final material
What does the FAB region do (what does it promote) , what would happen if we removed it ?
- the FAB region of an antibody is where antigens bind - it promotes neutralization and opsonization of the antigen , if we removed it, there would be none of this - maybe complement wouldn't be able to happen; because its needed for complement binding
why would using a rapid id test be better than the other lab tests we have discussed?
- use molecular detection tests for HARD TO GROW / cant grow microbes - no need to culture -results quicker because dont need to incubate
why is it not good to use a traditional transposable element, what is the ideal TE? what are the 2 ways we can get the ideal TE.
-Drawback to traditional TEs: very unstable, constantly move in the genome - the IDEAL TE is one that integrates in the genome and has no further movement n the genome: -Can get the Ideal TE with 1) Introduce the TE without the transposaze enzyme, only the marker is in it. add the transposaze enzymes in separately for a short while until have sufficient population of unique mutants 2) Use of a suicide vector to provide the transposase enzyme in TRANS; the plasmid CANNOT replicate. can only express the Transposase for a short amount of time, allowing for the marker TE to insert into the hosts genomes . The plasmid will be lost overtime with subsequent cell divisions and the TEs will be stuck in place
Define gene knockout
-gene inactivation to disable promoter, introduce frame-shifts, early stop codon, gene deletion, etc Loss of function of a gene due to manipulation of DNA. Cloned gene is a mutation that can inactivate function of a gene.
what are the ways we can identify pathogen , just list the ways we can and if it is conventional lab approach or rapid
1) Clinical lab approach : a) Determine by structure b) determine by metabolism 2) RAPID: A) Nucleic acid based techniques - still in lab though (PCR, QPCR) B) antibody via immunoglobulin use (ELISA OR Fluorescent antibody staining) - can also still be done in the lab C) POC test-
how can immunity prevent microbes from infecting
1) DIRECT killing mechanism via A) opsonization or phagocytosis, to cause a pH change and digest compounds or B) complement killing/lysis 2) Blocking via neutralization or just physical barriers like epithelium
what are the 4 antibiotic resistant mechanisms microbes can take (just list them)
1) Destroy the antibiotic 2) modify the antibiotic 3) modify/alter the target 4) Drug efflux pump
what does a (E) strip test do & determine ? what does the Kirby Bauer disk diffusion method determine
1) E-test:determines the MIC- gradient of antibiotic impregnated on a strip. check for zone of clearance. the lowest part of the teardrop is the MIC 2) Kirby: determines the MIC of a specific antibiotic on a disk. Discs have different levels of antibiotics
what are the 3 main concerns with the immune system
1) How does the immune system recognize self from non-self 2) how can immunity prevent microbes from infecting? 3) How can immunity protect against future infections?
how do we ID what genes cause what phenotypes (how do we go about assigning gene function?)
1) Inactivate a gene lead to loss of phenotype via mutation 2) re-activate the gene to restore phenotype via complementation to show gene important for phenotype
what are some chemical methods used to determine the efficiency of antibiotics/antimicrobials. what one is always higher for a specific microbe & drug how can you tell if a microbe is more resistant to the drug
1) MIC: minimal inhibiting concentration- the lowest concentration to PREVENT GROWTH- precision is dependent on dilutions. varies per species. the microbes are constant across the dilution, but the drug amount differs. MAY still have living cells but doesnt actively divide anymore 2) MLC: minimal lethal concentration- lowest concentration to kill all microbes in the media. SAME step as the MIC but you just plate the dilutions to observe if any are actively growing. The plate that gives no growth is the MLC the MLC will always be higher than the MIC for a drug & microbe the lower the MLC the less resistant, the HGIHER the MC the MORE resistant
what are the 6 ways bacteria can evade the host immune system,
1) PHASE VARIATION: change outer surface antigen to avoid immune detection via mutation = no memory now 2) BIND to FC region to prevent FAB binding of the antigen and complement binding 3) degrade antibodies with enzymes 4) Form structures to prevent access of immune cells (biofilms, capsule)- also allow resist to phagocytes bc pH resistant 5)go intracellular to avoid humoral immunity 6) kill immune/effector cells
List Molecular Koch's Postulates
1) The phenotype/property under investigation should be associated with pathogenic members of a genus or strains of a species. The ability to cause disease is associated with other pathogenic organisms and not seen in non pathogenic causing organisms. Can also work if narrow down gene for virulence too. 2) Specific inactivation of the gene(s) associated with the suspected virulence trait should lead to a measurable loss in pathogenicity or virulence. Break gene no longer shows phenotype: - break gene by: 1) adding in a TE without the transposase enzyme, but it does have a marker on it (antibiotic resistance or something to make it luminesce). add in the TE to the wild type population. Plate on the antibiotic to gather ALL of the mutants. Later, see if it knocked out the gene with a competition assay & calculating the competition index or if it has the same affect or not in its host when injected into the host -2) can also just sequence the genome, compare it to known databases of pathogenic strains, see which sequences are for virulence and compare/ search for homology in the known virulence genes - -measure virulence by competition assay & competition index 3) Reversion or allelic replacement of the mutated gene (COMPLEMENTATION) should lead to restoration of pathogenicity. The gene us restored, restoring phenotype. Can be expressed by using a strong promoter with the 1 gene encoding the knockout gene product (SG). If supplying this one product brings the phenotype back, it was the cause of the virulence factor. MAKE SURE to observe polar mutations. If knockout an entire operon with a mutation (polar mutation) you can add in one plasmid containing a strong promoter and just 1 gene from that broken operon.. if the supplied plasmid with the strong promoter restores function, that ONE gene was responsible for virulence, all downstream genes dont matter.
what are the 5 general steps to infection of microbes
1) attach & invade host tissues in host environment 2) suppress host defense (immune system) 3) Acquire nutrients from host 4) propagate in host 5) transmit to new host
what are the types of microbiomes based on their mechanism of action?
1) bactericidal: Kills the target organism 2) bacteriostatic: Prevent growth of target organism. May not directly kill vegetative cell but long term use can indirectly kill it usually target actively dividing cells, have to continue use until the cells leave the stationary phase
what are the types of antibiotics based on the targeted microbiome range?
1) broad spectrum- effect many organisms and species 2) narrow spectrum- effective against very few OR a single microbial species
explain what phagocytosis is the 7 steps
1) chemotaxis and adherence of the microbe to the phagocyte- PRR recognize pamp 2) Ingestion of microbe by the phagocyte 3) Phagosome is formed (intracellular compartment), and protons are pumped in to lower the PH 4) The pH change can kill or harm the antigens, and here it fuses with another compartment that can contain substances to further digest the microbe (lysosome) to form a phagolysosome 5) the lysozymes digest the antigen 6) the residual body is formed with the indigestible peices 7) discharge of wast/indigestible pieces into the environment
describe how microbes can be controlled with chemical agents (3 types) and how!! - the types of microbes they kill -where they are used in comparison to objects & or people -if their mode of control is selective or not
1) disinfectants - chemicals kill all microbes INDESCRIMINATLEY (no selective toxicity)- hurts human cells too so must be used on surfaces and usually not on/in humans (bleach, iodine, soap) 2) antiseptics- chemical can be applied on body surfaces NOT INSIDE THE BODY- to prevent infection of living tissues- i dont think it has selective toxicity 3) CHEMOTHERAPY- chemicals to kill/inhibit microbe growth WITHIN HOST TISSUES. such as antibiotics. has selective toxicity to only target bad microbes, not host
describe the growth curve of viruses (bacteriphages)
1) eclipse phase - the virion concentration FREE in the cell/culture is very low. because the virus is binding to the host cell & entering (looks low) - drop in initial [] 2) RISE period: after the eclipse period, the rise in virion concentration in the culture via LOG/exponential. rises until all bacteria are dead/lysed 3) END of rise: cell lysis is complete, hit the max point of [virions] in the culture bc killed all available hosts. cell lyses is complete time x axis pages/mL in culture in y axis
what are 3 mechanisms bacteria use to resit phages, list if they are active or indirect
1) genetic resistance (indirect)- dont have correct host cell receptor to initiate attachment and entry 2) restriction endonucleases- ACTIVE: control foreign DNA entering the cell via cleave viral DNA not methylated 3) CAS-9: also active: integrate phage DNA as a spacer, transcribe it, join w/ cas9, looks for homology DNA to destroy
in classification based on shape or structure, what are the 3 main types of shape/structures (symmetry)
1) icosahedral - spherical with rotational symmetry. has 20 different faces with many axis. 2) Filamentous helical symmetry 3) Asymmetrical- complex with many parts I.E pox vs bacteriophages
what are the 4 key things needed in order for an organism to be considered genetically tractable:
1) need to be grown in pure culture to preform tests in isolation 2) need to be able to introduce DNA into the cell (transform, conjugate, transduce) (plasmid, phage, TE) 3) express the TE enzyme (if add a promoter for the enzyme, need to be able to make it to be able to jump in) 4) need selectable phenotype markers for identification of mutants - insertion of a TE with antibiotic resistance etc
what is the immune system induced by? what is inflammation
1) pathogen- infectious agent 2) Allergin- substance that induces immune response against otherwise harmless stimuli 3) antigen- broad statement for anything that causes the production of antibodies all of these- can lead to a global response called inflammation, leading to the neutralization or killing of the foreign substance. After inflammation the system goes back to the quiescent state
how does the epithelium act as a barrier
1) physical barrier to stop passage of a pathogen 2) physical barrier of a pathogen via movement across the epithelium allowing the FLUSHING of microbes (create inhospitable environment) 1) chemical barrier to secrete chemical antimicrobials that degrade the integrity of bacterial cells
describe the 5 functions of epithelium
1) secrete things into the environment or onto host tissues (mucosa) 2) absorb nutrients, 3) transportation in and out 4) sensing the environment 5) 1st barrier of defense against invaders
1) what is the immune system 2) what is it called when the immune system targets its own healthy cells 3) due to the dangers of (#2 answer) how does the immune system normally function
1) the immune system is a complex system of numerous cell types to define self from non self 2) autoimmunity 3) due to the danger of autoimmunity, the immune system is kept closely regulated and its resting state is OFF (quiescent) , when it is activated, the cascading events are very regulated
What is a plaque assay?
A virological assay developed to count and measure active viruses. We count these up to determine how many viruses are present, as each virus copy infects one cell. each plaque represents one virus that replicated and lysed the surrounding cells to form a plaque (PFU) # of plaques (holes) = # of viruses in a sample mix virus with host cells, in different dilutions; pour onto plate, incubate , will form a lawn if no virions, - observe clearings after incubation , take the pate with 30-300 clearings, if not = too variable or could be prone to errors in counting -1 plaque formed made upon lyses from 1 virion propagating in host cells and lysing out USE A CONFLUENT MONOLAYER WITH ANIMAL VIRUSES
How is the Baltimore classification system organized? A) Virus replication mechanisms B) Whether viruses are enveloped or not C) The structure of the viral nucleocapsid D) The host tropism of viruses E) Viruses are classed as bacteriophages vs eukaryotic viruses
A) viral replication mechanism
describe the first step of the phage lifecycle
ATTACHMENT: the specific contact & attachment of virus to the host cell mediated by cell surface receptors of the host that bind to specific viral components these are conserved bacterial things usually for everyday functions (sugar receptor, flagella, etc) - they did not evolve to be targets, viruses evolved to target them
what is the API20E test?
Analytical profile index- trip technology combining many test at once . each well have a different bch test, put sample in each one. STEPWISE interpretation.
Describe the lysogenic cycle; how does it induce lytic cycle what is a bacterial cell that has one called?
Attachment → Entry of viral genome → Viral genome integrates itself into host DNA (now called prophage or provirus) → Host reproduces (reproducing own and viral genome) → Can induce lytic cycle based on certain factors (environment, ds breaks, SOS response) - can induce lytic cycle by excision (XIS) of the prophage -lambda phage (non contractile tail ) - cell with a prophage is called a lysogen -TEMPERATE ; stable host relationship,
Why might we use anti-viral treatments combining different targets of action? (select best answer) A) Not all treatments are safe for human use B) Viruses can mutate rapidly to avoid the action of some drugs C) We need to do this to control co-infections of different viruses D) Viruses commonly use enzymatic degradation of anti-viral agents in order to escape treatment E) Viruses can integrate into host genome
B) Viruses can mutate rapidly to avoid the action of some drugs ( target multiple parts of replication to reduce escape mutants)
what is the plague, what microbe is it, how is it spread (give the mode(s) of transmission) how the infection cycle works
Bacteria: yersinia pestis Gram - bacteria SPREAD: indirect from vectors on rats (zoonosis) then DIRECT human-human via pneumonic plague. Y-pestis multiplies in the stomach of the host, causing the flea to starve, the rat is the sylvatic cycle; when it spills over to the human population it is the urban cycle
A bacterial protein that binds antibody Fc would inhibit which immune functions? A) Complement activation B) Neutralization C) Co-stimulation of T-cells at the immune synapse D) A & B E) A & C F) NONE of the above
Binding to the FC region would inhibit complements binding AND would inhibit neutralization ANSWER: A & B IT WOULD ALSO inhibit opsonization but thats not on there
A phage genome integrated into the bacterial chromosome is known as a: A) intemperate phage B) temperate phage C) prophage D) pathogenicity island E) transposon
C) Prophage
You run an animal infection experiment with a mutant and wild-type pathogenic bacterium, mixed together and phenotypically tagged, in order to calculate a competitive index. The input ratio is 10/1 and the output ratio is 10/1. What can you conclude? A) The input ratio was miscalculated B) The wild-type is more pathogenic than mutant C) The mutant is more pathogenic than wild type D) The two genotypes have similar fitness in the host
D) The two genotypes have similar fitness in the host
What characterizes innate immunity? A) It is acquired after birth B) It is increases in strength in responses to infections C) It provides memory of previous infections D) It acts rapidly on initiation of infection
D) it acts rapidly to initiate infection
describe how animal DNA, RNA, and retroviruses replicate/reproduce in animals
DNA viruses replicate in the nucleus usually using the host machinery RNA viruses use RDRP encoded by the virus, then use HOST translational machinery Retroviruses: are RNA but turn into DNA via viral RTASE then insert into host chromosome as a pro-virus
Which of the following statements on drug MIC/MLC is true? A) MLC is the minimum drug concentration required to inhibit microbial growth B) MIC is the minimum drug concentration required to kill a microbial population C) We should inject disinfectants like bleach in order to control microbial infections D) MIC is typically higher than MLC E) An organism that has a higher calculated MLC for antibiotic 1 than antibiotic 2 is more resistant to antibiotic 1
E) An organism that ha a higher calculated MLC for antibiotic 1 than antibiotic 2, IS MORE resistant to antibiotic 1
What are virulence factors?
GENES that give properties of the pathogen that allow it to successfully invade and cause disease in a host > contribute to pathogenicity (virulence & or infectivity) > encoded by virulence genes; highly regulated, only on when needed > can be distributed across the genome or could be in a pathogenicity island
for HIV what is the genome describe attachment and host receptors how does it enter how is it expressed
HIV is a retrovirus, has a DNA intermediate virus attach to CD4 receptor using GP120 lead to fusion of cytoplasm allow +SSRNA genome enter host cell RTASE is used to turn the RNA to DNA the DNA is brought into the nucleus then inserts into the chromosome via integrase as a provirus gets transcribed and translated, forms enveloped , buds out
define an infection, does it always cause disease? define an infectious disease?
Infection- pathogen grows and multiplies within or on another organism, DOES NOT ALWAUS CAUSE DISEASE (severe symptoms_ infectious disease is due to presence/multiplication
what are the cells of the adaptive immunity and the innate immunity
Innate immunity: Phagocytes (neutrophils, macrophages, antigen presenting cells). NON specific defender cells against foreign PAA's but PRRs recognize PAAs to further activate components of adaptive immunity Adaptive immunity: T cells and B cells MAKE antibodies specific for antigen. USED TO CLEAR INFECTION. without= cant clear. need innate to induce
what is ID50?
Measure of infectivity: the infectious dose: # organisms required to colonize 50% of the host population, determines the # microbes required to cause symptoms in half of the experimental host population A LOWER NUMBER MEANS MORE INFECTIOUS /pathogenic
what is the NON SPECIFIC way to determine self vs non self
NON SPECIFIC PARTS OF THE innate immunity: A non-specific pattern recognition receptor (PRR) made by host cell recognize PAMPs (pathogen associated molecular patterns). PAMPS are conserved microbiological structures NOT made by host trying to rid of them (LPS, LTA, flagella, peptidoglycan) -NON specific complements of the ADAPTIVE immunity:
are all infections fatal? what are some other possibilities
NOT ALL ARE FATAL , can have host cell mechanisms to stop/eliminate infection - some infections can be dude to viruses with slow release that DO NOT LYSE the host but do cause filamentous extrusion
What does PCR do? what does the size of the fragment tell you what does PCR not tell you
PCR copies/amplifies DNA segments quickly and precisely - the size of the fragment can indicate the species -begin with specific primers, the sequence specific annealing allows for amplification - just tells you if the product is there, NOT how much product is there
what are the pros and cons of a competition assay
PROS: 1) no need to strictly normalize for colonization density across all animals 2) Ratios are immediately comparable across animals, experiments under similar conditions 3) Requires much fewer animals CONS: 1) possibility for trans-complementation of phenotype the willd type may be able to supply factors to the mutant to raise the CI artificially. May think the mutant is growing better and lead to a conclusion that the gene we knocked out is not a virulence gene when it is
what are the sequential sets of actions for our bodies defense (PLAN A-C)
Plan A- BLOCK infection- microbe cannot get past host chemical and physical barriers@ site of entry (epithelium). If unsuccessful it can cause infection inside host Plan B- destroy invaders via innate immunity (phagocytes). If it is unsuccessful in containing infection it continues to trigger the adaptive immune system Plan C- trigger adaptive immune response to make specific molecules to target the pathogens/antigen. If the amplification of the adaptive is unsuccessful DISEASE OR DEATH
What is POC testing, where is it done? what are the advantages what are the disadvantages
Point of care testing, at bedside not in the lab advantages: convenient, rapid, no need to culture, can guide immediate therapy, cheaper Disadvantages: less sensitivity for speedy results (cant get low [pathogen]), provides NO data on BCH properties (antibiotic resistance etc), leads to overlooking double infections, false + & -
Explain how the drug efflux pump of microbes work to gain antibiotic resistance
Pump antibiotics out of cell using specific OR non specific transporters. can cause MULTI drug resistance.
what are the 2 ways to measure fluorescence in the QPCR,
SYBR: NON SPECIFIC, just fluoresces with each DSDNA made TAQMAN- very specific- requires a custom probe covalently linking the reporter (R) gene flouraphore to a QUENCHER (Q). when they are linked, there is no signal, when they are broken via polymerase copying & amplifying the target DNA= signal shown/glow= specific. only when actively being amplified
what is the SPECIFIC way to determine self vs non self
Specific generation of antigen specific antibodies via the adaptive immunity which is part of the humoral immunity to protect against extracellular infections
Define sterilization. Define disinfection.
Sterilization is a measure that destroys all microorganisms, including pathogens & endospores Disinfection is a process that kills/removes pathogens or vegetative microbes, but not all pathogens. both usually on inanimate objects
What does the FC region do (what does it promote) , what would happen if we removed it ?
The Fc region is the constant region - where complements bind to destroy/kill the cell. NON SPECIFIC part of the antibody killing - if we removed FC= NO complement, no opsonozation, no neutralizing the antigen that grabbed it
What is Viral tropism?
The ability of a virus to infect some tissues and not others. This involves a cell surface receptor on the host cell. CONTRIBUTES TO VIRULENCE
what is the difference of innate immunity and adaptive immunity: - the type of response -when the immune system is acquired -type of diversity -type of specificity -if they have memory - if the response is fast or slow - if the response is constant &/or amplifies etc
The innate immunity: - is the initial response, present at birth, limited diversity, NON specific (broad), no memory, FAST response, constant during response & does not amplify, unchanged after infection Adaptive immunity: - Only acquired after initial contact w pathogens&antigens NOT acquired at birth, after induction has VAST diversity of responses to SPECIFIC antigens, is amplified during the response due to amplification of antibodies, has memory, initial response is slow, faster & greater response next time
list the ways we can determine the pathogens identification by structure what does this give you
VISUALIZATION via using staining, like gram staining. then look at them under a microscope to get gram type and shape. gives the 1st indication of possible microbes in the disease associated sample
what is a plaque forming unit
a measure of the number of particles capable of forming plaques per unit volume PFU: single virus deposited on a plate with its host (to infect as an intracellular parasite) that can propagate in the host cells & form new virions & lyse out to kill the host cells & form clear zones/plaques in the plate. Each plaque represents 1 virus that infected and lysed
What is pathogenicity? what aspects does it include ?
ability of a microorganism to cause disease (yes or no type situation) includes: 1) infectivity: how easily it spread from organism to organism 2) virulence: measure of degree/severity of the disease
What is AB toxin?
an exotoxin with 2 subunits. a monomer of A and a pentomer of B the A monomer has the toxic activity the B binds to the host receptor, brings into host cell via endocytosis, and protects A ; the A translocates to mediate toxic activity
define genetically tractable
an organism that can be changed or genetically manipulated
describe what antibodies are, the parts of them, what cells they are secreted out of, describe the parts of them
antibodies are glycoproteins secreted by b-lymphocyte cells (plasma cells) Has 2 heavy chains & 2 light chains held by disulfide bonds The FAB site: is on the top light chain- where ANTIGENS BIND--> has sequence variation for different antigens The FC site: constant region, no variability bc effector activity happens here.
what is antigen presentation? what is it for? what presents the antigens? are there different types of antigen presenting cells
antigen presentation is due to Major histocompatibility complex that present foreign antigens to the cells of the adaptive immunity to later induce production of antibodies specific to that antigen - MHC1- presents INTRACELLULAR antigen which can happen when microbe invade & produce proteins or when antigen is digested via phagocytosis . PRESENT outward CD8-CTL cells -MHC2- present extracellular antigen obtained by endocytosis (IN VESICLES)- CD4 & T-cells, whole/parts of antigens from outside
what type of microbial control does pasteurization & Ultra high temperature & autoclaving provide. describe them. do they sterilize or just disinfect
both controlling microbes via temperature mechanism 1) pasteurization: kill large proportion of initial microbes in a food sample to delay log time (lengthen lag). DOES NOT STERILIZE, will spoil eventually but doesn't change food taste/texture > 63ºC for 30 minutes or 72ºC for 15 seconds 2) Ultra high temperature (UHT); STERILIZING mechanism except maybe endospores. 150ºC for 3 seconds. Allows for shelf stable food 3) AUTOCLAVING:= control via temperature and pressure- STERILIZING to ALL microbes including endospores.
how can you calculate the LD50 or ID50 experimentally? what are the X and Y axis how are the curves generated
calculate them in a susceptible host model X-axis is LOG # of the dose of the given pathogenic organism/animal the Y axis is the % of animal infected (ID50) or dead (LD50) The curves are generated by inoculated many animals at EACH dose. Need a high number of animals for high precision. 10 animals is 10% precision 100 animals is 1% precision
explain how microbes can MODIFY their own targets (how does it do it)
change phenotype= change genotype. can be done via mutation or enzymatic process to change the structure that antibiotics bind to on the bacteria so it can no longer attach (MOST common streptomycin resistance mechanism)
what are the 4 pitfalls of the LD50 & ID50
convenient because easy to measure in shorthand 1) need lots animals for reasonable precision 2) Insensitive to disease cause or how fast the progression is or the symptoms 3) CANNOT do it for weak viruses, would take too many animals to measure the curves= would be way to high 4) need to define measurements clearly with time point checks defined and site of infection
for coronavirus: what is the genome describe the attachment. and host receptors how does it enter how is it expressed
corona virus is a enveloped +SSRNA attaches to host ACE2 receptor using spike glycoprotein taken into the cell via endocytosis forming an endosome. the capsid an receptor interaction allow for fusion with cytoplasm releasing the RNA into the cytoplasm is immediatley expressed by host translational machinery then do replication with those proteins made.
explain how microbes can destroy antibiotics (how does it do it)
destroy the antibiotic before it can target and act via enzymatic activity ex: bacteria make Beta-lactamase to destroy penicillin and analogs to make resistant strains and lower the [penicillin] nearby
explain fluorescent antibody staining
fluorescent die on the FC region of the antibody, glows hen antigen bind to FAB region , view under microscope
in terms of introducing mutations to organisms to make knockout mutants of interest, how do we go about doing this? what is the best way
get random mutations; - can do via chemical or UV mutagenisis BUT there is NO MARKER to track down gene location , too labor intensive -better to do via transposons. generate mutants one at a time with a marked transposon. each transposon inserts into an individual randomly , select the mutant colonies made using the marker on the TE, make a library of mutants, narrow down if its the one you want - the simplest TE has a gene encoding for the transposase with 2 inverted repeats, best to make a suicide TE in a vector with NO transposase gene , but add in the marker gene instead
what does it mean if the TSI agar slant or butt is yellow? what if it is red? how do we know if it produced gas? how do we know if it produced the gas we want?
if the agar is yellow, its showing that it uses either sucrose or lactose and ferments to lower the pH = acidic if the agar is RED/purple the bacteria uses proteins to make alkaline base (no ferment, produce amines= basic) we know if it produced gas via bubbles/cracks in the agar we know if it produced H2S if the agar is BLACK via reaction of the gas with ferrous sulfate
in order to fulfill Kochs 3rd postulate, what do we need
in order to inoculate the cultured microbe into the susceptible host and observe the same symptoms we must chose a good model organism - most of the time use different models for different aspects of infection or interaction with the host (some colonization, some pathogenicity, etc)
describe the lytic cycle of the phage lifecycle: what it does, what the genome does when it enters the cells,
lytic vs lysogenic is decided based on environment; phage quickly replicates, leaves via lysis to kill the cell, is called INTEMPERATE; (t4 phage): attaches, inserts, DNA CIRCULARISES, transcribe, translate, package, lyses
what is a competition assay? what are the steps:
method to determine effect of a specific mutation or strains on infection or colonization., and compare it to the wiltype. Based on a corrected ratio of strains after infection as a measure of pathogenicity/fitness in the host (able to establish within the host) 1) mix mutant and wildtype together, inject a mixed population into the susceptible host 2) observe the output of wildtype bacteria CFU and the mutant bacteria CFU. see how well the mutant does relative to the wild type
what is molecular kochs postulates , what is it used for, what is needed in order to fulfill it?
molecular koch's postulates is used to understand what true virulence genes are. Assigns a role of a gene or product to pathogenesis . NOT THE OVERAL organism, the gene of the organism need mutagenesis and complementation in order to say a gene is a virulence gene that makes a virulence factor contributing to pathogenesis in an organism
what are viruses
noncellular particles that MUST infect a host cell to reproduce (OIP) and an convert host cell machinery to produce virions consists of 1 nucleic acid type nucleocapsid = capsid + NA
explain how ELISA works what is the [color] dependent on -
only need very small amounts of antigen (nano-picogram) clinical samples are serially diluted in wells (antigens diluted bound to wells) add antibody with linked enzyme add substrate , if color change= positive for that antigen/antibody reaction the [color] being shown depends on the amount of antigens bound to the antibody, the more antigens bound the stronger the color. the more dilute the antigen the weaker the color
what type of control does a biological safety cabinet provide? what about filterization?
physical or mechanical barrier using airflow and hepa air filters to trap microbes >0.3 µM, it can trap &/or have properties to kill them. may not be completely sterilizing if there are smaller microbes; can be combined with Ionizing radiation and UC filterization may be a form of sterilization as it removes anything >0.22µM. is like a hepa filter for liquid media for heat sensitive media
define a genetic screen
procedure to create and detect mutant organism with the phenotype of interest
what are microbial toxins? what do they do and what are the 2 main types, are there any other types?
products of pathogens that can alter their hosts metabolism and damage them 1) endotoxins: intrinsic to bacterial cells; the bacteria can enter the blood stream, break down , and release the toxins causing sepsis. 2) exotoxins: proteins secreted out of the pathogen IN ENVIRONMENT via a seq system which can enter the host cell or damage the host cell other types include - toxins directly injected into host cell (T3SS, T6SS effector proteins these all can prevent immune activation or damage/kill host cells
What does qPCR do and what does it measure? -how do you know which sample has more of a given target sequence? - what is on the x-axis and what is on the y axis
quantifies the amount of specific DNA using targeted primers, not used to amplify per say but used to see how much of the sample you have -measures a fluorescent that increases with each target copy increased per cycle - if the sample reaches the target threshold quicker (amount target sequence) (takes less cycles to reach) then that sample has MORE target dna in it (lower number cycles, reach target threshold= faster -x-axis is the cycle number; y-axis is the log [fluorescent measured]
how are animal viruses different from prokaryotic viruses ; how are they similar
similar: need host attachment for entry, gene expression, virion assembly, and release DIFFERENT: Euk cells are more complex so they have a greater diversity in viral replication than phages due such as compartmentalization ; the animal virus enters as a complete virion into the host, does NOT inject like phages then has to uncoat
describe what viruses are MORE error prone , include the nucleic acids and their enzymes required what can this lead to?
ss/ds/DNA using host machinery and enzymes< RNA via RTASE< RNA using rdrp because RDRP and RTASE no proofreading ability lead to mutations that change tropism of virus creating new variants (escape mutants)
what is LD50?
the lethal dose: the # of microbes required to kill 50% of the host population. A low LD50 is MORE virulent. MORE pathogenic
what is a competitive index? what happens when CI=1 wha happens when CI<1 what happens when CI>1
the measurement of the competition assay, via a corrected ratio to determine the effects of bacteria virulence factors on infection/colonization When the CI=1: the wild type and the mutant have the same fitness, the gene that we knockout was not virulent gene. both same virulence when CI<1: it means the wildtype OUT COMPETES THE MUTANT AND IS MORE VIRULENT THAN THE MUTANT. This means that the gene that we knocked out in the mutant, knocked out a virulence gene and may be a candidate gene for virulence factors. when CI>1: it means that the mutant outcompetes the wildtype and is MORE virulent than the wiltype. THis could be because we may have knocked out a genes that suppresses/controls the virulence genes, without them, they are up-regulated
What is the complement system?
the non specific aspect of the humoral immunity- set of proteins activated by proteolytic cleavage can 1) bind to the FC region of the antibody , to promote phagocytosis (opsonization) or neutralization of the antigen in the FAB region or 2) directly bind to antigen and kill it via MAC system
describe the second stage, after attachment of phage lifecycle (entry)(is there any host suppression here??)
the phage injects their genome into the cell through the host envelope and the ghost phage remains outside is usually DNA. phages can prevent host gene transcription to reserve cell machinery to the virus (target host sigma factors, use own polymerases to produce transcripts)
What is a primary function of antibodies? A) Opsonization B) Phagocytosis C) PRR activation D) Viral receptor engagement E) Engagement of MHC in the immune synapse
the primary function would be Binding to the antigen> Neutralization> opsonization Answer: A) all viable options
Define mutagenesis
the process of changing genetic information of an organism induced by physical, chemical, biological, nucleic acid tools (make mutants)
what is the "infection cycle" what are the different modes
the route of transmission an organism takes to infect additional hosts direct transmission- via direct contact or aeresolization INDIRECT- animal or insect vectors and formites
What does TSI agar test for? list the different biochemical things it tests for what does the test contain
the triple sugar iron agar is used to differentiate enteric bacteria -what sugars are being fermented (if it uses alternative sugars) -if it ferments is gas produced? if so what kind of gas? - are proteins being metabolized instead of sugars? -contains a small amount of glucose to get the reaction growth going, the contains sucrose & lactose for other sugar option, then proteins incase it doesnt/cant use those sugar options, ferrous sulfate to help show if H2S is produced in gas producing molecules, then a pH indicator
explain how we can identify microbes using antibodies (the 2 ways) ;what these test largely depend on
the two tests are ELISA and fluorescent antibody staining depend on a specific antigen binding to the FAB domain on the antibody
how do we measure pathogenicity?
using ID50 and LD50 to measure infectivity and virulence respectively
explain how microbes can MODIFY the antibiotics (how does it do it)
via enzymatic activity- enzymes modify aminoglycosidase antibiotics to add acetyl/phosphoryl/adenyl groups. Makes it so the antibiotic can no longer interfere with its target
when a MHC is presenting antigens, how does the process work to induce the immune system? where does this occur? - explain the state of the immune system with each binding response
when MHC presents antigens, immune cells (effector cells) come by and BIND in the interface called the immune synapse - when the effector cell bind to the MHC antigen presenting portion of the host cell many connections are responsible for the complete activation of the immune system - the activation of the immune system is tightly regulated : NEED STIMULATION with antigen binding to effector cell & NEED CO-stimulation between APC and effector cell which is UP-REGULATED BY PRR activation as a danger signal so need initial stimulation & co-stimulation (PRR danger signal)
what happens when only the initial stimulation occurs without the co-stimulation ? what does this prevent
when the effector molecule only binds to the antigen via the antigen presenting cell WITH NO further PRR co-stimulation, this prevents autoimmunity and stays in an ANERGY state. no activation is taken . means that the antigen is not dangerous and is probably just a host-self antigen
what are the multiple killing mechanisms that can activate when the phagolysosome is formed
when the phagosome fuses with the lysosome, there are multiple killing mechanisms via oxidative and nonoxidative NONOXIDATIVE: antimicrobial peptides or lysosomal enzymes activated at low pH to target bacterial cell integrity OXIDATIVE: ROS & RNS causes DNA & protein damage of microbes
list the ways we can determine the pathogens identification by structure give the different tests what does this give you
you can 1) grow on selective media to remove non pathogenic (normal) flora from the sample to grow the disease microbe 2) grow on differential media to exploit the microbes unique BCH properties, gives off different products during growth cycle DO DIFFERENTIAL tests to combine multiple tests in the same sample until a single species is identified 1) TSI agar 2) API20E
