Standard microbiological procedures were used to inoculate samples onto 5% sheep Blood Agar Medium (HiMedia Pvt. Ltd.), Chocolate Agar (HiMedia Pvt. Ltd.), Mannitol Salt Agar (HiMedia Pvt. Ltd.), MacConkey Agar (HiMedia Pvt. Ltd.), and Bile Esculin Agar (HiMedia Laboratory Pvt. Ltd., Mumbai, India). The plates were aerobically incubated at 37°C for 24–72 hours, with the inoculated Chocolate Agar incubated in a candle jar for 72 hours at 35–37°C to provide a 5–10% CO2 concentration. Following a gram stain on each positive culture medium, morphologically identical 3–5 pure colonies of bacteria from overnight incubated agar media were suspended in nutrient broth using sterile wire loop with reference to 0.5 McFarland standards and incubated for up to 4 hours at 37 °C for further processing. Biochemical tests (HiMedia Laboratory Pvt. Ltd., Mumbai, India) like the indole test, methyl red/Voges Proskauer test, coagulase test, oxidase test, triple sugar iron, motility, citrate utilization, urease, gas production, and hydrogen sulfide production were done for Gram-negative bacteria species identification, while catalase and coagulase tests were used for Gram-positive bacteria species identification.23 ,24
Chocolate agar
Manufactured by HiMedia
Sourced in India
Chocolate agar is a type of growth medium used in microbiology laboratories. It is primarily used for the cultivation and isolation of fastidious microorganisms, particularly Neisseria species and Haemophilus influenzae.
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Lab products found in correlation
Macconkey agar, by HiMedia (7 mentions)
Blood agar, by HiMedia (7 mentions)
Sheep blood agar, by HiMedia (3 mentions)
Mannitol salt agar, by HiMedia (2 mentions)
Nutrient agar, by HiMedia (2 mentions)
Potato dextrose agar (pda), by HiMedia (2 mentions)
Tryptic soy agar, by HiMedia (1 mentions)
Amies transport medium, by Thermo Fisher Scientific (1 mentions)
Brain heart infusion broth bhi, by HiMedia (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Condalab (1 mentions)
Macerozyme r 10, by HiMedia (1 mentions)
Mueller hinton, by HiMedia (1 mentions)
Mili q, by Merck Group (1 mentions)
Vitek 2 system, by bioMérieux (1 mentions)
Sabouraud dextrose agar, by HiMedia (1 mentions)
Bhi broth, by HiMedia (1 mentions)
Mrs agar, by Acumedia (1 mentions)
17 protocols using chocolate agar
1
Bacterial Identification from Clinical Samples
2
Throat Swab Collection and Bacterial Culture
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Throat swabs were taken by the attending pediatricians from each patient using a sterile cotton swab. Visible exudates or hyperemic areas on the tonsillar walls were swabbed with a sterile cotton swab, while the tongue was depressed by a wooden spatula when necessary. All swab samples were immediately transported to the Microbiology Department of HGH using Amie's transport medium (Oxoid, England). Swabs were simultaneously plated onto Tryptic Soy Agar (Himedia, India) containing 5% sheep blood, chocolate agar (CA), and MacConkey (MAC) Agar (Himedia, India) and incubated for 48 h at 37°C. chocolate agar was incubated in a candle jar to get 5% CO2, while BA and MAC were incubated under a normal atmosphere.
3
Identification of E. coli and K. pneumoniae
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A total of 5,690 different clinical specimens that included urine (n = 2,710), sputum (n = 1,490), pus (n = 770), blood (n = 512), and body fluids (n = 208) were cultured on different agar media such as nutrient agar, MacConkey agar, cysteine–lactose electrolyte deficient medium, 5% sheep blood agar, chocolate agar, and brain heart infusion broth as well as bile broth (HiMedia, Mumbai, India) depending upon requirements and isolated following standard microbiological techniques [24 ]. The identification of E. coli and K. pneumonia was done by using standard microbiological techniques, which involved studying the colonial morphology, Gram staining, and various biochemical tests (indole, methyl-red, Voges-Proskauer, citrate utilisation, triple sugar iron, oxidase, catalase, oxidative/fermentative, motility, and urease) [24 ].
4
Diagnosis of Keratomycosis via Corneal Scraping
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Corneal scrapings were collected by an ophthalmologist from the patients with suspected keratomycosis at Aravind Eye Hospital and Postgraduate Institute of Ophthalmology (Coimbatore, Tamilnadu, India) during 2013-2015. The collected material was inoculated directly onto 5% sheep blood agar, Chocolate agar, brain heart infusion broth and potato dextrose agar (PDA) (HiMedia, Mumbai, India) and also spread on a glass slide for direct microscopy after 10% KOH wet mount. The Culture plates were incubated at 37 °C (for bacteria) and 27 °C (for fungi), examined daily, and discarded after 1 week if no growth were present. The fungi that were initially identified based on colony morphology on SDA were further characterized microscopically after lactophenol cotton blue staining (Harris, 2000 ). Suspected A. flavus isolates were further screened on Aspergillus differentiation agar (ADA) to differentiate other similar morphological species of Aspergillus genera (Rodrigues et al., 2007 ). All the isolates were stored in screw capped tubes containing 0.85% saline at 4 °C.
5
Aseptic Blood Culture Protocol
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About 1-2 ml of blood was drawn aseptically before starting antimicrobial therapy and directly inoculated into Brain Heart Infusion broth (BHI) (HiMedia, India) in a ratio of blood:BHI of 1:5. The blood culture bottles were immediately sent to the microbiology laboratory and incubated at 37°C for 24 hrs and subcultured on MacConkey agar, blood agar, and chocolate agar (HiMedia, India) daily for 7 days. The inoculated MacConkey agar plates were incubated aerobically, whereas blood agar and chocolate agar plates were incubated in CO2 enriched humid atmosphere using candle jar, at 37°C for 24-48 hours. Blood culture bottles showing no growth on subculture done after incubation of 7 days were reported as negative. All the collected blood samples were processed for culture and isolation by standard microbiological methods [9 ].
6
Nasopharyngeal Swab Sampling and Culture
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The swab was gently introduced along the floor of the nasal cavity, passing under the inferior turbinate until it reached the pharyngeal wall, with the tip of the nose raised. The swab was removed carefully once it had made contact with the pharyngeal wall. The swab was placed in liquid transport media and kept refrigerated at 2–8 °C until it was transported on ice to microbiology laboratory [33 , 34 ]. The nasopharyngeal swab was streaked onto Blood agar (HiMedia™), Chocolate agar, MacConkey agar (HiMedia™) and Mannitol salt agar (HiMedia™). The Chocolate agar was incubated in a candle jar at 37 °C for 24–48 h. Whereas, Blood agar and Mannitol salt agar was aerobically incubated for 24 h at 37 °C. Positive growth on Blood agar and Mannitol salt agar (HiMediaTM) was subculture onto Nutrient agar (HiMediaTM) for biochemical and antimicrobial susceptibility tests [35 , 36 ].
7
Blood Culture Inoculation and Incubation
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Venous blood (10, 2, and 1 mL from adults, children, and neonates, respectively) was aseptically collected. Immediately after collection, blood samples were added to tryptic soy broth (TSB) (Oxoid, Ltd.) in two bottles for each patient at approximately 1 h intervals [13 ] and incubated at 37 oC. If growth was observed, subculturing was performed on MacConkey agar (MAC) (HiMedia, India) and blood agar (BA) (HiMedia, India) plates. For chocolate agar (HiMedia, India) it was incubated in a 5–10% CO2 atmosphere using a candle jar at 37 °C for 24 to 48 h. When no visible growth was detected, the TSB tubes were incubated for 7 days before being reported as negative.
8
Microbiology Lab Processing of Respiratory Samples
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The TUTH laboratory has a full-time microbiology department, which receives all samples from both outpatient and inpatient departments, including the ICU. The specimens are transported to the microbiology laboratory directly after they are collected by ICU staff. Sputum specimens are inoculated onto 5% sheep blood agar, MacConkey agar and chocolate agar (HiMedia, Mumbai, India) and incubated at 35°C for 24–48 h.
9
Isolation and Identification of Fastidious Bacteria
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A loopful of specimen was inoculated immediately within 30 min into blood agar and chocolate agar (Hi Media Laboratories, Pvt. Limited, India) plates and incubated in candle jar (5–10% CO2) at 37 °C for 24 h. As these fastidious bacteria grow well in a humid atmosphere, a dampened paper towel was kept at the bottom of the candle jar. The moisture source was changed regularly to prevent contamination with molds [9 ]. Identification of bacterial isolates was done at National Public Health Laboratory (NPHL), Teku, Kathmandu, Nepal by standard microbiological techniques including observation of colony characteristics, Gram’s staining, catalase, oxidase and other biochemical tests [13 ].
10
Microbial Culture Media Preparation
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Blood agar and chocolate agar were procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India), MacConkey agar from Condalab (Madrid, Spain), and Triple Sugar Iron (TSI) Agar from MAST group Ltd. (Merseyside, UK), and Simmon’s Citrate Agar was obtained from Ltd. (Lancashire, UK). LB agar was prepared in the lab using peptone (Oxford, India), yeast extract (Lab M Limited, UK), NaCl (Biotech, Egypt), and Agar-Agar (B&V Ltd., Italy).
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