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Week 2 Bacterial Physiology, Metabolism, Genetics, B. CELL ENVELOPE Growth and Nutrition, and Bacterial Infection a. Located in gram negative...

Week 2 Bacterial Physiology, Metabolism, Genetics, B. CELL ENVELOPE Growth and Nutrition, and Bacterial Infection a. Located in gram negative bacteria: Periplasm and Outer membrane Prokaryotic Cell Structures 1. Plasma membrane (Cell Membrane) Phospholipid bilayer with embedded A. Prokaryotic Cell Components proteins that envelope the cytoplasm a. Cytoplasmic Structures - inner site of the cell b. Cell Envelope Structures - protects the inside 2. Cell wall / Cell envelope (peptidoglycan or of the cell murein layer) c. Surface Polymers or Appendages Maintains the shape of the cell and protects from osmotic pressure. A. CYTOPLASMIC STRUCTURES Repeating disaccharide attached by polypeptides. 1. Nuclear Area (Nucleoid) Single circular chromosome attached to a Component of peptidoglycan layer: mesosome. N-acetylglucosamine (NAG) N-acetytlmuramic acid (NAM) 2. Plasmids Side chain amino acid Small circular, (double stranded) dsDNA Cross-bridge amino acid molecule 3. Periplasm / Periplasmic space Antibiotic resistance Located between cell membranes and cell wall 3. Ribosome Site of protein synthesis Consist of gel like matrix containing nutrient binding nutrients Consist of RNA (ribosomal RNA) & Protein Contains enzymes for degrading and 4. Inclusions detoxifying macromolecules Metachromatic granules - represents reserves of polyphosphates used in the 4. Outer membrane synthesis of ATP. Cells initial barrier (certain antibiotics and erasion of phagocytes) Polysaccharide granules - consists of glycogen & starch granules Composed of Lipopolysaccharides (LPS), Lipoprotein and Phospholipids Metachromatic granules and Polysaccharide granules are stored energy, Contains Porins used by prokaryotic cells when they need Water-filled structures that control the energy. passage of nutrients and solutes 5. Spores Highly refractile resting cells that are highly durable and dehydrated with thick walls. Use for heat resistance, enzymatic degradation, and protection from disinfectants. Spores are used to withstand harsh environment. MED224 FGV T  Week 2 d. Peritrichous - flagella distributed over Table 1. Cell Envelope the entire cell (ex. Escherichia coli) Characteristics Gram Positive Gram Negative Peptidoglycan Thick Thin (single layer (multilayered) layered) Teichoic acids Present in many Absent Periplasmic Absent Present space Outer membrane Absent Present LPS content Virtually none High Lipid & High (due to 3. Fimbriae and Pili Low lipoprotein outer membrane) Hairlike appendages that are shorter, straighter, and thinner than flagella. C. SURFACE POLYMERS & APPENDAGES a. Fimbriae (sing. fimbria) - also called common pili, can occur at the poles or can be vent distributed 1. Glycocalyx (slime layer and capsule) from few to several hundred. General substances that surround cell b. Pili (pilus) - also called sex Made up of gelatinous polymer of (conjugation) pili, are hollow protein polysaccharide (CHO), polypeptide (CHON), and both tubes, longer than fimbria and number is 1 or 2 per cell. a. Capsule - substance is organized and firmly attached to cell wall Function: c. Fimbriae - for adherence of cells to b. Slime layer - unorganized and loosely one another and to environmental attached to the cell wall surfaces. d. Pili (pilus) - join bacteria, cell in Functions: (slime layer & capsule) preparation of DNA transfer from Prevents phagocytosis Attachment to various surfaces in its one cell to another. natural environment B. anthracis S. pneumoniae K. pneumoniae 2. Flagella Exterior protein filaments that rotate and cause bacteria to be motile 4. Axial filaments (endoflagella) Bundles of fibrins anchored to Four arrangement of flagella: one end of spirochete and spiral a. Monotrichous - single polar flagellum around the cell. (ex. Vibrio cholerae) The rotation of filaments propels b. Lophotrichous - two or more flagella the spirochete in a spiral motion at one pole of the cell (ex. Bartonella bacilifordhis) *in between the cell membrane & cell wall c. Amphitrichous - single/tuft of flagella at each end of the cell (ex. Spirillum serpens) MED224 FGV T  Week 2 BACTERIAL METABOLISM Butanediol Fermentation - end product are acetoin and 2, 3 butanediol Klebsiella spp., Enterobacter spp. Various biochemical pathways exist for substrate Serratia spp. breakdown in the microbial world, and the particular pathway used determines the end product and final CARBOHYDRATE UTILIZATION AND CATOSE pH of the medium. FERMENTATION DIAGNOSTIC SCHEMES Ability of m.o to use various sugar for growth is an integral part of most diagnostic identification 1. Utilization of various substances as carbon schemes source 2. Production of specific end products from Fermentation of sugar is usually detected by various substances acid production and a contaminant change of 3. Production of an acid or alkaline pH in the test color medium Due to pH indicator present in the culture medium, low pH FERMENTATION & RESPIRATION For Enterobacteriaceae: determination of the Fermentation - anaerobic process carried out by m.o ability to ferment lactose both obligate and facultative anerobes, Lactose fermenters, lactose non- End product: lactate, ethanol, butyrate, acetoin fermenters, late lactose fermenters Used for MRVP MacConkey agar (orig color is light pink) Respiration - efficient energy-generating process in which molecular oxygen is the final MacConkey inhibitor: electron acceptor bile salts and crystal violet (inhibits Obligate aerobes and facultative anerobes the growth of gram positive bacteria) GLUCOSE TO PYRUVIC ACID 2 Purpose of MacConkey: Selective - selection of certain Embden-Meyerhof-Parnas (EMP) Glycolytic bacteria (gram negative) Pathway (glycolysis) Differential - differentiates lactose Pentose phosphate Pathway fermenter to non-lactose fermenter Entner-Doudoroff Pathway the organism can utilize lactose and it will ANAEROBIC UTILIZATION OF PYRUVIC ACID produce acid, production of acid will result in color change Alcoholic fermentation - major end product is ethanol (beer or wine making) Used by yeast Homolactic fermentation - end product is almost exclusively lactic acid only Streptococcus spp. Lactobacillus spp. Heterolactic fermentation - end product are lactic acid, CO2, alcohols, formic acid, acetic acid Propionic acid fermentation - end product is propionic acid Carried by Propionibacterium acnes Mixed acid fermentation - lactic acid, acetic, succinic, formic acid Escherichia spp., Salmonella spp., Shigella spp. MED224 FGV T  Week 2 BACTERIAL GENETICS Saprophytes - obtain energy from decaying organisms (ex. mushroom and certain fungi) 1. Transformation - uptake and incorporation of naked DNA into a bacterial cell Parasites - compete for nutrients from their host, S. pneumoniae, N. gonorrhoeae, H. causes harm and diseases. Influenzae Oxygen Requirement 2. Transduction - transfer of bacterial genes by a bacteriophage (virus) Obligate aerobes - requires oxygen for growth. C. diphtheria Obligate anaerobes - grows in strict absence of 3. Coagulation - transfer of genetic material from oxygen. a donor bacterial strain to a recipient stain. Facultative anaerobe/Facultative aerobes - can grow without oxygen, but will utilize it upon its presence. Microaerophiles -requires small amount of oxygen. Aerotolerant - can grow without oxygen but can survive in the presence of it. Carbon Dioxide Requirement Capnophiles - require CO2 to grow ◦ Neisseria gonorrheae ◦ Haemophilus influenzae ◦ Brucella abortus ◦ Streptococcus pneumoniae ◦ Mycobacterium spp. Temperature Requirement Psychrophilic/ Cryophilic - low temperature BACTERIAL GROWTH & NUTRITION (10-20C) ex. Listeria, Legionella Nutritional Requirements Mesophilic -where most pathogen grow (30-40C or 20-45C) Autotrophic - organisms that produce their own most bacteria grows in human temperature food from inorganic substance like carbon (37 degrees C) dioxide. Thermophilic - heat loving (50-60C) Phototrophs - obtain energy from sunlight/ light. ex. Thermus aquaticus (ex. Cyanobacteria) TA Q p o l y m e r a s e e n z y m e i s extracted from Thermus aquaticus Chemotrophs - organisms that obtain energy by which is used for Polymerase chain oxidizing chemical compounds either organic or reaction or PCR because it can inorganic. (ex. lithotrophs - a subset of withstand high temperature. chemotrophs) Hyperthermophilic - (80-113C) Lithotrophs - uses inorganic compounds (ex. H2S, iron, thiosulfate) pH Requirement Heterotrophs/Organotrophs - uses organic Acidophilic - requires acid medium (pH 0-5.5) compounds pH is lower. ex. Lactobacilli MED224 FGV T  Week 2 Basophilic - (pH 8.5-11.5) pH is higher/alkaline. TYPES OF CULTURES AND CULTURE MEDIA ex. Vibrio cholerae Neutrophilic - optimum pH 6.5-7.5 of most Culture Media bacteria. Any substance that contains the nutritional Other Requirements requirements needed for bacterial growth. Hemidophilic - moisture content Culture - group of organisms obtained in a media. Halophilic - salt concentration 3 types of culture: Osmophilic - high osmotic pressure Pure Culture - 1 organism only Mixed Culture - 2 or more organisms Stock Culture - also a pure culture, BACTERIAL GROWTH CURVE purpose is control. Generation time of bacteria in a culture varies Classification of Culture Media according to their cellular properties. It takes 20 mins for fast growing bacterium (E. CONSISTENCY coli) or 24 hours for slow growing bacteria (M. Liquid Medium (broth) - does not contain any tuberculosis) amount of agar, allows growth of aerobes, anaerobes, and facultative anaerobes. Stages of Bacterial Growth Curve Commonly used liquid medium: 1. Lag Phase or Period of Rejuvenescence Trypticase soy broth - used for non ✓ Period when there is no cell division, start of fastidious bacteria. biosynthesis, adjustment phase to a new Brain Heart Infusion broth - used environment. for fastidious organism. Thioglycolate broth - used for 2. Log or Exponential Phase anaerobic bacteria or organism. Period when microorganisms are actively growing and dividing, phase where m.o. are Semi-solid medium - contains 0.5%-1% agar, utilized in physiological, biochemical, and used to observe bacteria, motility and detect antimicrobial testing. insole and sulfide production. Utilizing the substrate in the medium Sulfide Indole Motility Agar (SIM) 3. Stationary/ Plateau Phase Solid culture medium (agar) - 2% to 3% agar Balance between cell division and dying Nutrient Agar organisms, but number of viable organisms are constant COMPOSITION Synthetic culture medium (uses chemicals 4. Death or Decline Phase such as iron, H2S, thio in equal amounts) Period of cessation of bacteria, growth as the All components are known number of dead cells exceeds the living Research purposes microorganisms. Non-synthetic culture medium (contains plant extract, animal extract or yeast extract in an unequal amount) Some of the substances are unknown Isolation of medically significant bacteria Tissue culture media For obligate intercellular bacteria virus is non-culturable (uses tissue culture) utilizes cytopathic effect (changes in the cell) MED224 FGV T  Week 2 MEDIUM DISPENSED Chloral hydrate Plated Medium Tube Medium Substances that inhibit gram negative organisms Liquid - broth Potassium tellurite Slant Sodium azide Butt Neomycin and sodium azide Butt and slant Special/ selective culture medium Lowenstein-Jensen/ 7H-10 agar/ 7H-9 broth - M. tb Thayer-Martin - Neisseria group MacBridge Agar - L. monocytogenes Fletcher Medium - Leptospira W medium - Brucella Bordet-Gengou Agar - B. pertussis Mannitol Salt Agar - Staphylococci Saboraud Dextrose Agar - fungi Loeffler’s Medium/Tinsdake/Tellurite Media - C. diphtheriae Inoculation of Culture Media USE Simple media - routine in the lab, w/o additional TECHNIQUES supplements. Liquid medium - broth inoculation or common is nutrient agar ( to grow an suspension organism) Plated medium - streaking method Butt medium - stabbing method, anaerobically Enrichment medium (liquid-type media) - observed enhances the propagation Slant medium - streaking method, aerobically ex. BHI, TSB, THIO observed Butt/slant - stab the butt portion then stream the Enriched medium - with additional supplements slant medium (blood, vitamins, yeast) ex. BAP (blood agar plate), CAP (chocolate agar plate) Differential medium - distinguishes organisms growing together by their differences in cultural characteristics. ex. MacConkey Selective medium - promotes the growth of desirable organisms but at the same time inhibiting the growth of others. ex. has inhibitory agent METHODS OF STREAKING (for plated media) Special/ Selective culture medium Radial streak method ex. Thayer martin ( for Neisseria) Overlap streak method - for sensitivity testing Selective Medium Substances that inhibit gram positive organisms Multiple streak method - commonly used in the Gentian violet laboratory, not advisable Crystal violet Sodium desoxycholate and other bile salt Interrupted streak method Substances that prevent swarming of Proteus Multiple interrupted streak method - most spp. common and used to isolate pure culture Alcohol MED224 FGV T  Week 2 Incubation If CO2 incubator is not available, used candle gar or Gaspak jar with CO2 generator. OBTAINING PURE ISOLATED COLONIES Streak plate method - multiple interrupted Candle jar principle: if the candle fire dies - there streak method is no oxygen inside. Pour plate method - used to determine the Gaspak jar: the pack inside is a CO2 generator. appropriate number of viable microorganisms in a liquid medium Reports reported as number of colonies per mm of Save culture until satisfactory growth is obtained inoculum and final diagnosis is made. Using selective medium/containing antibiotics Some cultures maybe held for several weeks Animal inoculation test before final report is made. Blood culture - 7 to 10 days Anaerobic Culture (do not require oxygen) Brucella and M. tb - 4 weeks Culture media should be protected from oxygen L. monocytogenes - 4 months exposure, freshly prepared, and should be held All media except Thioglycolate broth should be in anaerobic condition until needed. stored in the refrigerator. Recommended methods: Thioglycolate broth The number of media stored should not exceed and anaerobic jar. 2-3 week supply. Use of anaerobic chamber with vacuum pump for All media must be checked before use. processing of sample and storage of culture media to inoculation. COLONIAL GROWTH Anaerobic Chamber: Sealed Glove Box and Gloveless Chamber Colony 1. Nitrogen gas - acts as a filler for the remaining Colonies - groups of bacteria forming on solid percentage of the anaerobic atmosphere media as a result of division of one or a few 2. Hydrogen and Carbon dioxide gas - facilitates organisms. the growth and isolation of anaerobes Types of Colonies: 3. Palladium catalyst pellets - remove residual S or smooth colonies - uniform texture & oxygen from the chamber homogeneity, easily emulsified in NSS, 4. Silica gel (desiccant) - absorbs the water that is virulent organisms. formed when hydrogen combines with free M or mucoid colonies - associated with oxygen capsulated and virulent m.o., slimy/ watery 5. Methylene blue/reazurin - oxygen-reduction (ex. Klebsiella pneumoniae). incubator R or rough colonies - granulated in appearance, hard to emulsify in NSS. MED224 FGV T  Week 2 Conventional Methods Blood Agar Plate and MacConkey are Biochemical characterization serves as the commonly used for these tests. fundamental source of information for ID of most Growth in BAP and MacConkey bacteria after microscopy. indicates a gram negative organism. Motility test - wet mount and hanging If there is growth in BAP and no drop preparation growth in MacConkey the organism is gram positive because MacConkey Staining - gram staining ( + or -), acid- has inhibitory agent. fast staining (for mycobacterium sp.), Gram (+) usual test are coagulase and structural staining catalase. To differentiate staph. and strep. Manual biochemical test - carbohydrate fermentation, (LIA - Lysine Iron Agar), catalase and coagulase tests Use of routine and selective media Analytical Profile Index Consists of plastic strips and microtubes that contain dehydrated biochemical substrates. Commonly used for gram (-) enteric bacteria Same principle with biochemical manual tubed method Automated Method for ID of Bacteria Patterns of microbial growth are compared with Principle: Biochemical substrates (pH-based the test organism using a computer software. substrates) are inoculated with pure culture Growth is detected through colorimetric, suspended in sterile physiologic saline and fluorometric, or turbidimetric analysis. incubated for 18-24 hours at 35C, some Used for various m.o. reagents may be added after incubation. Principle: pure bacterial isolate during incubation at 35C for 16-24hrs utilized the carbohydrate in Type of tests commonly done in the Microbiology the reagents, thus producing acid or alkaline end Laboratory to determine certain enteric bacteria products as early as 2 hours. sp. Mixed bacteria, isolate is not used because it can TSI or Triple Sugar Iron (butt/ slant media) cause interference with the result. identify if certain bacteria utilizes 3 sugars. BF Phoenix System, Vitek System LIA or Lysine Iron Agar (butt/ slant media) identify if certain bacteria will utilize Lysine MICROSCOPY and produce acidic environment. SIM (butt media) identifies three: (S) sulfite - Fundamental methods used for both the detection and characterization of bacteria. observe for blackening in the media, (I) indole - observe if certain organism will Microscope - vital in magnifying m.o. utilize tryptophan to indole, (M) motility - Remember: Unit of measurement in determine bacteria, observe if certain organism will hydrolyze size is micrometer (um) the gelatin. Ocular micrometer is located within the eyepiece, 10x magnification. Citrate - observe if the organism can utilize Refractive index (bending of light) - citrate as their carbon source, the media will measure of the relative velocity at which become highly alkaline if citrate is utilized light passes through a material or specimen. and it will produce carbonate then change Total magnification - product of the lens color from green to blue. and the objectives that are used, ocular x objective. Urease Test (slant media) observe if certain Organisms are observed under OIO only. organism will utilize urea, change color from orange to pink MED224 FGV T  Week 2 Types of Microscopes Staining Techniques 1. Light Microscope - visible light passes through the specimens then through the lenses that reflect Purpose light. To observe and appreciate the appearance Brightfield microscope (compound To differentiate one organism from another (gram microscope) - most commonly used positive and negative, AFB, NAF) microscope, forms a dark image against a To help in the identification of organism and their brighter background, can distinguish between special structures (flagella and capsule) 2 dots that are 0.2 um apart. Staining can be classified into basic or acidic (employed in differential staining) Phase contrast microscope - permits detailed examination of internal structures in Types of Stains living organisms, fungi, staining not included. Simple stains - uses 1 stain only, ex. Methylene Enhance contrast of clear/ transparent blue (MB) specimen. Indirect, Relief, or Negative Stains - stains background, microorganism will appear clear. Fluorescence microscope - involves (stains example such as India ink and nigrosin) excitation of fluorochromes (dye) using light. Special stains - capsular stain, cell wall stain, (Fluorescein - green apple color) metachromatic granules, flagellar Differential stain - uses 2 stains: basic and acidic Darkfield microscope - uses a dark field (ex. Gram stain and Acid fast stain) condenser that blocks light, used to observe spirochetes (Treponema pallidum). Uses stain General Rules for Gram Staining such as India ink (Cryptococcus neoformans All cocci are gram positive except Neisseria, that causes fungi meningitis) or nigrosin. Branhamella, Moraxella, and Velionella. All bacilli are gram negative except 2. Electron Microscope - utilizes electrons instead of Mycobacteria, Clostridium, Corynebacterium, light. Bacillus, Erysipelothrix, Listeria, Lactobacillus. Has electromagnetic fields instead of lenses to form images. Presence of built-in camera to capture images of cells in black-and-white space transmission. Transmission Electron Microscope (TEM) Allows visualization of the internal structures of cells Resolving power: 0.2 nm, magnification: 150,000x to 10Mx Used to examine very thin specimens and m.o. Scanning Electron Microscope (SEM) Scans the surface of the cells or specimens Resolving power: 200nm, magnification: 20x to 10,000x Specimen is positioned at the bottom of the column. Higher forms of organisms (Actinomyces, Streptomyces, yeasts, and molds) are gram 3. Digital Microscopy positive. A technology that utilizes the science of staining All spiral organisms are reported as gram like the gram reaction. negative. Capture cellular images through web-based interface. Uses an automated microscope and the interface Gram stain (V.I.A.S) to present images on screen or computer Crystal Violet monitor. Iodine 95% Alcohol MED224 FGV T  Week 2 Safranin - red or pink color Table 3 Purpose Ziehl-Neelsen Kinyoun’s Primary Stain Carbol Fuchsin Carbol Fuchsin (red) (red) Mordant Heat (5 mins.) Phenol, Tergitol (wetting agent) Decolorizer 3% acid alcohol 3% acid alcohol Counterstain Methylene blue Malachite green Ziehl-Neelsen is commonly used for DSSM (Direct Sputum Smear Microscopy) Table 2 Kinyoun’s is commonly used for tissue specimen (leprosy, causative agent M. leprae) Reasons why Gram (+) Reasons why Gram (-) Directions of use for Ziehl-Neelsen: becomes (-) becomes (+) 1. Flood slide completely with strong (con.) Carbol Overdecolorization Underdecolorization Fuchsin solution. 2. Heat the slide from below until fumes arise; don’t Smear too thin Smear too thick overheat or boil. 3. Repeat the process of heating twice Improper washing between 4. Allow the slide to cool and wash the slide with steps running water. 5. Was the slide well with running water. Too old culture 6. Pour few drops of 2% H2SO4 solution on the smear and let it act for 1-2 minutes. Impure or mixed culture 7. Wash the slide in running water 8. Repeat the decolorization and washing steps until Decolorization - crucial method in gram staining the smear is colorless or faintly pink. Too old culture - will not take up the stain 9. Wash the slide in running water. Underdecolorization - residue of primary stain is 10. Pour few drops of Loeffler’s methylene blue on the not removed entirely smear and let it act for a minute. 11. Wash the slide in running water, blot it dry and Acid-fast Stain (resist the acid alcohol observe under microscope. decolorizer) All bacteria are non-acid fast except Other staining methods: Mycobacteria group 1. LANA (L-alanine 4-nitroanilide) One does not belong to the Mycobacteria group 2. 3% KOH method (string test) - usually employed but is slightly acid fast: Nocardia asteroides in vibrio spp. Acid fast organisms (AFO) are very hard to stain To a drop of 3% KOH, emulsify a loop full of due to the presence of unsaponifiable wax called bacterial colonies mycolic acid or hydroxymethoxy acid. Stir continuously for 60 seconds with a wooden In staining, the mycolic acid is temporarily stick removed through the steaming process. Loop is gently pulled out Commonly used methods of acid-fast staining: BIOCHEMICAL TESTING 1. Ziehl-neelsen - hot method 2. Kinyoun - cold method Biochemical Tests Are never done in mixed plates Pure cultures are used Diagnostic test for Enteric Bacilli: Growth on Media CHO fermentation test MED224 FGV T  Week 2 TSI (most commonly used) - dispersed in conditions used peptones in the medium, slant slant butt becomes alkaline. Results and Observation: In TSI those organisms that fermentation Alkali pH - red (ALK) yellow (ACID) sucrose will have an A/A reaction: H2S production - indicated by A - (yellow) blackening K - (red) Gas production - indicated by Wherein: splitting, cracking, bubbling of agar A/A -2-3 sugars fermented K/A - glucose fermented K/K no sugar fermented, no acid formation EMB - Eosin Methylene Blue Mac - MacConkey XLD - Xylose Lysine Desoxycholate HEA - Hektoen Enteric Agar SSA - Salmonella Shigella Agar Readings/ Interpretations: Red slant-Red butt (K/K) Red slant-Yellow butt (K/A) Yellow slant-Yellow butt (A/A) Yellow slant-Yellow butt (A/A Vibrio - halophilic (salt loving) Red slant-Yellow butt (K/A) CARBOHYDRATE FERMENTATION Table 4. Carbohydrate fermentation NLF LF LLF Fermentation - utilization of CHO by anaerobic bacteria Edwardsiella Enterobacter Citrobacter In bacteriology, this process is detected by Hafnia Escherichia Arizona observing changes in pH indicator as acid Morganella Klebsiella products are formed Proteus *after 24 hrs Providencia color change in Basic Principle of Fermentation Salmonella agar Glucose is degraded through a series of Shigella enzymatic glycolytic cleavages and Yersinia *incubate for 24 transformations; the glucose molecule is split into Serratia hrs three carbon compounds, the most important is pyruvic acid. Result: Non-Fermenter (NF), Non-Lactose Fermenter (NLF), Lactose Fermenter (LF), Late Lactose Fermenter (LLF) Media used is: TSI (Triple Sugar Iron) - contains lactose, glucose, sucrose, and iron Bacteria species incapable of fermenting glucose cannot utilize lactose Phenol red - pH indicator Another media that can be used: Kligler’s Iron Agar - contains lactose, glucose, and iron salt If glucose is fermented, it becomes yellow, slant is initially yellow, since glucose is in low H2S production concentration, organisms under aerobic Indicators: sodium thiosulfate and ferrous sulfate MED224 FGV T  Week 2 (+) black color or black precipitate Reagent: Ehrlichs’ Reagent/ KOVAC’s / Paradimethylbenzaldehyde Gas Production Result: (+) pink to wine colored ring at the (+) bubble formation or splitting of the media or junction, (-) no color development complete displacement of the media from the Rapid Spor indole test (blue) - filter paper strips bottom of the tube impersonated with p-amminocinamaldehyde reagent, screening for indole production Beta-galactosidase and ONPG test (Orthonitrophenyl beta-D-galactopyranoside) Interpretation development of cherry red color at the Is a compound structurally similar to lactose test surface of the reagent and broth within seconds after that detects the enzyme beta-galactosidase adding KOVAC’s reagent indicates the presence of Used to distinguish enteric bacteria (Salmonella indole and the test is positive. If no color change is (-), Citrobacter (+)) and to identify observed, then the test is negative. Pseudomononas. (+) yellow, (-) no color change SIM TEST (H2S, Indole, Motility) Indole production - ability of organism to split tryptophan to form compound indole Result (+) pink to wine red colored ring Sulfide = (+) blackening of agar = motility (turbidity) METHYLENE RED TEST Determines the ability of microbes to oxidize glucose with production and stabilization of high content of acid end product Media: MRVP Borth or Clark Lubbs Broth IMViC TEST pH indicator: Methyl Red I - indole production from tryptophan M - Methyl Red T Test in which acidification of glucose broth during to formation of mixed carboxylic acids from pyruvate Vi - Viges-Proskauer test due to formation of acetone from pyruvate in glucose broth C - ability to utilize citrate as a single carbon source VOGES-POSKAUER TEST Principle: this test determines the capability of some organisms to produce non-acidic or neutral end products, such as acetylene methyl corbinol (acetoin), from the organic acid that results from glucose metabolism. This test identifies bacteria that ferments glucose and leading to 2,3- INDOLE TEST butanediol accumulation in the medium. Based on the ability of organism to produce Reagent: alpha naphthol and KOH (Barritt’s indole from tryptophan Method) - (+) pink to red color (E. cloacae)/ Media: Tryptophan broth/ SIM broth (-) no color change (E. coli) MED224 FGV T  Week 2 Reagent: alpha naphthol and 40% KOH in creatinine (Coblentz) - (+) red (S. mutants)/ (-) yellow (S. minis) PHENYLALANINE DEAMINASE TEST Principle: some organism can deaminate phenylalanine converting it to phenylpyruvic acid Phenylalanine is an amino acid CITRATE UTILIZATION TEST (+) dark green color after addition of ferric Principle: citrate will be the sole carbon source chloride. of the microorganism. Sodium citrate in the used in the initial differentiation of Proteus, Simmon’s Citrate Agar will be the carbon source Providencia, and Morganella while NH4+ as the nitrogen source. When Media: Phenylalanine agar or Tryptophan agar bacteria oxidize citrate, they remove it from the Reagent: 10% Ferric chloride medium and liberates CO2. CO2 combines with Result: sodium and water to form carbonate, an alkaline (+) green color on slant (P. vulgaris) product that raises the pH. The pH indicator (-) E. coli (bromthymol blue) will change the color of the media, indicating a positive result. OXIDASE TEST (CYTOCHROME OXIDASE/ INDOPHENOL BLUE) Principle: this test uses certain reagent dyes such as p-phenylenediamine dihydrochloride that substitute for oxygen as artificial electron acceptors. It is colorless in reduced state. In the presence of cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized forming indophenol blue. LYSINE DECARBOXYLASE TEST (+) bluish purple - P. aeruginosa Principle: some organisms can decarboxylate (-) no color change - E. coli lysine converting it to cadaverine Media: Lysine Iron Agar (LIA)/ Moeller’s Agar UREASE TEST Lysine deamination is an aerobic process that Principle: this test determines microorganisms occurs on the slant of the media. that can degrade urea by urease (enzyme Lysine decarboxylation is an anaerobic coming from the organism). Urease hydrolyzes process that occurs in the butt of the media. urea releasing ammonia which alkalinizes the medium by forming ammonium carbonate and CO2. Proteus, Morganella, Providencia - strong urease producer Klebsiella - weak urease producer Yersinia enterocolitica - frequently a urease producer Media: Christensen’s Urea Agar/ Stuart A. Lysine Iron Agar Test Urea Broth Determines whether g (-) can decarboxylate or pH indicator: phenol red deaminate lysine and forms H2SS Media: LIA - contains lysine, peptones, glucose, ferric NH4 citrate and sodium thiosulfate H2S indicator - ferric ammonium citrate MED224 FGV T  Week 2 Indicator - bromcresol purple P. vulgaris - - - When glucose is fermented, the butt becomes acidic (yellow) Shigella D - + + If decarboxylase is not produced, the butt remains purple. Shigella A-C - - - If oxidative deamination of lysine occurs, it will form a burgundy color on the slant. H2S PRODUCTION Differentiating test for Salmonella and Shigella Systems for H2S detection (contains iron thio ferrous): Lead acetate paper SIM tube Hektoen and SS agar XLD agar TSI (+) Salmonella, Edwardsiella, Citrobacter, Proteus Result: (+) production of black color MOTILITY TEST Ability of n organism to produce proteases that B. Decarboxylase Test (Moeller’s method) hydrolyzes gelatin and liquefy solid gelatin Test measures the ability of an organism to medium. decarboxylate an amino acid to form an amine Used in the identification of Clostridium, Serratia, pH indicator - bromcresol purple Flavobacterium, and Pseudomonas Decarboxylation of amino acids results in (+) result gel liquifies/ (-) gel solidifies alkaline pH change 3 decarboxylate broths: arginine, lysine, and NITRATE REDUCTION TEST ornithine Principle: This test determines microorganism It requires acid pH and anaerobic environment that can utilize nitrate as a terminal electron (+) result = purple alkaline color acceptor during anaerobic respiration. Nitrate is reduced to nitrite by nitrate reductase. Amino acid Decarboxylation If an Enterobacteriaceae contains amino acid Decarboxylase, amines produced by Decarboxylase action cause an alkaline pH LOA Test Important in the identification of Enterobacter, Klebsiella, Escherichia, and Salmonella Table 5 Lys Orn Arg Klebsiella + - - Enterobacter +/- + +/- Escherichia + +/- +/- Salmonella + + + E. aerogenes + + - E. cloacae - + + P. mirabilis - + - MED224 FGV T  Week 2 MUG TEST identifier of E. Coli O157H7 - will appear negative in mugtest- train of E.coli Uses 4-methylumbelliferyl-beta-D-glucoronide (+) blue fluorescence - E. coli (-) no fluorescence - P. aeruginosa KCN BROTH TEST This test determines whether the microbe can grow in a medium where potassium cyanide is present as a carbon and nitrogen source. Result (+) turbid; (-) clear IMMUNODIAGNOSIS Serological Test/IMMUNODIAGNOSIS Basic detection of antibody response from an antigenic, stimulation either through a single identification or serial dilution. The presence of antibodies implies either a pathogen exposure or a subclinical infection. Rickettsia and spirochetes - is not culturable - not growing in culture media. Utilized to assist in the detection of congenital infections in newborn TORCH - Toxoplasma gondii, Other organisms MALONATE UTILIZATION TEST ( Tre p o n e m a p a l l i d u m s u b s p. P a l l i d u m , Malonate test is a colorimetric test of the ability Parvovirus B19, Varicella- Zoster, etc) Rubella of bacteria to use malonate as a source of virus, Cytomegalovirus, and Herpes simplex carbon, the endpoint of which is the production of virus alkaline metabolites that induce a color change. Serology- identification of antibodies that are Result (+) blue; (-) green present in the serum. Immunudiagnosis- immune response of the body to a certain antigen. Antigenic- foreign substance that go inside in our body but will trigger immune response Torch- use for newborn Treponema pallium- causative agent of syphilis Parvovirus B19 - cause 5th disease in child Varicella- Zoster - chicken pox Rubella virus - causes German measles Cytomegalovirus - under herpes simplex virus Bacterial Agglutination Performed by adding antibodies (agglutinins) to STRING TEST the bacterial suspension that will bind to the For identification of Vibrio spp. surface of the organisms. Reagent: 0.5% sodium deoxycholate Used to identify bacteria that are difficult to Result: (+) string like cultivate in a culture medium (+) Result: Presence of visible clumps or agglutination MED224 FGV T  Week 2 Interpretation: Specific antibodies bind to the (-) Result: Occurrence of lysis with the red bacterial surface antigens, causing the organism blood cells and the absence of antibodies in the to clump. test serum Particle agglutination Ex. 2 test tube Uses artificial carriers, such as latex particles or treated red blood cells, or biological carriers 1st tube – presence of antibody – add as reagents, which can absorb test antigen and antigen – add compliment (will bind to react with the specific antibody present in the antigen) – rbc – result (will settle) patient's serum. (+) Result: Presence of visible clumps or 2nd tube – absence of antibody – add agglutination antigen - add compliment (will bind to Examples: MHA-TP, HATTS, and passive antigen) – RBC – result (cell lysis/ hemagglutination test for streptococci hemolysis) MHA-TP- For qualitative detection of Flocculation Test treponema pallium uses a soluble antigen that reacts with the HATTS- Confirmatory test for syphilis antibody in a serum sample. (+) Result: Occurrence of macroscopic or Latex Agglutination (Antigen Test) microscopic flocculation (precipitation) The antigen in the patient's specimen binds to Some examples of this test are venereal the antibody that is preset on the surface of the disease research laboratory (VDRL) test and latex beads (reagent). rapid plasma reagin (RPR) test. Also used for direct identification of group B VRDL and RPR- For syphilis streptococci Enzyme-linked Immunosorbent Assay (ELISA) The advent of the molecular assays limits the A sensitive and specific method for the utilization of this method for bacterial detection of antibodies against certain identification. pathogens. This method consist of enzyme-bound Coagglutination antibodies with the antibody- binding sites free Uses the antibody that is bound to a particle to to react with their specific antigen. increase the visibility of the agglutination reaction Utilized for the diagnosis of infectious diseases between an antigen and an antibody. such as those caused by Legionella Assist in the detection of N, meningitidis, Alkaline phosphatase or horseradish Hemophilus, and streptococci. peroxidase is used as an enzyme- conjugate The antibody (Fc portion) binds to the test-strain antibody reagent. while the test- (+) Result: Colored end product antibody (Fab portion)/ Immunofluorescent Assays S. aureus organism that contains protein A in its Commonly used for the rapid identification of cell walls is utilized in this procedure as part of bacterial and viral antigens in body fluids, the reagent. swabs, and infected cells. Te s t S t r a i n - S. a u r e u s o r g a n i s m Monoclonal or polyclonal antibodies, which are (specifically the coulon 1 strain) attached to fluorescent dyes, are applied to an (+) result – agglutination antigen that is previously treated with either formalin, acetone, or alcohol, ten the reaction is Complement Fixation visualized under the Composed of two parts: a test system which fluorescent microscope where final results can includes the antigen that causes the disease in be released within an hour. the patient serum and an indicator system Borrelia, Legionella, Mycoplasma pneumoniae, which consists of the sheep's red blood cells, Rickettsia, and TORCH complement-fixing antibody like the Example of fluorescent dyes: Auramine, immunoglobulin G(IgG), and an exogenous rhodamine, and fluorescein isothiocyanate complement. (FITC) (+) Result: Absence of lysis with the red blood (+) Result: Occurrence of fluorescence as seen cells which means presence of antibody in the under the microscope patient serum MED224 FGV T  Week 2 Types: Direct Fluorescent Antibody (DFA) Test This method creates unique mass spectral and Indirect Fluorescent Antibody (IFA) Test fingerprints that are compared to a massive database of mass spectra. Western Blot Confirmatory test for HIV Molecular Diagnosis Used to identify protein particle of an organism Considered the most important method for Based on the electrophoretic separation of microbial identification bacterial proteins in supporting media. Useful in confirming the taxonomy of the Used to confirm the presence of antibodies to emerging and re-emerging pathogens human immunodeficiency virus type 1 (HIV-1) Used to test genetic material(DNA/RNA) Supporting media: Agarose gel and polyacrylamide gel Polymerase Chain Reaction (Nucleic Acid Employs gel electrophoresis Amplification Assay) (+) Result: Bands on the strips (pattern of Most commonly used amplification technique in antibodies or molecular weight marker ) molecular diagnosis. Increases the nucleic acid of the test sample or Types of Western Blot target microorganism from a very small amount to a million copies Pulsed-field Gel Electrophoresis Utilized for rapid detection of nucleic acid in Enzyme-digested chromosomal fragments of biological samples, a very significant tool in the bacteria are isolated electrophoretically in this diagnosis of the etiologies of diseases. approach. Can be used in an outbreak investigation. Types of PCR Bacteriophage Typing Conventional PCR Based on the specificity of phage surface Principle: The DNA sequence is amplified receptors for cell surface receptors utilizing a Taq polymerase(Thermus aquaticus) A bacteriophage is a virus that attacks Clinical/Diagnostic Application: Microbial bacteria. detection, phylogenetic study, gene analysis, therapeutic cancer detection, therapeutic Chromatographic Method (Gas Chromatography management, and DNA profiling and cloning and High Performance Liquid Chromatography) Thermocycler- too amplify genetic material Analysis of microbial metabolites, cellular fatty Gel electrophoresis- to observe genetic material acids, and products of pyrolysis of the whole Southern blot- used to identify DNA bacterial cells. Northern blot- Used to identify RNA Gas-liquid chromatography is used to detect Western blot- Used to identify protein the cellular fatty acids of anaerobes. HPLC is utilized for the fatty acid analysis of Real-time PCR the Nocardia species. Known as quantitative PCR Quantifies the target nucleic acid after each Matrix-Assisted Laser Desorption lonization- replication cycle in the same PCR equipment Time-of-Flight Mass Spectrometry utilizing commercially available fluorescence Excellent tool for the rapid identification of a detecting thermocyclers wide range of pathogenic species Uses fluorescent dyes to mark specific DNA Utilized for species, subspecies, and strain Measure the amplified product and determine identification of bacteria. the number of copies of target substance Faster result in terms of bacterial identification present in the original specimen. (in mins.) Advantages: combines amplification and Components: lonization Phase and the TOF product detection at one time, used for multiple Phase sample analysis, reduces cross-contamination Procedure: bacterial isolate is ionized by with the amplified production, and lowers turn- transferring it from the culture plate to a metal around time. plate, where it is treated with matrix solution until it forms a crystallized microbial protein or Nested PCR (Single-tube Nested PCR or matrix lattice which is then analyzed using STNPCR) MALDI-TOF instrument. Effective for divergent nucleic acid samples such those isolated from formalin-fixed, paraffin-embedded tissue MED224 FGV T  Week 2 Reverse-Transcription PCR (RT-PCR) Transcribes RNA 'reversely' into complementary DNA by way of the reverse transcriptase enzyme, hence, transcription is the differential phase compared to the conventional PCR Re a l - t i m e RT- P C R / Reve rs e Tra n s c r i p t i o n Quantitative PCR Useful for measuring the abundance of certain RNAs to determine the gene expression. Gold standard method for the detection of SARS-CoV-2 Multiplex PCR Allows simultaneous amplification of multiple gene segments rather than separate test runs for each. MED224 FGV T