Tissue samples were processed according to the following protocol: after arrival at the Department for Infectious Diseases, Medical Microbiology and Hygiene of Heidelberg University, the tissue was ground using a sterile porcelain mortar, followed by the addition of 1 mL of 0.9% NaCl. This suspension was inoculated onto Columbia 5% sheep blood agar (BD life sciences, Heidelberg, Germany), chocolate agar, MacConkey agar, SCS agar, Schaedler Neo Vanco +5% sheep blood (SNVS) agar (all BioMérieux, Marcy, France), and thioglycolate broth (BD life sciences), and then Gram staining was performed. Samples were incubated under aerobic or anaerobic conditions, as appropriate, for 48 h (aerobic microorganisms) or 72 h (anaerobic microorganisms) at 36°C. If growth on plates was detected, identification of microorganisms was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker Daltonics, Bremen, Germany) as described elsewhere.24 (link) Susceptibility testing was performed using VITEK2 (Biomérieux, Nürtingen, Germany) or MIC test strips (Liofilchem, Piane Romano, Italy), respectively, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing clinical breakpoints.
Columbia 5 sheep blood agar
Manufactured by BD
Sourced in Germany
Columbia 5% Sheep Blood Agar is a microbiological growth medium used for the isolation and cultivation of a variety of microorganisms, including fastidious bacteria. It contains 5% defibrinated sheep blood, which provides essential nutrients and growth factors for the growth of many bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Thioglycolate broth, by BD (1 mentions)
Macconkey agar, by bioMérieux (1 mentions)
Scs agar, by bioMérieux (1 mentions)
Vitek 2, by bioMérieux (1 mentions)
Mic test strip, by Liofilchem (1 mentions)
Matrix assisted laser desorption ionization time of flight mass spectrometry, by Bruker (1 mentions)
Chocolate agar, by bioMérieux (1 mentions)
Ir biotyper, by Bruker (1 mentions)
Tryptic soy agar, by bioMérieux (1 mentions)
Sabouraud agar, by BD (1 mentions)
Allplex entero dr assay, by Seegene (1 mentions)
Vitek ms, by bioMérieux (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Bactectm fx system, by BD (1 mentions)
Anaerogen compact, by Thermo Fisher Scientific (1 mentions)
8 protocols using columbia 5 sheep blood agar
1
Microbial Isolation and Identification
2
IR-Biotyper Controls for Pneumococcus
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For all experiments, an IR-Biotyper™ (Bruker Daltonics, Bremen, Germany) was used with a 96-spot silicon microtiter plate. The following controls were included in each run: (a) Bruker Infrared Test Standards (IRTS), each of the two standards was applied twice per target and run (hardware control), and (b) S. pneumoniae ATCC 49619, fresh 24-h culture on Columbia 5% sheep blood agar (BD, Heidelberg Germany), grown at 36 °C in 5%CO2 for 24 h and applied in triplicate (in process control).
3
Microbial Assessment of Air Quality
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Three different media were used to grow a wide spectrum of microorganisms: Columbia 5% Sheep Blood Agar (CAB, Becton Dickinson), Tryptic Soy Agar (TSA) (BIOMÉRIEUX), and Sabouraud Agar (SAB, Becton Dickinson). A microbiological assessment of the air in the control and experimental rooms was performed twice (before and after the experiment), and was based on three replicates of each medium at three measurement points. The TSA plates were incubated for 72 h at 37° C and a further 14 days at 28 °C. Based on the plate analysis of the number of colony-forming units (CFU/m3), the number of microorganisms in 1 m3 of air was determined.
4
Carbapenem Resistance Profiling of Gram-negative Pathogens
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In the period under study Gram-negative bacterial pathogens recovered from clinical specimens were routinely identified via MALDI-TOF MS (VITEK MS, Biomerieux, Marcy-l’Etoile, France) and susceptibility-tested with the VITEK 2 system (Biomerieux, Marcy-l’Etoile, France). Carbapenem-resistant K. pneumoniae and P. aeruginosa isolates, or such with an unusual carbapenem-susceptibility profile (ertapenem/imipenem/meropenem) were routinely analyzed for the presence of common resistance genes, using the Allplex Entero-DR Assay (Seegene, Seoul, South Korea).
All VIM-encoding P. aeruginosa isolates and all NDM- and OXA-48-encoding K. pneumoniae isolates recovered from patients of oncology wards between September 2014 and November 2021 were traced in the laboratory information system, thawed and sub-cultured twice on Columbia 5% sheep blood agar (Becton Dickinson, Heidelberg, Germany) prior to testing and DNA extraction. Only first isolates were selected for each pathogen–patient combination. The environmental strains used for comparison had been isolated between Nov. 2016 and Sept. 2018 [17 (link)]
All VIM-encoding P. aeruginosa isolates and all NDM- and OXA-48-encoding K. pneumoniae isolates recovered from patients of oncology wards between September 2014 and November 2021 were traced in the laboratory information system, thawed and sub-cultured twice on Columbia 5% sheep blood agar (Becton Dickinson, Heidelberg, Germany) prior to testing and DNA extraction. Only first isolates were selected for each pathogen–patient combination. The environmental strains used for comparison had been isolated between Nov. 2016 and Sept. 2018 [17 (link)]
5
Microbial Enumeration in Bedding
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Samples of bedding were transferred to 500 ml of sterile phosphate-buffered saline solution (PBS, BTL) and shacked for 60 min to release the microbes in the solid components of the bedding into the solution. Next, serial dilutions (10^0 – 10^-6) of the solution were made using sterile PBS. Two independent series were prepared for each sample. A 100 µl sample of each dilution was cultivated on Columbia 5% Sheep Blood Agar (CAB, Becton Dickinson) and Sabouraud agar (SAB, Becton Dickinson). CAB plates were incubated for 48 h at 37 °C and then a further 48 h at room temperature. SAB plates were placed at 37 °C for 24 h and transferred to room temperature for the next four days. The resulting colonies were counted using a colony reader (Galaxy 330). Results were expressed as the number of colonies in 1 g of bedding.
6
Cryopreservation and Reactivation of Isolates
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After completion of the routine diagnostic procedures the isolates were cryo-conserved at −78 °C. For this study the isolates were thawed and sub-cultured twice on Columbia 5% sheep blood agar (Becton Dickinson, Heidelberg, Germany) prior to testing.
7
Identification of Bacterial Isolates from Blood Culture
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Two blood culture sets were usually drawn from an antecubital vein. Blood samples were inoculated into both aerobic and anaerobic blood culture bottles and incubated in the BD BACTECTM FX system (BD, Becton Dickinson) for a maximum of 5 days. Positive anaerobic bottles were inoculated onto aerobic and anaerobic blood agar (BD Columbia Agar 5% Sheep Blood, Becton Dickinson, Franklin Lakes, NJ, USA), incubating the plates at 35–37 °C for a maximum of 5 days. Anaerobic plates were incubated in an anaerobic atmosphere generated with the AnaeroGen Compact anaerobic system (Oxoid Ltd, Wide Road, Basingstoke, England) at 35–37 °C.
Identification of all isolates was carried out using MALDI-TOF MS (Bruker Biotyper, Bellerica, MA, USA), following the manufacturer’s recommendations. Only strains clinically relevant and with a log (score) ≥ 2.0 were included and interpreted with high confidence [17 (link)]. Some isolates (n = 5) were also identified by 16S rRNA gene sequencing, when the MALDI-TOF MS score was lower than 2.0.
Identification of all isolates was carried out using MALDI-TOF MS (Bruker Biotyper, Bellerica, MA, USA), following the manufacturer’s recommendations. Only strains clinically relevant and with a log (score) ≥ 2.0 were included and interpreted with high confidence [17 (link)]. Some isolates (n = 5) were also identified by 16S rRNA gene sequencing, when the MALDI-TOF MS score was lower than 2.0.
8
Identification of Clinical Isolates via MALDI-TOF MS
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All clinical samples were inoculated onto aerobic and anaerobic blood agar (BD Columbia Agar 5% Sheep Blood, Becton Dickinson, Franklin Lakes, NY), chocolate agar (BD Choco Agar, Becton Dickinson), mannitol agar (BD Mannitol Salt, Becton Dickinson), Mac Conkey agar (BD Mac Conkey II, Becton Dickinson), and thioglycolate broth (BD Fluid Thioglycolate Medium, Becton Dickinson), incubating all media at 35–37 °C for 5 days. Anaerobic plates were incubated in an anaerobic atmosphere generated with the AnaeroGen Compact anaerobic system (Oxoid Ltd., Wide Road, Basingstoke, England) at 35–37 °C. Identification of all isolates was carried out using MALDI-TOF MS (Bruker Biotyper, Bellerica, MA, USA), following the manufacturer’s recommendations. Only strains with a log (score) ≥2.0 were included and interpreted as high confidence [24 (link)] and identified only with this technique. Two isolates with the lowest log (score) were further identified by 16S rRNA gene sequencing [25 (link)].
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