this appears around the Kirby-Bauer antibiotic disk where the concentration is high enough to stop growth of the organism
zone of inhibition
any chemical used in the treatment, relief, or prophylaxis of a disease
chemotherapeutic drugs
natural antimicrobial agents produced by microorganisms
antibiotics
synthetic or natural agents that are used to treat bacterial infections
antimicrobials/ antimicrobics
minimal or no effect on host cells but maximum effect against the infecting microorganism
selective toxicity
this is a term used by microbiologists to describe the appearance of bacterial colonies when all the individual colonies on a petri-dish agar plate merge to form a field or mat of bacteria
bacterial lawn
-static means:
stops bacteria from dividing, but does not kill them
(bacteriostatic)
-cidal means:
drugs that kill the organism
(bactericidal)
MIC means:
minimum inhibitory concentration
it is where the junction of the zone of inhibition with growth is; the concentration of antimicrobics has become too low to effectively stop growth for that particular strain
Identify the factor that the Kirby-Bauer antibiotic disk diffusion test is designed to evaluate
it is a valuable tool for establishing the effectiveness of antimicrobics against pathogenic microorganisms in clinical laboratories
Identify variables in this method of antibiotic testing that the Kirby-Bauer method eliminates (controls).
Your lab book identifies seven.
1. use of mueller-hinton agar- 7.2-7.4 PH
2. Depth of agar used
4mm in 150mm or 100mm Petri dishes
3. use of 0.5 McFarland
turbidity Standard for broth culture
4. Disks dispensed onto
inoculated plate
5. Temperature at which incubated 35C
6.
Time incubated 16-18 hours
7. Clear zones measured after
incubation and compared to a standardized table of results
Evaluate the effectiveness of chemotherapeutic drugs by using the interpretive tables. Understand the meaning of resistance and sensitivity in this context
look at the table on pg 531
Identify bacterial and fungal species which are known for production of antibiotics. List the three genera that produce most antibiotics and one antibiotic originally isolated from each of the genera.
Penicillium (fungal)- penicillin
Streptomyces (fungal) -
streptomycin
Bacillus (bacterial) - bacitracin
Summarize and give an example of how most antibiotics accomplish selective toxicity.
Selective toxicity relies upon differences between the structure and
metabolism of the patient's cells and the structure of the microbe
being targeted.
They interfere with pathway with cell wall synthesis. since
mammals don't have cell walls; they also interfere with the nucleic
acid and protein synthesis
Distinguish a broad-spectrum antibiotic
effective against many types of microbes and tend to have higher toxicity to the host.
Distinguish a narrow-spectrum antibiotic
effective against a limited group of microbes and tend to exhibit lower toxicity to the host
Examine colonies within a zone of inhibition and classify them as resistant mutants or contaminants.
A resistance mutation is a mutation in a virus gene that allows the virus to become resistant to treatment with a particular antiviral drug.
The main source of antibiotic contamination is wastewater from antibiotic manufacturer, large scale animal feeding operations, households, and hospitals
all aspects of the Kirby-Bauer test are standardized to assure reliability
a.) what might be the consequence of pouring the plates 2mm deep instead of 4mm deep?
This would affect the distance the antibiotic diffuses from the disk. The thicker the agar, the more downward diffusion there is and the less antibiotic available to diffuse outward. Thus, the zone would be smaller.
all aspects of the Kirby-Bauer test are standardized to assure reliability
b.) the Mueller-Hinton II plates are supposed to be used within a specific time after their prep and should be free of visible moisture. What negative effect (s) might moisture have on the test?
The older the plates, the drier they become. This could affect the ability of the antibiotic to diffuse through the agar. Moisture might help spread the antibiotic further than diffusion alone.
In clinical applications of the Kirby-Bauer test, diluted cultures (for the McFarland standard comparison) must be used within 30 minutes. Why is this important?
The cells divide as time passes. What was equivalent to a .5 McFarland standard may be considered more dense after 30 min
E. coli and S. aureus were chosen to represent Gram (-) and Gram (+) bacteria, respectively.
For a given antibiotic, is there a difference in susceptibility between the - and + bacteria? If so, what differences do you see?
Yes. Antibiotics that affect the peptidoglycan of the cell wall are more effective against gram + cells because of their greater abundance of peptidoglycan
How does the antibiotic get from the disk into the agar?
The antibiotic diffuses out of the disk and into the agar. This diffusion can be affected by temperature and depth of agar in the plate.
Does the antibiotic extend into the agar beyond the zone of inhibition? How does your answer relate to the concept of MIC?
The edge of the zone of inhibition is not the limit of antibiotic diffusion. Diffusion occurs beyond the zone, but the concentration of the antibiotic is too low to be lethal. The edge of the zone represents the minimum inhibitory concentration (MIC) of the antibiotic.
Suppose you do this test on a hypothetical Staphylococcus species with the antibiotics penicillin and tetracycline. You record zone diameters of 20mm for the tetracycline disc and 25mm for the penicillin disc. Which antibiotic would be most effective against this organism? What does this tell you about comparing zone diameter to each other and the importance of the interpretive chart?
**look at chart**
According to the chart, a zone of 25mm for Staph around a penicillin disk indicates resistance to the antibiotic. A 20mm zone around a tetracycline disk for same species indicates susceptibility. Therefore, even though the zone is smaller, tetracycline would be the more effective antibiotic against this organism.
What is the purpose of inoculating TSA plates with samples from the zone of inhibition?
Any bacteria that grow in the zone of inhibition are resistant to the antibacterial used. By inoculating new plates with the bacteria, you will have a pure culture of resistant bacteria.
Understand the differences between fermentation and anaerobic respiration, between fermentation and oxidation of carbohydrates and between aerobic and anaerobic respiration.
...
List the primary types of products which bacteria may produce from fermentation
Lactic acid
Ethanol
Butyric acid
Propionic acid
2,3-Butanediol
Mixed acids
Understand why it is important to have nutrients other than the fermentable carbohydrate in a Phenol Red Carbohydrate Broth.
Because not all bacteria can utilize the fermentable carbohydrates.
The ability or inability of a particular species to ferment a particular carbohydrate depends on the presence of enzymes needed for a particular fermentation pathway
Understand the purpose of a durham tube in Phenol Red Broths.
Gas production from fermentation is indicated by a bubble or a pocket in the durham tube where the broth has been displaced
Understand why some microbiologists advocate using 1% carbohydrate rather than 0.5% carbohydrate in phenol red broths.
Some microbiologists prefer to use 1% rather than 0.5% to ensure against reversion of the reaction due to depletion of the carbohydrate (the organisms break down available amino acids after carbs are used, raises pH).
Understand why a Phenol Red Broth base medium would be inoculated in addition to the PRB tubes containing carbohydrates when examining the fermentative abilities of an organism.
To make sure that the environment remains anaerobic.
Understand how Kligler Iron Agar can be used to differentiate groups of species.
It is a differential medium for Gram-negative enteric organisms. It differentiates organisms based on their ability to ferment the lactose present in the media. (Note: all Gram-negative enteric organisms will ferment the glucose.)
Describe the difference between Kligler Iron Agar and Triple Sugar Iron Agar.
TSI contains three carbohydrates: glucose (0.1%), sucrose (1%), and lactose (1%). TSI is similar to Kligler's iron agar, except that Kligler's iron agar contains only two carbohydrates: glucose (0.1%) and lactose (1%)
Understand why the amount of glucose is limited as compared to lactose (and sucrose) in Kligler Iron agar and TSI agar.
The lower concentration of glucose allows for the detection of utilization of this substrate alone. Since glucose is a monosaccharide, it will be used first.
What two metabolic pathways lead to the production of hydrogen sulfide?
Hydrogen sulfide may be produced by the reduction of thiosulfate (acidic conditions must exist) in the medium or by the break down of cysteine in the peptone. Ferrous sulfate reacts with the H2S to form a black precipitate, usually seen in the butt (indication of sulfur reduction and fermentation).
these are bacteria that are a commonly used bacterial indicator of sanitary quality of foods and water.
They are defined as rod-shaped Gram-negative non-spore forming bacteria which can ferment lactose with the production of acid and gas when incubated at 35–37°C.[1]
___________ can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of warm-blooded animals. E. coli is one of these
coliform
are a large family of Gram-negative bacteria that includes, along with many harmless symbionts, many of the more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella.
Many live in the gut. They are facultative anaerobes, fermenting sugars to produce lactic acid and various other end products. Most also reduce nitrate to nitrite. Lack cytochrome c oxidase.
Enterobacteriaceae
Gut flora. They are rod-shaped gram-negative bacteria of the family Enterobacteriaceae; most occur normally or pathogenically in intestines of humans and other animals
enterics
know what IMViC stands for
Indole, Methyl Red, Voges-Proskauer and Citrate tests
Identify the group of organisms which can be differentiated by the IMViC tests.
the family Enterobacteriaceae and also from other Gram-negative rods
Understand the difference between mixed acid fermentation and butanediol fermentation. Know which portion of the MRVP reaction tests for each type of fermentation.
Mixed acid fermentation is an anaerobic fermentation
where the products are a complex mixture of acids, particularly
lactate, acetate, succinate and formate as well as ethanol and equal
amounts of H2 and CO2. It is characteristic for members of the
Enterobacteriaceae family. Methyl red indicator dye will change color
based on pH to test for fermentation.
Butanediol fermentation is anaerobic fermentation of
glucose with 2,3-butanediol as one of the end products. It's typical
for Klebsiella and Enterobacter. Acetoin reacts with reagents to turn
red.
2,3-butanediol fermentation produces smaller amounts of acid
than mixed acid fermentation, and butanediol, ethanol, CO2 and H2 are
the end products. While equal amounts of CO2 and H2 are created during
mixed acid fermentation, butanediol fermentation produces more than
twice the amount of CO2 because the gases are not produced only by
formate hydrogen lyase like they are in the mixed acid fermentation
understand why a citrate test in which the organism has grown, but has not changed color is still considered a positive test
in most cases, it is because it was not completely incubated
in the absence of color change, growth on the slant indicates that citrate is being utilized and is evidence of a positive reaction
understand why a light inoculum is used for the citrate test
to avoid confusion between actual growth and heavy inoculum, which may appear as growth
understand why citrate negative organisms will not grow on Simmons Citrate medium
bacteria that do not possess citrate permease will not grow on this medium
does the citrate test use a defined or undefined (complex) medium?
Simmons citrate agar is a defined medium (the amount and source of all ingredients are carefully controlled)
many bacteria that are able to metabolize citrate (as seen in the citric acid cycle) produce negative results in this test. why? be specific
citrate is the first intermediate of the citric acid cycle where it is ultimately catabolized to CO2 and oxaloacetic acid.
However, the citrate test does not detect the ability of an organism to perform the citric acid cycle.
explain how an organism that possesses the citrate lyase enzyme might not test positively on Simmons citrate agar. Is this a false negative result? why or why not?
Simmon’s Citrate agar is used to determine an organism’s ability to
use citrate as a sole carbon source.Simon’s citrate agar is a defined
medium in which sodium citrate is the sole carbon source, and ammonium
is the sole nitrogen source. Bromothymol blue (BTB) is included as a
pH indicator
The medium is initially at pH 6.9, at which BTB is
green; at a pH greater than 7.6 BTB turns a deep blue. The pH change
is induced by CO2, which is given off as a by-product of citrate
utilization. When it reacts with Na and H2O in the agar it raises the
pH above 7.6
The organism must contain the enzyme citrase to degrade citrate
(citrase)
Citrate --------->CO2 + Na HCO + H2O
I
(blue color change)
Now, coming to how a citrate test may give false results. There
are only 2 ways that can happen.
- Failure to incubate with
loose caps may result in a false-negative results.
- Avoid using
a large inoculum to streak the strand; an inoculum that is too heavy
may result in a false-positive test.
consider the uninoculated tube for the citrate test.
a) is it a positive or a negative control?
negative
identify the two enzymes that citrate positive organisms have that allow them to grow in this medium
citrate permease and citrate lyase
identify the two abilities required for an organism to be citrate-positive
growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue
Understand why both forms of fermentation may be tested using the same inoculated and incubated broth tube.
Because both are fermenting the nutrient medium which contains 0.5%
glucose. The way they ferment the medium determines the difference in
the tests MR and VP.
If the microbe performs mixed acids fermentation, the medium
will acquire an acidic pH. Addition of a pH indicator which changes
color below pH 4 indicates presence of the extreme acidity associated
with mixed acids fermentation. The reagent added to determine pH is
methyl red, and so the test is called the methyl red (MR) test.
2,3-butanediol is the end product of a long fermentation
pathway, but it is not easily detected. So, the test detects one of
the pathway intermediates instead: acetylmethylcarbinol (acetoin). The
production of 2,3-butanediol is thus indirectly detected when the
pathway intermediate acetoin reacts with reagents to turn red. The
test is named for its discoverers, as the Voges-Proskauer test.
understand the meaning of an orange color of the medium following addition of Methyl Red and a copper color in the VP test
both would be negative results for the test
Understand why it is necessary to repeatedly vortex the VP test over 30 minutes.
to oxygenate it
Prepare and evaluate cultures of each of the test media.
yep
Understand what the reversion of a reaction is and how it might affect the results of both the Phenol Red Broths and KIA (TSI) slants.
TSIA is inoculated with a glucose only fermenter. As the glucose diminishes, the organisms located in the aerobic region (slant) will break down amino acids, producing NH3 and raising the pH. This process takes 18-24 hours and only occurs in the slant because of the anaerobic conditions in the butt. This is reversion. A high pH will change the color of the broth and slant to make it appear that it did not ferment. Fermentation brings down the pH and changes the color. It can give a false reading.
Understand the purpose of an uninoculated control when running metabolic studies.
It shows the medium in an aerobic environment when uninoculated. It allows you to clearly see the fermentation in an anaerobic environment in contrast. Helps to ensure accuracy of interpretation.
Differentiate between the lack of sensitivity and the lack of specificity and how each might affect the interpretation of results of metabolic tests.
An inability to detect small amounts of the chemical or organism in
question would yield a false negative result and would be a result of
inadequate sensitivity of the test.
An inability to discriminate between the chemical or organism in
question and similar chemicals or organisms would yield a false
positive result and would be the result of inadequate specificity of
the test.
Sensitivity = True Positives / (True Positives + False
Negatives)
Specificity = True Negatives / (True Negatives + False
Positives)
The closer the sensitivity and specificity is to one, the more
useful the test is.
a metabolic process that converts sugar to acids, gases and/or alcohol. It occurs in yeast and bacteria, but also in oxygen-starved muscle cells, as in the case of lactic acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth medium.
fermentation
a form of respiration using electron acceptors other than oxygen. Although oxygen is not used as the final electron acceptor, the process still uses a respiratory electron transport chain; it is respiration without oxygen
anaerobic respiration
a form of respiration that requires oxygen as an electron acceptor in order to generate ATP.
aerobic respiration
the metabolic process by which an organic molecule acts as an electron donor (becoming oxidized in the process) and one or more of its organic products act as the final electron acceptor in respiration
oxidation of carbohydrates
consider the controls:
a) were the uninoculated controls positive or negative controls, and what purpose did they serve?
b) what purpose did the PR base serve?
a) the controls were negative, and served as color comparison for experimental tubes as well as verification for media sterility.
b) the base set lacks a sugar, so it slows that color change in other tubes truly due to fermentation.
early formulations of this medium used a smaller amount of carbohydrate and occasionally produced false (pink) results after 48 hours. This phenomenon is called a reversion.
a) why do you think it happened?
b) list at least two steps, as a microbiologist, you could take to prevent the problem
a)
b)
suppose you inoculate a PR broth with an organism known to be a slow-growing fermenter. After 48 hours, you see slight turbidity but score it as (--- l ---)
a) is this a result of a false positive or a false negative?
b) is this false result caused by poor specificity or poor sensitivity of the test system?
False Negative
Poor Sensitivity
some protocols call for a shorter incubation time for the MR and VP tests. Other protocols allow for up to 10 days incubation with virtually no risk of producing a false positive.
a) which of the two tests would likely produce more false negatives with a shorter incubation time?
b) which test would likely benefit most from a longer incubation time? Why?
a) (False negative= Culture comes back negative but not really negative) VP. Have to let organism run its metabolism for awhile.
b) VP. because it gives organism to build up acetone.
would a false negative result for the VP test more likely be attributable to poor sensitivity or to poor specificity if the test system?
poor sensitivity
Why were you told to shake the VP tubes after the reagents were added?
We needed oxygen. Color changes better in presence of oxygen.
why is the methyl red test read immediately after addition of methyl red reagent and the Voges-Proskaueh read up to 60 minutes after addition of VP reagents A and B?
MR tells us if its basic or acid. VP had to wait for chemical to react which is why we gave it some time.
Some microbiologists recommend reincubating organisms producing methyl red negative results for an additional 2-3 days. Why do you think this is done?
Gives organism enough time to lower pH to make sure we don't get a false negative result.
As mentioned in theory, the fermentation readings with TSIA and KIA must take place between 18 and 24 hours after incubation.
a) why is this true?
b) is timing as critical with H2S readings? Why or why not?
a) To make sure that carbohydrates are not depleted so the color change will still be present when the observations are made.
b) no; for interpretation of sulphur reduction, tubes can be reincubated if need be to find H2S production
You learned in theory that if the black precipitate obscures the color of the butt it must be acidic and scored as "A." Why do you think this is true?
indication of sulfur reduction and fermentation
acid conditions must exist for thiosulfae to reduce
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
a) 1% glucose is added rather than the amount specified in the recipe
take longer to exhaust glucose; yellow color
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
b) Ferrous ammonium sulfate (or ferric ammonium citrate in KIA) is ommitted
would not indicate sulfur reduction
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
c) Casein and animal tissue are omitted
no reversion would occur b/c no amino acids available to utilize
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
d) Sodium thiosulfate is omitted
substrate would be absent
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
e) phenol red absent
no color change; no differentiation
TSIA and KIA are complex media with many ingredients. what would be the consequences of the following mistakes in preparing this medium?
f) Initial pH is 8.2
Increase baseline for pH- raise bar for amount of acid needed to see color changed
Indicate the function of cytochrome c oxidase in a bacterial cell
It is the last enzyme in the chain, Complex IV, and is so named because it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm, to the oxygen inside the cell
Understand why all aerobic bacteria are not oxidase positive
Some bacteria are capable of aerobic respiration but have a different terminal oxidase system and give a negative result for the oxidase test.
the gain of electrons or a decrease in oxidation state by a molecule, atom, or ion
reduction
the loss of electrons or an increase in oxidation state by a molecule, atom, or ion.
oxidation
any enzyme that catalyzes an oxidation-reduction reaction involving molecular oxygen (O2) as the electron acceptor. In these reactions, oxygen is reduced to water (H2O) or hydrogen peroxide (H2O2)
oxidase
Differentiate: catalase and peroxidase
Catalase: enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen
Peroxidase: the plant enzymes that use peroxide to break down bacteria and other harmful material; they are located in the granules of neutrophils
Understand why aerotolerants would not use catalase. Identify an example of this characteristic.
they do not use oxygen, so they have no need to use catalase to convert hydrogen peroxide into water and oxygen
Identify sources of error that might produce false positives in the oxidase test.
Once the vial has been opened, oxidase reagent will turn purple over time due to oxygen in the air. To avoid false positives, be sure to: 1) use oxidase reagent that is still clear and 2) to read your test quickly once you have performed it.
Explain the function of zinc in the test for nitrate reduction
The addition of zinc dust is used to determine if the nitrates were reduced past nitrite stage. If a red color develops after the addition of zinc, the nitrates were not reduced to nitrites. If there is no color after the addition of zinc, the nitrates in the media were reduced beyond nitrites to ammonia or nitrogen gas
Understand the purpose of a durham tube in the nitrate test
tests for the presence of gas
Understand why the presence of a bubble in the durham tube of a nitrate broth is not definitive for nitrate reduction when working with an organism capable of fermentation
The source of the gas is unknown
Understand the relationship between the enzyme nitrate reductase and anaerobic respiration
anaerobic respiration involves the reduction of an inorganic molecule, something other than oxygen, as a final electron acceptor in the ETC. In this case, it is nitrogen that gets reduced and the nitrate reductase is what reduces nitrate to nitrite
Understand the nitrate/nitrite test and be able to accurately interpret the results
Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate. In this test, if the organism reduces he nitrate to nitrite, the nitrite reacts with the reagents sulfanilic acid and alpha-naphthylamine to produce a red color. No color after addition of these reagents indicates nitrates are not reduced or nitrites were reduced further. If the organism possesses a strong nitrate reductase it can further break down nitrites to ammonia or molecular nitrogen. The addition of zinc dust is used to determine if the nitrates were reduced past nitrite stage. If a red color develops after the addition of zinc, the nitrates were not reduced to nitrites. If there is no color after the addition of zinc, the nitrates in the media were reduced beyond nitrites to ammonia or nitrogen gas
Describe how the toxins hydrogen peroxide and superoxide radicals may be formed and how bacterial cells convert them to none toxic substances
Hydrogen peroxide and superoxides are produced when aerobes,
facultative anaerobes, and microaerophiles use the aerobic respiratory
pathway during degradation of carbohydrates for energy production.
Accumulation of these substances will result in death of the organism
unless they can be enzymatically degraded. Catalase or peroxidase
rapidly degrades H2O2 into H2O and O2.
All aerobes and some facultative anaerobes are catalase
positive. Superoxide dimutase is the enzyme responsible for
degradation of the superoxides in catalase negative aerobic organisms.
Strict anaerobes are not able to produce these enzymes. Therefore,
they cannot be cultivated in the presence of oxygen
Think about the advice given in the procedure to test a known catalase-positive organism along with an unknown
a) is this a positive or a negative control?
b) what information is provided by the results?
a) positive control
b) to make sure the hydrogen peroxide has not broken down and is still working
consider the step in the tube test where hydrogen peroxide is added to the uninoculated tube
a) is this a positive or a negative control?
b) what information is provided by the results?
a) To see if its catalase positive or negative
b) if positive, oxygen gas bubbles form immediately
when flavoprotein transfers electrons directly to oxygen, hydrogen peroxide is produced. What other consequences might result from electron carriers in the ETC being bypassed?
an increase in heat and a decrease in ATP production. This is analogous to releasing the energy from gasoline out of the context of an automobile engine. It would not be "productive"
Would a false positive from the reaction between the inoculating loop and hydrogen peroxide be caused by poor specificity or poor sensitivity of the test system? explain.
Specificity; the loop gives the same result as the enzyme catalase
think about the advice given in the procedure to test a known oxidase-positive organism along with an unknown.
a) is this a positive or a negative control?
b) what information is provided by the results?
a) positive: dark blue withing 20 seconds, cytochrome c oxidase is present
b) negative: no color change to blue within 20 seconds, cytochrome c oxidase is NOT present
think about the 20-second time limit on the oxidase test
a) what happens to the oxidase reagent after 20 seconds?
b) does this only happen after 20 seconds? If not, why is a 20-second time limit set?
a) the reagent can react with air and produce a false positive result
b) Read the reaction within 20 seconds (NOT after), usually it will change in less than 15 seconds. The oxygen will change the reagent color as time passes, so it must be read quickly
provide a possible explanation as to why this test identifies the presence of cytochrome c oxidase and not other oxidases (such as those of E. coli)
An oxidase test relies on a reagent that will change colors when it is oxidized
The reagent used in the test only reacts to cytochrome c oxidase by acting in place of oxygen to receive electrons from the cytochrome
transfer of electrons to 02, thus reducing it to water
consider the uninoculated tube:
a) is it a positive or a negative control?
b) what information is provided by the uninoculated control?
a) negative result
b) because it indicates that nitrate is still present in the tube after the addition of reagents
why is gas production not recognized as nitrate reduction when the organism is a known fermenter?
Organisms that ferment also produce gas. You cannot tell if the gas is from the fermentation or nitrate reduction.
suppose you remove your test cultures from the incubator and notice that one of them - a known fermenter - has a gas bubble in the Durham tube. Knowing that fermenters frequently produce gas, you ignore the bubble and proceed to the next step. Adding reagents produces no change, and neither does adding zinc. Is this occurrence consistent with what you have learned about this test?
would you change your answer to this is the control broth did not change color after the addition of reagents? what if the control broth had changed color only after the addition of reagents and zinc?
The gas bubble is nitrogen. if the reagents and zinc do not produce a change, then the nitrates have been used by the bacteria. If you were looking for aerobic and facultative anaerobic gram-negative microorganisms, then congratulations.
A bubble plus colour change would indicate some other kind of bacteria, inconsistent with the test.
when testing microaerophiles, some microbiologists prefer to use a semisolid nitrate medium that contains a small amount of agar. why do you think this is done?
Agar slows down the diffusion of oxygen into the medium
It is thin enough to allow the cells to move to the level of medium that has the right concentration of oxygen for their optimal growth, and thick enough for them to stay there and grow enough that they can be detected easily
SIM medium is used to differentiate what group of organisms?
Enterobacteriaceae
Know and understand the 3 tests contained within SIM medium
sulfur reduction (S), indole production form tryptophan (I), and motility (M)
understand the two ways in which sulfur reduction can occur in SIM medium
1. the enzyme cysteine desulfurase catalyzes the hydrolysis of the sulfur-containing amino acid cysteine to pyruvate and H2S during putrefaction
2. in a totally unrelated way, the enzyme thiosulfate reductase catalyzes the reduction of sulfur (in the form of sulfate) to H2S at the end of an anaerobic respiratory electron transport chain
understand which amino acid is broken down to produce indole,
what the source of this amino acid is in the medium,
and the presence of what enzyme is necessary for the pathway to operate
- tryptophan (contained in casein and animal protein); tryptophanase can hydrolyze tryptophan to pyruvate, ammonia (by deamination), and indole
- casein and animal protein
-tryptophanase
understand why indole is the preferred product to test for the presence of tryptophanase
the formation of red color in the reagent layer indicates a positive reaction and the presence of tryptophanase; no red color is indole negative
understand how the medium is designed to show motility
single stab in the medium; motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth extending outward in all directions from the central stab line
understand how to differentiate true motility from movement of the needle during the stab
growth that that radiates in ALL directions and appears slightly fuzzy throughout the tube is an indication of motility
Both SIM and KIA media test for 3 abilities of the organisms in a single tube. The MRVP tube has one tube for growth, but requires the transfer of medium to a separate tube, before the reagents are added. Why the difference?
I don't know
consider the uninoculated SIM tube
a) is it a positive or negative control in each test?
b) what purpose does it serve in the sulfur reduction test?
c) what purpose does it serve in the indole test?
d) what purpose does it serve int he motility test?
...
the sulfur reduction test is not able to differentiate H2S produced by anaerobic respiration and H2S produced by putrefaction. Is this inability the result of poor sensitivity or poor specificity of the test system?
poor specificity
List five groupings of microorganisms according to their oxygen tolerance. Know the characteristics of each group.
1. Obligate aerobes- require Oxygen to survive (ML, CV)
2. Facultative anaerobes- do not require oxygen
but can
use it (SA, SM)
3. Obligate anaerobes- oxygen is toxic (CS)
4. Aerotolerant anaerobes- oxygen not toxic but
can't use
it (EF)
5. Microaerophiles- High oxygen level is toxic but
need a
little oxygen
Understand the characteristics of capnophiles as they relate to aerotolerance
capnophiles are microaerophiles that can survive only if carbon dioxide levels are elevated
Prepare and evaluate the growth of organisms in agar shake cultures, stab cultures, Fluid Thioglycollate medium, candle jars and GasPaks, using organisms with various oxygen tolerances.
Shake: innocuate with loop then gently roll to spread
stabs:
using a needle and agar deep
Fluid Thioglycollate Medium:
innoculate with loop
candle jars: divide plate into sections, one
line of bacteria per section.place innoculated plate in jar, then
light candle and let the candle go out
Gas Packs: place
innoculated plates in bag or jar and open gas pack, seal immediately
Understand how each of the methods used create environments that are anaerobic and/or have low oxygen tension/increased carbon dioxide concentration.
Stab cultures heated or boiled drive oxygen out.
Old tubes will need to be reheated to remove oxygen that may
have gotten into it.
Water, phyglycolaid will reduce any oxygen
Resizure- indicator will turn pink to indicate how much air has
defused
Gas pack- white indicator button = no oxygen, blue=
contaminated. Methylene blue turns blue in presence of oxygen. sodium
borohydride and sodium bicarbonate react with water to produce
hydrogen and CO2 gases.
Accurately appraise the redox potential of a GasPak anaerobic system and tubes of Fluid Thioglycollate Medium. Identify the redox potential indicators.
Gas Pak - is a method used in production of an anaerobic environment.
It is used to culture bacteria which die or fail to grow in presence
of oxygen (anaerobes). Redox indicator is a paper indicator strip
saturated with methylene blue that changes color to blue in the
presence of oxygen and is colorless when reduced. Pak creates an
anaerobic, microaerophilic, or CO2 enriched conditions.
Fluid Thiglycollate Medium - used primarily to determine the
oxygen requirements of microorganisms. Sodium thioglycolate in the
medium consumes oxygen and permits the growth of obligate anaerobes.
This, combined with the diffusion of oxygen from the top of the broth
produces a range of oxygen concentrations in the media along its
depth. The oxygen concentration at a given level is indicated by a
redox sensitive dye like resazurine that turns pink in the presence of oxygen.
Know the purpose of catalase (peroxidase) and superoxide dismutase in bacterial metabolism and how these relate to aerotolerance.
catalase turns hydrogen peroxide into water and oxygen, superoxide dismutase is an enzyme thay breaks down superoxide into hydrogen peroxide. they protect the bacteria from
Understand why anaerobes are inhibited/killed by oxygen.
they do not have superoxide dismutase to protect them
Understand why tubed media are boiled for at least 10 minutes prior to inoculation or should be used soon after initial preparation. (agar shake cultures, stab cultures, Fluid Thioglycollate medium)
to remove air and oxygen
Name at least one bacterial genus that tolerates oxygen but does not produce catalase.
Streptococcus
Understand why it is important to time how long it takes for the candle in a candle jar to go out.
to ensure enough carbon dioxide has built up and oxygen has been removed
Understand why adding a small amount of agar to the Thioglycollate Medium would be beneficial. (Small amount would be enough to slightly thicken the medium but not solidify it.)
slows diffusion of oxygen into medium
Understand the meaning of the term “fastidious” as it relates to bacterial culturing.
picky as to requirements such as nutrients, temperature, PH, salinity, oxygen
Understand why water (moisture) accumulates in the candle jar after the lid has been tightened down.
Palladium catalyzes a reaction between the hydrogen and free oxygen in the jar to produce water. Removal of free oxygen produces anaerobic conditions in the jar within approximately an hour, as evidenced by a white indicator strip and moisture on the inside of the jar.
why is it important to use this medium (agar deep stabs) soon after preparation?
So clostridium sporogenes grow. Oxygen doesn't diffuse and ruin results
when inoculating agar deep stabs, why do you have to insert and remove the needle along the same stab line?
You don't want any more oxygen to get in and ruin results
suppose that after incubation you find a tube that indicates uniform growth through the agar
a) can the aerotolerance category of the organism be determined? explain
b) with which aerotolerance group are you most likely to confuse this tube and how will you distinguish between them?
a) It might be facultative
b)
if you inoculate an organism known to chemically reduce sulfur (as done by organisms capable of anaerobic respiration), where would you expect to see its growth in the test medium used today? could it be seen in more than one zone? explain
I would expect to see it in the bottom, where there is no oxygen
if you have no growth in the stab after 24 hours' incubation, what are some possible explanations?
Did not inoculate correctly, bacteria wasnt alive to begin with, medium was too hot
consider aerotolerance categories and their growth in FTB.
a) where would you expect to see growth of a strict aerobe?
b) anaerobe?
c) microaerophile?
d) facultative anaerobe?
e) aerotolerant anaerobe?
a) organisms that require oxygen for respiration, grow at the top where oxygen is most plentiful
b) organisms that grow without the presence of oxygen, grow in the lower part of the medium only
c) survive only in environments containing lower than normal levels of oxygen. Growth is in the middle or upper middle of the medium
d) grow in the presence OR absence of oxygen. They grow throughout the medium but appear denser at the top
e) don't require oxygen and are not affected by it. Grow uniformly throughout the medium
consider resazurin dye. Considering the major application of this medium, which is more desirable, a thick colored or a thin colored band?
Redox indicator like resazurin in medium that turns pinkwhen exposed to oxygen. The top turns pink bc of oxygen. Thin band is preferred , if it is thick, that means it has gone old and more oxygen has seeped down deeper.
a) what purpose does the dye serve in FTB?
b) where in the broth column is resazurin dye located?
c) why is the broth pink at its surface?
d) considering the primary application of FTB, which is more desirable, a thick or a think colored band at the surface?
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consider medium freshness:
a) why is it important that this medium be fresh?
b) which type of organism (obligate aerobe, obligate anaerobe, microaerophile, facultative anaerobe, aerotolerant anaerobe) would most likely be affected negatively by the use of old media?
c) which would most likely be affected positively by the use of old media?
d) which would be least affected by old media?
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Understand how UV light harms bacteria. (Understand the mode of action of ultraviolet light on bacterial growth.)
When DNA absorbs UV radiation at 254 nm, the energy is used to form new covalent bonds between adjacent pyrimidines: cytosine-cytosine, cytosine-thymine, or thymine-thymine. Collectively, these are known as pyrimidine dimers, with thymine dimers being the most common. These dimers distort the DNA molecule and interfere with DNA replication and transcription.
is a type of electromagnetic energy. like all electromagnetic energy, it travels in waves and is distinguishable by its wavelength (the distance between adjacent wave crests, measure in nanometers (nm))
UV light
Differentiate UV-A, UV-B and UV-C and their effects on living cells.
UV-A: the longest wavelengths, ranging from 315 to 400 nm
UV-B:
wavelengths between 280 and 315 nm
UV-C: wavelengths ranging from
100 to 280 nm
most detrimental to bacteria, exposure for more
than a few minutes usually results in irreparable DNA damage and death
of the organism
Understand the effects of UV irradiation on microorganisms that allow it to be used as for sterilization.
long and/or intense exposures to UV produce more damage than the cell can repair, making UV radiaiton lethal
Understand why UV irradiation has limited application for sterilizing materials.
it penetrates materials such as glass and plastic poorly, bacterial cells have mechanisms to repair UV damage
Differentiate spontaneous and induced mutations.
Spontaneous mutations occur on their own without any exposure and their cause cannot be pointed out while induced are the ones which occur due to exposure to mutagens (both chemical and physical)
Understand why some bacteria are less susceptible to UV irradiation than others. (For example: Understand why UV light is less successful at killing Bacillus spp. than Serratia marcescens)
Bacillus spp. forms spores which are less susceptible to UV irradiation
Define and describe photoreactivation.
the repair enzyme, DNA photolyase, is activated by visible light (340-400 nm) and simply monomerizes the dimer by reversing the original reaction. E. coli
Define and describe excision repair (dark repair) of DNA.
involves a number of enzymes. the thymine dimer distorts the sugar-phosphate backbone of the strand. this is detected by an endonuclease (UvrABC) that breaks two bonds. A helicase (UvrD) removes the 13-nucleotide fragment (including the dimer), leaving single-stranded DNA. DNA polymerase I inserts the old DNA which is then binded by DNA lygase and the repair is complete. E. coli
Interpret results of our experiment: How does UV irradiation affect Chromobacterium violaceum, Serratia marcescens and Bacillus subtilis var. niger? How does the ability to complete photoreactivation change those effects?
UV raditation affects all three but has the most affect on CV and Serratia. Completing photoreactivity by being put under the grow light, increased the amount of viable colonies. Grow light provides the energy for DNA photolyase.
Understand why a section of each experimental plate was not exposed to UV irradiation. (Understand why a portion of the plate remained covered with the cardboard.)
a portion remained uncovered as a control. In order for the results of the UV radation to be interpreted, there must be a control to compare them to.
the purpose of this exercise is to demonstrate the comparative effect of UV on two (or three) bacterial populations. This could have been accomplished without the cardboard cover. Why was the cover used?
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this is not a quantitative exercise. keeping this in mind, can you see a general trend between bacterial death and UV exposure time?
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which organism survived the longest exposure? why?
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why were you told to remove the plate covers prior to exposing them to UV?
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consider the exposed part of the plate.
a) what does the relative spareness of growth tell you about the effect of UV radiation?
b) what do individual colonies tell you about the cells from which they grew?
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what does the relatively heavy growth on the plates covered by the mask and lid during exposure tell you about UV radiation?
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what is the effect of longer UV exposure on Serrate marcescens? To answer this question, which plates should be compared?
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what is the effect of the poster board mask on UV radiation? to answer this question, which plates should be examined?
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what is the effect of the plastic lid on UV radiation? to answer this question, which plates should be compared?
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