Macconkey agar

Manufactured by bioMérieux

Sourced in France, United States, Germany, Italy

MacConkey agar is a selective and differential culture medium used in microbiology laboratories for the isolation and identification of Gram-negative enteric bacteria, particularly members of the Enterobacteriaceae family. It is designed to inhibit the growth of Gram-positive bacteria while allowing the growth of Gram-negative bacteria.

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45 protocols using macconkey agar

Isolation and Identification of E. coli from Fecal and Chicken Samples

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For each fecal sample, buffered peptone water (BPW) was added up to the 45 ml mark of the 50 ml Falcon tube and vortexed. One (1 ml) of the mixture was transferred to 9 ml of freshly prepared tetrathionate broth and incubated at 37°C for 24 hours. After 24 hours, a 10 ul loopful of incubated tetrathionate broth was streaked on MacConkey agar (Oxiod).
For each chicken sample, 25 g was weighed and placed in a sterile empty wide mouth container with screw cap followed by the addition of 9 parts (225 ml) of buffered peptone water (BPW) and incubated at 35°C for 24 hours. A loopful of BPW and sample mixture were streaked on MacConkey agar and incubated at 37°C for 24 hours.
Well isolated pinkish colonies on MacConkey agar, typical of E. coli were sub cultured on fresh MacConkey agar plates and identified using the Analytical Profile Index (API 20E, BioMérieux, France) isolate identification according to established protocol. All isolates were further confirmed using Matrix- Assisted Laser Desorption- Ionization Time of Flight Mass Spectrometry (MALDITOF-MS).

Mensah G.I., Adjei V.Y., Vicar E.K., Atsu P.S., Blavo D.L., Johnson S.A, & Addo K.K. (2022). Safety of Retailed Poultry: Analysis of Antibiotic Resistance in Escherichia coli From Raw Chicken and Poultry Fecal Matter From Selected Farms and Retail Outlets in Accra, Ghana. Microbiology Insights, 15, 11786361221093278.

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Bacterial Isolation from Hospital Surfaces

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Four samples collected during week six by swabbing and collecting water samples from the pipes, basin, and sink drain at the neonatal ward were also included in the study. Samples from hard surfaces were obtained with a cotton swab moistened with a sterile saline solution. The swabs were then inoculated in brain-heart infusion broth (Tec-Laim, Madrid, Spain), vortexed for 30 s, incubated overnight at 37 °C, and plated on MacConkey agar (Biomérieux, Marcy L’etoile, France). Water samples were obtained by first extracting water using a sterile urinary catheter and syringe. The water was then centrifuged at 3000 rpm for 5 min, and the precipitate was inoculated in brain-heart infusion broth, incubated overnight at 37 °C, and plated on MacConkey agar (Biomérieux, Marcy L’etoile, France).

Dahdouh E., Lázaro-Perona F., Ruiz-Carrascoso G., Sánchez García L., Saenz de Pipaón M, & Mingorance J. (2021). Intestinal Dominance by Serratia marcescens and Serratia ureilytica among Neonates in the Setting of an Outbreak. Microorganisms, 9(11), 2271.

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Microbial Culture and Identification from Specimens

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Specimens were placed in separate sterile containers and sterile normal saline was added to cover the sample completely. After vortexing for 30s, the sample was sonicated by use of an ultrasound bath (VWR, Milan, Italy) for 5 min at a frequency of 30 kHz and vortexed for another 30s [78 (link)]. The outside of the container was treated with 70% ethanol. The resulting sonication fluid was centrifuged at 4000 rpm for 15 min, and the sediment was used for subsequent microbiological cultures onto chocolate agar, Columbia blood agar, Mannitol salt agar, MacConkey agar, Sabouraud agar and Schaedler agar plates (Biomerieux, Marcy-l’Etoile, France) and inoculated into Brain Heart Infusion and Thyoglycollate broths (TermoFhisher, Cornaredo, Italy). Anaerobic and aerobic agar plates were incubated at 37 °C for 5 days while broths were incubated for 15 days in the same conditions. Microorganisms were identified using conventional methods.

Di Domenico E.G., Rimoldi S.G., Cavallo I., D’Agosto G., Trento E., Cagnoni G., Palazzin A., Pagani C., Romeri F., De Vecchi E., Schiavini M., Secchi D., Antona C., Rizzardini G., Dichirico R.B., Toma L., Kovacs D., Cardinali G., Gallo M.T., Gismondo M.R, & Ensoli F. (2019). Microbial biofilm correlates with an increased antibiotic tolerance and poor therapeutic outcome in infective endocarditis. BMC Microbiology, 19, 228.

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Comprehensive Microbiological Analysis of Implant Infections

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To conduct microbiological analysis, at least three dry swabs (MASTASWAB TM, Mast Group Ltd., Bootle, UK) were taken directly from the removed implant, the interface, and from macroscopically suspicious areas of the wound. The swabs were streaked out on Columbia agar with 5% sheep blood, chocolate agar, MacConkey agar, and thioglycolate broth (bioMerieux, Hazelwood, MO, USA). Samples were then incubated at 37 °C in 5% CO₂ or anaerobic conditions for 48 h for short-term culturing; morphologically distinct colonies were identified and antibiotic susceptibility to 28 antibiotics was determined using the Vitek2-machine (bioMerieux, Hazelwood, MO, USA) with standardized definition of minimum inhibitory concentration (MIC) and multi-drug resistance [14 (link)]. At least two tissue samples from the interface, non-union, or macroscopically suspicious areas were directly inserted into a sterile containment prefilled with 9 ml of thioglycolate broth (bioMerieux, Hazelwood, MO, USA). After incubation at 37 °C in 5% CO₂ or under anaerobic conditions for at least 14 days (long-term culturing), the suspension was additionally streaked out and proceeded as described above.

Kellermann F., Hackl S., Leister I., Hungerer S., Militz M., Stuby F., Holzmann B, & Friederichs J. (2023). Advances in the Treatment of Implant-Associated Infections of the Pelvis: Eradication Rates, Recurrence of Infection, and Outcome. Journal of Clinical Medicine, 12(8), 2854.

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Sonication-Based Microbial Isolation and Identification

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Clinical specimens were collected in sterile containers and then transferred to a microbiology laboratory for immediate processing. The samples were processed by sonication according to the methodology previously described by our group (44 (link)). In summary, 50 mL of phosphate saline buffer was added, and the tubes were vortexed and sonicated in an Ultrasons-H 3000840 low-power bath sonicator (J.P. Selecta, Barcelona, Spain) at 22°C for 5 min, followed by an additional centrifugation. After centrifugation, the resulting pellet was cultured in different growth agar plates: tryptic soy agar with 5% sheep blood, chocolate agar, MacConkey agar, Schaedler agar with 5% sheep blood, and Sabouraud-chloramphenicol agar, all from bioMérieux (Marcy-l’Étoile, France) and incubated at 37°C and 5% CO₂ for at least 24 h and up to 7 days, with the exception of Sabouraud-chloramphenicol agar, which was maintained at 30°C for 4 weeks. All of the microorganisms isolated were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (VitekMS, bioMérieux, Marcy-l’Étoile, France) and subsequently frozen and stored at –80°C.

Macias-Valcayo A., Aguilera-Correa J.J., Broncano A., Parron R., Auñon A., Garcia-Cañete J., Blanco A, & Esteban J. (2022). Comparative In Vitro Study of Biofilm Formation and Antimicrobial Susceptibility in Gram-Negative Bacilli Isolated from Prosthetic Joint Infections. Microbiology Spectrum, 10(4), e00851-22.

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Microbiological Analysis of Clinical Samples

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We analyzed samples according to standard microbiological procedures, as previously described, depending on the specimen (9 (link)–12 (link)). This process included systematic inoculation onto Columbia agar with 5% sheep blood (BioMérieux, Craponne, France), chocolate agar (BioMérieux) (excluding urine and fecal samples), and specific media such as colistin-nalidixic agar or MacConkey agar (both BioMérieux) for specimens potentially contaminated by resident flora. Blood cultures were incubated into a Bactec device (Becton Dickinson, Le Pont de Claix, France) and analyzed as previously described (9 (link)).

Dubourg G., Baron S., Cadoret F., Couderc C., Fournier P.E., Lagier J.C, & Raoult D. (2018). From Culturomics to Clinical Microbiology and Forward. Emerging Infectious Diseases, 24(9), 1683-1690.

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Standardized Microbial Infection Detection

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Tissue samples were processed according to the standardized setup of our microbiological department. Positive evidence for bacterial infection of the bone defect was defined if obligate pathogen bacteria were detected or ≥2 independent tissue samples were positive for the same facultative pathogen bacteria (eg, Staphylococcus epidermidis or Corynebacterium spp., which are known as commensals of the skin).21 (link) Tissue was further processed and cultivated in our microbiology department as previously described.22 (link) Samples were spread onto porcelain mortar, followed by the addition of 1 mL of 0.9% NaCl. This suspension was inoculated onto Columbia 5% sheep blood agar, chocolate agar, MacConkey agar, streptococcus-selective agar, Schaedler Neo Vanco +5% sheep blood agar (all Bio-Mérieux, Marcy-l’Étoile, France), and thioglycolate broth (BD Diagnostic Systems, San Jose, CA, USA), and then Gram staining was performed. Plates and broth were incubated until positive or for a maximum of 5 days at 36°C in 5% CO₂ or under anaerobic conditions. Identification of bacteria was done with a matrix-assisted laser desorption/ionization time-of-fight mass spectrometer (Microfex; Bruker Corporation, Billerica, MA, USA). Susceptibility testing was performed using a Vitek 2 microbial identification system (BioMérieux).22 (link)

Helbig L., Bechberger M., Aldeeri R., Ivanova A., Haubruck P., Miska M., Schmidmaier G, & Omlor G.W. (2018). Initial peri- and postoperative antibiotic treatment of infected nonunions: results from 212 consecutive patients after mean follow-up of 34 months. Therapeutics and Clinical Risk Management, 14, 59-67.

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Characterizing Lung Microbiome in Mice

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BAL was performed in 3-week-old mice under sterile conditions. Mice were deeply anesthetized via intraperitoneal injection with a combination of ketamine and xylazine (120 mg/kg and 16 mg/kg, respectively) and killed by exsanguination. A cannula was inserted into the trachea and whole lungs were lavaged 3 times with 300 μL PBS. The recovered BAL fluid was plated on columbia blood agar (Becton Dickinson, Heidelberg, Germany), chocolate agar, Mac Conkey agar, prereduced Schaedler agar and kanamycin-vancomycin blood agar plates (bioMérieux, Nürtingen, Germany). After 48 h of incubation at 37 °C, colony forming units were counted and classified by MALDI-TOF mass-spectrometry (Bruker Daltonik, Bremen, Germany). Then, 16S rRNA PCR was performed to detect non-culturable bacterial species [63 (link)].

Leitz D.H., Duerr J., Mulugeta S., Seyhan Agircan A., Zimmermann S., Kawabe H., Dalpke A.H., Beers M.F, & Mall M.A. (2021). Congenital Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Alveolitis and Pulmonary Fibrosis in Neonatal Mice. International Journal of Molecular Sciences, 22(11), 6146.

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Identification of Bacterial Pathogens

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To identify the bacterial pathogens, Columbia agar supplemented with 5% sheep blood and MacConkey agar (bioMérieux, France) were used. Both media were inoculated with 100 μL of milk. Plates were incubated at 37 °C for 48 h. To identify the bacteria species, VITEK 2 equipment was used (bioMérieux).

Reczyńska D., Zalewska M., Czopowicz M., Kaba J., Zwierzchowski L, & Bagnicka E. (2018). Small ruminant lentivirus infection influences expression of acute phase proteins and cathelicidin genes in milk somatic cells and peripheral blood leukocytes of dairy goats. Veterinary Research, 49, 113.

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Endotracheal Tube Microbiome Analysis

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ETTs were removed (extubation) by medical staff, immediately placed in 500 mL sterile glass bottles (Schott, Germany), and processed at the Microbiology Laboratory of the University Hospital Fundación Santa Fe. 10 cm from the distal part of each tube were cut and placed in 0.85% NaCl. Associated bacteria were dislodged using three vortex cycles (30 s) followed by sonication (60 s).
The suspension obtained from each ETT was used to recover microorganisms on blood agar (bioMérieux, France), MacConkey agar (bioMérieux, France) and Chocolate agar (bioMérieux, France), incubating at 37 °C for 18–24 h [14 ]. Bacterial identification was carried out employing the VITEK® 2 automated system (bioMérieux, France). All samples were handled by the same person, stored at 4 °C for 2–4 h prior to further analyses, taking care to minimize variability associated with sample handling.

Cifuentes E.A., Sierra M.A., Yepes A.F., Baldión A.M., Rojas J.A., Álvarez-Moreno C.A., Anzola J.M., Zambrano M.M, & Huertas M.G. (2022). Endotracheal tube microbiome in hospitalized patients defined largely by hospital environment. Respiratory Research, 23, 168.

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