Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

Micro Lab > Midterm 2 > Flashcards

Midterm 2 Flashcards

(110 cards)

Start Studying
1
Q

Which test panel bacteria were inhibited by antibiotic?

A

E. coli and S. marscens (this one was sensitive)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

In the chloroform killed bacteria experiment, the antibiotic producing bacterium was grown for ___ days. During this time, if an
antibiotic was produced, it likely ______ into the agar media. This typically produces a _______
of antibiotic concentration. The greatest concentration would be _______ the chloroformed-killed
bacteria. The lowest concentration would be at a point _____ from the chloroform-killed
bacteria.

A

two, diffused, gradient, nearest, farthest

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

16S rRNA genes have several properties that make them useful for analyzing
______ _______

A

bacterial phylogeny

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Properties of 16S rRNA genes (4)

A
  1. found in all living microorganisms (they are not found in viruses)
  2. they perform a similar function in all organisms
  3. They have regions that
    mutate slowly and quickly, enabling both long- and short-term evolutionary events to be
    examined
  4. they are of sufficient size (>1500 nucleotides) to allow most organisms to be
    differentiated
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How did we determine the nucleotide sequence of the 16S rRNA gene for our antibiotic producing bacterium?

A
  1. isolating DNA from the bacteria
  2. PCR amplifying the
    rRNA genes
  3. performing a DNA nucleotide sequencing reaction on this gene.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

In DNA Isolation, bacteria are lysed in _____ and ____ processes. What are these 2 processes specifically?

A

chemical (detergents in buffer) and physical (bead-beating)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

DNA is purified after lysing, and it preferentially binds to _____ (+ specific name) under ____ salt conditions

A

silica (Binding Matrix), high

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

DNA can be eluted under ___ salt conditions with ___ ___ ____ ____

A

low, Homemade DNA Elution Buffer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

The polymerase chain reaction (PCR) is a process by which specific DNA fragments
can be ______ _____

A

amplified exponentially

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the positive and negative controls (made by your TA) and why do they matter?

A

the positive control is E. coli and the negative control is water
the positive controls are analyzed to make sure that the gel electrophoresis ran correctly and accurately. The negative control is ensuring that there is no cross contamination.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Why are three different temperatures necessary in PCR?

A

The three different temperatures are necessary in PCR because of the ever changing state of DNA. Hydrogen bond formation requires lower temperature and high temperature is used for breaking these hydrogen bonds. These acts change the structure and state of DNA from being double or single stranded.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are you making copies of in PCR?

A

Copies of specific DNA segments

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Why are there NO colonies from the 1,000 ug/ml streptomycin + 100 ug/ml ampicillin dose?

A

E. coli most likely cannot grow in the presence of ampicillin because this antibiotic prevents E. coli from growing and creating a cell wall.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Two different methods to purify their PCR amplified 16S rRNA genes

A
  1. agarose electrophoresis
  2. enzymatic method (“ExoSAP-IT)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What does “ExoSAP-IT do?

A

It treats PCR products ranging in size from less than
100 bp to over 20 kb with absolutely no sample loss by removing unused primers and nucleotides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

When do you add ExoSAP-IT?

A

directly to the reaction products following PCR

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Is buffer exchange required for Exo-SAP IT? Why?

A

No, because it is active in commonly used PCR buffers

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How is ExoSAP-iT reagent inactivated?

A

heating to 80 deg Celsius for 15 min

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Exposure to ultraviolet (UV) light is _____ for most bacteria, depending on the
wavelength, distance from the UV bulb, exposure time, and the bacteria itself.

A

lethal

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Spectral analysis
of the ultraviolet range (_____nm - ______nm) reveals that a wavelength of _____ nm is most effective in
killing bacterial cells.

A

190-300 nm; 265 nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Why is 265 nm wavelength important?

A

This is also the wavelength at which DNA has its maximal absorption

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

UV light causes damage to DNA in the form of _____ ___ ______ _____ ____.

A

dimerization of adjacent pyrimidine bases.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Bacterial cells can
possess many different mechanisms that enable them to survive this UV irradiation: (3)

A
  1. thicker peptidoglycan layer
  2. more DNA repair enzymes
  3. upregulation of osmolytes (such as glycine)
    that can scavenge for reactive oxygen species created by UV light
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

Exposure of microorganisms to high temperatures can result in their ____

A

death

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Steam is more effective than ___ ___ because:
dry heat; In the presence of water, heat causes disruption of hydrogen bonding
26
Disruption of hydrogen bonding also causes:
denaturation of protein and DNA
27
Bacteria that are heat tolerant have a greater proportion of _____ ____ and _____ and/or a high __ + ___ content
hydrophobic proteins; lipids; G+C
28
Some pathogenic bacteria can survive over ___ hours of boiling in water.
10
29
Bactericidal or bacteriostatic antibiotics
Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria
30
Example of sulfa drugs
sulfanilamide
31
sulfa drugs are _____ ____
bacteriostatic agents
32
______ is a bactericidal agent produced by the bread mold ______
penicillin; penicillium
33
function of penicillin
inhibiting synthesis of the peptidoglycan cell wall.
34
Penicillin is less effective against _____ ______ bacteria because their cell wall is comprised of a ______ _____ and a relatively _____ amount of peptidoglycan occurs within the periplasm.
gram-negative; lipid bilayer, small
35
what happens when an bacterial infection is suspected as the causative agent?
then the organism should be isolated and its sensitivity to a range of antibiotics tested. This helps a physician choose the best antibiotic dose and duration of therapy. Unfortunately, costs, the severity of the infections, and the time required to make these determinations, frequently results in physicians making a best guess as to which antibiotic to use.
36
the use of antibiotics did not come into common usage until the ______
1940s
37
bacteria can become resistant to antibiotics through (3)
1. mutation 2. selection 3. acquisition of genes that confer resistance
38
What is generation time ?
the time required for the number of cells to double
39
Generation times of different organisms can vary widely and depend on environmental conditions such as _____, ______, and _____
temperature, pH, and nutrients
40
Knowledge of generation time is very important in (3)
estimating shelf life of foods, critical stages of infections, and productivity of ecosystems
41
What is the Ames test?
an assay to assess the ability of a substance to cause mutations. also provides a measure of the carcinogenicity of a substance
42
What are mutations?
e heritable changes in the nucleotide sequences of the genetic material (usually DNA).
43
If a ______ substance is added to the bacteria before they are spread on the streptomycin agar, this can lead to a greater number of mutations, and therefore a greater number of bacteria that are resistant to the streptomycin, and therefore a greater number of colonies.
mutagenic
44
Lectures
Lectures
45
How do we identify specific microbes? (You isolated an organism, so how do you tell what it really is? (3 ways)
1. physical (ex. gram stains) 2. biochemical (ex. what agar is grows on) 3. Molecular/genetic markers
46
What are the molecular fingerprints that a microbes gives for its identification? (5)
1. Must be able to align molecules to determine genetic relatedness (how similar) 2. Universal molecule found in all organisms 3. Has conserved functions in all organisms (doing same thing in organisms) 4. Strictly vertically transferred (generation to generation) 5. Can’t be picked up from the environment or different cells (horizontal gene transfer) 6. Constant substitution rate – sequence divergence proportional to time (slow evolving) [we wan't something that doesn't mutate very fast]
47
* The most widely used molecular “fingerprint” for microbial cell identification is the gene sequence encoding the:
small subunit rRNA (SSU rRNA)
48
2 examples of SSU rRNA
16S rRNA (bacteria) or 18S r RNA (eukaryotes)
49
How can we figure out what species we're looking at by using 16S rRNA?
The accumulation of inherited sequence differences (mutations) in 16S rRNA during evolution can be used to differentiate microbial species
50
16S and 18S rRNA are how long?
1.5 kB in length
51
16S rRNa is protein coating?
no
52
_______ is what makes 16S rRNA universal and conserved
translation
53
RNA bases can base-pair within the same molecule to then become ____ ______
secondary structures (which then react with other things in bacteria) *Structural basis for independent activity
54
How can the secondary structure of SSR RNA fall apart?
If enough double stranded regions are unable to form because of sequence mismatches due to mutations (translation then gets inhibited) Then, this mutation gene gets selected against through evolution
55
* Base pairing determines ____
structure * As long as function is necessary, sequences that contribute to base pairing will be conserved
56
Regions in 16S rRNA that are sensitive to mutations are called _______ and insensitive to mutations are called _____
- conserved regions (SENSITIVE) - variable regions (INSENSITIVE)
57
_______ regions are across all microbes while ______ regions are specific to species, genus, phylum, or domain
conserved, variable
58
_____ regions are used as a molecular identifier
variable
59
We can amplify the 16S gene using ____ ____ ___
polymerase chain reaction (PCR)
60
The product of PCR will then be ______ and compared to reference databases to identify a microorganism
sequenced
61
The general PCR cycle layout has several steps (3)
1. Melting: (take template DNA that's held together by H Bonds, and apply high temperature to it to melt to single strands to then act as a template) 2. Annealing: Bind primers to tempalte strands 3. Extension: DNA polymerase extends DNA from primers based on the sequence of the template (brought to a temperature to do so)
62
PCR starts with a double stranded DNA in which we add a MasterMix consisting of:
1. DNA polymerase (enzyme that does assembly of DNA) 2. Nucleotides (dATP, dCTP, dGTP, dTTP) 3. Ions that stabilize reaction (MG+2, K+) 4. Buffer to maintain optimal pH
63
The Mastermix is common to all our reactions, but we change our reactions based on the
template DNA of choice Primers that are specific to your target gene
64
What do you not add in PCR
Contamination
65
In the first cycle of PCR, how many copies are made?
two double stranded copies, it keeps doubling each cycle
66
What do we do after PCR amplification process?
1. PCR DNA product is cleaned up to remove excess primers and nucleotides 2. Concentration check - We want to check the concentration of our final DNA PCR amplification product 3. Quality check for contamination by other molecules (proteins or salt 4. 16S gene will then be sequenced
67
What mixture cleans up PCR reaction? Be specific in describing it
ExoSAP; ExoSAP is an enzymatic degradation of remaining primers and excess dNTPs that may interfere with sequencing
68
What temp does ExoSAP do its cleanup? What happens after?
37C; it is then heat inactivated at 80C to prevent interference with later steps
69
ExoSAP acts on protein (T or F)
FALSE
70
How do we check if our DNA is pure?
Quantify the amplified rRNA genes by measuring absorbance at 260nm (optimal wavelength that DNA absorbs
71
What are the two other wavelengths that are important?
230 nm -- salt 280 -- protein contaminants
72
What is Sanger sequencing?
Method of sequencing DNA using di-deoxy ddNTAPs nucleotide terminators
73
dNTP vs ddNTP
- dNTPs retain the 3' -OH for extension (we add these during PCR reaction so that DNA polymerase can extend it) - ddNTPs don't have this extension, rather is ENDS the extension reaction
74
In sanger sequencing, ___ reactions will be run, each beginning with a _____ specific to your target sequence
4, primer
75
In Sanger sequencing, each reaction contains a small amount of _____ ddNTP
ONE
76
In sanger sequencing, random incorporation of ______ will generate a product terminating at every T base in your template
ddTTP
77
In sanger sequencing, too much _____ will not give you enough bands
ddNTP
78
In sanger sequencing _____ _____ allows you to read the sequence by the order of the band size
size fractionation
79
3 bactericidal agents
1. UV Light 2. Heat 3. Antibiotics
80
UC light of ________ - ________ can be lethal because this is the wavelength at which DNA has its maximal absorbtion
260-265
81
UV damages DNA by causing pyrimidine dimers (C or T) which causes DNA ___ ____
replication errors
82
Bacterial strategies to reduce heat damage
1. heat shock response (production of chaperone proteins that help them fold properly) 2. heat stability can be conferred by structural changes in macromolecules 3. specific compounds can stabilize proteins (di-inositol phosphate, diglycerol phosphate, manosylglycerate)
83
What is an extreme way of dealing with heat/becoming heat resistant
Endospore formation (GRAM POS only)
84
Clear zones on Antibiotic (large clear circles) mean
the bacteria is sensitive to the antibiotic
85
Now that you have isolated an organism, how do you tell what that organism is?
- Physical – Gram stain and microscopy - Molecular/genetic – 16S - Combined biochemical tests and morphology (shape/appearance)
86
Traditional identification
The traditional method is based on Gram status, morphology, and physiological properties
87
Bergey's Manual is:
Using the Gram status & shape & Bergey’s Manual – you will be able to determine which taxonomic groups your antibiotic producer likely belongs to
88
4 bacterial growth curve steps
1. lag phase 2. log phase 3. stationary phase 4. death phase
89
3 things that affect lag phase length
-age of culture (old=longer) - growth substrates (complex rich media to defined media = longer) - environmental changes
90
Most bacterial doubling times are _____ hours in complex media: E. coli can divide in 20 minutes under optimal growth conditions. * Doubling time (td) varies according to growth conditions
1-3
91
How to determine doubling time? what's y and x axis?
generate a semi-log growth curve y axis is logarithmic scale (absorbance) x axis is linear scale (time)
92
Stationary Phase
Cells are alive, but can no longer be produced faster than others die * Many cells stop growing * Induction of genes for survival and maintenance * Substrate limitation and waste accumulation * Cells often become smaller; reduce number of ribosomes * Some species can make specialized cell structures in response to stationary phase (e.g. endospores)
93
Death Phase
Loss of viable cells due to accumulation of toxins and byproducts of metabolism, and absence of new nutrients * Decline in cell numbers highly variable and not predictable by any mathematical relationship * Sometimes accompanied by cell lysis * Viable, non-culturable phenomenon: * Live cells can be observed and counted (live/dead stain) * Number of colonies obtained after transfer of this mixture to agar plates may be lower
94
What is an Ames test?
The Ames test examines the ability of a chemical substance to cause mutations, or the strength of the mutagen tested
95
What are mutations?
heritable changes in the nucleotide sequences of the genetic material – DNA/RNA (can be beneficial or detrimental)
96
What is a mutant?
An organism that differs genetically from its parent
97
What is a mutagen?
A chemical (or a physical phenomenon) that causes changes in the nucleotide sequences of the genetic material
98
What are spontaneous mutations?
caused by DNA replication errors – these happen NORMALLY or background mutations that occur in all organisms (usually at a low rate in bacteria)
99
What are induced mutations?
by chemical, physical, or biological agents – for example – X-rays, cigarette smoke, some food preservatives, etc.
100
* Phenotypic changes resulting from point mutations depend upon: (30
Where the mutation occurs * What the base change is * What the DNA where the mutations occurs encodes
101
Point Mutations? Meaning and the 3 types
changes in a single base transition: purine --> purine transversion: purine <--> pyrimidine insertion and deletion of one or more bases
102
* Missense mutation
changes the amino acid sequence to another
103
Nonsense mutation
changes the amino acid sequence to a premature stop codon
104
* Silent mutation
does not change the amino acid sequence because of degeneracy in the genetic code (multiple codons can encode the same amino acid)
105
Frameshift Mutations
Changes the open-reading frame (ORF) of the gene meaning, insertions and deletions can disrupts the reading fram and the mutant gene is no longer in a multiple of 3
106
Streptomycin irreversibly binds to the ____ rRNA and ____ protein of the bacterial ribosome. It interferes with translation by disrupting ____ conformation
16S and S12, ribosome
107
Ames Test controls
1. Use of a control exposure (water) 2. plating mutagen-exposed bacteria on agar WITHOUT streptomycin (to make sure the cells were not killed by the mutagen) 3. see if the test substance contains contaminating microbes (should be no growth)
108
Clinical Microbiology helps anticipate likely _____ (condition that is results of disease/injury)
sequelae
109
GIGO
garbage in garbage out
110
selective media vs differential media
Selective media allows for the growth of specific organisms, while differential media is used to distinguish one organism from another.

Micro Lab (2 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions