To understand the mechanism(s) of T4 phage inactivation by EcN, E. coli strains were subjected to different treatments as mentioned below. (i) Heat-killing: 10 ml of 24 h static cultures were pelleted at 4696 × g for 10 min at RT and washed twice with 500 μl of 0.9% saline. The pellets were resuspended in LB medium to obtain 1010 Cfus/ml and thereon heat-killed for 1 h at 100°C. The supernatants of heat-killed cells were sterile filtered (0.22 μm PALL filter, Cat no: 514-4131, VWR). (ii) Concentrated supernatant: supernatants of 24 h E. coli static cultures were sterile filtered and concentrated approximately 10 times (10×) with Vivaspin Turbo 15 (MWCO: 5 KDa, Cat no: VS15T11, Sartorius). (iii) LPS destruction: Further to understand the involvement of LPS, E. coli heat-killed cultures (1010 Cfus/ml) or supernatants were incubated with 25 μg/ml polymyxin B (PMB) for 1 h at 37°C. 100 μl of the differentially treated E. coli strains or supernatants were added to 100 μl of phages, to a final volume of 1 ml with LB medium and incubated for 24 h at 37°C in a 24 well system. In the case of PMB treatment, 25 μg/ml PMB was used also in the coincubation.
Pall filter
Manufactured by Avantor
Sourced in Germany
PALL filters are a type of filtration device used in various laboratory and industrial applications. They are designed to remove particulates, microorganisms, and other contaminants from liquids and gases.
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6 protocols using pall filter
1
Mechanism of T4 Phage Inactivation by EcN
2
Screening for Lambdoid Phage Integration in E. coli
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E. coli strains were tested for a possible integration of the lambdoid phages. Therefore, E. coli strains were incubated static at 37°C with and without lambdoid phages until the plaque forming units (pfus) reached zero (44 h for stx-phages and 120 h for lambda-phages). At these timepoints, we could not detect any pfu in the sterile filtrate of EcN coincubation by PPA. 1 ml bacteria were washed twice with LB medium (13,000 × g, 5 min, RT) and resuspended in 1 ml LB medium. The washed bacteria were incubated for 14–16 h with 1 μg/ml MMC to induce lysis. The supernatant of the bacteria was sterile filtered (0.22 μm PALL filter/VWR, Darmstadt, Germany) and incubated for another 24 h, 37°C, static with MG1655 to amplify the phage signal by MG1655 lysis. The final phage titer was detected with the phage plaque assay.
3
T4 phage infection dynamics with bacterial strains
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T4 phage lysate was incubated with bacterial strains as described in Bury et al. (2018) (link). In short, the OD600 of bacterial overnight cultures (ONC) was determined and the cells were collected by centrifugation at 4696 × g for 10 min at RT. The bacterial pellet was resuspended in fresh LB medium to obtain ∼109 Cfus/ml. 100 μl of a phage extract (∼109 Pfus/ml) were used to set up mono; co; or tri-cultures with 100 μl of EcN and/or E. coli K-12 strains in a 24 well plate. Each well was adjusted to a final volume of 1 ml with LB medium. The plates were incubated in a static manner for the desired amount of time at 37°C. The Cfus/ml were determined by plating serial dilutions in 0.9% saline on 1.5% LB agar plates. The samples were sterile filtered (0.22 μm PALL filter, Cat No: 514-4131, VWR) and serially diluted in 0.9% saline for further analysis.
4
Quantifying Bacterial Co-Cultures and Phage Interactions
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The OD600 of bacterial overnight cultures (ONC) was determined and the cells were collected by centrifugation at 9,400 × g for 5 min at RT. The bacterial pellet was resuspended in LB medium to obtain ~108 CFUs/ml of the STEC strains and ~109 CFUs/ml of the commensal and E. coli K-12 strains. 100 μl of the STEC strains or 100 μl of a phage extract were used to set up mono-/co-/or tri-cultures with 100 μl of EcN and/or K-12 strains in a 24 well plate. The cell numbers used for co- and tri-cultures of STEC/phages: commensals/SK22D: K-12 strain was 1:10:10 = 107 CFUs or pfus/ml: 108 CFUs/ml: 108 CFUs/ml. Whenever the K-12 strains were used in co- and tri-cultures with stx-phages, 100 μl of a non-MMC induced phage lysate was used (~106 pfus/ml). The MOI of stx-phages: commensals/SK22D: K-12 strain was 1:100:100 = 106 pfus/ml: 108 CFUs/ml: 108 CFUs/ml. Each well was adjusted to a final volume of 1 ml with LB medium. The plates were incubated in a static manner for the desired amount of time at 37°C. The CFUs were determined by plating serial dilutions in 0.9% saline on LB agar plates with a selective antibiotic or on ECC plates (medco Diagnostika GmbH, Munich, Germany). The samples were sterile filtered (0.22 μm PALL filter/VWR, Darmstadt, Germany) for further analysis.
5
Induction of Bacteriophage Production
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The induction of the phage production was achieved by adding 1 μg/ml of mitomycin C (MMC/OMNILAB GmbH & Co. KG, Munich, Germany) to a lysogenic E. coli culture in its mid-log growing phase (OD600 0.3-0.5). The culture was further incubated in the dark for 6 h/24 h at 37°C in a rotary shaker (200 rpm) before the phages were isolated by sterile filtration (0.22 μm PALL filter/VWR, Darmstadt, Germany) of the supernatant (9,400 × g, 5 min, RT). stx-phages for the mono-/co-/and tri-culture studies with MG1655 were extracted from a 24 h shaking STEC culture without the addition of MMC.
6
Propagation and Concentration of T4 Phage
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The T4 phage was propagated by adding 100 μl of T4 phage stock (∼107 Pfus/ml) to 20 ml of E. coli K-12 MG1655 culture in its early log growing phase (OD600∼0.3). The culture was further incubated at 37°C in a rotary shaker (200 rpm) until clear lysis was observed (∼5 to 6 h). Chloroform (2%) was added to the lysate and incubated for 15 min at room temperature (RT) and centrifuged (4696 × g, 10 min, 4°C). The phages were then isolated by sterile filtration (0.22 μm PALL filter, Cat no: 514-4131, VWR) of the supernatant. For microscopy experiments, T4 phage lysate was further concentrated as described in Bonilla et al. (2016) (link). Briefly, phage lysate was concentrated by repeated centrifugation in Amicon® Ultra-15 centrifugal filter units, Ultracel 100 KDa membrane (Cat no: UFC910008, Millipore). By this technique, we were able to produce phage stocks of high concentration (up to 1015 Pfus/ml).
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