Micro Lab Exam 2 Flashcards
(125 cards)
1
Q
Microbes adapt to what environmental conditions? (5)
A
Temperature
pH
Osmotic Pressure
Atmospheric Gas
Availability of Nutrients
2
Q
Oxygen creates toxic byproducts that must be neutralized. What are they? (3)
A
Superoxide Ion
Peroxide
Hydroxyl Radicals
3
Q
What enzymes allow organisms to degrade the toxic byproducts of oxygen metabolism? (3)
A
Catalase
Superoxide Dismutase
Peroxidase
4
Q
What are the 5 classifications based on oxygen requirements?
A
Obligate Aerobe
Microaerophile
Facultative Anaerobe
Aerotolerant Anaerobe
Obligate Anaerobe
5
Q
Oxygen Requirements for Obligate Aerobe
A
Requires an oxygen rich environment
Can detoxify oxygen products
6
Q
Oxygen Requirements for Microaerophile
A
Requires some oxygen, but too much can damage it
It CAN NOT detoxify oxygen products
7
Q
Oxygen Requirements for Facultative Anaerobe
A
Can use oxygen if present, but can use anaerobic pathways if needed
Can detoxify oxygen productd
8
Q
Oxygen Requirements for Aerotolerant Anaerobe
A
Does not require oxygen, but no affected if oxygen is present
Can detoxify oxygen products
9
Q
Oxygen Requirements for Obligate Anaerobe
A
Requires an OXYGEN FREE environment
Can NOT detoxify oxygen products
10
Q
How can you control growth of microorganisms in a lab?
A
Manage their preferred growing conditions (incubator vs. fridge), manage oxygen preferences
11
Q
How do anaerobic jars work?
A
Create an anaerobic environment by using a chemical envelope that catalyzes a reaction to remove oxygen from the container
12
Q
How do you verify the anaerobic conditions in side the jar?
A
An indicator strip
13
Q
What happens with Obligate Aerobes on the agar plate? Obligate Anaerobes?
A
Aerobes will only grow on the plate when it is incubated at normal conditions
Anaerobes will only grow if the plate is incubated in the jar.
14
Q
Where will facultative organisms grow?
A
On both plates
15
Q
What can you use to grow microbes in anaerobic conditions?
A
Fluid Thioglycolate Medium (FTM)
Works for both aerobic and anaerobic
16
Q
What do the colors of FTM mean?
A
Top = slightly red = oxygen present
Middle and Bottom = Yellowish = Low to No Oxygen
17
Q
What does FTM contain and why?
A
A small amount of agar to localize organisms in places where the conditions are most favorable for growth
18
Q
Where do you expect Obligate Aerobes to grow in FTM?
A
Very Top
Aka the surface where oxygen levels are the highest
19
Q
Where do you expect Microaerophiles to grow in FTM?
A
A band about 1/3 down
Oxygen is present, but not in high amounts
20
Q
Where do you expect Facultative Anaerobes to grow in FTM?
A
Throughout the entire thing, but better at the top due to aerobic metabolism
21
Q
Where do you expect Obligate Anaerobes to grow in FTM?
A
The bottom half of the tube
22
Q
What does growth look like in FTM?
A
It’s lighter/brighter in color
Cloudy = turbid = growing
23
Q
What determines how the environmental factors affect microbial growth?
A
Enzymes
Environmental factors can disrupt the structures of enzymes, making them unable to function
24
Q
What are the three sets of a temperatures in which a microbe can survive?
A
Minimum - the temperature necessary for metabolic functions to continue
Maximum- the highest temperature before bonds start to break and enzymes lose function
Optimal- where metabolism and growth proceed at the highest level possible
25
What is a spectriphotometer?
An instrument that measures the amount of light that is absorbed or transmitted by molecules in a solution.
The amount of bacteria is related to the amount of light transmission.
This measures the turbidity
26
What is the value for measuring turbidity?
Optical Density
27
What is the turbidity of a tube no bacterial growth?
0
More growth = higher reading
28
How do you find a microbes optimal range?
Measure turbidity at different temperatures
29
What is a cuvette?
A straight-sided, optically clear container for holding liquid samples in a spectrometer
30
What else can temperature do to bacteria?
Other than influence growth rate, it can cause phenotypic changes
Serratia marcescens appears red at 25C, but not at 38C.
31
What type of organism would grow well at 55C?
Thermophile
32
What happens to microbes that get exposed to pH outside of their survival range?
Protein denaturation and ultimately cell death
33
What are the classifications for organisms' pH preferences?
Acidophile - optima pH <5.5
Neutrophile - optimal pH near 7
Alkalinophile - optimal pH above 8.0
34
Osmosis Definition
A movement of water across the cell membrane due to unbalanced solute concentrations
35
Isotonic Solution Definition
Equal Solute Concentrations inside and outside the cell, resulting in no net change in cell volume
36
Hypotonic Solution Definition
Lower solute concentration, resulting in water moving into the cell causing it to increase in volume
37
Hypertonic Solution Definition
Higher solute concentration, resulting in water moving out of the cell causing it to decrease in volume
38
Which solutions can bacteria survive in? Which can it not? Give Examples
Can survive in Isotonic, would be many body areas with normal biota
Can survive in hypotonic, would swell but cell wall keeps from bursting. This would be freshwater ponds and streams.
Can NOT survive in hypertonic. The water moving out causes shrinkage and irreversible damage. Example would be salt water.
39
What is it called when bacteria can grow in high-sugar environments?
Osmophile
40
Halophile definition
Growing in high-salt environments over 13%
Can be classified as oligate
41
Halotolerant definition
Growing in high-salt environments
42
What are the three approaches to identify microorganisms?
Phenotypic, genetic, or immunologic
43
What does the catalase test do?
Detects the presence of the enzyme catalase found in some bacteria
44
What is the function of catalse?
Convert H202 (Hydrogen Peroxide) to H2O and O2
45
How can you tell if a cell has catalase?
Add H2O2 and see if there is bubbling. If there is, that means O2 is being produced from the H2O2.
Any bubbles whatsoever = catalase positive
46
A specific bacteria that often gets the catalse test
Gram-Positive Cocci
47
What are three approach to identify a microbe?
Phenotypic, genetic, or immunologic
48
What does the oxidase test do?
Detects the presence of the enzyme cytochrome c oxidase in bacteria
49
What is cytochrome c oxidase?
A transmembrane enzyme found in the cell membrane. It is the last in the chain of proteins known as the electron transport system, aka the respiratory chain, of many aerobic bacteria.
50
What color does the microbe change to when cytochrome c oxidase is present?
Dark purple = positive
51
Why are sugar fermentation studies useful?
They're usually done first because sugar fermentation varies widely among bacteria.
They can determine gram-negative bacteria from gram-positive, but also from each other.
52
In bacteria cells, how are sugars metabolized?
Using the glycolytic pathways, resulting in pyruvate and ATP
When oxygen is limited, pyruvate is further metabolized into alcohol, gases, and acids. These are what are detectable in testing.
53
What is phenol red broth (PRB) used for?
To determine an organisms ability to utilize a specific sugar. The color of the PRB lets us know the pH of the solution.
54
What are the three most common sugars in PRB testing?
Glucose, lactose, and sucrose
55
What color does PRB change to if acidic products are present?
Yellow, indicating a positive result for sugar fermentation
56
What color does PRB change if it becomes alkaline?
A bright red, magenta, or fuchsia
This happens when bacteria catabolize the peptide amino acids into ammonia.
This signifies a negative result for usage of the sugar.
57
What is a Durham tube?
A small, inverted test tube sometimes used in PRB testing.
It traps gases that may be released during fermentation.
58
Why is it important to observe and record results of your phenol red broth (PRB) immediately after 24 hours of incubation?
Extended incubation can lead to reversion and inconclusive test results
59
What are the three characteristics that SIM medium can evaluate at once?
Sulfide, indole, and motility
60
What does the S in SIM Media indicate?
The medium's ability to detect the production of hydrogen sulfide (H2S).
Iron salt is added to the indicator, and H2S binds to that salt. A black color is formed by Iron Sulfide. A black color = positive for H2S production.
Any color other than black = negative for H2S production
61
What does the I in SIM Media indicate?
The indole test, which determines the ability of a bacterium to break down the amino acid tryptophan.
If a bacteria CAN break down tryptophan, then it will produce indole, pyruvic acid, and ammonia. This means it can produce the enzyme tryptophanase.
After inoculation, add Kovac's reagent to the surface of the agar tube.
If turns red = positive for indole.
If it remains the same color = negative for indole.
62
What does the M in SIM media indicate?
The motility of the bacteria
The lower concentration of SIM agar makes it easy for bacteria to swim around.
After stabbing the needle in, it will leave a streak line.
Non-motile bacteria will grown in a dense line where the streak line was. Motile bacteria will swim away from the stab line, appearing turbid and cloudy.
63
What is the triple-sugar iron test for?
To study the carbohydrate fermentation capabilities of an organism
64
What is in a TSI test?
Glucose, sucrose, and lactose
pH indicator
2 sulfur sources
an iron salt
65
What does a TSI result look like?
Organisms will use the sugar for energy, which produces color patterns.
If the organism produces gas during fermentation, cracks will form in the agar.
66
What other thing can TSI test for?
Just like the S in SIM testing, it can test for H2S.
Black = positive.
67
What is the rule of thumb if you see black in a TSI test?
the H2S will mask the color in the butt; however; sulfur needs an acidic environment, so if you see black then you can assume the butt is acidic (yellow).
68
What does No Change/No Change mean in the TSI test?
No fermentation
69
What does Red/Yellow (K/A) mean in the TSI test?
Glucose fermentation
70
What does Red/Yellow+ Bubbles (K/A,G) mean in the TSI test?
Glucose fermentation and gas produced
71
What does Yellow/Yellow (A/A) mean in the TSI test?
glucose plus lactose or sucrose fermentation
72
What does Yellow/Yellow + Bubbles (A/A,G)g mean in the TSI test?
glucose plus lactose or sucrose fermentation and gas produced
73
What does Yellow/Yellow + Black (A/A,H2S) mean in the TSI test?
glucose plus lactose or sucrose fermentation, hydrogen sulfide produced
74
What does Red/Yellow + Black (K/A,H2S) mean in the TSI test?
glucose fermentation, hydrogen sulfide produced
75
What does Red/Red (K/K) mean in the TSI test?
alkaline reaction due to protein catabolism
76
How do we identify DNA of a particular individual?
By detecting the pair of nitrogen-containing bases
77
Quick overview of DNA profiling
DNA is cut with enzymes that recognize specific sequences and then the fragments are separated on a gel to reveal a specific pattern for an individual.
This pattern is called a DNA fingerprint, since it is unique to an individual.
78
What enzymes are produced to cut the DNA of viruses that infect bacteria?
Restriction enzymes
79
What do restriction enzymes do?
They cut DNA at a specific site or sequence of base.
(Mutations can cause a different cutting pattern, and therefore different sizes of DNA fragments)
80
What does biotechnology do?
Focuses on the use of living organisms and their biological processes to make useful products and address practical problems.
81
DNA profiling can be used in clinical diagnostics, epidemiological tracking, forensics, and industry. What is it used for in our lab?
To determine the causative agent in a disease outbreak.
This can be used to control the spread of illness and prevent future outbreaks.
82
What are the three steps in DNA profiling?
1. Prepare a restriction enzyme digest of the DNA samples
2. Use an electrophoresis gel and chamber to separate the DNA fragments by size.
3. Use a staining process to visualize and analyze the DNA fragments in the gel.
83
Explain Restriction Enzyme Digest in DNA profiling
The DNA is first cut with restriction enzymes in a microcentrifuge tube. (First add isolated DNA, then restriction enzyme buffer, then restriction enzyme containing mixture.)
The centrifuge to pool and incubate in a water bath.
Add loading dye and glycerol to avoid samples floating at the top of the tube. This helps track the gel runs.
84
What dye is used in the restriction enzyme digest phase of DNA profiling?
Bromophenol blue and glycerol
85
Explain Electrophoresis Chamber Assembly in DNA profiling
First place the casted gel onto the platform of the chamber partially filled with buffer solution.
Orient the gel to the negative electrode side of the chamber.
The chamber is filled with buffer solution, which conducts the electrical current through the negative and positive ends.
One of the wells contains a known DNA sample. These spread thorough the gel and provide the key for the sample fragments to be compared against for length.
86
Explain Loading the Gel in DNA profiling
You use a micropipette that measures volume in microliters.
The tips are disposable and changed to prevent contamination.
It allows the sample to be placed in the gel without dispersing and keeps air out of the well.
87
Describe Running the Gel in DNA profiling
Place a lid on the chamber and turn on the electrical current.
The negative DNA fragments move toward the positive electrode.
Small fragments move faster than large ones.
They separate by size, which makes a pattern in the gel matrix.
88
Describe Analyzing the Gel/Visualizing in DNA profiling
Add a stain, like ethidium bromide, that binds to the DNA molecules and allows them to show up as bright lines in the gel.
You then analyze the patterns.
89
Define DNA
a double helix structure containing base pairs used as the basis of genetic information
90
Define DNA ladder
a solution of DNA fragments of known size used to compare unknown DNA to estimate fragment size
91
Define Electrophoresis Chamber
A buffer-filled box where a separation gel is placed and a current is applied to separate DNA fragments by size.
92
Define Ethidium Bromide
A molecule that binds to DNA bases and fluoresces under UV illumination to visually detect the location of DNA bands in the electrophoresis gel.
93
Define SYBR Green I
A safer alternative to Ethidium bromide used to visually detect the location of DNA bands in the electrophoresis gel.
94
Define Loading Dye
A solution added to an electrophoresis sample to give it color and density
95
Define microcentrifuge
Small tabletop centrifuge that uses centripetal forces to separate substances by density.
96
Define microcentrifuge tube
Small tube with a lid holding less than 2 mL used in a microcentrifuge.
97
Define micropipette
A device used to precisely transfer small volumes of liquid
98
Define micropipette tip
A disposable plastic tip holding different volumes of liquids
99
Define restriction enzyme. Include 2 examples.
An enzyme that cuts DNA at a specific recognition sites called restriction sites, EcoRI and Psti enzymes are examples.
100
What are the main purposes for adding a loading dye to the samples before they are loaded into the gel? (2)
- It contains glycerol, which allows the samples to sink into the wells.
- It contains a colored indicator, allowing migration of samples to be monitored during electrophoresis.
101
What would be the result of running the gel too long?
The DNA sample would move through the entire gel and exit at the bottom.
102
How do bacteria reproduce? How does that affect their genetic material?
They reproduce asexually, but simple division. The have to acquire their genetic material from other bacterial cells through a process called horizontal gene transfer.
103
What is transformation?
A type of horizontal gene transfer in which bacterial cells pick up bits of naked DNA from their environment. They can become part of their genome or exist separately as a plasmid.
104
What is transformation used for in a lab?
To insert medically useful genes into bacterial cells that then produce a specific protein, like insulin or growth hormone.
Typically inserted as a plasmid
105
Why are plasmids beneficial?
They are circular DNA found in some bacteria, but they can be engineered to carry certain genes and then inserted into bacteria to express those phenotypes.
106
How do you make cells competent? Why?
In order to get a recombinant plasmid through the bacterial cell wall, it needs to be made competent. Competent means that they are able to allow large DNA molecules to pass through.
You do this by exposing the cell to a solution of cold calcium chloride and then a heat shock treatment then right back to cold calcium.
107
What does the cold/hot/cold do when making cells competent?
The cold part is though to neutralize the negatively charged DNA to pass easier through a nonpolar cell membrane
The hot part is through to increase the permeability of the cell membrane
108
How can you tell which cells are transformed?
You have to use a selective medium that only allows transformed cells to grow. Oftentimes this is an antibiotic.
109
What are the 4 important sites on the recombinant plasmid, pGLO.
1. Ori Site - where DNA polymerase starts replication
2. Bla Gene - resists ampicillin, meaning this will allow us to see the transformed cell growth on a plate of selective medium containing ampicillin.
3. GFP gene - the gene of interest for our simulation
4. araC gene - regulates the transcription of the GFP gene
110
What is the purpose of the LB agar plate?
It is a non-selective medium for bacterial growth
111
What is the purpose of the LB/amp agar plate?
It is a selective medium containing an antibiotic to inhibit the growth of non-resistant cells.
112
What is the purpose of the LB/amp/ara agar plate?
It is a selective medium with an antibiotic which prevents growth of non-resistant cells and a sugar that allows for the expression of a plasmid gene.
113
What ways does the gene for sickle cell hemoglobin differ from the gene for normal hemoglobin?
There is a single nucleotide difference.
114
What would happen if the electrical current was not left on long enough during gel electrophoresis?
The fragments would not have time to separate very much.
115
What allows for the creation of an electric field?
Electrophoresis buffer
116
What test is used to distinguish between staphylococci and streptococci?
Catalase test
117
Distilled water would be ______ to bacterial cells
Hypotonic
118
an example of an organism that can tolerate and grow in a high temperature and highly acidic environment?
Solfolobus
119
An isotonic solution prevents water from crossing the cell membrane. (T/F)
FALSE
120
Bacteria in cured meats and jams would experience what type of environment?
hypertonic
121
A hypertonic solution is prepared by the addition of sucrose. Which of the following will happen to bacteria placed in this solution?
The bacterial cells will undergo plasmolysis.
122
Microorganisms growing on mucous membranes such as the oral mucosa or vaginal mucosa are most likely in a(an) _____ environment.
isotonic
123
Which of the following terms refers to microorganisms capable of growing in jams, corn syrup, and maple syrup?
osmophiles
can survive in high SUGAR environments
124
To promote the development of red-pigmented colonies by Serratia marcescens, cultures should be incubated at
25 degrees C.
125
The activated chemical pack envelope that is added to an anaerobe jar effectively removes
oxygen
