BACTE LEC 1.2 INTRO TO BACTERIOLOGY 2.pdf

BACTERIOLOGY BACTERIAL METABOLISM WEEK 2 LECTURE CARBOHYDRATES GLUCOSE PYRUVIC ACID ENERGY + BY-PRODUCTS GLUCOSE TO PYRUVIC ACID 1. EMBDEN-MEYERHOF-PARNAS GLYCOLYTIC PATHWAY major source (90% glycolysis) yields 2 ATPs ANAEROBIC 2. PENTOSE-PHOSPHATE (PHOSPHOGLUCONATE) PATHWAY 10% glycolysis y...

BACTERIOLOGY BACTERIAL METABOLISM WEEK 2 LECTURE CARBOHYDRATES GLUCOSE PYRUVIC ACID ENERGY + BY-PRODUCTS GLUCOSE TO PYRUVIC ACID 1. EMBDEN-MEYERHOF-PARNAS GLYCOLYTIC PATHWAY major source (90% glycolysis) yields 2 ATPs ANAEROBIC 2. PENTOSE-PHOSPHATE (PHOSPHOGLUCONATE) PATHWAY 10% glycolysis yields 38 molecules of ATPs AEROBIC PYRUVIC ACID TO ENERGY + BY PRODUCTS 1. RESPIRATION AEROBIC PROCESS Kreb's cycle or Tricarboxylic Acid Cycle - aerobic process carried out by obligate aerobes and facultative anaerobes Electron transport chain - aerobic process Glucose -> CO2 and H2O (in the presence of oxygen) Oxygen is the final electron transporter 2. OXIDATION AEROBIC PROCESS Glucose --> Acid 3. FERMENTATION ANAEROBIC PROCESS Anaerobic process carried out by both obligate anaerobes and facultative anaerobes Organic Molecule is the final electron transporter can be classified according to their end products ALCOHOLIC FERMENTATION ethanol (yeast) HOMOLACTIC FERMENTATION lactic acid only (Streptococcus and Lactobacillus) HETEROLACTIC FERMENTATION lactic acid, formic and acetic acid and alcohols and CO2 PROPIONIC ACID FERMENTATION propionic acid (P. acnes) MIXED-ACID FERMENTATION lactic acid, succinic acid, formic acid, acetic acid detected by METHYL RED TEST Ex. Enterobacteriaceae BUTANEDIOL FERMENTATION acetoin (acetyl methyl carbinol) and butanediol which are neutral end products acetoin production is detected by VOGESPROSKAUER TEST BUTYRIC ACID FERMENTATION butyric acid, acetic acid, carbon dioxide and hydrogen (Obligate anaerobes) BACTERIOLOGY BACTERIAL GENETICS WEEK 2 LECTURE BACTERIAL GENE TRANFER 1. CONJUGATION it involves cell-to-cell contact the sex pilus / transposon (donor) establishes a conjugative bridge that serves as the conduit for DNA transfer from donor to recipient cell. the plasmid (independent circular DNA, nonchromosomal element) may be transferred by conjugation, but not all plasmids are capable of conjugative transfer. BACTERIAL GENE TRANFER 2. TRANSDUCTION DNA of 2 bacteria will come together to form other traits this process is mediated by viruses that infect bacteria (bacteriophage) BACTERIAL GENE TRANFER 3. TRANSFORMATION involves recipient cell uptake of free DNA released into the environment when another bacterial cell (donor) dies and undergo lysis. BACTERIOLOGY BIOLOGICAL SAFETY LEVEL (BSL) WEEK 2 LECTURE BIOLOGICAL SAFETY LEVELS 1. BSL-1 Contaminants Those with no known potential of infecting healthy people Those with MINIMAL threat to laboratory workers Example: Bacillus subtilis, Mycobacterium gordonae, Naegleria gruberi 2. BSL-2 Those acquired thru INGESTION, MUCOUS MEMBRANE, PERCUTANEOUS EXPOSURE Those that may pose MODERATE threat to laboratory workers BSL-1 practices + lab coats, protective gloves, limited access, decontamination of all infectious waste and biohazard warning signs Example: Bacillus anthracis, Yersinia pestis, Staphylococcus, Enteric pathogen like Salmonella and Shigella; Viruses like HIV and HBV BIOLOGICAL SAFETY LEVELS 3. BSL-3 Those with a potential for AEROSOL TRANSMISSION (INHALATION) Those that may pose HIGH risk to lab workers BSL-2 practices + Special lab clothing and controlled access Example: Mycobacterium tuberculosis, Rickettsiae,Francisella tularensis, Brucella, Coxiella, fungi like Coccidiodes immitis (Systemic Fungi) and virus like Arbovirus, St. Louis Encephalitis Virus BIOLOGICAL SAFETY LEVELS 4. BSL-4 Those that may pose EXTREME to lab workers Can cause life threatening diseases Organisms encountered in RESEARCH institution BSL-3 practices + entrance to a separate room where street clothing is changed and replaced with lab clothing; includes decontamination of all personnel and material before leaving the area Example: MERS-COV, EBOLA, Filovirus, Arenavirus, Arbovirus, Small Pox Virus BIOLOGICAL SAFETY CABINETS CLASS 1 Negative pressure, ventilated cabinet. Unsterilized air entersand circulates within the cabinet, and the exhaust air from the cabinet is filtered by HEPA filter Has the least protection because it it entirely open Sterilizes the air to be EXHAUSTED Process NON-PATHOGENS/(BSL-1) LOW ONCOGENIC VIRUS/LOW TOXIC CHEMICALS/CARCINOGENS With 1 HEPA FILTER 0.3um pore size Air Velocity: 75 linear feet/min Protect the worker and environment NOT the organisms BIOLOGICAL SAFETY CABINETS CLASS 2 Also known as VERTICAL LAMINAR FLOW Sterilizes the AIR ENTERING and CIRCULATING within THE CABINET and the EXHAUST AIR Sterilizes air to be exhausted and the air that flows over infectious material Process BACTERIAL and FUNGAL PATHOGENS/MEDIUM RISK ONCOGENIC VIRUS/CHEMICALS/CARCINOGENS (BSL-2) TYPES: 2A: FIXED OPENING exhaust air inside the room 70% of air is recirculated 2B: VARIABLE SASH Exhaust air is discharged outside the building Air velocity: 75-100 linear feet/min Protects the environment, workers and culture BIOLOGICAL SAFETY CABINETS CLASS 3 Most effective because the system is entirely closed Infectious material is handled using gloves attached and sealed to the cabinet Highly toxic materials Entire room is with negative pressure Process viral pathogen (BSL-3) BACTERIOLOGY MICROBIAL CONTROL WEEK 2 LECTURE STERILIZATION complete destruction and removal of all forms of microbial life including their SPORES PHYSICAL METHOD 1. Moist Heat Autoclave Tyndallization Inspissation 2. Dry Heat Oven Incineration Direct Flaming Cremation 3. Filtration 4. Ionizing Radiation/Cold Sterilization CHEMICAL METHOD 1. Ethylene oxide (Gas Sterilant) 2. Formaldehyde vapor and vapor phase H202 3. 2% Glutaraldehyde 4. Peracetic acid DISINFECTION refers to the destruction and removal of pathogen but NOT NECESSARY ALL MICROORGANISMS AND THEIR SPORES PHYSICAL METHOD 1. Pasteurization 2. Boiling 3. UV CHEMICAL METHOD 1. Antiseptics Alcohols Iodophors Chlorhexidine Hexachlorophene 10% Hydrogen peroxide 2. Disinfectant HALOGENS (Chlorine, lodine, fluorine) Heavy metals Aldehydes QUATS Phenolics PHYSICAL METHOD OF STERILIZATION MOIST HEAT Destroys microorganism by COAGULATION OF ENZYME and STRUCTURAL PROTEINS and DEGRADATION OF NUCLEIC ACID 1. AUTOCLAVE Principle: steam under pressure PRESSURE is used to raise temperature, not to kill the organisms Acts by coagulating or denaturing enzymes and proteins. 121°C; 15Psi; 15-30mins sterilizing used and unused media Indicator: Bacillus stearothermophilus Not killed by autoclave are *PRIONS 2. DISCONTINUOUS/FRACTIONAL/INTERMITTENT METHODS A. TYNDALLIZATION Uses FLOWING STEAM Equipment used is Arnold Sterilizer 100°C, for 30mins for 3 consecutive days Used to sterilized media containing milk or serum. Process: Day 1: destroys vegetative cells Day 2: destroys spores Day 3: destroys remaining spores 2. DISCONTINUOUS/FRACTIONAL/INTERMITTENT METHODS B. INSPISSATION "Thickening through evaporation" Used for sterilization of culture media that are EGG-BASED AND OF HIGH PROTEIN content since extremely high temperature can denature protein 75-80°C for 2 hrs for 3 consecutive days DRY HEAT It kills microorganism by DENATURATION of proteins sterilization WITHOUT WATER. It is utilized for the sterilization of GLASSWARES, OIL PRODUCTS and POWDERS. 1. OVEN 160-180 degC for 1-2hrs Used to sterilize glasswares, metal instruments, oils and certain fatty substances which are not permeable to water (i.e. oil, petrolatum or powders) Biological Indicator: Bacillus subtilis var niger incubated at 35-37 degC 2. INCINERATION Most common method of treating infectious waste Burning to ashes, temperature of 870-980degC Disadvantage: production of toxic emission and presence of heavy metals in ash after incineration 3. DIRECT FLAMING For loops and needles 4. CREMATION To control diseases FILTRATION Sterilize heat sensitive materials; for antibiotic solutions, toxic chemicals, vaccine, carbohydrates, radioisotopes A. DEPTH FILTERS Consists of FIBROUS or GRANULAR material Example: Berkefield filter (Diatomaceous) ate ora Asbestos (Seitz) Chamberland filter (Unglazed porcelain) B. MEMBRANE FILTERS These are porous membranes 0.1mm thick to sterilize culture media, antibiotics Composed of CELLULOSE ACETATE and POLYCARBONATE Used to sterilize PHARMACEUTICALS, OPHTHALMIC SOLUTIONS, CULTURE MEDIA, ANTIBIOTICS and OIL PRODUCTS 1. Liquid Filtration It uses CELLULOSE ACETATE/CELLULOSE NITRATE membrane with vacuum 2. Air Filtration It uses HIGH-EFFICIENCY PARTICULATE AIR (HEPA) filters HEPA Filters - can remove objects larger than 0.3um; use in BSCs 3. Filtration of bacteria, yeast and molds It uses 0.45 um pores of membrane filters 4. Critical Sterilizing (Millipore Filter) It uses 0.22um membrane filters to remove vegetative cells but not viruses It can give 100% sterility IONIZING RADIATION/COLD STERILIZATIONTION Exposure to GAMMA RAYS; for DISPOSABLE syringe, gloves, catheters Uses short wavelength but high energy gamma rays Radiation Indicator: Bacillus pumilis CHEMICAL METHOD OF STERILIZATION 1. ETHYLENE OXIDE (GAS STERILANT) most commonly used chemical sterilant, used in gaseous form of sterilizing heat sensitive objects; DISINFECTANT FOR MACHINE THAT CANNOT BE AUTOCLAVED OR HEATED -INDICATOR: Bacillus subtilis var globijii 2. FORMALDEHYDE VAPOR AND VAPOR PHASE OF H2O2 used to sterilize HEPA filters in BSCs 3. 2% GLUTARALDEHYDE sporicidal; kills spores in 3-10 hours, for medical equipment such as bronchoscope 3. PERACETIC ACID is effective un the presence of organic material. PHYSICAL METHOD OF DISINFECTION 1. PASTEURIZATION Often used to destroy pathogen (such as Salmonella, Streptococcus, Brucella, Listeria, M. bovis) in milk and beverages Use to lower total bacterial count by 95-99% This method cannot eliminate bacterial endospores Phosphatase - test for success of pasteurization Grade A milk - 75,000 before pasteurization, 15,000 after pasteurization TYPES: 1. Ultra-High Temperature (UHT) 140°C for 3secs Milk can be stored for 2 months at Room Temp without affecting its flavor; applicable for COFFEE-CREAMER 2. FLASH/High Temp. Short Time (HTST): 72°C for 15secs 3. BATCH/Low Temperature Holding (LTH) 63°c for 30 mins. 2. BOILING At 100 degC for 15-30 minutes Kills all vegetative organisms but not all spores or viruses For surgical instruments 3. NON-IONIZING RADIATION Use MERCURY ARC LAMPS with wavelength of 240-280mu Do not penetrate well and organism must have direct surface exposure such as working surface of biologic safety cabinet, to be effective CHEMICAL METHOD OF DISINFECTION 1. ANTISEPTIC chemical germicide for use on the skin or tissue and not to be substituted for a disinfectant A. Alcohols (Non-sporicidal) 70% ethanol, isopropyl alcohol; 70% ETHANOL IS MORE EFFECTIVE THAN 95% ETHANOL B. lodophors lodine + Detergent/neutral polymer (I.e povidone) Iodine Tincture = 2% Iodine + 70% Alcohol 70% ETOH, followed by an iodophor is the MOST COMMON compound used for skin disinfection drawing blood for blood cultures or before surgery. C. Chlorehexidine D. Hexachlorophene E.. 10% hydrogen peroxide - is used for cleansing of wound. 2. DISINFECTANT applied to nonliving surfaces, such as countertops and handrails A. Halogens (Chlorine, iodine, fluorine) Sodium Hypochlorite (NaOCI) also known as Household Bleach recommended dilution is 1:10 Dilutions: 1:10 dilution on porous surface 1:100 on smooth, hard surfaces, 1:5 for large spills Inactivates HBV within 10 minutes and HIV within 2 minutes B. Heavy Metals Mercury active component of merthiolate (sodium ethyl mercuri-thiosalicylate); toxic to humans and environment Copper algaecide Silver in the form of 1% AgNO3; used in Crede's prophylaxis for infants with eye infection Manganese in KMN04 C. Aldehydes 1-8% formaldehyde or glutaraldehyde not used as surface disinfectants due to their irritating fumes Antibacterial effect: INACTIVATION OF PROTEINS AND NUCLEIC ACIDS D. QUATS Quaternary Ammonium Compounds Benzalkonium chloride/Zephiran low toxic, rapidly inactivated by organic substance; used to disinfect bench tops or other surfaces in the laboratory P. aeroginosa grown in ammonium acetate media Antibacterial effect: IT DISRUPTS CELL MEMBRANE, RESULTING IN LEAKAGE OF CELL CONTENTS E Phenol (Tuberculocidal) STANDARD DISINFECTANT in the laboratory in which other chemical disinfectant are compared Phenol coefficient refers to the expression of bacterial powder of a particular substance as compared to pure phenol Endpoint is the lowest concentration that kills test organisms in 10 minutes at 20 degC BACTERIOLOGY TYPES OF MICROSCOPY WEEK 2 LECTURE Brightfield Miscroscope GS, AFS, KOH, most common microscope Used for stained and unstained samples Magnification: COLOR OCULAR LENS TOTAL MAGNIFICATION 4X RED 10x 40x 10x YELLOW 10x 100x 40x BLUE 10x 400x 100x BLACK 10x 1000× OBJECTIVE LENSES Darkfield Miscroscope Motility of Spirochetes Also used for fluorescent stains Higher resolving power than bright field Phase-Contrast Miscroscope Inclusion bodies seen on virus and Chlamydia, living cells/natural state, Microlymphocytotoxicity test for HLA Good for KOH mount Fluorescent Miscroscope Requires fluorescent stain (Calcofluor white, acridine orange) UVL - provided by mercury arc lamp Substitute: Dark Field microscopy Electron Miscroscope viral morphology Resolution: 0.5nm Fixatives: Glutaraldehyde or Osmium Tetroxide Dehydrating Agents: Alcohol or Acetate Stain: Lead citrate and Uranyl acetate Transmission Electron Microscopy - internal structure Scanning Electron Microscopy - external structure