Sheep blood agar

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Sheep blood agar is a microbiological culture medium used for the isolation and identification of various bacteria. It consists of a base agar supplemented with defibrinated sheep blood. The blood agar supports the growth of fastidious microorganisms and can help differentiate bacteria based on their hemolytic properties.

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Lab products found in correlation

Chocolate agar, by HiMedia (3 mentions) Macconkey agar, by HiMedia (3 mentions) Potato dextrose agar (pda), by HiMedia (1 mentions) Mannitol salt agar, by HiMedia (1 mentions) Macconkey agar, by Thermo Fisher Scientific (1 mentions)

14 protocols using sheep blood agar

Evaluating Anti-Hemolytic Activity of Botanicals

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The effect of
the methanol extract of I. verum and
3-HBA on the β-hemolysis activity of S. aureus was examined. Briefly, the overnight culture of the test S. aureus strain (20 μL) was inoculated into
tryptic soy broth (TSB) (180 μL) with or without the crude extract
of I. verum (2.4 mg/mL) and with or
without 3-HBA (25 μg/mL) and incubated at 37 °C for 18
h. After overnight incubation, the control and the treated samples
were streaked on sheep blood agar (Himedia, Mumbai, India). The plates
were incubated at 37 °C, and the zone of red cell clearance was
observed after 24 h.

Ganesh P.S., Veena K., Senthil R., Iswamy K., Ponmalar E.M., Mariappan V., Girija A.S., Vadivelu J., Nagarajan S., Challabathula D, & Shankar E.M. (2022). Biofilm-Associated Agr and Sar Quorum Sensing Systems of Staphylococcus aureus Are Inhibited by 3-Hydroxybenzoic Acid Derived from Illicium verum. ACS Omega, 7(17), 14653-14665.

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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.

Balakrishnan Sangeetha A., Abdel-hadi A., Hassan A.S., Shobana C.S., Suresh S., Abirami B., Selvam K.P., Al-Baradie R.S., Banawas S., Alaidarous M., Alshehri B., Dukhyil A.A., Dhanasekaran S, & Manikandan P. (2019). Evaluation of in vitro activities of extracellular enzymes from Aspergillus species isolated from corneal ulcer/keratitis. Saudi Journal of Biological Sciences, 27(2), 701-705.

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Surveillance of Urinary VRE Isolates

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In total, 16 non-duplicate urinary VREfm stains were collected through 2016–2020 from three 500–800-bed tertiary hospitals in Thailand. A total of 1,507 urine samples were collected including hospital A (n = 462), hospital B (n = 378), and hospital C (n = 667). Each isolate of VREfm were cultured on sheep blood agar (HiMedia Laboratories Pvt. Ltd., Nashik, India), followed by incubation for 24 h at 37°C. The colonies with typical enterococcal morphological characteristics were first identified based on Gram staining and standard biochemical tests, including arabinose utilization, growth in 6.5% NaCl, bile esculin degradation, and pyrrolidonyl β-naphthylamide (PYR) degradation (Saenhom et al., 2022 (link)). All isolates were confirmed by species-specific multiplex polymerase chain reaction (PCR), enterococcal superoxide dismutase (sodA) gene is the identification marker according to previously described by Jackson et al. (2004) (link). Each isolate was stored in a freezer at −80°C.

Chopjitt P., Boueroy P., Jenjaroenpun P., Wongsurawat T., Hatrongjit R., Kerdsin A, & Sunthamala N. (2024). Genomic characterization of vancomycin-resistant Enterococcus faecium clonal complex 17 isolated from urine in tertiary hospitals in Northeastern Thailand. Frontiers in Microbiology, 14, 1278835.

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Enzymatic Profiling of Strain PAW1

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The proteolytic activity of PAW1 was assessed on gelatin agar and skimmed milk agar medium. Caseinase activity using 1% azocasein (Sigma-Aldrich, Germany) as substrate was determined as reported earlier [19 (link)]. Lipolytic activity of cell free supernatant of PAW1 was determined using p-nitrophenyl palmitate as the substrate [20 (link)]. Haemolysin and siderophore production ability of PAW1 was examined qualitatively by spot inoculating an overnight grown culture on 7% sheep blood agar (HiMedia, India) [21 (link)] and chrome azurol S (CAS) agar [22 (link)] respectively.

Tawre M.S., Kamble E.E., Kumkar S.N., Mulani M.S, & Pardesi K.R. (2021). Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1. PLoS ONE, 16(2), e0246020.

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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.

Ghimire R., Gupte H.A., Shrestha S., Thekkur P., Kharel S., Kattel H.P., Shrestha P.S., Poudel N., Shakya S., Parajuli S., Mudvari A, & Edwards J. (2021). High drug resistance among Gram-negative bacteria in sputum samples from an intensive care unit in Nepal. Public Health Action, 11(Suppl 1), 64-69.

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Isolation and Identification of Listeria monocytogenes

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Twenty-five grams of food sample was homogenized with 9 mL of nutrient broth using a blender. The harvested homogenate was firstly pre-enriched in 225 mL of Buffered Listeria Enrichment Broth with pyruvate and incubated at 30 °C for 48 h, then cultured in specific Oxford medium, CHROM agar and sheep blood agar (Himedia, India) for 48 h at 35 °C as described by FDA BAM and ISO 11290 method [14 (link)]. Characteristic colonies of L. monocytogenes were identified on different agar media. Morphological and biochemical characteristics of the bacteria were analyzed using Gram staining, catalase test, sugar fermentation test and motility test according to FDA BAM and ISO 11290 method [14 (link)]. The CAMP (Christie–Atkins–Munch-Peterson) test was performed as described previously [15 (link)] using standard hemolytic Staphylococcus aureus strain (MT211620), which was streaked on blood agar in a straight line across the center of the plate, then the L. monocytogenes strain was streaked in a direction perpendicular or vertical to S. aureus without touching the S. aureus culture. Then the plates were incubated at 37 °C for 18–24 h and checked for β-hemolysis which appeared as an arrowhead, circle or rectangle shape in CAMP-positive species.

Abdeen E.E., Mousa W.S., Harb O.H., Fath-Elbab G.A., Nooruzzaman M., Gaber A., Alsanie W.F, & Abdeen A. (2021). Prevalence, Antibiogram and Genetic Characterization of Listeria monocytogenes from Food Products in Egypt. Foods, 10(6), 1381.

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Identification of Staphylococcus aureus in Traditional Cheeses of Azerbaijan

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Traditional cheeses in Azerbaijan region are generally produced from goat or sheep's fatty milk with a temperature of 30°C by adding commercial rennet [14 ]. In the present study, one hundred samples of 20 grams were collected from local producers in the rural areas of East Azerbaijan from January through December 2012. For the primary culture, 50 μL of homogenized samples was surface-plated on nutrient agar and incubated at 37°C for 24–48 h under aerobic conditions. Colonies suspected as S. aureus were selected and transferred into individual tubes of nutrient broth. Colonies were sub-cultured on 5% sheep blood agar and mannitol salt agar (Himedia, India). Culture characteristics, Gram staining, and catalase and coagulase test utilizing fresh rabbit plasma were the criteria used for the identification of presumptive isolates. The carbohydrates differential fermentation was also used for confirmation of all coagulase positive (CP) isolates [15 ]. S. aureus ATCC 25923 and Escherichia coli ATCC 35218 served as reference strains for biochemical tests.

Shanehbandi D., Baradaran B., Sadigh-Eteghad S, & Zarredar H. (2014). Occurrence of Methicillin Resistant and Enterotoxigenic Staphylococcus aureus in Traditional Cheeses in the North West of Iran. ISRN Microbiology, 2014, 129580.

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Detecting gimB in E. coli from Animals

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In order to detect the presence and origin of gimB in E.
coli from different animal species, PCR was performed on DNA isolated
from clinical samples of swine, cattle, dogs and cats stored in the LABAC’s
collection, UFSM/RS (Table 1). These samples
were taken between 1990 and 2012 and the isolates were preserved by lyophilization.
The lyophilized samples were plated on 5% sheep-blood agar (Himedia, Mumbai, Índia)
and MacConkey agar (Himedia, Mumbai, Índia). Colonies were confirmed as E.
coli by Gram staining and biochemical characterization (Quinn et al., 1994 ). Subsequently, DNA was
extracted from the confirmed colonies (Cheng and
Jiang, 2006) and was used as a template for PCR assay with primers 6F and
6R (6F: 5′-GCGGGTGCCGATTATATTTC-3′ and 6R: 5′-CTTCGCGCTGCTATTGAA-3′) according to the
conditions described by Matter et
al. (2011). The 6F and 6R primers were designed with the
gimB sequence available (access number AY170898.1) using the
Primer3Plus program. PCR reaction resulted in an amplicon of 724 bp. In order to
verify the DNA quality for PCR, species-specific PCR for the detection of E.
coli was performed as well using the primer pair ECA75F
(5′-GGAAGAAGCTTGCTT CTTTGCTGAC-3′) and ECR619R (5′-AGCCCGGGGAT TTCACATCTGACTTA-3′)
(SABAT et al., 2000). The MT 78 strain was used as a positive
control in all assays (Matter et
al., 2011).

Matter L.B., Spricigo D.A., Tasca C, & de Vargas A.C. (2015). Invasin gimB found in a bovine intestinal Escherichia coli with an adherent and invasive profile. Brazilian Journal of Microbiology, 46(3), 875-878.

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Hemolysis Screening of LAB Isolates

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Sheep blood agar (Himedia Laboratories Pvt, Ltd.) was used for inoculation of selected LAB isolates and incubated for 48 h at 37˚C, and then plates were observed for α, β, or γ hemolysis (22 (link),23 (link)).

Vasudha M., Prashantkumar C.S., Bellurkar M., Kaveeshwar V, & Gayathri D. (2023). Probiotic potential of β‑galactosidase‑producing lactic acid bacteria from fermented milk and their molecular characterization. Biomedical Reports, 18(3), 23.

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Identification and Antimicrobial Susceptibility of Respiratory Pathogens

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The respiratory specimens were cultured on sheep blood agar, MacConkey agar, chocolate agar, and Burkholderia cepacia selective agar (Hi Media, Mumbai, India). Growth was identified by conventional microbiological methods. The test for antimicrobial susceptibility was carried out using the Kirby-Bauer method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute. 13

Sharma G., Lodha R., Shastri S., Saini S., Kapil A., Singla M., Mukherjee A., Jat K.R., Kabra M, & Kabra S.K. (2016). Zinc Supplementation for One Year Among Children with Cystic Fibrosis Does Not Decrease Pulmonary Infection. Respiratory care, 61(1).

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