Microbiology/Mycobacteriology PDF

142 INOCULATION 3. Number of colonies x 100 (.OJ loop) or 1. Streak for isolation wit h nichrome or number of colonies x 1000 (.001 loop) pl atinum, or disposable loops...

142 INOCULATION 3. Number of colonies x 100 (.OJ loop) or 1. Streak for isolation wit h nichrome or number of colonies x 1000 (.001 loop) pl atinum, or disposable loops 4. Read and report after 18-24 hrs 2. Calibrated 0.01 ml or 0.001 ml (. 001 ml for urine colony count plates) Stains Commonly Used in Microbio/ogy/Mycobacteriology PRIMARY DECOLORIZER COUNTERSTAIN RESULTS PRINCIPLE STAIN POS NEG Gram Stain Crystal Violet Alcohol/Acetone Safranin Purple Pink Iodine mordant Methanol or heat tix Violet dye & iodine form complex in cell; washes out of gram neg cells Kinyoun & Carbol Fuchsin Acid Alcohol Methylene Blue Pink Blue Acid tast Ziehl-Nielson For Mycobacteria Auramine- Auramine and Acid Alcohol Potassium Orange No For Mycobacteria Rhodamine Rhodamine Permanganate Fluoresc. Fluoresc. (Fluorescent Stain) Calcofluor Calcofluor White Bluish- No For yeast and fungi White + 10%KOH white Fluoresc. KOH to break down Fluoresc. debris and mucous Gram Positive Cocci STAPHYLOCOCCUS if methicillin-resistant S. aureus 1. "Grape-like" clusters (MRSA), vancomycin is drug of choice 2. S. a:ureus g. Laboratory diagnosis a. Coagulase positive ❖ BAP - soft, opaque, regular b. Most common pathogen of genus colonies 2-3 mm in diam; some are c. Common infections beta hemolytic and some have pale ❖ Furnncles (boils) and carbuncles golden color ❖ Biillous impetigo (blisters) ❖ Growth in 7.5% NaCl and ferment ❖ Paronycbia (nails) mannitol ❖ Post surgical wounds and ❖ Catalase positive and coagulase bacteremia positive d. Intoxications ❖ Pulse Field Gel Electrophoresis ❖ Scalded skin syndrome (PFGE) and susceptibility (exfoliatin - neonates) proflle for epidemiologi.c studies ❖ Toxic shock syndrome (TSST-1) - women ages 12-52 3. Coagulase negative Staphylococcus ❖ Food poisoning (en terotox.in) - a. Opportunist in symptoms in 1-5 hrs after immunocompromised hosts and ingestion (potato salad , cream patients with prosthetic valves and dislies) devices e. Exotoxins - h emolysins, leukocidins, coagulase and hyaluronidase (spreading factor), nuclease, protease and lipase f. Resistance/sensitivity ❖ Most resistant to penicillin due to Susceptibility Teating of plasmid mediated B-lactamase Staphylococcw ❖ Some sensitive to penicillinase- resistant penicillins (PRP's) (methicillin , oxacillin, etc);  143 Biochemical Tests COAGULASE TEST CATALASE TEST 1. Rea gent - EDTA rabbit plasma 1. Reagent: 3% H202 2. Bound coagulase - clumping on slide 2. Add one drop to colony on slide (plasma and colony) 3. Free coagulase - gels in tube test (0.5 ml 3. If catalase present, H 20 2 is broken plasma and colony; 35-37°C 4-12 hrs) down to water and 0 2 (which bubbles off) 4. Agglutination tests - detect coagulase and protein A 4. Positive: Staph; negative - Strep 5. Positive: S. aureus; negative: other 5. QC each day of use Staph (human pathogens) Staphylococcus (Catalase Positive) ORGANISM COAGULASE INFECTIONS INTOXICATIONS NOTES S. aureus + Carbuncles, furuncles, Scalded Skin Syndrome, Most beta lactamase +; paronychia, wounds, Toxic Shock Syndrome, many MRSA and bacteremia and Gastritis (enterotoxin, CA= community acquired 1-5 hours after eating) HA=hosptial aquired S. epidermidis - Endocarditis, prosthe- Most methicillin resistant; tic device infections sensitive to novobiocin S. saprophyticus - UTI in young women Resistant to novobiocin Coag Negative Staph: Opportunistic Infections in lmmunocompromised Patients and Those with Prosthetic Devices Staphylococcus aureus Staph aureus is my nickname Preparation of Kirby-Bauer In the Plates 1UJd bJoculum pathogen Hall of Fame Look for bubbles Organisms Used for QC in with catalase BUBCeptibility resting And for clots with coagulase. STREPTOCOCCUS Gram positive clusters everywhere Skin and gut, nose and hair. 1. Spherical or oval; chains or pairs Protein A is my main trait 2. Lancefield grouping based on C carbohydrate and Enterotoxin in what you ate. 3. S. p yogenes (Group A) Penicillin won't touch me a. Beta hemolytic Methicillin, well... maybe? b. Streptolysin S - stable in 0 2 ; non- Vancomycin killed in days of yore antigenic But now that drug is not for sure. c. Streptolysin 0 - oxygen labile; Wash your hands, don't pick your nose. antigenic d. Erythrogenic toxin - rash of scarlet Wear a lab coat over your clothes. fever 144 e. Highly sensitive to penicillin f. Infections ❖ Pharyngitis (Strep throat) ❖ Impetigo ❖ Erysipelas ❖ Wounds, burns ❖ Rheumatic fever (a utoimmune compound sequelae to infection with (etbylbydrocupreine Streptococcus g1·oup A) ❖ Lab diagnosis ~ Sensitive to 0.04 units Streptococcus bacitracin disc pyogenes I&' Typing Streptococcus 4. S. agalactiae (Group B) pyogenes a. arrow zone of beta hemolysis Penicillin will kill h. Neonatal sepsis and meningitis; with eose. UTI; vaginal infections c. Laboratory diagnosis But Protein M is ❖ Serotyping the biggest trick ❖ CAMP reaction (with S. aureus) To make throat ❖ Na hippurate positive and muscle sick. 5. Group D Antistreptolysin O a. S. bovis/ gallolyticus Will lay the heart and kidneys low. ❖ BEM positive Bacitracin is my sign ❖ No growth in 6.5% NaCl Zone of inhibition every time. ❖ Associated with colorectal cancer b. Enterococcus See what I do on 8-A-P ❖ Bile esculin medium (BEM) Destroy the sheep R-8-C. positive ❖ Growth in 6.5% NaCl ❖ UTI, bacteremia, others REMEMBER! 6. S. pneumoniae Staph and Strep a. Alpha hemolytic crater -like colonies STREP: or mucoid, "water drop" colonies Pairs or Chains b. Lancet-shaped diplococci Catalase - c. Check sensitivity to penicillin using Growth in 6.5% NaCl OX (oxacillin) disc(~ 20 mm= (Enterococcus) sensitive) d. Cau ses ❖ Primary lobar pneumonia (rusty sputum) Catalase neg ❖ Menin.gitis ❖ Bacteremia STAPH: ❖ Otitis media Clusters (Grape-Like) ❖ Conjunctivitis Catalase + e. Laboratory diagnosis Growth in 7.5% NaCl ❖ Typical colony mo1phology ❖ Quellung reaction ❖ Sensitive to optochin ❖ Bile soluble Catalase pos 7. Other alpha Streptococcus ( viridans group) - subacute bacterial Coagulase + endocarditis (SBE) (S. aureus) (See Streptococci Chart on next page.)  streptococci (Catalase Negative) Hemolysis Bacitracin Na Optochin Bile Bile Esculin 6.5% NaCl Infections Hippurate Solubility S.pyogenes B s R Pharyngitis, wounds, scarlet (group A) fever, impetigo, sequalae-rheumatic fever S. agalactiae B, y R + R Neon atal septicemia and (group B) meningitis, UTI (CAMP+) Entercoccus a, B, y R R + + UTI, endocarditis (treat with (group D) aminoglycoside+penicillin) Non-enterococcus a ,y R R + Endocarditis (rare), Strep. bovis (group D) associated with color cancer Pneumonia, meningitis, S. pneumoniae a ("water drop'1/ R s + bacteremia (screen for crater colonies) penicillin sensitive with Ox; > 20 mm= sensitive) S. viridans a,y R R Endocarditis (rare) I ~ -, er-t: ! a hemolysis = greening ar?und colony o hemolys1s = no hemolys1s B hernolysis = complete clearing around colony Shaded areas = Key Reactions (3 Growth Requirements 3r Strepiococci ~ REMEMBER! ~ Victory Belongs to Key Cbara(iteristics oi the Most Persevering. Pneumococcl.lB...... ~