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165 Cards in this Set
- Front
- Back
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CHO Tube
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Carbohydrate Fermentation
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Purpose of CHO Tube
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-Test of the ability to ferment certain carbohydrates
-Also for gas production |
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OF Test
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Oxidation-Fermentation Test
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Purpose of OF Test
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-Test of oxidative aerobic respiration versus fermentation
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CHO Indicator
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Brom Cresol Purple
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If a CHO tube is purple,
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-pH above 6.8
-no fermentation |
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If a CHO tube is yellow,
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-pH below 6.8
-fermentation -acids produced |
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What is the tube inside another called?
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Durham tube
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What is the electron acceptor with oxidative respiration?
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Oxygen
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What is carbohydrate oxidated to?
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CO2
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What is the electron acceptor with fermentation?
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organic molecule
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How much energy is generated with fermentation compared to respiration?
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Less energy
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What are some end products of fermentation?
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-acids: carbon dioxide
-aldehydes: methane -alcohols: hydrogen gas |
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O-F tube Indicator
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Brom thymol blue
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How are O-F tubes inoculated?
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-two tubes
-one covered with oil -other exposed to oxygen |
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When is there oxidative respiration with O-F tubes?
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When oil tube is bluish-green, and other is yellow at top
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When is there fermentation with O-F tubes?
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When both tubes are yellow
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If both O-F tubes are bluish-green,
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non-sacharolytic
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pH of O-F tubes
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-bluish green: 7.0-7.6
-yellow: 6.0 |
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Miniaturized multi-test systems
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-commercially prepared kits
-Combine several biochemical tests into a single inoculated in a simple manner -Use a computer (numeric code) to analyze the test results and provide specific identifications |
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API
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Analytical Profile Index
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Analytical Profile Index
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-different strips for enteric, nonfermenters, staphylococci, and streptococci
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What is Enterotube II used for?
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to identify enteric bacteria
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API 20e strips
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-Conducts 20 biochemical tests simultaneously
-Different strips for enterics (like the API 20E shown above), nonfermenters, staphylococci, anaerobes, yeasts, and also streptococci -Difficult to inoculate, requires much practice |
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Enterotube II
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The Enterotube II contains 12 different agars enabling the performance of a total of 15 biochemical tests as well as an enclosed inoculating wire
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How do you score Enterotube?
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-Score test s as + or – results (circle +’s)
-Add the scores (1, 2, 4) of the + tests in the each box -Continue adding scores of + tests for each box -Use the codebook for the appropriate test strip to look up the identification of the strain |
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Catalase Test
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-Tests for presence of catalase which converts hydrogen peroxide to water and oxygen (bubbles = + test)
-Indicates the ability of an organism to utilize oxygen; can differentiate Staphylococcus (catalase +) from Streptococcus (catalase -) |
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Oxidase Test
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-Tests for the presence of cytochrome aa3 in the electron transport chain of some bacteria (Pseudomonas)
-Immediate purple color of indicator = positive test for the presence of oxidase |
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Urea Agar Slant
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-Differentiates bacteria on the basis of the ability to produce urease (positive result = pink)
-Urease hydrolyzes urea to NH3 and CO2 and is indicative of Proteus |
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Kliger-Ion Agar Slant
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Characterizes bacteria on the basis of the ability to ferment glucose and/or lactose and hydrogen sulfide production (this is similar to TSI tubes except they lack sucrose)
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Phenylalanine Agar Slant
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-Characterizes bacteria on the basis of the ability to produce phenylalanine deaminase
-Typical of Proteus |
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Coagulase Test in Rabbit Plasma
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-Characterizes bacteria on the basis of the ability to produce coagulase
-Used to differentiate pathogenic strains of Staphylococcus from non-pathogenic strains |
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Phenylalanine Results
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-(Top) Greening of the medium =
phenylalanine was catabolized to phenolpyruvic acid and ammonia by phenylalanine deaminase -(Bottom) No color change of medium = phenylalanine was not catabolized |
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What must be added to phenylalanine test?
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FeCl2
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Coagulase Results
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-(Top) Fibrin clot forms (media gels) = coagulase present
-(Bottom) No clot forms (media liquid) = coagulase absent |
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What are IMViC tests used for?
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differentiating the Enterobacteriaceae family members Escherichia coli and Enterobacter aerogenes
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What is similar about E. coli and E. aerogenes?
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Similar morphology: gram negative rods
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E. coli
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-found in mammalian intestinal tract
-indicator of fecal contamination: water, food |
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E. aerogenes
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-found extensively in nature
-mainly associated with plants and plant products |
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What does IMViC stand for?
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I: Indole
M: Methyl Red V: Voges-Proskauer C: Citrate |
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Indole Production Test
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-test for indole production from tryptophan via the enzyme tryptophanase
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Methyl Red Test (MR)
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-test for production of large amounts of mixed acid end products from glucose fermentation
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Voges-Proskauer Test (VP)
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-test for the production of 2,3-butanediol from glucose fermentation
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Citrate Utilization Test
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-test for the ability to use citrate as a sole source of carbon
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What do you add to Indole test? When?
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-Kovac's
-after bacterial growth in tryptophan broth |
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Indole Test: Red at top
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indole has been produced
-tryptophanase present |
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Indole Test: No color change
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no indole production
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MR-VP Tests
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-each test is separate
-either two replicate cultures are grown, or one culture is grown and split in two for testing -reagents added after growth -most will test positive for one or the other, not both |
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What is pH indicator of Methyl Red?
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-Methyl Red
-yellow at pH 6.0 and above -red at less than 4.4 |
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Methyl Red Test Results=red
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-glucose fermented to large amounts of mixed acids
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Methyl Red Test results=yellow
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-no large amounts of acids made
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VP Reagents
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-alpha napthol
-KOH -react with acetoin and guanidine to produce a red color |
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VP Results=red
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-glucose fermented to 2,3-butanediol
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VP results=no color change
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-no 2,3-butanediol production
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Simmons Citrate Agar
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-ammonium salt (sole N-source)
-citrate (sole C-source) -brom thymol blue pH indicator -green at 7.0 blue higher than 7.6 |
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Citrate Results=green
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citrate not utilized
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Citrate results=blue
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citrate utilized, alkaline by-products
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E. coli v. e. aerogenes: indole
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-E. coli: +
-E. aerogenes: - |
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E. coli v. E. aerogenes: Methyl red
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-coli: +
-aer: - |
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coli v. aerogenes: VP
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-coli: -
-aer: + |
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E. coli v E. aerogenes: Citrate
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-coli: -
-aer: + |
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Waterborne Bacteria
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-E. coli
-Salmonella typhi -Vibria cholerae |
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Waterborne Viruses
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-Poliomyletis
-Hepatitis |
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Waterborne Protozoa
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-Giardia
-Amoeba -Cryptosporidium |
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Coliforms
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-facultative anaerobes
-Gram negative rods -non endospore forming -ferment lactose to acid + gas within 48 hours -common inhabitant of animal intestines |
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Why should we test for coliforms?
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-presence indicates possibility of fecal pollution in a water system
-not all coliforms are equal -E.coli and other pathogenic intestinal bacteria have limited survival outside of intestine -E. coli easily monitored and acts as indicator species of other pathogens |
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What should ideal detection of coliforms be?
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Easy, rapid, cheap, accurate
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What is the presumptive test for coliforms?
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Lauryl Tryptose Broth
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Lauryl Tryptose Broth (LTB)
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-selective: bile and detergent
-differential: lactose fermentation |
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What is the confirmed test of coliforms?
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Brilliant GreenLactose Bile Broth
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Brilliant Green Lactose Bile Broth (BGLB)
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-selective: brilliant green
-differential: lactosefermentation with gas at 57C -determines total coliforms present |
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What is the completed test for coliforms?
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-E. coli broth
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E. coli broth (EC)
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-selective: bile salts and increased temp
-differential: lactose fermentation with gas at 44.5C -determines only fecal coliforms present |
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MPN
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Most probable number
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Most probable number
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-statistical estimation of bacterial cell numbers
-replicate (5) tubes at three dilutions are typically done -tubes with growth in the presumptive test are transferred onto other tests |
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Statistical Average
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1100 coliforms/100 ml
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Minimum Level
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400 coliforms/100ml
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Maximum Level
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3000 coliforms/100ml
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US Drinking water safety limits
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-Total coliforms=0 per 100 ml
-Fecal coliforms=0 per 100 ml |
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Swimming pools and beaches
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Fecal coliforms=200 per 100 ml
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How many swimmers on average ccontract gastroenteritis traced to bacteria in swimming waters?
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8 out of 1000
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Phage Replication
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-phage only reproduce when they inhabit living cells
-lytic phage takes over host cell machinery to produce many copies of themselves, then burst the host cell to release the newly produced infective particles -300 viruses produced from a single virus inside ahost cell in about 20 minutes |
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What do lysogenic viruses do?
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-incorporate themselves into host chromosome and replicate as it does, allowing cell to continue living
-may be triggered to de-integrate from host chromosome and begin lyic cycle |
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Plaque (Lytic Phage) Assay
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-serves to enumerate the number of lytic phage present in a sample via serial dilution and production of a plaque in a bacterial lawn
-assumes that a single phage enters as individual bacterial cell and undergoes replication and lysis, then spreads to adjacent bacterial cells within the immediate radius -performed within an agar overlay to locate motile bacterial cells so that only 1 plaque will form per initial phage |
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Plaque Assay Assumptions
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-only enumerates phage which are undergoing lytic reproduction
-host bacterium must be susceptible to phage infection -some lysogenic phage infectionsconfer immunity from infection by other phages -some hosts do not have appropriate phage receptros on cell wall surfaces, so phage cannot attach and infect cell |
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What is transformation?
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-form of genetic recombination in which free DNA is taken up by a bacterial cell and incorporated into its genome
-cells musst be competent or able to take the DNA ito their cytoplasm so that recombination with homologous regions of its chromosome |
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What happens in a successful transformation?
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-transformed cells will inherit a new phenotpic trait, such as antibiotic resistance
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Where is normal flora found?
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-skin
-mucous membranes, including intestines |
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Do normal flora cause pathogenesis?
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-not generally
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Antagonistic mutualism
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mere presence there prevents harmful bacteria from colonizing the space
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Complex interaction of microbes
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-greater than 200 species of bacteria in some cases: bacteria and fungi
-variety depends on age, gender, stress, nutrition, and diet of individual |
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Staphylococcus
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-Gram positive cocci arranged in clusters
-nonmotile, facultative anaerobic, catalase positive, able to grow on media containing 10% sodium chloride -temp range 18-40C |
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Common Staphylococcus on skin
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S. aureus, S. epedermidis, S. saprophyticus
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Epidermal Staphylococcal Infections
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-impetigo
-scaled skin syndrome -folliculitis |
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Staphylococcal Abscesses
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-boils
-carbuncles |
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Staphylococcal Infections of internal organs and tissues
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-endocarditis
-pneumonia -osteomyelitis -cystitis -pyelonephritis -enteritis -septicemia -toxic shock syndrome |
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Staph aureus
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-yellow
-hemolysis + -anaerobic growth + -coagulase + -glucose fermentation + -mannitol fermentation + -DNAse + |
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Staph epidermidis
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-white
-hemolysis +/- -anaerobic growth + -coagulase - -glucose fermentation + -mannitol fermentation - -DNAse - |
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Staph saprophyticus
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-white
-hemolysis - -anaerobic growth +/- -coagluase - -glucose fermentation - -mannitol fermentation - -DNAse - |
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What kind of toxins do Staphylococcus have?
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-cytolytic toxins
-exfoliative toxins -toxic shock toxins -enterotoxins -hylaronidase -coagulase |
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cytolytic toxins
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-hemolysins: lyse red blood cells
-leukocidans: destroy white blood cells |
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exfoliative toxins
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-associated with scalded skin syndrome
-causes separation of keratinized skin cells from the living epidermal layer-redness of skin and peeling effect -toxin absorbed by the bloodstream and delivered throughout the body |
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toxic shock toxins
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-exotoxin causes the release of massive amounts of cellular cytokines by the immune cells
-results in a drop of blood pressure and kidney failure |
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enterotoxins
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-cause food poisoning: vommitting, diarrhea, fever
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hylaronidase
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-degrades host connectivetissues, enables spread
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coagulase
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-clots plasma and impedes white blood cells from reaching infection, traps bacteria within a "fort" ofinfectable area
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Streptococcus
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-Gram positive cocci arranged in chains
-catalase negative, facultative anaerobes with complex nutritional requirements |
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streptococcus classification schemes
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-clinical presentation
-serological properties -hemolytic patterns -biochemical properties |
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Clinical Presentation
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-pyogenic
-oral -enteric |
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Serological Properties
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Lancefield
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Hemolytic Patterns
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-alpha
-beta -gamma |
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Streptococcal Epidermal infections
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impetigo
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streptococcal oral infections
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-caries
-oral abcesses -strep throat -tonisilis -laryngitis |
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Streptococcal Infections of internal organs and tissues
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-endocarditis
-pneumonia -otitis media -sinusitis -pvelonephritis -septicemia -scarlet fever -rheumatic fever -glomerulonephritis |
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Streptococcus capsules
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help prevent phagocytosis by host's immune system
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Streptococcus cytolytic toxins
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hemolysins and leukocidans
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Does streptococcus have hylaronidase?
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yes
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Streptococcus streptokinase
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breaks down fibrin clots and enables spread
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Streptococcus pyrogenic exotoxins
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cause body to have fever response
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Streptococcus erythrogenic toxin
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-associated with scarlet fever
-exotoxin is secreted and gets into bloodstream causing a reddening of the skin and a white coat on the tongue |
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How can you control food spoilage?
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1. Removing contamination microorganisms
2. Inhibit growth of microorganisms 3. Ancient methods |
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How do you remove contaminating microorganisms?
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-heat, pasteurization, filtration, chemicals, irradiation
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How do you inhibit growth of microorganisms?
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-low temperature storage, dehydration, increasing solute concentration, pH reduction, chemical inhibitors (preservatives), altering storage conditions (O2 availabiltiy)
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Ancient methods
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-heat, salting, smoking, microbial fermentations
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Preventing spoilage with microbes
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-in use since at least 4000BC
-some common organisms: lactic acids & priopionic acid bacteria, yeasts |
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Sour cream, buttermilk
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-mesophilic lactic acid bacteria fermentation
-lactobacillus and lactococcus -occasionally streptococcus -Leuconostoc |
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Kefir
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-Lactobacillus brevius
-Streptococcus lactis -Saccharomyces delbrueckii |
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Where was yogurt first created?
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In Asian and Eastern European countries
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What kind of fermentation is used to produce yogurt?
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Thermophilic
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Yogurt production
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-traditionally a 1:1 ratio Lactobacillus an dStreptococcus
-probiotics add Bifidobacterium and others |
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Disinfectants and antiseptics
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-can have static or cidal effectson bacteria
-disinfectants used on non-living surfaces or formite -antiseptics used on living tissues |
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Disinfectant examples
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-Lysol, bleach etc
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Antiseptic examples
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-mouthwash, hydrogen peroxide, mercurochrome
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Antimicrobials can be
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bacteriostatic or bacteriocidal
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Bacteriostatic
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inhibit growth of bacteria
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Bacteriocidal
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kill or destroy bacteria
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Phenolic Compounds
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Lysol, Listerine
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Alcohols
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70% EtOH, Witch Hazel
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Synthetic Detergents
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Zephiran
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Heavy Metals
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Mercurochrome, Zinc oxide
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Oxidizers
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Bleach, peroxides
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Both antiseptics and disinfectants have a general affinity for ______
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organic matter (ex: microbe cell, flesh)
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What do organic materials do to antiseptics or disinfectants before they can react with microbes?
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react with and inactivate or dilute them
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Are disinfectants and antiseptics sterilizing agents?
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No; they do not always kill all fungal and bacterial spores and vegetative cells
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Chemical Treatment affected by concentration
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-100% EtOH is less effective than 70% EtOH
-requires water activity for reactions to occur=hydrolysis reactions |
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Chemical Treatment affected by application time
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must be sufficient for penetration of the material to be disinfected and the specific chemical/microbe interaction to be achieved
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Efficacy Testing by Disc Assay
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-make a lawn of culture on plate (TSAYE and BAP)
-soak a filter paper disc in disenfectant or antiseptic; drip/wick off excess liquid -place discs on plate and tap into place with sterilized forceps -incubate for 24 hours and then examine for zones of inhibition -judging: if growth occurs to the very edge of the disc=ineffective |
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Antibiotics
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natural substances isolated from a biological source (bacteria or fungi) that are antagonistic to microorganisms
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designer drugs
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antibiotics with synthetic chemicals
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Broad spectrum antibiotics
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acts effectively on both Gram negative and positive
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Narrow spectrum antibiotics
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primarily acts on a single group of organisms
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What percent of discovered antibiotics have proven clinically useful?
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less than 1%
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Antibiotics: Inhibition of Cell Wall Synthesis
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-bacteriocidal
-bind to transpeptidases which inhibits cross linking of cell wall; results in cell lysis -vancomycin inhibits synthesis of peptidoglcan precursors, which also weakens cell walls |
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Antibiotics: Cell Membrane Interference
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-bacteriocidal high concentration, bacteriostatic low concentration
-binds to LPS (G-), displacing Ca2+ and Mg2+ bridges that stabilize it -leads to changes in membrane permeability and inhibition of respiration |
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Antibiotics: DNA Gyrase Inhibition
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-bacteriocidal
-interact with DNA gyrase preventing it from supercoiling DNA -supercoiling is required for packaging DNA in the bacterial cell |
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Antibiotics: RNA Polymerase Inhibition
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-bacteriostatic
-rifampin and streptovaricin bind to the beta subunit of RNA pol, and inhibit mRNA synthesis -actinomycin inhibits RNA elongation by binding the major groove of DNA at G-C base pairs |
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Antibiotics: Inhibition of Translation
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-bacteriostatic
-binding to either subunit prevents the formation of the ribosome complex so that no protein synthesis can occur |
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Antibiotics: Inhibition of Folate Synthesis
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-sulfonamides are analogs of p-aminobenzoic acid and competitively bind dihyropteroate synthase
-trimethroprim competes with DHF in dihyrofolate reductase -both acctions inhibit folate synthesis, which is a precursor of nucleic acids -selectively toxic to bacteria -humans can take up folic acids from their diet, bacteria must synthesize |
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Measuring Antimicrobial Activity
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-minimal inhibitory concentration (MICs): the smallest amount of agent needed to inhibit the growth of a test organism
-important for dosage |
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Antibiotic Suceptibility Testing
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determine which antibiotics an infectious bacterium is susceptible to so that the prescribed antibiotic will be effective
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Do doctors usually test antibitoics?
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No, for the most part, doctors are aware of which antibiotics are effective against which pathogens and prescribe without testing
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What to do if antibiotic not showing effectiveness
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-perform test to challenge isolate with different antibiotics/concentrations
-whichever works best is prescribed |
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Kirby Bauer Method
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Standardized Disc Susceptibility Test as outlined by federal registrar
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Standards/Precautions of KB Method
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1. Culture medium must be Mueller-Hinton Agar at at depth of 4mm
2. Inoculum must be standardized against McFarland Standards and only pure cultures can be used 3. Plates must be incubated at 37C for 18-24 hours |
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KB Method Steps
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1. MHA inoculated with desired organism (spread plate)
2. Discs impregnated with antibiotics are placed on surface of medium using dispenser 3. Plates incubated for 18-24 hours at 37C 4. After incubation, plates are examined for zones of inhibition, which are the areas around the disc where no growth occurred 5. Zones of inhibition are measured and the diameter is used to determine resistance or susceptibility 6. Zone-Size Interpretive Chart 7. Numbers obtained by using quality controlled type strains and are dependent upon concentrations of the antibiotic |
