BACTE (LEC) WEEK 2_240826_075703 (1).pdf

BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, R...

BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT BACTERIAL CELL STRUCTURE, PHYSIOLOGY, METABOLISM AND GENETICS, IMMUNODIAGNOSIS LIPID & LOW HIGH (due to outer LIPOPROTEIN membrane) OUTLINE I. BACTERIAL CELL STRUCTURE SURFACE POLYMERS AND APPENDAGES A. CYTOPLASMIC ↳ GLYCOCALYX (Slime layer & capsule) – it evades 1. NUCLEAR AREA 2. PLASMIDS phagocytosis. 3. RIBOSOMES ✔ General substances that surround cells 4. INCLUSIONS 5. SPORES ✔ Gelatinous polymer of polysaccharide, B. CELL ENVELOPE polypeptide, or both. C. SURFACE POLYMERS AND APPENDAGES II. BACTERIAL METABOLISM ✔ Capsule – Substances is organized and firmly attached to III. PHYSIOLOGY cell wall (India ink & nigrosine dye). IV. GENETICS ✔ Slime layer – Unorganized and loosely attached to the CYTOPLASMIC cell wall. ↳ NUCLEAR AREA (NUCLEOID)- single circular ○ Prevents phagocytosis chromosome attached to a mesosome. ○ Attachment to various surfaces in its natural ↳ PLASMIDS- small, circular, dsDNA molecule environment. ✔ Antibiotic resistance ○ B. anthracis, S. pneumoniae, K. pneumoniae ↳ INCLUSIONS: ↳ FLAGELLA/FLAGELLUM – long filamentous appendages ✔ METACHROMATIC GRANULES- represents that propel bacteria, allowing bacterium to move towards reserves of polyphosphates used in the synthesis of ATP. favorable environment or away from adverse ones. ✔ POLYSACCHARIDE GRANULES- consists of ○ Monotrichous – single polar flagellum glycogen and starch granules. ○ Lophotrichous – 2 or more flagella at one pole ↳ SPORES- highly refractile resting cells that are highly of the cell durable and dehydrated with thick walls. ○ Amphitrichous – single/tuft of flagella at each ✔ Schaeffer-Fulton- most used endospore stain. end of the cell. CELL ENVELOPE ↳ PLASMA MEMBRANE – A phospholipid bilayer with ○ Peritrichous – flagella distributed over the embedded proteins that surrounds the cytoplasm. entire cell. ✔ Acts as an osmotic barrier. ✔ ↳ CELL WALL (peptidoglycan or murein layer) – A rigid structure that maintains the shape of the cell and prevents bursting of the cell from the high osmotic pressure inside it. ✔ Maintains the shape of the cell and protects the cell from osmotic pressure. ✔ Repeating disaccharide attached by polypeptides. ✔ Two major types of cell walls: 1. Gram-positive – With a very thick protective peptidoglycan (murein) layer. a. Many antimicrobial agents are effective against ↳ FIMBRIAE OR PILI– adhere some bacterial cells to one gram-positive organisms because of the another and to host cells. peptidoglycan layer. (ex: penicillin) ✔ Hairlike appendages that are shorter, straighter, 2. Gram-negative – have a thinner layer of peptidoglycan and thinner than flagella. and an additional outer membrane that is unique. ↳ FIMBRIAE (sing. fimbria) – “common pili”, can occur at the a. Less affected by the agents. poles or can be evenly distributed from few to several ↳ PERIPLASMIC SPACE – absent in gram-positive. hundred. ↳ OUTER MEMBRANE – Cells initial barrier (certain ✔ For adherence of cells to one another and to environmental surfaces. antibiotics and evasion of phagocytes) ↳ PILI (pilus) – “sex (conjugation) pili”, are hollow protein tubes, ✔ Composed of Lipopolysaccharides (LPS), longer than fimbria and number is 1 or 2 per cell. lipoproteins, and phospholipids. ✔ Join bacterial cell in preparation of DNA transfer ✔ Contains Porins – Water-filled structures that from one cell to another. control the passage of nutrients and solutes. ↳ AXIAL FILAMENT (endoflagella) – bundles of fibrils anchored at one end of spirochete and spiral around the cell. CHARACTERISTICS GRAM (+) GRAM (-) STAIN PURPOSE PEPTIDOGLYCAN Thick (multilayered) Thin (single layered) Acid-fast ✔ Bacteria with high (LAYER) cell wall lipid and wax content TEICHOIC ACIDS PRESENT ABSENT ✔ Do not stain well with traditional PERIPLASMIC ABSENT PRESENT bacterial stains. SPACE Acridine Orange ✔ Both gram-positive OUTER MEMBRANE ABSENT PRESENT and gram-negative bacteria, living or LPS CONTENT VIRTUALLY LOW HIGH dead. ✔ Binds to the nucleic FNAM 1  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT acid of the cell and fluorescent microscope is used. ✔ Bacteria in blood cultures. Calcofluor White ✔ Binds to chitin in fungal cell walls. ✔ “Bluing” in high-volume laundries to whiten yellow-appearing white cotton. GLUCOSE TO PYRUVIC ACID ✔ C. diphtheriae for Methylene Blue ↳ Embden-Meyerhof-Parnas (EMP) Glycolytic Pathway observation of ↳ Pentose Phosphate Pathway metachromatic ↳ Entner-Doudoroff Pathway granules. ✔ Counterstain in ANAEROBIC UTILIZATION OF PYRUVIC ACID FERMENTATION acid-fast staining procedures. FERMENTATION MAJOR END USED ✔ WBC in stool samples. PRODUCT Lactophenol Cotton Blue ✔ Cell walls of fungi ALCOHOLIC Ethanol Yeasts grown in slide HOMOLACTIC Almost exclusively Streptococcus spp., culture. lactic acid Lactobacillus spp. HETEROLACTIC Lactic acid, CO2, Some lactobacilli India Ink & Nigrosin (-) ✔ Capsules in alcohols, formic acid, Stains yeast. (ex: acetic acid Cryptococcus spp.) PROPIONIC ACID Propionic acid Carried by propionibacterium Endospore stain ✔ Bacterial spores acnes, some (Schaefer-Fulton spore ✔ The endospores anaerobic non-spore stain) appear green within forming, gram (+) pink-appearing or bacilli red- appearing MIXED ACID lactic , acetic, Escherichia spp., bacterial cells. succinic, formic acids Salmonella spp., Shigella. BUTANEDIOL Acetoin and Klebsiella spp., FERMENTATION 2,3-butanediol Enterobacter spp., Eubacterium spp. ↳ Anaerobic process carried out by both obligate and BUTYRIC ACID Butyric acid, Acetic Clostridium spp., facultative anaerobes. acid, CO2, hydrogen Fusobacterium spp., ✔ End products: Lactate, Ethanol, Butyrate, Eubacterium spp. Acetoin ✔ Used for MRVP CARBOHYDRATE UTILIZATION AND LACTOSE FERMENTATION ↳ Ability of microorganisms to use various sugars for growth RESPIRATION is an integral part of most diagnostic identification schemes. ↳ Efficient energy-generating process in which ↳ Fermentation of sugar is usually detected by acid molecular oxygen is the final electron acceptor. production and a concomitant change of color ✔ Obligate aerobes and facultative anaerobes. ✔ Due to pH indicator present in the culture medium ↳ For Enterobacteriaceae: determination of the m.o. Ability to ferment lactose ✔ Lactose fermenters, lactose non-fermenters, late lactose fermenters. MECHANISMS OF GENE TRANSFER ↳ TRANSFORMATION- uptake and incorporation of naked DNA into a bacterial cell. FNAM 2  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT ✔ S. pneumoniae, N. gonorrhoeae, H. influenzae ↳ Hemidophilic – moisture content ↳ TRANSDUCTION- transfer of bacterial genes by a ↳ Halophilic – salt concentration bacteriophage. ↳ Osmophilic – high osmotic pressure ✔ C. diphtheria ↳ CONJUGATION- transfer of genetic material from a donor STAGES OF BACTERIAL GROWTH CURVE bacterial strain to a recipient stain. ↳ Generation time of bacteria in a culture varies according to their cellular properties. ○ Fast growing bacterium (E. coli) – 20 mins ○ Slow growing bacteria (M. tb) – 24 hrs 1. Lag Phase or Period of Rejuvenescence a. No cell division b. Start of biosynthesis c. Adjustment phase to a new environment 2. Log or Exponential Phase a. Microorganisms are actively growing and dividing b. Utilized in physiological, biochemical, and antimicrobial testing. 3. Stationary/Plateau Phase a. Balance between cell division and dying organisms. b. Number of viable organisms are constant. 4. Death or Decline Phase a. Period of cessation of bacterial growth as the number of dead cells exceeds the living microorganisms. NUTRITIONAL REQUIREMENT ↳ Autotrophic- ↳ Phototrophs- ↳ Chemotrophs- ↳ Lithotrophs- ↳ Heterotrophs/Organotrophs- ↳ Saprophytes- ↳ Parasites- TYPES OF CULTURE MEDIA ↳ Culture Media – any substance that contains the OXYGEN REQUIREMENT nutritional requirement needed for bacterial growth. ↳ Obligate aerobes – requires oxygen for growth ↳ Culture – group of organisms obtained in a media. ↳ Obligate anaerobes – grows in strict absence of oxygen. TYPES OF CULTURE ↳ Facultative anaerobes/Facultative aerobes – can grow ↳ Pure culture – only one species. without oxygen, but will utilize upon its presence ↳ Mixed culture – more than one species. ↳ Microaerophiles – requires small amount of oxygen ↳ Stock culture – several species contained in separate ↳ Aerotolerant – can grow without oxygen but can survive culture medium (1 species/culture medium). in the presence of it. ✔ Should be grown in a large volume of broth then CO2 REQUIREMENT divide into a small freezer (lengthens the shelf ↳ Capnophiles – require CO2 to grow. life for a year). ✔ Neisseria gonorrhoeae ✔ For academic and industrial purposes. ✔ Haemophilus influenzae ✔ Brucella abortus CONSISTENCY ✔ Streptococcus pneumoniae ✔ Mycobacteria spp LIQUID MEDIUM No agar, allowas growth TEMPERATURE REQUIREMENT SEMI-SOLID MEDIUM 0.5% - 1% agar ↳ Psychrophilic/Cryophilic – low temp (10-20° C) ↳ Mesophilic – where most pathogens grow (30-44°C or SOLID MEDIUM 2% - 3% agar 20-45°C) ↳ Thermophilic – heat loving (50-60°C) ↳ Hyperthermophilic – (80-113°C) COMPOSITION ↳ ↳ Synthetic or defined medium pH REQUIREMENT ✔ All components are ↳ Acidophilic – requires acid med (pH 0 – 5.5) known. ✔ Lactobacilli ✔ Research purposes ↳ Basophilic – (pH 8.5 – 11.5) (liquid/solid forms) ✔ Vibrio cholerae ↳ Neutrophilic – optimum pH 6.5 – 7.5 of most bacteria. ✔ ü Isolation of cyanobacteria OTHER REQUIREMENTS and chemoorganotrophs FNAM 3  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT ↳ Non-synthetic or complex medium ✔ Some of the components are unknown. TECHNIQUES ✔ Isolation of clinically significant bacteria ↳ Liquid medium ↳ Tissue culture ↳ Plated medium ✔ For obligate intracellular organisms. ↳ Butt medium – stabbing ↳ Slant medium – streaking DISPENSING ↳ Butt/slant – stab the butt portion then streak the slant ↳ Plated medium – dispensed in a petri plate. medium. ↳ Tube medium – prepared as either liquid, agar, METHODS OF STREAKING FOR PLATED MEDIA butt/deep, agar slant, or agar slant and butt/deep. ↳ Radial streak method ↳ Overlap streak method – for sensitivity testing ↳ Multiple streak method ↳ Interrupted streak method ↳ Multiple interrupted streak method USE ↳ Simple media ✔ Routine in the lab without additional supplements. ✔ Composed of meat and soybean extracts. ANAEROBIC CULTURE ↳ Enrichment medium (liquid-type media) ↳ Culture media should be protected from oxygen ✔ Enhances the propagation exposure, freshly prepared, and should be held in ✔ To cultivate certain groups of bacteria from a anaerobic condition until needed. mixture of microorganisms ↳ Recommended methods: Thioglycolate broth & anaerobic ✔ Has specific components without additional jar supplements ↳ Use of anaerobic chamber with vacuum pump for ↳ Enriched medium processing of samples and storage of culture media prior ✔ With additional supplements (Ex: chocolate to inoculation. agar) ANAEROBIC CHAMBER: SEALED GLOVE BOX AND ↳ Differential medium GLOVELESS CHAMBER ✔ Distinguishes organisms ↳ Nitrogen gas – acts as a filler for the remaining percentage of the anaerobic atmosphere. ✔ Allows the visualization of metabolic ↳ Hydrogen and CO2 gas – facilitates the growth and differences between groups of bacteria. (Ex: isolation of anaerobes. Blood agar) ↳ Palladium catalyst pellets – remove residual oxygen ↳ Selective medium from the chamber. ✔ Promotes the growth of desirable organisms ↳ Silica gel (desiccant) – absorbs the water that is formed but at the same time, inhibiting the growth of when hydrogen combines with free oxygen. other. ↳ Methylene blue/resazurin – oxygen-reduction ✔ Substances that inhibit gram positive organisms incubator. ✔ Gentian violet REPORTS ✔ Sodium deoxycholate and other bile salt ↳ Save culture until satisfactory growth is obtained and ✔ Substances that prevent swarming of Proteus final diagnosis is made. spp. ↳ Some cultures may be held for several weeks before final Alcohol report is made. Chloral hydrate ✔ Blood culture – 7-10 days ✔ Substances that inhibit gram negative ✔ Brucella and M. tb – 4 weeks organisms ✔ L. monocytogenes – 4 months Potassium tellurite ↳ All media except thioglycolate broth should be stored in Sodium azide the refrigerator. ↳ Special medium ↳ The number of media stored should not exceed 2-3 ✔ Isolate organisms with special growth weeks supply. requirements ↳ All media must be checked before use. ✔ Lowenstein-Jensen / 7H-10 agar / 7H-9 broth COLONY – M. tb ↳ Colonies – groups of bacteria forming on solid media ✔ Thayer-Martin – Neisseria group because of division of one or a few organisms. ✔ MacBride agar – L. monocytogenes ↳ TYPES OF COLONIES ✔ Fletcher medium – Leptospira ✔ S or smooth colonies – uniform texture & ✔ W medium – Brucella homogeneity, easily emulsified in NSS, virulent ✔ Bordet-Gengou Agar – B. pertussis organisms. ✔ Mannitol Salt Agar – Staphylococci FNAM 4  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT ✔ M or mucoid colonies – associated with capsulated and virulent m.o., slimy/watery ✔ R or rough colonies – granulated in appearance, hard to emulsify in NSS. CONVENTIONAL METHODS ↳ Biochemical characterization serves as the fundamental source of information for ID of most bacteria after microscopy. ✔ Motility test – wet mount and hanging drop preparation. ✔ Staining – Gram staining, acid-fast staining, structural staining ✔ Manual biochemical test – carbohydrate fermentation (LIA), catalase and coagulase tests ↳ Use of routing and selective media ANALYTICAL PROFILE INDEX CARBOHYDRATE FERMENTATION ↳ Consists of plastic strips and microtubes that contain ↳ Result: dehydrated biochemical substrates. ○ Non-fermenter (NF) ↳ Commonly used for gram (-) enteric bacteria. ○ Non-Lactose Fermenter (NLF) ↳ Same principle with biochemical manual tubed method. ○ Lactose Fermenter (LF) ↳ Principle: Biochemical substrates (pH-based substrates) NON-LACTOSE LACTOSE LLF are inoculated with pure culture suspended in sterile FERMENTATION FERMENTATION physiologic saline and incubated for 18-24 hrs at 35°C, Edwardsiella Enterobacter Citrobacter Arizona some reagents may be added after incubation. Hafnia Morganella Escherichia TYPES OF STAIN Proteus Klebsiella ↳ Simple stains Providencia ↳ Indirect, Relief, or Negative Stains Salmonella Shigella ↳ Special Stains – Capsular Stain, Cell Wall Stain, Yersinia Serratia Metachromatic Granules, Flagellar ↳ Bacteria species incapable of fermenting glucose cannot ↳ Differential Stain utilize lactose. GENERAL RULES FOR GRAM STAINING ↳ Another media that can be used: Kligler’s Iron Agar ↳ All cocci are gram positive except Neisseria, ✔ contains lactose, glucose, and iron salt Branhamella, Moraxella, and Velionella. ↳ H2S production ↳ All bacilli are gram negative except Mycobacteria, ✔ Indicators: Sodium thiosulfate and ferrous Clostridium, Corynebacterium, Bacillus, sulfate Erysipelothris, Listeria, Lactobacillus. ✔ (+) Black color or black precipitate ↳ Higher forms or organisms (Actinomyces, ↳ Gas production ↳ Streptomyces, yeasts, and molds) are gram positive. ✔ (+) bubble formation or splitting of the media or ✓ All spiral organisms are reported as gram complete displacement of the media from the negative bottom of the tube. ↳ Beta-galactosidase and ONPG (Ortho Nitrophenyl beta-D-galactopyranoside) ✔ A compound structurally like lactose test that detects the enzyme beta-galactosidase. ✔ Used to distinguish enteric bacteria (Salmonella (-), Citrobacter (+)), and to identify Pseudomonas. ✔ Orthonitrophenol is a chromophore that is released into the medium and detected by a pale yellow color. ✔ (+) yellow, (-) no color change SIM TEST ↳ Indole production – ability of organisms to split tryptophan to form compound indole. ↳ Result (+) – pink to wine red colored ring ↳ Sulfide = (+) blackening of agar = Motility ACID FAST NON-ACID FAST 1 STAIN RED RED MORDANT RED RED DECOLORIZER RED COLORLESS 2 STAIN RED PURPLE-BLUE FNAM 5  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT iMViC TEST ↳ I – Indole production from tryptophan ↳ M – Methyl red test in which acidification of glucose broth due to formation of mixed carboxylic acids from pyruvate ↳ Vi – Voges-Proskauer test due to formation of acetoin from pyruvate in glucose broth ↳ C – ability to utilize citrate as a single carbon source. INDOLE TEST ↳ Based on the ability of organisms to produce indole from tryptophan. ↳ Media: Tryptophan broth/SIM broth VOGES-PROSKAUER TEST ↳ Reagent: Ehrlichs’ ↳ Principle: Determines the capability of some organisms Reagent/KOVAC’s/Paradimethylbenzaldehyde to produce non-acidic or neutral end products, such as ↳ Result: (+) pink to wine colored ring at the junction, (-) acetyl methyl carbinol (acetoin), from the organic acid no color development that results from glucose metabolism. ↳ Rapid Spot indole test (blue) – filter paper strips ↳ Identifies bacteria that ferments glucose and leading to impregnated with p-amylcinnamaldehyde reagent, 2,3-butanediol accumulation in the medium. screening for indole production. ↳ Reagent: alpha naphthol and KOH (Barritt’s Method) – Interpretation Development of cherry red color at the surface of (+) pink to red color (E. cloacae) / (-) no color change (E. the reagent and broth within seconds after adding KOVAC’s coli) reagent indicates the presence of indole – positive ↳ Reagent: alpha naphthol and 40% KON in creatinine No color change – negative (Coblents) – (+) red (S. mutans) / (-) yellow (S. minis) CITRATE UTILIZATION TEST ↳ PRINCIPLE ✔ Citrate – sole carbon source of the microorganisms. ✔ Sodium citrate (Simmon’s Citrate agar) – carbon source ✔ NH4+ - nitrogen source ↳ When bacteria oxidize citrate, they remove it from the medium and liberate CO2. ↳ CO2 combines with Na & H2O = Carbonate – an alkaline METHYL RED TEST product that raises the pH. ↳ Determines the ability of microbes to oxidase glucose ↳ Bromothymol blue (pH indicator) – change the color of with production and stabilization of high content of acid the media, indicating a positive result. end product. ↳ Key biochemical properties of Salmonella, Citrobacter, ↳ Media: MRVP Broth or Clark Lubbs Broth Klebsiella, Enterobacter, and Serratia. ↳ pH indicator: Methyl red FNAM 6  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT OXIDASE TEST (CYTOCHROME OXIDASE/INDOPHENOL BLUE) ↳ PRINCIPLE: Uses certain reagent dyes such as dihydrochloride that substitute for oxygen as artificial electron acceptors. ↳ Colorless – reduced state. ↳ Presence of cytochrome oxidase & atmospheric oxygen – p-phenylenediamine is oxidized forming indophenol blue. ✔ (+) bluish purple – P. aeruginosa ✔ (-) no color change – E. coli LYSINE DECARBOXYLASE TEST ↳ PRINCIPLE: Some organisms can decarboxylase lysine converting it to cadaverine ↳ Media: Lysine Iron Agar / Moeller’s agar ↳ Lysine deamination – anaerobic process that occurs on the slant of the media. ↳ Lysine decarboxylase - anaerobic process that occurs UREASE TEST in the butt of the media. ↳ PRINCIPLE: Determines microorganisms that can degrade urea by urease. ↳ Urease hydrolyzes urea releasing ammonia which alkalinizes the medium by forming ammonium carbonate and CO2. ✔ Proteus, Morganella, Providencia – strong urease producers ✔ Klebsiella – weak urease producers ✔ Yersinia enterocolitica – frequently a urease producer ✔ ↳ Media: Christensen’s Urea Agar / Stuart Urea Broth ✔ pH indicator: phenol red LYSINE IRON AGAR TEST ↳ Determines whether gram (-) can decarboxylate or deaminate lysine and forms H2SS ↳ Media: LIA – contains lysine, peptones, glucose, ferric NH4 citrate and sodium thiosulfate ↳ H2S indicator – ferric ammonium citrate ↳ Indicator – bromocresol purple ↳ Fermented glucose – butt becomes acidic (yellow) ↳ Decarboxylase is not produced – butt remains yellow. ↳ If oxidative deamination of lysine occurs – burgundy PHENYLALANINE DEAMINASE TEST color on the slant will form. ↳ PRINCIPLE: Some organisms can be deaminate phenylalanine converting it to phenylpyruvic acid. ✔ (+) dark green color after addition of ferric chloride ✔ Used in the initial differentiation of Proteus, Providencia, and Morganella ↳ Media: Phenylalanine agar or Tryptophan agar ↳ Reagent: 10% Ferric chloride ↳ Result: ✔ (+) green color on slant (P. vulgaris) ✔ (-) E. coli DECARBOXYLASE TEST (MOELLER’S METHOD) ↳ Measures the ability of an organism to decarboxylate an amino acid to form an amine. FNAM 7  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT ↳ pH indicator: bromocresol purple ✔ (+) blue fluorescence – E. coli ↳ Decarboxylation of amino acids results in alkaline pH ✔ (-) no fluorescence – P. aeruginosa change ✔ 3 decarboxylate broths: arginine, lysine, and ornithine ✔ Required acid pH and anaerobic environment. ✔ (+) result – purple alkaline color ↳ 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 H2S PRODUCTION KCN BROTH TEST ↳ Differentiating test for Salmonella and Shigella ↳ Determines whether the microbe can grow in a medium ↳ System for H2S detection: where potassium cyanide is present as a carbon and ✔ Lead acetate paper nitrogen source. ✔ SIM tube ↳ RESULT: (+) TURBID, (-) CLEAR ✔ Hektoen and SS agar ✔ XLD agar ✔ TSI ↳ (+) – Salmonella, Edwardsiella, Citrobacter, Proteus ↳ Result: (+) production of black color MOTILITY TEST ↳ Ability of an organism to produce proteases that hydrolyzes gelatin and liquefy solid gelatin medium. ↳ Used in the identification of Clostridium, Serratia, Flavobacterium, and Pseudomonas ✔ (+) result gel liquifies / (-) gel solidifies NITRATE REDUCTION TEST MALONATE UTILIZATION TEST ↳ Principle: Determines microorganisms that can utilize ↳ Colorimetric test of the ability of bacteria to use malonate nitrate as a terminal electron acceptor during anaerobic as a source of carbon. respiration. Nitrate is reduced to nitrite by nitrate ↳ ENDPOINT: production of alkaline metabolites that induce reductase. a color change ↳ RESULT: (+) blue, (-) green MUG TEST STRING TEST ↳ Uses 4-methylumbelliferyl-beta-D-glucuronide ↳ For identification of Vibrio spp. FNAM 8  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT ↳ REAGENT: 0.5 % sodium deoxycholate immunoglobulin G(IgG), and an exogenous ↳ RESULT: (+) string like complement. ↳ (+) Result: Absence of lysis with the RBC – means presence of antibody in the patient serum. ↳ (-) Result: Occurrence of lysis with the RBC and the absence of antibodies in the test serum. FLOCCULATION TEST ↳ Uses a soluble antigen that reacts with the antibody in a serum sample. ↳ (+) Result: Occurrence of macroscopic or microscopic flocculation (precipitation) ✔ Ex: Venereal disease research laboratory (VDRL) test and rapid plasma reign (RPR) test. ENZYME-LINKED IMMUNOSORBENT ASSAY ↳ A sensitive and specific method for the detection of antibodies against certain pathogens. IMMUNODIAGNOSIS ↳ Enzyme-bound antibodies with the antibody-binding sites free to react with their specific antigen. SEROLOGICAL TEST/ IMMUNODIAGNOSIS ↳ Utilized for the diagnosis of infectious diseases such as ↳ Basic detection of antibody response from an antigenic those caused by Legionella. stimulation either through a single identification or serial ↳ Alkaline phosphatase or horseradish peroxidase is used dilution. as an enzyme-conjugate antibody reagent. ↳ Antibodies – pathogen exposure or a subclinical ✔ (+) Result: Colored end product infection. IMMUNOFLUORESCENCE ASSAYS ↳ Rickettsia and spirochetes ↳ For the rapid identification of bacterial and viral antigens ↳ Utilized to assist in the detection of congenital infections in body fluids, swabs, and infected cells. in newborn. ↳ Monoclonal or polyclonal antibodies, which are attached ↳ TORCH – Toxoplasma gondii, other organisms to fluorescent dyes, are applied to an antigen that is (Treponema pallidum subsp. Pallidum, Parvovirus B19, previously treated with either formalin, acetone, or Varicella-Zoster, etc.) Rubella virus, Cytomegalovirus, and alcohol, then the reaction is visualized under the Herpes simplex virus. fluorescent microscope where final results can be BACTERIAL AGGLUTINATION released within an hour. ↳ Performed by adding antibodies (agglutinins) to the ✔ Borrelia, Legionella, Mycoplasma bacterial suspension that will bind to the surface of the pneumoniae, Rickettsia, and TORCH organisms. ↳ Example of fluorescent dyes: Auramine, rhodamine, ↳ Used to identify bacteria that are difficult to cultivate in a and fluorescein isothiocyanate (FITC) culture medium. ↳ (+) Result: Occurrence of fluorescence as seen under ↳ (+) Result: Presence of visible clumps or agglutination the microscope. ↳ Interpretation: Specific antibodies bind to the bacterial ↳ Types: Direct Fluorescent Antibody (DFA) Test and PARTICLE AGGLUTINATION Indirect Fluorescent Antibody (IFA) Test ↳ Uses artificial carriers, such as latex particles or treated WESTERN BLOT RBC, or biological carriers as reagents, which can absorb ↳ Based on the electrophoretic separation of bacterial test antigen and react with specific antibodies present in protein in supporting media. the patient’s serum. ✔ Used to confirm HIV Type 1 (HIV-1) ↳ (+) Result: Presence of visible clumps or agglutination ↳ Supporting media: Agarose gel & polyacrylamide gel ✔ Examples: MHS-TP, HATTS, and passive ↳ (+)Result: Bands on the strips (pattern of antibodies) hemagglutination test for streptococci. LATES AGGLUTINATION (ANTIGEN TEST) TYPES OF WESTERN BLOT ↳ The antigen in the patient’s specimen binds to the ↳ Pulsed-field Gel Electrophoresis antibody that is present on the surface of the latex beads ✔ Enzyme-digested chromosomal fragments of (reagent). bacteria are isolated electrophoretically in this ↳ Also used for direct identification of group B streptococci. approach. ↳ The advent of the molecular assays limits the utilization of this method for bacterial identification. ✔ Outbreak investigation. ↳ Bacteriophage Typing COAGGLUTINATION ↳ Uses the antibody that is bound to a particle to increase ✔ Based on the specificity of phage surface the visibility of the agglutination reaction between an receptors for cell surface receptors. antigen and an antibody. ↳ Bacteriophage – a virus that attacks bacteria ↳ Assist in the detection of N. meningitidis, Haemophilus, CHROMATOGRAPHIC METHOD (GAS CHROMATOGRAPHY and streptococci. AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY) ↳ The antibody (Fc portion) binds to the test-strain while the ↳ Analysis of microbial metabolites, cellular fatty acids, and test antigen (suspected pathogen) binds to the specific products of pyrolysis of the whole bacterial cells. target site in the antibody (Fab portion). ↳ Gas-liquid chromatography – used to detect the cellular ↳ S. aureus organism that contains protein A in its cell walls fatty acids of anaerobes. is utilized in this procedure as part of the reagent. ↳ HPLC is utilized for the fatty acid analysis of the Nocardia COMPLETE FIXATION species. ↳ 2 parts: ✔ A test system – includes the antigen that causes the disease in the patient's serum. ✔ Indicator system – consists of the sheep’s RBC, complement-fixing antibody like the FNAM 9  BACTERIOLOGY / MLSBACTC MED22BACTC-2P CLASS TYPE: LECTURE SCHOOL YEAR 2024-2025 PROFESSOR NAME : LANCE EDILBERT G. VELOSO, RMT MATRIX-ASSISTED LASER DESORPTION multiple sample analysis, reduces IONIZATION-TIME-OF-FLIGHT MASS SPECTROMETRY cross-contamination with the amplified production, and lowers turn- around time. ↳ Nested PCR (Single-tube Nested PCR or STNPCR) ✔ Effective for divergent nucleic acid samples such those isolated from formalin-fixed, paraffin- embedded tissue. ↳ 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. ↳ Real-time RT-PCR/Reverse Transcription Quantitative PCR ✔ Used 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. ↳ Rapid identification of a wide range of pathogenic species. ↳ Utilized for species, subspecies, and strain identification of bacteria. ↳ Faster result in terms of bacterial identification (mins) ↳ Components: Ionization Phase and TOF Phase ↳ Procedure: Bacterial isolate is ionized by transferring it from the culture plate to a metal plate, where it is treated with matrix solution until it forms a crystallized microbial protein or matrix lattice which is then analyzed using the MALDI-TOF instrument. ↳ Creates unique mass spectral fingerprints that are compared to a massive database of mass spectra. MOLECULAR DIAGNOSIS ↳ Confirmatory is PCR ↳ Most important method for microbial identification. ↳ Useful in confirming the taxonomy of the emerging and re-emerging pathogens. POLYMERASE CHAIN REACTION (NUCLEIC ACID AMPLIFICATION ASSAY) ↳ Used amplification technique in molecular diagnosis. ↳ Increases the nucleic acid of the test sample or target microorganism from a very small amount to a million copies. ↳ Utilized for rapid detection of nucleic acid in biological samples, a very significant tool in the diagnosis of the etiologies of diseases. TYPES OF PCR ↳ Conventional PCR ✔ Principle: The DNA sequence is amplified utilizing a Taq polymerase ✔ Clinical/Diagnostic Application: Microbial detection, phylogenetic study, gene analysis, therapeutic cancer detection, therapeutic management, and DNA profiling and cloning. ↳ Real-time PCR “Quantitative PCR” ✔ Quantifies the target nucleic acid after each replication cycle in the same PCR equipment utilizing commercially available fluorescence detecting thermocyclers. ✔ Fluorescent dyes – mark specific DNA. ✔ Measures the amplified product and determine the number of copies of target substance presence in the original specimen. ✔ Advantages: Combines amplification and product detection at one time, used for FNAM 10