Exam 2 Flashcards
What are the environmental conditions that a bacteria may encounter?
Different temps, pH levels, and osmotic pressures
What are a bacteria’s Cardinal temperatures?
The minimal, optimal, and maximal temperatures at which a bacterium can GROW.
Psychrophiles
Can grow ONLY at temps less than 20C (typically -5 to 20)
Psychrotrophs
Typical range from 0C to 30C
Psychrophiles continued
80% of all bacteria can grow at less than or equal to 10C.
Can be found in alpine soils, icefields, and oceans.
They are NOT human pathogens.
Mesophiles
Grow from 15C to 40C.
Optimal for growth is normal human body temperature.
CAN be human pathogens that cause disease.
Psychrotrophs continued
Found in soils, surface water, and in foods.
CAN be human pathogens.
Thermophiles
Typical growth at 40C to 75C.
Can be found in hot springs.
Hyperthermophiles or extreme thermophiles
Typically grow from 65C to 110C.
Can be isolated from ocean floor thermal vents/ridges.
What is pH?
The concentration of hydrogen ions in a solution.
Measured on a logarithmic scale 0-14
pH=-log[H+]
What are the three major categories bacteria can be placed in based on the pH at which they can grow?
Acidophile, neutrophile, and alkaliphile
Acidophiles
Grow in acidic environments
pH: < or = 4.5-5.5
Found in acid mine drainage and the stomach
Alkaliphiles
Grow in basic conditions
Ph: > or = 8-8.5
Found in soil of alkali flats or basic shores like Mono lake, CA
Neutrophiles
Grow at neutral ph between 5.5 and 8
Most human pathogens are neutrophiles since most body fluids are at a neutral ph (blood 7.4)
Why is water necessary for bacteria to survive
To maintain turgor pressure
Turgor pressure
The pressure inside a cell that is required for survival.
If not maintained, a cell could Lyse or or shrink (plasmolysis)
How do cells regulate turgor pressure
By maintaining high levels of potassium and sodium ions in their cytoplasm.
High concentration in the cell and low outside creates a concentration gradient. ==> leads to osmosis
What is osmosis
Movement of water from an area of low solute concentration to area of high solute concentration in order to achieve equilibrium
What is osmotic pressure?
The FORCE with which water flows from low solute concentration to high solute concentration
Hypotonic
Higher solute inside cell.
Low osmotic pressure.
Water moves into cell.
Cell will lyse
Isotonic
Equal solute inside and outside cell.
H2o moves into and outside cell at equal rate
Hypertonic
More solute outside cell than inside.
Water moves outside cell.
High osmotic pressure.
Cell will shrink (plasmolysis)
Salinity
Concentration of salt in a solution.
Can affect osmotic pressure.
Most bacteria grow under 3% salt.
Halophiles
Grow optimally at >3% salt concentrations.
Found in areas like the great salt lake in Utah and also used in the fermentation of food products (soy sauce, for example.
Extreme halophiles
Subgroup of halo phones that can ONLY survive at salt concentrations from 15 to 25%
Osmotolerant bacteria
Bacteria that can survive and grow over a wide range of salt concentrations
What are the direct methods that the growth of bacteria can be measured?
Plate counts, filtration, Most probable numbers (MPN), Direct Counting
Plate counts
measures bacterial population;
pros: measures number of VIABLE cells (CFU/ml)
cons: time consuming; assumes that EACH bacterium grows and divides to produce a SINGLE colony
Filtration
measures bacterial population; based on fact that bacteria CANNOT pass through pores of membrane filters.
pros: measures VIABLE cells in a small quantity of sample
cons: time consuming; assumes each bacterium grows/divides to produce a single colony
Filtration process
Apply membrane to screen platform, attach glass cylinder, add sample and vacuum, after filtration remove membrane, place membrane on agar plate and allow bacteria to grow.
Most probable numbers
statistical estimating method for measuring a bacterial population (MPN/100ml)
pros: can be used for bacteria that CANNOT grow on solid media.
cons: only a statement that there is a 95% statistical chance that a bacterial population falls within a certain range
direct counting
measures the # of bacterial cells in a sample; Manual counting of bacteria using a Petroff-Hausser counting chamber.
pros: very accurate and inexpensive
cons: very tedious and time consuming
Direct counting Continued
Electronic cell counter (Coulter counter)
pros: very accurate and results obtained quickly.
cons: expensive equipment that requires significant training to operate
What are indirect methods of measuring growth of bacteria?
Metabolic activity, dry weight, and turbidity.
Metabolic activity
estimates bacterial #’s by measuring the metabolic activity of a bacterial population (e.g., production of oxygen or acid)
assumes amount of metabolic product is directly proportional to # of bacteria present
Dry weight
estimates bacterial #’s by measuring weight of bacterial population.
Based on the fact that as a bacterial pop grows, there will be greater #’s of cells and therefore an increase in the entire weight of the pop over time
Turbidity
estimates bacterial numbers by measuring the turbidity of growing bacterial cultures in liquid medium.
One of the quickest, easiest, and most common methods for assessing bacterial growth
Relationship between turbidity and # of bacteria in a sample
As turbidity decreases, so does the number of bacteria
What is a spectrophotometer?
used to measure the turbidity in a liquid sample
Monochromatic beam of light (single wavelength) shone through a sample in order to measure the percent transmittance (%T) or the absorbance
What is transmittance?
Fraction of incident light that passes through a sample
T= Intensity of light out/intensity of light in
produces exponential curve when plotted
what is absorbance?
The Optical Density:
quantity of light that a sample neither transmits nor reflects; direct/linear relationship between absorbance and concentration
Absorbance range of spectrophotometer
From 0 to 1.99 A
If sample is 1.99 A or above, then it will need to be diluted before reading.
Absorbance directly related to concentration
If you know absorbance you can determine concentration through extrapolation
McFarland Standards
Set of reference samples of different turbidity that can be used to estimate the number of bacterial cells in a sample; Numbered such that the lowest standard number has the lowest level of turbidity and as the standard number increases so does the turbidity
McFarland Standards continued
Each McFarland standard represents a specific bacterial cell concentration
How do bacteria replicate?
Binary fission
What is doubling/generation time?
time taken to get from 2^x to 2^(x+1)
Nf= Ni(2^n)
Nf= # of cells at the final time point Ni= # of cells at the initial time point n= # of generations; growth time divided by doubling time
Closed-Growth System
an environment in which NO nutrients are added and NO waste products removed; 4 distinct phases of growth can be observed
What are the 4 phases of a closed-growth system?
Lag phase, log phase, stationary phase, and death phase
What happens during the lag phase?
Initial growth phase; acclimation (adjusting) to new environment; NO cell division but high metabolic activity in prep for growth
What happens during the Log phase (or exponential growth phase)?
Dividing begins; at maximum growth rate, division is exponential; the data will represent an ascending line on a graph; growth will continue while there are favorable conditions
What happens during the stationary phase?
Nutrients become limited; waste products accumulate leading to unfavorable environment; growth rate = death rate; therefore horizontal line on a graph
What happens during the death phase?
All nutrients depleted and waste products toxic; bacteria begin to die; death could happen rapidly or gradually;
Not all species will lyse, but lysis does cause absorbance to decrease which makes death phase easily observable
If lysis doesn’t occur during death phase how can it be observed?
Viable plate counts
What is aerotolerance?
The ability or inability to live in the presence of Oxygen
What are obligate aerobes?
MUST have Oxygen to survive and grow
What are facultative anaerobes?
can grow in the presence OR absence of Oxygen
What are aerotolerant anaerobes?
do NOT need Oxygen but can survive in the presence of Oxygen
What are microaerophiles?
survive ONLY when Oxygen levels are low
What are capnophiles?
survive ONLY when CO2 levels are high
What are obligate anaerobes?
ANY Oxygen will kill the bacteria
Why is autoclaving used during aerotolerance tests?
Autoclaving removes most free Oxygen from medium, but as the media cools, oxygen will diffuse back in. This diffusion allows for an Oxygen concentration gradient; surface has high Oxygen, bottom has low Oxygen
Where will bacteria grow within a tube during an aerotolerance test?
Bacteria will grow where the Oxygen concentration in the tube is optimal for that organism
What is an agar deep stab?
10 mL of Enriched TSA (allows more types of bacteria to grow); more media is used to ensure the bottom of the tube is anaerobic
What does the Sodium thioglycollate do in the fluid thioglycollate medium?
Reducing agent; reduces Oxygen to water
What does L-cystine do in fluid thioglycollate?
reducing agent; reduces Oxygen to water
What does Resazurin do in fluid thioglycollate?
oxidation-reduction indicator; red/pink in the presence of Oxygen; straw color in the absence of Oxygen
Can aerotolerance be examined on agar plates as well?
Yes, but requires that plates be incubated in both aerobic and anaerobic conditions; anaerobic conditions can be produced via a GasPak system or anaerobic bag
How can you produce anaerobic conditions and maintain them?
Chemical gas generating pack, which releases CO2; methylene blue indicator strip/tablet will confirm conditions (blue when O2 present, white when O2 is absent)
What are common reasons anaerobic conditions were not achieved?
Incomplete gasket seal on anaerobic jar, hole in anaerobic bag
Where is anaerobic bacteria typically researched?
Anaerobic chamber where the bacteria can be successfully grown and studied; completely devoid of O2
What does the Oxidation-Fermentation test determine?
What CATABOLIC pathway a bacterium can use to break down a CARBOHYDRATE
What two pathways can occur after glucose enters a cell?
Aerobic respiration and/or fermentation
Describe aerobic respiration pathway
Oxidation pathway; O2 final electron acceptor; O2 environment required
Describe fermentation pathway
Organic molecule final electron acceptor; can occur in the presence OR absence of O2
Is there a small or large amount of acidic compounds produced during aerobic respiration?
A small amount acidic intermediate compounds
Small or large amount of acidic compounds in fermentation?
LARGE amount of acidic compounds produced
What are the 2 important media components in the Huge & Leifson’s O-F medium?
A carbohydrate (like glucose) and the pH indicator Bromothymol Blue
What is the initial color of the OF test medium and what does color suggest about the media’s pH level?
Green is the initial color (pH of about 7.1, so neutral). Can change to YELLOW if it becomes ACIDIC (around 6.0)
What can be placed on top of the OF medium to promote fermentation?
Mineral oil which produces an anaerobic environment
What will OF media (both aerobic and anaerobic test tubes) look like if the organism can ONLY use the oxidation pathway?
Aerobic tube==>top part of tube will be yellow because O2 is available at top and glucose can be broken down, producing SMALL amount of acid turning green to yellow due to pH indicator.
Anaerobic tube will have no color change from green because no O2 present to break down glucose
What will happen to OF tubes if fermentation or fermentation AND oxidation can occur?
Fermentation will allow the anaerobic tube to turn yellow because the organism doesn’t need O2 to break down glucose and produce a lot of acids.
If oxidation is ALSO possible then the aerobic tube should have growth at the top and throughout the tube (because fermentation is also possible).
What happens if an organism is a weak/slow fermenter?
The glucose can SLOWLY be broken down, producing acids that lower the pH and turn both tubes yellow starting at the top of the tubes, but will remain mostly green throughout the rest of the tubes
What happens if the organism cannot metabolize the carbohydrate in the OF test AT ALL?
Both tubes will remain green (or bluish green if protein is broken down) because glucose will not be broken down and no acids will be produced which means pH indicator will not turn yellow.
What will the Phenol Red Broth test determine?
What CARB a bacterium can FERMENT
What are carbohydrates broken down into during glycolysis and what can that then be used to make during fermentation?
glucose and other carbohydrates are broken down into compounds that enter glycolysis, making pyruvate. pyruvate is used in fermentation to make alcohols, acids, and gases.
Is glucose the only carbohydrate that can be used in glycolysis?
No
Why is the Phenol red broth test considered differential?
Because the carbohydrate added to the media can be specifically selected by the person preparing the media
Does the base broth of phenol red come made with a carbohydrate already?
No, there is no carbohydrate mixed in yet so that a SINGLE one can be added later for testing
What are the other important components of the phenol red broth media?
Peptone (source of amino acids), pH indicator (phenol red), and a Durham tube (determines if gas if produced)
What does color suggest during the phenol red broth test?
A pH change: starts out as red (neutral 7.3), turns yellow if acidic (6.8), turns reddish-pink if more alkaline (>7.3)
What happens if the bacterium ferments the carbohydrate in the phenol red broth?
carb will be broken down in acidic products, pH will decrease causing phenol red indicator to turn yellow. gas could be present (there will be a bubble in the durham tube).
What if a bacterium CANNOT ferment a carb during the phenol red broth test?
no carb broken down, no acids produced, no drop in pH, media remains red in color, no gas produced
What if the bacterium CANNOT ferment the carb but it DOES break down the peptone in the media?
no carb broken down, so no acidic products produced. degradation of peptone which makes basic compounds raising the pH. color will be reddish-pink in color
Why are the results from the phenol red broth tests significant?
can produce “profiles” of carbs a bacterium can ferment which aids in identification. This is why the test is a differential test (different bacteria have different profiles)
What final electron receptors does anaerobic respiration use instead of O2?
nonoxygen receptors like nitrate (NO3-)
If nitrate is used as a final electron receptor then what can the bacteria perform (purpose of nitrate reduction test)?
Either nitrate respiration (denitrification) that produces nitrogen gas, OR ammonification that produces ammonia
What is denitrification?
multi-step process performed by a group of enzymes that leads to the production of nitrogen gas from nitrate
Step by step process of denitrification
NO3 (nitrate) > NO2 (nitrite)> NO nitric oxide > N2O nitrous oxide> nitrogen gas
what is ammonification?
group of enzymes work to produce ammonia from nitrate
NO3>NO2>NH4
what is the enzyme used to reduce nitrate to nitrite?
nitrate reductase
What are the key components to nitrate broth?
potassium nitrate (nitrate source) and a durham tube to monitor for gas; broth is initially clear cause there are no color indicators
How do you know if a nitrate reduction test should be continued with the rest of the steps after an overnight inoculation?
If a gas is produced: had to happen by either denitrification or fermentation; OR if there is growth but no gas production
What does it mean if the bacterium used during nitrate reduction test is a NON FERMENTOR
then the gas produced must be from denitrification and must be nitrogen gas: this means it is nitrate reductase POSITIVE
If growth occurred and no gas produced OR if the bacteria is a fermentor then what must be done?
2 reagents must be added: sulfanilic acid and naphthylamine
what happens if after the 2 reagents are added to the medium after the overnight inoculation and there is a red color produced?
nitrous acid must be present for reagents to react with, meaning nitrite was available to react with water to make nitrous acid; nitrate was apparently converted by NITRATE REDUCTASE to make nitrite, so POSITIVE
What if after adding the 2 reagents there is no red color produced?
no nitrous acid available for reagents to react with, no nitrite to react with water to produce nitrous acid, but there IS nitrate in the broth, so must continue with test
What are the next possibilities in the nitrate reductase test after the 2 reagents have been added and there is no red color?
either nitrate reductase is not present OR the nitrite was reduce to OTHER nitrogen products
Next, add ZINC POWDER
What does anaerobic respiration use as a final electron acceptor?
Non oxygen acceptors like NITRATE
If nitrate is used as final acceptor what 2 things can bacteria perform?
Nitrate respiration (denitrification) or ammonification
Multi-step process performed by enzymes that produces nitrogen gas
Denitrification
Multi-step process performed by enzymes that produce ammonia from NO3
Ammonification
What reduces nitrate to nitrite? (1st step)
Nitrate reductase
How is gas production seen in nitrate reductase test?
Bubble in Durham tube
If the organism is a non fermentor then the gas is produced through what and what is the gas?
Denitrification and nitrogen gas
If the organism used is a fermentor what type of gas is produced from nitrate reduction test?
Not known and continue testing
What is reagent A in nitrate reduction test?
Sulfanic acid
What is reagent B in nitrate reduction test?
Naphthylamine
If organism is red after both reagents added in nitrate reduction test what does that mean?
Positive for nitrate reductase
If organism is red after zinc powder in nitrate reduction test then?
Nitrate reductase negative
What 3 steps do aerobic and anaerobic respiration consist of?
Glycolysis, Krebs cycle, and ETC
What’s the difference between aerobic and anaerobic respiration?
The final electron carrier
What passes an electron down a chain of molecules that alternate being oxidized and reduced?
ETC
When is SUPEROXIDE produced?
Premature electron leakage
What is used to break down superoxide?
Superoxide dismutase
What is used to break down H2O2?
Catalase
What was the catalase test?
Smearing organism on slide and adding hydrogen peroxide to it
How is the presence of catalase determined?
Production of O2 gas
What’s the last mitochondrial complex? (Oxidase test)
Cytochrome C Oxidase
What is TMPD? (Oxidase test)
Chromogenic reducing agent
What does cytochrome c oxidase do?
Catalyzes the movement of and electron from one molecule to another
What is a CHROMOGENIC REDUCING AGENT? (Oxidase test)
Chemical that can donate an electron to another molecule and undergoes a color change
What did we do in the oxidase test?
Smeared organisms on to 4 different squares and watched for color change
When TMPD remains in reduced form and no color change?
Oxidase negative
When TMPD is oxidized and paper becomes blue where organism was smeared…
Oxidase positive
How quick should TMPD test be read? Why?
20-30 seconds because it’s unstable and can lose its electron no matter what
What is the protein found mostly in fibrous tissues?
Collagen
What is the product from the hydrolysis of collagen?
Gelatin
Gelatin is a large protein polymer made of numerous peptide subunits. Gelatin is an ____ source of ___ ___.
Gelatin is an abundant source of amino acids
Proteiolytic enzyme that BREAKS DOWN gelatin into individual amino acid components by cleaving PEPTIDE BONDS
Gelatinase
SECRETED from the bacteria
Gelatinase
Media used in the gelatin hydrolysis test
Nutrient Gelatin
What kind of media is gelatin? and What kind of agent is it?
A simple media that contains gelatin. And it is a solidifying agent.
Gelatin media is ___ and ___ in consistency prior to incoluation.
Firm and solid
What are the 2 possibilites for the gelatin hydrolysis test?
1) after inoculation the media remains solid, meaning gelatin (the solidifying agent) must be present and not broken down. Gelatinase negative. (media is firm and does not move when slant is slanted)
2) After inoculation the media becomes liquified, meaning the gelatin (solidifying agent) must be gone and broken down, so gelatinase must have been secreted. Gelatinase positive (media is liquid)
What is Casein?
Phosphoproteins that account for 80% of protiens in cow’s milk.
- Gives milk its white color
- contains between 200 and 220 amino acids.
- can provide source of amino acids for certain bacteria.
Problem with casein?
= It is too large to pass through the bacterial cell membrane
What is casease?
An enzyme that breaks down casein
What does casease do specifically?
It’s a secreted enzyme that can break the peptide bonds between adjacent amino acids. Breaking the peptide bonds produces smaller peptides and eventually individual amino acids that can be taken up by the bacterium.
Media used in casease test? and What is it the most important component?
Milk agar; powdered nonfat milk
2 possible results of the casease tests:
1) Casease Negative = no change in the color of the agar plate where the bacteria have grown, meaning no break down of case in (white color), and no casease produced
2) Casease positive = A zone of clearing around the bacteria, meaning the bactium secreted casease, diffused out through the agar to break down the casein in the agar, which loses its white color when broken down.
What is Urea?
the product of the decarboxylation (removal of carboxyl group) of certain amino acids
What is urease?
an enzyme present in certain bacteria that can hydrolyze urea into ammonia (alkaline compound that higher pH) and CO2.
What is urea broth and what is in it?
Urea broth is the media used to detect the presence of urease.
1) trace amounts of yeast extract (only nutrient source in media)
2) Urea (substrate for enzyme)
3) Potassium Phosphate buffer (strong enough to maintain a neutral pH unless large amounts of alkaline compounds are present)
4) Phenol Red pH indicator
2 possible results of urease tests
1) Urease Negative: Broth remains peach or turns slightly yellow in color, meaning urease is not products because urea in broth is not broken down, alkaline products ammonia not made, no pH change, and organism may die since there are only trace nutrients in broth
2) Urease Positive: Broth turns pink, meaning urease is produces because urea was broken down intro large amounts of alkaline (ammonia) compoud produced quickly, so much ammonia so fast overwhelms the pH buffering capacity of broth, pH of broth rises (becomes alkaline), and indicator turns pink in color.
What is starch?
Extremely large polysaccharide made of glucose moleculse linked by glycosidic bonds to form very long chains
2 types of molecules in starch:
1) Amylose - long chains of glucose
2) Amylopectin - branched chains of glucose
What’s the problem with using starch?
Starch is too large to pass through the bacterial cell membrane
What is a-amylase and what does it do?
Secreted enzyme that breaks down glycosidic bonds to make starch into usable glucose.
What’s the media used in starch hydrolysis?
Starch Agar, contains starch (substrate for a-amylase)
To determine if bacterium produces a-amylase:
Bacteria inoculated onto starch agar and grown overnight.
- a-amylase will be secreted from the cell and diffuse into the agar around bacteria and breaks down starch into glucose
- Flooding with iodine (which reacts with starch , not glucose, to make dark color)
- Starch stained black
- Glucose is clear
Possible results of starch hydrolysis test
1) a-amylase negative:
- whole plate is dark colored, even around the bacteria, meaning that no starch was broken down and there’s no a-amylase, no zone of clearing
2) a-amylase positive:
Zone of clearing around the bacterium, meaning that agar around the bacterium were not dark colored, so the starch were broken down into glucose around bacteria (by a-amylase)
What is the MR test used for?
Determine wether a bacteria can perform mixed acid fermentation
What is the VP test used for?
Determine whether a bacterium can perform 2,3-butanediol fermentation
What is MR-VP broth made of?
Peptone, glucose, and a phosphate buffer
Does the MR-VP pH indicator?
No
What color with MR test be at pH 4.4?
Red
What color will the MR test be at pH 6.2?
Yellow
What intermediate is pyruvate converted to in 2,3 butanediol fermentation?
Acetoin
What is acetoin converted to in the VP test?
2,3- butanediol
What does the VP test detect?
Acetoin
What 2 reagents must be added in the VP test to detect acetoin?
Alpha-naphthol and potassium hydroxide
What color will broth turn If acetoin is present in VP test?
Red at surface
What color will broth turn of no acetoin is present in VP test?
Copper at surface
If surface is copper in VP test then is diacetyl produced or is there a reaction with guanidine?
If surface is copper in VP test then is diacetyl produced or is there a reaction with guanidine?
If surface is red in VP test then is diacetyl produced or is there a reaction with guanidine?
Yes
Decarboxylation
Removal of the carboxyl group of an amino acid
Decarboxylase
Enzyme that catalyzes the removal of an amino acids carboxyl group
What is the coenzyme required for decarboxylase activity?
Pyridoxyl phosphate
What are the byproducts of decarboxylase reactions?
Alkaline compounds that neutralize and raise the intracellular pH
Specific decarboxylases for….
Specific amino acids
Decarboxylases are introduced under what conditions?
Acidic
What does the fermentation pathway of the decarboxylation test produce?
Large amounts of acidic products that can lower pH of the media
What condition is most favorable to promote glucose fermentation in decarboxylation tests?
Anaerobic
What does decarboxylase media contain?
Glucose, specific amino acid, coenzyme, and a pH indicator
What color is media if decarboxylase negative?
Copper
If media is copper in decarboxylation test can it ferment glucose under anaerobic conditions?
No
If media turns yellow in decarboxylation test then?
Can ferment glucose, lower pH, acidic products produced, decarboxylase negative
If media turns purple in decarboxylation test then?
Can ferment glucose anaerobicly, raise in pH, alkaline products made, decarboxylase positive
