Microbiology Lab Exam 3
Microbiology Lab Exam 3 BSC 242
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This 5 page Study Guide was uploaded by Haley Etheridge on Saturday April 23, 2016. The Study Guide belongs to BSC 242 at University of Alabama - Tuscaloosa taught by Dr. Daryl Lam in Spring 2016. Since its upload, it has received 166 views. For similar materials see Microbiology and Man in Biological Sciences at University of Alabama - Tuscaloosa.
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Date Created: 04/23/16
Microbiology Lab Exam 3 Ø Exercise 7-2: Antimicrobial Susceptibility Test • Antibiotics are natural antimicrobial agents produced by microorganisms. • Antimicrobials/Antimicrobics à describes any substance that kills microorganisms, natural or synthetic. • The Kirby-Bauer Test, also called the disk diffusion test, is a valuable standard tool for measuring the effectiveness of antimicrobics against pathogenic organisms. • In the test, antimicrobic-impregnanted paper disks are placed on a plate that has been inoculated to form a bacterial lawn. • The plates are incubated to allow growth of the bacteria and time for the agent to diffuse into the agar. • As the drug moves through the agar, it establishes a concentration gradient. • If the organism is susceptible to it, a clear zone will appear around the disk where growth has been inhibited. • Zone of Inhibition à on an agar plate, the area of nongrowth surrounding a paper disc containing an antimicrobial substance. • Minimum Inhibitory Concentration (MIC) à the lowest concentration of an antimicrobial substance required to inhibit growth of all microbial cells it contacts; on an agar plate, typically the outer edge of the zone of inhibition where the substance has diffused to the degree that it no longer inhibits growth. • The size of this zone of inhibition depends upon the sensitivity of the bacteria to the specific antimicrobial agent and the point at which the chemical’s MIC is reached. • Bactericidal à the zone may be produced as a result of killing the bacteria. • Bacteriostatic à the drug may only stop/slow bacterial growth without killing them. • Meuller-Hinton Agar, which has a pH between 7.2-7.4, is poured to a depth of 4 mm in either 150 mm or 100 mm Petri dishes. • The depth is important because of its effect upon diffusion. • Thick agar slows lateral diffusion and thus produces smaller zones than plates help to the 4 mm standard. • After 16 to 18 hours incubation, the plates are removed and the clear zone diameters are measured and recorded in millimeters. • In this exercise you will test the susceptibility of E. coli, S. epidermidis, and P. aeruginosa strains to different antimicrobials, antiseptics, and disinfectant. Ø Exercise 2-13: Chemical Germicides: Disinfectants and Antiseptics • Chemical germicides are substances designed to reduce the number of pathogens in a surface, in a liquid, or on or in living tissue. • Disinfectants à germicides designed for use on surfaces (tables and floors) or liquids. • Antiseptics à germicides designed for use on or in living tissue. • The Use-Dilution Test is a standard procedure used to measure the effectiveness of disinfectants. • In the standard procedure, glass beads or stainless steel cylinders coated with living bacteria are exposed to varying concentrations with living bacteria are exposed to varying concentrations (dilutions) of test germicides, then transferred to a growth medium. • After a period of incubation, the medium is examined for growth. • If a solution is sufficient to prevent microbial growth at least 95% of the time, it meets the required standards and is considered a usable dilution of that germicide for a specific application. Antimicrobials Antiseptics Disinfectants Ampicillin Iodine Lysol Tetracycline Mint Mouthwash Pinesol Gentamian Antiseptic Mouthwash Ethanol Tricarcillian Bactine - Streptomycin - - Novobiocin - - Ø Exercise 6-1: Standard Plate Count • The standard plate count is a procedure that allows microbiologists to estimate the population density in a liquid sample by plating a very dilute portion of that sample and counting the number of colonies it produces. • Serial Dilution à series of dilutions used to reduce the concentration of a culture and thereby produce between 30 and 300 colonies when plated, providing a means of calculation the original concentration. • The inoculum that is transferred to the plate contains a known proportion of the original sample because it is the product of a serial dilution. • Dilution Bank à a test tube containing a measured volume of sterile diluent (water, saline, or buffer) used to serially dilute a concentrated solution or broth. • A serial dilution is simply a series of controlled transfers down a line of dilution banks. • The series begins with a sample containing an unknown concentration (density) of cells and ends with a very dilute mixture containing only a few cells. • Each dilution bank in the series receives a known volume from the mixture in the previous tube and delivers a known volume to the next, typically reducing the cell density to 1/10 or 1/100 at each step. • A known volume of appropriate dilutions is then spread onto agar plates to produce at least one countable plate. • A countable plate is one that contains between 30 and 300 colonies. • A count lower than 30 colonies is considered statistically unreliable and greater than 300 colonies is typically too many to be viewed as individual colonies. • The first transfer in the series is a simple dilution, but that all successive transfers are compound dilutions. • Compound dilutions are dilutions of previously diluted samples. • Know this formula: V D 1 V1 2 2 • V1 ndD r1 the volume and dilution of the concentrated broth respectively, while V and D 2 2 are the volume and dilution of the completed dilution. • Undiluted samples are always expressed as 1. • Spreading a known volume of this dilution onto an agar plate and counting the colonies develop would give you all the information you need to calculate the original cell density (OCD). • Another Formula: OCD = CFU / (D)(V) • CFU (colony forming unit) is actually the number of colonies that develop on the plate. CFU is the preferred term because colonies could develop from single cells or from groups of cells, depending on the typical cellular arrangement of the organism. • D is the dilution tube from which the inoculum comes. • V is the volume transferred to the plate. **Look at the math example posted on BB by your TA** Ø Exercise 7-8: Making Yogurt • Several species of bacteria are used in the commercial production of yogurt. • One common pairing of organisms in commercial yogurt is that of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. • Yogurt gets its unique flavor from acetaldehyde, diacetyl, and acetate produced from fermentation of the milk sugar lactose. • Both species mentioned above contain constitutive β-galactosidase systems that break down lactose and convert the glucose to lactate, formate, and acetate via pyruvate in the glycolysis pathway. • Lactose is a disaccharide composed of glucose and glactose. • S. thermophilus does not posses the enzymes needed to metabolize galactose, and L. delbrueckii preferentially metabolizes glucose. • This results in an accumulation of galactose, which adds sweetness to the yogurt. • Acetaldehyde is produced directly from pyruvate by S. thermophilus and through the conversion of proteolysis products threonine and glycine by L. delbrueckii. • Some strains of S. thermophilus also produce glucose polymers, which give yogurt a viscous consistency. Ø Exercise 4-3: Mannitol Salt Agar • Mannitol Salt Agar (MSA) contains the carbohydrate mannitol, 7.5% sodium chloride (NaCl), and the pH indicator phenol red. • Phenol red is yellow below pH 6.8, red at pH 7.4 to 8.4, and pink at 8.4 and above. • Mannitol provides the substrate for fermentation and makes the medium differential. • NaCl makes the medium selective because its concentration is high enough to dehydrate and kill most bacteria. • Staphylococci thrive in the medium, largely because of their adaptation to salty habits such as human skin. • Phenol red indicates whether fermentation has taken place by changing color as the pH changes. • Most staphylococci are able to grow on MSA, but do not ferment the mannitol, so the growth appears pink or red and the medium remains unchanged. • Staphylococcus aureus ferments the mannitol, which produces acids and lowers the pH of the medium. The result is formation of bright yellow colonies usually surrounded by a yellow halo. • Mannitol Salt Agar is used for isolation and differentiation of Staphylococcus aureus. Result Interpretation Presumptive ID Poor growth or no Organism is inhibited by NaCl Not Staphlyococcus growth – PINK Good Growth Organism is not inhibited by NaCl Staphlyococcus Yellow Growth or Halo Organism produces acid from Possible pathogenic mannitol fermentation Staphlyococcus aureus Red Growth (no halo) Organism does not ferment mannitol. Staphylococcus other than S. No reaction. aureus. Ø Exercise 5-21: Blood Agar • Hemolysins à destroy red blood cells and hemoglobin. • Blood Agar, sometimes called Sheep Blood Agar, allows differentiation of bacteria based on their ability to hemolyze RBCs. • The three major types of hemolysis are β-hemolysis, α-hemolysis, and γ-hemolysis. • β-hemolysis à the complete destruction of RBCs and hemoglobin, results in a clearing of the medium around the colonies (YELLOW). • α-hemolysis à the partial destruction of RBCs and produces a greenish discoloration of the agar around the colonies (DARK GREEN). • γ-hemolysis à nonhemolysis and appears as simple growth with no change to the medium. • Hemolysins produced by streptococci are called streptolysins (type O and S). • Streptolysin O is oxygen-labile and expresses maximal activity under anaerobic conditions. • Streptolysin S is oxygen-stable but expresses itself optimally under anaerobic conditions as well. • Streak-Stab Technique à the Blood Agar plate is streaked for isolation and then stabbed with a loop. The stab encourages streptolysin activity because of the reduced ocygen concentration of the subsurface environment. Result Interpretation Symbol Clearing Around Growth Organism Hemolyzes RBCs Completely β-hemolysis Greening Around Growth Organism Hemolyze RBCs Partially α-hemolysis No Change in the Medium Organism Does Not Hemolyze RBCs γ-hemolysis Ø Exercise 9-6: Identification of Streptococcus and Streptococcus-like Organisms by Latex Agglutination: The Streptex Rapid Test • The genus Streptococcus is composed of Gram-positive, catalase-negative cocci in pairs or chains. • In this exercise, you will be identifying clinically significant “streptococci” based on cellular antigens. • The Streptex Rapid Test kit has six different antibody suspensions, each with a different antibody coating polystyrene latex particles and homologous to one of six different group-specific antigens. • In performing the test, first the antigen is extracted from a colony growing on an agar plate. • Then each antibody suspension is added to one of six circles on the reaction card, followed by a known volume of extracted antigen. • After mixing the antigen-antibody suspensions in each circle, the card is rocked gently for one minute. • A positive result shows agglutination. • A negative result retains a homogenous appearance. • The Streptex Test is most reliable when grouping β-hemolytic organisms, but it can be used to identify some other groups – most notably the enterococci, which typically are α – or – γ hemolytic. • The two most important streptococcal groups are Group A, B, C, D, F, and G. o Group A Steptococci (GAS) is composed of S. pyrogenes, the causative agent of bacterial pharyngitis (“strep throat”), impetigo, scarlet fever, necrotizing fasciitis, streptococcal toxic shock syndrome, and many other superficial and deep infections. In most cases, penicillin is still effective in treating S. pyrogenes infections. The Group A antigen is a branched polysaccharide or N-acetylglucosamine and the sugar rhamnose. o S. agalactiae is the only species of Group B streptococcus (GBS). It was first isolated from cows, where it causes bovine mastitis, but it also is a human pathogen, especially in neonates and immunocompromised adults. The Group B antigen is a complex of N- acetyglucosamine and the sugars galactose and rhamnose. o S. bovis group and some members of the genus Enterococcus compromise Group D streptococci. S. bovis is associated with bacteremia, endocarditis, and meningitis. The two most frequently recovered enterococcal species from fecal material from E. faecium and E. faecalis. A major cause of nosocomial infections, they are of increasing concern because of their resistance to multiple antibiotics and possession of several intrinsic resistance factors. The Group D antigen is a teichoic acid composed of glycerol phosphate and glucose. o Other β-hemolytic streptococci that posses group-specific antigens are the S. anginosus group, which have the A, C, F, and G antigens, and S. dysgalactiae with either C or G antigen. Although these species demonstrate antigen variability, each isolate will have only a single antigen. S. anginosus species cause abscesses, and S. dysgalactiae is a causative agent of pharyngitis and sometimes glomerulonephritis. o Although they are not classified by the Lancefield system, two other streptococcal groups are important. S. pneumonia is a α-hemolytic and is a causative agent of bacterial pneumonia, meningitis, otitis media, and bacteremia. Viridans streptococci comprise more than 20 α – or – γ hemolytic species and are so-named because of the greening they often produce on blood agar plates. They are normal inhabitants of the oropharynx, digestive tract, and urogenital tract but are responsible for diseases as varied as dental caries, endocarditis, and abdominal infections.
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