Unit 1 Lecture Notes.pdf

Introduction to Clinical Microbiology Role of the Clinical Microbiologist from Bailey & Scott’s Diagnostic Microbiology. “A Principal role of a clinical microbiologist is to consult with other health care professionals, to provide accurate infectious disease diagno...

Introduction to Clinical Microbiology Role of the Clinical Microbiologist from Bailey & Scott’s Diagnostic Microbiology. “A Principal role of a clinical microbiologist is to consult with other health care professionals, to provide accurate infectious disease diagnosis, to devise laboratory testing to assist in optimum management of infectious diseases in patients. Microbiologist communication and collaboration with the healthcare team helps provide diagnosis and optimal management of infectious disease, and helps to prevent spread of disease.” I. Diagnostic Microbiology A. Purpose 1. Provide relevant information needed to make clinical decisions 2. Establish guidelines for proper specimen collection, transport of specimen, and the isolation of the infectious agent. 3. Identify organisms rapidly (including presumptive & final), accurately, and definitively 4. Perform susceptibility testing for treatment 5. Establish close consultation between clinician and microbiologist 6. Being properly trained, having experienced microbiologists 7. Assist in control of nosocomial pathogens B. Detailed Duties 1. Having experienced microbiologist on duty for 24 hours/day so that results can be reported as soon as possible 2. Help the facility save money by organizing workflow *By identifying and controlling nosocomial pathogens and to quickly track organisms resistant to antimicrobial agents. Contacting infection control department as soon as resistance is known. Nosocomial infections occurring 72 hrs after admission to hospital. Major one urinary tract infection, also pneumonia and wound infection. 3. Coordinate the antimicrobial agents tested in the laboratory with those selected by the institutions pharmacy and therapeutic committee. 4. Serves the patients and clinicians by preparing and updating manuals regarding appropriate specimen collection, transport, and labeling. 5. Positive findings should be reported to the clinician. Results should be legibly handwritten or generated electronically using a computer. Documentation should include info given, who it’s reported to, tech initials, date, and time. 6. Microbiology technologists also use computers to help send information throughout the hospital 7. Quantitates bacterial culture results, distinguishing pathogens from normal flora, determining importance of different organisms in a mixed culture, aids in comparing numbers of organisms growing on plated media to that seen in direct gram stain to detect presence of anaerobes or fastidious organisms. II. Safety All personnel working with infectious agents should observe the appropriate safety precautions. All labs have eye wash stations, emergency showers, and fire extinguishers centrally located. A. Exposure Control Plan: mandated by OSHA The plan must be annually reviewed and updated. The plan must identify tasks that are hazardous to employees and promote safety through use of the following: 1. Employee education of possible hazards 2. Appropriate disposal of hazardous waste 3. Standard precautions 4. Enforcing safe work practices and having controls in place to ensure that rules are followed 5. Provide personal protective equipment (lab coat, mask, gloves, etc.) 6. Post-exposure plan- All employees should be aware of what steps to take in the event of an accident. A plan to investigate the incident and ways to prevent reoccurrence. B. BioSafety People are exposed in various ways to laboratory acquired infections. These include the following: 1. Rubbing or touching eyes, nose, or mouth with contaminated hands 2. Inhaling aerosols produced during centrifugation, vortexing, or spills of liquid cultures or body fluid 3. Inhalation of infectious agents such as fungi, Brucella, or Mycobacterium 4. Accidental needle stick 5. Contamination through open cut or wounds C. Protection from Infectious Agents 1. Biohazard symbol should be prominently displayed on lab doors and equipment 2. Negative pressure rooms: Ideally the microbiology lab should have negative pressure to prevent administrative areas from being exposed to toxic or pathogenic materials from escaping and injuring humans or contaminating the environment. Patients suspected of having TB are placed in negative pressure rooms. 3. Limitations are placed on visitors inside the laboratory, especially micro. 4. High Efficiency Particulate Air (HEPA) filters are supplied to phlebotomists who may enter potential TB patient’s rooms. Also supplied, if necessary for techs cleaning up spills. D. Biological Safety Cabinets (BSC) Defined as a device that encloses a workspace in such a way as to protect workers from aerosol exposure to infectious disease agents. Air that contains the infectious material is sterilized, either by heat, ultraviolent light, or most commonly by passage through a HEPA filter that removes most particles larger than 0.3um in diameter. There are several types of cabinets that include the following:  1. Class I cabinet (laminar flow BSC): allows room air to pass into the cabinet around the area and material within, sterilizing only the air to be exhausted. The air flows in sheets. 2. Class II cabinet: has a variable sash opening through which the operator gains access to work the surface. Air is passed through a HEPA filter before reaching workspace, and then again before being exhausted outside. Commonly used BSC in the micro department.( Mostly common used safety cabinet in micro) 3. Class III cabinet: has the highest level of protection. Air coming into and going out of the cabinet is filter sterilized and the infectious material within is handled with rubber gloves that are attached and sealed to the cabinet.( Higher level of protection). E. BioSafety Levels Based on the organism’s ability to cause disease and availability of treatment 1. BSL-1: Minimal (Medical records, receptionist, mantience) potential hazard. No known pathogenic potential for immunocompetent person. Work conducted on open bench tops with PPE. Ex. Bacillus subtilis (Found on arms,skin,outside) 2. BSL-2: Moderate potential hazard. Handling of specimens which may contain common infectious agents including HIV. Workers must be immunized. All PPE must be worn. (MLT’S) Ex. Staph, Neisseria, Cryptococcus 3. BSL-3: Potential for respiratory transmission. May cause disease that has the potential to be fatal. PPE plus full protective clothing and devices; additional air handling, etc. Must have negative air pressure. (Working in micro dept, nurses) Ex. Arboviruses, arenoviruses, M.tuberculosis (Mycobacterium, Tuberculosis) , Francisella 4. BSL-4: Potential for life-threatening transmissions through aerosol route, for which there is no available vaccines. Thorough training required. (CDC) Ex. Ebola, smallpox, arenaviruses or filoviruses (none in US) F. Special Precautions for Areas: 1. Bacteriology: study of bacteria a. Use biological safety cabinet (BSC) for: 1. All respiratory tract specimens ( EX. Lung fluid, fluidum, broncolonwashing, brushing get specimen, broccolini) 2. All tissue specimens (Piece of organ, piece of bone) 3. Blood culture systems- laminar flow cabinet when entering 4. Opening specimen containers b. Use Class II or higher BSC for processing cultures on lab media after incubation of: (Don’t open for anthrax) Yesinia pestis Coxiella sp. Brucella sp. Bacillus anthracis Francisella tularensis Pseudomonas pseudomallei Rickettsia 2. Mycobacteriology: study of mycobacteria  Mycobacteria also known as AFB = Acid fast bacilli EX. TB (Tubercolisis) a. All specimens in BSC b. Wear solid front gowns, masks c. Centrifuge in sealed tubes in BSC 3. Mycology: study of fungus (EX. Green stuff grown on bread or food) (Penicillum) a. Tape all inoculated plates shut or sealed b. Use BSC to work on mold like fungi, esp, fuzzy white 4. Parasitology: study of parasites (EX. Plasmodium, malaria, Enterobius, pinword) a. Wear gloves b. Use BSC for Pneumocystis carinii or flukes 5. Virology: study of viruses (Herpes simplex, HIV, CMV, Adevirus) a. Use BSC b. Wear gloves c. Use 4x4 gauze soaked in 10% bleach to cover rubber stoppers of serum tubes when removing stoppers III. Decontamination Procedures A. Important terms 1. Sterilization- removal of all forms of life including spores 2. Disinfection- removal of a wide range of organisms, but does not kill spores 3. Disinfectant- chemical agents applied to inanimate objects(Cleans like a counter) (Doesn’t kill spores) 4. Antiseptic- disinfectant applied to living tissue (skin), does not kill spores (Wash your hands, alcohol pad, iodine) 5. Bacteriostatic- inhibits growth but does not kill bacteria 6. Bacteriocidal- kills bacteria B. Factors Affecting Degree (Disinfectant) 1. Types of organisms 2. Number of organisms 3. Concentration of disinfecting agent & following proper directions 4. Amount of organic soil (material) present 5. Nature of surface to be disinfected 6. Contact time (critical) 7. Using chemicals together (one may inactivate the other) C. Selection of Disinfection/Sterilization for Medical Materials 1. Critical materials: invade sterile tissue or vascular system; need sterilization (Needle, catheter) (Have to be sterilized) 2. Semi critical materials: come in contact with mucous membranes; need high level disinfectant ( Cannoli – put in nose to give oxygen – can throw away – but if used again must be sterilized) 3. Noncritical materials: come in contact with skin; need intermediate to low level disinfectants (Turnic) D. Methods for Disinfection/Sterilization 1. Physical Methods A. Moist or dry heat (most common) 1. Boiling water a. 100°C, 15 min b. Kills vegetative forms, spores survive 2. Autoclave: must be quality controlled (Like a oven, applies to pressure to put whatever in there) a. 121°; 15 min.; 15psi b. Sterilizes; kills spores c. Commonly used to sterilize media, instruments equipment stc., and decontaminate waste 3. Dry heat; flame or dry air oven a. 160-180°C; 1.5-3 hours b. Sterilizes c. Dry oven when used materials could be harm by moisture d. Used for glassware 4. Pasteurization (Milk, Food, Cheese) a. Batch pasteurization: 63 °C; 30 min; disinfects; endospores survive b. Flash pasteurization: 72°C; 15 min; disinfects; endospores survive c. Used mostly for food products d. Destroys food pathogens B. Radiation 1. Ionizing: gamma rays or electron beams have short wavelength and high energy, used for disposable plastics and heat sensitive materials. Used commercially for (syringes, gloves, catheters, butterfly needles) 2. Non-ionizing: UV; long wavelength; low energy. Used for surfaces (Kill anything that its shining on) C. Filtration 1. Filters containing pores used for antibiotics, vaccines, etc. 2. Pore size 0.45um and 0.80um required to remove bacteria, yeast, molds 3. HEPA filters are used for air filtration in goods D. Chemical Methods (chemosterilizers) a. Regulated by EPA b. Modes of action 1. Destroys cytoplasmic membrane 2. Destroys cell proteins 3. Reacts with enzyme group 4. Damages DNA & RNA c. Types 1. Phenols: Very effective against vegetative bacteria, including mycobacteria. Spores are not affected. Ex. Orthophenylphenol in germicidal soaps 2. Alcohols: Spores and no affected. Bacteriocidal, fungicidal, tuberculocidal, virucidal. Must evaporate to be effective. Ex. 70% ethyl alcohol as skin antiseptic (Alcohol pads)  3. Iodophors: Used mainly in antiseptic, kills spores, bacteria, fungi, viruses. Formulations for antiseptics or disinfectants should not be interchanged. Ex. 2-3% iodine, povidine (used for blood culture collection) 4. Aldehydes: Kills spores, bacteria, viruses particularly HBV. Used to sterilize heat sensitive instruments and large areas, rooms. These are toxic to humans. Ex. Formaldehyde; glutaraldehyde 5. Quaternary ammonium compounds: Generally bacteriostatic, more effective against gram-positive bacteria. Does not kill spores or tuberculosis. Ex. Detergent 6. Hydrogen Peroxide: Mild oxidizing agent used frequently for antiseptic purposes. Kills bacteria and fungal spores. 7. Chlorine compounds: Oldest and most commonly used. Requires at least 3 minutes of contact Ex. Hypochlorite for disinfecting countertops Bacterial Classification I. Taxonomy A. Kingdom: Prokaryotae 1. Phylum 2. Class 3. Order 4. Family (-aceae) 5. Genus (capitalized) ex. Staph 6. species (lower case) ex aureus 7. Subspecies 8. subspecies may be further divided: a) servoarieties (serologic) b) biovarieties (biochemical testing) c) phage typing (susceptibility to phages) d) restriction fragment length polymorphism analysis (DNA, RNA analysis) B. Bacteria Terms 1. Pathogen: a microorganism, including bacteria, viruses, fungi, and parasites that cause disease or infection 2. Normal flora: microorganisms normally residing in a particular body sire; they do not generally cause infection  3. True pathogen: one that has the ability to infect those individuals with a healthy immune system as well as those who are immunosuppressed 4. Opportunistic pathogen: one that attacks an already debilitated host but usually presents no danger to an individual with an intact immune system 5. Infection: the entrance and multiplication of microorganisms in or in a host 6. Infectious disease: refers to an infection with functional and structural harm to the host that usually is accompanied by signs and symptoms C. Bacterial Transmission to Humans 1. Directly: congenital, sexual, inhalation droplet infection hand-to-hand, hand-to- mouth, etc. 2. Indirectly: fomites, food & water, animals, insects, or arthropod vectors D. Types of Cells 1. Prokaryotic: bacteria 2. Eukaryotic: algae, fungi, protozoa, animal cells 3. Archaebacterial: organisms grow under extreme environmental conditions II. Prokaryotic (bacterial) Cell Structures A. Cell envelope: consists of the cell membrane and cell wall 1. Cell membrane: phospholipid bilayer with embedded proteins; acts as a barriers & is where electron transport chain is located, which produces energy; controls nutrients & wastes in & out of cell 2. Cell wall: rigid structure that helps to maintain shape, acts as a barrier, allows nutrients to pass through, & helps attachment to host a) Gram positive: on thick peptidoglycan & outer layer consisting of proteins, phospholipids, and lipopolysaccharide; the lipopolysaccharide consists of antigenic O, core, & lipid A or endotoxin A b) Acid fast: gram positive cell wall with a waxy layer of glycolipids and fatty acids c) No cell wall: Mycoplasma & Ureaplasma have no cell wall, but have sterols in their cell membrane B. Cytoplasm: gelatinous material consisting of ribosomes, granules, endospores 1. Ribosomes: protein synthesis 2. Granules: storage deposits of glycogen or lipids 3. Endospores: small asexual develop inside bacterial for survival from chemicals, temperature, dehydration, etc. 4. Replication: binary fission 5. Plasmids: extrachromosomal DNA; transfer genetic info; aids in antibiotic resistance C. Surface polymers 1. Capsule: a polysaccharide polymer a) Inhibit phagocytosis b) May block antigen detection and need to be removed c) Appears as a clear halo when stained 2. Slime layer: similar to capsules a) Inhibit phagocytosis b) Aid in adherence 3. Flagella: rotates and helps organism to be motile  a) Polar: only at one end b) Peritrichous- all the way around the organism c) May not possess any d) Aid in identification upon staining 4. Fimbriae: sticky protein, hair-like, help in adherence 5. Pili: nonmotile long protein that connect two bacteria cells to allow DNA exchange III. Bacterial Metabolism: regulated by enzyme production and activity A. Fermentation and respiration: used to break down carbs for energy 1. Fermentation: anaerobic process in which an organic compound is the electron acceptor a) Produces less energy than reparation b) Produces lactate, ethanol, acetoin, butyrate end products c) Testing of end products help identification d) Carried out by obligated and facilitative anaerobes 2. Respiration (oxidation): energy producing process in which oxygen is the final electron acceptor a) Produces much more energy than fermentation b) Carried out by obligated aerobes and facultative anaerobes B. Glucose utilization: three pathways that all break down glucose to pyruvic acid 1. Embden- Meyerhof-parnas glycolytic pathways 2. Pentose- phosphate pathway 3. Entner- deidoroff pathway C. Fermentation of pyruvate: a) Alcohol b) Lactic acid c) Lactic acid, CO2, alcohols, formic acid, acetic acid d) Propionic acid e) Lactic acid, succinic acid, formic acid (mixed acid fermentation)—methyl red test f) Acetyl methyl carbinol (acetoin) and 2,3 butabediol (butanediol fermentation)—VP reaction g) Acetic acid and butyric acid D. Oxidation if pyruvate 1. Krebs cycle and tricarboxylic acid (TCA) a) Produces large amount of ATP b) Acid and carbon dioxide are produced E. Lactose fermentation 1. Ability of organism to ferment lactose, helps identification 2. Organisms that ferment lactose can also ferment glucose 3. First step requires beta-galactosidase permease to transport lactose through call wall 4. Second step requires beta-galactosidase to break the bond to release glucose to be fermented 5. Slow LFs possess only beta-galactosidase 6. ONPG test: detects bets galactosidase for slow LFs IV. Growth of Organisms A. Nutritional Requirements for Growth 1. Carbon & nitrogen (food), moisture, ATP, warmth, neutral pH 2. Trace elements (inorganic salts) 3. Enrichment factors (blood, or serum, hemin, iron, yeast extract provides vitamins, and carbohydrates, etc.) B. Energy sources: from oxidation-reduction reactions 1. Fermentation- utilization of carbohydrates under anaerobic conditions 2. Oxidation- utilization of carbohydrates in the presence of oxygen C. Growth Classification 1. Autotrophs- CO2 as carbon source; H2O, inorganic materials, energy from photosynthesis or oxidation of inorganic substances 2. Heterotrophs- organic substances are carbon source a) Energy from oxidation or fermentation b) Human pathogens are heterotrophs D. Typical growth curve in liquid media 1. Lag phase: getting ready to divide 2. Log (exponential) phase: division, numbers of bacteria increase exponentially 3. Stationary phase: numbers of bacteria remain the same, nutrients becoming limited 4. Decline: death phase V. Bacterial-Human Relationships A. Host-Parasite Terms 1. Commensalism: parasite living in host that causes no harm or benefit to host 2. Parasitism: living on or within a host, cannot live without 3. Compromised host: increased risk of infection when the host is weakened 4. Exogenous source: somewhere other than patient 5. Endogenous source: normal bacterial flora 6. Virulence: ability of organism to cause infection or disease 7. Toxigenicity: ability to produce toxins that damage the host 8. Invasiveness: measure of ability to invade & live in the host B. Immunologic Terms 1. Antigen- Antibody response: antibodies directed against bacteria esp. secretory IgA in respiratory, genital and digestive tract 2. Phagocytosis: neutrophils & macrophages kill & ingest bacteria 3. Bacteriolysis: rupture of bacteria, due to natural causes or by chemicals from neutrophils and antibiotics 4. Inactivation of toxin: IgG antibodies neutralize toxins by blocking active site on toxin molecule C. Bacterial Toxins: released by bacteria that causes damage 1. Exotoxin: 2 subunits, one binds to cells & is nontoxic, second is toxic 2. Endotoxin: GNRs, toxin composed of cell wall, causes BP changes, clotting, fever, septic shock, and immune system effects 3. Cytolytic toxins (cytotoxins): toxins that cause alterations in activity of cells VI. Microscopic Examination  A. Types of stains: direct specimen or from culture 1. Gram stain a) Used to distinguish Gram positive and Gram negative b) Steps: crystal violet, iodine, decolorizer, safranin c) Gram positive: purple, Gram negative: pink 2. Acid fast: mycobacteria a) Ziehl-Neelson: hot method, carbolfuschin, heat, acid alcohol, methylene blue b) Kinyoun: cold method, carbolfuschin, detergent, acid alcohol, methylene blue c) Positive: red, Negative: blue 3. Fluorescent stains: fluorescent microscope a) Auramine-rhodamine: binds to mycolic acid in cell wall, positive: orange-yellow b) Acridine orange: binds to nucleic acid, used in blood/CSF, positive: orange 4. Calcoflour white: fungal stain a) Binds to chitin in fungi b) Used in tissue and other specimens c) Positive: blue/white or apple green 5. Methylene blue a) Stains metachromatic granules b) Used for Corynebacterium diphtheriae 6. Lactophenol Cotton Blue: stains fungi 7. India Ink: negative stain; used for Cryptococcus 8. Fluorescein-conjugated stains a) antibodies bind to fluorescein-isothiocyanate, which binds to antigen if present b) Fluorescent scope required to see bacteria in direct specimens c) Positive: apple green d) Used for Bordetella pertussis, Legionella, Chlamydia, herpes, respiratory syncytial, rabies, treponemes 9. Enzyme-conjugated stains a) Enzyme horse radish peroxidase is bound to specific antibody. Conjugated stain will bind to antigen is present. b) Horse radish peroxidase produces orange-brown precipitate c) Measured photometrically d) Used to identify CMV and other viruses 10. Electron microscopy: greater magnification to study bacteria, fungi, parasites, viruses in more detail Specimen Collection and Examination I. Collection Procedures A. General Considerations 1. Collect before antibiotics a) May give false negative culture results b) Notify lab  2. Collect specimen from area most likely to have organism a) Avoid normal flora b) Cleanse area with alcohol or iodine 3. Types of specimens a) Blood, CSF, other fluids, urine, wounds, stool, throat, ear skin, etc b) Anywhere infection is suspected 4. Sterile collection containers 5. Transport media: preserves bacteria but prevents overgrowth, ex. Stuart broth, Amies, Cary-Blair 6. Sufficient quantity a) Adequate for complete exam b) Inadequate specimens marked QNS 7. Prompt delivery a) Prevents overgrowth of normal flora, causing pathogen not to grow b) Prevents organisms from dying (fastidious require special requirements) c) 2 hour limit between collection and receipt of specimen if not in media to preserve B. Specimen Rejection 1. Name/source/Id don’t match computer or requisition 2. Easily recollect: stool, urine, wound, sputum 3. Not easily recollected: blood culture, amniotic fluid, CSF, OR specimens 4. Stool with barium sulfate or urine 5. Syringe with needle attached 6. Leaking container 7. Refrigerated blood culture 8. Specimen in formalin 9. Dried specimen 10. Collector is notified if rejected, documentation of call, and asked to recollect II. Processing Specimens A. Gross examination: document description such as volume, clear/cloudy, dry swab, bloody, clots, etc. B. Direct examination 1. Slide for staining prepared a) Swabs b) Tissue: touch to slide c) Fluids: centrifuge, use sediment 2. Other methods a) Direct wet mount b) KOH prep (10%): fungal elements, protein is partially digested C. Slide Examination 1. Screen under low power, then oil immersion, examine large area of smear 2. Review slide for a) Bacteria, fungi, parasites: must be graded b) RBCs  c) Epithelial cells: contamination d) Curshmann spirals: in sputum in bronchospasms e) Intracellular organisms: seen in infection f) Mucous: surface irritation of gland g) WBCs: indicate infection Methods for Bacterial Growth and Identification I. Types of Media A. Classification of Media 1. Broth: liquid, gel, or semi-solid 2. Supportive: will grow most nonfastidious; ex. Nutrient agar, trypticase soy agar 3. Nonselective or enriched: nonfastidious media; ex. BAP (blood agar plate), Choc 4. Selective: supports growth on one type of bacteria; ex. Thayer Martin for Neisseria gonorroheae 5. Differential: distinguishes bacterial according to metabolic characteristics a) MacConkey: lactose fermenters and nonlactose fermenters; selective for GNRs b) Eosin methylene blue (EMB): selective and differential 6. Enriched substances can be added to media to support fastidious, ex. Choc 7. Enrichment broth: used with few bacteria are present to cause bacteria to increase in growth a) Selenite: allows isolation of Salmonella/Shigella; sodium hydrogen selenite b) GN broth: toxic to gram positives, inhibits normal flora 8. Broth medium: grows aerobes, anaerobes, microaerophilic 9. Antibiotic media: antibiotic inhibits gram negatives and allows gram positives; ex. CNA (colistin-naladixic acid) B. Routinely Used Media 1. SBA (sheep blood agar): made from TSB (tryptisoy sheep blood) and 5% sheep red cells; preferred media to observe for hemolysis 2. Choc (chocolate): similar to SBA except cells are lysed by heat, releasing NAD; also has dextrose, cysteine, B12, thiamine, ferric nitrate, supports fastidious such as H. influenzae and N. gonorrohoeae 3. MAC (MacConkey): contains bile salt, lactose, neutral red indicator, and crystal violet. Selective and differential: selects for GNRs to grow, differentiates lactose and nonlactose 4. THIO (thioglycolate) broth.: supports anaerobes, microaerophiles, aerobes; fastidious organisms may grow if serum or heme is added; serves as a backup to plated media. 5. Special Media a) Brucella: Castenada bottle (biphasic bottle) b) Bordetella pertussis: Bordet-Gengou c) Anaerobes: Schaedler, CDC, BBE  d) Clostridium botulinum: anaerobic e) Actinomyces: anaerobic f) Cornyebacterium diptheriae: Loeffler, tellurite g) Francisella: chocolate h) Haemophilus ducreyi: chocolate i) Leptospira: Fletcher’s media j) Vibrio: TCBS (thiosulfate-citrate-bile-sucrose) agar k) Streptobacillus moniliformis: serum supplemented media l) Neisseria gonorrhoeae: enhanced by Martin Lewis, Thayer Martin 6. Media selection: based on type of specimen II. Inoculation to media A. Plated media 1. Specimens are streaked for isolation 2. Four sections 3. Used to report a grade a) 1+ growth in primary quadrant b) 2+ growth in primary, secondary c) 3+ growth in primary, secondary, tertiary d) 4+ all four sections B. Broth media: used as backup; fluid is dropped in it or swab broken off; can be streaked to plate after incubation 18-24 hours III. Growth requirements A. Aerobic: with oxygen B. Anaerobic: without oxygen: incubator with gas provided, glove box or jar a) Gas Pak jars: contains dried chemicals that react with water to create anaerobic environment. Pallidum pellets used as catalyst. Methylene blue strips: turns from blue to white when oxygen is eliminated b) CO2 (5-10%): fastidious: Co2 gas pack, candle jars, CO2 incubator c) Temperature: most medically important organisms 35-37C 1. Mesophiles: grow at 20-40C 2. Thermophiles: growth at high temperatures 50-60C 3. Psychrophiles: grow at 10-20C d) Humidity: 70-80%; water is added e) Fungi: best at 30C f) pH 7.0-7.5 g) Special requirements: Campylobacter requires 42C incubator h) Gas classification 1. Strict aerobes: require O2 at 21% 2. Microaerophilic: 5-6% O2; prefer CO2 3. Faculatative anaerobes: grow under aerobic or anaerobic conditions 4. Strict anaerobes: no O2; may grow in very bottom of broth 5. Capnophilic: require CO2 6. Aerotolerant: anaerobes that may tolerate little oxygen, but grow better in strict anaerobic conditions IV. Bacterial Identification A. Initial Examination: after 18-24 hours of incubation 1. Growth requirements a) Aerobic, anaerobic, increased CO2 b) Appropriate media and temperature for specimen 2. Observe growth patterns 3. Type of hemolysis a) Alpha: partial hemolysis showing a green area around colony; Strep pneumoniae b) Alpha prime: zone of alpha hemolysis surrounded by a zone of beta hemolysis c) Beta: clearing around colony; complete hemolysis; Ex. Staph aureus d) Gamma: no hemolysis; ex. Staph epidermidis 4. Colony margin: smooth, rough, irregular, swarming 5. Elevation of colonies: raised, convex, flat, umblicate, umbonate 6. Color 7. Density: transparent, translucent, opaque 8. Odor 9. Broth morphology a) Enteric organisms: turbidity b) Strep: puff balls c) Staph: streamers or fingers B. Gram Stain 1. Gram positive cocci a) In chains: Strep or Enterococcus b) In pairs, tetrads, clusters: Staph 2. Gram positive rods, gram negative rods (bacilli) 3. Gram positive or negative coccobacilli 4. Gram positive or negative diplococci 5. Gram positive or negative filamentous rods 6. Gram negative fusiform rods 7. Spirochetes 8. Pleomorphism: variation in size and shape 9. Palisading: rods or cocci aligned side by side C. Biochemical Testing: tests done to identify organism D. Identification Methods 1. Multitest systems uses multi-well systems to identify organisms by using various tests set up all at once 2. API, Minitek, Enterotube E. Semi-Automated: relies on colorimetry or nephelometry 1. Panels are manually inoculated, bacteria grow, reactions read 2. Sensititre systems reads reactions by fluorometry F. Fully automated systems 1. Vitek: prefilled cards, bacteria inoculated, incubated, instrument reads reactions 2. Autoscan Walkaway: uses colorimetry and fluorometry  3. Aladin: video camera reads the area where reactions occur; uses pictures to id 4. Blood culture systems: ESP, BacT Alert, BacTec 5. MALDI-TOF: (matrix assisted laser desorption ionization-time of flight), uses mass spec to identify bacteria in less than 1 hour G. Immunoserological Methods 1. Agglutination 2. ELISA 3. Immunofluorescence 4. Molecular Technique: very popular now a) Uses DNA detection; organism can be detected even if dead; many molecular analyzers on market b) Genetic probes c) Nucleic acid probes d) Southern blot e) Western Blot f) PCR g) MALDI-TOF: (matrix assisted laser desorption ionization-time of flight), uses mass spec to identify bacteria in less than 1 hour, detects antibiotic resistance a. The sample for analysis by MALDI-TOF MS is prepared by mixing or coating with a solution of an energy-absorbent matrix which entraps and co-crystallizes the sample when dried. b. The matrix is ionized with a laser beam, and transfers the charge to the analytes, generating singly charged ions from analytes in the sample that are then accelerated. c. Ions are separated from each other on the basis of their mass-to-charge ratio (m/z) before being detected and measured using the TOF mass analyzer.