MLS Review - Bacteriology - Laboratory Protocols & Techniques

CLINICAL BACTERIOLOGY: LABORATORY PROTOCOLS & TECHNIQUES OUTLINE • Clinical Bacteriology Laboratory o Specimens & Specimen Processing o Culture Media o Biosafety o Methods of Microbial Control o Quality Control o Staining o Antibiotic Susceptibility Testing o Bacterial Identification CLINICAL BACTERIOLOGY LABORATORY SPECIMENS & SPECIMEN PROCESSING • • Blood – detection of bacteremia, sepsis (septicemia), fever of unknown origin o Bacteremia - presence of viable/living bacteria in blood ▪ Common agents: Coagulase Negative Staphylococci (CoNS: S. epidermidis & S. saprophyticus), E. coli, P. aeruginosa, S. aureus, B. fragilis o Septicemia - presence of viable bacteria in blood with accompanied systemic inflammatory response o Blood is a sterile specimen ▪ Specimen should not be contaminated and do not have normal flora ▪ For blood collection and phlebotomy • Sterilize the phlebotomy site, area, and materials using: 70-95% alcohol → Iodine scrub or chlorhexidine → alcohol rinse • Or 70% alcohol & chlorhexidine or iodophor for 1 minute o Common contaminants: S. epidermidis, Viridans Streptococci, P. acnes, Bacillus spp. (other than B. anthracis) and diphtheroids o Blood-Broth ratio is 1:10 o Media can be: TSB, BHI, Brucella broth o Anticoagulant: 0.025% SPS (sodium polyanethol sulfonate ▪ Inhibitory to: Neisseria, P. anaerobius, G. vaginalis, S. moniliformis ▪ Addition of 1% gelatin will neutralize effect of SPS o Other anticoagulants: sodium amylosulfate, Heparin, 0.5-1% sodium citrate ▪ Heparin - may inhibit some gram (-) and yeast • Usually used for viral culture o Not used for CULTURE: EDTA Sputum – to detect respiratory tract infections (RTIs), must be processed in BSC o May usually contain M. tuberculosis (#1 pathogen) ▪ MTB - classified under BSL3 and processed using BSC Class II • Acquired through inhalation o Agents of pneumonia: K. pneumoniae, S. aureus, P. aeruginosa in those with cystic fibrosis o Agents of ventilator associated pneumonia: S. pneumoniae, H. influenzae, S. aureus, P. aeruginosa, Klebsiella, Enterobacter, Serratia o Sputum is nonsterile ▪ Quality must be evaluated before culture Bartlett’s classification ▪ Purpose: determine between sputum vs saliva microscopically ▪ Sputum - less than 10 Sq. ECs and more than 25 PMNs are noted ▪ Saliva - more Sq. ECs than PMNs o For deep cough collection = 5-10 mL o Alveolar macrophage & columnar cells - presence in sputum is suggestive of poor collection o For M. tuberculosis detection, we do digestion & decontamination ▪ If specimen is sterile (eg. CSF), it may be processed immediately, centrifuge immediately ▪ If specimen is nonsterile (eg. sputum), do digestion and decontamination before processing ▪ Digestion - to liquefy the mucus; to free any trapped organism ▪ Decontamination - to remove contaminants as well as normal flora ▪ Gold standard for digestion & decontamination is N-acetyl-l-cysteine + NaOH • Nalc - digesting agent • NaOH - decontaminating agent ▪ Others: 5% oxalic acid, Zephiran Trisodium Phosphate (Z-TSP), sputolysin, 4% NaOH • 5% oxalic acid + Z-TSP - used for specimens likely to contain Pseudomonas Swab o Collection through swabbing is generally for aerobic bacteria o Least preferred for anaerobic culture o Throat swab ▪ S. pyogenes - major throat pathogen • Cause pharyngitis, scarlet fever, erysipelas • Disease & virulence factors: M protein o Major virulence factor o Encoded by emm gene o Antiphagocytic (prevent phagocytosis) ▪ Viridans streptococcus - Major throat flora • Cause Subacute Bacterial Endocarditis (SBE) • Alpha-hemolytic strep • Bile solubility test negative • Neufeld quelling negative • Optochin resistant o Nasopharyngeal swab ▪ For detection of carrier state of N. meningitidis • Cause meningitis in between 5-29 y.o. • N. meningitis - can be a normal flora in oropharynx and nasopharynx • To detect meningitis due to M. meningitidis, CSF is used • N. gonorrhea - always a pathogen ▪ For detection of B. pertussis (whooping cough), N. meningitis, H. influenzae and SARS COV-2, MRSA o Materials: Dacron fiber, Rayon fiber, Calcium alginate ▪ Cotton - toxic to Neisseria ▪ Calcium alginate - toxic to viruses o MEDICAL LABORATORY SCIENCE REVIEW • 1 • • • CSF o Manner of collection ▪ Collected through lumbar tap ▪ Collected in 3 tubes • Tube # 2 - used for microbiology ▪ CSF is examined ASAP • 1st Priority specimen • CSF is Level 1 • Levels of Prioritization in Microbiology Sample Processing o Level 1 - critical samples ▪ Do not refrigerate CSF if there is anticipated delay ▪ Storage temp: 37 degC ▪ Transport temp: RT (25 degC) o CSF is centrifuged prior to testing ▪ Sediments are used for culture and smears • Check for presence of C neoformans (encapsulated yeast) o Culture on BAP & CAP o Usual pathogens ▪ H. influenzae ▪ N. meningitidis ▪ S. pneumoniae ▪ L. monocytogenes ▪ S. agalactiae Stool o Purpose: to detect enteric pathogens like Salmonella o Usual media: Salmonella-Shigella Agar (SSA), EMB, MacConkey o Alternative specimen: rectal swab o Common pathogens: C. jejuni, Salmonella; Shigella; E. coli & its serotypes; Vibrio. Y. enterocolitica Urine o Submitted to detect UTI ▪ Usually for culture & sensitivity testing o E. coli - most common cause of UTI o S. saprophyticus - most common cause of UTI in young females o Process urine ASAP within 1 hr or refrigerate for not more than 24 hrs o Boric acid - preservative used to maintain accurate colony count o Urine more than 2 hrs old, 24 hr urine, Foley Catheter tips, urine bag for catheterized specimens – these specimens must be rejected o Clean catch midstream urine - preferred manner of collection of urine o Suprapubic urine - collected from infants or children o Urine Colony Count ▪ Use of Calibrated Loop (1 uL or 10 uL loop) • Inoculation on a plated media ▪ Centrifugation → Remove supernatant → Inoculate from Sediment → Incubation → Colony Count → Computation (CFU/mL) ▪ Count the number of colonies after incubation • CFU - colony-forming units ▪ To compute for the CFU/ml of urine, use formula: Colony counted x dilution factor = CFU/mL • Dilution factor is 1000 using 1 uL loop • Dilution factor is 100 using 10 uL loop ▪ Colony count of more than 100,000 is significant • (+) for UTI OTHER SPECIMENS • Wound & Tissue Specimens – we usually collect aspirates, tissue samples o For anaerobes we use aspirates or tissue sample ▪ Aspirate collected through aspiration ▪ Gaskpak jar is used for anaerobes; will drive off oxygen by palladium catalyst ▪ Indicators: • Methylene blue - blue in the presence of O2 • Resazurin - pink in the presence of O2 ▪ Most common failure of GASPAK JAR: inactivation of catalyst due to repeated use o Endotracheal aspirate specimen collected if the patient is intubated /on a ventilator CULTURE MEDIA • Agar - solidifying agent TYPES OF CULTURE MEDIA BASED ON CONSISTENCY • • • • Liquid media - 0% agar Semi solid media - 0.5-1% agar o Eg. SIM, Motility-Indole-Ornithine (MIO) Solid media - 2-3% agar o Solid liquefiable o Solid non-liquefiable Biphasic - both solid & liquie o Eg. Castañeda TYPES OF CULTURE MEDIA BASED ON PURPOSE • • • • • General purpose - contains only what is needed to support growth o Used for routine isolation o Eg. Nutrient agar, Nutrient broth, Sabouraud’s Dextrose Agar (for fungi) Enrichment media - used to enhance bacterial growth o Contains vitamins and supplements o Eg. Selenite Broth, Alkaline Peptone Water, Tetrathionate broth, Carrot broth Enriched media - to support growth of fastidious organisms o Fastidious organisms - not capable of growing in ordinary media (eg. Neisseria, Haemophilus) o Eg. BAP, CAP Selective media - use to support of the desired organisms while inhibiting growth of others may contain inhibitors o Intended for particular organism o May contain inhibitors to prevent the growth of undesired or unwanted organisms o Cystine Tellurite Blood Agar (CTBA) - C. diphtheriae ▪ Contains potassium tellurite to act as inhibitor (prevents growth of normal flora) o MacConkey Agar - for gram negative ▪ Contains crystal violet as inhibitor for gram(+) ▪ Used for Enterobacteriaceae (gram neg) o Lowenstein Jensen media - for Mycobacterium ▪ Contains malachite green as inhibitor for gram(-) Differential media - to differentiate organisms growing together o Selective Differential media: EMB, MacConkey, HEA, XLD, TCBS ▪ MacConkey - Differentiates lactose and nonlactose fermenters 2 • • • • • Transport media - to maintain organisms’ viability during specimen transport o Organisms requiring transport media: Neisseria o Eg. Transgrow (Neisseria), Cary Blair (Vibrio), Stuart’s (viruses) Antibiotic Susceptibility Test (AST) media - usual is MHA, for some fastidious, blood may be added o Mueller Hinton Agar (MHA) - usually used in KirbyBauer AST method Biochemical test media - for performance of biochemical tests which are needed to presumptively identify organisms o TSI, LIA, ONPG, Urea agar, Simmon citrate agar Motility test media - SIM & MIO Media for anaerobes - Bacteroides Bile Esculin, KVLBKanamycin Vancomycin Laked blood Level Description BSL-4 Poses extreme risk to laboratory personnel; Causes life-threatening diseases METHODS OF MICROBIAL CONTROL STERILIZATION • • BIOSAFETY BIOSAFETY CABINET • • • • • • Used in laboratory when performing microbio procedures A device that encloses a workspace Sterilizes air that contains infectious material thru filtration using High Efficiency Particulate Air (HEPA) Filter. o This can remove objects larger that 0.3 um (micra) BSC CLASS I - system is entirely open, sterilizes air to be exhausted; least effective BSC CLASS II - a.k.a laminar flow, the type that is used in most laboratories o Partly open and partly closed o Sterilizes the air that flows over infectious material and also the air to be exhausted o BSC CLASS IIA – exhaust air inside the room ▪ 70% of the air is recirculated o BSC CLASS IIB - exhaust air outside the building BSC CLASS III - most effective o Used with gloves attached & sealed to the cabinet o Entirely closed system, self-contained ventilated system • • BIOSAFETY LEVELS • • Based on the type of risk that microorganisms impose to the health of lab workers Also based on the mode of transmission of microorganisms Level Description BSL-1 Poses minimal risk to lab personnel; Do not cause disease in healthy people BSL-2 Poses moderate risk to lab personnel; Acquired thru ingestion, mucus membrane, & percutaneous exposure; Associated with laboratory-acquired infections BSL-3 Poses high risk to laboratory personnel; Acquired thru inhalation, possible for aerosol transmission Precautions Examples Practice of Standard Bacillus subtilis, Laboratory Techniques M. gordonae, Enterobacter aerogenes Those practices in Salmonella; BSL1 agents; Use of Shigella; BSC, gown, gloves, S. aureus; face shield (if needed); HBV; HIV Use of biohazard warning signs); Decontaminate all infectious waste Laboratory must have sustainable airflow in which air is drawn from clean area of the lab towards contaminated area; Use of PPE Agents of systemic mycoses; M. tuberculosis; Coxiella burnetiid; St. Louis encephalitis virus Precautions Examples Laboratory must be Arenaviruses; situation in a separate Margburg virus building; Use of PPE (full body) • • Complete destruction and removal of all forms of microbial life including the spores Physical Sterilization o Use of moist heat - autoclaving, tyndallization, inspissation ▪ Fractional/intermittent sterilization tyndallization, inspissation • Not done continuously; done in several days o Use of dry heat - oven, incineration, cremation, flaming of loops and needles o Filtration - Millipore filter, Nucleopore filter, High Efficiency Particulate Air (HEPA) filter ▪ For antibiotic solutions that cannot be heated ▪ HEPA - sterilization of air o Ionizing radiation - exposure to Gamma rays ▪ Used for those that can’t be filtered & heated Chemical Sterilization o Use of chemical sterilants/biocides ▪ Ethylene oxide Autoclaving - steam under pressure o To sterilize used and unused media - Carried out at 121 degC for 15-30 minutes at 15 psi o To sterilize contaminated microbiological materials Carried out at 121 degC for 30 mins at 15 psi o To sterilize infectious medical waste - Carried out at 132 degC 30-60 mins o Geobacillus stearrothermophilus (formerly Bacillus stearrothermophilus) ▪ Biological indicator of autoclaving ▪ For checking proper functioning of autoclave o Not killed by autoclaving: prions ▪ Prions - infectious protein particles causing neurologic disease in animals and man • Creutzfeldt-Jakob Syndrome, Mad Cow disease, Bovine spongiform encephalopathy (BSE) Target of sterilization: spores Fractional/Intermittent Method of Sterilization o Performed for several days o Tyndallization - Carried out at 100C, 30 mins, 3 days ▪ Principle: steam under pressure (autoclaving) ▪ Uses Arnold sterilizer ▪ Uses flowing steam o Inspissation - Carried out at 75-80C, 2 hrs for 3 days ▪ Involves thickening through evaporation ▪ To sterilize media with increased protein • Mycobacterium media/ egg-based media o Lowenstein-Jensen media, Petragnani’s media, ATS 3 • • • • • Biological Indicators o Geobacillus stearrothermophilus - autoclave o Bacillus subtilis var. niger - oven o Bacillus pumilus - ionizing radiation (gamma rays) o Bacillus subtilis var. globijii - for ethylene oxide (cold sterilization) Cold sterilization - use of ethylene oxide (chemical sterilant) Extended steam - Method to eliminate prions o Extended steam sterilization in conjunction with 1M sodium hypochlorite or sodium hypochlorite (2% available chlorine) Incineration - To dispose infectious materials o Burning materials into ashes o Exposure to very high temperatures Cremation - burning bodies into ashes EQUIPMENT QUALITY CONTROL • • • • • DISINFECTION • • • • • • • Destruction and removal of pathogens but not necessarily all microorganisms and their spores Physical o Boiling o Pasteurization - remove pathogens in food Chemical o Disinfectant - for surfaces o Antiseptic - for skin Disinfectants o Phenol - Standard disinfectant o Sodium hypochlorite - most commonly used disinfectant; o 10% Household Bleach Sodium hypochlorite ▪ Best to remove blood spills ▪ CDC recommends 1:10 dilution for porous surfaces ▪ 1:100 recommended for hard surfaces, smooth surfaces ▪ Vinegar - can be used as substitute ▪ HBV = 10 mins contact time ▪ HIV = 2 mins contact time Antiseptics o Best antiseptic is Iodophor o 70% alcohol - most commonly used o Iodophor = iodine + surfactant/detergent/neutral polymer ▪ Eg. Povidone iodine (betadine) o Tincture of iodine = iodine + 70% alchohol QUALITY CONTROL • • • • • • Handwashing - Most effective way to stop the chain of infection Universal Precaution o Type of precaution applied to all human blood & body fluids that contain visible blood Standard Precaution o To treat patient’s blood & body fluid as potentially infectious Donning: Gown → mask → goggles → face shield → gloves Doffing: Gloves → goggles → gown → mask, then do handwashing after As part of biosafety, autoclave material prior to disposal o Yellow bag - used for disposal Incubator, water bath, refrigerator, freezer, heating block - monitor temperature daily o Incubator - 35-37C o Microbio Ref - 2-8C Autoclave (efficiency/spore testing) - at least weekly Autoclave temperature - checked daily GASPAK Jar - checked every use o For anaerobic cultures ▪ Ensure that during incubation, organism should not receive even the smallest amount of air Centrifuge function/rpm - monitor efficiency every 6 months; some recommend monthly monitoring o CSF - centrifuged before processing; sediments are used for culture and smear preparations Microscopes - cleaned, maintained, and adjusted 4 times annually or as needed Weighing balance - check accuracy annually STAINING • • • Simple staining - use of one dye only o Color of the dye is also the resulting color o Eg. methylene blue Negative staining - only the background is stained; organism appears colorless o Aka indirect/relief staining Special staining - demonstrate special features of the cell o Special feature include flagella, spores, etc. DIFFERENTIAL STAINS • • Bacterial smear o Prepared before staining Gram Stain o Differentiate organisms between gram positive or negative o Primary dye: crystal violet o Mordant: Gram’s iodine o Decolorizer: alcohol (95% ethyl alcohol), acetone, acetone-alcohol mixture o Counterstain - Safranin ▪ Colors gram negative organisms ▪ Some gram negative may color pale pink ▪ Carbol fuchsin - alternative for safranin, to improve staining of some gram-negative organisms that are faintly stained with safranin o VIAS - Violet → Iodine → Alcohol → Safranin o Gram (+) - violet/purple; take the color of primary dye o Gram (-) - red o Decolorization - Most critical step in Gram staining ▪ Overdecolorization cause false negative ▪ Underdecolorization cause false gram positive o Hucker’s modification ▪ Gram staining technique for fungi ▪ Crystal violet + ammonium oxalate during primary dye ▪ Fungi in general are reported gram positive 4 • Acid Fast Stain (AFS) o Classifies acid-fast and non-acid fast organisms o Most common methods ▪ Ziehl Neelsen - hot method; uses steam or heat as mordant ▪ Kinyoun’s - cold method; uses tergitol as mordant • Tergitol - wetting agent o Primary dye - carbol fuchsin o Decolorizer - 3% acid alcohol (HCl + Ethanol) o Counterstain - methylene blue or malachite green ▪ Methylene blue - commonly used ▪ Malachite green - alternative o Acid Fast - appear red against blue background o Non-Acid Fast - appear blue/green depending on counterstain used o Size of AFB smear - 2x3 cm o Ziehl Neelsen - Best method for Direct Spum Smear Microscopy (DSSM ) o Kinyoun’s - Best to demonstrate AF organisms in tissues o Fite-Faraco method - used to demonstrate M. leprea ▪ Uses hematoxylin as counterstain TWO TYPES OF AST • • KIRBY BAUER DISK DIFFUSION METHOD • • • • • • • ANTIBIOTIC SUSCEPTIBILITY TESTING (AST) • • • • • • • • Carried out to determine susceptibility or resistance or organisms to antimicrobial agents o Uses antibiotics and antimicrobials Antimicrobials may be classified as o Bactericidal - can kill microorganisms ▪ Examples: Penicillin, Vancomycin, Aminoglycosides, Quinolones, Metronidazole o Bacteriostatic - can prevent/inhibit bacterial growth ▪ Examples: Tetracycline, Streptomycin, Chloramphenicol, Erythromycin Narrow and Broad spectrum o Narrow spectrum - antibiotics that can target limited range of disease-causing bacteria; limited range of axn o Broad spectrum - antibiotics that can target wide range of disease-causing bacteria Cell Wall Inhibitors o Kill bacteria by destroying the cell wall o Beta Lactams – Penicillin, Carbapenems, Caphalosphorins o Glycopeptides - Vancomycin Protein Synthesis Inhibitors o Kill bacteria by inhibiting protein synthesis o Macrolide - Erythromycin o Aminoglycosides - Gentamicin, Tobramycin o Tetracycline - Doxycycline o Chloramphenicol Beta Lactamase Inhibitors o Kill bacteria by inhibiting beta lactamase production o Tazobactam, Sulbactam, clavulanic acid Sulfonamides o SXT - Trimethoprim Sulfamethoxazole o Nitrofurantoin - Drug which can be used as treatment for UTI; inhibits bacterial enzyme Quinolones o Inhibition of DNA activity o Examples: Levofloxacin, Ciprofloxacin Dilution method o Quantitative technique Disk diffusion method o Qualitative o Eg. Kirby Bauer Disk Diffusion Method • • • • • • • • • • • • • • Media: Mueller-Hinton Agar (MHA) o Plated media pH: 7.2-7.4 Depth of agar: 4 mm Inoculum size: 1.5 x 108 CFU/mL o To achieve inoculum size, use a MacFarland standard o Use of 0.5 Mac Farland: 99.5 mL 1% sulfuric acid & 1.75% Barium chloride ▪ Appears turbid ▪ Organisms shall be as turbid as the media Size of filter paper (antibiotic) disk: 6 mm Use of antibiotic disk with specific concentration Storage temp for disk: o Working supply: 2-8 degC o Long term: -14 to -20 degC (freezing) Agar preparation → Inoculate → Antibiotic disk Incubate at 35-37C for 16-18 hrs in ambient air If plate size is 100 mm, place no more than 5 disks If plate size is 150 mm, place no more than 12 disks Distance of disk from center: 24 mm Distance between 2 disks:15 mm Manner of streaking using sterile cotton swab: overlap streaking method Waiting time prior to placement of disks: 3-5 minutes Use ruler or caliper when measuring zone of inhibition o Determines susceptible, resistant, or intermediate Swarming by Proteus: not measured; ignored When using sulfonamides like SXT, if in case there would be 2 concentric zones, measure the outer zone False susceptible: Too light inoculum, very dry agar, thin agar, larger zone False resistant: Too heavy inoculum, too much moisture on agar surface, thick agar Possible Results in AST: Resistant, Intermediate, Susceptible E-TEST • • • • Uses a strip with single antibiotic of decreasing concentrations along its length o Strip has calibrations Inoculation in MHA → Placement of antibiotic strip Dilution method for fastidious organisms (eg. Hemophilus) (+) RESULT: ellipse of growth inhibition BETA - LACTAMASE TEST / CEPHALOSPORINASE TEST • Detect the ability of organism to produce enzyme betalactamase o Eg. S. aureus is known to produce beta-lactamase, it is resistant to beta-lactams o Beta-lactamase - counteracts beta-lactams 5 • • • • Use of cefinase disk o Has impregnated substrate o Substrate: Nitrocefin o Commercially available Inoculate → wait for change of color (+) result: pink to red color To treat S. aureus, do not use penicillin or beta-lactams o Use penicillinase-resistant drugs (eg. methicillin, oxacylin) METHICILLIN-RESISTANT S. AUREUS (MRSA) TESTS • • • Ideally, S. aureus should be destroyed by penicillinaseresistant drugs o However, some S. aureus are resistant to penicillinase-resistant drugs Cause of MRSA / resistance of S. aureus o Presence of PBP2a in the cell wall which is encoded by mec A gene ▪ PBP - Penicillin-binding protein MRSA Detection o Use of CHROM agar ▪ Also commonly used technique ▪ CHROM - chromogenic agar ▪ CHROM agar → inoculation → incubate ▪ (+) result: rose/mauve colony ▪ (-) result: colorless/blue colony or no growth o Cefoxitin Disk test (30 ug) o Oxacillin Screen Agar – use of MHA with 6 ug Oxacillin with 4% NaCl ▪ (+) result: presence of growth o Molecular – PCR; gold standard ▪ Detects the mec A gene D-TEST • • • • • • To detect inducible clindamycin resistance among strains of S. aureus o Clindamycin and erythromycin have same action as protein-synthesis inhibitors o In cases of variable erythromycin and clindamycin resistance o D-test is done when initial test is discrepant where erythromycin and clindamycin resistance are different Confirms clindamycin resistance o When clindamycin is initially susceptible MHA → Inoculate → insert 2 disk Uses erythromycin & clindamycin o 15 ug erythromycin o 2 ug clindamycin These antibiotics are positioned 15 mm apart (+) RESULT: blunting or flattening of clindamycin zone to produce D -pattern. • • Interpretation of Result: o (+) D-test: we report it as Clindamycin resistant o (-) D-test: we report it as Clindamycin susceptible erm gene - Gene that activates resistance to clindamycin PENICILLIN RESISTANCE • • • • For Streptococcus pneumoniae o Alpha-hemolytic o Optochin-susceptible Uses MHA supplemented with sheep’s blood + 1 ug oxacillin disk Sensitive - Greater than 20 mm zone of inhibition Resistant - Less than 20 mm zone of inhibition MODIFIED HODGE TEST • • Screening test for detection of ability of organism to produce carbapenemase enzyme o Carbapenemase - makes beta-lactam antibiotics ineffective (+) RESULT: clover leaf like pattern of zone of inhibition BACTERIAL IDENTIFICATION • 3 ways o Manual o Semi-automated o Automated MANUAL • • Can be inaccurate o Prone to errors Considers motility, morphology, shape, size, arrangement SEMI-AUTOMATED METHODS FOR BACTERIAL ID • • • • Improves turnaround time (TAT) o Faster than manual ID Uses databases or numeric codes for identification o Utilizes a software to analyze API - uses microtubes with dehydrated substrates BBL crystal ID - involves one-step inoculation; no need to add reagents and aoil AUTOMATED SYSTEM • • VITEK - automation program for bacterial identification and susceptibility o Initially launched than MADI-TOF MADI-TOF - faster and quicker than VITEK; has same functions as VITEK • 6