Practical Exam - Microbiology Lab - Semester 3 PDF

Lab 1 – Gram-Positive Cocci I Tests: Catalase, Coagulase, Hemolysis pattern, Bacitracin, Novobiocin Organism Gram Stain Colonial Key Biochemical Key Considerations Morphology Reactions Staphylococcus...

Lab 1 – Gram-Positive Cocci I Tests: Catalase, Coagulase, Hemolysis pattern, Bacitracin, Novobiocin Organism Gram Stain Colonial Key Biochemical Key Considerations Morphology Reactions Staphylococcus Gram-positive Golden, creamy Catalase +, Pathogen often associated with skin aureus cocci (clusters) colonies, beta- Coagulase +, Beta- infections. Beta-hemolysis is clear on hemolytic hemolytic BAP. Staphylococcus Gram-positive White, non- Catalase +, Novobiocin-sensitive. Opportunistic epidermidis cocci (clusters) hemolytic, small Coagulase -, Non- pathogen often associated with colonies hemolytic prosthetic device infections. Streptococcus Gram-positive Large, clear zone Catalase -, Beta- Causes strep throat and other pyogenes cocci (chains) of beta hemolysis hemolytic, Bacitracin- pyogenic infections. Beta hemolysis sensitive distinct on BAP. Streptococcus Gram-positive Alpha-hemolysis, Catalase -, Alpha- Requires optochin sensitivity test for pneumoniae cocci greenish colonies hemolytic further confirmation. Commonly (pairs/chains) associated with pneumonia and meningitis. Lab 2 – Gram-Positive Cocci II Tests: Catalase, Coagulase, Hemolysis pattern, Novobiocin, Bacitracin Organism Gram Stain Colonial Key Biochemical Key Considerations Morphology Reactions Staphylococcus Gram-positive Golden, creamy Catalase +, Similar results as in Lab 1. Test aureus cocci (clusters) colonies, beta- Coagulase +, Beta- novobiocin to rule out S. hemolytic hemolytic saprophyticus, a common UTI pathogen. Staphylococcus Gram-positive White, non- Catalase +, Part of the normal skin flora. Distinct epidermidis cocci (clusters) hemolytic, small Coagulase -, Non- from S. saprophyticus based on colonies hemolytic novobiocin sensitivity. Streptococcus Gram-positive Large, clear zone of Catalase -, Beta- Important for differentiation from S. pyogenes cocci (chains) beta hemolysis hemolytic, Bacitracin- agalactiae (Group B streptococcus), sensitive which is bacitracin-resistant. Streptococcus Gram-positive Alpha-hemolysis, Catalase -, Alpha- Key test: optochin disk for sensitivity. pneumoniae cocci greenish colonies hemolytic Non-hemolytic on some BAP due to (pairs/chains) environmental variations. Lab 3 – Gram-Positive Bacilli Tests: Catalase, Motility, Spore stain, Biochemical tests (e.g., VP, Indole) Organism Gram Stain Colonial Key Biochemical Key Considerations Morphology Reactions Bacillus Gram-positive Flat, spreading, Catalase +, Spore- Environmental contaminant, rarely subtilis rods (spore- rough colonies former, Motile pathogenic. Beta-hemolytic on BAP. forming) Bacillus Gram-positive Dry, irregular Catalase +, Non- Non-hemolytic; forms Medusa head anthracis rods (spore- colonies motile colonies. Associated with anthrax. forming) Clostridium Gram-positive Double zone of Catalase -, Beta- Obligate anaerobe; spore former. Causes perfringens rods (spore- hemolysis hemolytic gas gangrene. forming) Clostridium Gram-positive Yellow, non- Catalase -, Produces toxins causing difficile rods (spore- hemolytic colonies Anaerobic, Spore- pseudomembranous colitis. Requires forming) former anaerobic growth conditions. Lab 4 – Gram-Negative Cocci Tests: Oxidase, Penicillin disk, Glucose fermentation, API NH, Tributyrin test Organism Gram Stain Colonial Morphology Key Biochemical Key Considerations Reactions Neisseria Gram-negative Gamma-hemolytic, gray, Oxidase +, Ferments Found intra- and extracellularly. meningitidis diplococci convex colonies on BAP; glucose and maltose Penicillin-resistant. Causes (GNDC) medium, gray colonies on meningitis and septicemia. CHOC Neisseria Gram-negative No growth on BAP; small, gray- Oxidase +, Ferments Intracellular pathogen. Requires gonorrhoeae diplococci white, shiny colonies on CHOC glucose only CO₂ for growth. Penicillin- (GNDC) sensitive. Causes gonorrhea. Moraxella Gram-negative Gamma-hemolytic, smooth, Oxidase +, Extracellular pathogen. Known for catarrhalis diplococci opaque gray colonies on BAP; Asaccharolytic, resistance to decolorization. (GNDC) older colonies resemble Positive Tributyrin test Common in respiratory infections. "hockey puck" Lab 5 – Gram-Negative Coccobacilli Tests: Oxidase, XV factor test, ALA test, Cefinase (if applicable) Organism Gram Stain Colonial Morphology Key Biochemical Key Considerations Reactions Haemophilus Gram-negative Large, translucent, moist Requires both X and Satellites around S. aureus on BAP. influenzae coccobacilli colonies on CHOC; V factors, ALA Major cause of respiratory infections pungent, mousey odor negative and meningitis in children. Haemophilus Gram-negative Similar to H. influenzae Requires V factor Satellites around S. aureus on BAP. parainfluenzae coccobacilli on CHOC; pungent odor only, ALA positive Part of the normal upper respiratory tract flora. Lab 6 – Gram-Negative Rods (Enterobacteriaceae) Tests: MAC, BAP, XLD, HEK, TSI, API (ONPG, IND, VP, others) Organisms/Tests Colonial Morphology Key Biochemical Reactions Organism ID - Organism #18: - MAC: Dry pink colonies (LF) - Oxidase negative Escherichia coli E. coli - BAP: Beta-hemolysis - ONPG positive - XLD: Yellow - Indole positive - HEK: Yellow orange - Ferments glucose, lactose, trehalose, and xylose - TSI: A/A, gas - Non-H2S producer Organism #19: MAC: Mucoid pink colonies - Oxidase negative Klebsiella pneumoniae Klebsiella (LF) - VP positive pneumoniae - BAP: Non-hemolytic - Non-motile - XLD: Yellow - Citrate positive - HEK: Yellow-orange - Indole negative (variable in some strains) - TSI: A/A, gas Organism #20: - MAC: Pink colonies (LF) - Oxidase negative Enterobacter cloacae Enterobacter - BAP: Non-hemolytic - VP positive cloacae - XLD: Yellow - Motile - HEK: Yellow-orange - ODC positive - TSI: A/A, gas - Citrate positive Organism #21: MAC: (LLF) - ONPG positive (LLF) Citrobacter freundii Citrobacter - XLD: Yellow or colorless with - H2S producer freundii black centers - Citrate positive - HEK: Yellow with black - Indole variable centers - Urease negative (variable) - TSI: A/A, H2S Organism #22: - MAC: Pink colonies (LLF) - ONPG positive Serratia marcescens Serratia - BAP: Non-hemolytic - VP positive marcescens - XLD: Yellow - Motile - HEK: Yellow-orange - Produces red pigment (prodigiosin) in some - TSI: K/A or A/A, gas strains Organism #23: - MAC: (NLF), may swarm - Urease positive Proteus mirabilis Proteus mirabilis - XLD: Colorless - H2S producer - HEK: Colorless - TDA positive (deaminase activity) - TSI: K/A, H2S - Indole negative Organism #24: - MAC: (NLF) - Urease positive Proteus vulgaris Proteus vulgaris - XLD: Yellow or colorless with - TDA positive black centers - Indole positive HEK: Colorless with black centers - TSI: A/A, H2S Organism #25: - MAC: NLF - Indole positive Providencia stuartii Providencia - XLD: Yellow or colorless - Citrate positive stuartii - HEK: Colorless - Urease variable - TSI: K/A Organism #26: - MAC: NLF - Urease positive Morganella morganii Morganella - XLD: Yellow or colorless - TDA positive morganii - HEK: Colorless - Indole positive - TSI: K/A Organism #27: - MAC: NLF - H2S producer Salmonella enteritidis Salmonella - XLD: Red colonies with black - Citrate positive enteritidis centers - Urease negative - HEK: Green with black centers - TSI: K/A, H2S Organism #28: - MAC: NLF - ONPG positive (LLF) Shigella sonnei Shigella sonnei - XLD: Red - Non-motile - HEK: Colorless - Indole negative - TSI: K/A Organism #29: - MAC: NLF - Motile at 22°C Yersinia enterocolitica Yersinia - XLD: Yellow or colorless - Urease positive enterocolitica - HEK: Colorless - Indole variable - TSI: K/A  Lab 7 – Gram-Negative Bacilli – Non-Fermenters Tests: Oxidase, OF test, Growth on MAC, TSI, Motility test Organism Gram BAP (Oxygen) MAC Oxidase TSI Colonial Cellular Key Characteristics Stain Morphology Morphology Pseudomo GNB or Beta-hemolytic; NLF; Positive K/K or Green/blue Gram- - Strict aerobic metabolism nas coccoba metallic turquoise/ K/NC colonies with negative - Motile at RT and 42°C aeruginosa cillus blue/green blue fruity odor rods - Catalase-positive sheen, pigment - Produces pyocyanin pigment flattened, (pyocyani - Associated with CF patients spreading n) colonies; grape- like or corn tortilla odor Stenotroph GNB Large, smooth NLF; Negativ Green/Gr Lavender, non- Gram- - Loves maltose (OF maltose: omonas colonies with lavender- e een pigmented negative Yellow/Green) maltophilia uneven edges; green colonies rods - Non-motile at 42°C lavender-green tinge - Resistant to many to light purple antimicrobials pigment Acinetobac Small Smooth, NLF; Negativ K/K or Non- Gram- - Non-motile ter GNB or opaque, creamy purplish- e K/NC pigmented, negative - Common in nosocomial baumannii coccoba gray-white blue tinge smooth rods infections cillus colonies; (may colonies - Highly resistant to smaller than appear antimicrobials Enterobacteriac LF-like) eae  Lab 8 – Anaerobes Organism Gram Stain BBE Thio Broth Disc Susceptibility Catalase Colonial Morphology Key Characteristics BAP (Oxygen) Cellular Morphology BAP (Anaerobic) Bacteroides Pleiomorphic, Gram- Bile-resistant, Growth Vancomycin Positive Non-hemolytic, - Anaerobic fragilis negative rods growth 3/4 up =R gray colonies, - Resistant to bile Gamma hemolysis produces dark tube, not Colistin = black pigment - Black pigment on Large, 1-2 mm gray-white precipitate; at the top R/Variable on BBE BBE media colonies, smooth, shiny, media turns (aerobic Kanamycin = medium - Common gut flora, opaque, similar to Group brown to black environm R Gram-negative potential opportunistic D Enterococcus around ent) rods pathogen colonies (pleomorphic) Clostridium GPB, boxcar-shaped, No dark Growth at Vancomycin Negativ Double-zone - Obligate anaerobe perfringens large rods precipitate; the =S e or beta - Double-zone Gamma hemolysis colonies bottom Colistin = R Variable hemolysis, hemolysis on BAP Double-zone beta convex, only Kanamycin = translucent - Boxcar morphology hemolysis: inner zone translucent (anaerobi Variable colonies - Associated with gas complete, outer zone c Gram-positive gangrene partial; large 5-10 mm environm rods (boxcar- translucent colonies, ent) shaped) rhizoid, filamentous Clostridium GPB, large rods Does not No Vancomycin Negativ Yellow, non- - Known for toxin difficile Non-hemolytic colonies show black growth in =S e hemolytic production Yellow, non-hemolytic pigment; aerobic Colistin = R colonies - Selective media colonies typical condition Kanamycin = Gram-positive required for isolation "ground glass" s Variable rods (large) - Causes antibiotic- appearance associated diarrhea Lab 9 – Candida spp. Organism Gram Stain Colonial Morphology Key Biochemical Reactions Key Considerations Candida Large, Gram- Cream-colored, dull, smooth Germ tube negative Produces pseudohyphae with tropicalis positive, ovoid colonies on SAB; may be (constriction at base); sausage-like constrictions; yeast cells wrinkled or rough. Creamy- pseudohyphae production opportunistic pathogen associated colored with foot-like projections with immunocompromised hosts; on BAP. potential for pigment production. Candida Large, Gram- Creamy, pasty colonies, smooth Germ tube positive (true germ Presumptive ID based on germ tube albicans positive, ovoid after 24-48 hours at 25-37°C on tube, no positivity; true hyphae parallel without yeast cells SAB; star-like projections on constriction);pseudohyphae and constrictions; major cause of BAP. true hyphae candidiasis; yeast-like odor.