LAB EXAM #1 experiments deck Flashcards
(90 cards)
1
Q
Aseptic technique:
A
Insures that there is no contamination of microorganisms
2
Q
Loops are used to:
A
Transfer from liquid media to liquid media or petri plates
3
Q
Needles are used to:
A
Transfer from solid media to solid media or petri plates
4
Q
Purpose of smear prep:
A
To prepare the organism for staining
5
Q
Smear preps are used to determine:
A
- Cell shape
- Arrangement of cells
- Internal structures
6
Q
Procedure of aseptic technique:
A
- Transfer org to slant
- Transfer org from slant to broth
- Transfer org from broth to plate
7
Q
Procedure of smear prep from a broth: (3)
A
- Using AT, transfer 3 loops of org to the middle of the slide
- Air dry
- Heat-fix slide by passing slide through the bunsen burner flame 5 times
8
Q
Procedure of smear prep from a solid media: (4)
A
- Using AT, place 3 loops of WATER onto center of slide
- Transfer org to the water
- Air dry
- Heat-fix the slide by passing through the bunsen burner 5 times
9
Q
Purpose of heat-fixation of smear preps?
A
Kills the organism and adheres them to the slide so they aren’t washed off during staining
10
Q
Purpose of simple staining procedure: (2)
A
- Used to make bacterial cells easier to visualize on the slide
- Used to determine bacterial cell morphology (cell shape)
11
Q
2 types of dyes for staining:
A
Basic dye
Acidic dye
12
Q
Basic dye:
A
- Positively charged chromophore
- Works well with bacterial cells because they have a negative charge
- Stains inside of cell
13
Q
Acidic dye:
A
- Negatively charged chromophore
- Repelled by the negative charge of the bacterial cell
- Stains outside of cell
14
Q
3 main shapes of bacteria:
A
- Rods (bacilli)
- Cocci (spherical)
- Spiral or curved
15
Q
Dye use in simple staining experiment:
A
Methylene blue
16
Q
Procedure for simple staining: (4)
A
- Using AT, make a smear prep by adding org to middle of slide
- Air dry and then heat-fix slide
- Add methylene blue
- Rinse slide and view under oil
17
Q
Purpose of negative stain:
A
To demonstrate the size and shape of bacteria
18
Q
Appearance of bacterial cell in a negative stain:
A
Negative stain stains the background of bacterial cell and bacterial cell will appear transparent against a dark background
19
Q
Dye used in negative stain experiment?
A
Nigrosin or india ink
20
Q
Procedure for negative stain experiment: (4)
A
- Add 1 drop of india ink near end of slide.
- Transfer org into india ink drop
- With another slide, place the edge of the new slide against the drop of india ink and push the spreader slide across the slide
- Air dry then view under oil
21
Q
2 types of gel-like layer on the outside of the cell wall:
A
- Capsule layer
2. Slime layer
22
Q
Capsule layer: (3)
A
- Gelatinous layer
- Attached tightly to bacteria
- Made of polysaccharides known as glycocalyx
23
Q
Slime layer:
A
- Layer made of ECM
- Loosely attached to bacteria
24
Q
Function of capsule and slime layer:
A
- Help cells from being engulfed or destroyed
- Attachment to solid surfaces
25
In the capsule staining experiment, what is the first step?
Negative staining procedure
26
Under oil, how do capsule appear?
Halos around pink/red cells with a dark background
27
Dyes used in capsule staining experiment:
Include charges
- Congo red: acidic dye, stains background
| - Maneval's: basic dye, stains inside of cell
28
Procedure of capsule staining experiment: (7)
1. Add 1 drop of congo red to end of slide
2. Transfer org into congo red
3. With another slide, place the edge of the new slide against the drop of congo red and push the spreader slide across the slide
4. Air dry
5. Add Maneval's, as the counterstain, to the congo red
6. Gently rinse
7. Air dry and view under oIL
29
Hans Christian Gram:
Danish physician that developed a staining technique to differentiate bacterial cells from eukaryotic nuclei
30
Purpose of gram stain:
Provides identification of unknown bacteria
31
Gram positive cells:
- Appear purple
| - 90% peptidoglycan 10% teichoic acids
32
Gram negative cells:
- Appear pink or red
| - 10% peptidoglycan 90% outer membrane
33
Purpose of crystal violet in gram stain?
Primary stain that gives it the purple color
34
Purpose of gram's iodine in gram stain?
Mordant- combines insoluble complex with crystal violet in gram positive cells
35
Purpose of 95% ethanol in gram stain?
Decolorizing agent- removes purple from gram negative cells
36
Purpose of safranin in gram stain?
Counterstain for gram negative cells so they may be seen as pink or red
37
Dyes used in gram stain experiment: (4)
- crystal violet
- gram's iodine
- 95% ethanol
- safranin
38
Procedure for gram stain experiment? (6)
1. Make a smear prep by adding org to middle of slide
2. Heat-fix
3. Cover with crystal violet then rinse
4. Cover with gram's iodine then pour off
5. Decolorize with 95% ethanol then rinse
6. Counterstain with safranin then rinse and view under oil
39
Purpose of acid-fast staining:
Used to identify Mycobacterium and Nocardia that have mycolic acid in cell wall
40
Acid-fast bacteria are stained:
Pink to red by the dye basic fuchsin
41
Non-acid fast bacteria are stained:
Blue with the counterstain methylene blue
42
Dyes used in the acid-fast staining experiment: (3)
- Carbolfuchsin
- Acid alcohol
- Methylene blue (counterstain)
43
Procedure for acid-fast staining experiment: (5)
1. Make a smear prep by adding org to middle of slide
2. Cover with carbolfuchsin for 5 min
3. Gently rinse
4. Decolorize with acid alcohol then rinse
5. Counterstain with methylene blue then rinse and view under oil
44
2 species of bacteria that form endospores are:
Bacillus and Clostridia
45
How to enter an endospore in endospore staining?
Heat needs to be applied to help the stain enter the endospore- mordant
46
In the endospore staining experiment, what color will the endospore and vegetative cell be?
Endospore: green due to malachite green
Vegetative cell: red due to safranin
47
Dyes used in the endospore staining experiment:
Malachite green
| Safranin-counter stain
48
Procedure for endospore staining experiment:
1. Make a smear prep by adding org to middle of slide
2. Cover with malachite green
3. Heat-fix and replenish malachite green as needed
4. Rinse
5. Counterstain with safranin then rinse and view under oil
49
Motility is found in:
Spirochetes and rod shaped bacteria
50
Purpose of motility experiment?
To identify the presence of flagella
51
Procedure for wet mount in motility experiment:
1. Make a smear prep by adding org to middle of slide
2. Cover slide with cover slip
3. View under 400x immediately
52
Procedure for motility agar in motility experiment:
1. Take one tube with TTC in it and stab needle into agar for org
2. Incubate media at 25C
53
What is TTC?
A colorless dye that when bacteria reduce it, it becomes red
54
Motile organisms:
Move away from stab line and have a fuzzy appearance
55
Motile organisms that can reduce the TTC:
Move away from stab line and have red fuzzy appearance
56
Non-motile organisms:
Will not move away from stab line and the rest of the media will be clear
57
Non-motile organisms that can reduce the TTC:
Red color in the stab line only and the rest will b e clear
58
Who was the first person to utilize the pure culture technique?
Robert Koch
59
Pure culture technique:
Helps obtain a single kind of organism from a mixed culture
60
2 commonly use methods of the pure culture technique:
1. Pour plate
| 2. Streak plate
61
Streak plate method for the pure culture technique experiment:
Streak TSA plate with org using a quadrant streak
62
Pour plate method for pure culture technique experiment: (6)
Day 1:
1. Obtain 3 petri plates
2. Obtain 3 TSA tubes from the water bath and transfer the mixed culture into tube #1, mix
2. Before pouring into petri plate, transfer 1 loop from tube #1 into tube #2
3. Pour tube #1 into petri plate
4. Transfer 1 loop from tube #2 into tube #3, then pour tube #2 into petri plate
5. Pour tube #3 into petri plate
6. Gently rotate plate to mix content then flip upside down when agar has solidified
Day 2:
1. Obtain 3 TSA slants and transfer 1 colony of each color onto each slant
2. Incubate at 25C
63
UV light falls between:
4nm and 400 nm
64
Purpose of UV light effects experiment:
To demonstrate bacteriostatic effect
65
3 ways that UV light damages our cells:
UVA: tanning beds
UVB: damaging cancer
UVC: germicidal lamps, hoods not seen in nature-> absorbed by O2
66
Procedure for UV light effects experiment:
1. Obtain TSA plate and sterile swab
2. Swab plate with organism
3. When it is your exposure time, place plate in biosafety cabinet with a flash card covering half of the plate
4. Flip over and examine number of colonies exposed to the UV light next lab period
67
Enumeraton:
Number of viable and non-viable microbes in a sample
68
Purpose of enumeration of bacteria experiment:
Determine the concentration of an organism
69
How is turbidity measured?
With a spectrophotometer where % transmittance or absorbance is obtained
70
Why is turbidity considered an indirect method?
It uses absorption of light to determine the amount of colloidal material in suspension
71
E. coli charge and shape:
Gram (-) rod
72
B. cereus: charge and shape:
Gram (+) rod
73
S. aureus charge and shape:
Gram (+) cocci
74
Primary lethal effect of UV light:
At 260nm, DNA absorbs the UV light and bonds are broken at its maximum-> pyrimidine dimers formed
75
Pyrimidine dimers:
Covalent bonds that form 2 adjacent thiamine or cytosine molecules of DNA-> changes shape of DNA-> can't replicate
76
Purpose of effects of temp on growth exp:
To see the effects of temp on growth of organisms
77
Procedure for effects of temp on growth experiment:
Day 1
1. Obtain 6 TSA tubes and label different temps
2. Inoculate each tube with org
3. Obtain 2 TSA slants and label different temps
4. Inoculate with 2nd org
Day 2
1. Measure %transmittance of each temp tube
2. Determine OD and pigment production for each temp tube
78
Procedure for antiseptics mouth experiment:
Day 1
1. Swab inside of mouth and place swab into TSA tube
2. Incubate
Day 2
1. Obtain TSA plate and divide into 3 quadrants
2. Label each with scope, listerine, and cepacol
3. Streak plate with mouth swab
4. Place disks into each quadrant
79
Procedure for antiseptics assigned org experiment:
1. Obtain TSA plate and divide into 3 quadrants
2. Label each with: 70% isopropanol, bactine, and povidone iodine
3. Streak plate with assigned org
4. Place disks into each quadrant
80
T or F
E. coli is apart of Enterobacteriaceoe
T
81
T or F
E. faecalis is apart of streptococci as well as an enterococci:
T
82
Most effective antiseptic:
Least effective antiseptic:
Scope
Cepacol
83
Most susceptible org:
Least susceptible org:
Bactine
70% isopropanol
84
Procedure for antibiotic sensitivity experiment: Kirby-Bauer Method
Day 1
1. Obtain a Mueller-Hinton plate (big plate)
2. Swab plate with org
3. Place abx disc on plate
Day 2
1. Measure zone of inhibition for each abx disc on plate
85
Indirect methods of enumeration of bacteria experiment:
Turbidity
86
Direct methods of enumeration of bacteria experiment:
1. bacterial count
2. standard plate count
3. most probable number
87
Why is turbidity not a good test?
Contribution of dead and living cells
88
Formula for OD:
OD= 2-log(%T)
89
What is the difference between the methods?
Turbidity determines if sample has bacteria in it
Direct methods are a bacterial count
90
Purpose of slant tube:
More surface area
