Lab Midterm Lecture Info Flashcards
(209 cards)
1
Q
first person to observe microbes, including bacteria, which he called “animalcules”
A
Antonie van Leeuwenhoek, 1600s
2
Q
the use of any kind of microscope that uses visible light to observe specimens
A
light microscopy/compound microscopy
3
Q
see living organisms, motility, bright objects on a dark background
A
dark-field microscopy
4
Q
blocks most of the light from the illuminator in dark field microscopy
A
opaque disk
5
Q
the only light that reaches the objective in dark field microscopy
A
refracted or reflected light by structures in the specimen
6
Q
causes syphilis
A
treponema pallidum
7
Q
two sets of light- one directly from the light source, one from light that is reflected or diffracted from a structure in the specimen
A
phase contrast microscopy
8
Q
structures that differ in features such as ___ ___ will differ in levels of darkness; phase microscopy
A
refractive index
9
Q
example- 40x objective lens
a. E- type apochromatic lens
b. 40x magnification
c. numerical aperture of 0.65
d. 160 mm mechanical tube length
e. 0.17 mm thickness cover slip
A
important markings on a light microscope objective lense
10
Q
___ lenses are made in such a way that chromatic aberration is reduced to a minimum
A
apochromatic
11
Q
describes the capacity of a microscope to enlarge an image
-objective and ocular
A
magnification
12
Q
the ability to distinguish two adjacent objects as distinct and separate
A
resolution
13
Q
light-gathering ability of the objective lense
A
numerical aperture
14
Q
__ wavelength = better resolution
A
shorter wavelength
15
Q
limit of resolution for a light microscope is about __ um
A
0.2
16
Q
objects closer together than this value cannot be resolved as distinct and separate
A
limit of resolution
17
Q
magnification of ocular lense
A
10x
18
Q
D = wavelength / NAcondenser + NAobjective
A
formula for calculating the actual limit of resolution for a microscope
19
Q
has the same refractive index as glass
A
immersion oil refractive index
20
Q
increases the maximum angle at which light leaving the specimen can strike the lense
A
immersion oil
21
Q
mostly UV or blue light
A
light source of fluorescent microscopy
22
Q
uses an electron beam to create an image, with electromagnets acting as lenses
A
electron microscopy
23
Q
image generated using flurescence
A
fluorescent microscopy
24
Q
uses electron beams to observe small, thin specimens such as tissue sections and sub-cellular structures
A
transmission electron microscope (TEM)
25
uses electron beams to visualize surface 3D surface details of specimens
scanning electron microscope (SEM)
26
used to clean all lenses
dry, clean lens paper
27
used to remove oil from the stage
ethanol
28
-low-power objective in position and body tube lowered completely
-centered mechanical stage
proper set up of microscope
29
coil the electric cord around the body tube and the stage
proper cord position during microscope transfer
30
three types of bacterial morphology
cocci, bacilli, spiral
31
example of diplococci
streptococcus pneumonia, Enterococcus
32
example of cocci clusters
staphylococcus aureus
33
example of cocci chains
streptococcus pyogenes
34
example of flagellate rods
salmonella typhi
35
example of bacilli chains
bacillus anthracis
36
example of spore formers
clostridium botulinium
37
example of spirochetes
treponema pallidum morphology
38
example of spirilla
helicobacter pylori
39
example of vibrios- aka curved rods
vibrio cholera
40
will occur if a culture plate is left open on a lab bench
microorganisms will contaminate
41
practices and procedures to prevent contamination from pathogens and minimize the risk of infection
aseptic technique
42
6 inches
stay within ___ inches of bunsen burner to minimize contamination
43
a liquid medium
broth
44
usually made with 1.0 % - 2.0% agar in plates or tubes
solid medium
45
usually made with 0.3%-0.5% agar in plates or tubes
semi-solid medium
46
refers to cultivating and growing microorganisms in the lab on various types of media
culturing
47
-solidifying agent
-allow surface growth or restrict mobility
-grow bacteria over a range of temperatures
-liquifies at 100 C and solidifies at ~42C
agar
48
organism, name, section number, date
tube labels
49
how to sterilize an inoculating loop/needle
-hold in blue cone of flame at 45 degree angle
-let cool before transfer
-re-flame when finished and place upright back in block container
50
considered contaminated at all times
wire holder of inoculating loop/needle
51
color of inoculating needle/loop for sterilizing in flame
orange/red incandescence
52
-abundance of growth
-pigmentation
-optical characteristics
-form
microbe culture characteristics of agar slants
53
-growth type and distance from stab
-pigmentation or appearance
microbe culture characteristics seen in agar deep tubes
54
-abundance of growth
-type of growth
microbe culture characteristics seen in broth medium
55
transfer from agar slant to sterile broth tube
use loop
56
from broth stock to sterile agar deep tube
use needle and stab inoculation
57
from broth stick to sterile agar slant tube
use loop and inoculate surface of slant
58
incubation temperature of bacillus cereus and Serratia marcescens
25 degrees C for 24 to 48 hours, exercise 2
59
incubation temperature for staphylococcus epidermidis and Pseudomonas aeruginosa
37 degrees C for 24 to 48 hours, exercise 2
60
bacteria (pure or mixed culture) are diluted until single cells are separated from one another/isolation of distinct colonies
streak plate definition (4 quadrant streak)
61
-to grow into isolated pure colonies/determine purity of culture
-color, morphology and other physical characteristics
-quick first step in the identification of the bacteria being studied
purposes of streak plate
62
heavy confluent growth, light growth, discrete colonies
types of growth on streak plate
63
streak plate but with 3 sections instead of 4
t-streak
64
1. sterilize loop
2. touch loop to bacterial culture (broth or colony)
3. streak heavy in one quadrant
4. flame loop
5. streak in and out of 1st quadrant a few times into 2nd quadrant then only streak in 2nd quadrant to fill
6. repeat steps for the 3rd and 4th quadrants
7. when done, sterilize loop
procedural steps for streak plate- isolating discrete bacterial colonies
65
-form
-elevation
-pigmentation/color
-size
-optical properties
microbe culture characteristics of agar plates
66
colonies are mucoid, raised, and shiny
Klebsiella pneumoniae agar plate properties
67
sample is pipetted onto surface of agar plate, sample is spread evenly over surface using sterile glass spreader, surface colonies seen
spread-plate method
68
sample is pipetted into sterile plate, sterile medium is added and mixed well, surface and sub-surface colonies seen
pour-plate method
69
24 to 48 hour nutrient broth cultures of a mixture of E. coli and B. subtilis
cultures for exercise 3
70
two trypticase soy (TS) plates per student
media used for exercise 3
71
incubation temperature for exercise
37 degrees celcius
72
practice aseptic technique to transfer cultures
-agar slant to terile broth tube (loop)
-broth to agar deep tube (needle and stab)
-broth to agar slant (loop surface)
exercise 2 culture transfer
73
perform two 3- or 4-quadrant streak plates and two spread plates using a mixed organism broth culture
exercise 3- isolating distinct colonies
74
picking up a single isolated colony
pure culture
75
to prepare a stock culture of an organism using isolates from the mixed cultures prepared on the agar streak plate and or the spread plate in exercise 3
purpose of exercise 4- preparation of pure cultures
76
solution consisting of a solvent (usually water or ethanol) and a colored molecule (often a benzene derivative)- the chromogen
stains
77
positive chromogen, stains cell
basic stain
78
negative chromogen, background is stained
acidic stain
79
auxochrome
provides covalent or ionic bonds in stain
80
colored compound, benzene (colorless) and chromophore (imparts color)
chromogen
81
developed the gram stain
Hans Christian Gram 1884
82
appear purple after staining
gram-positive bacteria
83
appear pink after staining
gram negative bacteria
84
differences in cell wall structure
reason for color difference in gram stain
85
has thick cell wall
gram-positive bacteria structure
86
has thin cell wall, with outer membrane
gram-negative bacteria structure
87
1. primary stain- crystal violet
2. mordant- iodine
3. decolorizing agent- alcohol-acetone
4. counterstain- safranin
order of application for gram stain
88
appears colorless after decolorizing agent
gram-negative cells
89
contains notable human pathogens such as M. tuberculosis, M. leprae
genus Mycobacterium
90
gram-positive bacteria that are acid-fast because of the waxy mycolic acid in their cell walls
mycobacteria
91
detects the presence of cell walls rich in mycolic acid
acid-fast staining protocol (Ziehl-Neelsen Method)
92
stains everything strongly in acid-fast staining
carbolfuchsin
93
decolorizing agent in acid-fast staining
acid alcohol
94
counterstain in acid-fast staining
-stains non acid-fast cells blue
methylene blue
95
structures that protect the bacterial genome in a dormant state when environmental conditions are unfavorable
endospore
96
bacillus and clostridium
endospore-forming, gram-positive bacteria genera
97
the process by which vegetative cells transform into endospores
sporulation
98
staining detects endospores
Schaeffer-Fulton method
99
malachite green and carbolfuchsin
need steam to enter cells for staining
100
1. primary stain- malachite green
2. spore retains malachite green while bacterias structures loose the stain- spores resist decolorization with water
3. counterstain- safranin
process of spore staining- Schaeffer-Fulton Method
101
non-specific media configured to culture a wide range of microorganisms without many restrictions
general growth media
102
designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes
selective media
103
make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate
differential media
104
Nutrient agar and Luria-Bertani (LB) Broth
examples of general growth media
105
similar to selective media but designed to increase numbers of desired microbes to detectable levels
enrichment
106
both selective and differential for staphylococcus aureus
mannitol salt agar
107
high salt concentration (7.5% NaCl) inhibits growth of most bacteria except staphylococci
what makes MSA selective for staphylococci
108
carbohydrate mannitol is fermented by S. aureus, resulting in acidic end products- turns phenol red indicator yellow around growth
MSA differential reaction of staphylococcus aureus
109
microbiologists often use this to identify bacterial species that destroy red blood cells
blood agar
110
alpha (a) hemolysis
partial/incomplete lysis of RBC, zone of partial clearing = green halo around colonies
111
beta (B) hemolysis
complete lysis of RBC, complete zone of clearing around colonies
112
gamma (Y) hemolysis
no lysis of RBCs, no clearing of medium surrounding colonies, no color change
113
tends to be used to recover fastidious bacteria, often Streptococcus species (pathogens)
blood agar bacterial species
114
blood agar is a ___ media, bacteria distinguished by ability to cause hemolysis
differential media (hemolysis)
115
selective and differential medium containing lactose, bile salts, neutral red, and crystal violet
MacConkey Agar
116
interferes with the growth of many gram-positive bacteria and FAVORS gram-negative bacterial growth, especially Enterobacteriaceae
MacConkey agar favorable growth
117
named enterics, reside in the intestine, are adapted to the presence of bile salts
Enterobacteriaceae
118
ph indicator in MacConkey agar, colorless above a pH of 6.8 and red at a pH below 6.8
- acid accumulating from lactose fermentation turns dye red
neutral red dye
119
-used for the isolation and identification fo fecal coliform bacteria
-sugars act as fermentable substrates, which yield acid by-products
-dyes inhibit growth of gram-positive bacteria and act as pH indicators (only used to see gram-negative)
Eosin Methylene Blue Agar
120
eosin and methylene blue inhibit growth of gram-positive bacteria
why eosin methylene blue agar is selective
121
distinguishes gram-negative bacteria that can ferment lactose from those that cannot
why eosin methylene blue agar is differential
122
lactose fermenters in eosin methylene blue agar
produce dark colonies- metallic green shade
123
non lactose fermenters in eosin methylene blue agar
produce opaque or translucent colonies
124
example of lactose fermenter
Escherichia. coli
125
small amounts of acid production in eosin methylene blue agar
results in pink coloration of the growth
126
an undefined, selective medium that allows growth of gram-positive organisms and stops or inhibits growth of most gram-negative orgamisms
phenylethyl alcohol agar
127
false negative results and low colony counts
viable but non-culturable (VBMN)
128
bacterial species that will grow within a temperature range of -5°C to 20°C
- all will grow between 0° and 5°
psychrophiles
129
bacteria species that will grow within a temperature range of 20°C to 45°C
-all can grow at human body temperatures (37°C) and are unable to grow above 45°C
-three distinct groups
mesophiles
130
optimal growth at 20-30°C
-mesophile
plant sacrophytes
131
optimum growth temperature at 35-40°C
-mesophile
organisms that grow in warm blooded hosts
132
optimum growth at 20-40°C but are capable of growing at 0°, typically found in soil and water habitats in temperate regions
-mesophile
psychrotolerant
133
bacterial species that will grow at 35°C and above
-two groups exist
thermophiles
134
thermophiles that will grow at 37°C but grow optimally at 45-60°C
facultative thermophiles
135
thermophiles that will grow only at temperatures above 50°C, optimum growth above 60°C
obligate thermophiles
136
detection of gas accumulation
air bubble in durham tube (within a culture tube)
137
enzyme that protects cell from toxic H2O2- hydrogen peroxide
catalase and peroxidase
138
enzyme that protects cell from toxic O2^- superoxide
superoxidedismutase
139
has no enzymes to protect against toxic oxygen- cannot grow in its presence
strict anaerobes
140
-requires the presence of atmospheric oxygen for growth
-their enzymatic needs to use oxygen as the final electron acceptor
aerobes
141
microaerophiles
-require limited amounts of atmospheric oxygen for growth
-excess oxygen blocks the activities of their oxidative enzymes and results in death
142
require the absence of free oxygen for growth because the require the presence of molecules other than oxygen to act as the final electron acceptor
-presence of oxygen is lethal
obligate anaerobes
143
fermentative organisms that do not use oxygen as a final electron acceptor
-produce enzyme so they can survive in the presence of toxic oxygen
aerotolerant anaerobes
144
can grow in the presence or absence of free oxygen
-preferentially use oxygen for aerobic respiration but can perform cellular respiration anaerobically if necessary
-can use nitrates or sulfates as final hydrogen acceptors or use a fermentative pathway
facultative anaerobes
145
proof of life/viable counts
show that an organism can replicate and form colonies on an agar plate
146
serial 10 fold dilutions
allow estimation of the number of live organisms in the initial sample
147
pros of serial dilution
allow for colony isolation, quantifies only live cells, controls for mixed cultures
148
cons of serial dilution
takes longer (incubation time), user error, material use
149
less than 30 colonies, TFTC
too few to count
150
more than 300 colonies, TNTC
too numerous to count
151
colony forming units / milliliter of initial culture
CFU/mL
how to present results of serial dilution viable counts
152
CFU calculation formula
(number of colonies on plate) x (reciprocal of dilution of sample)
153
standard CFU formula
(colony count on agar plate) / (total dilution of tube) x (amount plated)
154
bacteria normally reproduce by __ __
binary fission- how bacteria reproduce
155
intense activity preparing for population growth, but no increase in population
lag phase of the growth curve
156
logarithmic, or exponential, increase in population
log phase of the growth curve
157
period of equilibrium; microbial deaths balance production of new cells
stationary phase of the growth curve
158
population is decreasing at a logarithmic rate
death phase of the growth curve
159
the logarithmic growth in the log phase is due to reproduction by ___ ___ (bacteria) or __ (yeast)
binary fission, mitosis
160
the generation time of a culture
the growth rate of a culture, determined by the number of cells at several time points, only calculated during the log phase
161
equation of generation time
g = t/n
g - generation time (minutes)
t - time of exponential growth
n - the number of generations
162
turbidity- optical density
- 600 nm light source
detects "cloudiness" or transmittance through a medium
163
pros of optical density
quick and easy
164
cons of optical density
do not distinguish between live and dead cells, contamination not taken into account, does not allow for isolation of colonies
165
formula for n
(log N - log N0) / log 2
166
axes of graph converting optical density to cell number (original graph)
x- optical density
y- cell number
167
axes of graph used to determine generation time (new graph)
x- time
y- cell number
168
polymer of collagen that makes up connective tissues, liquid above 25°C
gelatin
169
break down collagen
-nutrient acquisition, virulence
gelatinases (collagenases)
170
liquefaction after growth followed by refrigeration indicates hydrolysis
gelatin hydrolysis test
171
iodine is used to detect the presence of starch, hydrolysis revealed as a clear zone around bacterial growth
amylase/starch hydrolysis test
172
tributyrin agar, lipase-positive organisms produce clear zone around growth
lipid hydrolysis
173
converts 1 molecule of glucose to 2 molecules of pyruvate
glycolysis
Embden-Meyerhof pathway
174
molecular oxygen as the final electron acceptor (more ATP generated)
aerobic cellular respiration
175
inorganic ions rather than oxygen are final electron acceptor
anaerobic cellular respiration
176
doesn't require oxygen and organic substrate is the final electron acceptor
fermentation
177
may cause acid and gas
carbohydrate fermentation
178
indicator of acid production (yellow = acid)
phenol red indicator
179
indicator of presence of air bubble
durham tube
180
indicates that an organism can metabolize sugar in the tube
color change red to yellow, acid production
181
fermentation and gas production
color change and bubble in the durham tube
182
color change to a dark pinkish-red for carbohydrate fermentation
indicates a basic or alkaline metabolic product due to utilization of the peptone rather than the sugar
183
reacts with indole to produce red/pink color
Kovac's reagent
184
tryptophanase
enzyme used to identify bacteria that produce indole
185
produced by certain enterobacteriaceae by two pathways
H2S (Hydrogen sulfide gas)
186
combines with hydrogen sulfide gas to form black sulfide precipitate
FeSO4 (ferrous ammonium sulfate)
187
a positive result for __ is indicated when radiating outward from the central stab; a negative result shows only along the stab line
motility
188
all ferment glucose to organic acids (the acid varies)
enteric microorganisms
189
differentiates between E. coli and K. aerogenes final end products
MR-VP tests
190
determine ability to oxidize glucose to make acid end products (E. coli)- makes red color
methyl red test
191
K. aerogenes converts acids to acetylmethylcarbinol raising the pH brining color back to yellow
-barritt's reagent turns pink with acetylmethylcarbinol
voges proskauer test
192
determine the ability of an organism to use the enzyme citrase to use citrate as a sole carbon source
citrate IMViC test
193
growth on the slant and blue color
means citrate was utilized as a carbon source
194
differentiate organisms based on their ability to hydrolyze urea with the enzyme urease
urease test
195
urinary tract pathogens from the genus __ may be distinguished from other enteric bacteria by their rapid urease activity
Proteus
196
broth tube turns bright pink
positive urease test
197
fissures or cracks in the clot of litmus tube
gas production in litmus tube
198
acid clots solidify the medium and can appear __ or __ with a pink band at the top depending on the oxidation-reduction status of litmus
pink, white
199
lactose fermentation acidifies the medium and turns the litmus pink
pink in litmus milk test
200
reduced litmus is white
white color in litmus test
201
oxidized litmus is purple
purple in litmus test
202
alkaline reaction for litmus test
blue/purple medium or blue band at the top
203
stormy fermentation
heavy gas production that breaks up the clot
204
digestion of peptone
milk protein completely digested, clear to brown fluid
205
acid clot formation
proteolysis
206
enzyme catalase
degrades hydrogen peroxide
207
performed on a glass slide, colony + hydrogen peroxide - bubbles form
positive catalase test
208
uses a chromogenic reducing agent as an indicator to detect bacteria that produce cytochrome oxidase
oxidase test
209
indicator in oxidase test
- donates electrons to cytochrome oxidase, becomes self oxidized changing from light pink reduced to a dark maroon almost black oxidized compound
p-aminodimethylalanine oxalate
