LAB 2: Staining and Streaking Protocols for Simple Stain, Gram Stain, Streak Plate Technique and Culture Maintenance
Total Page:16
File Type:pdf, Size:1020Kb
Load more
Recommended publications
-
Gst Gram Staining Learning Objectives the Student Will Use Aseptic Techniques in the Safe Inoculation of Various Forms of Media
GSt Gram Staining Learning Objectives The student will Use aseptic techniques in the safe inoculation of various forms of media. Follow oral and written instructions and manage time in the lab efficiently. Use the bright field light microscope to view microbes under oil immersion, make accurate observations and appropriate interpretations and store the microscope according to lab procedures. Properly prepare a bacterial smear for accurate staining and describe the chemical basis for simple staining and negative staining. Background/Theory Differential staining distinguishes organisms based on their interactions with multiple stains. In other words, two organisms may appear to be different colors. Differential staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining. This link to the OpenStax Microbiology text provides more detail on these differential staining techniques. (OpenStax CNX, 2018) The Gram stain is a differential staining procedure that involves multiple steps. It was developed by Danish microbiologist Hans Christian Gram in 1884 as an effective method to distinguish between bacteria containing the two most common types of cell walls. (OpenStax CNX, 2018) One type consists of an inner plasma membrane and a thick outer layer of peptidoglycan. The other type consists of a double phospholipid Figure 1 Simplified structures of Gram negative cells (left) and Gram positive bilayer with a thin layer of cells (right) peptidoglycan between the two. The Gram Staining technique remains one of the most frequently used staining techniques. The steps of the Gram stain procedure are listed below and illustrated in Figure. (OpenStax CNX, 2018) 1. -
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. -
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. -
Francisella Tularensis 6/06 Tularemia Is a Commonly Acquired Laboratory Colony Morphology Infection; All Work on Suspect F
Francisella tularensis 6/06 Tularemia is a commonly acquired laboratory Colony Morphology infection; all work on suspect F. tularensis cultures .Aerobic, fastidious, requires cysteine for growth should be performed at minimum under BSL2 .Grows poorly on Blood Agar (BA) conditions with BSL3 practices. .Chocolate Agar (CA): tiny, grey-white, opaque A colonies, 1-2 mm ≥48hr B .Cysteine Heart Agar (CHA): greenish-blue colonies, 2-4 mm ≥48h .Colonies are butyrous and smooth Gram Stain .Tiny, 0.2–0.7 μm pleomorphic, poorly stained gram-negative coccobacilli .Mostly single cells Growth on BA (A) 48 h, (B) 72 h Biochemical/Test Reactions .Oxidase: Negative A B .Catalase: Weak positive .Urease: Negative Additional Information .Can be misidentified as: Haemophilus influenzae, Actinobacillus spp. by automated ID systems .Infective Dose: 10 colony forming units Biosafety Level 3 agent (once Francisella tularensis is . Growth on CA (A) 48 h, (B) 72 h suspected, work should only be done in a certified Class II Biosafety Cabinet) .Transmission: Inhalation, insect bite, contact with tissues or bodily fluids of infected animals .Contagious: No Acceptable Specimen Types .Tissue biopsy .Whole blood: 5-10 ml blood in EDTA, and/or Inoculated blood culture bottle Swab of lesion in transport media . Gram stain Sentinel Laboratory Rule-Out of Francisella tularensis Oxidase Little to no growth on BA >48 h Small, grey-white opaque colonies on CA after ≥48 h at 35/37ºC Positive Weak Negative Positive Catalase Tiny, pleomorphic, faintly stained, gram-negative coccobacilli (red, round, and random) Perform all additional work in a certified Class II Positive Biosafety Cabinet Weak Negative Positive *Oxidase: Negative Urease *Catalase: Weak positive *Urease: Negative *Oxidase, Catalase, and Urease: Appearances of test results are not agent-specific. -
Biofire Blood Culture Identification System (BCID) Fact Sheet
BioFire Blood Culture Identification System (BCID) Fact Sheet What is BioFire BioFire BCID is a multiplex polymerase chain reaction (PCR) test designed to BCID? identify 24 different microorganism targets and three antibiotic resistance genes from positive blood culture bottles. What is the purpose The purpose of BCID is to rapidly identify common microorganisms and of BCID? antibiotic resistance genes from positive blood cultures so that antimicrobial therapy can be quickly optimized by the physician and the antibiotic stewardship pharmacist. It is anticipated that this will result in improved patient outcomes, decreased length of stay, improved antibiotic stewardship, and decreased costs. When will BCID be BCID is performed on all initially positive blood cultures after the gram stain is routinely performed and reported. performed? When will BCID not For blood cultures on the same patient that subsequently become positive with be routinely a microorganism showing the same morphology as the initial positive blood performed? culture, BCID will not be performed. BCID will not be performed on positive blood cultures with gram positive bacilli unless Listeria is suspected. BCID will not be performed on blood culture bottles > 8 hours after becoming positive. BCID will not be performed between 10PM-7AM on weekdays and 2PM-7AM on weekends. BCID will not be performed for clinics that have specifically opted out of testing. How soon will BCID After the blood culture becomes positive and the gram stain is performed and results be available? reported, the bottle will be sent to the core Microbiology lab by routine courier. BCID testing will then be performed. It is anticipated that total turnaround time will generally be 2-3 hours after the gram stain is reported. -
The Use of Matrix-Assisted Laser Desorption Ionization
Session: OS202 MALDI-TOF - diagnostics for the micro lab in the 21st century Category: 4b. Diagnostic bacteriology – non-culture based, including molecular and MALDI-TOF 25 April 2017, 14:30 - 14:40 OS1031 The use of matrix-assisted laser desorption ionization - time-of-flight mass spectrometry (MALDI-TOF MS) for rapid bacterial identification in patients with smear- positive bacterial meningitis - a study of diagnostic accuracy Boaz Bishop*1, Yuval Geffen2, Ola Kassis3, Adi Kamar4, Mical Paul5, Ami Neuberger6 1Rambam Medical Center; Department of Internal Medicine B 2Rambam Health Care Campus; Microbiology Laboratory 3Technion 4Rambam Medical Center; Microbiology Laboratory 5Rambam Health Care Center; Division of Infectious Diseases 6Rambam Medical Center; Unit of Infectious Diseases Background: Bacterial meningitis is a rapidly occurring fatal disease, thus immediate diagnosis and adequate therapy are crucial. Diagnosis is obtained by cerebral spinal fluid (CSF); cultures results are the gold standard for diagnosis, but are available only within 24-72 hours. MALDI-TOF-MS identifies a wide range of bacteria by examining protein profiles. Little data exists regarding the use of MALDI- TOF-MS for examining CSF samples. We aimed at assessing the accuracy of MALDI-TOF-MS in rapid identification of CSF pathogens in patients with meningitis. Material/methods: We conducted a prospective study examining samples with positive CSF Gram stains with MALDI-TOF-MS. The results were compared with the CSF culture; identification was considered accurate when identical to the CSF culture results (species and genus level). Lab workers preforming the MALDI-TOF MS and ones interpreting its results were blinded to the result of the direct smear, besides the fact of it being positive. -
Use of the Diagnostic Bacteriology Laboratory: a Practical Review for the Clinician
148 Postgrad Med J 2001;77:148–156 REVIEWS Postgrad Med J: first published as 10.1136/pmj.77.905.148 on 1 March 2001. Downloaded from Use of the diagnostic bacteriology laboratory: a practical review for the clinician W J Steinbach, A K Shetty Lucile Salter Packard Children’s Hospital at EVective utilisation and understanding of the Stanford, Stanford Box 1: Gram stain technique University School of clinical bacteriology laboratory can greatly aid Medicine, 725 Welch in the diagnosis of infectious diseases. Al- (1) Air dry specimen and fix with Road, Palo Alto, though described more than a century ago, the methanol or heat. California, USA 94304, Gram stain remains the most frequently used (2) Add crystal violet stain. USA rapid diagnostic test, and in conjunction with W J Steinbach various biochemical tests is the cornerstone of (3) Rinse with water to wash unbound A K Shetty the clinical laboratory. First described by Dan- dye, add mordant (for example, iodine: 12 potassium iodide). Correspondence to: ish pathologist Christian Gram in 1884 and Dr Steinbach later slightly modified, the Gram stain easily (4) After waiting 30–60 seconds, rinse with [email protected] divides bacteria into two groups, Gram positive water. Submitted 27 March 2000 and Gram negative, on the basis of their cell (5) Add decolorising solvent (ethanol or Accepted 5 June 2000 wall and cell membrane permeability to acetone) to remove unbound dye. Growth on artificial medium Obligate intracellular (6) Counterstain with safranin. Chlamydia Legionella Gram positive bacteria stain blue Coxiella Ehrlichia Rickettsia (retained crystal violet). -
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 -
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. -
CARBOL FUCHSIN STAIN (ZIEHL-NEELSEN) - for in Vitro Use Only - Catalogue No
CARBOL FUCHSIN STAIN (ZIEHL-NEELSEN) - For in vitro use only - Catalogue No. SC24K Our Carbol Fuchsin (Ziehl-Neelsen) Stain is Formulation per 100 mL used in the microscopic detection of acid-fast microorganisms such as Mycobacterium . SC25 Carbol Fuchsin Stain (Ziehl-Zeelsen) Acid-fast organisms such as Mycobacterium Basic Fuchsin ..................................................... 0.3 g have cell walls that are resistant to conventional Phenol ................................................................ 5.0 g staining by aniline dyes such as the Gram stain. Ethanol ............................................................ 10 mL However methods that promote the uptake of dyes De-ionized Water ............................................. 90 mL are available; once stained these organisms are not easily decolorized even with acid-alcohol or acid- SC26 Carbol Fuchsin Decolorizer acetone solutions therefore they are described as Hydrochloric Acid .......................................... 3.0 mL acid-fast. Their resistance to destaining is a useful Ethanol .......................................................... 97.0 mL characteristic in differentiating these organisms from contaminating organisms and host cells. SC27 Carbol Fuchsin Counterstain (Methylene Blue) The Ziehl-Neelsen staining procedure is often Methylene Blue ................................................. 0.3 g referred to as hot carbolfuchsin because of the need De-ionized Water ............................................100 mL to apply heat during the staining -
(BCID) Results Are “Not Detected”
Interpretation of Positive Blood Cultures When PCR Blood Culture Identification (BCID) Results are “Not Detected” Nebraska Medicine currently uses a multi-plex PCR-based blood culture identification (BCID) system that is able to identify 19 potential pathogens growing in blood culture. BCID generally detects over 90% of the most common causative agents in bloodstream infections; however, when microbes not included on the panel are present in a blood culture, it returns a result of “Not Detected.” This document aims to provide guidance in these scenarios supported by data collected at Nebraska Medicine from January 2018 to August 2019. Table 1: Recommendations for treatment of patients with blood cultures growing organisms not detected on BCID Gram Stain/Preliminary Likely Organism (% total BCID negative)* Recommended Treatment Culture Result Gram-positive: Aerobe Micrococcus sp. (18.1%) (most can also grow in Coagulase-negative Staphylococcus (9.3%) None anaerobic bottles) Diphtheroids (7%) None Peptostreptococcus sp. (4.4%) If therapy is desired: Anaerobe bottle only Lactobacillus sp. (2.6%) Metronidazole 500 mg PO q8h Clostridium sp. (2.6%) OR Penicillin G 4 million units IV q4h Gram-negative: Aerobe Acinetobacter sp. (1.8%) (most can also grow in Stenotrophomonas maltophilia (1.6%) Levofloxacin 750 mg IV/PO q24h anaerobic bottles) Pseudomonas fluorescens-putida group (1%) Bacteroides fragilis group (9.3%) Anaerobe bottle only Metronidazole 500 mg IV/PO q8h Fusobacterium sp. (4.7%) *A full list of isolated organisms can be found below in Table 2 Orange text = Cocci, Blue text = Bacilli (rods) Gram-Positives When BCID results as “Not Detected” but there is microbial growth, the organism is most frequently gram-positive (71%). -
Short Communication Is It Possible to Perform Bacterial Identification and Antimicrobial Susceptibility Testing with a Positive
Rev Soc Bras Med Trop 51(2):215-218, March-April, 2018 doi: 10.1590/0037-8682-0311-2017 Short Communication Is it possible to perform bacterial identification and antimicrobial susceptibility testing with a positive blood culture bottle for quick diagnosis of bloodstream infections? Tamily Cristina Lemos[1], Laura Lúcia Cogo[2], Adriane Cheschin Maestri[2], Milena Hadad[3] and Keite da Silva Nogueira[2],[4] [1]. Residência Multiprofissional em Atenção Hospitalar, Universidade Federal do Paraná, Curitiba, PR, Brasil. [2]. Hospital de Clínicas da Universidade Federal do Paraná, Curitiba, PR, Brasil. [3]. BioMérieux SA, Jacarepaguá, Rio de Janeiro, RJ, Brasil. [4]. Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba, PR, Brasil. Abstract Introduction: Bloodstream infections can be fatal, and timely identification of the etiologic agent is important for treatment. Methodology: An alternative method, consisting of direct identification and susceptibility testing of blood culture bottles using the automated VITEK 2® system, was assessed. Results: All 37 of the Gram-negative bacilli (GNB) identifications and 57.1% of the 28 Gram-positive cocci (GPC) identifications matched those obtained with standard methods. In susceptibility testing, the agreement was greater than 90%. Conclusions: This alternative methodology may assist in the early identification and susceptibility testing of GNB. Further research is necessary to develop appropriate methods for GPC. Keywords: Blood culture. Sepsis. Rapid diagnosis. Bloodstream infections (BSI) in general hospitals are The blood culture method is still considered the gold serious and life-threatening and are ranked as the third leading standard for the identification of bacteria in Bloodstream. cause of health care-related infections.