Microbiology Notes PDF

Slide 1 MICROBIOLOGY PREPARED BY: LAODICEA SY, RMT BACTERIA BACTERIA – is a minute, unicellular organisms that reproduces by binary fission. - Prokaryotes (due to absence of a nuclear membrane)...

Slide 1 MICROBIOLOGY PREPARED BY: LAODICEA SY, RMT BACTERIA BACTERIA – is a minute, unicellular organisms that reproduces by binary fission. - Prokaryotes (due to absence of a nuclear membrane) - possess both DNA and RNA Slide 3 BACTERIAL MORPHOLOGY I. ARRANGEMENT – affected by plane of division and position taken after cell division 1. Pairs – diplococci, diplobacilli 2. Chains – streptococci, streptobacilli 3. Grape-like clusters – staphylococci BACTERIAL MORPHOLOGY 4. Group of four – tetrads ex. Peptococcus 5. packet of eight – cuboidal ex. sarcinia 6. Chinese character – bend at the point of division ex. C. diphtheria BACTERIAL MORPHOLOGY II. CAPSULE - slimy layer that surrounds some bacteria; Virulence factor, Anti‐phagocytic. Ex. Cryptococcus neoformans Haemophilus influenza Klebsiella pneumoniae Neisseria meningitidis Salmonella typhi Streptococcus pneumoniae Pseudomonas aeruginosa Slide 6 BACTERIAL MORPHOLOGY III. CELL WALL – imparts in shape; protect bacteria from phagocytosis; correlated with virulence. Gram positive cell wall – consist of Peptidoglycan and techoic acid Gram negative cell wall – peptidoglycan, lipochon, outer membrane and lipopolysaccharide layers. IV. Nucleus – lacks nuclear membrane and mitotic apparatus; nucleoids or chromatin bodies (DNA)  BACTERIAL MORPHOLOGY V. Metachromatic granules/ Volutin granules – means accumulation of food reserves Corynebacterium diphtheriae – babes-Ernst granules Mycobacteria – Much granules VI. Spores/Endospores – highly resistant structure; withstand dryness, heat, poor nutrient. Bacillus anthracis – central spores Clostridium tetani – terminal Clostridium botulinum - subterminal Slide 8 BACTERIAL MORPHOLOGY VII. Pili/Fimbriae – hair-like microfibrils (flagellated g-); electron microscope Types: 1. Common/Ordinary Pili – for bacterial adherence 2. Sex pili – bacterial conjugation; transfer of genetic materials; less common Slide 9 BACTERIAL MORPHOLOGY VIII. Flagella – for motility; thread-like appendages; swarming of proteus Classification: 1. Amphitrichous – polar flagella on both ends 2. Monotrichous – 1 polar flagella 3. Lophotrichous – tuft of flagella at one end 4. Peritrichous – all around bacteria 5. Atrichous – no flagella Slide 10 BACTERIAL MORPHOLOGY IX. SHAPE 1. bacilli – rods 2. Cocci – spheres 3. Spiral shape – ex. a. Vibrio – straight rods or with single rigid curve b. Spirillum – rigid helical rod c. Spirochete – flexous helical rod 4. Pleomorphic organisms – vary in size and shape (ex. bacteroides) Slide 11 BACTERIAL MORPHOLOGY X. SIZE * micrometer – unit of measurement * Bacillus anthracis – largest pathogenic bacillus - 1 x 3-10um *Haemophilus – smallest pathogenic bacillus - 0.2 x 0.5um BACTERIAL PHYSIOLOGY I. Oxygen requirement: 1. Obligate aerobe – strict Oxygen required (free oxygen); has superoxide dismutase to protect from the toxic oxygen. 2. Microaerophiles – Campylobacter ( 5% O2, 10% CO2, 85% N2‐ campy gas) – requires reduced amount of free oxygen 3. Obligate anaerobe ‐ 5% CO2, 10% H2, 85% N2 (Gas Pak Jar) – can’t grow in the presence of oxygen; lack SD 4. Facultative anaerobe – With or without Oxygen; 5. Aerotolerant anaerobes‐ does not grow well but survives in the presence of free (atmospheric) oxygen Ex. Lactobacillus Slide 13 BACTERIAL PHYSIOLOGY II. Nutritional requirement A. autotrophs / lithotrophs – inorganic compound as carbon source ex. CO2 B. heterotrophs /organotrophs‐organic compound as carbon source ex. Glucose, amino acid - most medical important bacteria are heterotrophs BACTERIAL PHYSIOLOGY III. Temperature 1. Psychrophilic = 0‐20’C (refrigerator); blood bank contaminants ex. Listeria monocytogenes 2. Mesophilic = 20‐40’C (pathogenic); most pathogens 3. Thermophilic = 40‐60’C; basis of test for effective autoclaving IV. pH requirement:  Acidophilic ‐ Lactobacillus, Fungi  Neutrophilic‐ pathogenic bact. (pH7.2‐ 7.4)  Basophilic (alkaliphilic) ‐ Vibrio  BACTERIAL PHYSIOLOGY Humidophilic - moisture Halophilic – salt concentration Ex. Staph. aureus ‐ 7.5% NaCl, 6.5 % NaCl - Enterococci 3‐8% NaCl - Vibrio spp Slide 16 STERILIZATION I. STERILIZATION METHOD‐ moist heat (coagulates CHON) 1. AUTOCLAVE = uses steam under pressure at 121’C 15lbs psi 15 min  Best sterilization and for infectious waste disposal  Best for bacteriologic media sterilization  Uses Bacillus stearrothermophilus at 56 C (QC) 2. INSPISSATION = 75‐80’C 2hrs 3days – best for high protein media (L‐J, Loeffler’s)  STERILIZATION 3. TYNDALLIZATION = flowing steam 100’C 30min 3days 4. BOILING = 100’C 15-30 mins; non sporicidal; kills all vegetative organisms 5. PASTEURIZATION = milk and dairy products disinfection A. LTH/Batch (Low Temperature Holding) 62 oC for 30 mins B. HTST/Flash (High Temperature Short Time) 72 oC 15 secs Slide 18 STERILIZATION II. STERILIZATION METHOD‐ dry heat (sporocidal); kills by oxidation 1. HOT AIR OVEN = drying  Dry heat method; Dry hot air at 170‐180’C for 2hours;  Glass wares, cotton swabs, metallic instruments, oils, powders  Bacillus subtilis spores (QC) incubated at 35-37C 2. INCINERATION = Not used 3. CREMATION = control diseases Slide 19 STERILIZATION 4. Flaming = needles and inoculating loop III. GAS‐ ETHYLENE OXIDE (cold sterilization) = heat labile machine or rubber and plastic tubes; bacillus subtilis spores (QC) at 35-37 C Slide 20 STERILIZATION IV. RADIATION – ionizing radiation (high energy gamma rays); UV light: used for disposable plastic syringes, catheters and gloves. V. FILTRATION – method of choice for antibiotic solutions, toxic chemicals, radioisotopes, vaccine and CHO. - cellulose acetate or cellulose nitrate membrane with vacuum Slide 21 DISINFECTION DISINFECTION – the process of removing micro-organisms, including potentially pathogenic ones, from the surfaces of inanimate objects. 1. SODIUM HYPOCHLORITE (CLOROX)‐ 10% bleach ; best spillage disinfectant 2. IODINE/ IODOPHOR‐ best antiseptic; Iodine plus detergent ex. Povidone Iodine 3. 70% ETHYL ALCOHOL‐ non sporocidal 4. HYDROGEN PEROXIDE‐ cleansing of wound 5. 1% SILVER NITRATE‐ eye drops to prevent gonococcal opthalmia neonatorium Slide 22 DISINFECTION 6. FORMALDEHYDE‐ preservative; not good for bacteria culture 7. GLUTARALDEHYDE ‐ cold sterilant; 2% aqueous glutaraldehyde (for lensed instrument 8. PHENOL (CARBOLIC ACID)‐ standard disinfectant; 0.5%–3% solution for disinfecting; does not kill spores 9. LYSOL (CRESOL)‐ hard surface disinfectant 10. QUATS‐ surface disinfectant ( Zephiran) Slide 23 BIOSAFETY CABINET Slide 24 BIOSAFETY CABINET Class 1 Class 2 Slide 25 BIOSAFETY CABINET Class 3 Class 3 Slide 26 BIOSAFETY LEVEL Slide 27 CATEGORY SAFETY Slide 28 BACTERIAL GROWTH CURVE 1. LAG phase / adjustment phase/ adaptation phase  Period of adaptation; Increase in cell size not in numbers. 2. LOG phase / exponential phase  Increase in growth rate (cell division)  Susceptible to antimicrobial agents 3. Stationary / Plateau phase  Cell reproduction equals the rate of cell death 4. Death phase / period of decline  Increase death rate; cessation of multiplication Slide 29 STAINING PROCEDURE 1. Simple stains – one dye (ex. Crystal violet) 2. Differential stains – more than one dye A. Gram stain – bacteria w/thick cell walls containing Techoic acid, retain the crystal violet-iodine complex after decolorization. B. Acid fast – primary stain binds to Mycolic acid in the cell wall of mycobacteria and retained after decolorization. Slide 30 GRAM STAIN Reagent Purpose Gram + Gram - Crystal violet Primary dye Violet Violet Iodine Mordant Violet Violet 95% ethyl alcohol or decolorizer Violet Colorless acetone or combination Safranin Counterstain Violet Pink Slide 31 Gram Stain ‐ General Rule: Gram Positive bacteria 1. All cocci are gram (+) except Neisseria, Veilonella, Moraxella 2. All bacilli are gram (–) except Mycobacteria, Corynebacteria, Clostridia, Bacillus, Lactobacillus, Listeria, Erysiphilothrix, Nocardia, Actinomyces 3. All spiral org are reported as gram (– ) 4. Yeasts are Gram (+) Slide 32 Gram (+) becomes Gram (‐): 1. Over‐decolorization, Old,dying 2. Use of acidic iodine as mordant 3. Penicillin, Omit iodine Gram (‐) becomes Gram (+): 1. under‐decolorization; 2. thick smear Slide 33 ACID FAST STAIN Purpose Reagent Reagent Acid fast Non acid Ziehl-Neelsen Kinyoun organism fast Organism Primary stain Carbol fuchsin Carbol fuchsin Red Red Mordant Heat Phenol Red Red Decolorizer Acid alcohol Acid alcohol Red Colorless Secondary stain Methylene blue Methylene Blue Red Blue/green *best AFB stain * Modified AFS/Kinyoun: Uses 1% H2SO4 as decolorizer *tissue stain Slide 34 STAINING PROCEDURE 3. SPECIAL STAIN – used to emphasize certain special structures or features of organism.  A. Capsule – Negative stain (colorless object against dark background ex. India ink, nigrosin method * Capsular stain – Hiss, Gin’s, Welch’s, Anthony  B. Spore – Dorner, Wirtz Conklin, Scheffer‐Fulton, Heat and Acetic acid  C. Metachromatic granules ‐Albert’s, LAMB (Loeffler’s alk. Methylene blue), Neisser’s, Ljubinsky method  D. Flagellar stain‐ Leifson, Grays, Fisher & conn, Casares -Gil  E. Nucleic acid ‐ Fuelgen  F. Polar bodies‐ Wayson  G. Rickettsia‐ Gimenez, Giemsa , Macchiavelo  H. Spirochetes‐ Levaditi Slide 35 CULTURE MEDIUM Culture medium – any material containing necessary nutritional and environmental requirements for bacterial growth. Classification of Culture Media: A. Physical state/Consistency: 1. Liquid – no solidifying substances (ex. Nutrient broth, thioglycolate) 2. semi-solid – 0.5 – 1.5% of agar (ex. SIM) 3. solid – 2-3% of agar (ex. BAP, CAP, TSI) 4. Biphasic= both liquid and solid Slide 36 CULTURE MEDIUM TYPES OF CULTURE: 1. Pure Culture‐ one organism (ID and AST)  A. Streak plate – to isolate organisms in pure culture  B. Pour Plate – to determine the type of hemolysis produced by streptococci; determine approx. no. of viable organisms in liquid.  C. Selective medium – media containing antibiotics  D. Animal Inoculation 2. Mixed Culture‐ more than 2 organism 3. Stock Culture‐ Quality Control strain; used for source of supply in research, and student use. Slide 37 CULTURE MEDIUM TYPE EXPLANATION EXAMPLES Nutritive Supports growth of most nonfastidious bacteria Nutrient agar, trypticase soy agar Enrichment contains added growth factors, e.g., blood, vitamins, Sheep blood agar (SBA), yeast extract chocolate agar (CHOC), brain heart infusion, buffered charcoal yeast extract (BCYE) agar Selective Contains additives such as dyes, bile salts, alcohols, Columbia colistin–nalidixicacid acids, or antibiotics to inhibit growth of certain (CNA) agar, eosin methylene blue bacteria (e.g., gram pos) (EMB), MacConkey (MAC), Hektoen enteric (HE), xylose lysine deoxycholate (XLD), Thayer Martin Differential Contains compounds that allow certain bacteria (or EMB, MAC, HE, XL even species) to be visually differentiated (e.g., lactose fermentation, hydrogen sulfide [H2S] production) Slide 38 HEMOLYTIC REACTIONS ON SHEEP BLOOD AGAR HEMOLYSIS DESCRIPTION EXPLANATION EXAMPLES Alpha Green zone around Partial lysis of red blood Streptococcus pneumoniae colony cells (RBCs) and viridans streptococci Beta Clear zone around Complete lysis of RBCs Group A strep (GAS), group colony B strep (GBS), Listeria monocytogenes Gamma (non No zone of hemolysis No lysis of RBCs Enterococcus faecali hemolytic) Slide 39 HEMOLYTIC REACTIONS ON SHEEP BLOOD AGAR Alpha hemolysis Beta hemolysis Slide 40 HEMOLYTIC REACTIONS ON SHEEP BLOOD AGAR Gamma hemolysis Slide 41 CULTURE MEDIUM FOR AEROBES & ANAEROBES MEDIUM TYPE FOR ISOLATION OF DESCRIPTION SBA (Blood agar) E, D Most nonfastidious bacteria Tryptic soy agar with 5% sheep blood. Allows differentiation of hemolysis. Chocolate Agar E Fastidious organisms; Supplies X (hemin) & V (NAD) Haemophilus & Neisseria factors. Incubate in 5% co2 CNA agar (Columbia S Gram positive; wounds, stool Colistin & nalidixic acid suppress Colistin Nalidixic acid) culture most gram negative (GN). Contains 5% sheep blood but should not be used to observe hemolytic reactions Phenylethyl alcohol agar S GPC & anaerobic gram Phenylethyl alcohol inhibits enteric (PEA) negative rods (GNR) GNR. Contains 5% sheep blood. Slide 42 Chocolate Agar CNA Slide 43 CULTURE MEDIUM FOR AEROBES & ANAEROBES MEDIUM TYPE USE DESCRIPTION Brain heart infusion E, S Fastidious and Useful for culturing streptococci, agar nonfastidious; anaerobes pneumococci, and meningococci; included Can add vancomycin to detect vancomycin resistant enterococci(VRE) EMB (Eosin Methylene S, D Enteric GNR Eosin & methylene blue inhibit BLue) gram positive (GP). Lactose fermenters(LF) green black or purple. E. coli produces green metallic sheen. Nonlactose fermenters (NLF) colorless or light purple. MAC agar S, D Enteric GNR Bile salts & crystal violet inhibit (MacConkey) most GP. LF will be pink. NLF colorless. Slide 44 EMB BHI MAC Slide 45 CULTURE MEDIUM FOR AEROBES & ANAEROBES MEDIUM TYP USE DESCRIPTION E Sorbitol MacConkey S E. coli O157:H7 E. coli O157:H7 doesn’t ferment sorbitol. (SMAC) agar Colorless colonies. Some labs have stopped using because non O157 serotypes can be pathogens HE agar (Hektoen S, D Salmonella & Shigella in Bile salts, bromothymol blue, & acid fuchsin inhibit Enteric) stool normal gastrointestinal(GI) flora. Nonpathogensor ange to salmon pink. NLF green to blue green. H2S pos colonies have black precipitate. XLD (Xylose lysine S, D Salmonella & Shigella in Deoxycholate inhibits many GNR & GP. 4 types deoxycholate) stool of colonies: yellow (e.g., E. coli), yellow with black centers (e.g., some Proteusspecies), colorless or red colonies (e.g., Shigella),red colonies with black centers (e.g., Salmonella). (Some shigellae may be inhibited. Some salmonellae Salmonella Shigella (SS) S Salmonella & Shigella in Brilliant green & bile salts inhibit other enterics. agar stool Salmonella & Shigella don’t ferment lactose (colorless colonies). Salmonella produces H2S (black center). Slide 46 SSA HE XLD (Salmonella) Slide 47 CULTURE MEDIUM FOR AEROBES & ANAEROBES MEDIUM USE USE DESCRIPTION Deoxycholate citrate S Salmonella & Shigella Other nonpathogenic enterics inhibited agar Thioglycolate medium E Campylobacter from stool, Subculture to Campy selective agar wound cultures after overnight incubation at 4°C. Campylobacter blood E, S Campylobacter from stool Incubate plates in 5% CO2 at 42°C agar (Campy BAP) Slide 48 Deoxycholate Citrate Agar Thioglycolate Medium Slide 49 CULTURE MEDIUM FOR ANAEROBES MEDIUM USE Blood agar, anaerobic CDC Enrichment medium for isolation of fastidious anaerobes (yeast extract, L-cysteine, hemin, vit. K) Bacteroides Bile Esculin (BBE) S, D for isolation & identification of Bacteroides fragilis; hydrolysis of esculin – blackening of agar; Bile salts, & gentamicin act as inhibitors. Egg yolk Agar (EYA) Det. Of lecihinase & lipase production of Clostridia & fusobacteria. Cooked Meat Medium Isolation of anaerobes, especially Clostridium. Laked Kanamycin-vancomycin Selective medium for Bacteroides & Prevotella. Blood agar (LKV) Phenylethyl Alcohol Agar (PEA) Inhibits enteric G(-) rods & swarming by some Clostridia. Slide 50 CULTURE MEDIUM FOR ANAEROBES MEDIUM USE Cycloserine Cefoxitin Fructose Egg Selective for Clostridium difficile. yolk Agar (CCFA) Thioglycolate Broth (THIO) All purpose medium (support most growth of aerobes & anaerobes); TOP – aerobes, BOTTOM – strict anaerobes, THROUGHOUT – facul. Anerobes; store at room temp. (boil & cool prior to use). Slide 51 CULTURE MEDIUM FOR ISOLATION OF NEISSERIA GONORRHOEAE AND NEISSERIA MENINGITIDIS MEDIUM COMMENTS Modified Thayer Martin Vancomycin, colistin, nystatin, & trimethoprim(TMP) inhibit growth of (MTM) normal genital flora. Incubate in ↑ CO2. Some N. gonorrhoeae may be inhibited. Martin Lewis Similar to Thayer Martin, but different antibiotics. Inhibits yeast better. Incubate in ↑ CO2. New York City medium (NYC) Incubate in ↑ CO2. Some N. gonorrhoeae are inhibited by antibiotics. Genital mycoplasmas will grow. GC LECT Antibiotics to inhibit GN & GP bacteria & yeast. JEMBEC plates For transportation& growth of N. gonorrhoeae. Plates contain Neisseria selective medium & come with resealable polyethylene bag & CO2 generating tablet. No need to transfer to culture plate. Slide 52 NYC MEDIUM Modified Thayer Martin JEMBEC plates Slide 53 SPECIAL BACTERIOLOGIC MEDIA Slide 54 CIN agar (Yersinia) Alkaline peptone water Slide 55 LOEFFLER Cystine Tellurite Agar Tindale agar (Corynebacterium) Slide 56 SPECIAL BACTERIOLOGIC MEDIA Slide 57 BCYE TCBS Bordet-Gengou Agar & Regan Lowe Slide 58 SPECIMEN AND COLLECTION Slide 59 COLLECTION Kinds of Swab: 1. Cotton tipped swab – most commonly used; Cotton‐ good for virus, toxic to Neisseria (use charcoal to detoxify) 2. Calcium alginate tipped swab – excellent substitute for cotton swab; don’t use in Herpes virus (inhibit replication); good for Neisseria 3. Polyester tipped swab – Dacron or Rayon; good for bacteria and virus Slide 60 SPECIMEN TRANSPORT Transport medium 1. Cary Blair‐ rectal swab 2. Stuart’s‐ good for bacteria and virus 3. Amies – respiratory sample 4. Transgrow ‐ Neisseria 5. JEMBEC – Neisseria 6. Todd Hewitt – vaginal carriage Slide 61 REMEMBER: Organisms Requiring Incubation in Increased CO2 : 1. Campylobacter(10%–15% CO2) 2. Haemophilus(5%–10% CO2) 3. Helicobacter(5%–10% CO2) 4. Moraxella catarrhalis(5% CO2) 5. Mycobacterium(5%–10% CO2) 6. Pathogenic Neisseria (5%–10% CO2 Slide 62 ANTIMICROBIAL SUSCEPTIBILITY TEST Slide 63 ANTIMICROBIAL SUSCEPTIBILITY TEST Methods: I. Dilution Tests 1. Broth/tube dilution method – bets method but time consuming; MIC &MBC 2. Agar dilution method – agar plates inoculated with Steers-Foltz replicator; research lab. Technique. 3. Disk agar diffusion – utilizes a filter paper disk (impregnated w/specified amt. of antimicrobial agents). A. The Kirby-Bauer Technique - aerobic and facultative bacteria Medium – Mueller-hinton agar Temperature – 35‐37’C (MRSA‐35’C) pH – 7.2 – 7.4 Depth – 4 mm Standard Inocolum – 1.5 x 108 Antibiotic disc= 6mm Slide 64 Kirby Bauer Technique Mueller Hinton agar Slide 65 ANTIMICROBIAL SUSCEPTIBILITY TEST Condition – Aerobic, No CO2 Incubation – 16-18 hours Standard = 0.5 McFarland (1%H2SO4 and 1.175% BaCl2) FALSE SENSITIVE: 1. Light inoculum 2. Thin medium FALSE RESISTANT: 1. Heavy inoculum, Thick medium 2. Delay in application 3. Ca/ Mg ‐ aminoglycoside (P. aeruginosa) 4. Thymine‐Thymidine‐ SXT (Enterococci) Slide 66 BIOCHEMICAL IDENTIFICATION OF BACTERIA 1. Catalase test Catalase – produces water and oxygen from hydrogen peroxide (H2O2). * drops of H2O2 + bacterial smear on a microscope slide = bubbles (+) (If catalase is present, h20 & o2 (bubbles) will form) Staphylococci (+) Streptococci (-) 2. Coagulase test a. Clumping factor (slide coagulase): Formerly slide coagulase tests; used rabbit plasma (S. lugdunensis. & S. schleiferi can also be (+). * Based on Latex agglutination – high sensitivity & specificity for S. aureus; detects protein A Slide 67 BIOCHEMICAL IDENTIFICATION OF BACTERIA b. Tube coagulase test – uses rabbit plasma; incubated at 37°C for up to 24 hours. - Tests must be checked at 4 hours for clot formation (some produce staphylokinase which can cause false (-) result.) * S. aureus (+) * S. intermedius & S. hyicus – animal pathogens; (+) Slide 68 BIOCHEMICAL IDENTIFICATION OF BACTERIA 3. PYR test - detects the enzyme L-pyrrolidonyl arylamidase. - colony is placed on filter paper + pyrrolidonyl-a-naphthylamide (PYR) = red color (after the addition of /p-dimethylaminocinnamaldehyde (DMACA)) Streptococcus pyogenes and Enterococcus spp. (+) differentiate S. aureus (-) from S. lugdunensis & S. schleiferi (-) Slide 69 BIOCHEMICAL IDENTIFICATION OF BACTERIA 4. Bile solubility test Streptococcus pneumoniae colonies – soluble in sodium deoxycholate (bile). In the presence of the bile at 37°C, colonies autolyse within 30 minutes, and disappear from the agar surface. 5. Hippurate hydrolysis test - detects the bacterial enzyme hippuricase (which hydrolyzes hippurate to glycine and benzoic acid. ) after addition of ninhydrin – Purple color (+) Group B streptococci (+) differrentiate Campylobacter jejuni (+) from most other Campylobacter spp. Slide 70 Bile Solubility Test Hippurate Hydrolysis Test Slide 71 BIOCHEMICAL IDENTIFICATION OF BACTERIA 6. Oxidase test - detects cytochrome oxidase (used in the electron transport system) * drops of oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) + filter paper w/bacterial colonies or plate colonies = Purple color (10-15 sec) * Media with a high concentration of glucose can inhibit oxidase activity. * use colonies from nonselective, nondifferential media. Slide 72 CRITERIA FOR REJECTION OF SPECIMENS IN MICROBIOLOGY Unlabeled or improperly labeled specimen Syringes with needles attached Improper collection site Culture for anaerobes requested on inappropriate sources or not received in Prolonged transit (over 2 hrs without anaerobic transport tube preservation) Specimen received in formalin Improper temperature during transport or storage Saliva instead of sputum Leaking specimens Foley catheter tip Specimens in nonsterile containers Insufficient quantity Improper transport medium Formed stool for C. difficile toxin testing Dry swab Swab for acid fast bacilli (AFB) or fungal smear and culture Improper swab, e.g., wood or calcium alginate for viruses or Chlamydia Slide 73 GRAM POSITIVE COCCI STAPHYLOCOCCUS * non motile, spherical, grape-like clusters * aerobic or facultative anaerobic * catalase positive (differentiates staph from strep)  gram positive cocci in clusters, pairs  catalase positive, “pinhead” colonies  facultative anaerobes, Culture medium: BAP, CAP  Selective medium: PEA, Chapman, Vogel‐Johnson, Columbia CAN Slide 74 GRAM POSITIVE COCCI Differentiate Micrococcaeae Micrococcaeae Lysostaphin and Furazolidone Staphylococcus Modified Oxidase (‐) S Micrococcus Modified Oxidase (+) R Stomatococcus Modified Oxidase (‐) R Slide 75 STAPHYLOCOCCI A. Staphylococcus aureus I. Antigenic structure: 1. Teichoic acid – contains ribitol techoic acid in c.w. (s. aureus) 2. peptidoglycan – protects from lysis; adherence 3. Protein A – prevents phagocytosis by PMN 4. Clumping factor – clumping of staph. in the presence of plasma (s. aureus) 5. Capsular polysaccharide – protects from phagocytosis Slide 76 STAPHYLOCOCCI II. Extracellular enzymes 1. Hyaluronidase – digests connective tissue; spreads infxn. 2. Staphylokinase – dissolves clots 3. DNase – digests DNA 4. Lipases – degrades fat/oil; colonizes skin 5. Penicillinase ‐ inactivates penicillin 6. Gelatinase‐ liquify gelatin Slide 77 STAPHYLOCOCCI III. Toxins Leukocidin – lyses WBC; panton-valentine Enterotoxin – food poisoning; antibiotic induced pseudomembranous colitis Exfoliative toxin (epidermolysin) – skin scalded syndrome Toxic shock syndrome toxin (TSST)=shock/organ failure (tampon usage) IV. Infections * No. 1in skin infections – Carbuncles, furuncles, Folliculitis, cellulitis, wound, impetigo. * No.1 in Osteomyelitis, No.1in Nosocomial infection * Scalded skin syndrome, toxic shock syndrome, food poisoning Slide 78 TAMPON Skin Scalded Syndrome Toxic shock syndrome Slide 79 STAPHYLOCOCCI Lab. Diagnosis: 1. Gram stain - 2. Catalase Test – Catalase enzyme on 3% H2O2  (+) gas bubbles; (+) Staphylococcus; (‐) Streptococcus 3. Coagulase test Bacterial colonies Clot (4hrs.) A. Slide (screen)‐ rapid screening; cell bound coagulase;30 seconds B. Test tube (confirm) – confirm slide neg. result; extracellular coagulase Medium: rabbits plasma with EDTA; (+) Staph. aureus Slide 80 STAPHYLOCOCCI 4. Mannitol Fermentation test Mannitol Salt agar (7.5% NaCl) (+) = Yellow and (‐) = red 5. DNASE test= Detects deoxyribonuclease * Two methods: A. Toluidine blue – pink zone;Methyl green‐ clear zone B. HCL precipitation – no precipitation after 1N HCL; Dnase (+) = colorless or Clear zone 6. Novobiocin Test  CNS differential test, ID of Staph. saprophyticus  R= less than 16mm; S= equal or more than 16mm Slide 81 STAPHYLOCOCCI 7. Modified Oxidase Test Rgt: Tetramethyl p‐phenylene diamine dihydrochloride in DMSO. Matrix Assisted Laser Desorption/Ionization Time Of Flight (MALDI TOF) mass spectrometry - ID of staphylococci at species level is >97% but lower for coagulase negative staph. - analyzing peptides, lipids, and saccharides. Slide 82 COAGULASE NEGATIVE 2. Staphylococcus epidermidis - cause infection to immuno-compromised host - skin flora; like to attach in prosthetic heart valves, catheters, CNS shunt, hip prosthesis - Novobiocin sensitive 3. Staphylococcus saprophyticus - UTI in young sexually active women; Novobiocin Resistant Slide 83 SUMMARY OF TESTING Test Staph. aureus Staph epidermitidis Staph. saprophyticus Catalase + + + Coagulase + - - Colony Yellow White white DNAse + - - Gelatinase + + + Mannitol + - +/- Novobiocin S S R Phosphatase + + - Slide 84 STREPTOCOCCI - facultative anaerobe; gram positive cocci in chain, pairs; catalase (-) - pinpoint colonies; Capnophilic ( 5‐10% CO2) According to Classification: 1. Smith and Brown classification – based on hemolytic reaction in BAP A. Alpha streptococci – greenish zone (inc. hemolysis) * S. pneumoniae, Viridans Streptococcus B. Beta streptococci – Colorless zone (complete hemolysis) * S. pyogenes, S. agalactiae, Grps C, F, G C. Gamma streptococci – no zone (no hemolysis) * E. fecalis, E. faecium, S. bovis (grp D) Slide 85 STREPTOCOCCI 2. Lancefield Classification – based on cell wall antigen or antigenic characteristic (cell wall polysaccharide). A. Group A streptococci – Streptococcus pyogenes * M protein – major virulence; resistant to phagocytosis * Hemolysins: Streptolysin O (SLO) – antigenic toxin; o2 labile; sub surface hemolysis Streptolysin S (SLS) – non-antigenic; o2 stable; surface hemolysis * Erythrogenic Toxin – Scarler fever * Hyaluronidase – spreading factor * Streptokinase – dissolves clot Slide 86 STREPTOCOCCI Disease and Lab. Identification: 1. Acute Pharyngitis 2. skin: impetigo, erysipelas, wound infection 3. Scarlet Fever * Dick’s test – skin test * Schultz-Charlton – blanching phenomenon; antitoxin is injected (neutralization) 4. Toxic Shock Syndrome Slide 87 STREPTOCOCCI Laboratory Identification: 1. Gram stain – gram (+) cocci in chain 2. Culture – BAP 3. Catalase – negative (no gas bubbles) 4. Basitracin (Taxo A)‐ sensitive (any zone) 5. SXT test ‐ resistant 6. PYR test – positive (red) Slide 88 STREPTOCOCCI B. Group B streptococci – Streptococcus agalactiae * beta hemolytic; NF of G.I., genitourinary tract and vagina, pharynx * Bovine mastitis; (1)neonatal sepsis; meningitis; UTI * penicillin – antibiotic of choice Lab. Identification: 1. CAMP test – arrow head zone of BH 2. Hippurate Hydrolysis Test ‐ purple Slide 89 STREPTOCOCCI C. group C Streptococci - Strep. equisimilis – source of streptokinase Strep. Equi – dse. In horses Strep. Dysagalactiae * Beta hemolytic; Basitracin Resistant; SXT sensitive D. group D streptococci – Normal fecal and oral flora; Alpha-hemolytic or nonhemolytic; Colonies are gray to white, translucent, round, and convex. 1. Enterococcus 2. Non enterococcus Slide 90 STREPTOCOCCI A. Enterococcus  E. faecalis – UTI, wound  E. faecium, E. durans B. Non enterococcus Strep. bovis Strep. Equinus –UTI Laboratory Identification: 1. Bile Esculin Positive Bile esculin medium (blackening); Differentiates GRP D from other Strep S 2. 6.5% Nacl test – (+) enterococcus 3. PYR test ‐ (+) enterococcus Slide 91 TEST TO DIFFERENTIATE ENTERO TO NON Enterococcus Non-enterococcus 1. Growth in 6.5% NaCl + - 2. PYR & Hippurate + - Slide 92 STREPTOCOCCI Streptococcus pneumoniae * Encapsulated gram (+) cocci; lancet shaped/bullet shaped; facultative anaerobe * Requires choline; glistening dome shaped young colonies (BAP), doughnut/checker/nail head (older colonies). * important cause of community-acquired bacterial pneumonia. * Adult bacterial meningitis (no.1), pneumococcal pneumonia, otitis media (most common cause) *Pneumolysin O – O2 sensitive; cytolytic for cells C substance – react with CRP Slide 93 STREPTOCOCCI Laboratory Diagnosis: 1. Gram stain - gram (+) diplococcic 2. Culture - SBA (alpha, mucoid) 3. Optochin Sensitivity Test – presumptive test; Taxo P; (+) >14 mm zone 4. Bile Solubility – lysis of colony (+) 5. Neufeld Quellung/ Capsular precipitation Rxn. – rapid method; capsular swelling 6. Mouse virulence test – death 7. Francis test – skin test *Penicillin – antibiotic of choice Slide 94 STREPTOCOCCI Viridans Streptococci * NF of mouth, nasopharynx, GI tract, female genital tract * Not classified under Lancefield; Optochin resistant 1. Streptococcus sanguis – bacterial endocarditis 2. Streptococcus mutans – dental carries 3. Streptococcus mitis Slide 95 TEST TO DIFFERENTIATE VIRIDANS STREP Group A Group B Group C, F, G Bacitracin S R R SXT R R S CAMP - + - Hippurate - + - PYR + - -