MLS Review - Bacteriology - Introduction Recall

MEDICAL LABORATORY SCIENCE REVIEW • CLINICAL BACTERIOLOGY: INTRODUCTION TO BACTERIOLOGY RECALL OUTLINE • IMPORTANT BACTERIAL STRUCTURES • Introduction to Bacteriology o General Bacterial Characteristics o Important Bacterial Structures o Growth Requirements o Stages of Bacterial Growth o Critical Values in Microbiology INTRODUCTION TO BACTERIOLOGY • GENERAL BACTERIAL CHARACTERISTICS • • • • • Average size: 0.4 - 2 micra o Genus Mycoplasma - Smallest living organisms o Bacillus anthracis - Largest pathogenic bacterium Prokaryotes - With no true nucleus Compared w/ viruses, bacteria have both RNA & DNA With cell wall except Genus Mycoplasma and Ureaplasma (cell wall-less) o Cell wall - aka peptidoglycan layer or Murein layer ▪ Peptidoglycan - main component ▪ Also contain disaccharides (n-acetylglucosamine & n-acetylmuramic acid) o Functions of cell wall ▪ Gives shape to organism • 3 morphologic forms: cocci, bacilli, spiral ▪ Provides attachment/anchorage for flagella • Where flagella are attached ▪ Used as basis for Gram staining • Gram (+) cell wall - Teichoic acid & thicker peptidoglycan • Gram (-) cell wall - no teichoic acid, thinner peptidoglycan, with Lipopolysaccharide (LPS) & periplasmic space ▪ Used as primary target in development of antibiotics (damages/destroys cell wall) Some may produce spores o Spores - aka endospore ▪ Resistant structures enabling the organism to resist/withstand unfavorable/serious conditions ▪ Dipicolinic acid - responsible for making spores resistant ▪ Autoclaving - best way to destroy spores ▪ Can be round or oval in shape ▪ Located at the center, terminal or subterminal of the organism • C. tetani - have terminal swollen spores • B. anthracis - spores centrally located • C. botulinum - spores are subterminal o Spore Stain: Schaeffer & Fulton; Dorner’s stain ▪ Schaffer & Fulton - requires Malachite green & safranin • Malachite green - primary dye • Safranin - counterstain • Spores appear green o Genus Clostridium and Bacillus - genera considered as spore formers ▪ Bacillus - aerobes ▪ Clostridium - mostly anaerobes • Some are aerotolerant Cell membrane with sterols except in Mycoplasma • • Pili (Pilus - singular) o Usually found in gram negative organisms o Common Pili - for attachment/adherence to host cell ▪ Can serve as virulence factor ▪ Attachment to host cell - 1st step in infection process ▪ N. gonorrhea - organism with common pili o Sex Pili - for gene conjugation (transfer for genetic material) Capsule o Some bacteria may have capsule o Usually found in gram negative organisms o Antiphagocytic (prevents phagocytosis) o Can be a virulence factor o S. pneumoniae, K. pneumoniae, N. meningitidis have polysaccharide capsule o B. anthracis - polypeptide-D-glutamic acid; not polysaccharide in nature o Pasteurella multocida - hyaluronic acid capsule ▪ Important pathogen that causes animal bite wound in man o H. influenzae - polyribosyl ribitol phosphate ▪ Cause meningitis in children below 5 years old ▪ Serotype A, B, C, D, E, F • Serotype B - most common; implicate din cases of meningitis o P. aeruginosa - alginate capsule o Detection of Capsule ▪ Stain for capsule - Capsular stains: Hiss, Anthony’s, Tyler, India ink (C. neoformans) ▪ Capsular swelling test - Neufeld Quellung Test ▪ Colony characteristic of those with capsule development of mucoid colonies Inclusion Bodies o Represents stored food o Babes-Ernst bodies - C. diphtheriae inclusion bodies ▪ Culture media to enhance formation of BabesErnst - PAI’s slant, Loeffler’s serum ▪ Stains: Leoffler’s Methylene Blue, Albert, Neisser • Allow demonstration of inclusion bodies o Halberstaedter Prowazek – glycogen containing inclusion bodies that can be noted in C. trachomatis ▪ Stains: Gimenez, Machiavello, Castañeda o Much granules - M. tuberculosis (appears granulated/beaded under microscope) o Bipolar bodies - seen in Y. pestis, gives safety pin appearance ▪ Stain: Wayson stain or Methylene blue • Wayson - preferred; best demonstrates safety pin appearance Flagella - organ for locomotion o Not all bacteria are motile o Bacilli & spiral - usually motile o Cocci - usually nonmotile o Tumbling motility - aka end over end motility; seen in Listeria monocytogenes ▪ L. monocytogenes - primarily an animal pathogen; causes meningitis in immunocompromised, elderly, stillbirth, sepsis, food poisoning of soft cheese and coleslaw 1 • ▪ o o o o o o B. cereus - cause food poisoning in consumption of contaminated rice meals Gliding/ sliding motility - Capnocytophaga gingivalis (requires increased CO2) Darting motility - Campylobacter Twitching motility - Kingella kingae (part of HACEK group that causes SBE) ▪ Kingella kingae - not a common lab ▪ HACEK - Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella ▪ SBE - subacute bacterial endocarditis Shooting star motility - Vibrio cholerae Corkscrew motility - spiral organisms Detection of Flagella ▪ Flagellar stains: Gray’s, Leifson ▪ Hanging Drop - presumptive test for Listeria (tumbling motility) ▪ H antigen • Found in organisms with flagella • Flagellar antigen • in Enterobacteriaceae ▪ • • • GROWTH REQUIREMENTS • • • Halophilic - requiring increased salt concentration o 7.5% - S. aureus ▪ S. aureus - significant pathogen in genus Staphylococcus • Normal biota/flora • Can cause a number of toxin & non-toxin mediated disease • Toxin - food poisoning, toxic shock syndrome, scalded skin syndrome • Non-toxin-mediated - carbuncle, furuncles, boils, cellulitis, impetigo, sty • Coagulase test - definitive test o 6.5% - Enterococci ▪ Screening test: • Bile esculin test o Uses bile esculin media o Inoculate → Incubate → Observe color o (+) - Blackening of media o Indicator: ferric ammonium citrate ▪ Causes blackening ▪ Causes UTI in hospitalized patients • Agent of nosocomial UTI o 8-10% - required by most Vibrios except V. cholerae & V. mimicus Capnophilic - requires increased CO2 o i.e., Neisseria, Streptococci, Helicobacter, Haemophilus, HACEK o Use of Candle jar ▪ Candle jar - supply increased CO2 to organism Obligate anaerobe o i.e., Prevotella, Porphyromonas, Fusobacterium o Not requiring oxygen for growth o In the presence of oxygen, they die o Requires: 0% Oxygen, 5% CO2, 10% H2, 85% N2 o Use of Gaspak Jar ▪ Gaspak Jar - contains indicator to determine presence of O2 (Resazurin or Methylene blue) • Methylene blue - blue in the presence of O2 • Resazurin - pink in the presence of O2 In the absence of O2, Resazurin and Methylene blue are colorless • • Gaspak Jar Contains palladium catalyst • To remove oxygen ▪ Common failure of Gaspak jar: inactivation of catalyst due to repeated use Microaerophilic o Requires: 5-10% oxygen, 10% CO2, 85% N2 o i.e., Campylobacter, Treponema, Helicobacter, Borrelia Aerophilic / Aerobes o Requires: 21% oxygen Cold Enrichment o Usual bacterial and viral culture is incubated at 35-37C ▪ Fungal culture is at RT (25-30C) o Cold Enrichment is incubation at 4C for several weeks o Can tolerate cold enrichment: Y. enterocolitica & L. monocytogenes o Refrigerated Blood Bag (4C) Contaminants: Y. enterocoliitca, P. fluorescens, S. liquefaciens ▪ Listeria is not included because it is primarily an animal pathogen o Blood bag contaminants at RT: S. epidermidis, B. cereus and Cutibacterium acnes TCBS - selective and differential media for Vibrios o Contains sucrose as fermentable carbohydrate o Indicators: bromthymol blue & thymol blue o Sucrose fermenters = yellow colonies o Non-sucrose fermenters = green colonies Alkaline peptone water o Enrichment media for genus Vibrio STAGES OF BACTERIAL GROWTH • • • • Lag or Adjustment phase - no cell division, increase in size, no increase in number or organisms, adaptation o Bacteria adapts to their new environment o Growth rate at 0% Log or Exponential phase - start of cell division o Number of bacteria starts to increase o Bacteria become susceptible to antibiotics Plateau or Stationary phase - balance between dead cells and living cells (equal number) o Spores start to develop Death phase or phase of decline o Bacteria dies due to unfavorable conditions, pH, accumulation of toxic waste CRITICAL/PANIC VALUES IN MICROBIOLOGY 1. 2. 3. 4. 5. 6. 7. 8. • Positive blood culture Positive cerebrospinal fluid Gram stain or culture Positive cryptococcal antigen test or culture Positive blood smear for malaria Streptococcus pyogenes from a sterile site Positive acid-fast smears or positive Mycobacterium culture Streptococcus agalactiae or herpes simplex virus from genital site of a pregnant woman at term Detection of significant pathogen (i.e., Bordetella pertussis, Brucella, Legionellai) RULE: Any findings of these must be reported immediately (communicate to the members of healthcare team ASAP) 2