MALDI-TOF MS) Test Procedure
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
Catalase Test: Lab-3380
Standard Operating Procedure Subject Catalase Test Index Number Lab-3380 Section Laboratory Subsection Microbiology Category Departmental Contact Sarah Stoner Last Revised 9/18/2019 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS) and/or COLA. Applicable To Employees of Gundersen Health System Laboratory, Gundersen Tri-County, Gundersen St. Joseph, Gundersen Boscobel Hospital, and Gundersen Palmer Lutheran Hospital Laboratories. Detail PRINCIPLE: The breakdown of hydrogen peroxide into oxygen and water is mediated by the enzyme catalase. When a small amount of an organism that produces catalase is introduced into hydrogen peroxide, rapid elaboration of bubbles of oxygen, the gaseous product of the enzyme’s activity, is produced. CLINICAL SIGNIFICANCE: This test is used as an aid in distinguishing between Staphylococci and Streptococci. All members of the genus Staphylococcus are catalase (+), where as members of the genus Streptococcus are catalase (-). Listeria monocytogenes {catalase (+)} can be distinguished from beta-hemolytic streptococcus {catalase (-)}. Most Neisseria sp. are catalase (+). Catalase can also help distinguish Bacillus sp. {catalase (+)} from Clostridum sp. {mostly catalase (-)}. SPECIMEN: Isolates preferably grown on non-blood containing media not older than 24 hours old. REAGENTS AND MATERIALS: 1. 3% hydrogen peroxide (from stock bottle). Store 2o – 25o C. Do not freeze or overheat. Light sensitive, store in brown bottle. 2. Clean microscope slide or glass test tube 3. Wooden applicator stick EQUIPMENT/INSTRUMENTATION: N/A QUALITY CONTROL: Each new lot and shipment or once a month, perform QC on reagent with stock organisms of S aureus (positive) and Beta strep group A (negative). -
Mass Spectrometry-Based Microbiological Testing for Blood
Nomura et al. Clin Proteom (2020) 17:14 https://doi.org/10.1186/s12014-020-09278-7 Clinical Proteomics REVIEW Open Access Mass spectrometry-based microbiological testing for blood stream infection Fumio Nomura1* , Sachio Tsuchida1, Syota Murata2, Mamoru Satoh1 and Kazuyuki Matsushita2 Abstract Background: The most successful application of mass spectrometry (MS) in laboratory medicine is identifcation (ID) of microorganisms using matrix-assisted laser desorption ionization–time of fight mass spectrometry (MALDI-TOF MS) in blood stream infection. We describe MALDI-TOF MS-based bacterial ID with particular emphasis on the methods so far developed to directly identify microorganisms from positive blood culture bottles with MALDI-TOF MS including our own protocols. We touch upon the increasing roles of Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) as well. Main body: Because blood culture bottles contain a variety of nonbacterial proteins that may interfere with analysis and interpretation, appropriate pretreatments are prerequisites for successful ID. Pretreatments include purifcation of bacterial pellets and short-term subcultures to form microcolonies prior to MALDI-TOF MS analysis. Three commercial protocols are currently available: the Sepsityper® kit (Bruker Daltonics), the Vitek MS blood culture kit (bioMerieux, Inc.), and the rapid BACpro® II kit (Nittobo Medical Co., Tokyo). Because these commercially available kits are costly and bacterial ID rates using these kits are not satisfactory, particularly for Gram-positive bacteria, various home-brew protocols have been developed: 1. Stepwise diferential sedimentation of blood cells and microorganisms, 2. Combi- nation of centrifugation and lysis procedures, 3. Lysis-vacuum fltration, and 4. Centrifugation and membrane fltra- tion technique (CMFT). -
Helicobacter Pylori Infections: Culture from Stomach Biopsy, Rapid Urease Test (Cutest®), and Histologic Examination of Gastric Biopsy
Available online at www.annclinlabsci.org 148 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 An Efficiency Comparison between Three Invasive Methods for the Diagnosis of Helicobacter pylori Infections: Culture from Stomach Biopsy, Rapid Urease Test (CUTest®), and Histologic Examination of Gastric Biopsy Avi Peretz1, Avi On 2, Anna Koifman1, Diana Brodsky1, Natlya Isakovich1, Tatyana Glyatman1, and Maya Paritsky3 1Clinical Microbiology Laboratory, 2Pediatric Gastrointestinal Unit, and 3Gastrointestinal Unit, Baruch Padeh Medical Center, Poria, affiliated to the Faculty of Medicine, Bar Ilan University, Galille, Israel Abstract. Background. Helicobacter pylori is one of the most prevalent pathogenic bacteria in the world, and humans are its principal reservoir. There are several available methods to diagnose H. pylori infection. Disagreement exists as to the best and most efficient method for diagnosis. Methods. In this paper, we report the results of a comparison between three invasive methods for H. pylori diagnosis among 193 pa- tients: culture, biopsy for histologic examination, and rapid urease test (CUTest®). Results. We found that all three methods have a high sensitivity and specificity for the diagnosis of infections caused by H. pylori. However, the culture method, which is not used routinely, also showed high sensitivity, probably due to biopsies’ seeding within 30 minutes, using warm culture media, non-selective media, and longer incuba- tion. Conclusions. Although not a routine test, culture from biopsy can be meaningful in identification of antibiotic-resistant strains of H. pylori and should therefore be considered a useful diagnostic tool. Keywords: Helicobacter pylor, Culture, Urease test, Gastric biopsy. Introduction Helicobacter pylori is one of the most prevalent Recently, a close association was found between H. -
Gram Stain Workshop for the Laboratory Generalist
Gram Stain Workshop for the Laboratory Generalist Karen Stiles, SM(ASCP)CM State Training Coordinator Assistant Chemical Terrorism Coordinator Nebraska Public Health Laboratory 402-559-3590 [email protected] 1 GRAM STAIN OBJECTIVES: Upon completion, the participant will be able to: 1. Explain the principle of the Gram stain procedure, including what elements can affect staining results 2. Correlate the most common pathogens with positive Gram stains from blood cultures and direct specimen sterile body fluid smears 3. Perform and interpret Grams stains 2 Purpose of Gram Stain Classify bacteria based on form, size, cellular morphology, Gram reaction Assess quality of specimen Identify specific infectious agent from morphology and Gram reaction Correlation with culture growth Correlation with culture-independent methodologist Guide presumptive antibiotic therapy 3 Principle of Gram Stain Cell wall composition Gram positive – think peptidoglycan layer with teichoic acid Gram negative – high in lipid content Basic premise Crystal Violet – all cells take up primary stain Gram’s iodine – mordant to form complex Decolorizer – mixture of acetone and alcohol Dehydrate lipids in Gram negative cell walls, wash out complex Gram positive cells resistant, retain stain complex Safranin - counterstain 4 Gram negative cells take up counterstain Preparation of Samples Specimen Type Preparation CSF/sterile body fluids Cyto/Centrifuge Blood Culture Broth Drop to slide Tissue Touch prep Tissue homogenate Drop to slide Swabbed material Roll -
Diagnostic Microbiology 170-1 Provides an Overview of the Most Commonly Used Stains
PART Infectious Diseases XVII Section cells is used to judge the suitability of a specimen for culture. For 1 example, the presence of more than 10 epithelial cells per low-power field in a sputum specimen is highly suggestive of a specimen contami- General Considerations nated with oral secretions. In addition, a preliminary assessment of the etiologic agent can be made based upon the morphology (e.g., cocci vs rods) and stain reaction (e.g., Gram-positive isolates are purple; Gram- negative are red) of the microorganisms. However, a negative Gram stain does not rule out infection as 104 to 105 microorganisms per mL Chapter 170 in the specimen are required for detection by this method. In addition to the Gram stain, many other stains are used in micro- biology, both to detect organisms and to help infer their identity. Table Diagnostic Microbiology 170-1 provides an overview of the most commonly used stains. Carey-Ann D. Burnham and Isolation and Identification Gregory A. Storch The approach to isolation of microorganisms in a clinical specimen will vary depending on the body site and pathogen suspected. For body sites that are usually sterile, such as cerebrospinal fluid, nutrient-rich media such as sheep blood agar and chocolate agar are used to aid in Laboratory evidence to support the diagnosis of an infectious disease the recovery of fastidious pathogens. In contrast, stool specimens may be based on 1 or more of the following: direct examination of contain abundant amounts of commensal bacteria and thus to isolate specimens using microscopic or antigen detection techniques, isola- pathogens, selective and differential media must be used. -
Identification and Antimicrobial Susceptibility Testing of Anaerobic
antibiotics Review Identification and Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Rubik’s Cube of Clinical Microbiology? Márió Gajdács 1,*, Gabriella Spengler 1 and Edit Urbán 2 1 Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary; [email protected] 2 Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, 6725 Szeged, Hungary; [email protected] * Correspondence: [email protected]; Tel.: +36-62-342-843 Academic Editor: Leonard Amaral Received: 28 September 2017; Accepted: 3 November 2017; Published: 7 November 2017 Abstract: Anaerobic bacteria have pivotal roles in the microbiota of humans and they are significant infectious agents involved in many pathological processes, both in immunocompetent and immunocompromised individuals. Their isolation, cultivation and correct identification differs significantly from the workup of aerobic species, although the use of new technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, whole genome sequencing) changed anaerobic diagnostics dramatically. In the past, antimicrobial susceptibility of these microorganisms showed predictable patterns and empirical therapy could be safely administered but recently a steady and clear increase in the resistance for several important drugs (β-lactams, clindamycin) has been observed worldwide. For this reason, antimicrobial susceptibility testing of anaerobic isolates for surveillance -
Use of Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry Analyser in a Diagnostic Microbiology Laboratory in a Developing Country
African Journal of Laboratory Medicine ISSN: (Online) 2225-2010, (Print) 2225-2002 Page 1 of 6 Original Research Use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry analyser in a diagnostic microbiology laboratory in a developing country Authors: Background: Rapid and accurate identification of pathogens is of utmost importance for 1 Atang Bulane management of patients. Current identification relies on conventional phenotypic methods Anwar Hoosen1 which are time consuming. Matrix-assisted laser desorption/ionisation-time of flight mass Affiliations: spectrometry (MALDI-TOF MS) is based on proteomic profiling and allows for rapid 1Department of Medical identification of pathogens. Microbiology & Virology, University of the Free State, Objective: We compared MALDI-TOF MS against two commercial systems, MicroScan Bloemfontein, South Africa Walkaway and VITEK 2 MS. Corresponding author: Methods: Over a three-month period from July 2013 to September 2013, a total of 227 bacteria Atang Bulane, and yeasts were collected from an academic microbiology laboratory (N = 121; 87 Gram- [email protected] negatives, seven Gram-positives, 27 yeasts) and other laboratories (N = 106; 35 Gram-negatives, Dates: 34 Gram-positives, 37 yeasts). Sixty-five positive blood cultures were initially processed with Received: 07 Dec. 2016 Bruker Sepsityper kit for direct identification. Accepted: 30 June 2017 Published: 08 Dec. 2017 Results: From the 65 blood culture bottles, four grew more than one bacterial pathogen and MALDI-TOF MS identified only one isolate. The blood cultures yielded 21 Gram-negatives, 43 How to cite this article: Bulane A, Hoosen A. Use of Gram-positives and one Candida. There were 21 Escherirchia coli isolates which were reported matrix-assisted laser by the MALDI-TOF MS as E. -
BBL MR-VP Broth
BBL™ MR-VP Broth ! L007474 • Rev. 06 • May 2006 QUALITY CONTROL PROCEDURES I INTRODUCTION MR-VP Broth is a medium which aids in the differentiation of bacteria by means of the methyl red and Voges-Proskauer reactions. II PERFORMANCE TEST PROCEDURE 1. Inoculate representative samples with the cultures listed below. a. Inoculate duplicate tubes using a 0.01 mL calibrated loop with 10-1 dilutions of 18- to 24-h Trypticase™ Soy Broth cultures. b. Incubate tubes with loosened caps at 35 ± 2°C in an aerobic atmosphere. 2. Examination of tubes for growth and determination of reactions. a. Voges-Proskauer Reaction—Read tubes after 18–24 h of incubation for growth and reaction. To perform the reaction proceed as follows (using one half of the duplicate tubes): 1) Add 0.6 mL of alpha-naphthol solution (see Barritt reagent, below) to each tube including uninoculated controls. 2) Add 0.2 mL potassium hydroxide solution to each tube including uninoculated controls. 3) Vortex each tube for several seconds. 4) Positive reactions occur at once or within 5 min. 5) Read for the development of a faint pink to deep red color. The appearance of a pink to red color indicates the presence of acetylmethylcarbinol. b. Methyl Red Reaction—Read the second half of the duplicate tubes after 5 days incubation for growth and reaction. Proceed as follows: 1) Add five drops of the methyl red indicator solution (see “Test Procedure”) to the test media (including the uninoculated controls). 2) Read the reactions immediately. The appearance of a distinct red color indicates the presence of a high degree of acidity and is regarded as a positive reaction. -
Laboratory Diagnosis of Sexually Transmitted Infections, Including Human Immunodeficiency Virus
Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus human immunodeficiency including Laboratory transmitted infections, diagnosis of sexually Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus Editor-in-Chief Magnus Unemo Editors Ronald Ballard, Catherine Ison, David Lewis, Francis Ndowa, Rosanna Peeling For more information, please contact: Department of Reproductive Health and Research World Health Organization Avenue Appia 20, CH-1211 Geneva 27, Switzerland ISBN 978 92 4 150584 0 Fax: +41 22 791 4171 E-mail: [email protected] www.who.int/reproductivehealth 7892419 505840 WHO_STI-HIV_lab_manual_cover_final_spread_revised.indd 1 02/07/2013 14:45 Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus Editor-in-Chief Magnus Unemo Editors Ronald Ballard Catherine Ison David Lewis Francis Ndowa Rosanna Peeling WHO Library Cataloguing-in-Publication Data Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus / edited by Magnus Unemo … [et al]. 1.Sexually transmitted diseases – diagnosis. 2.HIV infections – diagnosis. 3.Diagnostic techniques and procedures. 4.Laboratories. I.Unemo, Magnus. II.Ballard, Ronald. III.Ison, Catherine. IV.Lewis, David. V.Ndowa, Francis. VI.Peeling, Rosanna. VII.World Health Organization. ISBN 978 92 4 150584 0 (NLM classification: WC 503.1) © World Health Organization 2013 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for non-commercial distribution – should be addressed to WHO Press through the WHO web site (www.who.int/about/licensing/copyright_form/en/index.html). -
Best Practice No 167 the Laboratory Diagnosis of Urinary Tract Infection
J Clin Pathol 2001;54:911–919 911 Best Practice No 167 J Clin Pathol: first published as on 1 December 2001. Downloaded from The laboratory diagnosis of urinary tract infection J C Graham, A Galloway Abstract women develop a UTI during their lifetime; the Urinary tract infection is common, and it incidence increases at puberty and remains is not surprising that urine specimens high throughout adult life, only after the age of make up a large proportion of those sam- 50 years is a similar incidence seen in males. ples submitted to the routine diagnostic UTI accounts for approximately 23% of all laboratory. Many of these specimens will hospital acquired infections.2 Although the show no evidence of infection and several incidence of infection is high, most specimens methods can be used to screen out received will show no evidence of infection and negative samples. Those that grow bacte- several methods have been developed to screen ria need to be carefully assessed to out negative samples to minimise expense and quantify the degree of bacteriuria and improve turnaround times. These will also be hence clinical relevance. To influence reviewed. treatment, a final report should be pro- Most infections at all ages are the result of duced within 24 hours of specimen re- enteric bacteria, especially Escherichia coli, ceipt, with turnaround times continuously which colonise the perineum and then ascend monitored. Much work needs to be done to the urethra to multiply and infect the bladder, http://jcp.bmj.com/ determine the cost eVectiveness involved kidney, and adjacent structures.3 The most in processing urine specimens and the common site of infection is the bladder. -
BD™ Columbia Agar with 5% Sheep Blood
INSTRUCTIONS FOR USE – READY-TO-USE PLATED MEDIA PA-254005.06 Rev.: Apr 2013 BD Columbia Agar with 5% Sheep Blood INTENDED USE BD Columbia Agar with 5% Sheep Blood is a highly nutritious general purpose medium for the isolation and cultivation of nonfastidious and fastidious microorganisms from clinical specimens. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Ellner et al.1 in 1966 reported the development of a new blood agar formulation, which has been designated as Columbia Agar. BD Columbia Agar with 5% Sheep Blood derives its superior growth-supporting properties from the combination of two peptones, and yeast extract as a supplier of the B complex vitamins. Corn starch is included to absorb toxic by-products contained in the specimen and serves as an energy source for organisms possessing alpha- amylases. Sheep blood allows detection of hemolytic reactions and supplies the X factor (heme) necessary for the growth of many pathogenic species. On this medium, colonies tend to be larger and growth is more luxuriant than on media containing other blood agar bases. Columbia Blood Agar is recommended as a primary isolation medium in the MiQ standards and in other diagnostic manuals.2,3 In many European countries, this medium has become the most frequently used primary isolation medium for clinical specimens. REAGENTS BD Columbia Agar with 5% Sheep Blood Formula* Per Liter Purified Water Pancreatic Digest of Casein 12.0 g Peptic Digest of Animal Tissue 5.0 Yeast Extract 3.0 Beef Extract 3.0 Corn Starch 1.0 Sodium Chloride 5.0 Agar 13.5 Sheep Blood, Defibrinated 5 % pH 7.3 ± 0.2 *Adjusted and/or supplemented as required to meet performance criteria. -
Mikrobiologi Klinik FK UNUD
CHAPTER 8 Use of Colony Morphology for the Presumptive Identification of Microorganisms George Manuselis, Connie R. Mahon* CHAPTER OUTLINE ■ IMPORTANCE OF COLONIAL MORPHOLOGY AS A Form or Margin DIAGNOSTIC TOOL Elevation ■ INITIAL OBSERVATION AND INTERPRETATION OF Density CULTURES Color ■ GROSS COLONY CHARACTERISTICS USED TO Consistency DIFFERENTIATE AND IDENTIFY MICROORGANISMS Pigment PRESUMPTIVELY Odor Hemolysis ■ COLONIES WITH MULTIPLE CHARACTERISTICS Size ■ GROWTH OF ORGANISMS IN LIQUID MEDIA OBJECTIVES After reading and studying this chapter, you should be able to: 1. Describe how growth on blood, chocolate, and MacConkey agars is 4. Using colonial morphology, differentiate among the following used in the preliminary identification of isolates. microorganisms: 2. Differentiate α-hemolysis from β-hemolysis on blood agar culture • Staphylococci and streptococci medium. • Streptococcus agalactiae and Streptococcus pyogenes 3. Associate the colony characteristics shown on blood, chocolate, and • Neisseria spp. and staphylococci MacConkey agars with the microscopic findings on direct smear, and • Yeast and staphylococci use the information in the presumptive identification of • “Diphtheroids” and staphylococci microorganisms. • Lactose fermenters from lactose nonfermenters • “Swarming” Proteus species from other Enterobacteriaceae Case in Point with a surrounding pink precipitate and a few clear nonlactose fermenting colonies. Based on the Gram stain results and colo- An exudate from a sacral decubital ulcer on a 65-year-old hos- nial characteristics of the isolates, appropriate biochemical tests pital inpatient was cultured on blood agar plate (BAP), chocolate and antibiotic susceptibilities were performed to identify the (CHOC), and MacConkey (MAC) agars. Direct smear examina- causative agents of the ulcer. tion showed many white blood cells, a moderate number of gram-positive cocci in pairs and clusters, and a few gram- negative bacilli.