Haemophilus influenzae

Manufactured by American Type Culture Collection

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Haemophilus influenzae is a type of bacteria that can be used in laboratory equipment and research. It is a Gram-negative, pleomorphic, non-spore-forming coccobacillus.

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10 protocols using haemophilus influenzae

Antimicrobial Susceptibility Testing Protocol

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Broth microdilution minimum inhibitory concentration (MIC) testing was performed in 96-well microtiter plates according to Clinical Laboratory Standards Institute guideline M7-A7.¹⁷ MIC values were determined for E. coli (ATCC 25922), E. coli tolC mutant (W4573:tolC::Tn10), Enterococcus faecalis (ATCC 29212), Haemophilus influenzae (ATCC 49766), P. aeruginosa (ATCC 47085), P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain (ATCC 35151), Staphylococcus aureus (ATCC 29213), and Streptococcus pneumonia (ATCC 49619).
Time-kill studies were performed according to the CLSI document M26-A¹⁸ using S. aureus and the P. aeruginosa hypersensitive strain for compounds BT02D04, BT08E04, and BT09C11, and S. aureus and H. influenzae for compound BT02C02 based on the MIC assay results. Growth media was haemophilus test medium for H. influenzae and brain heart infusion for P. aeruginosa and S. aureus (Becton, Dickinson and Company, Franklin Lakes, NJ). The same growth media was used in MIC and time-kill studies.

Hu Y., Palmer S.O., Robles S.T., Resto T., Dean F.B, & Bullard J.M. (2017). Identification of Chemical Compounds That Inhibit the Function of Histidyl-tRNA Synthetase from Pseudomonas aeruginosa. SLAS discovery : advancing life sciences R & D, 23(1), 65-75.

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Bacterial Strain Cultivation and Assay

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The following four bacterial standard strains from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used: Haemophilus influenzae ATCC 49247, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Streptococcus pyogenes ATCC 19615. Cultivation and assay media (broth/agar) were Mueller–Hinton (MH) complemented by Haemophilus Tested Medium (H. influenzae), MH (S. aureus), and Brain Heart Infusion (S. pneumoniae and S. pyogenes). The pH of the broths was equilibrated to a final value of 7.6 using Trizma base (Sigma-Aldrich, Praha, Czech Republic). All microbial strains and cultivation media were purchased from Oxoid (Basingstoke, UK).
Stock cultures of bacterial strains were cultivated in broth medium at 37 °C for 24 h prior to testing. For the preparation of inoculum, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard using a Densi-La-Meter II (Lachema, Brno, Czech Republic) to obtain a final concentration of 10⁸ CFU/mL.

Houdkova M., Chaure A., Doskocil I., Havlik J, & Kokoska L. (2021). New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro. Molecules, 26(14), 4179.

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Antibiotic Susceptibility Profiling of Bacterial Strains

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Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method performed in 96-well microtiter plates according to the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guideline M7-A7.⁶ MIC values were determined for E. coli (ATCC 25922), E. coli tolC mutant (W4573:tolC::Tn10), Enterococcus faecalis (ATCC 29212), Haemophilus influenzae (ATCC 49766), P. aeruginosa (ATCC 47085), P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain (ATCC 35151), Staphylococcus aureus (ATCC 29213), and Streptococcus pneumoniae (ATCC 49619).
Time-kill studies were performed using H. influenzae and S. aureus as previously described,^{7 (link)} according to CLSI document M26-A. Growth media was Haemophilus test medium for H. influenzae and brain heart infusion for S. aureus (Becton, Dickinson and Company, Franklin Lakes, NJ). These same growth media were used in MIC and time-kill studies.

Corona A., Palmer S.O., Zamacona R., Mendez B., Dean F.B, & Bullard J.M. (2017). Discovery and Characterization of Chemical Compounds That Inhibit the Function of Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa. SLAS discovery : advancing life sciences R & D, 23(3), 294-301.

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Antibiotic Susceptibility Profiling

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Broth microdilution minimum inhibitory concentration (MIC) testing was performed in 96-well microtiter plates according to Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guideline M7-A7.¹⁴ MIC values were determined for E. coli (ATCC 25922), E. coli tolC mutant, Enterococcus faecalis (ATCC 29212), Haemophilus influenzae (ATCC 49766), Moraxella catarrhalis (ATCC 25238), P. aeruginosa (ATCC 47085), P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain (ATCC 35151), Staphylococcus aureus (ATCC 29213), and Streptococcus pneumonia (ATCC 49619).
Time-kill studies were performed using E. faecalis and S. pneumoniae for compound BT_03F04 and S. aureus and S. pneumoniae for compound BT_04B09 based on the MIC assay results, according to CLSI document M26-A.¹⁵ Growth media was Brain Heart Infusion and Trypticase Soy Broth from Remel (Lenexa, KS) with or without 3% lysed horse blood (Hemostat Laboratories, Dixon, CA).

Hu Y., Guerrero E., Keniry M., Manrrique J, & Bullard J.M. (2015). Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa. Journal of biomolecular screening, 20(9), 1160-1170.

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Microbiological Susceptibility Testing

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Broth microdilution MIC testing was carried out in 96-well microtiter plates according to Clinical Laboratory Standards Institute guideline M7-A7 [26 ]. MIC values were determined for E. coli (ATCC 25922), E. coli tolC mutant, Enterococcus faecalis (ATCC 29212), Haemophilus influenzae (ATCC 49766), Moraxella catarrhalis (ATCC 25238), P. aeruginosa (ATCC 47085), P. aeruginosa PAO200 (efflux pump mutant), P. aeruginosa hypersensitive strain (ATCC 35151), S. aureus (ATCC 29213), and S. pneumonia (ATCC 49619) from the American Type Culture Collection (Manassas, VA). For quality control (QC) of MIC determination and culture purity, MICs were determined for antibiotics specific for each bacterial strain. The antibiotic and concentration range for each bacteria was: E. coli (ampicillin, 0.125-128 μg/ml), E. faecalis (vancomycin 0.125-128 μg/ml), H. influenzae (ampicillin, 0.03125-32 μg/ml), M. catarrhalis (ampicillin, 0.015625-16 μg/ml), P. aeruginosa (ampicillin, 0.125-128 μg/ml), S. aureus (oxacillin, 0.03125-32 μg/ml), S. pneumoniae (penicillin, 0.0625-64 μg/ml).

Hu Y., Palmer S.O., Munoz H, & Bullard J.M. (2015). High Throughput Screen Identifies Natural Product Inhibitor of Phenylalanyl-tRNA Synthetase from Pseudomonas aeruginosa and Streptococcus pneumoniae. Current drug discovery technologies, 11(4), 279-292.

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Nephrotoxicity Assay with Kidney Cell Lines

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Human embryonic kidney (HEK293) cells and Madin-Darby Canine Kidney (MDCKII) cells were selected to perform the nephrotoxicity assay. Each cell line was cultured in DMEM medium containing 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified incubator containing 5% CO₂. HEK293 and MDCKII cells were grown as an adherent monolayer. Target bacterial strains, Escherichia coli (ATCC 25922, ATCC 4157, ATCC 12435, and ATCC 10798), P. aeruginosa (ATCC 27853), Haemophilus influenzae (ATCC 49247), Staphylococcus epidermidis (ATCC 12228), Sta. aureus (ATCC 12600), Enterococcus faecalis (ATCC 29212), and Streptococcus pneumoniae (ATCC 49619), were purchased from ATCC. All bacterial strains were grown in LB medium at 37°C in an incubator, with shaking at 200 rpm.

Kong J., Wu Z.X., Wei L., Chen Z.S, & Yoganathan S. (2020). Exploration of Antibiotic Activity of Aminoglycosides, in Particular Ribostamycin Alone and in Combination With Ethylenediaminetetraacetic Acid Against Pathogenic Bacteria. Frontiers in Microbiology, 11, 1718.

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Preparation of Bacterial and Parasitic Lysates

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Ehrlichia chaffeensis (ATCC 1455VR/Lot 1 W), Bartonella henselae (ATCC 49882/1436056), Bartonella elizabethae (ATCC 499271/1192868), Bartonella quintana (ATCC 51694/026125), Bartonella clarridgeiae (ATCC 51734/1284170), Staphylococcus aureus (ATCC 25923/1958041), Escherichia coli (ATCC 25922/1980205), Pseudomonas aeruginosa (ATCC 27853/1906403), Haemophilus influenzae (ATCC 19418/1574755), Staphylococcus bovis (ATCC 1165750), Listeria monocytogenes (ATCC 191115/20122438), Plasmodium falciparum (ATCC 30932/SF 2195) and Babesia microti infected hamster cells (AS 101901) as frozen cells were purchased from American Type Culture Collection (ATCC). The frozen cells were washed twice in PBS, followed by proteinase-K treatment at 60 °C for 1 h. Proteinase K was then inactivated by heating at 100 °C for 10 min. The processed cell lysate was diluted serially in TE-glycogen (10 mM Tris, 1 mM EDTA and 1% glycogen, pH 8.0).

Shah J.S., D’ Cruz I., Ward S., Harris N.S, & Ramasamy R. (2017). Development of a sensitive PCR-dot blot assay to supplement serological tests for diagnosing Lyme disease. European Journal of Clinical Microbiology & Infectious Diseases, 37(4), 701-709.

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Culturing Diverse Bacterial Species

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Bacteria, including Azoarcus oleivorans (ATCC2411), Aeromicrobium fastidiosum (ATCC12713), Acidovorax avenae (12530), Capnocytophaga canimorsus (ATCC35979), Fusobacterium nucleatum (ATCC25586), Haemophilus influenzae (ATCC51907), Neisseria meningitidis (ATCC13077), Streptococcus pneumoniae (ATCC6303), Selenomonas noxia (ATCC43541), and Veillonella parvula (ATCC 17742), were purchased from American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and cultured as previously described [4 (link),12 (link)].

Zhou H., Liao J., Leng Q., Chinthalapally M., Dhilipkannah P, & Jiang F. (2023). Circulating Bacterial DNA as Plasma Biomarkers for Lung Cancer Early Detection. Microorganisms, 11(3), 582.

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Assessing Specificity of COVID-19 Test Kit

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A total of 27 organisms were obtained and tested to evaluate the specificity of the kit. Human coronavirus 229E, human coronavirus NL63, MERS-coronavirus (irradiated), adenovirus, human metapneumovirus (hMPV), human parainfluenza virus 1, human parainfluenza virus 2, human parainfluenza virus 3, human parainfluenza virus 4a, human parainfluenza virus 4b, influenza B, enterovirus, and respiratory syncytial virus A were from BEI resource, and human coronavirus OC43, influenza A, Rhinovirus, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes, pooled human nasal wash, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pnuemophila, Staphylococcus aureus, Staphylococcus epidermidis, and Candida albicans were obtained from ATCC. Three replicates of each organism stock were tested.

Ma L., Fan Y., Kong X., Jiang Y., Huang H., Zhao M., Liu Y., Gao P., Cui Y, & Cao Y. (2023). Fast and Sensitive Detection of SARS-CoV-2 Nucleic Acid Using a Rapid Detection System Free of RNA Extraction. International Journal of Analytical Chemistry, 2023, 8053524.

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Antimicrobial Activity Evaluation

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The bacterial (n=13) and fungal reference strains (n=2) included methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA-44, Escherichia coli ATCC 25922, Enterococcus faecium ATCC 35667, Streptococcuspyogenes ATCC 19615, Pseudomonas aeruginosa ATCC 27853, Streptococcus mutans ATCC 25175, Haemophilus influenzae ATCC 10211, Streptococcus pneumoniae ATCC 49619, Streptococcus sanguinis ATCC 10556, Klebsiella pneumoniae ATCC BAA-2146, Streptococcus agalactiae ATCC 27956, Staphylococcus epidermidis ATCC 12228, Candida albicans ATCC 10231 and Candida glabrata ATCC15126. Strains were grown on Tryptone Soya Agar (TSA) plates at 37ºC under aerobic conditions.

Tan E.L, & Johari N.H. (2021). Comparative in vitro evaluation of the antimicrobial activities of povidone-iodine and other commercially available antiseptics against clinically relevant pathogens. GMS Hygiene and Infection Control, 16, Doc05.

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