Microbiology 2 Flashcards
1
Q
Anabolism
A
biosynthesis reaction
taking smaller molecules and bringing them together with energy to create larger molecules
hydrolysis
2
Q
Endergonic reaction
A
require more energy than they produce
3
Q
Catabolism
A
break a large molecule down into smaller molecules to create a lot of energy
hydrolysis
an exergonic reaction
coupled to ATP synthesis
4
Q
Enzyme characteristics
A
mostly proteins
catalysts(speed up a reaction)
not consumed by the reaction
lower activation energy
5
Q
Components of holoenzyme (active enzyme)
A
Apoenzyme
Coenzymes
Cofactors
6
Q
Apoenzyme
A
protein component of holoenzyme
7
Q
Coenzymes
A
vitamin derived, organic, carbon containing
Accept electrons or donate electrons
8
Q
Cofactors
A
low molecular weight
metal ions
9
Q
Enzyme inhibition
A
both can bind reversibly or irreversibly (suicide inhibitor)
10
Q
Competitive inhibition
A
occurs at the active site
sulfa drugs compete with PABA (para-aminobenzoic acid) at the active site of an enzyme that converts PABA to folic acid
Is a substrate analog (look a-like) that binds to the active site
Overcome by increasing substrate concentration
11
Q
Non-competitive inhibitor
A
inhibits the reaction
do not compete with substrate at binding site
adheres to another binding site to distort the substrate binding site
12
Q
Feedback inhibition or end product inhibition
A
Reversible, non-competitive inhibition
End product accumulates and inhibits the first enzyme of a metabolic pathway which shuts down the pathway
13
Q
Reaction rate influences
A
temperature
pH
substrate concentration
14
Q
Temperature effect on reaction rate
A
a catalyst up to a point then hinders the reaction by enzyme denaturation
15
Q
pH effect on reaction rate
A
too high or too low causes denaturation of H+ compete with hydrogen and ionic bonds in an enzyme
16
Q
Substrate concentration effect on reaction rate
A
saturation is when an active site of an enzyme always has a substrate bound
The more substrate there is, the faster the reaction until saturation occurs, and then there is a plateau
17
Q
Oxidation
A
loss of electrons gains oxygen loses hydrogen loss of energy exothermic/exergonic reaction
18
Q
Two key players to the oxidation of organic compounds
A
Dehydrogenases-enzymes
Coenzymes
19
Q
Coenzymes
A
vitamin derived organic molecules
a. Nicotinamide adenine dinucleotide (NAD) b. Flavinadenine dinucleotide (FAD)
20
Q
Reduction
A
gain of electrons loss of oxygen gain of hydrogen gain of energy endothermic/endergonic reaction
21
Q
Redox
A
combination of oxidation and reduction reactions they are used simultaneously
22
Q
Substrate level phosphorylation
A
ATP is generated when a phosphate is transferred from an organic compound to ADP
23
Q
Oxidative phosphorylation
A
uses chemiosmosis and electron transport chain to phosphorylate ADP
24
Q
Total ATP generated
A
38 ATP per glucose molecule
NAD produces more ATP than FAD
25
Fat catabolism
a fat is a lipid consisting of glycerol + 3 fatty acids-can be broken into this using a lipase
Krebs cycle is used
26
Protein catabolism
uses the Krebs cycle
| proteins are too large to pass through plasma membranes and must be broken down first
27
Deamination
Used in protein catabolism
| can use protease to break protein into individual amino acids that are able to enter the Krebs cycle
28
Metabolism
the sum of all chemical reactions occurring within an organism
balances energy
two classes of reactions, gaining or losing energy
29
Fermentation
an anaerobic pathway
generally does not use oxygen
does not use Krebs cycle
electron transport chain
cannot occur in presence of O2
can still perform glycolysis to get 2 ATP, 2 NADH NAD+
organic molecule is the final electron acceptor-is pyruvate or a derivative of pyruvate
30
Homeolactic acid fermentation
one glucose molecule is converted to two molecules of lactic acid
31
Alcohol fermentation/ethanol fermentation
sugars such as glucose, fructose, and sucrose are converted into cellular energy and produce ethanol and carbon dioxide as metabolic waste products
32
Lactic acid fermentation
glucose, fructose, and sucrose are converted into cellular energy and the metabolite lactate
Lactate dehydrogenase catalyzes the conversion of pyruvate and lactate with concomitant conversion of NADH and NAD+
33
Nutritional factors effecting the growth of bacteria
```
carbon
energy
sulfur
nitrogen
phosphorus
trace elements
oxygen
```
34
Carbon source
cell needs external carbon source
this makes up 50% of the cell’s dry weight
users can be heterotrophic or autotrophic bacteria
35
Heterotrophic bacteria carbon use
carbon comes from organic compounds like lipids, proteins and carbohydrates
36
Autotrophic bacteria carbon use
they can use inorganic carbon like from carbon dioxide
| they can fix O2 from the atmosphere
37
Uses for carbon
required for all organic compounds
38
Categories of energy users
phototrophic bacteria
| chemotrophic bacteria
39
Phototrophic bacteria
organisms that use light
40
Chemotrophic bacteria
organisms that get energy from the oxidation of organic compounds and inorganic chemicals
41
Energy uses
metabolism, all cellular processes
42
Sulfur sources
```
sulfate ion (inorganic)
proteins that have sulfur containing amino acids (organic)
```
43
Sulfur uses
sulfur containing amino acids for protein synthesis
| thymine and biotin synthesis
44
Nitrogen sources
organic: proteins (contain amino acids), amino acid is denatured into NH2
inorganic: nitrogen gas (N2), NH4+, NO3
45
Nitrogen uses
required for synthesis of amino acids
synthesis of nitrogenous bases in DNA
synthesis of amino sugars—NAN, NAG
46
Phosphorus source
inorganic only—PO4 3-
47
Phosphorus uses
make ATP
nucleic acid backbone—synthesis of nucleotides
synthesis of phospholipids
48
Trace elements sources
inorganic only
| ex: iron, copper, zinc, magnesium
49
Trace elements uses
cofactor for enzymatic reaction
required for synthesis of B12
bacteria have iron containing cytochrome in the electron transport chain
50
Superoxide dismutase
works of superoxide free radical to eliminate toxic forms of oxygen
51
Catalase
breaks down hydrogen peroxide to water and hydrogen gas
if bubbles form, catalase is present
2H2O2 2H2O + O2
52
Peroxidase
breaks down hydrogen peroxide
no O2 gas is produced
H2O2 + 2H+ 2H2O
53
Obligate anerobe
does not have superoxide dismutase, catalase or peroxidase
54
Facultative anerobe or aerobe
have superoxide dismutase, catalase and peroxidase
55
Physical factors affecting bacterial growth
```
some enzymes can adapt to be able to live in extreme environments like high salt, high heat and high or low pH
temperature
pH
oxygen requirements
osmotic pressure
```
56
Temperature affect on bacterial growth
there is an ideal temperature with steep drops on either end
57
Psychophile
optimal growth at 15C
| ex: organisms that grow deep in the ocean
58
Mesophile
optimal temp = 37C (25-40 is ok too)
| is most common type of microbe
59
Thermophile
heat loving microbes
optimal temp=50-60C
ex: microbes that grow in a compost heap
60
Neutrophiles
like a neutral pH
| most bacteria like pH 6.5-7.5
61
Acidophiles
like pH 6 and below
| ex: helicobacter pylori can grow at pH 2
62
Alkeliphiles
can grow at pH 7-11.5
63
Non-halophiles
grow at normal sodium levels
| cannot survive high salt concentrations
64
Moderate/facultative halophiles
will grow at concentrations of 2-15% salt
doesn’t require high salt but can grow at high salt
Ex: pseudomonas, organisms involved in bioremediation (cleaning oil spills)
65
Obligate halophiles
require high salt concentration for growth
66
Extreme halophiles
require even higher salt concentration, > 15%
| Ex: organisms from the dead sea grow at salt concentrations of 30%
67
Defined culture media
when we need to know everything that is in the growth media
we don’t use this in our labs
chemically defined is for research purposes only
68
Glucose culture media
carbon and energy source
69
Ammonium phosphate
nitrogen source and phosphate source
70
MgCl2
magnesium chloride provides trace metals
71
Sodium chloride
provides osmotic balance
72
K2HPO4
a buffer
| can accept or donate protons to keep pH neutral
73
Fastidious
an organism that requires one or more growth factors (an essential organic compound that the bacterium cannot synthesize)
74
Complex culture media
chemically undefined
easier to grow than defined media
chemical compound of soy, beef, peptones or yeast extract-can vary
75
Agar
isolated from seaweed which can vary from batch to batch
| any solid media is chemically undefined
76
Peptones
partially digested proteins that are easier for the bacteria to take up
if we add peptones, we add lots of different amino acids
77
TSA
tryptic digest of soy
| trypsin is a protease, cleaves/digests soy proteins
78
Beef extract
adding this adds amino acids and vitamins
79
Selective media
ex: PEA will suppress the growth of certain microbes
| suppresses growth of G- and gives G+ a chance to grow
80
Differential media
ex: Chrom agar E, causes pathogens to have different colors allowing for identification
ex: MSA, manitol salt agar, is also a selective media, has a pH indicator in it (phenol red), media is red at pH>6.8, media is yellow at pH <6.8, manitol ferments to change pH
81
Enrichment media
gives extra nutrition to fastidious organisms
| ex: blood agar, does not suppress growth
82
Gas pak
has packet that when activated removes all O2 and creates H2 with it
83
Candle jar method
reduces O2 concentration
| increases CO2 concentration
84
Binary fission
bacteria double in size before they split
is cell division in prokaryotes
is analogous to mitosis in eukaryotes
is asexual
85
Binary fission steps
i. DNA synthesis and elongation of the cell
ii. Cell begins to elongate
iii. Cross wall forms --> separates the 2 sets of chromosomes
iv. Binary fission/cell separates into 2 cells, all cells are clones of each other
86
Bacterial growth curve
lag phase
log phase
stationary phase
death phase
87
Lag phase
cells do not immediately divide
is not a resting phase
active metabolism occurs, “active metabolism”
88
Log phase
```
period of active growth
exponential growth
no nutrients are limiting or cell’s won’t divide as quickly
optimal conditions for cell growth
rapid and constant growth rate
can determine doubling time/growth rate
```
89
Log phase examples
ex: E. coli doubles every 20 min
Bacillus sterothermophilis doubles every 6 min, is a thermophile, likes 55C
Mycobacterium teburculous doubles every 360 min
90
Stationary phase
stabilization of growth
# of cells dividing = # of cells dying
usually begins when key nutrient is depleted
pH may have dropped
accumulation of toxic byproducts of metabolism and harmful changes to pH
91
Death phase
lacks all nutrients needed for growth
# of deaths far exceeds # of new cells
media is totally exhausted
environment is toxic
92
Measuring bacterial growth directly
standard plate count
direct microscopic count
filtration
93
Direct microscopic count advantage
no waiting period
can count cells immediately
no incubation period
94
Direct microscopic count disadvantage
can’t tell if cells are dead or alive so you count both dead and living bacteria
95
Petroff-Hauser cell counter
is a microscope slide with a fixed cover slip grid on it
| can count bacteria in certain # of squares and multiply by the conversion factor
96
Filtration
used when bacterial concentration is very small, <= 1 bacteria/mL
97
Filtration application
water testing for possible fecal contamination
98
Filtration steps
1: 100ml H2O sample and filter it
2: transfer filter to pad in petri dish soaked in nutrient broth
3: grow overnight
4: count # of bacteria and report # of bacteria/100mL
99
Measuring bacterial growth indirectly
turbidity/spectrophotometric
cell mass
metabolic activity
100
Turbidity/spectrophotometric
absorbance or transmittance readings can be taken
| measure the turbidity to indicate cell growth
101
Turbidity/spectrophotometric disadvantage
bacteria must be growing at high numbers to be turbid enough to read
102
Cell mass
a certain volume of culture would be filtered
| dried and weighed to get # of bacteria/mL
103
Metabolic activity
measure O2 consumed or measure amount of acid produced
104
Disinfection
reduction in # of bacteria
105
Disinfectant
chemical agent that results in a reduction in # of bacteria
106
Antiseptic
disinfectant that can be used on the skin
107
Sterilization
removal of all pathogens including those with endospores
108
Filter paper
discs soaked in chemical agents placed on a lawn of bacteria
create a zone of inhibition
the widest zones do not necessarily indicate the most effective chemical agent
109
Phenol coefficient
used by Lister to sterilize or used as a standard to compare other disinfectants
coefficient of 1 in other disinfectants means it is as effective as phenol
> 1 = better, < 1 =worse
110
Phenol mechanisms of action
protein damage/denaturation
membrane damage
damage to nucleic acids/chemical groups
111
Protein damage/ denaturation
ex- heat denaturation
hydrolysis (breaks bonds)-strong acids and bases
oxidation (strip electrons)
attachment of atoms or chemical groups
112
Bactericidal
kills the microbe
113
Bacteriostatic
inhibits the growth of the microbe
114
Membrane damage
will kill the cell because cell contents will leak out
115
Phenol coefficient dilutions use
dilute phenol and dilute the chemical to compare to phenol
| make 2 dilutions
116
Phenol coefficient dilutions steps
1. Add standard amount of bacteria to each dilution tube
2. Take samples out of each tube after a set amount of time and plate onto fresh media, let them grow 12-48 hours
3. You are looking for the highest dilution (lowest concentration) that killed the bacteria
117
Disinfectant types
```
surfactants
heavy metals
halogens
alcohols
phenolics
oxidizers
alkylating agents
```
118
Surfactants
decrease surface tension, effect the cell membrane
| ex: soaps/detergents, anionic sanitizers, quaternary ammonium compounds--none of these are effective against spores
119
Soaps/detergents
good de-germing agents
don’t kill bacteria very well
they mechanically remove bacteria, oil and debris
120
Anionic sanatizers
are negatively charged
they are less effective on bacteria that quaternary ammonium compounds(which are cations) because bacteria are also negatively charged and are repelled
used to sanitize utensils and equipment
121
Quaternary ammonium compounds
have a valence of 4, much better surfactants, especially against G+ bacteria
122
Quaternary ammonium compounds mechanism of action
causes changes to cell permeability, effects the plasma membrane of the cell
123
Heavy metals
can be used as a disinfectant
| ex-silver, copper, zinc, mercury
124
Heavy metals mechanism of action
protein denaturation
can be biocidal (kill bacteria)
metals can combine with sulfhydryl groups on proteins and denature them
125
Halogens
Iodine
| Chlorine
126
Iodine
good antiseptic
| effective on G+, G- and some spores
127
Iodine mechanism of action
alters membrane permeability and disrupts protein synthesis
128
Betadine
iodine combined with an organic molecule (iodophore)
129
Chlorine
a good disinfectant
| effective against G+, G- and some spores
130
Mechanism of action of bleach
denature proteins
131
Hypochlorious acid
Cl2+H2O -> <-H+Cl-+HOCl
132
Hypochlorite ion
has strong oxidizing agent
| HOCl -> <- H+ +OCl-
133
Alcohols
```
denature proteins
disrupt membranes
needs water to denature proteins
100% alcohol is less effective than 70% alcohol
is not a good antiseptic
```
134
Phenolics
derivatives of phenol
135
Bisphenols
```
have 2 rings
effective against G+ and mycobacterium
ex: triclosan
can disrupt lipid wall of mycobacteria
used in disinfection of organic compounds like pus, saliva, vomit and feces
```
136
Oxidizers
H2O2 (hydrogen peroxide) is an oxidizing agent
is not a good antiseptic because our cells contain catalase
is a good disinfectant at high concentration and can be effective against spores
137
Oxidizers method of action
oxidation
| H2O2 ->O2 + H2O
138
Alkylating agents
Formaldehyde
| Glutoraldehide
139
Formaldehyde
used in vaccines to inactivate bacteria and viruses
140
Glutoraldehide
disinfectant for hospital equipment
kills spores but takes 3-10 hours to do it so it is not a stearalizing agent (must kill spores in <30min)
bactericidal, virucidal, tuberuclocidal, sporucidal (under the right conditions)
141
Physical antimicrobial agents
```
heat application
refrigeration
freezing
desiccation
freeze drying
osmotic control
radiation
filtration
```
142
Heat application
dry heat
autoclave
pasteurization
143
Dry heat
used when we flame our loops
| denaturing proteins through oxidation by incineration which achieves sterility
144
Autoclave
```
moist heat under pressure
achieves sterility
kills spores
reaches temperatures over 100C
can go above boiling point by adding pressure
```
145
Autoclave specifications
15lbs pressure, 121C for 20 min
146
Pasteurization
kill pathogens
does not sterilize
want to kill mycobacteria and salmonella
increases shelf life
147
Phosphate test
used to determine if pasteurization has worked
| should cause phosphatase to be inactivated
148
High temperature/low time pasteurization method
is the most common method
flash method
71.6C for 15 sec
149
Holding method for pasteurization
62.9C for 30 min
150
Sterilized milk
ultra-high temperature treatment
140C for 4 sec
milk can then be stored at room temperature
this can cause loss of flavor
151
Referigeration
does not kill bacteria
bacteriostatic effect
slowing metabolic rate so they don’t produce toxins
152
Freezing
not killing bacteria
| bacteria are dormant
153
Desiccation
removal of water which is required for metabolic processes
this stops metabolism
does not kill bacteria
bacteria can remain viable and desiccated state for years
when we re-hydrate, bacteria will grow and divide
154
Freeze drying
aka lypohilization
a good way to preserve microbes
Mycobacterium tuberculosis can remain viable in this state for several months
155
Osmotic control
hypertonic environment is when solute concentration is higher outside the cell
water moves out of the cell
this causes the cell membrane to shrivel which is called plasmolysis
156
Radation
UV radiation
| Ionizing radiation
157
UV radiation
can cause thymine dimers
UVB absorbed by nitrogenous bases
thymine dimers can inhibit correct replication of DNA
158
UV radiation, mechanisms of repair
light repair
| dark repair
159
Light repair
light activates photolysis which breaks bond between thymine dimers
160
Dark repair
can occur with or without light
| several enzymes repair the dimer
161
Endonuclease
can cut into DNA and repair around the site of mutation
162
Exonuclease
chews nicked part from the end to remove it
163
DNA polymerase and DNA ligase
required for light and dark repair
164
Ionizing radiation
radioactive element emitting gamma rays, X rays or electron beams, they dislodge electrons to create ions
To dislodge electrons from H2O (ionize it), we form a toxic form of O2 (hydroxyl radical which damages DNA and cell components)
165
Filtration
can achieve sterility with membrane filtration
0.22 or 0.45 um filter can trap bacteria on the surface of the filter because bacteria are larger than 0.22um
Viruses can still get through unless you use 0.1 um filter pore size which will block viruses
166
Chemotherapeutic agents
antibiotics
synthetic drugs
semi-synthetic drugs
167
Antibiotics
microbe made
| natural anti-microbial
168
Synthetic drugs
man made chemicals
169
Semi-synthetic drugs
can be produced by microbes if microbes are fed a synthetic precursor
170
Properties of antibicrobial agents
selective toxicity
| spectrum of activity
171
Selective toxicity
want it to be selectively harmful and toxic to bacteria but not to us
172
Chemotherapeutic index
maximum tolerable dose of antibiotic per kg of body weight divided by minimum effective dose per kg of body weight, we want this number to be high
173
Spectrum of activity
Broad
| Narrow
174
Broad spectrum of activity
works on a large range of taxanomic groups
175
Drug mechanisms of action
```
Inhibition of cell wall synthesis
Disruption of cell membrane function
Inhibition of protein synthesis
Inhibition of nucleic acid synthesis
Antimetabolites
```
176
Side effects of broad spectrum antibiotics
Disruption of micro flora
| Can lead to super infection
177
Resistance
genetic
non-genetic
plasmid borne
178
Non-genetic
an organism evades antibiotic based on its location
179
Genetic
resistance due to mutations
180
Plasmid borne
extrachromosal DNA, contain R plasmids that can carry 6-7 genes that have resistance to antibiotics
181
Superbugs
resistant to several antibiotics, multidrug resistance
182
Mechanisms of drug resistance
```
Mutations in target molecules
Alterations in membrane permeability
Enzyme development
Enzyme activity changes
Alterations of metabolic pathways
```
183
Alterations in membrane permeability
if there is a change in the DNA that effects cell wall permeability, could affect cell wall protein causing the cell to no longer be permeable to the antibiotic
184
Enzyme activity changes
could develop enzyme with a higher affinity for another substance
Ex: PABA when treated with sulfonamide
185
Alterations of metabolic pathways
normally, bacterium creates its own folic acid but pathway may have been altered and is unable to use readymade folic acid
186
Determining microbial sensitivities
disk diffusion or Kirby-Bauer method
| dilution method
187
Disk diffusion or Kirby-Bauer method
Kirby Bauer assay: there are standard measurements for the zone diameter, bigger zone does not necessarially indicate the best treatment, the different treatments are then labeled sensitive, moderately sensitive or resistant
188
Dilution method
Minimal inhibitory concentration (MIC)
| Minimal bacterial concentration (MBC)
189
minimal inhibitory concentration (MIC)
use dish with many wells and increase dilution, looking for the highest dilution where you do not see any growth
190
Minimal bacterial concentration (MBC)
highest dilution that kills the bacteria
191
Stages of cellular respiration
Glycolysis
Krebs cycle
Electron transport chain
192
Glycolysis location
takes place in the cytoplasm of the cell
193
Glycolysis
anerobic metabolism
oxidizes glucose
NAD+ are reduced to NADH
194
Glycolysis summary
Input: 2 ATP
Output: 4 ATP
Net: 2 ATP, 2 NADH, 2 pyruvate
195
Krebs cycle location-Eukaryotes
takes place in the inner space of the mitochondria
196
Krebs cycle pre-step
the oxidation of pyruvate, this must occur for pyruvate to enter the Krebs cycle
197
Krebs cycle location-Prokaryotes
occurs in the cytoplasm of the cell
198
Glycolysis location
takes place in the cytoplasm of the cell
199
Glycolysis
anerobic metabolism
oxidizes glucose
NAD+ are reduced to NADH
200
Glycolysis summary
Input: 2 ATP
Output: 4 ATP
Net: 2 ATP, 2 NADH, 2 pyruvate
201
Krebs cycle location
takes place in the inner space of the mitochondria
202
Krebs cycle pre-step
the oxidation of pyruvate, this must occur for pyruvate to enter the Krebs cycle
203
Classes of electron carriers (electron transport chain)
cytochromes
flavoproteins
ubiquinomes of coenzyme Q
204
Krebs cycle
aerobic metabolism
uses substrate level phosphorylation
is a highly oxidative pathway
GTP preforms decarboxylation
205
Krebs cycle summary
Input: 0 ATP
Output: 2 ATP, 2 FADH2, 8 NADH (including prep-step)
Pre-step makes 2 NADH
206
Chemiosmosis/electron transport chain location
occurs in the cellular membrane
207
Chemiosmosis/ electron transport chain
makes the most ATP
3rd stage of respirtation
electrons are passes from coenzymes to electron carriers which releases energy to make ATP
208
Chemiosmosis/ electron transport chain summary
Input: 10 NADH, 2 FADH2
Output: 30 ATP, 4 ATP (respectively)
209
Classes of electron carriers (electron transport chain)
cytochromes
flavoproteins
ubiquinomes of coenzyme Q
210
Cytochromes
contain a heme group (iron), all need to be able to exit at the oxidized and reduced state
211
Flavoproteins
are a co-enzyme
212
Ubiquinones or coenzyme Q
non protein carriers
213
Beta oxidation
the process where fatty acid molecules are broken down in the mitochondria to generate acetyl-coA, which enters the krebs cycle
