Midterm Flashcards
(176 cards)
1
Q
Light Microscopes
A
- use visible light/UV rays to illuminate specimens
- include brightfield, darkfield, phase-contrast, and fluorescent instruments
2
Q
fluorescent microscopes
A
- use ultraviolet radiations whose wavelengths are shorter than those of visible light and are not directly perceptible to the human eye
3
Q
electron microscopes
A
use electron beams (instead of light rays) and magnets (instead of lenses) to observe submicroscopic particles
4
Q
Brightfield Microscope
A
- two lens systems used for magnifying specimens (ocular and objective)
- tungsten light focused on stage by condenser
- specimen dark against bright background
- limitation: absence of contrast
- performed on nonviable, stained preparations
5
Q
Darkfield microscope
A
- condenser is modified so that the specimen is not illuminated directly
- condenser directs light so it is deflected/scattered from specimen
- bright against dark background
- living specimens observed more readily
6
Q
phase-contrast microscope
A
- can look at unstained microorganisms
- allow visualization of cellular components that differ only slightly in their refractive indexes
- light refracted by slight variations in density and thickness of cellular components
- dark against light background
7
Q
fluorescent microscope
A
- visualize specimens that are chemically tagged with fluorescent dye
- UV light
- used primarily for the detection of antigen-antibody reactions
- antibodies are conjugated with fluorescent dye that becomes excited in the presence of UV light and the fluorescent portion of the dye becomes visible against black background
8
Q
stage
A
- fixed platform with opening in the center that allows for the passage of light from illuminating source below to lens system above the stage
- can place slide over central opening
- stage can be moved vertically/horizontally
9
Q
illumination
A
- light source is in the base of the instrument
10
Q
abbe condensor
A
- found directly under the stage
- contains two sets of lenses that collect and concentrate light as it passes upward from light source to lens systems
- has iris diaphragm, shutter controlled by lever that is used to regulate the amount of light entering the lens system
11
Q
body tube
A
- above stage, attached to arm of microscope
- houses lens system that magnifies the specimen
- upper end contains the ocular/eyepiece lens
- lower portion consists of moveable nosepiece containing objective lenses
12
Q
total magnification
A
- calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens
- the magnification of these lenses are printed directly on the lens housing
13
Q
resolution
A
- how well two objects can be seen as distinct and separate
14
Q
resolution formula
A
R= wavelength/(NAobj + NAcond)
NAobj= numerical aperture of objective lens
NAcond= numerical aperture of the condensor
15
Q
refractive index
A
determines how much the path of light is bent/refracted when entering a material
- when wavelength is decreased, resolving power increases
- lower NA= lower resolving power
- air had refractive index of 1.0
- immersion oil= 1.5
- pure water= 1.333
16
Q
immersion oil
A
- using immersion oil results in light bending that is less than when the light is passed through air
- immersion oil has the same refractive index as glass
- refraction of light is at a minimum when using immersion oil
17
Q
pure culture
A
culture containing a single unadulterated species of cells
18
Q
mixed culture
A
culture containing multiple species of microorganisms
19
Q
culture medium
A
- a solution containing nutrients
- microorganisms need adequate supply of nutrients and favorable growth environment
- liquid, semisolid, solid
- liquid lacks solidifying agent
20
Q
broth
A
- liquid medium
- lacks solidifying agent
21
Q
agar
A
- extract of seaweed
- complex carb, no nutritional value
- liquifies at 100 degrees C, solidifies at 40 degrees C
- completely solid: concentration between 1.5-1.8%
- less than 1%: semisolid
- solid medium: presents hardened surface in which microorganisms can be grown
22
Q
agar slants
A
- while in liquified state, solid media can be placed in test tubes, which are then allowed to cool and harden in a slanted position
- useful for maintaining pure cultures
23
Q
agar deep tubes
A
- tubes allowed to harden in a upright position
- used primarily for the study of gaseous requirements of microorganisms
- may be liquified in boiling water bath and poured into Petri dishes producing agar plates
24
Q
sterilization
A
rendering a medium or material free of all forms of life
- need to use sterile equipment and sterile technique
25
sterilization techniques
- heat: dry(hot air) and moist (wet air)
- filtration: removal of organisms from thermolabile solutions by passage through filters that retain bacteria
- chemicals
26
subculture
microorganisms transferred from one medium to another
27
incubator
used to maintain optimum temperature during the necessary growth period
- thermostatically controlled so the temperature cam be varied depending on the requirements of specific microorganisms
- moist environment retards dehydration of medium
28
filiform
- growth on agar slant
- continuous, threadlike growth with smooth edges
29
echinulate
- growth on agar slant
- continuous, threadlike growth with smooth edges
30
beaded
- growth on agar slant
- nonconfluent to semiconfluent colonies
31
effuse
- growth on agar slant
- thin, spreading growth
32
arborescent
- growth on agar slant
- treelike growth
33
rhizoid
- growth on agar slant
- rootlike growth
34
circular
- form of agar plate colony
- unbroken, peripheral edge
35
irregular
- form of agar plate colony
- indented, peripheral edge
36
rhizoid
- form of agar plate colony
- rootlike, spreading growth
37
entire
- margin of colony on agar plate
- sharply defined, even
38
lobate
- margin of colony on agar plate
- marked indentations
39
undulate
- margin of colony on agar plate
- wavy indentations
40
serrate
- margin of colony on agar plate
- toothlike appearance
41
filamentous
- margin of colony on agar plate
- threadlike, spreading edge
42
flat
- elevation of agar plate colony
- elevation not discernible
43
raised
- elevation of agar plate colony
- slightly elevated
44
convex
- elevation of agar plate colony
- dome-shaped elevation
45
umbonate
- elevation of agar plate colony
- raised, with elevated convex central region
46
uniform fine turbidity
- growth in broth media
- finely dispersed growth throughout
47
flocculent
- growth in broth media
- flaky aggregates dispersed throughout
48
pellicle
- growth in broth media
- thick, pad like growth on surface
49
sediment
- growth in broth media
- concentration of growth at the bottom of broth may be granular, flaky, or flocculant
50
crateriform
- gelatin liquefaction growth
- liquified surface area is saucer-shaped
51
napiform
- gelatin liquefaction growth
- bulbous-shaped liquefaction at surface
52
infundibuliform
- gelatin liquefaction growth
- funnel-shaped
53
saccate
- gelatin liquefaction growth
- elongated, tubular
54
streak plate
- method is a rapid qualitative isolation method
- dilution technique that involves spreading a loopful of culture over the surface of an agar plate
55
spread plate
- previously diluted mixture of microorganisms used
- cells spread over surface of a solid agar medium with sterile, L-shaped bent rod while Petri dish is spun on lazy-Susan turntable
56
pour plate
- diluted inoculum is added to a molten agar medium in a Petri dish, mixed, and allowed to solidify
57
stock culture
- prepared using isolates from mixed cultures prepared on agar streak-plate and/or spread plate
58
pure culture
single bacterial species
59
Chemical composition of a stain
- benzene: organic colorless solvent
- chromophore: imparts color to benzene
- auxochrome: conveys ionization to the chromogen, enabling it to form salts and bind to fibers/tissues
- benzene + chromophore = chromogen
60
acidic stains
- anionic (chromogen has negative charge)= strong affinity for positive parts of cell
- proteins readily bind and accept color
61
basic stains
- cationic (chromogen has positive charge)= strong affinity for negative cell
- nucleic acids will readily bind
62
simple staining
- use of single stain
- for visualization of morphological shape and arrangements
63
differential staining
- use of two contrasting stains
- separation into groups: gram stain and acid-fast stain
- visualization of structures: flagella stain, capsule stain, spore stain, nuclear stain
64
heat fixation
- used so that the bacterial stain will not wash away during staining
- coagulates bacterial proteins and fixes them to the glass surface
- rapid passage of the air-dried smear two or three time over the flame of the Bunsen burner
65
primary stain
- first reagent
- imparts its color to all cells
66
decolorizing agent
- used to establish color contrast
- based on chemical composition of cellular components, may or may not remove primary stain from entire cell or only certain cell structures
67
counterstain
- final reagent
- has a contrasting color to the primary stain
- if primary stain was not washed out by decolorization, the counterstain cannot be absorbed and cell will retain color of primary stain
- if primary stain is removed, the cell will be stained contrasting color
68
mordant
- traps stain in microorganism
- ex: Gram's iodine
69
Gram-positive
- thick peptidoglycan layer
- cocci
70
Gram-negative
- much thinner peptidoglycan layer
- rods
71
What to note when observing gram stained cultures (and microbial cultures in general)
- size
- shape
- arrangement
- the appearance of the stain
72
Steps of Gram stain
1) prepare smear (heat fix)
2) flood with crystal violet
3) rinse with water (everything purple)
4) flood with Gram's iodine
5) rinse with water
6) rinse with alcohol
7) rinse with water (only gram positive are colored)
8) flood slide with safranin
9) rinse with water (gram positive are purple, gram negative are pink)
10) place slide between two pieces of Bibulous paper to absorb moisture
11)view with microscope
73
mycolic acid
- waxy polymer that makes the cell envelope of Mycobacteria significantly less permeable than other groups of bacteria
- also makes them resistant to staining (which can help differentiate these from other bacteria)
74
what organisms are we trying to identify with acid fast stain
- genus Mycobacterium
- these are the causative agents of tuberculosis and other respiratory diseases in humans and animals as well as leprosy
75
primary stain of acid-fast
- carbolfuchsin (can penetrate mycolic acid)
- penetration is enhanced by steam heating (makes mycolic acid more fluid)
- after application, all cells will be red
76
decolorizing agent of acid-fast
- acid alcohol
- decolorizes all but mycolic acid containing bacteria
77
vegetative cells
metabolically active
78
counterstain of acid-fast
- methylene blue
- stains non-acid fast bacteria
- non-acid-fast bacteria will be blue, while acid-fast bacteria will be red
79
spores
- highly resistant
- metabolically inactive
80
primary stain of spore stain
- malachite green
- binds weakly to bacteria
- driving stain into bacterial endospores with aid of steam heat= stains spore and other bacterial structures
81
counterstain of spore stain
- safranin
- once cooled, spore retains malachite green while other bacterial structures readily lose the stain
- endospore containing bacteria have a green spore inside red vegetative cell
82
decolorizing agent of spore stain
- water
- the stain does not have a strong affinity for vegetative cell components--> water removes it
- cells will be colorless, spore will be green
83
common sporulating bacteria
clostridium and bacillus
84
nutritional needs of microorganisms
- supplied through media
- carbon (central to all cell structures and functions)
- nitrogen (in many cellular macromolecules, esp proteins and nucleic acids; some use atmospheric nitrogen others use inorganic compounds)
- nonmetallic elements ( sulfur= amino acids, phosphorous= formation of nucleic acids)
- metallic elements (osmoregulation, regulation of enzyme activity, electron transport)
- vitamins (cell growth)
- water
- energy
85
autotrophs
organisms that can be cultivated in medium consisting solely of inorganic compounds (they use inorganic carbon in the form of carbon dioxide)
86
heterotrophs
- cannot be cultivated in medium consisting solely of inorganic compounds
- must be supplied with organic nutrients, primarily glucose
87
phototrophs
use radiant energy as sole energy source
88
chemotrophs
- depend on oxidation of chemical compounds as energy source
- some use glucose, others use inorganic compounds like H2S or NaNO2
89
temperatures
- influences rate of chemical reactions
- low temps slow/inhibit enzyme activity, impede cell metabolism, and cell growth
- high temps cause coagulation and can denature thermolabile enzymes
- temps around 70 degrees C will generally destroy most essential bacterial enzymes causing cell death
90
pH of extracellular environment
- optimal pH around 7
- inc/dec in acidity can be detrimental--> slows rate of chemical reactions because of destruction of cellular enzymes (affects rate of growth and survival)
91
gaseous requirement
- most cells need atmospheric oxygen for respiration
- vital in ATP formation
- others lack enzyme systems for respiration in the presence of oxygen and must use an anaerobic form of respiration/fermentation
92
selective media
- restrict the growth of particular organisms while allowing others to grow
- typically has a growth-inhibiting additive which will limit the growth to only desired organisms
93
differential media
- contain chemical compounds that cause an observable change in the medium or the medium surrounding the bacteria when a particular biochemical reaction occurs
- can distinguish among morphologically and biochemically related organisms
94
defined media
composed of known quantities of chemically pure, specific organic and/or inorganic compounds
- requires knowledge of organism's specific nutritional needs
95
complex media
- composed of a number of complex substances, plant and animal extracts, whose exact chemical compositions are not known
- can support growth of most heterotrophs
96
enriched media
- subset of complex media
- allows for cultivation of fastidious microorganisms, organisms that have highly elaborate and specific nutritional needs
- may not grow/grow poorly on basic artificial medium and require the addition or one or more growth-supporting substances, such as additional extracts, vitamins, or blood
97
mannitol salt agar
- selective for staphylococcus species (high salt inhibits growth of most bacteria except staphylococci)
- differential for staphylococcus aureus (mannitol is fermented by S aureus which turns the phenol red indicator yellow around S aureus growth; for other Staphylococcus species, there is no change in medium)
98
Why does the media change color in the presence of S. aureus (Mannitol Salt)
- the carbohydrate, Mannitol is fermented by S. aureus
- this produces acidic end products that turn the phenol red indicator yellow
99
blood agar
- used to recover fastidious bacteria, often Streptococcus sp. (pathogens)
- bacteria are differentiated based on their ability to cause hemolysis of RBCs
- hemolytic streptococci snd certain members of Enterococcus and Aerococcus
100
how is blood broken down on blood agar plates
- important human pathogens are capable of producing extracellular enzymes that break down blood cells and degrade hemoglobin
- ex: streptococci
- lysis will result in clearing of the medium
101
alpha hemolysis
- es: Streptococcus pneumoniae
- partial/incomplete lysis of RBCs
- ne of partial clearing= green halo around bacterial colonies
102
beta hemolysis
- ex: streptococcus pyogenes
- complete lysis of RBCs
- complete zone of clearing around colonies
103
gamma hemolysis
- usually non-pathogenic bacteria
- no lysis of RBCs
- no clearing of the medium surrounding colonies
- no color change
104
MacConkey Agar
- selective for gram negative enterics (bile salts and crystal violet in the medium inhibit growth of Gram+ organisms which are sensitive to these agents due to the lack of outer membrane to protect the cell wall, allowing for isolation of Gram negative bacteria)
- differential: distinguishes coliforms (ferment lactose) from non-coliforms (do not ferment lactose)
- neutral red is indicator dye that turns red at pH below 6/6 (production of acid by lactose fermentation lowers pH--> changes pH indicator to red)
105
Why does the medium change for MacConkey agar
- lactose and neutral red pH indicator permit differentiation between different enteric bacteria based on their ability/inability to ferment lactose
- neutral red is colorless above a pH of 6.8 and turns red at pH below 6.8
- production of acid by lactose fermentation leads to a lowering of the pH of the medium and causes a change in the indicator to red
- lactose fermenters will have a red/pink coloration on their surface (E. coli will turn the medium around it red)
- non-lactose fermenters will have uncolored colonies
106
Eosin-Methylene Blue
- selective for Gram-negative enterics (eosin and methylene blue inhibit growth of most gram positive organisms, isolated gram negative)
- differntial: distinguishes enteric coliforms from non-coliforms)
- lactose fermenters: thick, mucoid purple/pink colonies due to dec pH from lactose fermentation
- E. coli: colonies are blue-black with metallic green sheen
- non-lactose fermeters: no change, colorless colonies
107
Why do EMB plates change color
- used for identification of fecal coliform bacteria
- contain peptone, lactose, sucrose, and eosin Y dye and methylene blue dye
- sugars are fermentable substrates that yield acid byproducts
- dyes inhibit growth to Gram + and are also pH indicators
- eosin Y and methylene blue will precipitate in the presence of high amounts of acid and will impart green metallic sheen
- low acid= methylene blue will be red
- limited lactose/sucrose fermentation= pink/redcolor
108
Phenylethyl Alcohol Agar
- Selective: partially inhibitory for Gram-negative organisms
- gram-negative organisms may form visible colonies but the size and number are smaller than on other media
- gram-positive will predominate
109
minimum growth temperatures
- lowest temperature at which growth will occur
- below this temp, enzyme activity is inhibited, cells are metabolically inactive,
110
maximum growth temperature
- highest temperature at which growth will occur
- above this temperature, most cell enzymes are destroyed, and the organism dies
111
optimum growth temperature
- temp at which the rate of reproduction is most rapid
- not necessarily optimum/ideal for all enzymatic activities of the cell
112
psychrophile
- bacterial species that will grow within temp range of -5-20 degrees D
- all psychrophiles will frow between 0 and 5 degrees C
113
mesophile
- bacterial species that will grow within a temp range of 20 degrees to 45 degrees C
- all can grow at human body temp (37 C) and unable to grow at temps above 45 C
114
thermophile
bacterial species that will grow at 35 C and above
115
facultative thermophiles
will grow at 37 C but grow optimally at 45 C to 60 C
116
obligate thermophiles
organisms that will grow only at temperatures above 50 C with optimum growth temps above 60 C
117
Durham tube
- inverted inner vial within culture tube
- used to detect accumulated gas
118
aerobe
- require the presence of atmospheric oxygen for growth
- enzymes necessitate use of O2 as final electron acceptor in oxidative degradation of high-energy molecules like glucose
119
microaerophile
- require limited amounts of atmospheric oxygen for growth
- excess oxygen blocks the activities of their oxidative enzymes and results in death
120
obligate anaerobes
- require absence of free oxygen for growth because their oxidative enzyme system requires the presence of molecules other than O2 to act as final electron acceptor
- oxygen forms toxic end products like superoxide but these organisms lack the enzymes that degrade this
121
aerotolerant anaerobe
- fementative organisms that do not use O2 as final electron acceptor
- produce catalase/superoxide dismutase= not likely to be killed in presence of oxygen
122
facultative anaerobes
- can grow in presence or absence of oxygen
- preferentially use oxygen for aerobic respiration
- in oxygen-poor environment, cellular respiration may occur anaerobically, using nitrates or sulfates as final electron acceptors, or via fermentative pathway
123
viable plate count method
- most common method used to determine the number of viable microorganisms in a sample
- two types: spread plate method and pour plate method
124
CFU/mL
- colony forming units per milliliter
-regular associations of cells that will multiply to form a single colony
- not always the case that a single colony represents the progeny of a single cell
125
range of colonies per plate for reliable counts
30-300
126
TFTC
- too few to count
- plates with fewer then 30 colonies
127
TNTC
- too numerous to count
- more than 300 colonies
128
lag phase
- cells are adapting to their new environment and transition from maintenance metabolism to growth metabolism
- cellular metabolism is accelerated and enzymes necessary for cell growth start to be synthesized in preparation for next phase
129
log phase
- cells transition to growth metabolism
- cell number doubles regularly until max number of cells is reached
- length of phase depends on organism and medium
130
stationary phase
- cells transition to maintenance metabolism
- number of cells undergoing division is equal to number of cells that are dying
- nutrients in metabolism are exhausted and toxic end products start to accumulate
131
decline/death phase
- waste products start to build up
- cells in culture begin to die at rapid/uniform rate
132
generation time calculation
- growth rate expressed as generation time
- g= t/n
t= time of exponential growth
n= number of generations
g= generation time of a culture (usually in mins)
133
number of generations calculation
n = (logN- logN0)/log2
134
optical density
related to the concentration of bacteria
- measure turbidity to determine number of cells present
135
cellular metabolism
- sum of all chemical reactions
136
extracellular enzymes
- exoenzymes
- act on substance outside of the cell
- mainly hydrolytic enzymes that reduce high-molecular weight materials into their building blocks by introducing water into molecule, liberating smaller molecules, which may then be transported into cell
137
intracellular enzymes
- endoenzymes
- function inside cell
- mainly repsonsible for synthesis of new protoplasmic requirements and production of cellular energy from assimilated materials
138
starch hydrolysis
- starch agar used to demonstrate hydrolytic activity of amylase
- nutrient agar supplemented with starch
- starch test used to determine presence/absence of starch in medium after growth
- absence of starch/negative result: blue/black medium because of starch in presence of iodine
- positive result/starch hydrolyzation: clear zone of hydrolysis will surround growth of organism
139
lipid hydrolysis
- tributyrin agar used to demonstrate hydrolytic activities of exoenzyme lipase
- following inoculation and incubation, organisms excreting lipase will show zone of lipolysis (clear areas around bacterial growth)
- loss of opacity is due to hydrolytic activity (positive result)
- no exoenzyme= no clearing (negative)
140
gelatin hydrolysis
- use nutrient gelatin deep tubes to demonstrate hydrolytic activity of gelatinase
- following inoculation and incubation, cultures are placed in refrigerator
- cultures that are liquifies produced gelatinase and demonstrate gelatin hydrolysis
- cultures that are solid are negative for gelatinase
141
amylase
- breakdown of starch
- involved in hydrolysis into shorter polysaccharides and ultimately into maltose molecules
142
lipase
- exoenzymes
- hydrolyze triglycerides
143
gelatinase
- extracellular enzymes that degrade collagen into individual amino acids that
can then be taken into cell and used as a source of energy for biosynthesis
144
starch agar
- used to demonstrate the hydrolytic activity of amylase
- medium is composed of nutrient agar supplemented with starch
- following growth, the detection of hydrolytic activity is made by performing the starch test to determine the presence/absence of starch in the media
145
starch test
- used to determine the presence/absence of starch in starch agar
- starch in the presence of iodine will impart blue-black color to the medium, indicating the ABSENCE of starch-splitting enzymes= negative result
- if the starch was hydrolyzed, a clear zone of hydrolysis will surround the growth of the organism= POSITIVE result
146
Tributyrin agar
- used to demonstrate the hydrolytic activities of lipase exoenzyme
- medium is composed of nutrient agar supplemented with tributyrin--> opaque medium
- organisms excreting lipase will show a zone of lipolysis (clear area around bacterial growth)--> loss of opacity is the result of hydrolytic reaction yielding glycerol and fatty acids= POSITIVE reaction
- no exoenzyme= no clearing= NEGATIVE result
147
gelatin agar
- demonstrates hydrolytic activity of gelatinase
- high gelatin= stiff medium
- cultures that turned liquid= produced gelatinase= demonstrate gelatin hydrolysis= POSITIVE result
- cultures that are solid= NEGATIVE for gelatinase
148
fermentation
substrates such as carbs and alcohols undergo anaerobic dissimilation and produce an organic acid that may be accompanied by gases such as hydrogen or carbon dioxide
- does not require oxygen in which an organic substrate serves as final electron acceptor
149
carbohydrate fermentation test
- acid production produces color change from red to yellow (fermentation)
- gas trapped in Durham tube= gas production
- no color change: sugar not utilized by organism
- color change to dark pinkish-red: alkaline/basic metabolic product due to utilization of peptone, not sugar
150
IMViC Test
indole, methyl red, Voges-Proskauer, and Citrate
- differentiates members of Enterobacteriaceae
151
indole test
- used to assay for the presence of bacteria able to hydrolyze the amino acid tryptophan
- involves inoculation of SIM media
- tryptophanase positive bacteria will de-aminate tryptophan producing indole
- red reagent layer following addition of Kovac's reagent: indole positive
- absence of red coloration indicates tryptophan was not hydrolyzed (indole negative)
152
tryptophanase
converts tryptophan to indole plus pyruvic acid and ammonia
153
indole
the nitrogen-containing heterocyclic ring that forms the R-group of tryptophan
154
SIM media
- indole test
- tryptophanase positive bacteria will de-aminate tryptophan producing indole
- when Kovac's reagent is added, indole is extracted from the medium into the reagent--> forms a cherry red color in the ring around the top of the medium
- cultures with a red reagent layer following addition of Kovac's reagent= indole POSITIVE
- absence of red coloration= tryptophan was not hydrolyzed= indole NEGATIVE
155
Methyl Red Test
- determines ability to oxidize glucose with production of high concentrations of acid end products
- methyl red detects presence of large concentrations of acidic end products
- separates E coli and K aerogenes
- red is positive(less than 4.4), yellow is negative (pH greater than 6), orange is pH between two
- K aerogenes will be yellow, E coli will be red
156
Mixed acid fermentation
- determined by the Methyl Red test
- methyl red= determines ability yo oxidize glucose to make acid end products
- below 4.4= red
- above 6.2= yellow
- between 4.4 and 6.2= orange
157
Voges-Proskauer Test
- determines capability of some organisms to produce nonacidic or neutral end products
- Barritt's reagent used
- development of deep rose color after addition of Barritt's reagent is indicative of the presence of acetylmethylcarbinol and represents positive result
- absence of rose coloration= negative result
158
citrate utilization test
- differentiation among enteric organisms on basis of their ability to ferment citrate as a sole carbon source
- use of enzyme citrase
- indicator is Bromthymol blue which will turn from green at neutral pH to blue when pH is higher than 7.6
- pH is elevated by conversion of ammonium phosphate to ammonia and ammonium hydroxide
- positive result= blue
159
Bromothymol Blue
- indicator in the Citrate utilization test
- acid-base indicator dye
- will turn from green at neutral pH to blue when pH is higher than 7.6
- pH is elevated by conversion of ammonium phosphate to ammonia and ammonium hydroxide
- the conversion only occurs with growth in the tube, which requires the organism to uptake citrate
- a change to blue= POSITIVE result for citrate utilization
160
Simmon's citrate agar
- defined medium containing sodium citrate as sole carbon source and ammonium ion as sole nitrogen source
- if citrate is utilized, growth will produce alkaline products--> changes medium from green to blue
- positive: growth on slant
- negative: no growth, medium remains green
161
hydrogen sulfide test
- SIM medium used to detect enzymes involved in hydrogen sulfide gas production by Enterobacteria from substrates like sulfur-containing amino acids or inorganic sulfur compounds (reduction of sulfur)
- differentiates enteric organisms
- also used to test for motility
- FeSO4 in medium is indicator by combining with gas, forming black ferrous sulfide precipitate which is indicative of H2S production (positive)
- absence of precipitate: negative
162
FeSO4
- SIM medium contains peptone, sodium thiosulfate, FeSO4
- FeSO4 is the H2S indicator
- H2S gas is colorless, FeSO4 serves as indicator by combining with the gas
- forms a black ferrous sulfide precipitate that is seen along the line of stab inoculation= H2S production= POSITIVE reaction
- absence of precipitate= NEGATIVE reaction
163
motility
- SIM agar
- lower agar concentration= limited movement of motile bacteria from area of stab
- detectable as diffuse growth from stab line
164
urease test
- determines ability to degrade urea by means of the enzyme urease
- phenol red indicator turns dark pink/magenta when pH is greater than 8.4
- as urea is split, ammonia creates alkaline environment that turns phenol red to magenta (positive)
- no color change= negative
165
urease
hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia
166
Phenol red
- presence of urease is detectable when the organisms are grown in urea broth containing pH indicator phenol red
- turns dark pink (magenta) when pH is greater than 8.4
- as urea is split, ammonia creates alkaline environment that causes the phenol red to turn magenta= POSITIVE for urease
- no color change= NEGATIVE reaction
167
litmus milk test
| litmus reduction, proteolysis, alkaline reaction
- differentiates among microorganisms that enzymatically transform different milk substrates into varied metabolic end products
- presence of lactic acid: litmus is purple at neutral pH and turns pink when medium is acidified to pH of 4
- litmus reduction: while in oxidized state, litmus is purple, when it accepts hydrogen from substrate it will become reduced and turn white--> also produces gas
- curds
- proteolysis: litmus turns deep purple in upper portion of tube, medium begins to lose body and produces translucent, brown whey-like appearance as protein is hydrolyzed into amino acids
- alkaline reaction: color of medium remains unchanged or changes to a deeper blue (partial degradation of casein into shorter polypeptide chains, releases alkaline end products)
168
acid curd
- lactic acid/other organic acids cause precipitation of milk protein casein as calcium caseinate to form an insoluble clot
- hard
- will not move away from walls of test tube if inverted
169
rennet curd
- some organisms produce rennin which acts on casein to form paracasein, which in presence of calcium ions is converted into calcium para caseinate and forms insoluble rennet clot
- soft, semisolid clot
- will flow when tube is tilted
170
lactose
milk sugar
171
casein
milk proteins
172
nitrate reduction test
- determines ability of microorganisms to reduce nitrates to nitrites or beyond to N2 using nitrate reductase
- any gas production in form of N2, nitric oxide, or nitrous oxide will collect as bubble in durham tube--> indicates that organism can carry out complete reduction of nitrate to gaseous nitrogen
- no gas detection: add solution A and solution B--> red color change shows nitrite is present; no color change indicates nitrates were not reduced OR nitrates were reduced to ammonia
- zinc added: medium turns red ONLY in presence of unconverted nitrate; no color change after addition of zinc= ammonia was formed
173
catalase test
- determines ability of microorganisms to degrade hydrogen peroxide by producing the enzyme catalase
- catalase production determined by adding a few drops of H2O2 to loopful of solid culture on glass slide
- catalase present: bubbles form (positive)
- absence of bubble formation= negative catalase test
174
superoxide dismutase
- enzyme
- aerobic organisms lack catalase that can degrade toxic superoxide using the enzyme superoxide dismutase
- end product of superoxide dismutase is H2O2 which is less toxic to bacterial cells than superoxides
175
oxidase test
- used to identify bacteria containing the respiratory enzyme cytochrome oxidase
- following addition of p-aminodimethylaniline oxalate, the development of pink, then maroon, and finally black coloration on surface of the colonies is indicative of cytochrome oxidase production (positive)
- no color change/light pink coloration= absence of oxidase activity (negative)
176
cytochrome oxidase
- catalyzes the oxidation of a reduced cytochrome by molecular oxygen (O2)
- results in formation of H2O or H2O2
- aerobic bacteria and some facultative anaeobes/microaerophiles exhibit oxidase activity
