Antimicrobial discs

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Antimicrobial discs are a type of laboratory equipment used to test the susceptibility of microorganisms to various antimicrobial agents. These discs are impregnated with specific concentrations of antimicrobial compounds and are placed on a culture medium inoculated with the target microorganism. The resulting zone of inhibition around the disc indicates the sensitivity or resistance of the microorganism to the tested antimicrobial agent.

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Antimicrobial susceptibility test discs (13 citations) Antimicrobial susceptibility discs (4 citations) Antimicrobial impregnated discs (3 citations) Oxoid™ Antimicrobial Susceptibility Discs (3 citations) Standard antimicrobial discs (2 citations) Antimicrobial agent discs (2 citations)

28 protocols using antimicrobial discs

Antibiotic Susceptibility Profiling of K. pneumoniae

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Detection of antibiotic susceptibility profiles of K. pneumoniae isolates was executed by Kirby Bauer disc diffusion procedure [24] and interpreted as per the Clinical and Laboratory Standards Institute (CLSI) recommendations [25] . For examination of K. pneumoniae isolates susceptibility, subculturing of the preserved isolates in brain heart infusion (BHI) broth (Oxoid, UK) with glycerol was performed onto MacConkey's agar and the inoculated plates were incubated at 37°C for 24 h. After that, spreading of the prepared 0.5 MacFarland bacterial suspension onto Mueller Hinton agar (MHA) (Oxoid, UK) plates was carried out by a sterile cotton swab and then the antimicrobial discs (Oxoid, UK) were placed onto those plates. After incubation of the plates at 37°C for 24 h, the inhibition zone around each antimicrobial disc was measured. The used antimicrobial discs (Oxoid, UK) were ampicillin (AMP, 10 µg), amoxicillin/ clavulanate (AMC, 20/10 µg), amikacin (AK, 30 µg), azithromycin (AZM, 15 µg), aztreonam (ATM, 30 µg), cefepime (FEP, 30 µg), chloramphenicol (C, 30 µg), ciprofloxacin (CIP, 5 µg), imipenem (IPM, 10 µg), nalidixic acid (NA, 30 µg), tetracycline (TE, 30 µg) and trimethoprim/ sulphamethoxazole (SXT, 1.25/23.75µg).
Determination of multiple antibiotic resistance (MAR) index [26] MAR index for the antimicrobial = MAR index for the isolate =

Gomaa N. (2021). Prevalence, antimicrobial resistance, and biofilm formation of Klebsiella pneumoniae isolated from human and cows. Zagazig Veterinary Journal/Zagazig Veterinary Journal (Online), 49(1), 27-41.

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Antibiotic Susceptibility Testing Protocol

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Antibiotic susceptibility testing was performed as per prior study protocols [15 (link), 32 (link)]. Colonies from a 24-h incubation on Columbia Blood Agar (CBA) were suspended in 0.85% saline solution to a 0.5 McFarland standard and spread on the Mueller-Hinton Blood Agar (MHBA) plates. The following antimicrobial discs (from Oxoid, Nepean, Ontario) were used: penicillin G (P, 10 U), ceftriaxone (CRO, 30 μg), ceftazidime (CAZ, 30 μg), azithromycin (AZM, 15 μg), erythromycin (E, 15 μg), clindamycin (CDA, 2 μg), and levofloxacin (LEV, 15 μg). After a 24-h incubation, zones of clearance were measured and resistance was assigned in accordance to breakpoints as provided by the Clinical and Laboratory Standards Institute (CLSI) [33 ]. The “D-test” for inducible clindamycin resistance was performed.

Skolnik K., Nguyen A., Thornton C.S., Waddell B., Williamson T., Rabin H.R, & Parkins M.D. (2017). Group B streptococcus (GBS) is an important pathogen in human disease- but what about in cystic fibrosis?. BMC Infectious Diseases, 17, 660.

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Antimicrobial Susceptibility Testing by Disk Diffusion

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Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method as described by the Clinical Laboratory Standards Institute [15 (link)]. In brief, the pure culture was transferred to a tube containing 5 ml of sterile normal saline (0.85% NaCl) and mixed gently until it formed a homogeneous suspension. The turbidity of the suspension was adjusted to an optical density equivalent to 0.5 McFarland standards. A sterile cotton swab was then dipped into the suspension and the excess was removed by gentle rotation of the swab against the surface of the tube. The swab was distributed evenly over the entire surface of the Mueller–Hinton agar (Oxoid, UK). The inoculated plates were left at room temperature to dry for 3 to 5 minutes. Six antimicrobial discs (Oxoid, UK) such as ampicillin (10 μg), trimethoprim-sulfamethoxazole (23.75/1.25 μg), ciprofloxacin (5 μg), gentamicin (10 μg), ceftriaxone (30 μg), and Ddxycycllin (30 μg) were placed aseptically on the inoculated plate using sterile forceps. After 24 hours of incubation at 37°C, the zone of inhibition including disk was measured by using a digital caliper to the nearest whole millimeters and interpreted as sensitive, intermediate, or resistant based on interpretive breakpoints [15 (link)].

Gemechu T., Eshetu T., Kassa T, & Jarso H. (2022). Assessment of Intestinal Parasites, Enteric Bacterial Infections, and Antimicrobial Susceptibility among Street Food Handlers in Jimma Town, Southwest Ethiopia. Journal of Tropical Medicine, 2022, 5483367.

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Antimicrobial Activity of B. amyloliquefaciens ST34 Extract

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The susceptibility of the reference, clinical and environmental bacterial strains to the crude extract (10.00 mg/mL) obtained from the B. amyloliquefaciens ST34 strain was determined using the method outlined by Ndlovu et al. [27 ]. Briefly, the lyophilised ST34 crude extract was dissolved in 15% (v/v) methanol to obtain a concentration of 10.00 mg/mL. Antimicrobial discs (6 mm; Oxoid, USA) were placed on the MHA after the test isolates were spread plated onto the media and were impregnated with 50 μL of the surfactin crude extract (with approximately 9.98 μg surfactin in the crude extract). For each assay, a surfactin negative and positive control was included in the experiment for each of the test organisms. The negative control consisted of the test microorganism spread plated onto MHA (Merck) with 15% (v/v) methanol impregnated filter paper discs, while the positive control consisted of the reference test organisms [AB 1, EC 1, KP 1 and PA 1] spread plated onto MHA with commercial surfactin (1.00 mg/mL) (Sigma, USA) impregnated filter paper discs added on top of the media. All the agar plates were incubated for 24–48 h at 37 °C, whereafter the diameters of the zones of inhibition around the inoculated paper discs were measured and recorded.

Havenga B., Ndlovu T., Clements T., Reyneke B., Waso M, & Khan W. (2019). Exploring the antimicrobial resistance profiles of WHO critical priority list bacterial strains. BMC Microbiology, 19, 303.

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Antimicrobial Susceptibility Testing Protocol

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Antimicrobial susceptibility testing was performed using the disc diffusion method as described by the Clinical Laboratory Standards Institute [14 ]. In brief, the pure culture was transferred to a tube containing 5 ml sterile normal saline (0.85% NaCl) and mixed gently until it formed a homogeneous suspension. The turbidity of the suspension was adjusted to the optical density equivalent to 0.5 McFarland. A sterile cotton swab was then dipped into the suspension and the excess was removed by gentle rotation of the swab against the surface of the tube. The swab was distributed evenly over the entire surface of Mueller-Hinton agar (Oxoid, Ltd., UK).
The inoculated plates were left at room temperature to dry for 3 to 5 minutes. The antimicrobial discs (Oxoid, Ltd., UK) representative of the penicillin group (Amoxicillin 10 μg, Augmentin 30 μg, and Ampicillin 10 μg), Cephalosporin (Ceftriaxone 30 μg), aminoglycosides (Gentamycin 10 μg), Maxolid (Erythromycin 15 μg), Lincosamide (Clindamycin 2 μg), Sulfonamide (Cotrimoxazole 25 μg, and Tetracycline 10 μg were placed on the inoculated plates and incubated at 37°C for 18–24 hours.

Edae M., Teklemariam Z., Weldegebreal F, & Abate D. (2020). Asymptomatic Bacteriuria among Pregnant Women Attending Antenatal Care at Hiwot Fana Specialized University Hospital, Harar, Eastern Ethiopia: Magnitude, Associated Factors, and Antimicrobial Susceptibility Pattern. International Journal of Microbiology, 2020, 1763931.

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Antimicrobial Susceptibility Testing of APEC

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The susceptibility of isolates to 10 different antimicrobial agents was determined by disc diffusion assay as described by Clinical and Laboratory Standards Institute (CLSI) [7 ]. According to the measurement of inhibition zones, tested strains were evaluated as susceptible or resistant. The following antimicrobial discs (Oxoid, Basingstoke, UK) were used: gentamicin (GN, 10 μg), kanamycin (KAN, 30 μg), ampicillin (AMP, 10 μg), cefotaxime (CTX, 30 μg), nalidixic acid (NA, 30 μg), ciprofloxacin (CIP, 5 μg), tetracycline (TET, 30 μg), sulfamethoxazole-trimethoprim (STX, 25 μg), chloramphenicol (C, 30 μg), and streptomycin (S, 10 μg). The isolates were defined as multidrug-resistant APEC if they exhibited resistance to at least one agent in three or more antimicrobial categories [8 (link)]. Escherichia coli strain ATCC 25922 was used as a reference strain for susceptibility test. All cefotaxime-resistant isolates were confirmed for the production of extended-spectrum beta-lactamase (ESβL) by using the double-disk synergy test (DDST) following recommendation and interpretations of CLSI standards [7 ].

Awad A., Arafat N, & Elhadidy M. (2016). Genetic elements associated with antimicrobial resistance among avian pathogenic Escherichia coli. Annals of Clinical Microbiology and Antimicrobials, 15, 59.

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Antibiotic Susceptibility Profiling of Klebsiella pneumoniae

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The Kirby-Bauer disk diffusion method was conducted for all 230 K. pneumoniae isolates using Mueller-Hinton agar and the results were recorded in accordance with CLSI 201516 guidelines. The used antimicrobial discs (Oxoid; UK) included beta-lactams as ampicillin (10µg), amoxicillin (10µg), amoxicillin/clavulanic acid (20/10µg), ampicillin/sulbactam (10/10µg), ticarcillin/clavulanic acid (75/10µg), piperacillin/tazobactam (100/10µg), meropenem (10µg), imipenem (10µg), cefoxitin (30µg), cefotetan (30µg), cefazolin (30µg), cefuroxime (30µg), ceftriaxone (30µg), cefotaxime (30µg), ceftazidime (30µg), cefepime (30µg), aztreonam (30µg); aminoglycosides as gentamicin (10µg), tobramycin (10µg), amikacin (30µg) and other antimicrobials including chloramphenicol (30µg), ciprofloxacin (5µg), tetracycline (30µg) and trimethoprim-sulfamethoxazole (1.25/23.75µg),. Susceptibility to tigecycline was detected using Vitek2® compact system (BioMérieux®). Minimum inhibitory concentrations (MICs) were detected for all CR-KP isolates using Vitek2® compact system (BioMérieux®). Any isolate would be recorded to have MDR profile if it showed non-susceptibility to ≥3 different antimicrobial classes4 .

Ahmed El-Domany R., El-Banna T., Sonbol F, & Abu-Sayedahmed S.H. (2021). Co-existence of NDM-1 and OXA-48 genes in Carbapenem Resistant Klebsiella pneumoniae clinical isolates in Kafrelsheikh, Egypt. African Health Sciences, 21(2), 489-496.

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Antimicrobial Resistance Profiling of Salmonella and E. coli

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Antimicrobial susceptibility studies were carried out on the bacterial isolates using commercially available antimicrobial discs (Oxoid) by the Kirby Bauer disc diffusion technique. Lauria-Bartani (LB) broth (Himedia, Mumbai, India) containing 0.5 McFarland (MF) turbidity (0.14–0.17 OD₆₀₀) of bacterial culture was swabbed on Mueller Hinton agar plates. antimicrobial discs were placed on prepared plates about 20 mm apart, inhibition zone diameter was measured after 16–18 h incubation at 37 °C, and the results were interpreted as per Clinical and Laboratory Standard Institute guidelines [38 (link)]. antimicrobial discs from seven major antimicrobial groups were included for phenotypic susceptibility testing as given in Table 1.

Table 1

Phenotypic antimicrobial resistance profiles of clinical isolates used in current study

Sr. No.	Antimicrobials	Code-Disc potency (μg)	S. typhimurium	S. typhi	E. coli.
1	Sulfamethoxazole /Trimethoprim	SXT-23.75/1.25	R	R	R
2	Nalidixic acid	NA-30	R	R	R
3	Ampicillin	AMP-10	S	S	R
4	Chloramphenicol	C-30	S	S	R
5	Aztreonam	ATM-30	IR	IR	IR
6	Amoxicillin/ Clavulanic acid	AMC-30	IR	S	IR
7	Gentamicin	GEN-10	S	R	R
8	Ceftriaxone	CRO-30	IR	IR	R
9	Ciprofloxacin	CIP-5	R	S	R

R Resistant: IR Intermediate resistant: S Sensitive

Kiran S., Tariq A., Iqbal S., Naseem Z., Siddique W., Jabeen S., Bashir R., Hussain A., Rahman M., Habib F.E., Rauf W., Ali A., Sarwar Y., Jander G, & Iqbal M. (2024). Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae. BMC Complementary Medicine and Therapies, 24, 93.

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Antibiotic Susceptibility Testing of CoNS

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An inoculum of CoNS isolates, adjusted to an optical density of 0.5 in McFarland scale, were swabbed onto the Mueller–Hinton agar (Merck, Darmstadt, Germany), and antimicrobial discs (Oxoid, Basingstoke, UK) were placed on the plate, incubated for 24 h at 37 °C. The inhibition zone was interpreted according to the Clinical Laboratory Standards Institute (CLSI, 2020) guidelines. The tested antibiotics were: penicillin (P–10U)—beta-lactam antibiotic, cefoxitin (FOX-30 μg)—beta-lactam antibiotic, gentamicin (CN-10 μg)—aminoglycosides, erythromycin (E-15 μg)—macrolides, tetracycline (TE-30 μg)—tetracyclines, ciprofloxacin (CIP-5 μg)—fluoroquinolones, nitrofurantoin (F-300 μg)—nitrofurantoins, clindamycin (DA-2 μg)—lincosamides, trimethoprim/sulfamethoxazole (SXT-1.25/23.75 μg)—sulfonamides, chloramphenicol (C-30 μg)—phenicols, rifampin (RD-5 μg)—ansamycins, quinupristin/dalfopristin (QD-15 μg)—streptogramins, linezolid (LZD-30 μg)—oxazolidinones and fusidic acid (FD-10 μg)—fusydates. Staphylococcus aureus ATCC 25923 reference strain was used for antimicrobial disc control.

Chajęcka-Wierzchowska W., Gajewska J., Zadernowska A., Randazzo C.L, & Caggia C. (2023). A Comprehensive Study on Antibiotic Resistance among Coagulase-Negative Staphylococci (CoNS) Strains Isolated from Ready-to-Eat Food Served in Bars and Restaurants. Foods, 12(3), 514.

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Antimicrobial Susceptibility Testing of Bacteria

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Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method on Muller-Hinton agar (MHA) plates following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) using commercially available antimicrobial discs (Oxoid, UK). Twenty-one antimicrobial discs were used in this study: norfloxacin (NOR, 10 μg), enrofloxacin (ENR, 5 μg), levofloxacin (LEV, 5 μg), ciprofloxacin (CIP, 5 μg), ofloxacin (OFX, 5 μg), penicillin G (P, 10 μg), ampicillin (AMP, 10 μg), amoxicillin/clavulanic acid (AMC, 30 μg), cephalothin (KF, 30 μg), cephazolin (KZ, 30 μg), cefoxitin (FOX, 30 μg), ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), cefepime (FEP, 30 μg), imipenem (IPM, 10 μg), meropenem (MEM, 10 μg), chloramphenicol (C, 30 μg), tetracycline (TE, 30 μg), streptomycin (S, 10 μg), gentamicin (CN, 10 μg), and amikacin (AK, 30 μg). E. coli ATCC 25922 was used as a control strain. The standard of antibiotic susceptibility for CLSI were list in Additional file 5: Table S5.

Zhu Z., Shi Y., Zhou X., Li B, & Zhang J. (2018). Molecular characterization of fluoroquinolone and/or cephalosporin resistance in Shigella sonnei isolates from yaks. BMC Veterinary Research, 14, 177.

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