Biobanked isolates were removed from − 80 °C freezer (Thermo Scientific), thawed and sub-cultured unto three standard growth media: Blood Agar (BA – Columbia agar base supplemented with 5% sheep blood), chocolate agar (CA) and macConkey agar (Mac) (BD, Franklin Lakes, New Jersey, USA) under sterile working condition. All the plates were incubated aerobically overnight at 35 °C–37 °C except for CA plates which was incubated in 5% CO2 for microaerophilic condition.
Chocolate agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States, Germany, China
Chocolate agar is a microbiological culture medium used for the isolation and cultivation of fastidious microorganisms, particularly Haemophilus influenzae and Neisseria species. It is prepared by adding defibrinated blood to a base agar, which gives the medium a characteristic chocolate-brown color. Chocolate agar supports the growth of these organisms by providing necessary growth factors and nutrients.
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Lab products found in correlation
Macconkey agar, by Thermo Fisher Scientific (33 mentions)
Blood agar, by Thermo Fisher Scientific (22 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (9 mentions)
Sheep blood agar, by Thermo Fisher Scientific (6 mentions)
Schaedler agar, by Thermo Fisher Scientific (5 mentions)
Columbia blood agar, by Thermo Fisher Scientific (5 mentions)
Sabouraud agar, by Thermo Fisher Scientific (3 mentions)
Maldi tof ms, by Bruker (3 mentions)
Xylose lysine deoxycholate agar, by Thermo Fisher Scientific (3 mentions)
Pseudomonas selective cetrimide agar, by Thermo Fisher Scientific (2 mentions)
Maldi biotyper, by Bruker (2 mentions)
Nutrient broth, by Thermo Fisher Scientific (2 mentions)
Columbia agar, by Thermo Fisher Scientific (2 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (2 mentions)
Vitek ms, by bioMérieux (2 mentions)
Vitek 2, by bioMérieux (2 mentions)
Api 20ne, by bioMérieux (2 mentions)
Api 20e, by bioMérieux (2 mentions)
Thayer martin agar, by Thermo Fisher Scientific (2 mentions)
Blood agar base, by Thermo Fisher Scientific (2 mentions)
71 protocols using chocolate agar
1
Thawing and Culturing Biobanked Isolates
2
Sputum Collection and Microbiome Analysis
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Sputum samples were collected after patients rinsed their mouths out with sterile water. If spontaneous sputum was not possible, induced sputum was systematically performed according to international recommendations [31 (link), 32 ].
Extended culture analysis was performed on the sputum. After liquefaction by N-acetylcysteine, serial dilutions (1/1000, 1/10,000, and 1/100,000) were made and cultured in Columbia blood agar, chocolate agar, Schaedler agar, and Pseudomonas selective cetrimide agar (Thermo Fisher Scientific, USA), at 37 °C for 48 h for aerobic and 5% CO2 cultures and 5 days for anaerobic cultures. All colonies that appeared to be morphologically distinct were quantified as colony-forming unit (CFU) per milliliter and identified by matrix-associated laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI Biotyper®, Bruker Daltonics, Bremen, Germany). The α-diversity of the airway microbiota was evaluated with the Shannon index (a marker of intra-individual diversity).
Chronic P. aeruginosa infection was defined by the isolation of P. aeruginosa in two or more cultures, at least 3 months apart in a consecutive period of 12 months at a stable state [33 (link)].
Extended culture analysis was performed on the sputum. After liquefaction by N-acetylcysteine, serial dilutions (1/1000, 1/10,000, and 1/100,000) were made and cultured in Columbia blood agar, chocolate agar, Schaedler agar, and Pseudomonas selective cetrimide agar (Thermo Fisher Scientific, USA), at 37 °C for 48 h for aerobic and 5% CO2 cultures and 5 days for anaerobic cultures. All colonies that appeared to be morphologically distinct were quantified as colony-forming unit (CFU) per milliliter and identified by matrix-associated laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI Biotyper®, Bruker Daltonics, Bremen, Germany). The α-diversity of the airway microbiota was evaluated with the Shannon index (a marker of intra-individual diversity).
Chronic P. aeruginosa infection was defined by the isolation of P. aeruginosa in two or more cultures, at least 3 months apart in a consecutive period of 12 months at a stable state [33 (link)].
3
Wound Culture Identification Protocol
4
Monitoring ALA Biofilm Fluorescence
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Experiments were performed to determine the behaviour and timeline of ALA uptake and subsequent biofilm fluorescence. Three biofilms that had been exposed to ALA for the last 24 hours of incubation (“ALA endogenous”) were transferred to a chocolate agar (Thermo Scientific, does not contain ALA) to observe relative decreasing fluorescent signal over time. Three biofilms grown in the absence of ALA were transferred to Porphyrin Test Agar (Remel, Thermo Scientific), which contains ALA, the essential precursor for porphyrin production, to observe the potential uptake of ALA over time (“ALA exogenous”). As a negative control, three ALA negative biofilms were transferred onto chocolate agar to confirm no fluorescence induction. All were imaged with the MolecuLight i:X device (standard and fluorescent modes) at 24, 48, 72, and 96 hours.
5
Acute Pharyngotonsillitis Microbiological Analysis
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A 39-year-old man presented with acute pharyngotonsillitis accompanied by a fever up to 39.8 °C. His throat swabs were cultured on blood agar and chocolate agar (Thermo Fisher Scientific, Prague, Czech Republic) and the isolates were identified by standard bacteriological procedures [27 ] and MALDI-TOF mass spectrometry (Microflex LT, Bruker Daltonics, Bremen, Germany). Antimicrobial susceptibilities were determined by the disc diffusion method (discs from Oxoid, Brno, Czech Republic) and the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) procedures and criteria [28 ]. The blaTEM-1 gene was detected by PCR followed by digestion of 600 bp amplicon with MboI (New England Biolabs, Frankfurt am Main, Germany) as described previously [29 (link)].
6
Characterization of NTHI Strain 86-028NP
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For routine culturing, strains were grown on chocolate agar (Thermo Fisher Scientific, Waltham, MA). All growth was performed at 37°C in 5% CO2. For routine liquid culture, NTHI was grown in BHI supplemented with 2 μg/mL of NAD and 2 μg/mL of heme (sBHI). NTHI strain 86–028NP is a minimally passaged, fully sequenced, clinical isolate that has been extensively characterized in the chinchilla model of human otitis media (Harrison et al., 2005 (link); Mason et al., 2006 (link); Sirakova et al., 1994 (link)). During the course of the study, the input strain of 86–029NP was subjected to whole genome sequencing for comparison with the adapted strains. The 86–028NP input strain displayed the expected sequence from prior studies.
7
Pancreatic Fluid Microbial Analysis
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A drop of pancreatic fluid was taken for smear microscopy. A 50 μL sample from each pancreatic fluid sample was then inoculated onto the plates of blood agar, chocolate agar, and MacConkey agar (Thermo Fisher Scientific Inc., Shanghai, China), respectively. The plates were incubated at 35°C in a 5% CO2 atmosphere and observed after 72 h. Several colonies were selected by observation through the naked eye. Gram staining was performed based on the standard operation protocol of our laboratory, and biochemical identification was performed using VITEK® 2 COMPACT (bioMérieux, Marcy-l’Étoile, France). Anaerobic culturing was not performed because the samples from surgery or drainage did not guarantee an anaerobic environment.
8
Profiling Airway Microbiota Diversity
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Endobronchial samples (bronchial aspiration or BALF, n = 15) were collected, and extended microbiological culture was performed, as previously described [26 (link),27 (link)]. The samples and their dilutions (1/1.000 for bronchial aspiration) were cultured in Columbia blood agar, chocolate agar, Schaedler agar, and Pseudomonas selective cetrimide agar (Thermo Fisher Scientific, Waltham, MA, USA), at 37 ℃ for 48 h for aerobic and 5% CO2 cultures and 5 days for anaerobic cultures. All colonies that appeared to be morphologically distinct were quantified as colony-forming unit (CFU) per mL and identified using MALDI-TOF mass spectrometry (MALDI Biotyper®, Bruker Daltonics, Billerica, MA, USA). The α-diversity of the airway microbiota was evaluated with the Shannon index (a marker of intra-individual diversity).
9
Characterization of NTHI Strain 86-028NP
10
Diagnosing Infective Endocarditis through Blood Culturing
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A 63-year-old male presented to Qilu Hospital at Shandong University, Jinan, China, on April 26, 2016 with intermittent fever, fatigue, and chest distress for the previous 10 months. Ultrasonic cardiogram revealed infective endocarditis with valvular disease. Blood samples were sent to the microbiology laboratory for culturing on April 30, 2016. The blood cultures were incubated in the Bactec system (Becton Dickinson, Franklin Lakes, NJ) until a positive result was obtained. The positive blood cultures were inoculated onto Columbia blood agar, MacConkey agar, and Chocolate agar and incubated (Thermo Fisher Scientific, USA) at 35 °C until visible colonies appeared. Colonies were purified using blood agar for further analysis.
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