Kirby-Bauer Disk Diffusion Test

Total Page：16

File Type：pdf, Size：1020Kb

Lab Protocol Updated: October 4th, 2016 iGEM Stockholm 2016 Kirby-Bauer Disk Diffusion Test Aim Preparing plates with bacterial inoculum and adding filter papers soaked in samples to assess whether the samples have a bactericidal or bacteriostatic function. Procedure 1. Prepare sterile Whatman filter paper with a hole punch and also antibiotic free agar plates. For better visualisation MacConkey or Endo Agar can be used. Up to 6 samples or controls can be tested on one plate. 2. Streak 50 µl of a bacterial culture with approximately OD600 0.6 onto the plates. After streaking in one direction, rotate the plate by 90◦ and repeat the spreading (without applying more liquid culture) until the plate has completed a full turn. This step is essential to assure even distribution of the bacteria! 3. Do not turn the plates upside down as usual, instead let dry at RT while preparing samples. 4. When preparing samples, make sure to include proper positive and negative controls. For cell lysates, a good negative control is lysate from untransformed cells as well as sterile water. Kanamycin at 35 ug/ml (1:1000 dilution from stock) serves as a positive control. 5. Use tweezers to place filter papers onto an empty petri dish and pipet 20 µl of sample / control onto each paper. Let soak for a bit before ’dragging’ the papers out of the liquid and dabbing them onto an empty part of the dish to remove excess liquid. 6. Place filter papers onto agar, making sure to use proper sterile technique. 7. Let the plates dry as in step (3) or place them in the incubator lid facing up if drying at RT takes too long. 8. Check your plates after 12 h (ideally) and 24 h for bacterial growth and to check for halos. Take pictures of the halos and measure their radius. Note! It is very important to work with sterile equipment and reagents in this case to assure that no con- taminants influence bacterial growth. Sources http://www.globe-network.org/sites/default/files/sites/default/files/documents/disk-diffusion-susceptibilities- en_0.pdf retrieved 25.09.2016) http://www.antimicrobialresistance.dk/data/images/protocols/sop_dd_2010gfnlab002_v2.pdf retrieved 25.09.2016) https://www.addl.purdue.edu/newsletters/1997/spring/dds.shtml retrieved 25.09.2016) Page 1 of 1.

Copy Link

Recommended publications

Antibiotic Susceptibility of Bacterial Strains Causing Asymptomatic Bacteriuria in Pregnancy: a Cross- Sectional Study in Harare, Zimbabwe
MOJ Immunology Antibiotic Susceptibility of Bacterial Strains causing Asymptomatic Bacteriuria in Pregnancy: A Cross- Sectional Study in Harare, Zimbabwe Abstract Research Article Background and objective antibiotic susceptibility pattern: Effective among treatmentisolated bacterial of asymptomatic species among bacteriuria pregnant in Volume 6 Issue 1 - 2018 pregnancy requires susceptible drugs. The aim of this study was to determine womenMaterials with and asymptomatic Methods bacteriuria. : This study was conducted at 4 selected primary health 1Department of Nursing Science, University of Zimbabwe, care facilities in Harare, including pregnant women registering for antenatal Zimbabwe care at gestation between 6 and 22 weeks and without urinary tract infection 2Department of Medical Microbiology, University of Zimbabwe, symptoms. Asymptomatic bacteriuria was diagnosed by culture test of all Zimbabwe 3 midstream urine samples following screening by Griess nitrate test. Susceptibility Department of Obstetrics and Gynaecology, University of Zimbabwe, Zimbabwe test was done for all positive 24 hour old culture using the disk diffusion test. The resistant and intermediate. 4Institute of Clinical Medicine, University of Oslo, Norway minimum inhibitory concentration was measured and categorized as susceptible, Results *Corresponding author: : Tested antibiotics included gentamycin (88.2%), ceftriaxone (70.6%), Department of Nursing Science,Judith Mazoe Musona Street, Rukweza, PO Box nitrofurantoin (76.5%), ciprofloxacin (82.4%), ampicillin (67.6%) and norfloxacin University of Zimbabwe, College of Health Sciences, (61.8%). Prevalence of asymptomatic bacteriuria was 14.2% (95% CI, 10.28% to 19.22%). Coagulase negative staphylococcus was the most popular (29.4%) A198, Harare, Zimbabwe, Tel: 00263773917910; Email: bacteria followed by Escherichia coli (23.5%). Gentamycin (83.3%), ciprofloxacin Received: | Published: (75%) and ceftriaxone (70.8) overally had the highest sensitivity.
[Show full text]
Research Article Integrating Bacterial Identification and Susceptibility
Hindawi BioMed Research International Volume 2019, Article ID 8041746, 6 pages https://doi.org/10.1155/2019/8041746 Research Article Integrating Bacterial Identification and Susceptibility Testing: A Simple and Rapid Approach to Reduce the Turnaround Time in the Management of Blood Cultures Dariane C. Pereira1 and Luciano Z. Goldani 2 1Microbiology Unit, Laboratory Diagnosis Service, Hospital de Cl´ınicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil 2InfectiousDiseases Unit, Hospital de Cl´ınicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Correspondence should be addressed to Luciano Z. Goldani; [email protected] Received 1 June 2019; Revised 15 August 2019; Accepted 16 September 2019; Published 3 October 2019 Academic Editor: Jiangke Yang Copyright © 2019 Dariane C. Pereira and Luciano Z. Goldani. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We evaluated a rapid bacterial identification (rID) and a rapid antimicrobial susceptibility testing by disk diffusion (rAST) from positive blood culture to overcome the limitations of the conventional methods and reduce the turnaround time in bloodstream infection diagnostics. -e study included hemocultures flagged as positive by bacT/ALERT , identification by MALDI-TOF MS, and rAST. -e results were compared to identification and antimicrobial susceptibility testing (AST)® results by current standard methods, after 24 h incubation. For rAST categorical agreement (CA), very major errors (VME), major errors (ME), and minor errors (mE) were calculated. A total of 524 bacterial samples isolated from blood cultures were obtained, including 246 Gram-negative (GN) and 278 Gram-positive (GP) aerobes.
[Show full text]
Central Asian and European Surveillance of Antimicrobial Resistance
Central Asian and European Surveillance of Antimicrobial Resistance CAESAR Manual Version 3.0 2019 Central Asian and European Surveillance of Antimicrobial Resistance CAESAR Manual Version 3, 2019 Abstract This manual is an update of the first edition published in 2015 and describes the objectives, methods and organization of the Central Asian and European Surveillance of Antimicrobial Resistance (CAESAR) network. It details steps involved for a country or area wanting to enrol in CAESAR, as well as steps involved in routine data collection for antimicrobial resistance (AMR) surveillance. It contains the protocols and AMR case definitions used by the network. Key updates involve the addition of Salmonella species to the list of pathogens under surveillance, as well as updates related to the European Committee on Antimicrobial Susceptibility Testing categories. CAESAR continues to coordinate closely with the European Antimicrobial Resistance Surveillance Network (EARS-Net) and strives for compatibility and comparability with EARS-NET, as well as the Global AMR Surveillance System coordinated by WHO headquarters. Keywords DRUG RESISTANCE, MICROBIAL ANTI-INFECTIVE AGENTS INFECTION CONTROL POPULATION SURVEILLANCE DATA COLLECTION Address requests about publications of the WHO Regional Office for Europe to: Publications WHO Regional Office for Europe UN City, Marmorvej 51 DK-2100 Copenhagen Ø, Denmark Alternatively, complete an online request form for documentation, health information, or for permission to quote or translate, on the Regional Office website (http://www.euro.who.int/pubrequest). © World Health Organization 2019 All rights reserved. The Regional Office for Europe of the World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full.
[Show full text]
The Genus Staphylococcus 19 171
43038_CH19_0171.qxd 1/3/07 3:53 PM Page 171 THE GENUS STAPHYLOCOCCUS 19 171 The Genus 19 Staphylococcus embers of the genus Staphylococcus are gram-positive spherical organisms about 1 micrometer in diameter. They M occur singly, in pairs, and in irregular clusters, and form yel- low, orange, or white colonies on agar media. They are salt tolerant and grow on ordinary bacteriological media as well as on the selective media used in this exercise. Up to three species of Staphylococcus are studied in this exercise. Cer- tain strains of Staphylococcus aureus are the cause of food poisoning and toxic shock syndrome. They also are the cause of boils and carbuncles. A second species, S. epidermidis, usually is a saprobe of the skin that is rarely involved in human infection. The third species, S. saprophyticus, is an opportunistic species that may cause urinary tract infections in women of childbearing years. In this exercise, staphylococcal species will be isolated from the body’s environment and their properties examined. A. Isolation of Staphylococci Species of Staphylococcus are tolerant to salt and, therefore, they can be PURPOSE: to isolate and selected out from a mixture of bacteria in a high-salt medium. In addition, identify staphylococcal species from the nasal cavity S. aureus ferments mannitol, an alcoholic derivative of the hexose mannose, and other environments. while S. epidermidis and S. saprophyticus do not. Therefore, if the differential medium contains mannitol, the two species may be differentiated from one another. In this section, we will use mannitol salt agar, a medium that is both selective and differential.
[Show full text]
Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation
City University of New York (CUNY) CUNY Academic Works Open Educational Resources Queensborough Community College 2016 Laboratory Exercises in Microbiology: Discovering the Unseen World Through Hands-On Investigation Joan Petersen CUNY Queensborough Community College Susan McLaughlin CUNY Queensborough Community College How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/qb_oers/16 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation By Dr. Susan McLaughlin & Dr. Joan Petersen Queensborough Community College Laboratory Exercises in Microbiology: Discovering the Unseen World through Hands-On Investigation Table of Contents Preface………………………………………………………………………………………i Acknowledgments…………………………………………………………………………..ii Microbiology Lab Safety Instructions…………………………………………………...... iii Lab 1. Introduction to Microscopy and Diversity of Cell Types……………………......... 1 Lab 2. Introduction to Aseptic Techniques and Growth Media………………………...... 19 Lab 3. Preparation of Bacterial Smears and Introduction to Staining…………………...... 37 Lab 4. Acid fast and Endospore Staining……………………………………………......... 49 Lab 5. Metabolic Activities of Bacteria…………………………………………….…....... 59 Lab 6. Dichotomous Keys……………………………………………………………......... 77 Lab 7. The Effect of Physical Factors on Microbial Growth……………………………... 85 Lab 8. Chemical Control of Microbial Growth—Disinfectants and Antibiotics…………. 99 Lab 9. The Microbiology of Milk and Food………………………………………………. 111 Lab 10. The Eukaryotes………………………………………………………………........ 123 Lab 11. Clinical Microbiology I; Anaerobic pathogens; Vectors of Infectious Disease….. 141 Lab 12. Clinical Microbiology II—Immunology and the Biolog System………………… 153 Lab 13. Putting it all Together: Case Studies in Microbiology…………………………… 163 Appendix I.
[Show full text]
(IQC) Antimicrobial Susceptibility Tests Using Disk Diffusion
Internal Quality Control (IQC) Antimicrobial Susceptibility Tests Using Disk Diffusion National AMR Surveillance Network, NCDC National Programme on Containment of Antimicrobial Resistance National Centre for Disease Control, New Delhi April 2019 CONTENTS I. Scope .......................................................................................................................................................... 2 II. Selection of Strains for Quality Control ..................................................................................................... 2 III. Maintenance and Testing of QC Strains..................................................................................................... 3 Figure 1: Flow Chart: Maintenance of QC strains in a bacteriology lab......................................................... 4 IV. Quality Control (QC) Results—Documentation - Zone Diameter ............................................................. 5 V. QC Conversion Plan ................................................................................................................................... 5 1. The 20- or 30-Day Plan .......................................................................................................................... 5 2. The 15-Replicate (3× 5 Day) Plan ......................................................................................................... 5 3. Implementing Weekly Quality Control Testing ..................................................................................... 6 4.
[Show full text]
A Ketoconazole Susceptibility Test for Malassezia Pachydermatis Using Modified Leeming–Notman Agar
Journal of Fungi Article A Ketoconazole Susceptibility Test for Malassezia pachydermatis Using Modified Leeming–Notman Agar Bo-Young Hsieh 1,2, Wei-Hsun Chao 3, Yi-Jing Xue 2 and Jyh-Mirn Lai 2,* 1 Lugu Township Administration, Nanto County 55844, Taiwan; [email protected] 2 College of Veterinary Medicine, National Chiayi University, No. 580, XinMin Rd., Chiayi City 60054, Taiwan; [email protected] 3 Department of Hospitality Management, WuFung University, Chiayi City 60054, Taiwan; [email protected] * Correspondence: [email protected]; Tel.: +886-5-2732920; Fax: +886-5-2732917 Received: 18 September 2018; Accepted: 14 November 2018; Published: 16 November 2018 Abstract: The aim of this study was to establish a ketoconazole susceptibility test for Malassezia pachydermatis using modified Leeming–Notman agar (mLNA). The susceptibilities of 33 M. pachydermatis isolates obtained by modified CLSI M27-A3 method were compared with the results by disk diffusion method, which used different concentrations of ketoconazole on 6 mm diameter paper disks. Results showed that 93.9% (31/33) of the minimum inhibitory concentration (MIC) values obtained from both methods were similar (consistent with two methods within 2 dilutions). M. pachydermatis BCRC 21676 and Candida parapsilosis ATCC 22019 were used to verify the results obtained from the disk diffusion and modified CLSI M27-A3 tests, and they were found to be consistent. Therefore, the current study concludes that this new novel test—using different concentrations of reagents on cartridge disks to detect MIC values against ketoconazole—can be a cost-effective, time-efficient, and less technically demanding alternative to existing methods.
[Show full text]
LAB 2: Staining and Streaking Protocols for Simple Stain, Gram Stain, Streak Plate Technique and Culture Maintenance
LAB 2: Staining and Streaking Protocols for Simple stain, Gram Stain, Streak Plate Technique and Culture Maintenance Lab 2a: INTRODUCTION To Staining Live specimens are difficult to see with the bright field microscope. The contrast between a cell, which is primarily water, and the background, which is water, is poor. Staining is used to increase contrast and can be employed to provide information about the chemistry of a specimen. Stains, or dyes, are salts in which one of the ions is colored. In a basic stain, the color is in the positively charged ion. In an acidic stain the color is in the negatively charged ion. Bacterial surfaces have a slight negative charge. Thus, there is an affinity between a positively charged color ion and the negatively charged bacterial cell. In the Direct or Positive Staining Procedure a cell takes up a positively charged dye and becomes stained. Methylene blue, crystal violet, and safranin, are all basic dyes. In the Indirect or Negative Staining Procedure, a cell is immersed in a negatively charged dye. As the cell will repel the dye, the cell appears clear in a background of color. Nigrosine is an example of an acidic stain. Staining procedures that use one dye to increase contrast between specimen and background are simple staining procedures. Complex staining procedures employ a series of stains and chemical reagents to increase contrast and reveal information about the specimen. Any staining procedure that allows differentiation of one type of bacterium from another is a differential staining procedure. For all staining procedures, it is first required that cells be fixed to the slide.
[Show full text]
Rapid Identification and Antimicrobial Susceptibility Testing of Positive
Rapid identification and antimicrobial susceptibility testing of positive blood cultures using MALDI-TOF MS and a modification of the standardised disc diffusion test: a pilot study. Item Type Article Authors Fitzgerald, C;Stapleton, P;Phelan, E;Mulhare, P;Carey, B;Hickey, M;Lynch, B;Doyle, M Citation Rapid identification and antimicrobial susceptibility testing of positive blood cultures using MALDI-TOF MS and a modification of the standardised disc diffusion test: a pilot study. 2016 J. Clin. Pathol. DOI 10.1136/jclinpath-2015-203436 Publisher BMJ Journal Journal of clinical pathology Rights Archived with thanks to Journal of clinical pathology Download date 26/09/2021 22:03:45 Link to Item http://hdl.handle.net/10147/620889 Find this and similar works at - http://www.lenus.ie/hse Rapid Identification and Antimicrobial Susceptibility testing of Positive Blood Cultures using MALDI-TOF MS and a modification of the standardized disk diffusion test - a pilot study. Fitzgerald C1, Stapleton P1, Phelan E1 , Mulhare P1, Carey B1, Hickey M1, Lynch B1, Doyle M1. 1Microbiology Laboratory, University Hospital Waterford, Dunmore road, Waterford, Ireland Corresponding author: [email protected] Telephone: 00353-51-842488 Fax: 00353-51-848566 1 Keywords: blood culture, MALDI-TOF MS, rapid identification, rapid antimicrobial susceptibility testing, disk diffusion, clinical impact Word count: 3,000 Abstract Aims In an era when clinical microbiology laboratories are under increasing financial pressure, there is a need for inexpensive, yet effective, rapid microbiology tests. The aim of this study was to evaluate a novel modification of standard methodology for the identification and antimicrobial susceptibility testing (AST) of pathogens in positive blood cultures, reducing the turnaround time of laboratory results by 24 hours Methods 277 positive blood cultures had a gram stain performed, were o subcultured and incubated at 37 C in a CO2 atmosphere for 4 to 6 hours.
[Show full text]
Medical Bacteriology
LECTURE NOTES Degree and Diploma Programs For Environmental Health Students Medical Bacteriology Abilo Tadesse, Meseret Alem University of Gondar In collaboration with the Ethiopia Public Health Training Initiative, The Carter Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education September 2006 Funded under USAID Cooperative Agreement No. 663-A-00-00-0358-00. Produced in collaboration with the Ethiopia Public Health Training Initiative, The Carter Center, the Ethiopia Ministry of Health, and the Ethiopia Ministry of Education. Important Guidelines for Printing and Photocopying Limited permission is granted free of charge to print or photocopy all pages of this publication for educational, not-for-profit use by health care workers, students or faculty. All copies must retain all author credits and copyright notices included in the original document. Under no circumstances is it permissible to sell or distribute on a commercial basis, or to claim authorship of, copies of material reproduced from this publication. ©2006 by Abilo Tadesse, Meseret Alem All rights reserved. Except as expressly provided above, no part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission of the author or authors. This material is intended for educational use only by practicing health care workers or students and faculty in a health care field. PREFACE Text book on Medical Bacteriology for Medical Laboratory Technology students are not available as need, so this lecture note will alleviate the acute shortage of text books and reference materials on medical bacteriology.
[Show full text]
1815-IJBCS-Article-Sylvester Okorondu
Available online at http://ajol.info/index.php/ijbcs Int. J. Biol. Chem. Sci. 7(4): 1668-1677, August 2013 ISSN 1991-8631 Original Paper http://indexmedicus.afro.who.int Prevalence and antibiotic sensitivity profile of urinary tract infection pathogens among pregnant and non pregnant women S. I. OKORONDU 1* , C. O. AKUJOBI 1, C. B. NNADI 1, S. O. ANYADO-NWADIKE 2 et M. M. O. OKORONDU 3 1Department of Microbiology, Federal University of Technology Owerri, P.M.B. 1526, Owerri, Nigeria. 2Department of Biotechnology, Federal University of Technology Owerri, P.M.B. 1526, Owerri, Nigeria. 3Department of Biochemistry, Federal University of Technology Owerri, P.M.B. 1526, Owerri, Nigeria. * Corresponding author; E-mail: [email protected] ABSTRACT The prevalence and antibiotic sensitivity profile of urinary tract infection isolates from 100 pregnant women attending antenatal clinic in Owerri General Hospital, Nigeria was assessed. The prevalence of UTI isolates from the pregnant women was compared with that in non-pregnant women. The organisms isolated include: Escherichia coli, Staphylococcus aureus , Coagulase negative Staphylococcus, Klebsiella spp , Pseudomonas spp, Proteus spp and Streptococcus spp. Antibiotic sensitivity pattern of the isolates were also determined using disk diffusion test. One hundred (100) women were tested; 40% had bacteriuria as against 31% in non-pregnant women. The most sensitive isolate was E. coli, while the least was Streptococcus spp. The most effective antibiotics were Gentamycin, Tarivid and Ciprofloxacin, while the least occurred with Chloramphenicol, Ampicillin, Septrin, Ampiclox. Improvement on personal hygiene and diagnostic screening and treatment will help to reduce the prevalence of bacteriuria in pregnancy.
[Show full text]
Use of Susceptibility Testing in Veterinary Medicine
Use of Susceptibility Testing in Veterinary Medicine Peter D. Constable, BVSc, MS, PhD, DACVIM Department of Veterinary Clinical Medicine, University of Illinois, Urbana, IL 61 802 Abstract three categories, susceptible (sensitive), intermediate and resistant, using recommendations from the National There has been increased interest in optimizing Committee for Clinical Laboratory Standards (NCCLS) treatment protocols for antimicrobial agents, with sub for testing Veterinary Pathogens.4 The intermediate stantial reliance on susceptibility testing of bacterial term indicates an MIC value that is close to the pathogens isolated from diseased cattle. Antimicrobial breakpoint.5 susceptibility testing of bovine bacterial pathogens has The broth dilution method provides a direct mea traditionally used the agar diffusion (Kirby-Bauer) surement of MIC, and determines the ability of the method, which was designed to reflect the antibiotic pathogen to grow in the presence of a known antibiotic concentration in serum and interstitial fluid of human concentration. The broth dilution test is usually per patients. The validity of agar diffusion susceptibility formed as a commercially available microdilution test breakpoints derived from humans to the treatment of (Sensititre, Westlake, OH) in a 96 well microtiter plate mastitis, diarrhea and respiratory disease in cattle has that permits the testing of 12 antibiotics in a range of not been established. The use of susceptibility testing eight 2-fold dilutions.5 The microdilution method starts to guide treatment decisions for individual cattle is not by using a sterile loop to remove 3-5 representative colo recommended until the breakpoints have been validated nies from a 24-hour bacterial culture plate (use of mul as being predictive of treatment outcome.
[Show full text]