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Lab Quiz #4 Flashcards

1
Q

What do T helper cells produce after being activated by an MHC/peptide complex?

A

Cytokines

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2
Q

T cell activation assay: what type of T helper hybridoma used? How created?

A

KZH
Fusing normal T helper cells from mice immunized with antigen lysozyme with T lymphoma cell transfected with reporter construct

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3
Q

KZH hybridoma T cells have what gene that encodes what enzyme under transcriptional control of what transcriptional regulator?

A

lacZ gene
Encodes beta galactosidase
Cytokine IL-2 gene promoter

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4
Q

What happens when KZH cells are activated by antigenic peptide/MHC complex?

A

Transcription of IL-2 promoter and lacZ gene, producing IL-2 and beta galactosidase

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5
Q

How can beta-galactosidase be detected in T cell assay?

A

Lysing cells with detergent Triton X-100 and adding chromogenic substrate chlorophenol red galactoside (CPRG)
CPRG turns from yellow to red when hydrolyzed by beta galactosidase

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6
Q

What was the antigen presenting cell used in the T cell assay and what was the antigen?

A

APC: LK35.2 (mouse B cell hybridoma)
Antigen: lysozyme

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7
Q

Culture of KZH and LK35.2 cells was done using what type of incubator? What is flushed through incubator? What does this do for cells?

A

CO2 incubator
5% CO2 is flushed through incubator
CO2 establishes equilibrium with bicarbonate in growth medium, forming pH buffer of 7.4 to support optimal growth of cells

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8
Q

What type of medium was used for T cell assay? What did it contain?

A

RPMI-1640

Metabolic building blocks, buffer, antibiotics

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9
Q

Why was centrifugation step necessary in T cell assay?

A

Culture medium contained phenol red dye, which would interfere with color produced in assay
Pelleted cells stayed in plates while supernatant (medium) was removed

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10
Q

Antibiotics have what microbial origin? How do they affect other microbes?

A

Metabolic products of microbes

Toxic or inhibitory to other microbes

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11
Q

Antibiotics can inhibit what in microbes?

A

Steps in metabolic processes

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12
Q

Are antibiotics specific? What does that mean for effectiveness?

A

Antibiotics are specific

They are inhibitory only to microorganisms dependent on affected metabolic process

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13
Q

Narrow-spectrum antibiotics

A

Effective only against a limited number of pathogens

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14
Q

Broad-spectrum antibiotics

A

Attack many different kinds of bacteria

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15
Q

Cidal

A

Antibiotics that kill microorganisms

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16
Q

Static

A

Antibiotics that inhibit growth only

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17
Q

Useful antibiotics act against ___ ___ and not ___ ___.

A

Pathogenic microorganisms

Body cells

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18
Q

First medically useful antibiotic

A

Penicillin

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19
Q

Penicillin is produced by what 2 species of fungus?

A

Penicillium notatum

Penicillium chrysogenum

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20
Q

What genus of what bacteria produces antibiotics?

A

Streptomyces

Actinomycetes

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21
Q

Beta lactams: generic name (2)

A

Penicillins

Cephalosporins

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22
Q

Cephalosporins are produced by what type of fungi?

A

Cephalosporium

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23
Q

How do beta-lactams work?

A

Inhibit synthesis of bacterial cell wall peptidoglycan

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24
Q

Macrolides: generic name

A

Erythromycin

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25
Macrolides (erythromycin) are produced by what bacteria?
Streptomyces erythreus
26
How do macrolides work?
Inhibit rRNA associated with the 50S ribosome
27
Aminoglycosides: generic name (3)
Streptomycin Neomycin Gentamicin
28
Streptomycin is produced by what bacteria?
S. griseus
29
Neomycin is produced by what bacteria?
S. fradiae
30
Gentamicin is produced by what bacteria?
Micromonospora purpurea
31
How do aminoglycosides work?
Inhibit 30S ribosome function
32
Tetracyclines: generic name
Tetracycline
33
Tetracycline is produced by what bacteria?
S. aureofaciens
34
How do tetracyclines work?
Inhibit binding of aminoacyl-t RNAs to ribosomes
35
2 antibiotic-producing microbes used in cross-streak plate experiment
Penicillium notatum | Streptomyces griseus
36
4 bacteria tested in cross-streak plate experiment
Escherichia coli Bacillus subtilis Staphylococcus aureus Micrococcus luteus
37
Kirby-Bauer method uses what 4 things to test for antibiotic sensitivity?
Standardized inoculum Mueller-Hinton or Trypticase soy agar plates High potency antibiotic discs Exact measurement of inhibition zone
38
Minimal inhibitory concentration of antibiotic: how measured
Diameter of zone of growth inhibition around disc when placed on lawn of bacteria
39
Minimal inhibitory concentration of antibiotic: what is it
Lowest concentration of antibiotic that will prevent growth of a particular microorganism
40
Minimal inhibitory concentration of antibiotic provides a measure of what
Susceptibility of microorganism to antibiotic
41
McFarland standards are used for what purpose in antibiotic sensitivity testing?
Standardize bacterial inoculum used for antibiotic sensitivity testing
42
McFarland standards adjusts what?
Turbidities of bacterial cultures to be tested
43
What McFarland standard were bacterial cultures adjusted to in antibiotic sensitivity experiment? This is comparable to a bacterial suspension of how many CFU/mL?
0.5 | 10^8 CFU/mL
44
Horizontal gene transfer
Process by which genes are transferred from one mature, independent organism to another
45
3 mechanisms of horizontal gene transfer in prokaryotes
Conjugation Transformation Transduction
46
Conjugation
Genetic transfer by direct cell-cell contact between donor and recipient cells
47
Transformation
Free DNA is taken up by a cell and integrated into its genome
48
Transduction
Transfer of genes between prokaryotic cells by bacteriophages
49
In transformation, competent cells can do what?
Take up high molecular weight DNA across cell wall and plasma membrane
50
How transformation produces genetic diversity
DNA from donor strain contains different phenotypic characteristics: recipient integrates DNA through genetic recombination, then displays those phenotypic characteristics
51
Genus and species of bacteria used in transformation experiment: naturally or artificially competent, environment
Acinetobacter calcoaceticus Naturally competent Soil and water
52
Donor vs recipient strains of Acinetobacter calcoaceticus: designation and what gene DNA encodes
Donor: Streptomycin-resistant (Str^r) Recipient: Streptomycin-sensitive (Str^s)
53
Detergent used in transformation experiment to release DNA from donor Acinetobacter calcoaceticus
Sodium dodecyl sulfate
54
Is conjugation used to transfer large segments of bacterial chromosome, plasmids, or both?
Can be both
55
R factors
Plasmids that encode antibiotic resistance genes
56
Conjugative plasmid transfer occurs from donor designated ____ to recipient.
tra
57
Some tra genes in conjugative plasmid transfer encode what type of pilus? What does this pilus do?
F (sex) pilus | Mediates contact between donor and recipient
58
Steps of conjugative plasmid transfer (5)
1. Donor and recipient cells are drawn together 2. Formation of channel through which plasmid DNA is transferred 3. Plasmid DNA is cut at origin of transfer (oriT) 4. Cut strand is transferred to recipient with simultaneous synthesis of new complementary strands via rolling circle replication 5. Ends of strands are ligated to form complete plasmids in both donor and recipient cells
59
Conjugation experiment utilized what plasmid?
pBBR1MCS-2
60
Plasmid pBBR1MCS-2 encoded what 4 genes?
``` Kanamycin resistance gene lacZ gene (beta-galactosidase) mob region (region for conjugation) Multiple cloning site (contained numerous restriction sites) ```
61
Restriction sites are recognized by what enzymes that do what?
Restriction endonucleases | Cut DNA into pieces
62
What genus and species of bacteria was used in conjugation experiment?
E. coli
63
Donor strain of E. coli in conjugation experiment was designated what? It contained what genes for what purpose? Sensitive to what antibiotic, but resistant to what antibiotic?
S17-1 tra genes for plasmid transfer Sensitive to nalidixic acid Resistant to kanamycin
64
Recipient strain of E. coli in conjugation experiment was designated what? Sensitive to what antibiotic, but resistant to what antibiotic through what mechanism? Had mutation in what gene that was complemented by plasmid DNA?
DH5alpha Sensitive to kanamycin Resistant to nalidixic acid through mutation Mutation in lacZ gene
65
In conjugation experiment, what substrate was used for beta galactosidase? What color does it produce when hydrolyzed by beta galactosidase?
X-gal | Blue
66
Viruses of bacteria are called what?
Bacteriophages
67
Bacteriophage is made up of what 2 components?
Nucleic acid with protein coating
68
Steps of bacteriophage injection of host cell (2)
1. Phage binds via tail fibers to cell receptor | 2. Phage genetic material is injected into bacterial cell
69
What happens in the bacterial cell after being injected with bacteriophage genetic material (2 possible options)?
1. Virus genetic material converts all cell machinery over to manufacture of new virus particles, causing lysis of bacteria 2. Virus genetic material is inserted into bacterial chromosome, becoming a prophage
70
Bacteriophages whose genetic material brings about lysis of infected cells are referred to as ___ or ___.
Lytic | Virulent
71
Bacteriophages whose genetic material is integrated into infected cells are referred to as ___ or ___.
Temperate | Lysogenic
72
Prophage
Latent form of bacteriophage genome that remains within host but does not destroy it
73
Phage typing
Detecting and differentiating certain pathogenic strains of bacteria by their pattern of susceptibility to specific bacteriophages
74
Source material for E. coli bacteriophage
Raw sewage
75
E. coli bacteriophages: how separated from bacteria?
Filtration
76
Type of E. coli phage used in bacteriophage experiment: name, lytic or lysogenic?
T phage | Lytic
77
Phage (viral) titer
Concentration of infectious phage particles per milliliter of growth medium
78
How were T phage titers determined in bacteriophage experiment?
Serial dilutions of stock phage culture mixed with E. coli and grown on agar plates Counting zones of clearing (plaques) Calculating plaque forming units (PFUs) per mL by multiplying average # of plaques counted by dilution factor and dividing that number by volume plated
79
Designation of E. coli strain used in bacteriophage experiment
Beta
80
In lysogenic bacteriophage cycle, bacteria infected by masked temperate phages are termed ___ bacteria. Do they show overt evidence of infection?
Lysogenic | No overt evidence of infection
81
Name of temperate bacteriophage that infects E. coli
Lambda
82
Can lambda bacteriophage only enter lysogenic cycle?
No-can enter either lytic or lysogenic
83
What type of protein prevents lambda bacteriophage from entering lytic cycle? How does it do this?
Repressor protein called cI | Maintains prophage DNA in integrated state with bacterial DNA
84
Name of the lambda DNA site at which repressor protein cI binds
Operator
85
Lysogenic cells are referred to as ____.
Immune
86
Superinfection: what is it, how is it prevented
``` Subsequent bacteriophage infection of a lysogenic bacterium Protein repressor (like cI) prevents expression of all DNA molecules of the same type of immunity region regardless of whether they are integrated into the host chromosome ```
87
Resistance to bacteriophages: how it works
Phage is prevented from absorbing or from injecting DNA into the cell or is blocked at some other stage of the lytic cycle
88
Cro: what is it, what cycle does it promote in lambda phage
Regulatory protein | Lytic cycle
89
Does Cro bind to same or different operator sites as cI?
Same
90
Between cI and Cro, which binds more weakly and must rise to a higher level to exert its effects?
Cro
91
Phage attachment to receptors on the surface of bacterial cells is facilitated by the presence of ____ ____.
Magnesium ions

Microbiology (24 decks)

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