Practical 2 Flashcards
(107 cards)
1
Q
what is thioglycolate broth and what is it used for?
A
- Thio broth contains nutrients needed for bacterial growth.
- Thio broth is a differential medium, not selective.
2
Q
What is the role of thioglycolate in the media?
A
Contains nutrients for growth
3
Q
what does inoculation mean?
A
Introduction of microbes into any growth medium
4
Q
What is the role of bromothymol blue in the media?
(thio broth)
A
O2 indicator
5
Q
what is the consistency of this media?
(thio broth)
A
broth
6
Q
what is the gas Pak jar, how it’s used and what it is used for?
A
It is used to create an anaerobic environment for bacteria to grow
7
Q
Why is oxygen toxic to some bacteria?
A
It is anaerobic and cannot tolerate it
8
Q
How do aerobic cells deal with those toxic byproducts of aerobic respiration?
A
Catalase and SOD
9
Q
what is a strict anaerobe?
A
- no oxygen at all (clostridium) BOTTOM GROWTH
10
Q
what is an aerobe?
A
- needs oxygen TOP GROWTH
11
Q
what is a microaerophile?
A
- lower concentration of oxygen GROWTH JUST BELOW SURFACE
12
Q
what are facultative anaerobes?
A
- can grow both ways (prefer O2) GROWTH THROUGHOUT
13
Q
what are aerotolerant anaerobes?
A
- can grow both ways GROWTH THROUGHOUT
14
Q
what are cardinal temperatures?
A
The range of temperatures that bacteria will grow
15
Q
What makes a particular temperature an optimal temperature for a microbe?
A
Allows for the best growth due to max efficiency of enzymes and reproduction
16
Q
what are psychrophiles?
A
- 5 degrees C -> very cold
17
Q
what are psychrotrophs ?
A
- 25 degrees C -> cold
18
Q
what are mesophiles?
A
- 37 degrees C -> body temp
19
Q
what are thermophiles?
A
- 55 degrees C -> hot
20
Q
how was our temperature lab done?
A
We used nutrient broth to assess turbidity and cloudiness
21
Q
The red pigment of Serratia marcescens was produced at what temp(s)?
A
25°C and 37°C
22
Q
What classification do human pathogens fall into?
A
We are exposed the most to Mesophiles
23
Q
What classification do microbes that can grow in the refrigerator fall in?
A
Psychrophiles
24
Q
what thermophile did we use in PCR?
A
Taq polymerase
25
Why are serial dilutions done?
- To count colonies
- by obtaining 30-300 colonies
26
what is viable cell count?
- the # of living cells in a culture that can form colonies on plates
27
what is total cell count?
- the # of living and dead cells (requires a microscope)
28
did we use viable or total cell count during serial dilution lab?
viable cell count
29
What is the number range of bacteria on a plate appropriate for determining cell count/ml?
30-300
30
What is SV / TV?
Dilution:
- SV = sample volume
- TV = total volume
31
If I ask you to make a 1-20 dilution and you have 1 ml of culture, how much of the
diluent do you need?
19 ml
32
How did we do this experiment?
(serial dilution)
Transferred 1 mL of E-Coli with a sterile pipette into 9 mL of sterile water and repeated
33
What bacteria did we use? What did we observe about the culture?
- E-Coli
- we observed that the #3 tube was the one with a viable count
34
which dilutions have more cells per ml and which have less – what is the pattern?
1-10 dilution has more cells than a 1-100 dilution, which has more cells than a 1-1000
dilution.
35
What does PCR stand for?
Polymerase Chain Reaction
36
What is the goal of PCR?
To amplify or copy DNA
37
Why did we wear gloves for this experiment?
(PCR)
Because we are working with DNA
38
What did we use to measure volumes of liquids for this experiment?
(PCR)
Micropipettes with sterile tips
39
Why is a thermostable DNA polymerase required for PCR?
we heat up the DNA in the denaturing phase
40
What are dideoxy nucleotides?
The nucleotides are A, C, T and G
41
List what is needed (recipe) to do a PCR reaction, and the role of each.
2 tubes:
- one with white ball of Taq polymerase
- primer
- water
- control DNA
42
What are the 3 steps of a single PCR cycle?
- Denaturing: heat it up
- Annealing: cooling so primer sticks
- Extension: building back up
***Each cycle amplifies the sequence that is being copied.
43
What is the purpose of agarose gel electrophoresis?
Separation of nucleic acid fragments based on their size
44
Where are the PCR product (DNA) samples loaded into the gel?
Into the wells
45
Give an example of how PCR can diagnose a disease
By replicating the genetic material
46
Which size DNA fragments will migrate faster?
(smaller or larger)
The smaller move fastest (to the bottom)
47
On the gel the DNA fragments migrate in what direction? (cathode or anode)
- negative fragments migrate cathode (black)
- positive fragmentsmigrate
towards the anode (red)
48
What was used to stain / visualize DNA bands in the gel?
Ethidium Bromide
49
What is a thymine dimer? (what is thymine?)
The hydrogen bonds are broken, and a covalent bond is made forming a dimer
When exposed to UV, thymine dimer’s kink the DNA making replication and transcription
difficult. This can lead to mutations in DNA (cell death or cancer)
49
What does –cidal mean?
kills microorganisms
49
What does –static mean?
inhibiting microbial growth
50
Contrast a disinfectant and an antiseptic.
- Disinfectant is for non-living surfaces
- Antiseptic is applied to living surfaces
51
What chemicals were tested in this lab?
Phenols, Halogens, Alcohols, QUATS
52
What is the mechanism by which alcohols are bacteriocidal?
They dissolve lipids
53
What is the mechanism by which Halogens are bacteriocidal?
They denature proteins
54
What is unusual about the use of alcohol as a disinfectant?
Alcohol is more effective at killing microbes at 70% diluted with water than at a higher %
55
The size of the zone of inhibition does NOT matter
56
what do phenols and halogens do?
denature proteins
57
what do alcohols do?
dissolve lipids
58
what do QUATS do?
breakdown cell membranes
59
What is an antibiotic?
A medicine that inhibits the growth of or kills bacteria
60
List of 3 known targets of antibiotics (modes of action).
- Inhibits cell wall synthesis
- Inhibits protein synthesis
- Injures the cell wall
61
What is a zone of inhibition?
The clear area around the disk
62
How are they measured?
Across the center from side to side, in mm
63
If a clear zone of inhibition is present, does that mean the bacteria is sensitive to the
antibiotic?
No, it must be measured to determine that
64
Resistant
Intermediate
Sensitive
Resistant:
- antibiotic is not effective
Intermediate:
- antibiotic is somewhat effective
Sensitive:
- antibiotic is effective
65
How do the following antibiotics kill / stop growth of bacteria?
Penicillin:
- Inhibits cell wall synthesis
Cephalosporin:
- Inhibits cell wall synthesis
Erythromycin
- Inhibits protein synthesis
66
Which of the above antibiotics are broad spectrum antibiotics?
Penicillin
67
What antibiotic class is often useful for upper respiratory infections?
Penicillin
68
How is UV light damaging to humans? What tissues?
UV light can lead to burns, cancer and blindness (mutations- changes in DNA)
69
What is a pyrimidine dimer?
It is a structure that forms between nucleotide bases
70
What does the photoreactivation photolyase enzyme do to thymine dimers?
This is an enzyme that can repair DNA to its original pyrimidine bases after exposure to
UV light (present in white light)
71
What is the most effective wavelength of UV light for killing microbes?
UVC or the germicidal wavelength
72
What bacteria was used?
Serratia marcescens
73
What about the plate whose lid was on for the UV light treatment?
The lid blocked the UV
74
Plates immediately wrapped in foil after UV treatment were expected to show less
survivors in the kill zone than the plates that were exposed to light after UV treatment –
why?
There is less repair because there is no exposure to white light after UV light
75
What is a pure culture?
Only one organism is present
76
How do you do a streak plate?
On a TSA plate, start in quadrant one and make several light passes. Sterilize your loop again and do not get more sample. Drag from your initial quadrant into quadrant 2.
77
Why do you do a streak plate?
To isolate individual organisms for proper identification
78
What does selective mean?
Selective media inhibits the growth of one type of bacteria and promotes the growth of
the other
79
What does differential mean?
Differential media allows us to distinguish between two similar organisms based on their ability or inability to metabolize the ingredients of the medium. Colonies look different.
80
What will grow on each selective medium?
- MAC: E. Coli
- EMB: E. Coli
- PEAB: Staph aureus
- MSA: Staph
81
What can be observed on each differential plate?
MAC:
- Fermenters (Pink and Purple)
- Non-fermenters (Agar turns yellow/tan)
EMB:
- Strong F (metallic green)
- Mild F (pink/purple)
- Non-F (normal color)
PEAB:
- Alpha (green halos)
- Beta (clear halos)
- Gamma (Growth/no color change)
MSA:
- Staph aureus (yellow)
- Other Staph (remains pink)
82
Which type of plate was used for this exercise?
(staph)
Mannitol salt agar
83
Contrast transient and resident flora.
- Transient is present for several days to months and disappears.
- Resident is what makes
up our normal microbiome and is not harmful.
84
Why is E. coli classified as skin transient flora?
It doesn’t normally live on our skin
85
What does Staphylococcus epidermidis look like when grown on an MSA Plate?
- doesn’t ferment mannitol
- does not produce an acid
- produces pink colonies
86
What does Staphylococcus aureus look like when grown on an MSA Plate?
- ferments mannitol
- produces an acid
- produces yellow colonies
87
Why does the agar change to yellow color where Staphylococcus aureus is growing?
It is coagulase positive
88
Name 3 physiological (body action) effects that interfere / inhibit growth on the skin.
- Sebum
- sweat
- normal microbiota
89
Which type of plate was used for this exercise and why?
(staph and strep)
Blood agar because it allows B-hemolysis
90
How is this media differential? (blood agar)
Be able to identify the different types of hemolysis.
Alpha hemolysis:
- partial destruction of RBCs
- green-brown halos
Beta hemolysis:
- complete destruction of RBCs
- clear halos (looks yellow)
Non-hemolysis:
- organisms will grow but will not destroy the RBCs
- No halos or color
change
91
Name 3 bacteria present in normal / resident throat flora.
Streptococcus, Staphylococcus and Hemophilus
92
Are tests available that can differentiate between Staphs and Streps? Name one.
Catalase test using hydrogen peroxide
93
How do you do a catalase test?
- Drop hydrogen peroxide onto the beta hemolytic colonies.
- Vigorous quick bubbling
indicates a positive for Staph
94
Which genus is catalase positive?
Staph
95
what genus is Catalase negative?
strep
96
T / F Staphs and Streps can be pathogenic.
TRUE
97
What does ELISA stand for?
Immunosorbent Assay
98
What part of the immune system makes antibodies?
Plasma cells
99
direct and indirect ELISA tests.
Direct:
- using the secondary antibody
- more common.
Indirect:
- using the primary antibody
-is rare.
- (estimating antibodies)
100
Where does the primary antibody come from in an indirect ELISA?
Immunoglobulins
101
What does it specifically bind to in the ELISA test?
the antigen
102
What is the job of the secondary antibody in ELISA?
The secondary antibody either binds to the immobilized primary antibody by an enzyme link or does not bind and is removed
103
When was the chromogen added?
Chromogen is added after the secondary antibody
104
What did addition of the chromogen cause?
Color change
105
Know how to read the results of an ELISA test.
Look at the test group and compare it to the Negative control and Positive Control
