Clinical Bacteriology - 01 Bacterial Cell and Identification

o o CLINICAL BACTERIOLOGY PARTS OF BACTERIAL CELL AND TECHNIQUES IN BACTERIAL IDENTIFICATION OUTLINE • • Parts of Bacterial Cell o General Characteristics of Bacteria o Important Parts of Bacterial Cell o Requirements for Bacterial Growth o Stages of Bacterial Growth Techniques in bacterial Identification o Bacterial Identification o Staining o Culture Media LESSON 1 - PARTS OF BACTERIAL CELL GENERAL CHARACTERISTICS OF BACTERIA • • • • • • Prokaryotic Has both RNA & DNA Multiplies by binary fission o Produces 2 daughter organism (2n) Colonies - groups of bacteria in millions to billions o Slow/late growers - requires more than 24hrs to observe visible colonies ▪ TB bacteria - 6 weeks each stage • First symptom of HIV; decreases life expectancy o Rapid growers - colonies can be observed within 1824 hrs ▪ Some bacteria can multiply within 10 minutes Measured in um o Average size: 0.4-2.0 um o Mycoplasma - Smallest living organism o Bacillus - Largest organisms ▪ anthrax Produce either exotoxins or endotoxins o Toxins are biologically produced poisons o Exotoxins - usually produced by gram (+) organisms except Listeria spp.; excereted by living bacterial cells ▪ Diptheria toxin - Corynebacterium diphtheriae ▪ Coagulase - Staphylococcus aureus • One of the 1st bacteria to be observed (bacilli) ▪ Botulinum toxin - Clostridium botulinum • Most potent toxin known to man • 1 lb - enough to eradicate mankind • Can be seen in canned food • Anaerobic o Endotoxin - Usually produced by gram negative; released only ff: cell death • ie. Shigella dysenteriae IMPORTANT PARTS OF BACTERIAL CELL • Flagella - organ for locomotion o Motile - moves o Non-motile - without flagella o Atrichous - absence of flagella (non-motile) o Monotrichous - single flagellum at one end ▪ Some can be slow/fast moving ▪ V. cholera - very fast motility • • • Amphitrichous - single flagellum at both ends Lophotrichous - tuft/bundle of flagella at one or both ends o Peritrichous - bacterium surrounded with flagella o Cocci - usually non-motile (due to circular shape) o Bacilli and spiral organisms - motile o Escherichia coli - most abundant bacteria in intestine; normal intestinal flora ▪ Supplies Vit K (blood clotting) ▪ Lubricates food/stool for smooth defecation ▪ When lost in genitourinary tract, can cause UTI in women (70-80%) o Ways to demonstrate motility ▪ Use of flagellar stain - Gray’s and Leifson (stains for flagella) ▪ Use of semi-solid media - SIM • Sulfur Reduction, Indole Reduction, Motility • Included in biochemical testing ▪ Hanging Drop - presumptive test for Listeria monocytogenes ▪ L. monocytogenes - tumbling motility at room temp ▪ Campylobacter spp. - darting mobility (straight) ▪ Capnocytophaga gingivalis - gliding/sliding motility Pili/Fimbriae - usually found among gram negative organisms, o Can be a virulence factor which increases the ability of organism to cause disease o ie. Neisseria gonorrhaoeae (gram negative cocci) ▪ Women are asymptomatic due to hormones ▪ Men - symptomatic ▪ Gonorrhea - treatable o Common pili - for adherence to host cells; 1st step in establishing infection o Sex pili - used for copulation; ▪ Gene conjugation and transfer of genetic material Capsule - slimy area that covers the cell walls o “Mucoid” colonies o Anti-phagocytic - virulence factor; not common to all ▪ Cannot be detected by WBCs o Neufeld Quellung Test - capsular swelling test o N. meningitidis, H. influenzae, S. pneumonia polysaccharide capsule (sugar-like coating) o Bacillus anthracis - D-glutamate capsules o Can be used in food products (nata de coco) Cell wall aka Murein Late or Peptidoglycan layer o Gives shape to the organisms o Site of antibiotic action o Basis of gram staining ▪ Gram (+) - w/ thicker peptidoglycan layer ▪ Gram (-) - w/ thinner peptidoglycan layer o Provides attachment for flagella o Peptidoglycan - main component o Cell wall less - Genus Mycopalsma & ureaplasma o Parts external to cell wall = flagella; pili; capsule REQUIREMENTS FOR BACTERIAL GROWTH • • • Endospores/spores - resistant structures that enables the bacteria to withstand injurious conditions o Aka spores; could be round to oval o Location - central, terminal, or subterminal o Spore formers - Genus Bacillus (aerobes) and Clostridium (anaerobes) ▪ Clostridium tetani - binds with iron o Best way to destroy spores - autoclaving (means to sterilize); 121 psi (pound per square inch) o Component of spores: Calcium dipicolinate/dipicolinic acid o To demonstrate spores: Schaeffer and Fulton - Green (malachite green) o Clostridium tetani - terminal swollen spores (drumstick, lollipop) ▪ Aka Drumstick or Lollipop bacillus o Bacillus anthracis - central spores ▪ Suspected to be used biological weapon o Clostridium botulinum - subterminal ▪ Most potent form of toxin • • • • • • Inclusions/Metachromatic granules o Much granules - Mycobacterium spp. o Babes Ernst granules - Corynebacterium diptheriae Nucleoid - contains the genetic materials Plasmid - small DNA molecule that carries a resistant gene confers resistance to antibiotics Ribosome Plasma membrane • • Nutritional Requirements o Phototrophic — uses light as energy source ▪ Convert chlorophyl to bacterial nutrition o Chemotrophic- uses chemical compounds as energy source ▪ Autotrophic/Lithotroph - uses inorganic compounds as carbon source ▪ Heterotrophic/Organotroph - uses organic compounds (glucose) as carbon source ▪ Some may require CO2 as nutrient Oxygen Requirements o Obligate aerobe — require oxygen for growth ▪ Ex: Mycobacterium, Pseudomonas and Bacillus o Obligate anaerobe — cannot grow in the presence of oxygen ▪ Ex: Bacteroides and Clostridium ▪ Gaspak Jar - to ensure anaerobiasis o Facultative anaerobe — can live with presence or absence of oxygen ▪ Ex. Staphylococcus and Streptococcus, E. coli, Salmonella o Aerotolerant — can grow in the presence of oxygen, but grow best in an anaerobic environment ▪ Ex. Lactobacillus ▪ Yakult - L. casei (normal intestinal flora) • Interacts with pathogenic bacteria o Microaerophilic — bacteria grow in a reduced level of oxygen. ▪ Ex: Helicobacter and Campylobacter ▪ H. pylori - causative agent of ulcer (60-70%) ▪ Campylobacter - come from lower animal which may infect human from peculiar domestication pH requirements o Neutrophile - Most bacteria grow well at 7.0 to 7.5 o Acidophile — grows at an acidic pH = pH 3.0 (Lactobacillus) ▪ L. acidophilus - maintain acidic environment of vagina to prevent growth of pathogenic bacteria o Alkalophile/basophile — grows at an alkaline pH = pH 10.5 (Vibrio) ▪ Can survive in high concentrations of salt solutions (crustaceans) Temperature o Psychrophilic — 0-20 degC o Mesophilic — 20-45 degC ▪ Most pathogenic bacterias o Thermophilic — 50-125 degC o Thermoduric — not growing well in the presence at high temperature but can withstand exposure to high temperature ▪ Sulfur-containing bacteria Salt, Carbon Dioxide Requirements o Capnophilic- require concentrations of 5% to 10% CO2 for optimal growth o Ex: Streptococcus and Neisseria - Candle jar o Halophilic — High salt concentration Ex: Vibrio (7.5%) ▪ Vibrio - requires TCBS media ▪ Most bacteria don’t survive in high salt conc. o Microaerophilic & Capnophilic - Campylobacter spp. o o o o • • STAGES OF BACTERIAL GROWTH • • • • Lag Phase/Adjustment Phase/Adaptation phase o Initial phase: no cell division yet, growth rate is 0%; increase in size o Detoxifying medium o Turning on enzymes to utilize medium o Number of cells at beginning equal number of cells at the end of lag phase Log Phase/Exponential Phase o Logarithmic phase o Rapid exponential growth; cell division starts; growth rate starts to increase o Bacteria become susceptible to antibiotics Stationary Phase/Plateau o Plateau - balance between dead cells and living cells o Nutrients are used up o Toxic products accumulate o Sporulation begins to occur - spores are produced o New cells equal the number of dying cells Death phase/Stage of decline o Characterized by complete cessation of bacterial growth o Death of cells • • Cocci - round Bacilli - elongated Spirilla - spiral Others: coccobacilli, filamentous ▪ Coccobacilli - round & elongated ▪ Filamentous - growth similar to fungi o True motility is due to flagella Arrangement - singly, pairs, chains, tetrads Motility - motile or non-motile o Brownian Movement - movement of non-motile organism caused by the movement of molecules surrounding them; this is not true motility Staining characteristics o Gram Positive or Gram negative o Acid Fast or Non-Acid Fast Cultural characteristics (Use of Selective media & biochemical tests) o Salmonella Shigella (SS) Agar ▪ Salmonella - has black central spot STAINING • Process of artificially coloring the organism with dyes/stains STAINING TECHNIQUES • • Simple Staining - Methylene Blue o Uses only 1 dye, color of the dye is the resulting color Indirect/Relief/Negative - India ink, Nigrosin o Background and not the organism is stained; bacteria will appear colorless o Used for detecting fungal infection in brain o India ink - more commonly used Special Staining - staining technique used to demonstrate special features of the cell o Capsular stains - Hiss, Anthony’s o Spore stains - Dorner’s, Schaeffer and Fulton, Wirtz Conklin ▪ Schaeffer and Fulton - spores will appear green ▪ Malachite green - primary dye ▪ Counterstain - safranin o Flagellar stain - Gray’s, Firsher & Conn, Leifson o Metachromatic granules - Albert’s, Neisser, Ljubinsky, Ponder, Methylene Blue Smear preparation - heat fixation • o Calibrated loop can be used for both liquid & solid o Distilled water is used on slide o Allow slide to warm up but don’t boil to evaporate water Differential Staining - gram stain and acid fast stain • • LESSON 2 - TECHNIQUES IN BACTERIAL IDENTIFICATION BACTERIAL IDENTIFICATION • • • Manual (reference and accurate method) o Labor-intensive Semi-automated thru API (Analytical Profile Index) Automated thru VITEK, Maldi Tof, o VITEK - automation program for bacterial identification and susceptibility MANUAL IDENTIFICATION • • Size Shape - cocci, bacilli, spirilla GRAM STAINING • • Primary Dye - crystal violet Mordant - Gram’s Iodine o Improves binding of dye to peptidoglycan layer of gram (+) • • o Decolorizer - acetone, alcohol or alcohol acetone mixture o Thick peptidoglycan - retains violet pigment o Thin peptidoglycan - become colorless Counterstain - safranin o Thin peptidoglycan layer absorb pink pigment o o Higher forms of organisms like Actinomyces, Streptomyces, yeast and molds are gram (+) ▪ Actinomyces - presented as fungi ▪ Streptomyces - source of streptomycin (potent antibiotic) All Spiral organisms are reported as Gram (-) Not gram stained are Rickettsiae; Chlamydia, Mycoplasma, Ureaplasma (wall less) ACID FAST STAINING • • • • • VIAS o Crystal Violet (1m) → Iodine (1m) → Alcohol wash (30s) → Safranin (1m) Procedure: o Apply crystal violet to the specimen and let sit for 1 minute. o Wash the slide with distilled/tap water. o Flood the specimen with mordant (iodine) and let sit for 1 minute. o Wash the slide with distilled/tap water. o Flood the specimen with decolorizer (alcohol) and let sit for 30 seconds. o Wash the slide with distilled/tap water. o Add the counterstain (safranin) with slide and let sit for 1 minute. o Wash the slide with distilled/tap water. Rules in Gram Staining o All are Gram Positive Cocci ▪ Except Neisseria, Veillonella, Branhamella, Moraxella (gram -) ▪ Neisseria - medically important pathogen • N. gonorrhea - gonorrhea • N. meningitidis - meningococcemia ▪ Moraxella cataralis - rare ▪ Veillonella, Branhamella - normal flora o All are Gram Negative Bacilli ▪ Except Mycobacterium, Clostridium, Corynebacterium, Bacillus, Erysipelothrix, Listeria, and Lactobacillus (gram +) ▪ L. doderlein bacilli - L. acidophilus in vagina ▪ Boas Opplers bacilli - L. acidophilus in stomach • • Acid Fast Organisms (AFO) - organisms that are difficult to stain but once stained, they are difficult to decolorize because of Mycolic acid or hydroxymethoxy acid o Acid Fast Bacilli (AFB) Ways to Facilitate Acid Fast Staining o Steaming process (heat fix) o Increasing concentration of phenol and basic fuchsin ▪ Facilitative staining o Prolonging contact pf stain with the material o Addition of wetting agent (TERGITOL) prior to the stain solution Two Most Common Methods of Acid Fast Staining o Ziehl Neelsen Method - hot method; best method for direct sputum smear microscopy (DSSM) ▪ Standard - 3 specimen o Kinyoun’s Method - cold method; best stain for AFO in tissues Procedure o Gram Staining = VIAS o Acid Fast Staining = mnemonic = CAM ▪ Ziehl Neelsen ▪ Kinyoun’s method o Primary dye: carbol fuchsin o Mordant: help carbol fuchsin penetrate wall ▪ Ziehl neelsen= steam/heat ▪ Kinyoun’s = wetting agent i.e. tergitol o Decolorizer: 3% acid alcohol (HCl + ethanol) o Counterstain: methylene blue (if not available, we use MALACHITE GREEN) o AFO = will take the color of the primary dye ▪ red against a blue background o NAFO = will take the color of the counterstain = blue/ green o o o o o o Primary dye - Carbol Fuchsin Mordant - will depend on the method Decolorizer - 3% acid alcohol (HCl+ ETOH) ; Counterstain - Methylene Blue / Malachite green Acid Fast Organisms - Red against blue background Non-AFO - Blue or green • Other Methods of Acid Fast Staining o Pappenheim’s ▪ To differentiate Mycobacterium smegmatis (blue) from M. tuberculosis (red) o Baumgartens ▪ To differentiate M. leprae (red) from M. tuberculosis (blue) o Fite Faraco’s - M. leprae (uses hematoxylin as counterstain) o Fluorochrome Staining - most sensitive method of AF staining ▪ Uses Auramine-Rhodamine dye - primary dye ▪ No mordant ▪ Uses 0.5% acid alcohol - decolorizer ▪ 0.5% Potassium permanganate counterstain/quenching agent ▪ Result: AFO will appear yellow black against black background CULTURE MEDIA • • • Culture media - medium that contains all nutritional requirements to support bacterial growth Culture - organisms which have grow on media Types as to Physical State o Liquid (broth) - no solidifying agent; 0% agar ▪ ie. Trypticase Soy Broth, Alkaline peptone water, Brain Heart Infusion o Semi-solid - 0.5-1% agar ▪ ie. SIM media (for motility) o Solid - agar ▪ Solid Liquefiable - EMB, MacConkey, Mannitol, Salt Agar • EMB - ethylene methylene blue agar ▪ Solid Non-Liquefiable - Rice Medium o Biphasic - both solid and liquid ▪ Ie. - Castaneda’s HBT • Types as to Use/Purpose o General purpose/general isolation media - contains only the necessary nutrition to support bacterial growth, for routine cultivation of bacteria ▪ Nutrient agar/nutrient broth ▪ Agar - solid ▪ Broth - liquid** o Enrichment media - to enhance bacterial growth ▪ Selenite broth for Salmonella, Shigella growth ▪ Tetrathionate broth - Salmonella & Proteus o Enriched media - used to support growth of fastidious organisms ▪ Fastidious organism - requires specific media ▪ Sheep blood agar (BA) - Streptococci ▪ Horse BA - Haemophilus (infant meningitis) ▪ Human BA - Gardnerella vaginalis (vaginal inf) ▪ Chocolate agar plate (CAP) - Neisseria ▪ Priority for collection in making BA 1. Sheep blood - no contaminant 2. Horse blood 3. Human blood o Selective media - promotes the growth of the desired organism while inhibiting the growth of others, contains inhibitors (antibiotics) ▪ Cystine tellurite Blood agar (CTBA) - C. diphtheriae (Potassium tellurite) ▪ Lowenstein Jensen Media - M. tuberculosis (Malachite green) ▪ Thayer Martine Media - Neisseria spp. (antibiotics) ▪ MacConkey agar - Gram negative bacilli (crystal violet) • o o o • Mac Conkey agar – Lactose fermenters: Pink-purple colonies • While Non-Lactose fermenters - Colorlesspink colonies; pathogenic bacteria Differential media - used to differentiate organisms growing together ▪ Ethylene methylene blue (EMB) - to differentiate gram negative bacilli as to LF/NLF; uses stool sample ▪ Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) - to differentiate Vibrio as to Sucrose or Nonsucrose fermenters Selective and Differential media ▪ Ie. EMB, TCBS, MacConkey Transport media - to maintain viability of organisms during specimen transport ▪ JEMBEC - for Neisseria ▪ Cary Blair - for stool pathogens like Vibrio ▪ STUART’s - Viral transport media o o o Media for Antibiotic Susceptibility Testing Mueller Hinton Agar Genus ▪ Staphylococcus ▪ Neisseria ▪ Corynebacterium ▪ Mycobacterium Species: ▪ Staphylococcus aureus ▪ Neisseria meningitidis ▪ Corynebacterium diphtheriae ▪ Mycobacterium tuberculosis