Clinical Bacteriology: Medical Technology Exam Review PDF

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – CLINICAL MICROBIOLOGY CLINICAL BACTERIOLOGY Lecturer: Cristina SJ Liwanag, RMT, RN, Ph.D, M.A., MSMLSc Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus,...

MEDICAL TECHNOLOGY LICENSURE EXAM REVIEW – CLINICAL MICROBIOLOGY CLINICAL BACTERIOLOGY Lecturer: Cristina SJ Liwanag, RMT, RN, Ph.D, M.A., MSMLSc Notes by: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King IMPORTANT CHARACTERISTICS OF BACTERIA 4. PILI OR FIMBRIAE - Prokaryotic (organisms with no true nucleus) - Not common to all bacteria - Has both RNA and DNA - Shorter than flagella and usually uniform in length - Multiplies by Binary fission - Common pili: for adherence or attachment to host cells - Measured in μm; Average size: 0.4-2 μm - Sex pili: for gene conjugation (transfer of genetic material) - Smallest living organism: genus Mycoplasma - Usually found in gram (-) bacteria like E. coli, N. gonorrhoeae, Pseudomonas - Largest living organism: genus Bacillus - Produce either exotoxins or endotoxins; one of their virulence factors. 5. ENDOSPORES - Toxins: are biologically produced poisons. - Not common to all bacteria EXOTOXINS ENDOTOXINS o Bacteria with spores: Bacillus and Clostridium - Released by all major gram positive - Usually produced by gram  C. tetani: has terminally located spores organisms except: Listeria negative organisms  C. botulinum: has subterminal spores  B. anthracis: has centrally located spores - Gram negative said to produce exotoxin: Vibrio and E. coli - Resistant structure enabling bacteria to withstand adverse conditions; target of sterilization - Usually excreted or released by living - Released only when cells are bacterial cells; does not require cell death destroyed (cell death/lysis) - Best way to destroy spores is by autoclaving for release - Resistant because of calcium dipicolinate or dipicolinic acid - Composition: protein - Composition: polysaccharide 6. FLAGELLA - Effect: systemic - Effect: local - Toxicity: high - Toxicity: low - Not common to all bacteria - Stability to heating: unstable - Stability to heating: stable - For locomotion; longer than pili - Many bacilli and spiral are motile while true motility is seldom observed in cocci - Stimulates antitoxin production once - Stimulates antitoxin released into the host production once released MOTILE NON-MOTILE into the host E. coli C. diphtheria - Examples - Limulus lysate test C. tetani C. perfringens o Diphtheria toxin by C. diphtheria o Used to detect the H. pylori B. anthracis o Botulinum toxin by C. botulinum presence of endotoxin Staphylococci o TSST-1 by Staphylococcus aureus o Uses aqueous extract of - Motility is best seen at room temperature o Enzymes such as Coagulase by S. blood cells of horse shoe aureus crabs - True motility: is due to the presence of flagella - Types o (+) result: clumping - Brownian movement: movement of non-motile organisms due to the o Cytotoxin: kills host cells movement of molecules surrounding them o Enterotoxin: damages cells of the GIT - Axial filaments/periplasmic flagella in spiral organisms (i.e. E. coli and S. aureus) - Microscopes used to visualize spirochetes: (1) Fluorescence microscope & (2) - Neurotoxin: interferes with nerve impulses Dark field microscope - Ways to demonstrate motility o Hanging drop: place a loopful of inoculum on a cover slip; invert the IMPORTANT PARTS OF A BACTERIAL CELL WALL coverslip on a depression slide and observe under the microscope using LPO 1. CAPSULE o Flagellar stains: Gray’s & Leifson - Slimy area surrounding the cell wall o Semi-solid media: SIM media (dispensed as butt; inoculated by stabbing; - Responsible for mucoid colonies (encapsulated bacteria) motile if growth is found outside/beyond the line of inoculation) - Function: prevents phagocytosis - Classification of bacteria according to flagella o Monotrichous: single flagellum at one end; i.e. Vibrio and P. aeruginosa - Neufeld Quellung Test o Amphitrichous: single flagellum at both ends; i.e. C jejuni o A capsular swelling test o Atrichous: absence of flagella o Used to determine if an organism is encapsulated o Lophotrichous: tuft of flagella at one or both ends; i.e. S. maltophilia - Capsule (+): o Peritrichous: surrounded with flagella; i.e. E. coli o Bacillus anthracis - Motility o Klebsiella pneumoniae o With gliding motility: Capnocytophaga o Haemophilus influenzae o With darting motility: Campylobacter o Streptococcus pneumoniae o With tumbling motility: Listeria o With twitching motility: Kingella kingae 2. CELL WALL (MUREIN LAYER, PEPTIDOGLYCAN LAYER) o With shooting star motility: Vibrio cholerae - Defines the shape of bacteria; main component is peptidoglycan o With cork-screw motility: Spiral organisms or Spirochetes - Point of anchorage for flagella - Site of antibiotic action Board Exam Must-Know! - Basis of gram staining; determines gram positivity/negativity of an organism - Parts external to the cell wall: Capsule, Pili, & Flagella o Gram (+) with rigid peptidoglycan layer  Impermeable to alcohol 7. METACHROMATIC GRANULES/INCLUSION BODIES  With teichoic acid - Serves as energy source; these are food reserves  Sugars: n-acetylglucosamine, and n-acetyl muramic acid o Gram (-) with thinner peptidoglycan layer - Corynebacterium diphtheriae: Babes Ernst Granules  Permeable to alcohol - Mycobacterium tuberculosis: Much granules  No teichoic acid - Yersinia pestis: Bipolar bodies  With CHON (proteins), phospholipids, and LPS (lipopolysaccharide) - Nocardia and Actinomycetes: Sulfur granules  Lipopolysaccharide: used for evasion of host defenses - No cell wall: 8. NUCLEOID o Mycoplasma and Ureaplasma - The DNA in the bacterial cell is generally confined to this central region - M protein: major virulence factor of S. pneumoniae and used to prevent phagocytosis 9. RIBOSOME - Mycolic acid is responsible for the acid-fastness of Mycobacterium and prevents digestion during phagocytosis - Protein synthesis - Gives the cytoplasm of bacteria a granular appearance in EM 3. PLASMA MEMBRANE Additional notes! - Surrounds the cytoplasm; site of energy synthesis Plasmid - Transport of nutrients in and out of the cell; function as golgi apparatus and - Extrachromosomal DNA carries an antibiotic resistant gene lysosome - Can replicate independently - Types: o Large plasmid: confers resistance to beta lactams o Small plasmid: confers resistance to tetracycline and chloramphenicol  BACTERIAL PHYSIOLOGY/REQUIREMENTS FOR BACTERIAL GROWTH ANAEROBIC CULTURE MICROAEROPHILIC (Gas Pak Jar) (Candle jar) 5% CO2 5% O2 1. NUTRITIONAL REQUIREMENTS 10% H2 10% CO2 - Phototrophic: uses light as energy source 85% N2 85% N2 - Chemotrophic: uses chemical compounds as energy source; Types: Autotrophs Difficult to perform Intended for Neisseria and Heterotrophs Autotrophs/Litotrophs Heterotrophs/Organotrophs 4. pH REQUIREMENT Uses inorganic compounds as Uses organic compounds as carbon ACIDOPHILIC BASOPHILIC NEUTROPHILIC carbon source i.e. carbon dioxide source i.e. glucose - Requiring acid - Requiring alkaline - Those able to grow in medium for growth medium for growth medium with a pH 2. OXYGEN REQUIREMENT - Lactobacillus - Vibrio (medium: alkaline between 7.2-7.4 peptone water) Obligate Organisms requiring oxygen growth i.e. Brucella spp., *pH of media for fungi: 5-6 Aerobes Francisella, Mycobacterium Obligate Organisms not requiring oxygen for growth i.e. Veillonella, 5. MOISTURE Anaerobes Actinomyces Facultative Organisms that can live with or without air i.e. family - 70% moisture is needed to prevent drying (drying deters survival of organisms) anaerobes Enterobacteriacea - Humidophilic: those requiring increased (>70%) moisture Organisms requiring small amount of air i.e. Campylobacter Microaerophilic 6. SALT CONCENTRATION spp. Does not grow well but survives in the presence of air i.e. - Halophilic: those requiring high salt concentration; i.e. Vibrio Aerotolerant o 6.5%: Enterococci Lactobacillus and Propionibacterium Extremophiles: able to survive on unusual conditions i.e. o 8-10%: Vibrio Extremophiles absence of oxygen and increased temperature i.e. Bacillus o 7.5%: Staphylococcus aureus infernus 7. CARBON DIOXIDE Obligate Facultative Obligate - Capnophilic: require increased carbon dioxide; i.e. Neisseria and Haemophilus Examples Microaerophiles aerobes anaerobes anaerobes influenzae B. cereus Staphylococcus, Streptococci Clostridium B. anthracis, Oxygen Carbon dioxide Gram positive Corynebacterium, Aerobes 21% 0.3% Listeria, Anaerobes 0% 5-10% Actinomyces Capnophilic 15% 5-10% Neisseria, Most gram- Spirochete, Bacteroides Microaerophilic 5-10% 8-10% Pseudomonas, negative rods Campylobacter Gram Bordetella, negative Legionella, STAGES OF BACTERIAL GROWTH Brucella Acid fast/ Mycobacterium LAG PHASE LOG PHASE PLATEAU DEATH PHASE slightly & Nocardia Rejuvenescence Exponential Stationary Phase of decline AF phase phase phase - No cell division - Start of cell - Characterized - Increased death 3. TEMPERATURE during this division by a balance rate stage because - Growth rate in the number - Causes of Psychrophilic Mesophilic Thermophilic Thermoduric the organism is starts to (equal) of bacterial death 0-20oC 20-45 oC 50-150 oC still adapting to increase dead cells o Food shortage Cold-loving Organisms that Organisms that Bacteria which do not the - Organism and living o Increased bacteria grow at grow at high usually grow at high environment becomes cells concentration moderate temperature temperature but can - Increase in size susceptible - Decreased of toxic temperature withstand exposure but not in to antibiotics nutrients, products to high temperature number increased o Development Many bacteria - Growth rate: toxic waste of unfavorable are mesophilic 0% pH - Optimum temperature: defined as the temperature at which an organism grows best; temperature best suited for bacterial growth - Cold enrichment: incubation at 4oC for several weeks - Can tolerate cold enrichment: Yersinia enterocolitica and Listeria - Thermophilic organisms: o Bacillus stearothermophilus: biological indicator of autoclave o Thermus aquaticus: source of DNA polymerase used in PCR - Incubation temperature for most bacteria and viruses: 37oC - Incubation temperature for fungi: room temperature (27-30 oC) - Incubation temperature and time for aerobes: 37oC for 18-24 hours - Incubation temperature and time for anaerobes: 37oC for 24-48 hours Board Exam Must-Know! Blood bag contaminants at 4oC o Pseudomonas fluorescens o Yersinia enterocolitica o Serratia liquefaciens *Listeria, although able to tolerate cold temperature (4oC), is not considered as a blood bag contaminant because it is an animal pathogen BIOLOGICAL SAFETY LEVEL/BIOLOGICAL AGENTS/HAZARDS BSL-1 BSL-2 BSL-3 BSL-4 - Those that may pose - Those that may pose moderate threat to laboratory - Those that may pose high risk to - Those that may pose extreme risk minimal threat to laboratory workers laboratory workers to lab workers workers - Those associated with laboratory acquired infections - Those with a potential for aerosol - Can cause life threatening diseases - Those with no known - Those acquired through ingestion, mucus transmission (inhalation) - Organisms encountered in potential of infecting healthy membrane, and percutaneous exposure - i.e. Mycobacterium, Coxiella, all research institutions (RITM, PGH) people - i.e. Bacillus anthracis and Yersinia pestis; Enteric agents of systemic mycoses, - i.e. MERS-CoV, Ebola virus - i.e. Bacillus subtilis and pathogens such as Salmonella & Shigella; S. aureus; Rickettsiae, F. tularensis, Brucella Mycobacterium gordonae HIV and HBV  BIOLOGICAL SAFETY CABINETS 2. DRY HEAT A device that encloses a workspace USE OF OVEN INCINERATION DIRECT CREMATION Sterilizes air with infectious material; Sterilizes air through filtration using HEPA filter FLAMING Prevents laboratory acquired infections (aerosols) - Often used in a - Temperature to burn - For - Burning of CLASS I CLASS II CLASS III microlaboratory materials into ashes is loops the body - Least effective - Sterilizes air entering and - The most - 160-180oC for 300-400oC and into ashes because it has an circulating within the cabinet effective 1-2 hours - To dispose hazardous needles - To control open front and the exhaust air - The system is - Used to waste: 870-980oC disease - Negative pressure, - Sterilizes air to be exhausted entirely close sterilize - Not done in the ventilated cabinet and the air that flows over - The infectious glasswares Philippines because it is - Unsterilized air infectious material material is - Indicator: not environment friendly enters and - A.k.a laminar flow handled using Bacillus subtilis - Dry sterilization method circulates within the - Process bacterial and fungal gloves attached var. niger that can eliminate prions cabinet, & the pathogens, medium risk and sealed to the exhaust air from the oncogenic virus, chemicals, cabinet 3. FILTRATION cabinet is filtered by carcinogens - Can - For materials that are heat-sensitive HEPA filter - For BSL-2 & 3 organisms process/handle - For antibiotic solutions, toxic chemicals - Sterilizes air to be highly toxic - Consist of granular material exhausted Types: chemicals and Depth filter - i.e. Berkefield filter, asbestos - Process non- viral pathogens pathogens, BSL-1, IIA - For BSL-4 - These are porous membranes 0.1mm thick to sterilize Membrane filter culture media, antibiotics Low oncogenic - With fixed opening organisms virus, low toxic - Composed of cellulose acetate and polycarbonate - 70% of air is recirculated chemicals, IIB - Millipore filter with a pore diameter of 0.22µm can give Millipore filter carcinogens 100% sterility - With variable sash opening - With 1 HEPA filter HEPA filter - Can remove objects larger than 0.3µm - Exhaust air is discharged outside the building 4. IONIZING RADIATION/COLD STERILIZATION - Exposure to gamma rays CATEGORIES OF POTENTIAL INFECTOUS AGENTS OF BIOTERRORISM - Sterilize disposable gloves, catheters, and syringes - Indicator: Bacillus pumilus CATEGORY A CATEGORY B CATEGORY C - These are agents - Agents with moderate - Emerging pathogens CHEMICAL METHODS OF STERILIZATION that pose the morbidity and low that can be a. ETHYLENE OXIDE (GAS STERILANT) greatest public mortality engineered for mass - Most commonly used chemical sterilant health threat - Not easily transmitted spread in the future - Used in gaseous form for sterilizing heat sensitive objects - Easily transmitted as category A agents - Disinfectant for machine that cannot be autoclaved and highly contagious - Indicator: Bacillus subtilis var globijii - Can cause high b. FORMALDEHYDE VAPOR AND VAPOR PHASE HYDROGEN PEROXIDE mortality rate - Used to sterilize HEPA filters in BSCs - i.e. Bacillus anthracis - i.e. Rickettsia, Coxiella, - i.e. MERS-CoV, c. GLUTARALDEHYDE (2%) and Francisella Brucella, E. coli SARS-CoV, MDR- - Sporicidal; kills spores in 3-10 hours tularensis O157:H7 Tuberculosis, H1N1 - For medical equipment *Samples for possible bioterrorism must be processed within BSC Class-II d. PERACETIC ACID METHODS OF MICROBIAL CONTROL Board Exam Must-Know! - Sterilization: refers to the complete destruction and removal of all forms of - Cold sterilization: use of glutaraldehyde or peracetic acid microbial life, including their spores - Chemical sterilants a.k.a biocides - Disinfection: destruction and removal of pathogens but not necessarily all microorganisms and their spores B. DISINFECTION A. STERILIZATION PHYSICAL METHODS OF DISINFECTION PHYSICAL METHODS OF STERILIZATION - Purpose: remove pathogens in food - Use of moist heat, use of dry heat, filtration, and exposure to ionizing radiation - 63oC, 30 minutes (low temperature holding): VAT pasteurization or batch method PASTEURIZATION 1. MOIST HEAT - 72oC, 15 seconds: High temperature short time (HTST) - 121oC, 15 psi, 15-30 minutes: to sterilize used and unused media or Flash method - 132oC, 15 psi, 30-60 minutes: to sterilize medical waste - 140oC, 3 seconds: Ultra-High temperature short time AUTOCLAVE - Indicator: Bacillus stearothermophilus (New taxonomy: Geobacillus - 100oC, 15-30 minutes stearothermophilus) BOILING - For surgical instruments - Principle: steam under pressure - Not killed by autoclaving are: Prions (infectious protein particles said to UV - Non-ionizing radiation using UV light cause neurologic diseases in animals and man; Mad cow disease and CHEMICAL METHODS OF DISINFECTION CJD) 1. ANTISEPTIC: chemical germicide for use on the skin or tissue and not to be - Intermittent or fractional sterilization (process is not continuous; done for substituted for a disinfectant TYNDALLIZATION several days) - Alcohols - Uses: flowing steam o Commonly used antiseptic (but not the best): 70% ethyl alcohol or - Equipment used is Arnold sterilizer isopropyl alcohol - 100oC for 30 minutes, for 3 consecutive days - Iodophors o Regarded as the best antiseptic o Iodine + detergent - Intermittent or fractional sterilization - Chlorhexidine INSPISSATION - Principle: thickening through evaporation - Hexachlorophene - For sterilizing media with increased protein, like: Lowenstein-Jensen - 10% hydrogen peroxide is used for cleansing of wounds media (contains egg; for M. tuberculosis) - 75-80oC for 2 hours, for 3 consecutive days Board Exam Must-Know! - 70% ethanol is more effective than 95% EA as disinfectant - Ethyl alcohol and isopropyl alcohol are non-sporicidal Fractional sterilization will kill: - Iodine is prepared as tincture with alcohol or as an iodophor o 1st day: Vegetative cells o 2nd day: Spores o 3rd day: Remaining cells 2. DISINFECTANTS: for non-living things; for surfaces, used to remove blood spills D. CLINICAL SPECIMENS - Halogens (chlorine, iodine, fluorine) - Heavy metals - Avoid normal skin flora while collecting blood for culture - Aldehydes - The collection site must be cleansed with 70-95% alcohol, followed by - QUATS – Quaternary Ammonium Compounds iodine scrub, then alcohol rinse o i.e. Benzalkonium chloride/zephiran - Most common anticoagulant: 0.025% SPS o P. aeruginosa grown in ammonium acetate media is said to be o Inhibits Neisseria, G. vaginalis, P. anaerobius resistant to QUATS o To counteract, add: 1% gelatin - Phenolics - Dilution factor of blood to medium: 1:10 Blood - Amount (adult): 10mL (5mL for aerobic culture and 5mL for anaerobic Additional notes! culture) - Best disinfectant for blood spillage sodium hypochlorite/ household bleach/ - Amount (children): 1-5mL Clorox (10-30 minutes contact); the CDC recommends 1:10 dilution of bleach - Routine blood culture bottles are held for 5-7 days (TAT) - If not available, can be used as substitute is vinegar o Signs of growth: bubbles, hemolysis, cloudiness, pellicle - Standard precaution: require that blood and body fluids from every patient be o Brucellosis: held for 3-4 weeks treated as potentially infectious; set of preventive measures applied to all patients o Leptospirosis: held for 8 weeks designed to reduce risk of infection in health care setting based on the premise - Usually collected using 3-4 tubes Cerebrospinal fluid that all blood and other body fluids are infectious - Tube #2 is for microbiology, gram stain & culture, if with 4th tube, use - Universal precautions: precaution on all human blood and all other body fluids the 4th for better exclusion of skin contaminants (least contaminated) that contain visible blood; set of preventive measures designed to reduce - Examine immediately; priority/1st to process or held either at 37oC transmission of blood-borne pathogens storage temp; transport temperature: room temperature - Process: Centrifuge, use the sediments for smear and culture Examples of bacteria which remain active in a dry environment: - Common pathogens: Haemophilus influenzae and Neisseria - N. gonorrhoeae: viable for 1 hour meningitidis - M. tuberculosis: viable for several months - Usually for the detection of Streptococcal infection - Bacillus and Clostridium: viable for 10 years - Medium of choice: Sheep’s blood agar (SBA) - Most abundant throat flora: Viridans Streptococci COLLECTION, TRANSPORT, AND SPECIMEN PROCESSING - Most common throat pathogen: Streptococcus pyogenes (Group A Throat swab A. SPECIMEN COLLECTION AND TRANSPORT beta-hemolytic Streptococci) - Collect before antibiotic therapy - Culture on Todd-Hewitt broth for fluorescence microscopy of beta Streptococci - Ensure aseptic collection and quantity must be sufficient - Must be placed in sterile containers Nasopharyngeal swab - Ideal specimens should be transported to the lab within 2 hours of collection - Submitted for the detection of Bordetella pertussis - The specimen of choice to detect carrier state of N. meningitidis and to TRANSPORT MEDIA detect the presence of B. pertussis and H. influenzae - Non-sterile specimen; may often be contaminated with normal flora so - Used to maintain the viability of the organism during transport it is important to evaluate the quality of the specimen - With nutrients to maintain the growth of organism, buffer to maintain pH, small - Note the number of squamous epithelial cells/LPF and PMNs to amount of gas to maintain moisture evaluate the acceptability of the specimen Sputum - Stool pathogens: Cary Blair Transport media - >25 PMNs, 1 = sputum - Transgrow: Transport media for Neisseria - A gram stain is performed on all sputum samples - JEMBEC: Transport media for Neisseria - Collected ideally in the morning when it is most concentrated - Specimen of choice for bacterial culture is clean-catch midstream; COLLECTION TECHNIQUE/SAMPLE TYPE catheterized for those who are unable to void  For aerobic culture: collected through swabbing or needle aspiration - Must be preserved or refrigerated if not processed, suitable  For anaerobic culture: needle aspiration, tissue preservative: boric acid (to maintain accurate colony count)  Urine, CSF, Serous fluid, stool, sputum: place in a sterile container - Major cause of UTI: E. coli  Use sterile cotton tip applicator for throat, nose, eyes, wound, abscess - Cause of UTI among young females: S. saprophyticus  Sterile bottle broth with SPS: for blood culture - Suprapubic urine: specimen preferred for anaerobic culture, infants,  For fungal culture, specimen should never be collected through swabbing and children - MAC and BAP are suitable combination Urine B. ANTICOAGULANTS/PRESERVATIVE - Colony count should be performed on all urine sample; when - To maintain accurate colony count, if there is delay in processing of urine, this performing colony count, use calibrated loop (1 µl or 10 µl) preservative may be added: boric acid - Formula to compute for colony count per mL of urine: - 0.025% sodium polyanethol sulfonate/SPS: for blood specimens o # of colonies counted x 1,000 (if 1 µl loop was used) = colony - Heparin: anticoagulant used in viral culture, may inhibit gram positive organisms count/mL of urine and yeast o # of colonies counted x 100 (if 10 µl loop was used) = colony - Viral PCR anticoagulant: EDTA count/mL of urine - Not to be used in microbiology: EDTA and Citrate o Considered significant for UTI: >1.0 x 105 CFU/mL o 1.0 x 103 or 1.0 x 105 CFU/mL: contaminated or specimen C. SPECIMEN STORAGE collected during recovery following treatment - All specimens submitted for culture must be processed immediately, if not, - For the detection of enteric pathogens adequate storage must be observed - Stool specimen not processed within 2 hours of collection should be - CSF (1st priority) should be kept at 37oC if there is delay in processing; transport placed on a transport media like Cary Blair temperature: room temperature - This specimen is routinely screened for Salmonella, Shigella, and - Urine, stool, viral specimens, sputum, swab: placed inside the refrigerator (4oC) Campylobacter Stool - Serum for serology: -20oC for 7 days (1 week) - Maybe directly plated on differential plate EMB or Mac and onto a - Tissue/specimens for long term storage kept frozen at: -70oC selective media HEA or SSA and enrichment i.e. tetrathionate - Specimen suspected to contain anaerobe: should never be refrigerated; should - Number of quadrants streaked: 4 be kept at room temperature - Fecal pathogens - Anaerobic culture is seldom performed (difficult) o Oxidase (+): Vibrio, Aeromonas, Campylobacter, Plesiomonas o Oxidase (-): Enterobacteriaceae (Salmonella and Shigella) Ways to facilitate anaerobic cultivation/anaerobic atmosphere: 5% CO2, 10% - Used to detect urogenital pathogens H2, 85% N2 (Gas Pak Jar) Genital tract specimens - To detect presence of N. gonorrhoeae, G. vaginalis, and C. trachomatis - Boiling of culture medium to remove oxygen which is a common cause of cervicitis - Use of gaspak jar with palladium catalyst (remove oxygen) - Indicators: Resazurin (pink) or Methylene blue (blue) in the presence of air; in the absence of air they become: colorless - Anaerobic cultures are usually incubated at 37oC Board Exam Must-Know! - Ambient air: organism supplied with 21-22% oxygen during incubation - If the patient is in a ventilator (intubated), the preferred specimen is endotracheal aspirate Board Exam Must-Know! - Centrifugation rate and duration: - Most common failure of gaspak jar: inactivation of catalyst due to repeated o CSF & Urine: 1000-1500 rpm for 10-15 minutes use o Sputum: 3,000 rpm for 15 minutes  BACTERIAL IDENTIFICATION - Not gram stained: - Manual/Traditional: Gram staining, selective media, biochemical tests o Intracellular: Rickettsiae, Chlamydia - Semi-automated: uses API (Analytical profile index) o No cell wall: Mycoplasma, Ureaplasma - Automated: Vitek o Cant resolved by bright field: Spirochetes MORPHOLOGY Reasons why Reasons why - Consider the following: size, shape, arrangement, motility, and staining Gram (+) becomes Gram (-) Gram (-) becomes Gram (+) characteristics 1. Overdecolorization 1. Underdecolorization 2. Use of acidic gram’s iodine (alkaline 2. Use of thick bacterial MOTILITY gram’s iodine must be used) smears - Motile: those with flagella - Non-motile: no flagella but may show Brownian motility ACID FAST STAIN STAINING CHARACTERISTICS - To distinguish acid-fast from non-acid fast organisms - Staining: process of artificially coloring the organism with dyes/stains - Acid fast organisms: are organisms that are very hard to stain but once stained, - Purpose of staining: they are very hard to decolorize because of mycolic acid/hydroxymethoxy 1. To observe and appreciate the appearance of bacteria acid that envelopes the bacteria 2. To differentiate one organism from the other 3. To reveal the chemical nature of bacteria - Rule: all bacteria are non-acid fast except: o Mycobacterium and Nocardia (slightly acid fast) STAINING TECHNIQUES - Simple staining Ways to facilitate Acid-Fast staining o Uses only 1 dye; the color of the dye is also the resulting color - Ways to remove mycolic acid temporarily: o All organisms would retain the same color; less differentiation o Steaming process o Ex. Methylene blue o Addition of wetting agents (tergitol) prior to the stain solution - Indirect/Relief/Negative - Increasing concentration of phenol (accentuator; increase the staining power of o The background, not the organism is stained o The organism appears colorless the dye) and basic fuchsin o Ex. India ink/Borris method, Nigrosin o Acid fast organisms are hard to identify under the microscope, even at OIO - Special staining (used to demonstrate the special features of the cell) magnification, especially if they are few in number o Capsular stains = Hiss, Anthony’s, Tyler, Muir - Prolonging contact of stain with the material o Spore stains = Dorner’s, Schaeffer and Fulton, Wirtz and Conklin  Schaeffer and Fulton  Primary dye: Malachite green  Counterstain: Safranin  Spores will take the color of the primary dye which is green o Flagellar stain = Gray’s, Fisher and Conn, Leifson o Metachromatic granules = Albert’s, Neisser, Ljubinsky, Ponder, Methylene blue, Lindergran, Burke’s technique o Nucleic acid = Feulgen o Polar bodies = Wayson o Spirochetes = Levaditi, Warthin Starry, Fontana-Tribondeau - Differential staining o Utilizes more than one dye; used to differentiate one organism from another o The two most common differential stains are Gram’s stain and Acid-fast stain ZIEHL NEELSEN/ KINYOUN’S / AURAMINE GRAM STAIN HOT METHOD COLD METHOD- RHODAMINE PURPOSE Considered as the best Best method to PURPOSE REAGENT GRAM (+) GRAM (-) for DSSM (direct sputum stain AF organisms (FLUOROCHROME) Primary stain/ Most sensitive method smear microscopy) in tissues Crystal violet Appear violet Appear violet Initial stain Primary Auramine- Retain the violet Retain the violet Carbolfuchsin Carbolfuchsin Mordant Gram’s Iodine stain Rhodamine dye color color Mordant Steaming or heat Tergitol None 95% alcohol, Become 3% acid alcohol 3% acid alcohol Acetone, or Remain violet; not Decolorizer 0.5% acid alcohol Decolorizer colorless; (HCl + ethanol) (HCl + ethanol) Alcohol-acetone decolorized decolorized 0.5% potassium mixture Counterstain Methylene blue Methylene blue Counterstain/ Remain violet; permanganate Secondary Safranin red Does not absorb Becomes red Acid fast: red against a blue background stain safranin Result Non-acid fast: blue May be Malachite green (NF = green) - Most critical step in gram staining: used as decolorization substitute - Hucker’s modification: gram staining for for fungi; ammonium oxalate is methylene incorporated to crystal violet blue - Carbolfuchsin method of gram counterstain staining is used (counterstain = - Size of AFB smear: 2x3 cm Carbolfuchsin): to improve staining of - AF organism in tissues is best stained using: Kinyoun’s method gram negative organisms; used on - AFB smear can be fixed using a slide warmer set at: 65oC for 2 hours organisms which are poorly stained by - Following staining, we do microscopy: safranin such as Legionella, Brucella, Brightfield/ Fluorescence Darkfield EM Campylobacter, Fusobacterium, Light Microscope Bacteroides Bacteria + + + + Fungi + + - + RULES Viruses + + - + 1. All COCCI are gram (+) except Neisseria, Branhamella (Now: Moraxella), and Parasites - +/- - + Veillonella 2. All BACILLI are gram (-) except: Other methods of Acid-Fast staining o Mycobacterium, Corynebacterium, Clostridium - Pappenheim’s (urine): to differentiate M. smegmatis (blue) from M. tuberculosis o Bacillus, Lactobacillus o Erysipelothrix (red) o Listeria - Baumgarten’s (tissue): to differentiate M. leprae (red) from M. tuberculosis 3. Higher forms of organisms like Actinomyces, Nocardia, Streptomyces, yeast and (blue) molds are gram (+) - Fite Faraco’s: M. leprae (used as counterstain is hematoxylin) 4. All spiral organisms are reported as gram (-) STUDY OF CULTURAL/ COLONIAL CHARACTERISTICS/ PHENOTYPICAL 3. ENRICHED MEDIA ORGANIZATION - Type of media used for fastidious organisms (organisms which are unable - Culture media: contains the cultural requirements needed for bacterial growth to grow in ordinary media) - Culture: growth of microorganism in culture medium - Usually contains blood - Types of culture - Fastidious organisms: Haemophilus and Neisseria 1. Pure culture: contains only 1 species of organism - Sheep’s blood agar: for Streptococci 2. Mixed culture: contains more than 1 species - Horse blood agar: for Haemophilus 3. Stock culture: often used in research - ATCC: American-type culture collection - Human blood agar: for Gardnerella vaginalis - Best way to obtain pure culture is through streak plate method - Chocolate blood agar: for Neisseria - Other methods of obtaining pure culture: pour plate, serial dilution 4. SELECTIVE MEDIA - Steps to prepare media: - Promotes the growth of the desired organism while inhibiting the growth o Plated: initially weigh, dissolve, sterilize, dispense of others o Tubed: weigh, dissolve, dispense, sterilize - Contains inhibitors (to make the media selective) - Water to make agar: deionized/distilled water - i.e. antibiotics ACCORDING TO PHYSICAL STATE/CONSISTENCY Examples of inhibitors for Examples of inhibitors for gram positive organisms gram negative organisms Solidifying Examples - Bile salts: such as sodium - Potassium tellurite agent/agar desoxycholate None - Brain-heart infusion - Dyes: such as crystal violet Liquid 0% agar - Nutrient broth (incorporated in MacConkey agar) - Alkaline peptone water Semi-solid 0.5-1% agar - SIM Examples of selective media: 2-3% agar Liquefiable Lowenstein Jensen for M. tuberculosis; inhibitor is Malachite Green - When heated again, they liquefy Cystine Tellurite Blood for C. diphtheriae; inhibitor is Potassium tellurite - EMB, Mac-Conkey, MSA, SSA Agar (CTBA) Solid For Streptococci Non-liquefiable Gentamicin blood agar Bacitracin Chocolate agar – for Haemophilus - When heated again, they no longer liquefy Blood agar with Aeromonas - Rice medium or Rice grain agar (for fungi) ampicillin - Both solid and liquid Chloral hydrate Prevent swarming organisms Biphasic - Castaneda (for Brucella) - Human blood bilayer tween 5. DIFFERENTIAL MEDIA - Agar - Used to differentiate organisms that are growing together o Usual solidifying agent o Usually derived from red algae - Ex. EMB and MacConkey (differentiation of lactose and non-lactose o Solidifies at 40-50oC fermenting organisms), TCBS (differentiate Vibrios which are sucrose and o Melts at: 80-90oC non-sucrose fermenters) o Cooling temperature for distribution of media to plate: 55-60oC 6. SELECTIVE AND DIFFERENTIAL o Amount dispensed: 25mL per plate - EMB & MacConkey: Selective because they only allow the growth of gram negative bacilli; Differential because they can differentiate lactose and ACCORDING TO COMPOSITION non-lactose fermenters 1. SYNTHETIC/DEFINED 7. TRANSPORT MEDIA - All components are known to the user - May be required to maintain the viability of organisms during transport - For research - Maintains moist environment, with nutrients to maintain growth - Ex. BG-11 for the isolation of Cyanobacteria o Transgrow/JEMBEC: for Neisseria 2. NON-SYNTHETIC/COMPLEX o Stuart’s: Viral transport media - Composed of some unknown substance (peptone, meat) o Cary Blair: for stool pathogens - Useful for bacterial isolation 8. MEDIA FOR SUSCEPTIBILITY TESTING - EMB, MacConkey, Blood agar, MSA - Mueller-Hinton Agar 3. TISSUE CULTURE - For viral culture; for virus isolation we use living cells to allow growth of OTHERS viruses 1. TODD-HEWITT BROTH - In tissue culture, we wait for: cytopathic effect (CPE) - Selective enrichment media for S. agalactiae in female genital specimens o Cytopathic effect: change in the nucleus/cytoplasm of cells due to the presence of virus - Used to detect genital carriage of Group B Streptococci during pregnancy - Types of tissue culture: 2. CNA (COLUMBIA-COLISTIN-NALIDIXIC ACID) o Primary cell line – uses normal mature adult cells (ex. Primary monkey - For gram positive organisms kidney) o Semi-continuous/diploid – uses embryonic or fetal cells Other commonly used media: o Continuous/heteroploid – uses malignant, cancerous, or tumor cells  HeLa cells: uses cervical cancer cells 1. SHEEP’S BLOOD AGAR  A549: uses cells from lung carcinoma - For Streptococci  Hep2 cells: uses laryngeal cancer cells - To determine hemolytic pattern - Vero cells: from African green monkey kidney cells 2. PEA (PHENYL ETHYL ALCOHOL) AGAR - McCoy cells: for Chlamydia - For gram positive cocci and other anaerobic gram negative rods ACCORDING TO USE Additional info! 1. GENERAL PURPOSE/GENERAL ISOLATION MEDIA - Contains only the necessary nutrition to support bacterial growth - Bacterial cultures are held for: 48 to 72 hours - Does not contain any supplement o Anaerobes and broth culture are held for: 5 to 7 days - For routine cultivation of bacteria - In THIOGLYCOLLATE – enrichment broth - I.e. nutrient agar and nutrient broth o Gram negative facultative anaerobes: diffuse even growth all throughout the 2. ENRICHMENT MEDIA media o Strict aerobes: growth towards the surface - Type of media used to enhance bacterial growth o Anaerobes: growth at the bottom - When we place organisms in this type of media, bacterial yield is also increased - Ex. Alkaline peptone water for Vibrio, and medias for the isolation of gram negative bacteria such as Selenite broth and Tetrathionate broth  SUSCEPTIBILITY TESTING Possible Sources of error - To detect ability of antimicrobial agent to inhibit bacterial growth in vitro - To determine susceptibility/resistance of organism against antimicrobial agents FALSE SENSITIVE FALSE RESISTANT 1. Too light inoculum 1. Too heavy inoculum METHODS 2. Thin agar 2. Thick agar 1. Dilution method 3. Very dry agar 3. Too much moisture on surface - Standard inoculum for broth dilution: 5 x 105 CFU/mL 4. Acid pH affect result in of agar Tetracycline, Novobiocin, 4. Prolonged incubation - Standard inoculum for agar dilution: 1 x 104 CFU/mL Methicillin - Total broth volume 5. Alkaline pH affect result in o Microdilution: 0.05-0.1 mL aminoglycosides/erythromycin o Macrodilution: 1mL or greater - TUBE DILUTION - Use of mixed culture o A quantitative technique in which measured amounts of antibiotics are prepared by serial dilution to which is added specific concentration of - Improper storage of disk the suspected bacteria o Indicator of improper storage: Penicillin and Methicillin o Serial dilution of antibiotic is prepared, to each tube a uniform amount - Storage temp for antibiotics of inoculum is added o WORKING SUPPLY o Process: o Kept inside the refrigerator (4-8oC or 2-8oC) or -20oC in a desiccator  Several tubes are prepared for every antibiotic to be tested - If there is swarming: ignore  Prepared different concentrations of antibiotics through serial - When using SULFONAMIDES (such as SXT) if there are 2 concentric zones dilution measure the outer zone  Least diluted is tube 1, most diluted is tube 10  Add the same amount of inoculum/organism to all tubes Additional notes!  Incubate at 37oC - Antibiotics (derived from bacteria or fungi): Streptomycin and Penicillin  Identify tube with no growth/no turbidity = MIC - Chemotherapeutics: chemically produced (ex. SXT) o MIC: Minimum inhibitory concentration; lowest concentration of - Narrow: has limited range of action antibiotic that inhibited bacterial growth - Broad spectrum: can act against a number of organisms o MBC/MLC: Minimum bactericidal concentration/minimum lethal - Bacteriostatic: inhibit bacterial growth (ex. chloramphenicol, tetracycline, concentration; lowest concentration of antibiotic that killed the clindamycin) organism - Bactericidal: kill the organism (ex. beta lactams) - AGAR DILUTION METHOD o Same procedure as tube dilution but must be done on petri dishes What is "D" test? 2. Disk diffusion method - Not usually done in the lab; susceptibility - A qualitative technique and sensitivity test - Kirby Bauer Method - Double disk diffusion test KIRBY BAUER METHOD - To detect inducible clindamycin resistance among strains of S. aureus 1. Preparation of media - Uses: 15 ug erythromycin + 2 ug - Media: MHA (Mueller Hinton Agar) clindamycin - Depth of agar: 4mm - Positive result: appear as if it is - pH of agar: 7.2-7.4 sensitive; blunting/flattening of - Uses filter paper disks/size: 6mm clindamycin zone to have “D pattern” - MHA: standard media for Disk diffusion method - Negative: erythromycin zone should - MHA w/ 5% sheep blood: for susceptibility testing of Streptococcus and appear as if it is resistant Neisseria meningitidis - Report: - MHA w/ 2% NaCl: susceptibility testing of MRSA and other Staphylococci o Negative = clindamycin sensitive - Middlebrook 7H10: isolates Mycobacteria and susceptibility testing of o Positive = clindamycin resistant Mycobacterium only What is "E" test? 2. Preparation of inoculum - A susceptibility test, a quantitative method, and a dilution method - Throat swab on BHI/TSB; Incubate for 24 hours at 37oC - a.k.a. MIC on a stick o Turbid = (+) growth - Uses a strip with single antibiotic of decreasing concentration along its length - Subculture 4-5 colonies on TSB - For fastidious organisms and anaerobes - Incubate at 37oC for 3-5 hours or until the turbidity of the subculture - Positive result: ellipse of growth inhibition matches the turbidity of the McFarland standard o Too turbid = dilute - Compare turbidity of subculture with McFarland standard purpose: in order to standardize the inoculum - 0.5 McFarland Standard o 0.5 mL of 1.175% Barium chloride o 99.5 mL of 1% sulfuric acid o Equivalent of McFarland Standard: 1.5 x 108 CFU/mL - If turbidity is ok, inoculate on MHA (using sterile cotton swab) - If too turbid, dilute using NSS or distilled water (overlap streaking) - Wait for 3-5 minutes before applying the disk 3. Plate size & number of disks; distance of disks BACTERIAL IDENTIFICATION - If plate size is 150 mm place no more than 12 disks CONVENTIONAL - Microscopy is performed - If plate size is 100 mm place no more than 5 disks o Motility - Distance of disk from center is 24 mm, between 2 disks is 15mm o Staining - Invert plates and incubate 35- 37oC; incubation time: 16-18 hours - Use of selective medium - Aerobic incubation no CO2 - Manual biochemical tests - Measure zone of inhibition using a ruler or caliper, at the underside of the ANALYTICAL - Semi-automated procedure plate, and interpret if S, R or intermediate PROFILE INDEX - Biochemical tests are still performed but made smaller - For media with blood: measure the ZOI from the top with cover removed - Uses plastic strips and microtubes with biochemical - For Streptococci, MHA is supplemented with 5% Sheep's blood substrates - The biochemical substrates are inoculated with pure culture suspension - Ex. API 20E (for the identification of Enterics) AUTOMATED - Vitek 1 & 2 (Automation program for bacterial identification and susceptibility) ANTIBIOTICS Erythromycin Macrolide Clarithromycin Bacteriostatic MODE Azithromycin REPRESENTATIVE Inhibit protein synthesis OF CLASS For intracellular ANTIBIOTICS ACTION organisms, spirochetes Penicillin V Allergic reaction Tetracycline Natural Tetracycline Bacteriostatic Penicillin G is a common side Doxycycline penicillins Can stain teeth, effect abnormal bone growth Methicillin Gentamycin Oxacillin Amikacin Toxic to kidneys and Synthetic Aminoglycosides Ampicillin Tobramycin ears penicillins Carbenicillin Kanamycin Piperacillin Chloramphenicol Inhibit cell wall synthesis Cephalexin Ceftriaxone Inhibit folic Cephalosporins synthesis Bacteriostatic Beta lactams Cefotoxin Sulfamethoxazole acid Cefepime Sulfonamides Used primarily for UTI Trimethoprim Carbapanems Imipenem Monobactams Aztreonam Drug of choice synthesis for MRSA; Some Ciprofloxacin Inhibit Ciprofloxacin used for DNA strains of Quinolones Levofloxacin anthrax Enterococcus are Ofloxacin now resistant Bacitracin Glycopeptides Vancomycin membrane inhibitors Colistin Cell Polymixin Amphotericin B Nystatin RECALLS FROM PREVIOUS BOARD EXAMS Fluorescent Critical step in stain will require Darkfield Bacteria with Ethylene oxide Chemical sterilant Decolorization Campylobacter gram staining what type of microscope darting motility Bench marking Peer microscope Increase in size Lag phase is also known as comparison Unique in cell Subacute CNA is for Gram (+) cocci wall of Mycolic acid Sulfuric acid + 0.5 McFarland S. viridans bacterial Mycobacteria Barium chloride endocarditis CSF storage Schaeffer and 37oC Spore stain 121oC, 15 psi, Species are temperature Fulton Autoclaving Lowercase 15 minutes written in Tumbling motility Listeria JEMBEC Neisseria Incubation at 35- Bacterial and viral Water used to at 25oC Deionized water 37oC culture make agar Best disinfectant Bacterial 10% household Bacteria are To measure Use calipers or for blood VITEK system identification and Moisture bleach attracted to zone of inhibition rulers spillage sensitivity Automation Urine CS for Dye retained by Carbolfuchsin program for VITEK Soap Germicidal ambulatory Random urine AFO susceptibility patients Bacteria with the Treatment for Urine CS for Clean catch Vancomycin Kirby Bauer Filter paper disk same size as Mycoplasma MRSA women midstream virus Smears of CSF Commonly used Cold AF stain Kinyoun’s are prepared Sediments B. anthracis to test for from BSC Class 2 VITEK uses sensitivity Possible result Susceptible, Purpose of nowadays in antibiotic intermediate, boiling Drive off oxygen Alternative if susceptibility 10% household resistant thioglycollate Disinfectant there is no Vinegar testing bleach Clorox in the lab Source – mode Iodine + Todd Hewitt Chain of of transmission Throat pathogen S. pyogenes Iodophor S. agalactiae detergent broth is for infection & susceptible Negative stain host