Microbiology Review PDF

MICROBIOLOGY Medical Laboratory Science Review College of Allied Medical Professions Lyceum of the Philippines University - Batangas RHC2019  Microorganisms Acellular Cellular Viruses Prokaryote...

MICROBIOLOGY Medical Laboratory Science Review College of Allied Medical Professions Lyceum of the Philippines University - Batangas RHC2019  Microorganisms Acellular Cellular Viruses Prokaryotes Eukaryotes W/o True Nucleus With True Nucleus A.Eubacteria – true bacteria A.Protozoa B.Cyanobacteria – B.Fungi blue-green algae C.Algae C.Archaebacteria – can survive in extreme environments (e.g. hot springs)  COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELLS PROKARYOTE EUKARYOTE Nucleus bound by Nucleoid NOT bound by Nuclear body membrane; consists of the membrane; consists of one or more pair of one chromosome (DNA chromosomes (DNA and and histone-like proteins) histones) Cell division Usually BINARY FISSION MITOSIS A.) EUBACTERIA – with PEPTIDOGLYCAN rich cell Animals, protozoans – no Cell wall wall EXCEPT Mycoplasma cell wall and Ureaplasma Plants, fungi – with cell B.) ARCHAEBACTERIA – with wall (cellulose and cell wall resembling chitin) peptidoglycan  COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELLS PROKARYOTE EUKARYOTE Fluid phospholipid Cytoplasmic bilayer without CHO membrane Fluid phospholipid and sterol except bilayer with CHO and Mycoplasma and sterol Ureaplasma (with CHO and sterol) Cell organelles Absent Present Site of energy Cytoplasmic production Mitochondria membrane Rough Endoplasmic Site of protein synthesis Free Ribosomes Reticulum (RER) BACTERIAL CYTOLOGY Bacterial cell - 70% Water; 30% CHO, CHONs, Lipids, etc ◻ CELL WALL Main constituent: Peptidoglycan Functions: 1) Protects internal structures 2) Provides shape of bacteria Some components are responsible for pathogenicity: M protein – S.pyogenes prevents phagocytosis Mycolic acid – MTB inhibits digestion during phagocytosis  GRAM STAIN GRAM STEP REAGENTS GRAM POSITIVE NEGATIVE Primary / Crystal Violet Violet Violet Initial Stain Mordant Gram’s Violet Violet Iodine Decolorizer Acetone alcohol (95% Violet Colorless Ethanol) Secondary Safranin Stain **for C. jejuni use Violet Red / Pink carbol fuschin  MUST KNOW! ◻ Gram positive bacilli (aerobes): , Bacillus, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Nocardia ◻ Gram positive bacilli (anaerobes): Actinomyces Clostridium Propionibacterium Bifidobacterium Eggerthella  MUST KNOW! ◻ Gram negative bacilli (aerobes): 1. Fermentative: ENTEROBACTERIACEAE*** 2. Nonfermentative: Pseudomonas, Acinetobacter, Stenotrophomonas, Burkholderia, Alcaligenes, Achromobacter, Flavobacterium, Chryseomonas, Acidovorax, Brevundimonas, Ralstonia 3. HACEK group: Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella 4. OTHERS: Vibrio, Campylobacter, Aeromonas, Helicobacter, Legionella, Bordetella, Pasteurella, Gardnerella, Capnocytophaga, Calymmatobacterium*, Francisella, Streptobacillus ENTEROBACTERIACEAE ◻ Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia, Klebsiella, Morganella, Plesiomonas, Proteus, Providencia, Salmonella, Serratia, Shigella, Yersinia  NOTE!!! HACEK ◻ HACEK - bacteria associated with SUBACUTE BACTERIAL ENDOCARDITIS ◻ Haemophilus spp. H. influenzae, H. parainfluenzae ◻ Aggregatibacter A. aphrophilus (Haemophilus), A. actinomycetemcomitans (Actinobacillus) ◻ Cardiobacterium hominis ◻ Eikenella corrodens ◻ Kingella kingae  MUST KNOW! Gram negative bacilli (anaerobes): Bacteroides, Bilophila, Porphyromonas, Prevotella, ◻ Fusobacterium IMPORTANT!!! Calymmatobacterium reclassified into enterobacteriaceae family ◻Spirochetes are GRAM NEGATIVE  Bacterial Cell Wall ◻ Gram positive = with THICK peptidoglycan, retains crystal violet Plasma membrane < periplasmic space < peptidoglycan (THICK) I- bind with CV+ forming CV-I complex which is trapped within the cell wall ◻ Gram negative = with THIN peptidoglycan, decolorized Plasma membrane < periplasmic space, peptidoglycan (THIN) < periplasmic space < OUTER MEMBRANE  MUST KNOW! ◻ Gram positive cocci (aerobes): Micrococcus, Staphylococcus, Streptococcus ◻ Gram positive cocci (anaerobes): Peptococcus, Peptostreptococcus, Sarcina ◻ Gram negative cocci (aerobes): Branhamella, Neisseria, Moraxella ◻ Gram negative cocci (anaerobes): Veilonella (red fluorescence under UV light), Acidaminococcus, Megasphaera  ACID FAST STAIN ◻ Ideal size: 2 x 3 cm ◻ Bailey and Scotts: Gargle with WATER or SALINE ◻ Removal of adherent sputum: Wash sands or small glassbeads with 90-95% spirit or 5% cresol (also known as sand alcohol) ◻ Two types of AFB stains CARBOL FUSCHIN FLUOROCHROME STAINS (auramine-rhodamine and auramine-O) - more sensitive than carbol fuschin, preferred method in detecting AFB  ACID FAST STAIN ZIEHL-NEELSE Non-Aci STEP KINYOUN Acid Fast N d Fast Primary Carbol fuchsin Red Red Stain Steam / Mordant Tergitol Red Red Phenol Acid Alcohol Decolorizer (3% HCl in 95% ETOH) Red Colorless Counter Methylene blue or Blue or stain Malachite green Red Green  NOTES! ◻ MYCOBACTERIUM The ONLY genus that is Acid-fast Gram positive Actual: Gram ghost / neutral Cell wall with: Mycolic Acid stored in Much granules Hard to stain once stained, resists decolorization with an acid (ACID FAST) ◻ Partially acid fast organisms: Nocardia asteroides, Rhodococcus, Legionella micdadei, Isospora, Cryptosporidium Mycobacterium (Acid-fast staining) Serpentine cords (right) -characteristic of M. tuberculosis BACTERIAL CYTOLOGY ◻ CYTOPLASMIC MEMBRANE Selectively permeable membrane Site of energy production ◻ MESOSOMES Point of attachment for chromosome Extension of cytoplasmic membrane ◻ RIBOSOMES Site of protein synthesis BACTERIAL CYTOLOGY ◻ INCLUSIONS Serves for nutrient storage Examples: Much granules → contain lipids (MTB) Babes-Ernst / Metachromatic / Volutin granules → contain polyphosphates (Corynebacterium diphtheriae) Stains: LAMB (Loeffler’s Alk. Met. Blue) Burke’s Modification of Gram stain Bipolar bodies → Y.pestis (“safety-pin appearance” on Wayson’s stain)  BACTERIAL CYTOLOGY ◻ ENDOSPORE Resting cell; highly resistant to dessication, heat and chemical agents Composition: CALCIUM DIPICOLINATE or DIPICOLINIC ACID Bacterial genera with spores: Bacillus – sporulate AEROBICALLY, Catalase (+) Clostridium – sporulate ANAEROBICALLY, Catalase (-) Stains: Shaeffer & Fulton Dorner’s Wirtz & Conklin Identify the bacteria with round and terminal spores ☺ Stained endospores  BACTERIAL CYTOLOGY ◻ CAPSULE Increase virulence by preventing phagocytosis Antigenic; on the basis of serotyping by K antigen Demonstration : Animal tissues and fluids Media containing milk or serum Colonies often slimy Stains: Hiss stain India ink / Nigrosin (Negative/Relief staining) ◻ PILI (Fimbriae) Ordinary pili: adherence of bacteria to host cell Sex pili: bacterial conjugation Identify Klebsiella pneumoniae on what culture medium?  BACTERIAL CYTOLOGY ◻ GLYCOCLAYX Capsule or slime layer A. Capsule - organized material firmly attached to the cell wall Antigen: K Ag Salmonella typhi – Vi (virulence) Ag Antigen specificity detected by Quellung (Neufeld) test ◻ Example organisms: N.meningitides, H.influenza, S.pneumoniae, B.anthracis, K. pneumoniae B. Slime layer - unorganized material not attached to the cell wall Example organism: S. epidermidis S. pneumoniae. “Quellung” German swelling BACTERIAL CYTOLOGY ◻ FLAGELLA For locomotion Antigen: H Ag Atrichous - no flagellum Monotrichous - flagellum on one pole (example: Vibrio) Amphitrichous - single flagellum on each pole Lophotrichous - tuft of flagella at one or both poles Peritrichous - flagella all over the organism (ex.: Enterobacteriaceae except Klebsiella & Shigella) Flagella Flagella Identify Identify Axial filaments of spirochetes BACTERIAL CYTOLOGY ◻ FLAGELLA Periplasmic flagella – axial filaments or periplasmic flagella LAB: Motility 1. Hanging drop preparation Tumbling – Listeria Darting – Campylobacter Gliding - Capnocytophaga 2. Semisolid medium Example: Sulfide Indole Motility (SIM) Optimum temp: 25 degC or RT Note: 37 degC is inhibitory for Listeria & Yersinia Transport (semi-solid) media  BACTERIAL CYTOLOGY ◻ Stains for flagella Contains TANNIC ACID to precipitate and coat flagella Examples: Leifson, Gray, Fisher & Conn, Cesares-Gil, Van Emerson BACTERIAL VIRULENCE FACTORS ◻ Adherence factors - pili ◻ Antiphagocytic factors capsule, some cell wall components ◻ Enzymes ex: Coagulase, Fibrinolysin, Hyaluronidase ◻ Toxins - Exotoxin and Endotoxin  Exotoxin vs Endotoxin COMPARISON EXOTOXIN ENDOTOXIN SOURCE Both Gr(+) & Gr(-) Gram (-) only Metabolic product RELEASE Released upon released by living cell lysis cell COMPOSITION Protein, peptides Lipids (Lipid A) HEAT STABILITY Heat LABILE (except Staph Heat STABLE enterotoxin) IMMUNOLOGIC Converted to toxoids; Not conv.to toxoids, easily neutralized w/ NOT easily neutralized anti-toxin w/ anti-toxin  Exotoxin vs Endotoxin COMPARISON EXOTOXIN ENDOTOXIN Specific Cytotoxin – kills host PHARMACOLOGIC cells General, nonspecific: Enterotoxin-damages -Fever GIT cells -Septic shock Neurotoxin – interferes -DIC w/ nerve impulse transmission TOXICITY HIGH LOW LETHAL DOSE Smaller dose Higher/Larger dose EXAMPLE DISEASES Tetanus/Lock jaw UTI, Typhoid Botulism MUST KNOW! ◻In Tetanus/Lock jaw – Clostridium tetani produces Tetanospasmin (neurotoxin) associated with spasmodic contraction ◻In Botulism – Clostridium botulinum produces Botulinum toxin (most potent exotoxin) which prevents the release of acetylcholine from axons causeing Flaccid Paralysis TEST TO DETECT ENDOTOXIN IN BODY FLUIDS AND SURGICAL INSTRUMENTS ◻ LIMULUS LYSATE TEST- detects endotoxin in body fluids and surgical instruments Reagent: Blood of horseshoe crab (Limulus polyphemus) Principle: In presence of endotoxin, amoebocytes (wbcs) will release lysate (protein) Positive result: CLUMPING Who am I?  BACTERIAL GROWTH FACTORS 1. NUTRIENTS ◻Carbon, Nitrogen, Minerals ◻Salt Halophilic organisms (“salt-loving”) ex: Staphyloccocus (7.5% NaCl), Enterococcus (6.5% NaCl), Vibrio spp. Except V.cholera & V.mimicus ◻Others ◻ MTB (↑ protein) ◻ Haemophilus (X & V factor) ◻ F. tularensis (Cysteine/cystine) ◻ Mycoplasma, Ureaplasma (Sterols)  BACTERIAL GROWTH FACTORS 2. OXYGEN AND CARBON DIOXIDE AVAILABILITY ◻Obligate/strict aerobe Grow only in presence of oxygen WITH Catalase & Superoxide Dismutase (SOD) that converts toxic products to non-toxic subs. Ex: Pseudomonas, Neisseria, Brucella, Bordetella, Francisella, Mycobacterium, Nocardia, most fungi Obligate/strict anaerobe ◻ Grow only in absence of oxygen WITHOUT Catalase and SOD Ex: Bacteroides, Clostridium  BACTERIAL GROWTH FACTORS 2. OXYGEN AND CARBON DIOXIDE AVAILABILITY ◻Facultative anaerobe Aerobe that can grow in absence of O2 (ex: Staph and Strep) ◻Aerotolerant anaerobe Anaerobe that can grow in presence of O2 ◻Microaerophilic (5% O2, 10% CO2, 85% N2) Requires less O2 (ex: Campylobacter – 5-6% O2) ◻Capnophilic Requires 5-10% CO2 (ex: Neisseria & HACEK) Look!  NOTES! ◻ Aerobes grow in ambient air containing 21% Oxygen and small amount (0.03%) Carbon dioxide ◻ Anaerobes cannot grow in the presence of oxygen. The atmosphere in anaerobe jars, bags, or chambers is composed of 5 to 10% Hydrogen, 5 to 10% Carbon dioxide, 80 to 90% Nitrogen, and 0% Oxygen. (Catalyst: PALLADIUM) NOTES! ◻ Microaerophiles grow under reduced Oxygen (5 to 10% Oxygen) and increased carbon dioxide (8 to 10% Carbon dioxide); can also be obtained in specially designed jars or bags ◻ Capnophiles requires increased concentrations of Carbon dioxide (5 to 10%) and approximately 15% oxygen; atmosphere can be achieved by a candle jar or Carbon dioxide incubator NOTES! ◻ Indicator of Anaerobiasis 1. Methylene blue Colorless / White : O2 absent BLUE : O2 present 2. Resazurin White : O2 absent PINK : O2 present Look! Look! BACTERIAL GROWTH FACTORS 3. TEMPERATURE ◻Psychrophilic / Cryophilic Cold temp 0.3 um ◻Viruses: 0.01 to 0.3 um in diameter (small size, pass thru filters) II. CHEMICAL METHODS 1. Ethylene oxide 2. Formaldehyde vapor and vapor phase H2O2 - sterilize HEPA filter 3. Glutaraldehyde - for medical instruments (ex. Bronchoscope) - does not corrode metal and rubber 4. Peracetic acid  DISINFECTION ◻ Destruction of pathogens I. PHYSICAL METHODS 1. Boiling ◻ Temp : 100 degC FOR 15 mins ◻ Destroys vegetative cells 2. Pasteurization ◻ Destroys food pathogen in milk and dairy products ◻ Batch / LTH : 63 degC for 30 mins ◻ Flash / HTH : 72 degC for 15 seconds 3. Non-ionizing radiation ◻ Long wavelength, low energy UV light ◻ Example: Mercury lamps DISINFECTION II. CHEMICAL METHODS ◻ANTISEPTIC - for living tissues ◻DISINFECTANT - for non-living things 1. Alcohol - considered an ANTISEPTIC ◻Ex: 70% Ethyl alcohol / Isopropyl alcohol 2. Quaternary Ammonium Compounds (QUATS) ◻Ex: Benzalkonium Chloride (Zephiran) ◻Inactivated by ORGANIC SUBSTANCES ◻Disadvantages: Nonsporicidal, nontubercoidal DISINFECTION 3. Halogen A. Iodine - remain on skin for 60 seconds ◻Ex: Alcohol-iodine, iodophor (iodine+detergent), iodine tincture (iodine in alcohol) B. Chlorine - active component of NaCl ◻can be neutralized with sodium thiosulfate DISINFECTION 4. Heavy metal ◻Mercury - active ingredient of merthiolate ◻Copper - algicide ◻Silver - 1% Silver Nitrate / Crede’s Prophylaxis in GON 5. Phenol - standard disinfectant PHENOL COEFFICIENT ◻ Expression of the bactericidal power of a particular substance as compared to pure phenol Formula: PC = Highest dilution of disinfectant that can kill org @ given time Highest dilution of phenol that can kill org @ given time Organism used: S.typhi, S. aureus ◻ Interpretation: ◻ PC > 1 : DISINFECTANT BETTER ◻ PC < 1 : phenol better ◻ PC = 1 : same efficiency BIOLOGICAL INDICATORS ◻ Autoclave: Bacillus stearothermophilus ◻ Ionizing radiation: Bacillus pumilis ◻ Dry heat oven: Bacillus subtilis var.niger ◻ Ethylene oxide (ETO): Bacillus subtilis var.globigii BIOLOGICAL SAFETY CABINETS CLASS I BSC ◻Open-fronted, negative pressure, ventilated cabinets ◻Unsterilized room air enters and circulates within the cabinet and exhaust air from cabinet is filtered by HEPA filter CLASS II BSC ◻Sterilize both the air entering and circulating the cabinet and exhaust air ◻Used by most hospital microbiology laboratories ◻Also known as LAMINAR FLOW BSC BIOLOGICAL SAFETY CABINETS CLASS III BSC ◻Provide the highest level of safety ◻All air entering and leaving the cabinet is sterilized with HEPA filter ◻System is entirely close and all infectious material are handled with rubber gloves sealed to the cabinet Class I Open Front  30% 70%  70% 30% 100% CLINICAL SPECIMENS BLOOD CULTURE ◻ Antiseptic: alcohol→iodine→alcohol ◻ Anticoagulant: 0.025% SPS Neutralizes bactericidal effect of human serum Prevents phagocytosis ◻ Blood to CM ratio – Adult- 1:10, Children- 5:10 ◻ Delost: 2 – 3 sx from different venipuncture sites at least 1 hour apart THROAT AND NASOPHARYNGEAL CULTURES ◻ Most abundant normal flora: Viridans Strep. (a-haemolytic strep) ◻ Most common pathogen: Group A Strep (S.pyogenes) ◻ Cultures must include anaerobic conditions for beta Streptococcus (some are Non-hemolytic unless conditions are anaerobic) ◻ Culture on Todd-Hewitt broth for fluorescence microscopy of beta Streptococcus ◻ Nasopharyngeal swab: Haemophilus influenzae, Neisseria meningitidis, Bordetella pertussis SPUTUM CULTURES ◻ Deep cough and examine immediately ◻ Gargle with WATER or SALINE ◻ Examine wet mount before culturing ◻ Specimens with TOO MANY SQUAMOUS EPITHELIAL CELLS and/or few PMNs are NOT SUITABLE for culture ◻ Use BARTLETT’S CLASSIFICATION 25 PMNs / LPF URINE CULTURES ◻ Midstream cleancatch urine ◻ Catheterized urine ◻ Suprapubic urine ◻ Preservative : Boric acid ◻ Colony count 10^5 bacteria/mL indicates infection (done in BAP) ◻ Use calibrated loop A. 1 uL loop (0.001 mL) - factor: 1000 B. 10 uL loop (0.01 mL) - factor: 100 NOTES! Colony count = # of colonies x factor In cases of UTI, ≥ 100, 000 CFU / mL ≥ 1 x 10^5 CFU / mL Escherichia coli - 90% UTI cases Staphylococcus saprophyticus – young females, NOVOBIOCIN RESISTANT CEREBROSPINAL FLUID ◻ Examine immediately or hold in incubator for no longer than 1 HOUR ◻ Centrifuge. Use sediment for: Smears, Gram stain, India ink Culture - Hemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae *Tube 2 – MICRO!!! Tube 4 (if available) for better exclusion of skin contamination *If only 1 tube – MICRO FIRST! ☺  MAJOR LABORATORY RESULTS FOR THE DIFFERENTIAL DIAGNOSIS OF MENINGITIS BACTERIAL TUBERCULAR VIRAL FUNGAL Elevated WBC Elevated WBC Elevated WBC Elevated WBC count count count count Neutros present Lymphos and Lymphocytosis Lymphos and Marked protein monos present present monos present elevation Moderate to Moderate proteinModerate to Markedly marked protein elevation marked protein decreased elevation Normal elevation glucose level Decreased glucose, normal Decreased Lactate level > glucose level glucose lactate level 35mg/dL Lactate level Lactate >25mg/dL Positive Gram stain >25mg/dL Positive India ink and culture Pellicle with Cryptococcus Positive Limulus formation neoformans lysate test with Positive gram negative immunologic test for Cryptococcus organisms neoformans NOTES! BACTERIAL MENINGITIS ◻Birth → 1 month: Group B Strep (S.agalactiae) ◻1 month → 5 years old: H.influenzae ◻5 years old → 29 years old: N.meningitidis ◻> 29 years old: S.pneumoniae ◻Listeria monocytogenes : infants, elderly and immunosuppressed GENITAL AND STOOL CULTURE GENITAL CULTURES ◻Specimens: cervical (female), urethral (male), rectal, throat ◻STD include infections caused by Treponema pallidum, Neisseria gonorrhoeae, Chlamydia trachomatis, Gardnerella vaginalis, Trichomonas vaginalis, Candida albicans and Herpes Simplex Virus STOOL CULTURE ◻For enteric pathogens NOTES! Incubation Period (Culture) ◻Routine: 7 days ◻Brucellosis (Blood, BM): 3-4 weeks ◻Leptospirosis (Blood, Urine): 6-8 weeks ◻Tuberculosis: 8 weeks ◻Signs of Growth ◻Turbidity ◻Bubbles ◻Clots SPECIMEN STORAGE 1. CSF : 37 deg C 2. Urine, stool, swab, viral specimens, sputum, foreign devices such as catheters : 4 deg C ◻Serum for Serology : -20 deg C for 1 week ◻Tissues or specimens for long-term storage : : -70 de C Note: Viral specimens (storage)4 deg C ; (transport) -70 deg C “BELIEVE YOU CAN AND YOU’RE HALFWAY THERE.” ~THEODORE ROOSEVELT #RMT2022