Microbiology Presentation 2025 - PDF

MICROBIOLOGY JA BUNAG 2025  Microorganisms Acellular Cellular Viruses Prokaryotes Eukaryotes  COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELLS PROKARYOTE EUKARYOTE Nuclear body Cell division Cell wall  COMPAR...

MICROBIOLOGY JA BUNAG 2025  Microorganisms Acellular Cellular Viruses Prokaryotes Eukaryotes  COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELLS PROKARYOTE EUKARYOTE Nuclear body Cell division Cell wall  COMPARISON BETWEEN EUKARYOTIC AND PROKARYOTIC CELLS PROKARYOTE EUKARYOTE Cytoplasmic membrane Cell organelles Site of energy production Site of protein synthesis BACTERIAL CYTOLOGY CELL WALL Main constituent: Peptidoglycan Functions: 1) Protects internal structures 2) Provides shape of bacteria Some components are responsible for pathogenicity: M protein Mycolic acid  GRAM STAIN GRAM STEP REAGENTS GRAM POSITIVE NEGATIVE Primary / Violet Initial Stain Mordant Decolorizer Secondary Stain ENTEROBACTERIACEAE Citrobacter, Edwardsiella, Enterobacter, Escherichia, Ewingella, Hafnia, Klebsiella, Morganella, Plesiomonas, Proteus, Providencia, Salmonella, Serratia, Shigella, Yersinia 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)  ACID FAST STAIN Non- ZIEHL- STEP KINYOUN Acid Fast Acid NEELSEN Fast Primary Stain Mordant Decolorizer Counter stain Mycobacterium (Acid-fast staining) Serpentine cords (right) -characteristic of M. tuberculosis BACTERIAL CYTOLOGY CYTOPLASMIC MEMBRANE Selectively permeable membrane MESOSOMES Point of attachment for chromosome Extension of cytoplasmic membrane RIBOSOMES BACTERIAL CYTOLOGY INCLUSIONS Serves for nutrient storage Examples: Much granules Babes-Ernst / Metachromatic / Volutin granules Bipolar bodies  BACTERIAL CYTOLOGY ENDOSPORE Resting cell; highly resistant to dessication, heat and chemical agents Composition: CALCIUM DIPICOLINATE or DIPICOLINIC ACID Bacterial genera with spores: Bacillus Clostridium BACTERIAL CYTOLOGY CAPSULE Increase virulence by preventing phagocytosis Antigenic; on the basis of serotyping by K antigen Colonies often slimy PILI (Fimbriae) Ordinary pili Sex pili  BACTERIAL CYTOLOGY GLYCOCLAYX Capsule or slime layer A. Capsule - organized material firmly attached to the cell wall B. Slime layer - unorganized material not attached to the cell wall S. pneumoniae. “Quellung” German swelling BACTERIAL CYTOLOGY FLAGELLA For locomotion Antigen: H Ag Atrichous Monotrichous Amphitrichous Lophotrichous Peritrichous BACTERIAL CYTOLOGY FLAGELLA Periplasmic flagella – axial filaments or periplasmic flagella LAB: Motility 1. Hanging drop preparation Tumbling – Darting – Gliding - 2. Semisolid medium Example: Sulfide Indole Motility (SIM) Optimum temp: Note: Transport (semi-solid) media BACTERIAL CYTOLOGY Stains for flagella Contains TANNIC ACID to precipitate and coat flagella BACTERIAL VIRULENCE FACTORS Adherence factors Antiphagocytic factors Enzymes Toxins  Exotoxin vs Endotoxin COMPARISON EXOTOXIN ENDOTOXIN SOURCE RELEASE COMPOSITION HEAT STABILITY IMMUNOLOGIC  Exotoxin vs Endotoxin COMPARISON EXOTOXIN ENDOTOXIN PHARMACOLOGIC TOXICITY LETHAL DOSE EXAMPLE DISEASES  BACTERIAL GROWTH FACTORS 1. NUTRIENTS Carbon, Nitrogen, Minerals Salt Halophilic organisms Others MTB Haemophilus F. tularensis Mycoplasma, Ureaplasma  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 Aerotolerant anaerobe Anaerobe that can grow in presence of O2 Microaerophilic campylobacter 5-6% O2 (5% O2, 10% CO2, 85% N2) Requires less O2 Capnophilic Requires 5-10% CO2  neisseria and HACEK Look! Indicator Methylene blue + colorless - blue Resazurin BACTERIAL GROWTH FACTORS 3. TEMPERATURE Psychrophilic / Cryophilic Mesophilic Thermophilic BACTERIAL GROWTH FACTORS 4. pH Optimum: Neutral or Slightly Alkaline Acidophile – pH 3.0 Alkaliphile – pH 8 to 10 GROWTH CYCLE CULTURE MEDIA Nutrient media for growing microorganisms in a laboratory environment CLASSIFICATION ACCORDING TO : I. PHYSICAL STATE / CONSISTENCY II. COMPOSITION III. DISPENSING / DISTRIBUTION IV. FUNCTION AND USE I. ACCORDING TO PHYSICAL STATE / CONSISTENCY Liquid Semisolid Solid II. ACCORDING TO COMPOSITION Synthetic / Chemically-defined Exact composition is known Complex / Non-synthetic Contains at least 1 component w/c is not chemically defined Tissue For organisms that cannot grow on cell-free medium III. ACCORDING TO DISPENSING / DISTRIBUTION 1. Plated 2. Tubed A. Weigh A. Weigh B. Dissolve B. Dissolve C. Sterilize C. Dispense D. Dispense D. Sterilize IV. ACCORDING TO FUNCTION AND USE 1. SIMPLE / BASAL / SUPPORTIVE / GENERAL ISOLATION / GENERAL PURPOSE MEDIA Supports the growth of most nonfastidious bacteria 2. ENRICHED MEDIA Contains nutrient supplements for fastidious bacteria BAP IV. ACCORDING TO FUNCTION AND USE 3. ENRICHMENT MEDIA Enhance the growth of an organism Selenite broth, Tetrathionate broth, Alkaline peptone water 4. SELECTIVE MEDIA Select the growth of a particular organism Contains inhibitors EXAMPLES OF SELECTIVE CULTURE MEDIA For MTB Medium: Inhibitor : For Corynebacterium diphtheriae Medium: Inhibitor : For gram positive bacteria EXAMPLES OF SELECTIVE CULTURE MEDIA Selective for Neisseria gonorrhoeae Enriched CAP Contains antibiotics as inhibitors Thayer-Martin Modified Thayer-Martin Martin-Lewis New York City Agar  Thayer Modified Martin New York Martin Thayer Lewis City agar Martin Gram (+) Vancomycin Vancomycin Vancomycin Vancomycin inhibitor Gram (-) Colistin Colistin Colistin Colistin inhibitor EXCEPT N. gonorrhea Antifungal Nystatin Nystatin Inhibitor of NONE Trimethoprim Trimethoprim Trimethoprim Proteus lactate lactate lactate swarming  EXAMPLES OF SELECTIVE CULTURE MEDIA Selective differential for Staphylococcus spp. Medium : Inhibitor : CHO : pH indicator : EXAMPLES OF SELECTIVE CULTURE MEDIA Selective differential for Vibrio spp. Medium: CHO : pH indicator : IV. ACCORDING TO FUNCTION AND USE 5. DIFFERENTIAL MEDIA Provides distinct colonial appearances of microorganisms to aid in their identification ENTEROBACTERIACEAE RAPID LACTOSE FERMENTERS 1. 2. 3. IV. ACCORDING TO FUNCTION AND USE LATE LACTOSE NON LACTOSE FERMENTERS FERMENTERS 1. 1. 2. 2. 3. 3. 4. 4. 5. 5. 6. 6. 7. SELECTIVE DIFFERENTIAL MEDIA FOR GRAM NEGATIVE ENTERIC BACILLI EOSIN-METHYLENE BLUE (EMB) AGAR Inhibitors of Gram (+) bacteria: CHO : LF : Non LF: SELECTIVE DIFFERENTIAL MEDIA FOR GRAM NEGATIVE ENTERIC BACILLI MAC CONKEY (MAC) AGAR Sel/Diff for Gram negative Enteric bacilli Inhibitor for Gram POSITIVE: CHO : pH indicator : LF : Non LF : SELECTIVE DIFFERENTIAL MEDIA FOR GRAM NEGATIVE ENTERIC BACILLI HEKTOEN ENTERIC AGAR (HEA) Sel/Diff for Gram negative Enteric bacilli Inhibitor for Gram POSITIVE : CHO: pH indicator : H2S indicator : SELECTIVE DIFFERENTIAL MEDIA FOR GRAM NEGATIVE ENTERIC BACILLI SALMONELLA SHIGELLA (SS) AGAR Sel/Diff for Salmonella and Shigella CHO: pH indicator : H2S indicator : Salmonella – Shigella – IV. ACCORDING TO FUNCTION AND USE 6. TRANSPORT MEDIA Semisolid media cont. CHARCOAL w/c absorbs fatty acids toxic to some bacteria 7. MEDIUM FOR SUSCEPTIBILITY TESTING Mueller-Hinton Agar (MHA) Agar depth : pH :  IV. ACCORDING TO FUNCTION AND USE 8. MEDIA FOR BIOCHEMICAL TESTING Examples: Triple Sugar Iron (TSI) agar, Lysine Iron Agar (LIA) 1. TRIPLE SUGAR IRON AGAR Considered an initial step in the identification of Enterobacteriaceae Composition: Protein source CHO: pH indicator : H2S indicator : Sulfur source :  MUST KNOW! TSI REACTIONS TYPICAL ORGANISMS A/A G H2S (-) K/A G H2S (+) K/A H2S (-) K/K H2S (-) MEDIA FOR BIOCHEMICAL TESTING 2. LYSINE IRON AGAR Can be used to determine the ability of the organism to deaminate lysine, decarboxylate lysine and produce H2S Composition: Small amount of protein, LYSINE CHO: pH indicator : H2S indicator : MEDIA FOR BIOCHEMICAL TESTING 2. LYSINE IRON AGAR Slant : Positive org.: Butt :  MUST KNOW! LIA REACTIONS TYPICAL ORGANISMS K/K H2S (+) K/A H2S (-) R/A H2S (-) SUSCEPTIBILITY TESTING 1. TUBE OR MICROPLATE DILUTION Serial dilution of antibiotic is prepared; to each tube, a uniform amount of inoculum is added Minimum Inhibitory Concentration (MIC) Minimum Bactericidal Concentration / Minimum Lethal Concentration SUSCEPTIBILITY TESTING 2. AGAR DILUTION Varying concentration of antibiotic is incorporated to appropriate plating media 3. DISK DIFFUSION (KIRBY BAUER) MUELLER HINTON AGAR (MHA) Depth of agar : pH of agar :  STEPS Pick 4-5 colonies into TSB. Incubate at 37degC for 2-5 hours. Inoculate MHA. Apply antibiotic disks. (24 mm from disk center to center) *Wait for 3-5mins., not longer than 15mins before inverting plates. Invert plates and incubate at 37degC for 16 to 18 hours. Measure zone of growth inhibition. Interpret susceptibility from standard chart zone. SUSCEPTIBILITY TESTING 4. E-TEST Strip with varying concentration of antibiotic along its length 5. AUTOMATED Vitek System : ANTIMICROBIAL AGENTS 1. CELL WALL SYNTHESIS Beta-lactams: penicillins, cephalosporins, carbapenems, monobactams Glycopeptides: vancomycin, teicoplanin Cycloserine Bacitracin 2. PROTEIN SYNTHESIS Aminoglycosides: gentamicin, tobramycin, kanamycin Tetracyclines Chloramphenicol Erythromycin Fusidic acid ANTIMICROBIAL AGENTS 3. NUCLEIC ACID SYNTHESIS Sulfonamides Trimethoprim Quinolones Rifampicin 4. CELL MEMBRANE FUNCTION Polymyxins: colistin Antifungals: Amphotericin B STERILIZATION Complete destruction of microbial life Pathologic and non-pathologic (vegetative cells and spores) I. Physical methods II. Chemical methods I. PHYSICAL METHODS 1. HEAT MOIST HEAT A. Autoclaving Principle: Standard: Most effective method Used for culture media and surgical instruments Biologic indicator : I. PHYSICAL METHODS 1. HEAT MOIST HEAT B. Fractional Alternate heating (kill veg cells), incubation (spores germinates), heating (kill remaining cells) Tyndallization: Fractional Intermittent / Discontinuous Sterilization Inspissation: for culture media with INCREASED PROTEIN Principle: THICKENING THROUGH EVAPORATION 75 to 80 degC for 2 hours, 3 consecutive days I. PHYSICAL METHODS 1. HEAT DRY HEAT A. Oven Temp : Biologic indicator : B. Incineration For infectious wastes; bringing materials into ashes Temp : Banned in the Philippines due to environmental concerns  I. PHYSICAL METHODS 2. Ionizing Radiation Plastic syringes, catheter or gloves Short wavelength, high energy gamma rays 3. Filtration For culture media which cannot be heated Antibiotic solution, CHO solution, vaccines Liquid: Pulling solution through cellulose acetate or cellulose nitrate medium with vaccum Air: Removes >0.3 um Viruses: 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 : 2. Pasteurization Destroys food pathogen in milk and dairy products Batch / LTH : Flash / HTH : 3. Non-ionizing radiation Long wavelength, low energy UV light DISINFECTION II. CHEMICAL METHODS ANTISEPTIC – DISINFECTANT - 1. Alcohol - considered an ANTISEPTIC Ex: 70% Ethyl alcohol / Isopropyl alcohol 2. Quaternary Ammonium Compounds (QUATS) Ex: Benzalkonium Chloride (Zephiran) Inactivated by: Disadvantages: DISINFECTION 3. Halogen A. Iodine - remain on skin for 60 seconds B. Chlorine - active component of NaCl DISINFECTION 4. Heavy metal Mercury Copper Silver - 5. Phenol 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: Interpretation: PC > 1 : PC < 1 : PC = 1 : 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: Anticoagulant: Blood to CM ratio – Adult- Children- THROAT AND NASOPHARYNGEAL CULTURES Most abundant normal flora: Most common pathogen: 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 URINE CULTURES Midstream cleancatch urine Catheterized urine Suprapubic urine Preservative : Colony count 10^5 bacteria/mL indicates infection (done in BAP) Use calibrated loop CEREBROSPINAL FLUID Examine immediately or hold in incubator for no longer than 1 HOUR Centrifuge. Use sediment for: Smears, Gram stain, India ink Culture – *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 organisms for Cryptococcus neoformans 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 “BELIEVE YOU CAN AND YOU’RE HALFWAY THERE.” ~THEODORE ROOSEVELT #RMT2025