1
Q
Elevated Temperatures
A
- kills most microorganisms because of susceptibility of macromolecules to heat
- proteins denature and unfold
- damage to nucleic acids which prevents cell division and protein synthesis
- damage to coenzymes
2
Q
Endospores
A
- resistant to heat and allow organisms to live in high temperatures
- belong to genera Bacillus and Clostridium
- lower water content so macromolecules are less susceptible to denaturation
- water is unavailable for chemical reactions that can damage macromolecules, coenzymes and other essential small molecules
- contain calcium dipicolinate that provides resistance to oxidizing agents that cause destruction
- specific proteins found in endospore can bind to nucleic acids and prevent denaturation
3
Q
Thermal Death Point
A
-lowest temperature at which a population of a target organism is killed in 10 minutes
4
Q
Thermal Death Time
A
-shortest time required to kill a suspension of cells or spores under defined conditions at a given temperature
5
Q
Temperature Lethal Effects Lab
A
- subject cultures of 3 different bacteria to temps of 60, 70, 80, 90, and 100 C
- at intervals of 10 minutes samples of test bacteria are removed and plated out to determine number of survivors
- Bacillus megaterium forms endospores and survives high temperatures
- strep faecalis is thermoduric
6
Q
Antimicrobials
A
- compounds that kill or inhibit microorganisms
- agents can vary in their effectiveness against various pathogenic bacteria
- some are narrow in their spectrum and some may be more effective against gram-negative bacteria
- broad-spectrum antimicrobials are effective against both kinds of organisms
7
Q
Antibiotics
A
- antimicrobials, usually of low molecular weight produced by microorganisms that inhibit or kill other microorganisms
- penicillin and streptomycin
- often chemically altered to make them more effective–>semi-synthetics
- synthetics are chemically synthesized in a lab and not produced by microbial biosynthesis
8
Q
MRSA
A
- methicillian-resistant S. aureus
- responsible for many health-care acquired infections
9
Q
Determination of Spectrum of Antimicrobials
A
- depends on mode of action and ability to be transported into the cell
- have different modes of action and affect different aspects of bacterial cell metabolism
- can target cell wall synthesis, DNA and RNA synthesis, protein synthesis, and vitamin synthesis
- permeability also has effect on ability-less permeable bacteria restrict entry of antimicrobials into cell
10
Q
Kirby-Bauer Method
A
- used to determine sensitivity or resistance of bacterium to an antimicrobial
- standardized test procedure
- streak plate uniformly
- place paper discs with specific concentrations of an antimicrobial or antibiotic deposited on agar surface forming a concentration gradient
- zone of inhibition forms if drug is effective
- measure zone and compare to chart to find if antimicrobial was resistant, sensitive, or indifferent.
- inoculated with cotton swab from broth culture
- incubate 16-18 hours then measure
- measure all the way across from one edge of zone to other
11
Q
Mueller-Hinton II Agar
A
- recommended for Kirby-Bauer Method
- pH 7.2-7.4
- uniform thickness of 4 mm
- for certain fastidious microorganisms 5% defibrinated sheeps blood is added to medium
12
Q
What factors influence the size of the zone of inhibition for an antibiotic?
A
- size of drug
- how well it diffuses out into the medium
- the resistance of the microorganism
13
Q
Antiseptics
A
- substances that inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue
- don’t destroy endospores
14
Q
Disinfectants
A
- chemical agents that are applied to inanimate objects
- more harsh than antiseptics and damaging to living tissue
15
Q
Sterilants or Sporcides
A
- destroy all microbial life, including endospores
- type of disinfectant
16
Q
Sanitizers
A
-reduce microbial numbers to a safe level but do not completely eliminate all microbes
17
Q
Bacteriostatic
A
-inhibits growth of bacterial cells but does not kill them
18
Q
Bacteriocidal
A
-agents that kill bacterial cells
19
Q
Evaluation of Antiseptics
A
- compare antiseptics against each other
- streak plate covering completely
- soak paper discs half in antiseptic
- place on dish
- incubate 24-48 hours
- measure zones of inhibition and compare to each other
- measure from edge of disc to edge of zone
20
Q
UV Light
A
- nonionizing short wavelength radiation that falls between 4 nm and 400 nm in visible spectrum
- the shorter the wavelength, the more damaging to the cell
- so UV light is very germicidal than visible light and infrared radiation
- kills most bacteria and is used in sterilization
- most germicidal at 260 nm because this is the wavelength at which DNA maximally absorbs UV light and this causes pyrimidine dimers
- deforms DNA so polymerase can’t read past it
21
Q
SOS System
A
- enzymatically removes dimers and inserts new pyrimidine molecules
- can move beyond point where dimers have formed in molecule
- unable to fix all the massive amounts of dimers formed via UV light and makes errors inserting wrong bases eventually resulting in cell death
22
Q
Killing properties of UV light factors
A
- time of exposure
- presence of materials that will block radiation from reaching cells
- presence of endospores-protected by small acid soluble proteins that bind to DNA and alter conformation, thereby protecting it from photochemical damage and unique spore photo-product is generated by UV light in endospores that functions in enzymatic repair of damaged DNA during endospore germination
23
Q
Staphylococcus
A
- means bunch of grapes
- gram positive, spherical that divide into more than one plane to form irregular clusters of cells
- non-motile, non-spore forming and able to grow in high salt concentrations
- most are facultative anaerobes
24
Q
S. aureus
A
- 20-30% of population carries this
- responsible for many serious infections (MRSA)
- most clinically significant staphylococcal pathogen
- can cause skin infections, wound infections, bone tissue infections, scalded skin syndrome, TSS and food poisoning
25
Virulence Factors of S. aureus
- coagulase positive-may cause clot to form around staphylococcal infection thus protecting it from host defenses
- DNase
- produces alpha toxin that causes a wide clear zone of beta hemolysis on blood agar but also damages leukocytes, heart muscle, and renal tissue
- pigment that has antioxidant properties which prevent reactive oxygen produced by the host immune system from killing the bacteria
- ferments mannitol to produce acid
26
Mannitol Salt Agar
- production of acid lowers the pH of the medium, causing the phenol red indicator to turn from red to yellow
- differential because of mannitol fermentors vs. non fermenters
- selective because of increase salt and selects for s. aureus because it survives in high salt
27
Coagulase Negative Staphylococci
- CNS
- don't produce coagulase or DNase or alpha toxin
- all people have this on their skin
- unpigmented and appear opaque when grown on blood agar and staphylococcus 110 plates
28
What are health-care-acquired infections?
-infections that patients develop during the course of receiving healthcare treatment
29
Streptococci
- most isolates occur in chains rather than in clusters
- lack catalase
- gram-positive
- facultative anaerobes
- generally non-motile
- very similar to enterococci
30
Beta-hemolysis
- complete lysis of red blood cells in the blood agar
| - clear zone surrounding colonies
31
Alpha-hemolysis
- partial breakdown of hemoglobin inside red blood cells
| - produces a greenish discoloration around colonies
32
Gamma-hemolysis
-have no effect on red blood cells in blood agar plate
33
Beta-hemolytic Groups
-Lancefield serological groups that fall into this are A, B, and C and some of group D
34
Group A Streptococci
- main rep is strep pyogenes->humans primary reservoir
- when grown on blood agar->small, transparent to opaque, and domed
- produce hemolysins which rapidly injure cells and tissues which result in complete lysis of red blood cells around each colony producing a clear zone at least 2-4 times the size of the colony
- spherical cocci, arranged in short chains in clinical specimens and longer when grown in broth
35
Group B Streptococci
- S. agalactiae is only recognized species of Lancefield group B
- important cause of serious neonatal infection involving sepsis and meningitis
- in adults infections consist of abscesses, endocarditis, septicemia, bone and soft tissue infections, and pneumonia
- large colonies with a narrow zone of beta-hemolysis
- spherical to ovoid and occur in short chains in clinical specimens and long chains in culture
36
Group C Streptococci
- uncommon human pathogens but may be involved in zoonoses
- s. dysgalactiae is important member
- can cause pharyngitis, endocarditis, and meningitis but most clinical infections from this group occur in patients with underlying illness
- produce large colonies with large zone of beta-hemolysis on blood agar
37
Alpha-hemolytic Groups
- not as clear cut
| - some groups exhibit weak alpha-hemolysis and a few alpha-hemolytic types may exhibit variable hemosysis
38
Group D Enterococci
- considered variably hemolytic
- e. faecalis and e. faecium
- don't possess many virulence factors, but are important pathogens in hospitalized patients where they can cause UTI's, bacteremia, and endocarditis
- form large colonies that can appear nonhemolytic, alpha-hemolytic, or rarely beta-hemolytic
- grow ad diplococci or short chains in culture
- enterococci can grow under extreme conditions and this phenotype can be exploited to help differentiate them from various streptococcal species
39
Group D Nonenterococci
- s. bovis is only clinically relevant non enterococcal species of group D
- has been implicated as a causative agent of endocarditis and meningitis and is associated with malignancies of the gastrointestinal tract
- appear large, mucoid, and either nonhemolytic or alpha-hemolytic
40
API Staph System
- reliable method for idnetifying 23 species of gram-positive coccis including 20 clinically important species of staphylococci
- 19 microcupules that contain dehydrated substrates and/or nutrient media
- make saline suspension of organism from isolated colony
- staph strip placed in tray that has small amount of water added to provide humidity during incubation
- pasteur pipette is used to dispense 2-3 drops of bacterial suspension into each microcupule
- covered in incubated aerobically for 18-24 hours
- 7 digit profile number obtained and used to determine identity of organism in appendix
41
Enterics
- 1 of 3 groups of gram negative rods
- gram negative, nonsporeforming, facultative anaerobic rods that ferment glucose with the production of acid and gas and can grow in the intestinal tracts of warm blooded mammals
- three groups of intestinal pathogens in this group: salmonella, shigella, and vibrio->all manifested in diarrhea
42
Salmonella typhi
- causes typhoid fever
- last several weeks and has two stages
- stage one: low-grade fever, constipation, organisms can only be cultured from blood
- stage two: liquid, bloody stools, high fever, organisms can only be cultured from stool
43
Salmonella typhimurium
- causes gastroenteritis (food poisoning)
- irritate mucous membranes and diarrhea results
- only lasts a few days
44
Shigella
-causes bacillary dysentery (diarrhea containing white blood cells or pus)
45
Vibro cholorea
- causes cholera
- produces powerful toxin that radically upsets ability of intestinal cells to contain water, hypersecretion of water results causing diarrhea
- leads to dehydration, acidosis, shock, and death
- characterized by rice water stools
46
Pyrogenic
- all gram negative rods are this
| - cause fever due to par tof cell wall called lipopolysaccharides
47
MacConkey Agar
- selective due to bile salts and crystal violet and differential because it contains the pH indicator, neutral red
- tests for lactose fermentation and indicates that organism is a gram negative rod
- streak organisms for isolated colonies
- growth=gram negative rod
- positive=red colonies=lactose fermenter
- negative=colorless colonies=non-lactose fermenter
- all pathogens (salmonella and shigella) are lactose negative
48
TSI Agar
- Triple Sugar Iron Agar
- differentiates gram negative enteric bacteria
- stab loop down to center of butt all the way to bottom (inoculation for anaerobic growth)
- pull loop out of agar and zigzag streak up slant (inoculate for aerobic growth)
- incubate for 24 hours at 37 C
- time is critical after 24 hours tubes are unreliable
- TSI contains 3 sugars-10X more lactose and sucrose than glucose; sodium thiosulfate-detects organisms that can produce H2S and phenol red-alkaline Red=K and acid yellow=A
- first letter refers to top half and second refers to bottom stab (K/A A/A and K/K)
49
Reactions of TSI Stabs
- K/A=only glucose fermented-top red, bottom yellow
- A/A=glucose and lactose or sucrose fermented-all yellow
- K/K no sugar fermented (non fermentors)-all red; not enterics
- gas production is detected by gas bubbles in the agar
- so K/A + gas or A/A + gas
- some organisms produce H2x (salmonella and shigella) produces black precipitate so K/H2S or A/H2S
50
Urease
- splits urea into CO2 and ammonia
- ammonia cause pH to rise above 8.4 where phenol red, is a deeper red
- produced by some of the gram-negative enteric bacteria (proteus, providencia, morganella) which can be differentiated from the to others by this test
- inoculate urease test medium which is a light pink slant
- positive turns hot pink
- negative is light pink
51
Indole
- organisms that possess tryptophanase can cleave tryptophan to indole
- use Kovac's reagent to detect which produces cherry red color
- use indole test medium (tryptone broth which is pale yellow) and kovac's reagent
- inoculate broth and incubate at 37 C for 24-48 hours
- add 5 drops Kovac's reagent and wait
- positive=cherry red ring
- negative=yellowish ring
52
Citrate
- test medium contains ammonium phosphate which when utilized results in release of ammonium ions and an increase in pH.
- pH indicator Bromothymol Blue changes from green to blue above pH 7.8
- use simmons citrate slant which is green agar
- inoculate sland and incubate for 24 hours at 37 C
- positive=growth and slant turns blue
- negative=no growth
53
Non Fermenters
- second group of gram negative rods
- aerobes
- Pseudomonas, alcaligenes, acinetobacter, and moraxella
- opportunistic pathogens
- not part of normal flora
54
Pseudomonas
- causes UTIs, septicemia, upper and lower respiratory tract infections and colonizes wound and burn infections
- grown on light-colored media will produce a diffusible water soluble, green pigment called pyocyanin and a grape like odor
- oxidase positive
55
Acinetobacter
- isolated less frequently but can cause same type of infections but is less resistant to antibiotics
- Moraxella and alcaligenes are isolated even less frequently
56
Oxidase Test
- tests for presence of cytochrome C
- all organisms that can grow in the presence of air have cytochromes but they may differ
- oxidase reagent detects only C cytochrome
- thaw oxidase reagent
- wet very tip of q-tip
- touch tip to bacterial colony to be tested
- observe for color change and time reaction
- positive=turns dark purple in less than ten seconds
- negative=no color change or turns purple after twenty seconds
- questionable=turns purple at 10-20 seconds
57
Metabolism
-the sum total of chemical reactions that occur in a cell
58
Exoenzymes
- function outside of cell to degrade large macromolecules
- break down proteins and polysaccharides and monosaccharides which are then transported into the cell for metabolic needs
59
Coenzymes
- assist other enzymes in catalytic reactions
| - transfer small molecules from one molecule to another
60
Oxidative Phosphorylation
-shuttling of electrons down an electron transport chain involving cytochromes facilitates the movement of the proteins to the outside of the cell
61
Substrate Level Phosphorylation in Fermentation
- metabolic intermediates in pathways directly transfer high energy phosphates to ADP to synthesize ATP
- fermentation much less efficient in producing energy relative to respiration because the use of metabolic intermediates as electron acceptors leaves most of the available energy in molecules that form the end products
62
Tryptophan Degredation
- degradation of tryptophan by this enzyme produces indole, ammonia, and pyruvic acid
- pyruvic acid can then be used by organism for various metabolic purposes
- degradation of tryptophan by enzyme can be detected with Kovac's reagent which forms a deep red color if indole is present
- tryptone broth used for test because it has high amounts of tryptophan
63
Urease Medium
- contains yeast extract, urea, a buffer, and the pH indicator phenol red
- when urease is produced by an organism the ammonia causes pH to become alkaline and phenol red changes from yellow to bright pink
64
API 20E System
- mini version of conventional tests used for identification of members of the family Enterobacteriaceae and other gram-negative bacteria
- saline suspension made then filled with bacterial suspension
- incubate and record
- results tabulated and profile number generated to be compared to known numbers to determine organism
65
Enterotube II
- mini multitest system
- 15 tests and 12 different media to be simultaneously inoculated
- pull wire through to inoculate
- positive reactions given numerical values to generate a code to be compared to an index
66
Bacteriophages
- viruses that infect bacterial cells
- obligate intracellular parasites-all viruses lack metabolic machinery like energy systems necessary for independent replication so they must use host cell's metabolic machinery to synthesize various component parts
67
Capsid
-protein coat that surrounds one type of nucleic acid in a virus
68
Host Specificity
- viruses exhibit this
- certain bacteriophage my only infect specific strain of bacterium
- can be used in phage typing of pathogens
69
Phage Composition
- nucleocapsid-nucleic acid and protein capsid
- protein sheath-contractile and contains hollow tube in center
- base plate to which tail fibers and spikes are attached
- most of phage structure is necessary for delivery of the phage nucleic acid into host
- single virus or phage particle is called viron
70
Steps in Lytic Phage Infection
- adsorption
- entry of viral genome
- synthesis of phage components
- release of virus
71
Adsorption
-bacteriophage recognizes host by tail fibers binding to chemical groups associated with receptors on the surface of the host cell
72
Entry of Viral Genome
- phage particle settles onto surface of host bacterial cell and lysozyme begins to erode local area of cell wall
- sheath contracts forcing hollow core connected to phage into cell
- viral genome injected into cytoplasm
73
Synthesis of Phage Components
- only phage nucleic acid enters host cell
- lytic infection
- to incapacitate host cell and ensure phage components are synthesized a viral nuclease injected with the viral DNA begins to degrade DNA so host cell can't carry out own metabolic functions
- viruses leave intact host cell metabolic machinery for producing energy and synthesizing nucleic acids and proteins
- eclipse period because no mature virus can be detected
- once all parts synthesized they come together by self-assembly to form mature phage particles
74
Productive Infection
-new phages are made and released from cell caused by lytic cycle
75
Lysogenic
- infect bacterial cell, phage genome can integrate into host DNA to become stable genetic element in host genome
- each time bacterial genome replicates phage DNA also replicated and all progeny contain phage DNA as part of genetic makeup
- integrated phage genome is known as prophage or provirus
76
Release of Virus
- combo of weakened cell wall resulting from lysozyme plus pressure of phage particles on cell wall causes cell to burst releasing mature phages
- lytic phages produce new lytic infections and lysogenic produce more lysogenic
- burst size is the number of virons that come out of a single host cell
77
Confluent Lawn
- produces when bacterial cells are mixed with bacteriophage in soft agar
- phages infect cells causing them to undergo lysis and form clear areas in lawn called plaques
- each plaque is formed by the progeny of a single viron that has replicated and lysed bacterial cells
78
PFU's
-plaque forming units can be counted to determine number of viral particles in a suspension of phage
79
Host Specificity
- viruses exhibit this
- certain bacteriophage my only infect specific strain of bacterium
- can be used in phage typing of pathogens
- such that it is possible to differentiate strains of individual species of bacteria based on their susceptibility to various kinds of bacteriophages
- since most bacteria are probably infected by bacteriophages it is theoretically possible to classify each species into strains based on their phage susceptibility
80
Bergey's Manual of Determinative Bacteriology
-four volume set separates bacteria into groups by phenotypic characteristic like gram stain reaction, cell shape, oxygen requirements, and metabolic properties, but it also includes detailed information on the relationships between organisms
81
Methyl Red Test
- measures mixed-acid fermentation
- some bacteria lower pH of broth to 5 or less so color change takes place to indicate mixed acid fermentation
- methyl red added to medium
- mixed acid fermentors also generally produce gas because they produce formic hydrogenlyase which splits formic acid to produce CO2 and H2
- add 5 drops methyl red to MR-VP tube after 48 hours
82
Voges-Proskauer Test
- indicates glucose fermentation to produce limited amounts of organic acids and a more neutral end product 2,3-butanediol
- 2,3-butanediol not detected directly but must be converted to acetoin by oxidation
- acetoin reacts with Barritt's reagent
- add 10 drops of Barritt's A and 10 drops of Barrett's B to MR-VP tube after 48 hours
- if positive tube turns pink
Microbiology M-315
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