Microbiology Week 1 PDF

INTRODUCTION TO MICROBIOLOGY Professor Hannah Reilly, MS, MLS, PA (ASCP) MICROBES  MICROSCOPE The inventor of the first microscope is unknown Estimated invention was in the early 1600’s Early microscope was two lenses in three sliding tubes Antonie van Leeuwenhoek and Ro...

INTRODUCTION TO MICROBIOLOGY Professor Hannah Reilly, MS, MLS, PA (ASCP) MICROBES  MICROSCOPE The inventor of the first microscope is unknown Estimated invention was in the early 1600’s Early microscope was two lenses in three sliding tubes Antonie van Leeuwenhoek and Robert Hooke observed the first microorganisms From there lots of theories of life emerged!  MICROBIOME Humans harbor a complex ecosystem of normal flora Play an important role in our health Form on the skin surface, nose, mouth, intestinal and genitourinary tract Most of these commensal organisms coexist happily with their human hosts and are not pathogenic Disruption of our normal flora can lead to disease GENERAL CONCEPTS The term pathogenic refers to the ability of microbes to cause disease Healthy individuals have natural and physical barriers to prevent disease Immune system Intact skin, normal flora, enzymes, secretions and Ph levels  MOST INFECTIOUS DISEASES ARE CAUSED BY …. Pathogenic, non-commensal organisms, which exhibit a wide range of virulence Highly infectious microbes produce disease in a high fraction of healthy individuals, sometimes at “doses” of only a few organisms. Minimally pathogenic microbes, require large exposures and major breaches of host defenses to cause disease Microbes can enter the host by breaching epithelial surfaces, inhalation, ingestion, or sexual transmission  SKIN Most skin infections are initiated by mechanical injury of the epidermis Minor trauma Large wounds Burns Pressure ulcers Needle sticks Bites  GASTROINTESTINAL Most gastrointestinal pathogens are transmitted by food or drink contaminated with fecal material When hygiene fails, diarrheal disease becomes rampant Acidic gastric secretions are particularly important since they are highly effective at killing certain organisms  RESPIRATORY TRACT A plethora of microorganisms, including viruses, bacteria, and fungi, are inhaled daily via dust and aerosol particles Innate barriers prevent diseease: Large particles are trapped in the mucociliary blanket that cilia move particles back up of the throat swallowed and cleared Particles smaller than 5 microns are carried into the alveoli, where they are phagocytosed by resident alveolar macrophages or by neutrophils  RESPIRATORY TRACT Failure in Defenses: Some respiratory viruses attach to and enter epithelial cells in the lower respiratory tract and pharynx ( influenza) Some release toxins that enhance their ability to establish an infection by impairing ciliary activity Another important mechanism, primary resistance to killing following phagocytosis. A classic example is Mycobacterium tuberculosis, which gains a foothold in alveoli by surviving within the phagolysosomes of macrophages  UROGENITAL TRACT Urinary tract pathogens almost always gain access via the urethra and must be able to adhere to urothelium to avoid being washed away Women have more than 10 times as many urinary tract infections as men because the distance between the urinary bladder and skin Obstruction of urinary flow or reflux of urine compromises normal defenses and increases susceptibility to urinary tract infections Minor cervical trauma may expose immature proliferating epithelial cells that are susceptible to infection by HPV or STDS Defenses: Vagina is protected from pathogens by lactobacilli (low pH=suppressed growth)  PATHOGEN SPREAD Pathogens can spread within the body in several ways: Some extracellular pathogens secrete enzymes that break down tissues, allowing the organisms to advance virtually unimpeded Lymphatics to regional lymph nodes and the blood, potentially leading to bacteremia and spread to distant organs Peripheral nerves and then traveling intracellularly along axons Most common and efficient mode of microbial dissemination is through the bloodstream  RELEASE FROM THE BODY Infectious microbes use a variety of “exit strategies” to ensure their transmission from one host to the next Depending on the location of of infection, release may be accomplished by: Skin shedding Coughing and sneezing Voiding of urine or feces During sexual contact Through insect vectors Some pathogens are released for only brief periods of time or periodically during disease flares  TRANSMISSION Most pathogens are transmitted from person to person by respiratory, fecal-oral, or sexual routes: Viruses and bacteria spread by the respiratory route are infectious only when lesions are open to the airways Most enteric pathogens are spread by the fecal-oral route, that is, by ingestion of stool-contaminated water or food Sexual transmission often entails prolonged intimate or mucosal contact and is responsible for spread of a wide variety of pathogens, including viruses  HOST DEFENSES AGAINST INFECTION The outcome of infection is determined by: Virulence of the microbe Nature of the host immune response, which may either eliminate the infection or, in some cases, exacerbate or even be the principal cause of tissue damage  IMMUNE EVASION BY MICROBES Most pathogenic microbes have developed one or more strategies that allow them to evade host defenses Some salient examples of immune evasion by microbes are as follows: Antigenic variation Inactivating antibodies or complement Resisting Phagocytosis (producing a capsule) Suppressing the host adaptive immune response (MHC expression or interfering with cytokines) Establishing latency (viral silent state)  BACTERIA Professor Hannah Reilly MS, MLS, PA (ASCP)  PROKARYOTIC ORGANISMS Simple unicellular organisms Single, circular DNA compressed into nucleoid Lack nucleus and membrane organelles Metabolism occurs in the cytoplasm  CELL WALL Provides mechanical stability Allows for the exchange of nutrients and waste Peptidoglycan forms the basis of cell wall Component of almost all bacterial walls (exception: mycoplasmas) Thickness of cell wall determines Gram staining  PLASMA MEMBRANE Analogous to prokaryotic membranes Composed of proteins and phospholipids Unlike eukaryotic organisms, lacks sterols Envelops the cytoplasm of cells Transport of matter across cell membrane via simple and facilitated diffusion  FLAGELLA AND PILI Flagella: Approximately 50% of prokaryotes move with help of flagella Consist of the protein, flagellin, and are not covered by cell membrane Pili (fimbriae): Structurally similar to flagella, but much smaller Aid in adherence to host/other bacteria = virulence factor Used to exchange genetic information during conjugation  CELLULAR STRUCTURES Nucleoid: Functional equivalent of prokaryotic nucleus Lacks membrane Contains genetic material: Usually, 1 chromosome May have additional plasmids Inclusions and vesicles: Gas vesicles allow photosynthetic bacteria to float in water Ribosomes for photosynthesis and chemosynthesis Storage granules (e.g., sulfur, phosphate, calcium, glycogen)  REPRODUCTION Reproduce via binary fission  EXCHANGE OF GENETIC MATERIAL Bacteria can exchange genetic material: Can integrate foreign DNA into their genome Can recombine in the existing gene pool Can transfer genetic traits between each other: Conjugation: parasexual transfer through contact via pili Transduction: through bacteriophages (viruses that infect bacteria) Transformation: introduction of free, isolated, foreign DNA into genome  MECHANISMS OF BACTERIAL INJURY Bacterial Virulence Mobile genetic elements Bacterial Adherence Bacterial Toxins  BACTERIAL VIRULENCE Bacteria can be virulent or opportunistic pathogens Virulent pathogens cause disease based on their virulence factors Virulence factors evade the immune system and promote the survival of bacteria Bacterial damage to host tissues depends on the ability of the bacteria to adhere to host cells, to invade cells and tissues, or to deliver toxins Pathogenic bacteria have virulence genes that encode proteins that confer these properties Opportunistic pathogens take advantage of pre-existing conditions such as immunosuppression  MOBILE GENETIC ELEMENTS Plasmids and bacteriophages can transmit functionally important genes to bacteria, including genes that influence pathogenicity and drug resistance Genes for acquired antibiotic resistance traits are more frequently found on plasmids For example, a plasmid with genes for vancomycin resistance can spread not only between species of Enterococcus, but also to more distantly related (and virulent) S. aureus Mechanism Virulence factors Function Colonization Teichoic acid (primarily in gram- Allows attachment to and positive organisms) invasion of host cell Adhesins surfaces Biofilms Biofilms protect from Flagella antibiotic penetrance Motility Avoiding the immune Capsule Creates physical barrier system IgA protease blocking opsonization and Protein A phagocytosis Bacterial nutrition Siderophores Chelate iron Iron absorption Antigenic variation Pili Camouflage of surface Capsule and flagella expression molecular markers that Antigenic drift allow evasion of the immune system Intracellular survival Inhibition of phagosome- Prevents intracellular lysosome fusion destruction of the bacteria Exiting phagosomes before fusion occurs Invasins Type III secretion Injectisome Allows bacteria to inject system toxins into host cells Inflammatory Bacteria-specific antibodies Mimic host cells response Immune complexes Delayed hypersensitivity Peptidoglycan and teichoic acid reactions  TOXINS Endotoxins: Generated during the breakdown of bacterial cell wall when bacteria die Activate host complement and coagulation cascades Cause septic shock Non-disease-specific symptoms: Fever, pain, shock, fatigue & discomfort Exotoxins: Produced and secreted Can result in severe, disease-specific symptoms 3 main categories: Enterotoxins, neurotoxins & cytotoxins  CLASSIFICATION OF BACTERIA Shape Coccus, bacillus, spiral Gram Stain Gram-positive and gram-negative Biochemical and Growth Aerobic and anaerobic Spore formation Biochemical profile Growth and metabolic properties Antigenic Structure Antigens in cell body, capsule, flagella  MODERN CLASSIFICATION Taxonomy now based on genomic sequence particularly 16S ribosomal RNA Also based on array of proteins and peptides Tests are automated using MALDI-TOF-MS (matrix assisted laser desorption/ionization time of flight mass spectrometry)  MACROSCOPIC DISTINCTION Initial distinction of bacteria can be made by growth characteristics on different media Growth characteristics: Ability to resist antibiotics Metabolize sugars Lyse erythrocytes Hydrolyze lipids Colony characteristics: Color, size, shape, and smell  MEDIA Artificial media is designed to meet the growth needs for microorganisms Types: General purpose media Enriched media Selective media Differential media  SHEEP BLOOD AGAR An enriched nutritive media Allows growth of many bacteria Is a tryptic soy agar base to which 5% sheep blood has been added  MACCONKEY AGAR (MAC) Selective and differential Gram negative rods grow on mac Lactose and non-lactose fermenters are differentiated due to lactose in the agar and neutral red as an indicator. Lf: form acids, pink or red on agar NLF: Colorless on agar  COLUMBIA CNA AGAR Selective and differential Grows gram positive organisms Contains colistin and naladixic acid which inhibits the growth of gram negative rods Also contains blood which allows differentiation based on hemolysis patterns  CHOCOL ATE AGAR Non-selective media for the isolation and cultivation of fastidious microorganisms Especially Neisseria and Haemophilus species, from a variety of clinical specimens  QUESTIONS: 1. What kind of media is blood agar? 2. What is blood agar enriched with? 3. What are the 3 hemolysis patterns found on blood agar? 4. Is macconkey agar selective or differential? Why? 5. What color are lactose fermenters and non-lactose fermenters on mac agar? 6. What kind of media is Cna? 7. What grows on cna media? 8. What does cna media contain to inhibit the growth of gram negative rods? 9. What type of organisms grow best on chocolate agar?  ENRICHMENT BROTH Broth used to enhance bacterial growth Subculture to solid media for isolation of bacteria Example: GN and selenite broths to enhance recovery of Salmonella and Shigella from stool  ANEROBIC MEDIA/PRE-REDUCED MEDIA Oxygen is removed from media Allows growth of anaerobic bacteria Example: thioglycollate broth (thio) Boil prior to use to drive off oxygen Store at room temp in the dark for a week to decrease O2 reabsorption Bottom of broth has no oxygen, anaerobes can be recovered  MAINTENANCE MEDIA Supports bacterial cells but discourages log phase growth Used to maintain stock cultures in lab Examples: Nutrient agar Tryptic soy agar  TRANSPORT MEDIA Used to keep bacteria alive during transport from patient to plating in lab Does not promote growth Examples Culturette swabs Cary-Blair media for stool bacteria  DIFFERENTIAL BIOCHEMICAL TESTS TSI and KIA ONPG Decarboxylases Nitrate reduction Indole Motility Voges Proskauer PAD Citrate Urease  TSI TEST Valuable tool for identifying enterbacteriaceae & other fermenting organisms Tube gives info about the ability of the organism to: Ferment glucose, lactose, and sucrose Produce h2s Produce co2 gas  TSI Nutritionally rich and contains no inhibitors All but the most fastidious organisms will grow on it Media contains lactose and sucrose, each in a 10:1 ratio to glucose Phenol red is incorporated as the ph indicator Turns yellow when acids are produced from the sugar fermentation Ferrous sulfate is used as the h2s production indicator The slant portion, exposed to oxygen, is aerobic The lower portion, called butt or deep, is anaerobic  HOW TO INOCULATE TSI SLANT: 1 2 3 Inoculate the slant by Stab the colony to the Incubate the slant picking up a single bottom of the deep, (with a loose cap) for colony with a long, then pull up, and 24 hours in 35 °C straight wire. streak over the slant. incubator.  INTERPRETING RESULTS: A = Acid (yellow) K = Alkaline (red) a/a: Glucose and lactose/sucrose fermented. (E coli and Klesiella sp) K/a: Glucose fermented; lactose/sucrose not (shigella and serratia) K/a h2s+: Glucose fermented, lactose was not, h2s is prodced. (salmonella and Proteus) K/k: No carbohydrate fermentation. Is not in the enerbacteriaceae family (pseudomonas sp)  KLIGER IRON AGAR (KIA) SLANT Similar to TSI slant Difference is lacks sucrose  QUESTIONS: 1. What does tsi mean? 2. What information does the tsi provide to the tech? 3. What is the ratio of lactose and sucrose to glucose? 4. Is the slant portion aerobic or anaerobic? What about the butt? 5. What color result do you get for acid vs. alkaline? 6. How does kia differ from tsi?  D E C AR BOXY L AT ION OF LYS IN E , AR GIN IN E & OR N IT H I N E Used to determine if the bacterium possesses the specific enzymes necessary to decarboxylate specie amino acids Forms an amine end product of co2 Bromcresol purple is the indicator used Oil is used to make an anaerobic enviroment, activateing the proper enzymes  INOCUL ATION OF DECARBOXYL ATION TUBE: Inoculate 1-2 colonies from a fresh culture sub with a loop mixing throughout the medium Overlay with 1ml of sterile mineral oil Incubate with caps tightened at 35°C for 18-24 hours If negative re-incubate another 24 hours Positive = purple Negative = yellow  INDOLE TEST Bacteria are grown on media containing tryptophan If bacteria possesses the enzyme tryptophanase: The tryptophan is broken down into indole and other products Types of tests: Kovacs or ehrlichs indole test: Uses the addition of the reagent p-diemethylaminobenzaldhyde to detect indole Positive result: red Spot indole test: Rapid test preformed on filter paper or with a sterile swab Indole is detected with the reagent paradimethylcinnemaldehyde Positive result: blue  VOGES-PROSKAUER TEST Detects bacteria which produce acetylmethyl carbinol (acetoin) as a product of glucose fermentation Acetoin is detected in mrvp broth by observing a red color after adding 1 drop of 5% a-naphthol and 1 drop of 5% KOH Positive result: red Negative result: no change  CITRATE UTILIZATION Determines the ability of the bacterium to utilize sodium citrate as its sole carbon source. The medium contains citrate, various salts, and bromthymol blue as the ph indicator. Make sure that cap is loose when incubating. Positive result: blue Negative result: green  QUESTIONS: What indicator is used for the decarboxylase test? What needs to be added to the tubes to activate the enzymes and achieve an anaerobic enviroment? What color is a positive reaction? What tests can be used to determine indole production?  QUESTIONS CON’T……. What is a positive reaction for kovacs and ehrlichs indole vs spot indole? What is vp test used for? What color is a positive reaction for vp? What is the citrate test used for ? Should a citrate tube cap be tight or loose during incubation?  BIOCHEMICAL TESTS Urease test: Bacteria will hydrolyze urea to ammonia and co2 if it possesses the enzyme urease Incubate test for 24 hours in 35°c incubator Strong urea producers can become positive in 30 minutes to 4 hours of incubation Positive result: pink-red PAD test Determines if bacteria are capable of deaminating phenylalanine with the production of phenyl-pyruvic acid Incubate test overnight in 35°c Add 10% ferric chloride Positive result: green  HYDROGEN SULFIDE PRODUCTION (H2S) Test the ability of bacteria to form h2s from the enzymatic breakdown of sulfur-containing amino acids Various indicators are used Positive result: black Negative result: no change  SIM (SULFIDE, INDOLE, MOTILITY) MEDIA Designed to allow the detection of sulfide production, indole formation and motility Using isolated colonies from culture on solid media, inoculate the SIM Medium by stabbing the center of the medium to a depth of 1/2 inch Incubate the inoculated medium aerobically at 35ºC for 18-24 hours Observe for H2 S production and motility Once H2 S and motility reaction have been read and recorded, apply three drops of Kovacs Reagent to the surface of the medium Observe for the development of a pink to red color  BIOCHEMICAL TESTS ONPG test: Detects organisms which have the enzyme b-galactosidase Breaks down ONPG into galactose Citrobacter & enterobacter may appear lactose negative, but are onpg positive Salmonella and most shigella are onpg negative Positive result: yellow Nitrate reduction: Determines if bacteria can reduce nitrate Bacteria are grown overnight in nitrate broth, add sulfanilic acid and n,n-dimethyl-l-naphthylamine Positive = red color within 30 minutes Negative = no change—add a pinch of zinc dust: Following zinc dust: Positive = no color change Negative = red color  MOTILITY Some bacteria display motility: Need pure culture that is 18-24 hours old Use nonselective media: blood agar Mix organism in drop of normal saline on a glass slide Examine under 40x with reduced light  BROWNIAN MOTILITY Normal movement of particles in fluid NOT true motility Detected by all particles moving in same direction  QUESTIONS: What is the urease test used for? What color is a positive result? What are pad + bacteria capable of doing? What reagent is added to the pad test and what color is a positive test? What organisms are positive for pad?  MORE QUESTIONS: How old should a culture be that is used for motility? What type of media should be used for motility? What objective so we look at motility? What is Brownian motion? What is sim media? What does onpg stand for?  MICROSCOPIC EXAMINATION Size, shape, configuration of organisms (cocci, rods, curved) Ability to retain gram stain +/-  MORPHOLOGY Bacteria display a wide diversity of shapes and sizes. Most common morphologies are cocci and bacilli, which can occur in many arrangements Arrangement: Pairs Tetrads Clusters Chains Palisades  SHAPE & CONFIGURATION Coccus (spherical) Bacillus (rod shaped) Clusters: Staphylococci Square ends: Bacillus anthracis Chains: Streptococci Rounded ends: Mycobacterium Pairs: Diplococci tuberculosis Club shaped: Corynebacterium Kidney bean-shaped, in pairs: Neisseriae Fusiform: Fusobacterium Spiral organisms Comma shaped: Vibrio Tightly coiled: Treponema pallidum Relaxed coil: Borrelia  GRAM STAIN Used to differentiate bacteria based on the differences in the constituents of their cell walls Both gram-positive and gram-negative bacterial cell walls contain peptidoglycan layers The layer in gram-negative bacteria is much thinner Gram-negative bacteria make up for this deficit by having another membrane layer outside the peptidoglycan layer  GRAM STAIN Process: Bacteria treated with crystal violet dye Stain is washed with alcohol Cells are counterstained with red safranin stain Staining of gram-positive bacteria: Cell wall contains a thick layer of peptidoglycan Retain crystal violet stain and are not affected by safranin counterstain Appear purple-blue Staining of gram-negative bacteria: Cell wall has a thin layer of peptidoglycan Do not retain crystal violet stain, but retain safranin counterstain Appear pink-red  GRAM NEGATIVE BACTERIA Gram negative cocci Neisseria Gram negative bacilli Enterobacteriaceae Vibrio Campylobacter Helicobacter Aeromonas  GRAM POSITIVE BACTERIA Gram positive cocci Staphylococcus Streptococcus Enterococcus Gram positive bacilli Bacillus Clostridium Listeria Nocardia  Enterobacteriaceae ferment glucose & are oxidase negative  MAIN CONCEPTS Basic characteristics of bacteria Virulence factors Media and biochemical tests Gram stain