Microbiology Lecture Notes PDF

Identifying and classifying microorganisms (Chapter 10) Identification methods based on phenotype Phenotypic characteristics can be used in the identification of microorganisms and these methods do not require sophisticated equipments. Microscopic morphology Determine siz...

Identifying and classifying microorganisms (Chapter 10) Identification methods based on phenotype Phenotypic characteristics can be used in the identification of microorganisms and these methods do not require sophisticated equipments. Microscopic morphology Determine size, shape & staining characteristics of a microbe. Microscopic exam may provide enough information to make a presumptive identification e.g.  Trichomonas (protozoa) & yeast in vaginal secretion.  Round worms' eggs in stool - identified based on their shape and size. Identification methods based on phenotype Gram staining is a rapid differential method, but it is generally not sensitive & specific enough to diagnose the cause of infections. It is a useful tool in narrowing the possible identities of a microbe. 2. Neisseria gonorrheae Gram staining may give (Gram -ve diplococci in enough information to start a urethral secretion). appropriate antimicrobial therapy e.g. Some microbes have unique characteristics 1. Streptococcus pneumoniae that can be detected (Gram +ve encapsulated with special staining diplococci) e.g., M. tuberculosis (acid-fast stain).  Culture characteristics Colony morphology: gives initial ideas for the identification of certain microbes. – Colonies of Streptococci are generally small relative to many other bacteria such as staphylococci. – Colonies of Serratia marcescens are red when incubated at 220C (production of a pigment) & white when incubated at 370C. – Pseudomonas aeruginosa usually produces a soluble green greenish pigment which discolors the growth media and has a distinct fruity odor. Use of selective and differential media:  Blood agar media: It is a differential media.  MacConkey agar is both selective & differential media.  Metabolic capabilities Culture characteristics can give ideas about the identities of bacteria, but biochemical tests are generally necessary for a more conclusive diagnosis. Biochemical tests: (Check table 10.3 for reference) Some are quite rapid as they detect presence of enzymes. However, most tests require at least 18hrs incubation. Catalase: Most bacteria which grow in the presence of oxygen are catalase +ve & breakdown H2O2 to release O2 which cause bubbling. Important exception are lactic acid bacteria which include Streptococcus. +ve -ve If a throat culture has beta-hemolytic colonies on blood agar but are catalase positive, then Streptococcus pyogenes is ruled out.  Biochemical tests Most biochemical tests are based on a pH indicator or chemical reaction that results in color change when a compound is degraded e.g. Uninoculated control No color change (Ist tube) indicate that sugar is not used. Fermentation of sugar results in acid production (lowers pH) which causes a color change from pink to yellow and gas production. Urease enzyme degrades urea to produce CO2 & ammonia, & medium turns bright pink in alkaline conditions.  Biochemical tests cont’d Strategy: Use of a dichotomous key, for identifying bacteria which is a flow chart of tests that give either a positive or negative result. The biochemical tests are usually initiated simultaneously to speed identification.  Biochemical tests cont’d In certain cases, biochemical test can be done without culturing the organism e.g.  Breath test, which assays for the presence of urease, is done to detect Helicobacter pylori.  Urea containing drink is labeled with an isotope of carbon. If H. pylori is present, labeled carbon dioxide is released which is measured in the exhaled air.  Breath test is less invasive and much cheaper and faster than the stomach biopsy. Commercial modifications of traditional biochemical tests:  RapID  API test strip  Biolog microtiter plate  Vitek 2 automated system (uses miniature card).  Serological testing In some cases, proteins & polysaccharides present on the bacterium surface can be used as identifying markers. The most useful of these are the molecules that make up surface structures i.e. Cell wall, capsule, flagella & pili. Antibodies to surface proteins & polysaccharides are frequently used to identify various bacteria. Methods which use antibodies for the detection of antigens are called serology. Some serological tests, such as used to identify Streptococcus pyogenes (cell wall components), are quite specific, simple and rapid.  Protein profile A relatively new technology that determines an organism’s protein profile is revolutionizing microbial identification because of its fast speed (