearson, 2000).
5. Compare results obtained for B. bacteriovorus and for E. coli by dividing B. bacteriovorus MIC values for each antimicrobial to the corresponding E. coli MIC value. A ratio higher than 1 indicates a higher resistance of B. bacteriovorus against an antimicrobial in comparison to E. coli and vice versa.
  
  Figure 3. MIC determination of antimicrobials for E. coli. Antimicrobial sensitivity test scheme (left panel). The calculation of the MIC of Aztreonam is shown as an example (right panel). Alphanumeric characters enclosed by circles indicate the corresponding steps in the text.

Notes

To speed up the cooling of melted agar, sealed bottles can be held manually at a 45° angle under running tap water while rotating along the long axis and mildly shaking them for a short time. Keeping the bottles in the water bath ensures, after a reasonable period of time, that they reach the set temperature of 55 °C (which can be determined by a thermometer placed in the water bath). The use of a higher temperature is not recommended as it kills bacteria. On the other hand, too low a temperature will cause the agar to solidify and result in an uneven surface of the cast agar.

Recipes

Note: Sterilize all buffers and broths by autoclaving.

CaCl₂ stock solution
200 mM CaCl₂
MgSO₄ stock solution
100 mM MgSO₄
YT broth
5 g/L yeast extract
10 g/L tryptone
5 g/L NaCl
2 mM CaCl₂
1 mM MgSO₄
Dissolve ingredients except CaCl₂ and MgSO₄ and adjust pH of the medium to 7.6 with NaOH. After autoclaving add CaCl₂ and MgSO₄ from 200 mM and 100 mM sterile stock solutions
YPSC broth
1 g/L yeast extract
1 g/L peptone
0.5 g/L anhydrous sodium acetate
2 mM CaCl₂
1 mM MgSO₄
Dissolve ingredients except CaCl₂ and MgSO₄ and adjust pH of the medium to 7.6 with NaOH. After autoclaving add CaCl₂ and MgSO₄ from 200 mM and 100 mM sterile stock solutions
Ca/Mg/HEPES buffer
25 mM HEPES pH 7.6
2 mM CaCl₂
1 mM MgSO₄
Bottom agar
25 mM HEPES pH 7.6
1% agar
Top agar
25 mM HEPES pH 7.6
0.6% agar
LB agar
5 g/L yeast extract
10 g/L tryptone
5 g/L NaCl
1% agar

Acknowledgments

This work was supported by funding from DFG-EXC115 (Cluster of Excellence Macromolecular Complexes at the Goethe University Frankfurt). E.M. was supported by a stipend from the FAZIT Foundation, Frankfurt am Main, Germany. This protocol was adapted from previous work (Marine et al., 2020). We thank Prof. Sockett RE, Lambert C, Milner D and their research group for the transfer of knowledge on B. bacteriovorus culture methods.

Competing interests

The authors declare no competing interests.

References

Im, H., Choi, S. Y., Son, S. and Mitchell, R. J. (2017). Combined application of bacterial predation and violacein to kill polymicrobial pathogenic communities. Sci Rep 7(1): 14415.
Lambert, R. J. and Pearson, J. (2000). Susceptibility testing: accurate and reproducible minimum inhibitory concentration (MIC) and non-inhibitory concentration (NIC) values. J Appl Microbiol 88(5): 784-790.
Lambert, C. and Sockett, R. E. (2008). Laboratory maintenance of Bdellovibrio. Curr Protoc Microbiol Chapter 7: Unit 7B 2.
Madhusoodanan, J. (2019). Inner Workings: Probing predatory bacteria as an antibacterial remedy. Proc Natl Acad Sci U S A 116(46): 22887-22890.
Marine, E., Milner, D. S., Lambert, C., Sockett, R. E. and Pos, K. M. (2020). A novel method to determine antibiotic sensitivity in Bdellovibrio bacteriovorus reveals a DHFR-dependent natural trimethoprim resistance. Sci Rep 10(1): 5315.
Pasternak, Z., Njagi, M., Shani, Y., Chanyi, R., Rotem, O., Lurie-Weinberger, M. N., Koval, S., Pietrokovski, S., Gophna, U. and Jurkevitch, E. (2014). In and out: an analysis of epibiotic vs periplasmic bacterial predators. ISME J 8(3): 625-635.
Rendulic, S., Jagtap, P., Rosinus, A., Eppinger, M., Baar, C., Lanz, C., Keller, H., Lambert, C., Evans, K.J., Goesmann, A., Meyer, F., Sockett, R.E. and Schuster, S.C. (2004). A predator unmasked: life cycle of Bdellovibrio bacteriovorus from a genomic perspective. Science 303(5658): 689-92.
Slonczewski, J., Foster, J. and Zinser, E. (2020). Microbiology: An evolving science. 5th Ed. W.W Norton& Company p. 131.
Tyson, J. and Elizabeth Sockett, R. (2017). Nature knows best: employing whole microbial strategies to tackle antibiotic resistant pathogens. Environ Microbiol Rep 9(1): 47-49.
Wiegand, I., Hilpert, K. and Hancock, R. E. (2008). Agar and broth dilution methods to determine the minimal inhibitory concentration(MIC) of antimicrobial substances. Nat Protoc 3(2): 163-175.