Bacterial Identification Methods PDF

CONVENTIONAL METHODS For Bacterial Identification Prepared by: JOEHN KERWAYNE M. VILLALUNA, RMT Faculty Member / Document Officer Matias H. Aznar Memorial College College of Medical Technology LEARNING OUTCOMES: At the end of this lecture, the students should be able to demonstrate th...

CONVENTIONAL METHODS For Bacterial Identification Prepared by: JOEHN KERWAYNE M. VILLALUNA, RMT Faculty Member / Document Officer Matias H. Aznar Memorial College College of Medical Technology LEARNING OUTCOMES: At the end of this lecture, the students should be able to demonstrate the following: 1. Understand the importance of accurate bacterial identification to diagnostic bacteriology; 2. Recall, identify, and correctly apply the most commonly used bacterial identification methods; 3. Correctly identify the name of a bacterial isolate when given a list of laboratory test results based on phenotypic criteria.  ACCURATE BACTERIAL IDENTIFICATION: ITS IMPORTANCE ▪ Determining the clinical significance of a particular bacterial isolate ▪ Guiding physician care of the patient ▪ Determining whether laboratory testing for detection of antimicrobial resistance is warranted  ACCURATE BACTERIAL IDENTIFICATION: ITS IMPORTANCE ▪ Determining the type of antimicrobial therapy that is appropriate ▪ Determining whether the antimicrobial susceptibility profiles are unusual or aberrant for a particular bacterial species ▪ Determining whether the infecting organism is a risk or may pose problems for infection control, public health, or laboratory safety  PRINCIPLES OF IDENTIFICATION ▪ Various laboratory tests generate information that provide characteristic profiles of bacteria ▪ Gram staining is an integral part of many schemes used for identifying a wide variety of bacteria ▪ Identification schemes – used for organism identification IDENTIFICATION SCHEMES GENOTYPIC PHENOTYPIC Analysis of Analysis of gene bacterial products genome (genes) Nucleic acid Physical testing Biochemical  PHENOTYPIC CRITERIA 1. Colony / macroscopic morphology 2. Microscopic morphology 3. Staining characteristics 4. Environmental requirements for growth 5. Resistance or susceptibility to antimicrobial agents 6. Nutritional requirements 7. Metabolic capabilities 8. Antigenic makeup (immunologic diagnosis)  MACROSCOPIC MORPHOLOGY Evaluation of colony morphology: ▪ Colony size ▪ Shape ▪ Elevation ▪ Surface or margin appearance ▪ Color or pigment production ▪ Any changes that colony growth produces surrounding the agar medium NOTE: macroscopic characteristics usually are not sufficient for establishing a final or definitive identification MACROSCOPIC MORPHOLOGY  ENVIRONMENTAL REQUIREMENTS FOR GROWTH INCUBATION ATMOSPHERE INCUBATION TEMPERATURE Organisms that grow Aerobes at 37oC Strict Anaerobes Organisms that grow at 0oC (-20 to +10oC) Facultative Anaerobes Organisms that grow Microaerophiles at 42oC (41 to 122oC)  MICROSCOPIC MORPHOLOGY ▪ Microscopic evaluation of bacterial cell morphology provides the most basic and important information on which final identification strategies are based. ▪ Gram stain – the first step in any bacterial identification scheme. ▪ In most instances, identification schemes for final identification are based on morphologies and staining characteristics of bacteria.  MICROSCOPIC MORPHOLOGY ▪ Microscopic evaluation of bacterial cell morphology provides the most basic and important information on which final identification strategies are based. ▪ Gram stain – the first step in any bacterial identification scheme. MICROSCOPIC MORPHOLOGY  MICROSCOPIC GROUP MORPHOLOGY REPRESENTATIVE ORGANISMS Gram-positive Staphylococcus, Micrococcus, cocci Streptococcus, Enterococcus Gram-negative Neisseria, Moraxella cocci Bacillus, Clostridium, Lactobacillus, Gram-positive Listeria, Corynebacterium, bacilli Erysipelothrix, Nocardia, Streptomyces Enterobacteriaceae, Acinetobacter, Pseudomonas, Gram-negative Campylobacter, Helicobacter, bacilli Legionella, Bordetella, Francisella, Spirillum, Streptobacillus  GRAM STAINING ▪ Hans Christian Gram – created a differential staining technique called “Gram staining” ▪ Gram staining – routinely performed in the microbiology section ▪ Gram-positive = blue/purple ▪ Gram-negative = red/pink  GRAM STAINING PROCEDURE Decolorization Gram-positive = blue/purple Gram-negative = red/pink  FLOW CHART FOR BACTERIAL IDENTIFICATION GRAM-POSITIVE Gram-Positive Cocci Gram-Positive Bacilli Catalase Spores (+) Staphylococci (+) Bacillus spp. Catalase (–) Streptococci or (+) Corynebacterium Enterococci Listeria, others (–) Lactobacillus Actinomyces  FLOW CHART FOR BACTERIAL IDENTIFICATION GRAM-NEGATIVE Gram-Negative Bacilli Gram-Negative Cocci Growth on MacConkey Agar Growth on Thayer-Martin Agar (+) Enterobacteriaceae (+) Pathogenic (–) Other Pseudomonas Neisseria spp. Neisseria spp. (–) Haemophilus Brucella, Legionella Oxidase (+) Pseudomonas (–) Enterobacteriaceae  NUTRITIONAL REQUIREMENTS AND METABOLIC CAPABILITIES ▪ Biochemical tests - is the most common approach used for determination of bacterial genus and species ▪ Methods share many commonalities but also have some important differences ▪ It should be used in combination with other methods or tests  ENZYME-BASED TESTS TEST ENZYME ▪ Catalase test Catalase ▪ Oxidase test Cytochrome oxidase ▪ Indole test Tryptophanase ▪ Urease test Urease ▪ PYR test L-pyrroglutamyl aminopeptidase ▪ Hippurate hydrolysis Hippuricase  CATALASE TEST  FLOW CHART FOR BACTERIAL IDENTIFICATION GRAM-POSITIVE Gram-Positive Cocci Gram-Positive Bacilli Catalase Spores (+) Staphylococci (+) Bacillus spp. Catalase (–) Streptococci or (+) Corynebacterium Enterococci Listeria, others (–) Lactobacillus Actinomyces CATALASE TEST  OXIDASE TEST OXIDASE TEST  FLOW CHART FOR BACTERIAL IDENTIFICATION GRAM-NEGATIVE Gram-Negative Bacilli Gram-Negative Cocci Growth on MacConkey Agar Growth on Thayer-Martin Agar (+) Enterobacteriaceae (+) Pathogenic (–) Other Pseudomonas Neisseria spp. Neisseria spp. (–) Haemophilus Brucella, Legionella Oxidase (+) Pseudomonas (–) Enterobacteriaceae  INDOLE TEST INDOLE TEST  UREASE TEST UREASE TEST  TRIPLE SUGAR IRON TEST Principle: The triple sugar iron medium is used to differentiate organisms based on their ability to metabolize glucose, lactose, and sucrose in either aerobic or anaerobic conditions. Ferrous sulfate creates a black precipitate when the organism produces hydrogen sulfide Lactose or sucrose-fermenting organisms turn the slant and butt portions yellow. Organisms that can only ferment glucose turn the slant red and the but yellow.