Bacteriology Complete Notes PDF

lOMoARcPSD|47212494 Bacteriology Complete Notes Med. Lab. Science Research (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Ada Mercado (aanjme...

lOMoARcPSD|47212494 Bacteriology Complete Notes Med. Lab. Science Research (Our Lady of Fatima University) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 COMPILED NOTES FOR MICROBIOLOGY Anton van Leeuwenhoek MAHONE 6th ED  Was the first to experiment with microbes: “wee beasties” “animalcules” INTRODUCTION  Father of protozoology and bacteriology Microbiology OVERVIEW  Study of the living organisms that are too small  Bacteria to be visible with the naked eye. - Unicellular organisms that lack nuclear  These include bacteria, archaea, viruses, fungi, membrane and true nucleus. prions, protozoa, and algae (collectively referred - Prokaryotes to as microbes).  Parasites  Organisms that can only be observed with the - Eukaryotic Unicellular Ex. Protozoa use of various types of microscopes. - Multicellular Ex. Tapeworm Two major categories - Categorized by locomotive structures: Flagella (Lat. whiplike), pseudopodia (Gk.  Acellular Infectious Agents (Prions, Viruses) false feet), or cilia (Lat. eyelash)  Cellular microorganisms  Fungi (Prokaryotes: Archaea, Bacteria) - Heterotrophic eukaryotes (Eukaryotes: Algae, Fungi, Protozoa) - Yeast - unicellular fungi, asexual Microbes reproduction (incubator/human temperature) - True yeast do not form hyphae or mycelia  Pathogens - disease-causing microorganisms - Mold - filamentous form, reproduce sexually  Nonpathogens - microbes that do not cause and asexually (room temperature) disease  Virus Importance - Smallest infectious particle - Neither prokaryotic nor eukaryotic  We have microbes normally living on and in our - DNA or RNA, not both bodies (Normal or indigenous microflora) - Acellular surrounded by protein coat  Opportunistic pathogens (Opportunists) Do not - Obligate intracellular parasite cause disease under ordinary conditions, but have the potential to cause disease should the CLASSIFICATION/TAXONOMY opportunity present itself.  Taxonomy -Gk. taxes: arrangement;  Microbes are essential for life on this planet Ex. Some microbes produce oxygen by the  nomos: law process known as photosynthesis  Orderly classification and grouping of organisms (Cyanobacteria) into taxa  Many microbes are involved in decomposition  Based on similarities and differences in (Saprophytes -organisms that lives on dead or genotype and phenotype decaying organic matter) - Genotypic: Ex. Sequencing of DNA and  Many microbes are essential in various food and RNA; DNA base composition ratio beverage industries (Biotechnology - the use of - Phenotypic: Ex. Colony morphology, living organisms or their derivatives to make or bacterial size, arrangement, staining modify useful products or processes) characteristics, nutritional requirements,  Some bacteria and fungi produce antibiotics that biochemical characteristics, susceptibility or are used to treat patients with infectious resistance to antibiotics diseases (Antibiotic - a substance produced by a  Species - the proper word for the name of the microbe that is effective in killing or inhibiting the species is an epithet growth of other microbes) Classification by Cellular Type  To understand the processes involved in diseases  Archaea - Microorganisms that grow under Two categories of diseases extreme environments  Infectious disease - a pathogen colonizes the - Cell walls lack peptidoglycan body and subsequently causes disease - Extremophiles  Microbial intoxication - a person ingests a toxin Prokaryotic cell structure that has been produced by a microbe Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 1. Cytoplasmic structures - Capsule: usually made of Bacteria do not contain a membrane polysaccharide polymers act as bound nucleus virulence factor (evade phagocytosis) - Genome consists of a single circular - Cell appendages chromosome a. Flagella: exterior protein filaments, - Bacterial ribosomes (consists of RNA cause bacteria to be motile and protein) are found free in the b. Pili: AKA conjugation pili nonmotile, cytoplasm and attached to the long, hollow protein tubes that cytoplasmic membrane. connect two bacterial cells and 2. Cell envelope structures mediate DNA exchange - Plasma membrane (Cell membrane) - c. Fimbriae: nonflagellar, sticky, phospholipid bilayer with embedded proteinaceous, hairlike appendages proteins that envelop the cytoplasm. that adhere some bacterial cells to a. Prokaryotic plasma membrane is one another made of phospholipid and proteins but does not contain sterols (except for mycoplasma) BACTERIAL MORPHOLOGY b. Plasma membrane acts as an osmotic barrier and the location of electron transport chain - Cell wall - rigid structure that maintains the shape of the cell and prevents bursting of the cell due to high osmotic pressure inside. - Mycobacteria have a modified gram positive cell wall called acid fast cell wall - ***Mycoplasma have no cell wall a. Gram positive cell wall – Composed of very thick protective peptidoglycan (murein) layer as principal component. Peptidoglycan layer, teichoic acid and lipoteichoic acid unique b. Gram negative cell wall - Composed of two layers: inner peptidoglycan layer (thinner than Gram +) and an outer membrane which contains Microbial growth and nutrition proteins, phospholipids, and lipopolysaccharides (LPS) Three Major nutritional needs for growth: LPS -contains three regions: antigenic O-specific polysaccharide, 1. A source of carbon (for making cellular core polysaccharide, and inner lipid constituents). A (endotoxin) 2. A source of nitrogen (for making proteins) Periplasmic space - between the 3. A source of energy (ATP) outer and inner membrane absent in Nutritional Requirements for Growth Gram positive bacteria. c. Acid Fast cell wall - Mycobacterium 1. Autotrophs (lithotrophs) and Nocardia have a gram positive - Carbon dioxide as the sole source of cell wall structure but contain a carbon waxy layer of glycolipids and fatty - Obtain energy either photosynthetically acids (mycolic acid) bound to the (phototrophs) or by oxidation of exterior of the cell wall. inorganic compounds d. Absence of cell wall - Mycoplasma (chemolithotrophs) and Ureaplasma lack a cell wall and 2. Heterotrophs contain sterols in their cell - Require more complex substances for membranes growth. 3. Surface polymers Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - Organic source of carbon, such as 4. Bacterial Growth - The time required for one cell glucose to divide into two cells is called the generation - Obtain energy by oxidizing or fermenting time or doubling time organic substances 5. Growth curve - all bacteria that inhabit the human body a. lag phase - bacteria are preparing to divide, b. log phase - bacteria numbers increase TYPES OF GROWTH MEDIA logarithmically c. stationary phase - nutrients are becoming 1. Minimal medium - contents are simple; not limited and the numbers of bacteria remain usually used in diagnostic microbiology constant laboratory d. death phase - the number of nonviable 2. Nutrient media - more complex; made up of bacterial cells exceeds the number of viable extracts of meat or soybeans (nutrient broth, cells trypticase soy broth)  Determination of cell numbers 3. Enriched media - growth medium that contains a. Direct counting under the microscope: used added growth factors (e.g. blood, vitamins, yeast to estimate the number of bacteria present extracts) in a specimen. 4. Selective media - Media containing additives b. Direct plate count: By growing dilutions of that inhibit the growth of some bacteria but allow broth cultures on agar plates, one can others to grow (e.g., MacConkey agar [MAC] determine the number of colony forming selective for gram-negatives while inhibiting units per milliliter (CFU/mL). gram positives. c. Density measurement: The density (referred 5. Differential media - Ingredients in media that to as cloudiness or turbidity) of a bacterial allow visualization of metabolic differences broth culture in log phase can be correlated between groups or species of bacteria. to CFU/mL of the culture MacConkey agar distinguishes between lactose fermenters (pink) and non-lactose fermenters BACTERIAL BIOCHEMISTRY AND METABOLISM (clear). 6. Transport medium - when a delay between Biochemical Pathways from Glucose to Pyruvic collection of the specimen and culturing the Glucose - starting carbohydrate for bacterial specimen is necessary. Stuart broth and Amies fermentations or oxidations and Cary-Blair transport media. Three major biochemical pathways bacteria use: Environmental Factors Influencing Growth 1. Embden- Meyerhof-Parnas (EMP) glycolytic 1. pH (most pathogenic bacteria =neutral pH) pathway 2. Temperature - The major pathway in conversion of a. Psychrophiles - cold temperatures (optimal glucose to pyruvate growth at 10° to 20° C). - Generates reducing power in the form of b. Mesophiles - moderate temperatures NADH2 (optimal growth at 20° to 40° C). c. Thermophiles - high temperatures (optimal - Generates energy in the form of ATP growth at 50° to 60° C). - Anaerobic; does not require oxygen 3. Gaseous composition of the atmosphere - Used by many bacteria, including all a. Obligate aerobes require oxygen for growth. members of Enterobacteriaceae b. Aerotolerant anaerobes (facultative aerobes) 2. Pentose phosphate pathway can survive in the presence of oxygen but - Alternative to EMP pathway do not use oxygen in metabolism. - Produces glyceraldehyde-3-phosphate, c. Obligate anaerobes cannot grow in the which can be converted to pyruvate presence of oxygen. - Generates NADPH d. Facultative anaerobes can grow either with - May be used to generate ATP (yield is or without oxygen. less than with the EMP pathway) e. Capnophilic organisms grow best when the - Used by heterolactic fermenting atmosphere is enriched with extra carbon bacteria, such as lactobacilli which lacks dioxide (5% to 10%). some of the enzymes required in the f. Microaerophilic - requires reduced oxygen to EMP pathway grow. 3. Entner-Doudoroff pathway - Converts glucose-6-phosphate (rather Terms than glucose) to pyruvate Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - Generates one NADPH per molecule of Genetic Elements and Alterations glucose but uses one ATP 1. Bacterial genome - Aerobic process - Bacterial chromosome - Consists of a Anaerobic Utilization of Pyruvic Acid (Fermentation) single, closed, circular piece of double- stranded DNA that is supercoiled in 1. Alcoholic fermentation: Major end product is order to fit inside the cell. ethanol. Pathway used by yeasts when they 2. Extrachromosomal elements ferment glucose to produce ethanol. - Plasmids - extra information on small 2. Homolactic fermentation: End product = lactic circular pieces of extrachromosomal, acid. All members of the Streptococcus genus double-stranded DNA and many members of the Lactobacillus genus - Genes that code for antibiotic resistance ferment pyruvate using this pathway. (and sometimes toxins or other 3. Heterolactic fermentation: mixed fermentation virulence factors) are often located on pathway, in addition to lactic acid, the end plasmids. products include carbon dioxide, alcohols, formic - Located in the cytoplasm of the cell and acid, and acetic acid. are self-replicating 4. Propionic acid fermentation: Propionic acid is - May also be passed (nonsexually) from the major end product of fermentations carried one bacterial species to another through out by Propionibacterium acnes conjugation. 5. Mixed acid fermentation: Escherichia, 3. Mobile Genetic Elements Salmonella, and Shigella within the - Jumping genes - certain pieces of DNA Enterobacteriaceae; end products—lactic, are mobile and may jump from one acetic, succinic, and formic acids. (MR) place in the chromosome to another 6. Butanediol fermentation: Klebsiella, place. Enterobacter, and Serratia within the - Insertion sequence (IS) element - when Enterobacteriaceae; end products are acetoin (acetyl methyl carbinol) and 2,3-butanediol. (VP) an IS element inserts itself into the 7. Butyric acid fermentation: Certain obligate middle of a gene, it disrupts and anaerobes, including many Clostridium species, inactivates the gene. This can result in Fusobacterium, and Eubacterium, produce loss of an observable characteristic. butyric acid - Transposons - Often carry antibiotic- resistance genes and are usually Aerobic Utilization of Pyruvic Acid (Oxidation) located in plasmids. - Mutations - Changes that occur in the 1. Krebs or TCA (tricarboxylic acid) cycle DNA code and often results in a change Pyruvate is oxidized in the coded protein or in the prevention Electrons donated by pyruvate are passed of its synthesis. It could be: a change in through an electron transport chain and used to one nucleotide base (a point mutation) generate energy in the form of ATP. This cycle that leads to a change in a single amino results in the production of acid and the acid within a protein insertions or evolution of carbon dioxide deletions in the genome that lead to Carbohydrate Utilization and Lactose Fermentation disruption of the gene. 1. Fermentation of sugar Mechanism of Gene Transfer - Usually detected by acid production and 1. Transformation - Free or “naked” DNA is taken a concomitant change of color resulting up by a competent bacterial cell. After uptake, from a pH indicator present in the the DNA may take one of three courses: culture medium. - it is integrated into existing bacterial - Microorganisms ability to ferment genetic material lactose - it is degraded, or - Lactose fermenters V. Lactose - If it is a compatible plasmid, it may nonfermenters replicate in the cytoplasm. - Lactose - disaccharide; glu+gal linked 2. Transduction - A phage injects DNA into the by galactoside bond bacterial cell. - Two steps involved: β-galactoside 3. Conjugation - An F+ cell connects with an F– cell permease and β-galactosidase via sex pili. DNA is then transferred from one cell BACTERIAL GENETICS to the other. Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 MICROSCOPE wall-deficient bacteria incapable of retaining dyes Optical instrument that is used to observe tiny objects, b. Auramine Rhodamine - binds often objects that cannot be seen at all with the unaided mycolic acid in cell wall of human eye mycobacteria; mycobacterial cells  Resolving power or resolution - limit as to what appear bright yellow or orange can be seen against a greenish background  Resolution - Extent to which detail in the c. Calcofluor white - binds cell walls of magnified object in maintained. fungi  Contrast - Make objects stand out from the 5. Transmission Electron Microscope (TEM) background - Specimen is viewed on a screen; allows 1. Simple microscope examination of cellular and viral - A microscope containing only one ultrastructure; reveals internal features magnifying lens of thin specimen - Magnifying glass (3-20x) 6. Scanning Electron Microscope (SEM) - Anton van Leeuwnehoek - used simple - Specimen is viewed on a screen; gives microscopes to observe many tiny the illusion of depth (3D); for examining objects, including bacteria and protozoa surface features of cells and viruses 2. Compound microscope STAINS - Microscope that contains more than one magnifying lens  Germ theory of disease - States that many - x1000 diseases are caused by the presence and actions of specific microorganism Louis Pasteur Types of Microscopes and Robert Koch  The staph – 1880 Staphylococcus is identified 1. Brightfield as a cause of wound infection by Scottish - Visible light is passed through the surgeon Sir Alexander Ogston. specimen and then through a series of  Gram stain (1884) Christian Gram lenses that reflect the light in a manner that results in the magnification of the Common stains organisms present in the specimen 2. Darkfield 1. Gram stain - Condenser does not allow light to pass - Most commonly used directly through the specimen but directs - Differential stain the light to hit the specimen at an - Gram positive (blue to purple) oblique angle. - Gram negative (pink) - Used to detect spirochetes - Four (4) sequential components (fixed 3. Phasecontrast with either heat or methanol) - Contrast enhancing technique a. Crystal violet -primary stain (1 - Beams of light pass through the minute) specimen and are partially deflected by b. Iodine -mordant or fixative (1 the different densities or thickness minute) (refractive index) of the microbial cells in c. Alcohol or alcohol acetone solution the specimen -decolorizer (quick on and rinse) - Allows observation of viable organisms damages thin lipid walls - Used to identify medical important fungi d. Safranin -counterstain (30 seconds) grown in culture **rinsing with water between each 4. Fluorescence step - Certain dyes (fluors or fluorochromes) - Gram positive bacteria with thick cell can be raised to a higher level after walls containing teichoic acid remain absorbing UV light (excitation). crystal violet-iodine complex dye after - Fluorescence: when dye molecules decolorization appear deep blue return to normal (lower energy state), - Gram negative Bacteria with thinner they release excess energy in the form walls containing LPS do not retain the of visible (fluorescent) light. dye a. Acridine orange - binds to nucleic acid (nonspecific); detection of cell Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - All cocci are Gram positive, except NVM Neisseria, Veillonela, Moraxella MICROBIAL FLORA (Branhamella) - All bacilli are Gram negative, except  Microbes that colonize the human body during Actinomyces, Atopobium, Bacillus, birth or shortly thereafter, remaining throughout Bifidobacterium, Clostridium, life Corynebacterium, Erysipelothrix,  Microorganisms that are commonly found on or Gardnerella, Listeria, Lactobacillus, in body sites of healthy persons are called Mycobacterium sp. (other than M. normal, usual, or indigenous flora. tuberculosis), Nocardia and Proprionibacterium Factors That Determine the Composition of the Usual 2. Acid fast stain Microbial Flora - Stains bacteria with high lipid and wax 1. Nutritional and Environmental Factors content in their cell walls 2. pH - Differential stain - Acid fast bacteria -red Composition of Microbial Flora at Different Body Sites - Non acid fast -blue 1. Usual Flora of the Skin - Acid fastness - mycolic acid renders the - Organisms concentrate the most in cells resistant to decolorizer areas that are moist such as the armpit, a. Carbol fuschin (red dye)-primary groin, and perineum. stain - The presence of skin bacteria inhibits b. Acidified alcohol -decolorizer the growth of more pathogenic bacterial c. Methylene blue -counterstain species, thus providing benefits to the ***Ziehl-Neelsen -uses heat (hot host. method) ***Kinyoun -uses detergent Mechanisms to prevent infection (cold method) a. mechanical separation of microorganisms 3. Acridine orange from the tissues, - Fluorochrome dye b. presence of fatty acids that inhibit many - Stains both G+ and G- bacteria, living or microorganisms dead c. excretion of lysozyme by sweat glands, - Binds nucleic acid of cell Fluoresce d. desquamation of the epithelium. bright orange (Fluorescent microscope) 2. Usual Flora of the Respiratory Tract 4. Calcofluor white - Upper respiratory tract -mouth, - Fluorochrome dye nasopharynx, oropharynx, and larynx - Binds chitin in fungal cell wall - Lower respiratory tract -trachea, - Fluoresce bright apple-green or blue- bronchi, and pulmonary parenchyma. white 3. Usual Flora of the Gastrointestinal tract 5. Methylene blue - Esophagus, stomach, small intestine, - Stains Corynebacterium diphtheriae for and colon observation of metachromatic granules 4. Usual Flora of the Genitourinary tract - Counterstain in acid fast - Kidneys, bladder, and fallopian tubes 6. Lactophenol cotton blue are normally free of microorganisms - Stains cell walls of medically important fungi grown in slide culture Role of the Microbial Flora in the Pathogenesis of 7. India ink Infectious Disease - Negative stain  Certain members of the usual flora are - Visualize capsules in certain yeasts opportunists (Cryptococcus spp.)  Cause disease when their habitat is damaged, - dark background, clear capsule disturbed, or changed, or when the host’s 8. Endospore stain immune system is weakened or compromised a. Malachite green -primary stain;  Development of immunologic competence heated to steaming for 5 minutes  Block colonization of extraneous pathogens Washed for 30 seconds b. Safranin -counterstain PATHOGENESIS OF INFECTION ***Endospores appear green within pink- or red-appearing bacterial cells 1. Pathogenicity is the ability of a microbe to produce disease in a susceptible individual. Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 a. True pathogens are organisms recognized  Specimens should be in sterile containers, to cause disease in healthy immune- except for stool competent individuals.  Generally, swabs are not recommended -do not b. Iatrogenic infection is an infection that provide sufficient quantity, easily contaminated, occurs as the result of medical treatment or can become dried out procedures.  Swabs -respiratory tract, external ear, eye, and 2. Virulence is the relative ability of a genital tract microorganism to cause disease or the degree of pathogenicity. Usually measured by the Patient Collected Specimen numbers of microorganisms necessary to cause  Urine -clean-catch midstream (representative of infection in the host the bladder) Microbial Virulence Factor  Sputum -for diagnosis of bacterial pneumonia; expectorated sputum and induced sputum 1. Ability To Resist Phagocytosis  Stool -for detection of gastrointestinal a. Capsule -masking the cell surface structures pathogens; should not be contaminated with -inhibits the activation of complement urine b. Protein A -cell wall of Staphylococcus aureus -interfering with the binding of the Clinical Specimens host’s antibodies to the surface of the 1. Blood organism. - Sterile c. Hemolysins by Streptococci -lyse red cells, - Bacteremia - presence of bacteria in the induce toxic effect on WBC and bloodstream macrophages. - Septicemia - serious disease d. Leukocidins by pathogenic staphylococci characterized by chills, fever, (Panton-Valentine) -lethal to leukocytes prostration, and the presence of bacteria 2. Surface Structures that Promotes Adhesion to or their toxins in the bloodstream Host Cells and Tissues 2. Urine a. Adhesins -fimbriae (pili) and/or - Sterile while in urinary bladder polysaccharides - Ideal specimen = clean-catch, 3. Ability to Survive Intracellularly and Proliferate Prevent fusion of phagosomes and lysosomes midstream Resistant to the effects of lysosomal contents - Other specimens = catheterized or Escape from phagosome into the cytoplasm suprapubic needle aspirate to obtain a. IgA protease (H. influenzae, N. sterile specimens gonorrhoeae, and N. meningitidis) degrades 3. CSF the IgA found at mucosal surfaces. - Sterile 4. Ability to Produce Extracellular Toxins and - For diagnosis of meningitis, Enzymes encephalitis, and meningoencephalitis a. Exotoxins -produced by both G+ and G- - Collected via lumbar puncture (spinal bacteria tap) b. Endotoxins -composed of the LPS portion of - Should be processed immediately the outer membrane on the cell wall of gram (STAT) negative bacteria LPS contains three 4. Sputum regions: an antigenic O–specific - Mucus coughed from lower airways polysaccharide, a core polysaccharide, and - For diagnosis of pneumonia, an inner lipid A (also called endotoxin). tuberculosis, or other lower respiratory infections Common Routes of Transmission 5. Throat swab  Airborne - Diagnosis of strep throat (pharyngitis)  Transmission by Food and Water caused by bacteria Streptococcus  Close Contact pyogenes  Cuts and Bites 6. Wound specimen  Arthropods - Should be aspirated rather than  Zoonoses swabbed - Must indicate what type of wound and SPECIMEN COLLECTION AND PROCESSING its anatomical location 7. Stool Collection Procedures Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - Should be collected and processed - Oxgall (bile salts) selective ingredient; immediately to prevent decrease in inhibits the growth of most gram-positive temperature, which allows pH to drop, organisms causing the death of bacteria Shigella - Esculin is the differential component. and Salmonella Esculin hydrolysis produce iron salts - Identification of result in blackening of medium a. Intestinal parasites (Ascaris 3. Bismuth Sulfite Agar lumbricoides, Trichuris trichiura) - Selective medium for the isolation of b. Intestinal protozoa (Giardia lamblia, Salmonella spp. Entamoeba histolytica)\ - Bismuth sulfite and brilliant green c. Fungi (Candida) -selective ingredients d. Bacteria (Escherichia coli, - Inhibit the growth of gram-positive Salmonella, Shigella) bacteria, most lactose-fermenting intestinal normal flora, and Shigella. Specimen Storage - Salmonella serotype typhi colonies are 1. Refrigerate black and surrounded by a metallic - Catheter (IV) sheen - CSF (virus) - Outer ear - Unpreserved feces - Feces for Clostridium 4. Sheep Blood Agar - Sputum - Routine medium used to cultivate a wide - Unpreserved urine variety of moderately fastidious bacterial 2. Room temperature organisms - Abscess, lesion, wound - 5 - 10% sheep, rabbit, or human blood - Body fluids - Sheep blood -most versatile - CSF (bacteria) - Allows detection and characterization of - Inner ear hemolysis - Preserved feces a. Beta – complete hemolysis - Genital Group A: Strep. pyogenes - Nasal Group B: Strep agalactiae - Tissue b. Alpha – partial hemolysis - Preserved urine Strep. Pneumonia Strep. Viridans CULTURE MEDIA c. Gamma – no effect on rbc Types of Culture Media Entero faecalis 5. Blood Phenylethyl Alcohol Agar 1. Nonselective media -support the growth of - Selective enrichment medium nonfastidious microbes (Ex. SBA) - Bacteroides, Prevotella, and other 2. Selective media -support the growth of one type obligate anaerobes or group of microbes but not another (Ex. MAC- - Phenylethyl alcohol -inhibits facultative selective for enteric G-) Gram negative anaerobes by 3. Differential media -allow grouping of microbes suppressing DNA synthesis and cell based on different characteristics demonstrated division on the medium 6. Bordet-Gengou Blood Agar Commonly Used Culture Media - Selective enrichment medium - Bordetella pertussis and Bordetella 1. Alkaline peptone water (APW) parapertussis - Enrichment medium 7. Buffered Charcoal-Yeast Extract Agar - Recovery of Vibrio and Aeromonas spp. - Enrichment medium from stool specimens - Legionella spp 2. Bile Esculin Agar 8. Chocolate Agar - Selective and differential - Enrichment agar - Identify group D streptococci and - Useful in promoting the growth of enterococci Haemophilus and other fastidious bacterial species Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - Variation of SBA -released Hemoglobin - Lactose -sole carbohydrate source and NAD Gram-negative rods that ferment lactose 9. Simmons Citrate Agar produce pink or red colonies - Useful in differentiating gram-negative - Non–lactose-fermenting gram-negative enteric bacilli. rods produce colorless or transparent - Based on the isolate’s ability or inability colonies. to use citrate as its sole source of - Neutral red dye –indicator carbon. 13. Mannitol Salt Agar (MSA) - At alkaline pH, the incorporated pH - Selective and differential indicator bromthymol blue shifts from - Recovery and identification of green to blue. staphylococci 10. Kligler’s Iron Agar (KIA) - High salt concentration (7.5%) inhibits - Used to determine whether a gram- most gramnegative and gram-positive negative rod is a glucose or lactose bacteria except Staphylococcus spp. fermenter or both - S. aureus is able to ferment mannitol, - Also tests for gas production during the sole carbohydrate. This lowers the carbohydrate fermentation and pH and changes the color of the pH hydrogen sulfide production indicator, phenol red, to yellow. - Contains: 14. Methyl Red-Voges-Proskauer Medium a. Glucose and lactose - Useful in distinguishing among (Carbohydrates) members of the Enterobacteriaceae. For b. Phenol red (pH indicator) example, E. coli is methyl red positive c. Peptone (carbon/ nitrogen source) and Voges-Proskauer negative d. Sodium thiosulfate plus ferric - MR positive -lactic, formic, succinic, and ammonium sulfate (sulfur source acetic; acidic pH; positive red color and hydrogen sulfide indicator, - VP positive -acetoin; neutral; positive respectively). pink-red color e. Slant/butt; “K” is used for alkaline 15. Mueller-Hinton Agar (red) and “A” for acid (yellow) - Testing susceptibility of organisms to 11. Lysine-Iron Agar antibiotics - Purple color denotes an alkaline 16. Cefsulodin-Irgasan Novobiocin (CIN) Agar environment (“K”) - AKA Yersinia-selective agar, is used to - “R” is used for the Bordeaux red color select for the isolation of Yersinia - “A” acid –yellow enterocolitica in stool samples. - Lysine decarboxylation to cadaverine 17. Phenylalanine Deaminase Agar (alkaline) - Used to detect an organism’s ability to - Three (3) parameters useful in deaminate phenylalanine identifying species of - positive reaction is most useful for Enterobacteriaceae: distinguishing Proteus, Providencia, and a. lysine decarboxylation Morganella spp. b. lysine deamination, and - + dark green slant on addition of ferric c. hydrogen sulfide production chloride - Contains 18. Salmonella-Shigella Agar a. lysine (amino acid) - Used to select for Salmonella and some b. Glucose (carbohydrate source) strains of Shigella from stool specimens c. Small amount of protein - Contains bile salts, sodium citrate, and d. Bromcresol purple (pH indicator) brilliant green, which inhibit the growth e. Sodium thiosulfate/ferric ammonium of gram-positive and many citrate (sulfur source and hydrogen lactosefermenting gram-negative rods sulfide indicator) normally found in stool. 12. MacConkey Agar - Lactose is the sole carbohydrate source - Selective and differential in the medium - Bile salts and crystal violet inhibit most - Neutral red is the pH indicator (pink - gram-positive organisms but permit red) growth of gram-negative rods. - Sodium thiosulfate -source of sulfur Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 - Shigella -colorless; do not ferment - Accomplished by physical or chemical lactose or produce hydrogen sulfide methods: Dry heat, autoclave (steam - Salmonella -colorless with black center; under pressure), ethylene oxide gas, do not ferment lactose but produce various liquid chemicals (formaldehyde). hydrogen sulfide H2S Terms 19. Thiosulfate citrate bile salts (TCBS) sucrose agar  Disinfection - elimination of most or all - Selective medium used to isolate Vibrio pathogens (except bacterial spores) from spp. from stool specimens nonliving objects - pH indicator: bromthymol blue  Pasteurization - method of disinfecting liquids; 20. Triple-Sugar Iron Agar (TSI) to eliminate pathogens from milk and most other - Determine whether a gram-negative rod beverages is a glucosefermenter or non–glucose-  Disinfectants - chemicals used to disinfect fermenter inanimate objects; do not kill spores - Contains glucose, sucrose, and lactose;  Antiseptics - solutions used to disinfect skin and - phenol red (pH indicator); other living tissues - sodium thiosulfate plus ferric ammonium  Sanitization - reduction of microbial populations sulfate (sulfur source and hydrogen to levels considered safe by public health sulfide indicator, respectively) standards Inoculation of Culture Media Using Physical Methods to Inhibit Microbial Growth 1. Heat  In a solid or plated medium  Use of a sterile inoculating loop to apply a - Most common type of sterilization for portion of the specimen to the surface of the inanimate objects able to withstand high medium (streaking temperatures - Most practical, efficient, inexpensive Importance of Aseptic Technique method of sterilization  Prevent Microbiology professionals from a. Dry heat - Incineration (burning) is becoming infected an effective means of destroying  Prevent contamination of their work environment contaminated disposable materials  Prevent Contamination of clinical specimens and b. Moist Heat - Heat applied in the cultures presence of moisture (boiling or steaming) Autoclave. Set to run 20 Techniques minutes at a pressure of 15 psi and a temperature of 121.5C 1. Incubation - After media are inoculated, they must be incubated (placed into a chamber CLINICALLY SIGNIFICANT MICROORGANISMS [incubator] that contains appropriate atmosphere, moisture level, and temperature) STAPHYLOCOCCI - To culture most human pathogens, incubator is set at 35C to 37C General Characteristics - Three types of incubators used in  Catalase-producing clinical microbiology laboratory:  Gram-positive cocci a. CO2 (carbon dioxide) incubator - to  Spherical cells that appear singly, in pairs, and in maintain a CO2 concentration of clusters about 5 - 10%  Staphylococcus Gk. staphle “bunches of grapes” b. Non-CO2 incubator - contains about  Nonmotile, non–spore-forming, and aerobic or 20 - 21% oxygen facultatively anaerobic, except for S. c. Anaerobic incubator - containing an saccharolyticus, which is an obligate anaerobe atmosphere devoid of O2  Colonies appear cream-colored, white, or rarely 2. Sterilization light gold and “buttery-looking” - Destruction or elimination of all  Species of staphylococci are differentiated by microbes the coagulase test - Sterile = devoid of microbial life  Positive test = clot formed in plasma  Active enzyme = Staphylocoagulase Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494  Coagulase-positive staphylococci = S. aureus only found associated with Panton-  Staphylococci that do not produce coagulase Valentine leukocidin (PVL) are referred to as coagulase-negative d. Panton-Valentine Leukocidin (PVL) staphylococci (CoNS) Exotoxin lethal to - CONS = S. epidermidis and S. polymorphonuclear leukocytes saprophyticus Suppresses phagocytosis Clinically Significant Staphylococci - ENZYMES 1. Staphylococcus aureus - Important cause of a. Staphylocoagulase is produced nosocomial or hospital-acquired infections mainly by S. aureus b. Hyaluronidase hydrolyzes *Virulence factors* hyaluronic acid present in the - ENTEROTOXINS intracellular ground substance that Heat-stable exotoxins that cause a makes up connective tissues, variety of symptoms including diarrhea permitting the spread of bacteria and vomiting during infection Stable at 100° C for 30 minutes, c. Lipases act on lipids present on the reheating contaminated food will not surface of the skin, particularly fats prevent disease. Staphylococcal food and oil secreted by the sebaceous poisoning is most commonly caused by glands enterotoxins A, B, and D - TOXIC SHOCK SYNDROME TOXIN-1 - PROTEIN A TSST-1 is a superantigen that Ability to bind the Fc portion of stimulates T cell proliferation and the immunoglobulin G (IgG). Binding IgG in subsequent production of a large this manner neutralizes IgG and can amount of cytokines block phagocytosis. - EXFOLIATIVE TOXIN AKA epidermolytic toxin Epidemiology It causes the epidermal layer of the skin to slough off and is known to cause  Primary reservoir for staphylococci is the staphylococcal Scalded Skin Syndrome nares, with colonization also occurring in the (SSS), sometimes referred to as Ritter axillae, vagina, pharynx, and other skin disease. surfaces  Transmission of S. aureus may occur by direct contact with unwashed, contaminated - CYTOLYTIC TOXINS hands and by inanimate objects (fomites) hemolysins and leukocidins Infections caused by S. aureus a. α-Hemolysin - lyses erythrocytes,  Skin and wound infections damages platelets and Suppurative. Abscess is filled with pus and macrophages and cause severe surrounded by necrotic tissues and tissue damage damaged leukocytes. b. β-Hemolysin (sphingomyelinase C) a. Foliculitis is a relatively mild Acts on sphingomyelin in the inflammation of a hair follicle or oil gland plasma membrane of erythrocytes b. Furuncles (boils) are large, raised, AKA “hot-cold” lysin. Enhanced superficial abscesses hemo- lytic activity on incubation at c. Carbuncles larger, more invasive lesions 37° C and subsequent exposure to develop from multiple furuncles, which cold (4° C). This hemolysin is may progress into deeper tissues exhibited in the Christie, Atkins, and d. Bulous impetigo larger and surrounded Munch-Petersen (CAMP) test by a small zone of erythema (highly c. δ-Hemolysin - Considered less toxic contagious) to cell structure than either α- or e. Scalded Skin Syndrome AKA Ritter βhemolysins. γ-Hemolysin is often disease - exfoliative dermatitis that Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 occurs primarily in newborns and Staphylocoagulase reacts with previously healthy young coagulase-reacting factor (CRF) to Children caused by exfoliative or form coagulase-CRF complex which epidermolytic toxin resembles thrombin and indirectly f. Toxic Shock Syndrome Rare but converts fibrinogen to fibrin potentially fatal, multisystem disease - Look for clot formation after 4 hours of characterized by a sudden onset of incubation at 37° C. fever, chills, vomiting, diarrhea, muscle - If no clot appears, the tube should be aches, and rash, which can quickly left at room temperature and checked progress to hypotension and shock the following day. g. Food poisoning - Novobiocin susceptibility - h. Staphylococcal pneumonia Zone of inhibition of ≤16 mm =resistant i. Staphylococcal bacteremia S. saprophyticus = resistant j. Staphylococcal osteomyelitis S. epidermidis = susceptible 2. Staphylococcus epidermidis - Most common Antimicrobial Susceptibility cause of hospital-acquired UTIs, Prosthetic 1. Penicillin-resistant strains require treatment with valve endocarditisinfections have been penicillinase-resistant penicillins, such as associated with intravascular catheters, nafcillin or oxacillin cerebrospinal fluid shunts, and other prosthetic 2. Methicillin-Resistant Staphylococcus aureus devices (MRSA) - Use of vancomycin for MRSA remains the treatment of choice 3. Staphylococcus saprophyticus - Associated with - Oxacillin-salt agar plate can be used to UTIs in young women screen for MRSA 4. Staphylococcus lugdunensis - An important - Most oxacillin resistance is due to the pathogen in infective endocarditis, septicemia, gene mecA, which codes for an altered meningitis, skin and soft tissue infections, penicillin-binding protein (PBP) called urinary tract infections and septic shock. PBP2a Laboratory Diagnosis - The altered PBP does not bind oxacillin - The “gold standard” for MRSA detection Isolation and Identification is the detection of the mecA gene by Cultural Characteristics using nucleic acid probes or polymerase chain reaction (PCR) amplification - Round, smooth, white, creamy colonies on SBA after 18 to 24 hours of Clinically significant Streptococcus and Strep-like incubation at 35° to 37° C organisms - S. aureus may produce hemolytic zones 1. Streptococcus pyogenes around the colonies and may exhibit pigment production (yellow) Virulence factors - S. epidermidis colonies are usually a. M protein - for adherence; resists small- to medium-sized, nonhemolytic, phagocytosis white to gray colonies b. Protein F - adherence molecule c. Capsule - prevents opsonisation Identification methods d. DNase, Streptokinase, Erythrogenic toxin - Coagulase test = identifies S. aureus e. Erythrogenic toxin/Streptococcal pyrogenic a. SLIDE coagulase test exotoxin - causes scarlet fever Clumping factor (cel-bound f. Hemolysins: coagulase) agglutinates human, - Streptolysin O - O2 labile, determine rabbit, or pig plasma recent infection (ASO), hemolysis on Directly converts fibrinogen to fibrin SBA anaerobically Screens catalase-positive colonies - Streptolysis S - O2 stable, surface that morphologically resemble S. hemolysis on SBA aerobically aureus b. TUBE coagulase test Staphylocoagulase (fee coagulase) Clinical infections causes a clot to form when bacterial cells are incubated with plasma Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 a. Bacterial pharyngitis: S. pyogenes, most 4. Viridans Streptococci - Viridans means green common pathogen (Strep throat) = alpha-hemolysis many species exhibit b. Pyodermal infections: Impetigo, Erysipelas, cellulitis, Scarlet fever Clinical infections c. Necrotizing fasciitis: flesh easting disease a. Most common cause of subacute bacterial d. Streptococcal Toxic Shock syndrome endocarditis e. Post-Streptococcal sequelae: Rheumatic b. Gingivitis, dental caries fever, acute glomerulonephritis Laboratory Diagnosis Laboratory diagnosis a. On SBA - small, transparent, smooth beta- a. PYR: negative hemolytic b. LAP: positive b. Key tests: Bacitracin susceptibility (TAXO A): c. 6.5% NaCl: negative (Group D) S. pyogenes (S) vs other beta-hemolytic (R) d. Bile esculin: positive c. PYR hydrolysis: S. pyogenes (+) vs other beta-hemolytic (-) 5. Enterococcus E. faecalis and E. faecium 2. Streptococcus agalactiae Virulence factors: Virulence factors a. Gelatinase a. Capsule, Hemolysins, CAMP factor, b. cytolysin neuraminidase Clinical infections a. Frequent cause of nosocomial infection (UTI Laboratory Diagnosis from catheterization) a. On SBA -grayish white mucoid, beta- hemolytic Laboratory diagnosis b. Key tests: Hippurate hydrolysis and CAMP a. Trypticase soy or Brain heart infusion agar test (+) supplemented with 5% sheep blood to c. StrepB Carrot Broth = + red or orange culture enterococci pigment b. Bile esculin: positive c. 6.5% NaCl: Enterococcus (+) vs Group D (-) 3. Streptococcus pneumoniae d. PYR: Enterococcus (+) vs Group D (-) Virulence factor: capsular polysaccharide 6. Streptococcus-like organisms Clinical infections - Vancomycin resistant: Leuconostoc and a. Recurrent otitis media (3 y.o) - S. Pediococcus pneumoniae -most common isolate - Vancomycin susceptible: Aerococcus b. Bacterial pneumonia- S. pnumoniae - Aerococcus =opportunistic; assoc. with -number one cause in elderly bacteremia, endocarditis, and UTI in c. Lobar pneumonia (Rust-colored sputum) immunocompromised; d. Meningitis - Grows in 6.5% NaCl - Lactococcus =assoc. with UTI and Laboratory diagnosis endocarditis a. Neufield test: Positive capsular swelling (Quellung reaction) Laboratory Diagnosis b. Gram stain -slightly pointed, oval or lancet 1. Bacitracin susceptibility (TAXO A) shape/bullet shape Identify S. pyogenes (Grp. A) c. Capsule -stained by capsule stain On SBA: beta-hemolytic colonies, bacitracin (S), d. On SBA - round, glistening, wet, mucoid, SXT (R) = S. pyogenes dome-shaped, alpha-hemolytic On SBA: beta-hemolytic colonies, bacitracin (R), e. Key test: Optochin susceptibility (TAXO P)= SXT (R) = S. agalactiae S. pneumoniae (S) vs other alpha hemolytic 2. CAMP test (R) Identify beta-hemolytic S. agalactiae (Grp B) f. Bile solubility = S. pneumoniae (+) Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 Utilizes β-lysin producing S. aureus = (+) *Clinical infections (2 forms)* arrowhead-shaped hemolysis pattern  Respiratory diphtheriaSpread by droplet 3. Hippurate hydrolysis infection or hand-to-mouth contact Differentiates S. agalactiae (+) from other beta- Most common site of infection: tonsils or hemolytic Strep (-) pharynx Enzyme hippuricase in S. agalactiae  Cutaneous diphtheria- consists of non 4. PYR hydrolysis healing ulcers with dirty gray membrane Positive = S. pyogenes (Grp. A) and nonhemolytic Grp. D (Enterococcus, *Laboratory diagnosis* Aerococcus, Gemella)  Microscopy: pleomorphic; palisades or V 5. LAP and L formation; “chinese letters” Positive = Streptococcus, Enterococcus,  Methylene blue: beaded appearance Pediococcus (red color) (Babes-Ernst granules-metachromatic Negative = Aerococcus, Leuconostoc area) 6. Bile Esculin and Salt tolerance Bile esculin: Positive = Grp. D Strep and *Culture* Enterococcus (blackening of agar)  best grown on medium containing blood 6.5% NaCl: Enterococcus (+) vs Grp. D Strep (-) or serum- Loeffler serum or Pai agar 7. Optochin susceptibility (Ethylhydrocuprein  On SBA: small zone of beta-hemolysis hydrochloride/ TAXO P)  Cystine-tellurite blood agar (CTBA; For alpha-hemolytic Strep selective and differential): contains On SBA: S. pneumoniae (S) vs. Viridans Strep sheep RBC, bovine serum, cystine and (R) potassium tellurite (inhibits non- 8. Bile solubility coryneform bacteria) (+) S. pneumoniae =active enzyme amidase  CTBA= (+) Corynebacteria- black or 9. Antibiotics susceptibility brownish colonies (from reduction of tellurite) with brown halo (Tinsdale halo- differentiates C. diphtheriae from Staph and Strep) *Identification*  Ferments: glucose and maltose (+ acid without gas)  Reduces nitrate to nitrite *Test for toxigenicity* AEROBIC GRAM POSITIVE BACILLI: Non-Spore-  Elek test: detects diphtheria toxin in vitro Forming, Non-branching Catalase-positive Bacilli I. CORYNEBACTERIUM 2. Corynebacterium jeikeium  Normal flora of the skin General characteristics  Infections are typically limited to immunocompromised  Medically important: Catalase positive  Most common cause of diptheroid  Nonmotile prosthetic valve endocarditis in adults  Divided into nonlipophilic and lipophilic  Causes septicemia, meningitis, and species (growth is enhanced if lipids are prosthetic joint infections on addition to included) endocarditis Lipophilic and strict aerobe  Gram stain: slightly curved Gram positive  Nonhemolytic bacilli “club shape” appearance or  Urease (+) coryneform  Reduces nitrate to nitrite  Drug of choice: vancomycin 1. Corynebacterium diphtheriae 3. Corynebacterium pseudodipthericum  Normal flora of nasopharynx *Virulence factors*  Causes respiratory tract infections in  Diphtheria toxin- potent and lethal; immunocompromised individuals blocks protein synthesis  Causes endocarditis, UTI, and cutaneous wound infections in Downloaded by Ada Mercado ([email protected])  lOMoARcPSD|47212494 immunocompromised patients (ex. AIDS)  Reduces nitrate to nitrite  Hydrolyze urea 4. Corynebacterium pseudotuberculosis