Lab Practical Study Guide

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1
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What level of BSL precaution do we follow?
BSL-2
2
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why do we wear lab coat, closed toed shoes, and have hair tied back
prevent contaminating the working space and for our own protection
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when should we not wear gloves?
around a flame; they could melt and injure the hands
4
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when is eye protection worn?
staining and using chemical reagents
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where do used test tubes, media, and other contaminated glass re-useable (slides, flasks) go?
red autoclave cart
red autoclave cart
6
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where do contaminated disposables (gloves, paper towels) and ALL agar plates go
into a biohazard bin
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where do uncontaminated gloves and paper towels go
into regular trash cans
8
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where do contaminated sharps (broken wet-mount slides, broken tubes with culture) go
in the biohazard sharps bin after dousing with 70% EtOH
in the biohazard sharps bin after dousing with 70% EtOH
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where do clean uncontaminated sharps (razor blades, broken glass) go
into regular sharps bin
10
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what should always be on a label
GREEN
Lab section (S2)
Date you inoculated (month/day)
Incubation temperature (in Celsius)
Culture source
Species if known (never write out full genus name; B. subtilis)
Genus if known, but species not known (ex. Staphylococcus sp. )
If neither species or genus is unknown (name of environmental source)
SOMETIMES include:
First name ONLY
Experimental conditions (streak #, controls)
11
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what should be on the label for microscope slides
source, stain type, date
12
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What is it and what are the pros and cons
What is it and what are the pros and cons
TSA broth
Pros: Grows bacteria in large quantities, quickly, under uniform conditions
Cons: Easily contaminated
13
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what are the different types of solid media
slant, deep, and plate
14
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what is the function of agar
Agar: the gelling agent used in most solid media
Cheap, sustainable, no nutritive value (few bacteria can digest it)
15
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what is it and what is it used for
what is it and what is it used for
solid Petri dish media for isolation of pure cultures and various tests
16
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What is it and what are the pros and cons
What is it and what are the pros and cons
TSA slant: solid test tube media for long-term storage of pure cultures and biochemical tests

Pro over broth: easy to catch contamination; look for unexpected growth off the S-path
Pro over plates: smaller surface for air exchange, so less likely to be contaminated and do not dry out as quickly
Con: not enough room to isolate pure culture; only used when pure culture has been isolated
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What is it and what is it used for
TSA deeps: solid test tube media for long-term storage of pure anaerobic cultures and oxygen requirement tests (stabs)
18
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what is defined/minimal/synthetic media
media that contains nutrient in pure chemical form and does not contain any plant or animal tissue; the exact chemical composition of the media is known
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what is complex/undefined media
complex/undefined media: media that contains nutrients from plants or animal extracts; the exact chemical formula is not unknown
20
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what are non-fastidious microorganisms
microorganisms that grow on general purpose media like TSA (our lab stuff)
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Fastidious microorganisms
microorganisms that require specific nutrients or growth factors that are not found in general purpose growth media (pathogenic)
22
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Autoclave
a machine that uses pressurized steam at high temperatures for a defined period of time; 121 degrees Celsius with steam pressure of 15 psi for 15 minutes
23
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do long autoclave times increase sterilization
no, it might even burn and caramelize the sugars in the media
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Why do we autoclave media, equipment
media ingredients/equipment are not sterile and will be contaminated with microbes from surrounding air, glassware, spatulas, and so on
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Why do we autoclave biohazard
to kill any remaining microbes in the bags, safer disposal
26
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Why autoclave and not boil?
Autoclaving can generate higher temperatures where microbes that are boiling-resistant cannot survive
27
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what shape is this
what shape is this
spindle
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what shape is this
what shape is this
filamentous; usually fungi
29
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what are the different types of whole colony forms
circular, spindle, punctiform, irregular, wrinkled, filamentous, rhizoidal
circular, spindle, punctiform, irregular, wrinkled, filamentous, rhizoidal
30
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what are the different types of colony elevation
flat, raised, convex, umbonate, convex with papilate surface
flat, raised, convex, umbonate, convex with papilate surface
31
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what are the different types of colony margin forms
what are the different types of colony margin forms
entire, undulate, lobate, crenate, serrate, filamentous, rhizoidal
32
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what kind of surfaces have high microbial load and diversity
soft, porous, natural material wha
33
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what kinds of surfaces have lower microbial load and diversity
Hard, metal, non-porous, man-made surfaces
34
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what are basic experimental design with control and hypothesis
Ask a scientific question
Form a hypothesis
Design an experiment to test your hypothesis
Collect results and analyze data
Draw a conclusion to support or not support your hypothesis
35
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how to do a T-streak
how to do a T-streak
a T-Streak as we taught it
Incinerator for 5 seconds, brand loop for 5 seconds, touch center of the colony (don’t scoop)
Streak outside to inside, incinerator for 5 seconds, turn the plate, brand loop into agar to cool, 2nd streak crosses 1st streak, incinerator for 5 seconds, brand loop into agar to cool, 3rd streak crosses 2nd streak, incinerator for 5 seconds, set sterile loop on the bench
36
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what is this
what is this
turbid liquid culture
37
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what is turbidity
microbes grow throughout the medius, making it cloudy
38
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WWW: if there's no growth
the initial culture was dead or you killed the inoculum with a hot loop, or the loop did not touch the inoculum
39
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what is Aseptic transfer:
the prevention of contamination of the culture and the microbiologist with unwanted microorganisms from the environment or other cultures
40
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what is the Purpose of aseptic methods:
maintaining stock cultures and transferring pure cultures from one vessel to another
41
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what is the purpose of Broth inoculation:
impossible to tell by eye if there’s a single species, must be determined microscopically; good for growing up pure cultures or cultures quickly
42
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what is the purpose of slant inoculation
inoculation of a “S” patten up the agar surface; great for pure cultures with lower risk of contamination
43
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what is isolation streaking
for obtaining pure culture from mixture of the environment; used to keep pure cultures alive and pure (subculturing); necessary for pure culture but careful of contamination
44
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what is the only way you can confirm pure culture
checking microscopically for the same cell morphology and gram type
45
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WWW culture transfers
WWW: don’t shake loop around, don’t leave plates open, don’t go back for more inoculum
46
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purpose of broth to broth
grow cultures more rapidly
47
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purpose of plate to plate
pure culture isolation
48
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what are the four objective lenses
4x, 10x, 40x, 100xw
49
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what are the two lenses
ocular len and objective len
50
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a microscope that is ready to be put away
Set the scope to 4x BEFORE removing your slide
Lower the stage
Clean all objective lenses and ocular lenses
Wrap the power cord around the base
Replace dust cover
51
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what is it and what does it do
what is it and what does it do
Condenser: collects light; moves up and down to focus the light on the specimen
Condenser: collects light; moves up and down to focus the light on the specimen
52
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what is it and what does it do
what is it and what does it do
Iris diaphragm: located between the lamp and the condenser, controls the amount of light that passes through the specimen
Iris diaphragm: located between the lamp and the condenser, controls the amount of light that passes through the specimen
53
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what is it
what is it
Objective lens: (4x, 10x, 40x, 100x) lenses closest to the specimen
54
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what is it
what is it
ocular lens: eyepiece
55
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how to calculate total magnification
Total magnification: 10x (ocular lens) times the objective lens
56
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what mag to determine size
1000x
57
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how to determine size
1 ocular unit is 10 microns (ocular units), one tick mark is 1 micron (always report in microns!!)
diameter/width and length of a cell
Might have to provide with a range for the length
58
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Why do we need immersion oil to observe bacteria?
Resolving power: ability to distinguish two adjacent points as distinct and separate
Numerical aperture (NA): the cone of light that enters the lens; short wavelength is the best
Immersion oil improves the numerical aperture and hence the resolving power of the lens; it has a refractive index similar to glass
59
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label this microscope
label this microscope
left
1. ocular lens
2. nosebridge
3. objective collar/wheel
4. objective lens
5. stage clips
6. stage
7. iris diaphragm
8. condenser
9. light source

right
1. NA
2. arm
3. stage
4. coarse knob
5. fine knob
6. stage control
7. base
8. light intensity dial
9. power
left
1. ocular lens
2. nosebridge
3. objective collar/wheel 
4. objective lens
5. stage clips
6. stage
7. iris diaphragm 
8. condenser
9. light source

right
1. NA
2. arm
3. stage
4. coarse knob
5. fine knob
6. stage control 
7. base
8. light intensity dial
9. power
60
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what stain is this
Crystal Violet: gram positive
Crystal Violet: gram positive
61
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what stain is this
what stain is this
Safranin: gram negative
62
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what stain is this
what stain is this
Malachite green: endospores
63
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what stain is this
what stain is this
Methylene blue: acid-fast stain
64
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Prokaryotic vs. Eukaryotic cells
Prokaryotic vs. Eukaryotic cells
left: eukaryotes (HUGE) right: prokaryotes (smol)
65
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Rods vs. Cocci
NOTE: short rods can be mistaken for cocci; gram negative cocci are rare, it's probably a dehydrated rod
NOTE: short rods can be mistaken for cocci; gram negative cocci are rare, it's probably a dehydrated rod
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Pairs vs. Tetrads
knowt flashcard image
67
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chains vs. single
knowt flashcard image
68
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How do you make a simple stain? What can go wrong?
smear a single-cell thick film on top of the slide (broth or water/solid), smear is air dried and heat fixed, slide is flooded with stain then rinsed with water
WWW: did not heat dry properly so cells washed off with the water, aimed the water at the cells
NOTE: timing isn’t that big of a deal with simple stains
69
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what is a simple stain
Simple stain: a single stain is used, all cells and structures staint he same color
70
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What is a positive vs. negative stain?
Positive stain: the stain attracts to the cells
Negative stain: the stain stains the background of the slide
71
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Cheek swab -- differentiate prokaryotic vs. eukaryotic cells.
Eukaryotes (10 - 100 microns) are much larger than prokaryotic cells (0.1 - 5 microns)
72
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Be able to heat fix and stain a sample of bacteria
Label: source, first name, date, simple, stain abbreviation (CV, Saf, MG, MB)
Smears: solid media (drop of water, spread over slide), liquid media (one drop, spread over slide)
Set hot plate to 3 and warm for at least a minute
Stain time:
Crystal violet: 10 seconds
safranin/malachite green: 30 seconds
Methylene blue: 60 seconds or longer
Rinse with DI water, aim for the labeled part
Blot dry with bibulous paper
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crystal violet stain time
10 seconds
74
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safranin/malachite green stain time
30 seconds
75
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methylene blue stain time
60 seconds or longer
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Be able to name and recognize the 4 simple stains we used
Crystal Violet, Safranin, Methylene Blue, Malachite Green
77
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Be able to tell yeast and bacteria apart.
Be able to tell yeast and bacteria apart.
Yeast are huge!! Bacteria are tiny
78
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what is it
what is it
Rods: hot-dog-shaped
NOTE: gram-negative coco are rare; you probably have a short gram-negative rod
79
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what is it
what is it
Cocci: spherical
Tetrads: groups of 4
Cuboidal: packets of 8
NOTE: can appear oval just before cell division
80
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what is it
what is it
Irregular rods: dumbbell-shaped, club shaped, cells can change shape over time
81
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what is it
what is it
Vibrio: curve rods like a comma
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what is it
Spiral: curly like a corkscrew
Spiral: curly like a corkscrew
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what is it
Spirochetes: spiral-shaped bacteria with many, many turns
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what is it
what is it
Pleomorphic: change shape over time, making X’s, Y’s and other irregular shapes
85
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trust wet mount or simple stain?
trust wet mount
86
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Growth on a 12-well NaCl plate
Growth on a 12-well NaCl plate
NaCl tolerance starts from the lowest concentration of NaCl with growth to the highest concentration with growth
optimum NaCl concentration: the one with the most total growth
87
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what is it
what is it
TSA deep: a confirmatory test for the Gas Pak anaerobic system
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how to interpret results for oxygen deep
Aerobic: growth on the surface of the deep and top part of the line
Anaerobic: growth at the bottom of the line only
Facultative anaerobe: growth all the way down the stab line and growth on surface
Microaerophiles: no growth on top and growth in the middle (not top or bottom of line)
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tolerant vs philic
Tolerant: doesn’t die
Philic: grows
90
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what is strenotherms:
organisms that function over a narrow range of temperature
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what is eurythermal:
microorganisms that grow over a wide range of temperatures (30 - 40 degrees)
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what is stenothermals:
microorganisms that grow within a very narrow (
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what is psychrophiles:
optimal growth less than 15 degrees Celsius; those that can grow below 15 but have a higher optimal temperature don’t count
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what is mesophile:
optimal temperature between 20 to 40 degrees
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what is thermophile:
optimal temperature between 45 and 80 degrees
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what is hypterthermophile:
optimal temperature above 80 degrees
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what is a halophiles:
bacteria that require high concentration of NaCl to grow
98
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what is a hypertonic solution:
greater concentration of solutes than bacterium; water moves out of the bacterium into the hypertonic solution; cell shrinks and die
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what is a hypotonic solution:
smaller concentration of solutes than bacterium; water moves into the bacterium; cell swells and bursts
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what is a isotonic:
no water moves, bacteria can live