CFU assay was performed to quantify viable cells of P. gingivalis or P. intermedia in planktonic or biofilm states. After the treated bacteria were transferred to liquid medium and serially diluted, the samples were plated on blood agar for further counting by using easyspiral Pro® (Interscience, St. Nom La Bretèche, France).
Easyspiral pro
Manufactured by Interscience
Sourced in France
The EasySpiral Pro is a laboratory equipment designed for automated spiral plating. It is capable of inoculating and spreading bacterial samples onto agar plates in a spiral pattern, enabling efficient colony counting and enumeration.
Automatically generated - may contain errors
Lab products found in correlation
Scan 1200, by Interscience (2 mentions)
Maximum recovery diluent, by Thermo Fisher Scientific (2 mentions)
Tryptone soya agar, by Thermo Fisher Scientific (2 mentions)
Scan 4000 ultra hd, by Interscience (2 mentions)
Bagfilter p, by Interscience (2 mentions)
Bag mixer 400 lw, by Interscience (2 mentions)
Streptomycin, by Merck Group (1 mentions)
10 protocols using easyspiral pro
1
Quantifying Bacterial Viability in Planktonic and Biofilm States
2
Quantifying Viable MRSA Remaining on Surfaces
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Under sterile conditions, three independent droplets of 10 µL (around 108 CFU) were inoculated on the tested surface and then incubated for two hours at room temperature. Bacteria of each inoculum were then carefully harvested using a sterile swab moistened with 50 µL of peptone water. The swab was firmly applied on area of the inoculum and rotated. The swab was then placed in a 50 mL sterile tube containing 7.5 mL of peptone water. Tubes were placed in ultrasonic bath (35 kHz) during 2 min and then briefly vortexed for 30 s. Serial dilutions of each tube suspension were performed in peptone water and 100 µL of each dilution was exponentially seeded (easySpiral Pro, Interscience, Saint-Nom-la-Bretèche, France) on TSA. Plates were incubated at 37 °C for 24 h. Colonies were counted with the Interscience Scan 1200 to determine the number of viable MRSA remaining in each inoculum. For each test, these numbers were streamlined to the mean number of viable MRSA remaining on the glass slide.
3
Recombinant Production Assay for Bacteria
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Isolates to be tested for the production of recombinants were grown overnight on LB agar plates and then inoculated and grown in 2 mL of LB until the cultures reach an optical density at 600 nm (OD600) of 1. The bacterial broths were then diluted to an OD600 of 0.01 in phosphate-buffered saline (PBS). Then equal volumes of bacterial suspensions were mixed by pairs, each consisting of an imipenem-resistant (Imir) and rifampicin-resistant (Rifr) isolate. The mixture (2.5 μL) was deposited on the surface of 1 mL of tryptone-NaCl medium solidified with 2% agarose D3 (Euromedex) poured into 2-mL microtubes or in wells of 24-well plates and incubated overnight at 37°C. Bacteria were then resuspended in PBS and were plated onto LB agar plates without antibiotics and LB agar plates containing rifampicin (100 μg/mL) and imipenem (1.6 μg/mL) with either beads or easySpiral Pro (Interscience). Recombinant frequencies were determined through calculation of the ratio of the number of CFU on rifampicin and imipenem plates to the total number of CFU on plates without antibiotics.
4
CRISPR Efficiency Quantification in E. coli
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The host strains E. coli K12 MG1655 carrying pdCas9-J23109 or pCas9-J23109 were used to prepare competent cells using the method described above (quantification of mismatch effects on binding activity). Plasmids carrying the sgRNA expression cassette (pTargetF) were transformed by electroporation into the prepared competent cells expressing Cas9 or dCas9. The electroporation was performed via a BTX Harvard apparatus ECM 630 High Throughput Electroporation System using an optimized parameter setting (2.1 kV, 1 kΩ, 25 μF). The transformed cells were incubated in LB medium (four times the volume of the competent cells) for 1 h at 37°C for recovery. We streaked the resulting culture onto the LB agar plates (with kanamycin and ampicillin) automated by EasySpiral Pro (Interscience). The colonies were counted after overnight cultivation. The survival rate for each sgRNA was calculated by comparing the CFU of Cas9-expressing cells with the CFU of dCas9-expressing cells. This ratio was further normalized by determining the colony number after transformation with a negative control sgRNA plasmid to minimize the impact of differences in electroporation efficiency that were due to competent cell preparation (Equation 5 ).
5
Glass Slide Decontamination by Plasma
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Contaminated glass slides were exposed either to O2, N2 or Ar plasma treatments for varying exposure times of 5, 15, 30, 45, 60 and 120 min. For each gas plasma treatment and each time period, at least six independent experimentations were performed in triplicate. The controls consisted of non-exposed samples to plasma as well as samples exposed only to low pressure and not to the ionized gas. Bacteria were collected from glass slides by mechanical agitation in buffered peptone water. These soft sonication and vortex procedures were previously demonstrated to be without effect on bacteria viability [27 (link)]. Both bacterial dilution and seeding on nutrient agar plates were performed with EasySpiral Pro® (Interscience, France). After a 24h-incubation at 37°C, the colonies on agar plates were counted automatically with GammeScan®1200 (Interscience, France).
6
Enumeration of LGG and E. coli CFUs
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LGG and E. coli were cultured in MRS broth and LB broth at 37°C
in accordance with the ATCC guidelines, respectively. In brief, the bacteria
were harvested from broth by centrifugation. Colony forming units (CFUs) were
counted by dilution and streaking on agar plates at 37°C overnight using Easy
Spiral Pro (Interscience, St. Nom, France).
in accordance with the ATCC guidelines, respectively. In brief, the bacteria
were harvested from broth by centrifugation. Colony forming units (CFUs) were
counted by dilution and streaking on agar plates at 37°C overnight using Easy
Spiral Pro (Interscience, St. Nom, France).
7
Enumeration of Aerobic Mesophilic Bacteria in Bedding
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Samples of 25 g of each material were weighed in sterile filter bags (BagFilter P, Interscience, Saint Nom la Bretêche, France) containing 300 mL of Maximum Recovery Diluent (Oxoid, Hampshire, UK) and ground in a stomacher homogeniser (Bag Mixer 400 LW, Interscience, Saint Nom la Bretêche, France). Series of ten-fold dilutions were made from the suspension of bedding after grounding and inoculated directly on the surface of the appropriate selective medium by an automatic spiral plater (easySpiral®Pro, Interscience, Saint Nom la Bretêche, France). Each sample dilution (0.05 mL) was surface inoculated on tryptone soya agar (Oxoid, Hampshire, UK) for a total count of aerobic mesophilic bacteria and incubated at 30 °C for 48 h in an air-jacketed incubator (NuAire 5831, NuAire Inc., Plymouth, MN, USA). Counting of colonies was performed with an automatic colony counter Scan 4000 Ultra HD (Interscience, Saint Nom la Bretêche, France). Finally, the results were expressed as the logarithm of the count of Colony Forming Units in one gram of bedding material (log CFU/g).
8
Mutagenic Effect of CNA on P. aeruginosa
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The mutagenic effect of CNA was determined by calculating the rate of mutants resistant to streptomycin as a result of point mutations occurring in the rpsL gene [19 (link)]. An overnight culture of P. aeruginosa PA14 was diluted into 20 mL MHB to reach A600 = 0.2 and incubated at 37 °C and 220 rpm for 3 h with a subinhibitory concentration of CNA (500 µg/mL); a similar assay was carried out in parallel with ciprofloxacin at 0.5 CMI (=0.006 µg/mL) as a positive control. Then, 50 µL volumes of the cultures were spread with a spiral plater (EasySpiral Pro, Interscience, Saint-Nom-la-Bretèche, France) on both MHA plates, and MHA plates supplemented with 8-fold the streptomycin MIC (final concentration: 512 µg/mL) (Sigma-Aldrich, Saint-Quentin Fallavier, France). The plates were incubated at 37 °C for 48 h, and the colonies were counted. Mutant rates were calculated by dividing the number of CFU developing on the streptomycin plates (mutation events) by the number of CFU on antibiotic-free plates [35 (link)]. The values presented are means of 3 independent experiments.
9
Quantification of Microbial Contamination in Bedding
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Samples of 25 g of each bedding were weighed in sterile filter bags (BagFilter P, Interscience, Saint Nom la Bretêche, France) containing 300 mL of Maximum Recovery Diluent (Oxoid, Hampshire, UK) and ground in a stomacher homogeniser (Bag Mixer 400 LW, Interscience, Saint Nom la Bretêche, France). Series of ten-fold dilutions were made from the suspension of bedding after grounding and inoculated directly on the surface of the appropriate selective medium by an automatic spiral plater (easySpiral®Pro, Interscience, Saint Nom la Bretêche, France). Each sample dilution (0.05 mL) was surface inoculated on tryptone soya agar (Oxoid, Hampshire, UK) for a total count of aerobic mesophilic bacteria, Sabouradud’s medium with chloramphenicol (Oxoid, Hampshire, UK) for a total count of yeasts and moulds and incubated at 30 °C for 48–72 h in an air-jacketed incubator (NuAire 5831, NuAire Inc., Plymouth, MN, USA). Counting of colonies was performed with an automatic colony counter Scan 4000 Ultra HD (Interscience, Saint Nom la Bretêche, France). Finally, the results were expressed as the logarithm of the count of Colony Forming Units in one gram of bedding material (log CFU/g).
10
Bacterial Detachment and Enumeration
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For each gas plasma treatment and each time period, seven independent experimentations were performed. Glass slides were submitted to mechanical agitation [1 min of vortex, 1 min in a ultrasonic bath (VWR TH USC 300 THD, 45 KHz), and 1 min of vortex] in 15 mL distilled water to detach bacteria and obtain homogenous suspensions. Both bacterial dilution and seeding on nutrient agar plates (trypto-casein soy agar, Biokar, France) were performed with EasySpiral Pro® (Interscience, France). After 24 h of incubation at 37°C, the colonies on agar plates were counted with the Scan®1200 (Interscience, France). A second count was made after 48 h to verify if new colonies could be visualized.
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