Micro LAB EXAM 2

Thermal Death Lab. Because this is a quantitative procedure, why is it not necessary to know the volume of broth in the 30 nutrient both tubes?

The quantitative aspect of this part of the lab is the time, not the number of cells. We are looking only for growth or nongrowth in the nutrient broths.

HE Agar. In your own words, what are the roles of lactose, sucrose, and salicin in HE agar?

These sugars are rarely fermented by Salmonella and Shigella species, which differentiates them from coliforms.

1. Which of the microscope images shows clearest evidence of a mixed-species culture? (a) J (b) M (c) O (d) P (e) Q

A

A ______ wavelength is likely to have more germicidal effect on bacteria. a) shorter b) longer c) wider d) slower

A

A chemical that punctures cell walls would most likely be considered: a) Bactericidal b) Bacteriostatic c) Toxic to animal cells d) A narrow spectrum antibiotic e) All of the answers shown would be true

A

HEA agar is designed to isolate and differentiate among enterics, meaning organisms which live in the intestines. Examples of enterics that can grow on HEA are: a) Staphylococcus epidermidis b) Escherichia coli c) Shigella flexneri d) Salmonella typhimurium e) C and D f) B, C, and D g) None of the above

F

cCNA Test. In your own words, what is the role of sheep blood in CNA agar?

Sheep blood supplies iron-containing heme. Iron is an essential element for growth. In addition, inclusion of sheep blood makes this a differential medium based on the degree of hemolysis each organism produces

UV Lab Why were you told to remove the plate covers prior to exposing them to UV?

The UV light has trouble penetrating the plate lid so organisms wouldn't have received much/any exposure if the lid was still on

Urea. Explain any differences.

Unexpected results can be caused by an number of factors, including the strain used and the amount of inoculum.

13. A disc containing 1 μg of penicillin was measured to have a volume of 2 μL. Calculate the concentration of penicillin in this disc (in grams per liter). a. 0.05 b. 0.5 c. 5 d. 50 e. 500

b

18. Which of the following results from cell staining would be considered most conclusive/reliable? a. cell-free (no microbes present) b. gram-negative c. non-spore forming d. spore-positive

d

14. Bile salts in Hektoen Enteric Agar help_______growth of most species of Gram-positive cocci. a. stimulate b. kill c. immobilize d. precipitate e. inhibit

e

cCNA Test. Which ingredient(s) in Columbia CNA plus 5% sheep blood agar supply(ies): Carbon? Nitrogen?

1) Casein (mild protein), animal tissue, yeast extract, beef extract, and corn starch can all supply carbon in a variety of forms. 2) Organic nitrogen is found in proteins and nucleic acids. Casein, animal tissue, yeast extract, and beef extract all have protein, and the latter three have nucleic acids. Corn starch is a polysaccharide and doesn't contain nitrogen

Thermal Death Lab. How long to kill a population of S. epidermidis with the same density?

...

The results for a particular species of bacteria in various diagnostic, morphological, and metabolic assays can be found in Bergey's Manual of Systematic Bacteriology. Why then should the class work together to build a control profile for bacteria that will eventually be used in th e"Mixed Double Unknown Project"? a) to confirm the behavior of the strain being used - some strains may produce the opposite results of expected for the species b) because an older edition of Bergey's manual will have outdated and useless information c) to avoid confusion between organisms with similar names d) because the media being used for the project may produce different results than those listed for the assay in Bergey's manual.

A

UV light is a mutagen that damages both bacterial and human DNA. a) True b) False

A

What happens to the bacteria on a molecular level when they are exposed to extreme heat? a) Their proteins denature which causes them to become ineffective b) It causes their genetic material to be erased c) The heat causes bacterial cells to fuse together d) Nothing, most bacteria can handle extreme heat just fine

A

What is the D-Value? a) the time required to kill 90% of a bacterial species at a certain temperature b) The time required to kill 70% of a bacterial species at a certain temperature c) The temperature that kills bacteria most effectively d) None of the above.

A

Which of the following are important to keep in mind while taking samples of the bacteria during the experiment? a) Remembering to use a fresh sterile pipette tip each time you get a new sample b) Always go to the second stop on the pipette to make sure you're collecting an accurate amount c) All of the above d) None of the above

A

How does UV light damage bacteria? a) It breaks down the cell membrane b) By creating pyrimidine dimers out of thymine base pairs in the bacterial DNA c) It just kills them d) It doesn't damage bacteria, it makes them healthier depending on the species

B

The bacteria we work with in this class (and most human pathogens) would be considered: a) Psychotrophs b) Mesophiles c) Thermophiles d) Extreme thermophiles

B

What color will the broth be if ammonia is created during the urease test? a) yellow b) pink c) no color change d) blue

B

UV Lab Which organism survived the longest exposure? Why?

B. megaterium survived the longest because it contains spores, which are resistant to heat.

HE Agar. In your own words, what is the role of bile salts in HE agar?

Bile salts make the medium selective, because they inhibit Gram positives (and to some extent, Gram negatives)

EMB Test. In your own words, what are the roles of eosin Y and methylene blue in EMB agar?

Both act as selective agents against Gram positives. They also are responsible for the color changes produced by lactose fermenters

cCNA Test. In your own words, what are the roles of colistin and nalidixic acid in CNA and how does each work?

Both are antibiotics that affect Gram-negative cells. Colistin disrupts membrane integrity and nalidixic acid affects DNA replication

4. Which of the microscope images could be consistent with Gram-stained E. aerogenes or E. coli? (a) K (b) M (c) O (d) P (e) Q

C

6. Which single microscope image would best demonstrate that the Gram stain is functioning properly? (a) J (b) K (c) L (d) M (e) P

C

7. Based on the phenol red broth results shown (see image on separate page), which sample(s) were positive for acid generation but negative for gas production? a. sample on the left side b. sample on the right side c. sample in the middle d. middle and right samples e. none of the above

C

Colonies that produce hydrogen sulfide on Hektoen Enteric Agar will turn: a) blue-green b) pink c) black d) yellow

C

HE Agar. Which ingredient(s) in HE agar supplies: Carbon? Nitrogen?

Carbon - Yeast extract, peptic digest of animal tissue, lactose, sucrose, salicin, and to some degree bile salts can provide carbon Nitrogen - Organic nitrogen is generally found in proteins and nucleic acids. Yeast extract and peptic digest of animal tissue contain these biochemcials. In addition, some lipid components of bile contain nitrogen.

EMB Test. Which ingredeint(s) in EMB agar supply(ies): Carbon? Nitrogen?

Carbon: gelatin and lactose contain usable carbon. Nitrogen: Organic nitrogen is generally found in proteins and nucleic acids. Gelatin is derived from the protein collage and is the sole source of nitrogen.

cCNA Test. Why might collisitin affect Gram-negative bacteria more severly than Gram-positive bacteria? ??

Colistin affects the membrane, which invariably interferes with an organism's ability to maintain osmotic homeostasis. With its thicker peptidoglycan wall, Gram-positive cells are more likely to delay lysis when the membrane falls.

Mike Robe takes his gelatinase test out of the incubator. He notices that all of the tubes are liquid, including his negative control. What can he conclude? a) Well he done messed up b) All of the samples are gelatinase positive c) All of the samples are gelatinase negative d) The heat from the incubator melts the gelatin. He should place his tubes in the fridge until the negative control becomes solid. Then, he can read his results.

D

Suppose you would like to make 2 liters of media with a working concentration of 100 micrograms/mL ampicillin. How much ampicillin would you add to 2 liters of media to obtain this concentration? a) 50 micrograms b) 100 micrograms c) 100 milligrams d) 200 milligrams e) 400 milligrams

D

The enzyme necessary to hydrolyze urea into ammonia and carbon dioxide is a) Deaminase b) Decarboxylase c) Ammoniase d) Urease

D

The germicidal effect of UV-C is related to: a) Lamp intensity b) The distance to the target c) Duration of exposure d) All of the above

D

Kirby-Bauer Test. How does the antibiotic get from the disk into the agar?

Diffusion

HE Agar. Write a description of an organism whose colonies are green with black centers on HE agar.

Green colonies on HE agar is consitent with the organism not fermenting lactose, salicin, or sucrose, so it is likely to be a Shigella, Salmonella, or Proteus species. The presence of a black center is an indication of sulfur reduction and is consistent with either a Salmonella or a Proteus species, which is as far as these data will take you.

EMB Test Suppose a mistake is made in preparing a batch of EMB agar and the starting pH is 7.6 instead of 6.9-7.3. Would that affect the medium's sensitivity or specificity? Explain your answer

If a batch of EMB were made at a higher than normal pH, it would take more acid production from lactose fermentation to produce the dark purple/green color of a positive. It is possible then, that a true positive for lactose fermentation might not change the medium's color in the allotted incubation time and would be incorrectly recorded as a negative. This would affect the medium's sensitivity.

EMB Test. Is EMB agar a define or an undefined medium? Provide the reasoning behind your choice and explain why this formulation is desirable.

It is undefined due to the gelatin in the mediu. Gelatin is a family of related biochemicals, and the exact chemical formula of the gelatin used in making Levine EMB is not necessarily known. An undefine medium is desirable to support growth of a wide variety of the organisms being selected for.

HE Agar Is HE agar a defined or an undefined medium?

It is undefined due to the yeast extract and peptic digest of animal tissue in the medium. An undefined medium is desirable to support growth of a wide variety of the organisms being selected for.

HE Agar In your own words, what is the application (purpose) of HE agar?

It is used to isolate Gram negatives and differentiate Salmonella and Shigella species from other Enterobacteriaceae.

HE Agar. Compare the recipes of nutrient agar and HE agar. If an organism can grow on both media, on which would you expect it to grow better?

It would probably grow better on the HE, because there are a greater variety of carbon sources than in NA

EMB Test. Why wouldn't it be advisable to compare growth of the organisms on each plate to each other? There are at least two answers to this question.

On the NA plate, "good growth" for one organism may be very dense, whereas "good growth" for another might be fairly thin. In the context of this exercise you want to compare the amount of growth of each organism on the selective medium against its growth on NA to see if inhibition occurred. You wouldn't want to compare growth of the organisms on the EMB plate to each other because you wouldn't know which ones were inhibited and which were normal

Urea. Suppose you ran this test with Providencia stuartii but it took 48 hours to turn pink. Do you think this is a false result?

Providencia stuartii is a rapid urea hydrolyzer; however, this result would tell you that the organism is a slow urea hydrolyzer. And although the result does, in fact, tell you that the organism utilizes urea, it does not give you a very important piece of information.

Urea. Explain why it is acceptable to record positive tests before the suggested incubation time is completed but it is not acceptable to record a negative result early.

When a positive result is seen in urea media, it can be recorded because there will be no further reaction. As always, the complete incubation time must pass before a negative result is recorded.

Kirby-Bauer Test. Does the agar have an antibiotic beyond the zone of inhibition? How does your answer tie in with MIC?

Yes, the antibiotic diffuses beyond the zone of inhibition, but the concentration is not high enough to have an effect. The edge of the zone occurs because it is the minimum inhibitory concentration (MIC).

Gelatin. If the control is solid and an inoculated tube is liquid, is it acceptable to read the result before the complete incubation time has elapsed? Explain.

Yes. For a change, the uninoculated control is not for color comparison. In this case, it is used to verify that liquefaction of the inoculated tube is due to gelatinase and not to melting. If the control is solid and the experimental tube is liquid, the result is a true positive. because there is no further reaction, it is acceptable to read a positive result before the incubation time has elapsed - as long as the control is solid

For a given antibiotic, is there a difference in susceptibility between the Gram-positive and Gram-negative bacteria? If so, what difference(s) do you see?

Yes; Antibiotics that affect the peptidoglycan of the cell wall (e.g., penicillin) are more effective against Gram-positive cells because of their greater abundance of peptidoglycan.

Urea. Did you expect them to? Why or why not?

You should expect a difference between the media only for slow-urease positive organisms, which will produce a positive result in urea agar after several days of incubation but won't produce a postive result in urea broth because of its shorter incubation time. Otherwise, rapid urease-positive and urease-negative organisms should produce identical results in both media.

Urea. If so, is it a false positive or a false negative? If not, why not? Give some possible reasons for this occurence.

we should not quibble over whether the result is a false positive or a false negative, but that the sensitivity of the medium was not adequate in this case. There are many reasons why the medium (or test) may have failed, mostly relating to human error - poor quality or expired medium, improper handling of the medium or sample, incubating at a suboptimal temp, etc.

Match the following terms with where they should be properly disposed of in the lab: a) Glass tubes - metal bins at the front of the room b) Plastic plates (petri dishes) - large red biohazard bins at the fron of the room c) Plastic microcentrifuge tubes (used for serial dilutions) - small red biohazard binds at the front of the room d) Cotton swabs - small red biohazard bins on the benchtops e) Plastic pipette tips - small red biohazard bins on the benchtops f) Gloves - large red biohazard bins at the front of the room g) Microscopic slides - small red biohazard bins on the benchtops

...

Thermal Death Lab. How long would it take to kill a population of E. coli with a density of 10^3 cells/mL?

...

Gelatinases function to a) hydrolyze gelatin into amino acids b) hydrolyze casein into amino acids c) hydrolyze agar into amino acids d) solidify gelatin

A

Suppose you want to see how susceptible the pathogen Staphylococcus aureus is to the antibiotic Chloramphenicol. You streak the sample onto a plate and put a disk containing Chloramphenicol on the plate. After a 48-hour incubation, you see that the Zone of Inhibition was 11 mm. What does this say about Chloramphenicol and its ability to treat S. aureus infections? (Hint: use table 7-4 in your book) a) Chloramphenicol is ineffective at treating this strain of S. aureus b) Chloramphenicol is effective at treating this strain of S. aureus c) Chloramphenicol is ineffective at treating all strains of bacteria d) Chloramphenicol is effective at treating all strains of bacteria

A

The Kirby-Bauer test is used for: a) Determining the dose of antibiotic to use for an infection b) Testing for pathogenic bacteria c) Determining the susceptibility of a microbe to an antimicrobial agent d) Testing the effectiveness of disinfectants

C

In the Kirby-Bauer antibiotic disk sensitivity testing procedure, which of the following processes creates a gradient of antibiotic concentration? a) Osmosis (flow of substances across membrane, down the concentration gradient) b) Passive diffusion (random movement of substances through semi-solid media) c) Electrical current applied to the media (external mechanical processes) d) Active transport (flow of substances across membrane, up (or against) the concentration gradient) e) Bacterial flagellar motion (biological processes within the media)

B

What pH indicator is used for the urease test? a) Mannitol b) Phenol red c) Brom thymol blue d) Methyl red

B

What is the minimum inhibitory concentration? a) The minimum concentration of bacteria needed ot infect a host cell b) The minimum concentration of bacteria needed to bypass the immune system c) The minimum concentration of antibiotic needed to inhibit bacterial growth d) The minimum concentration of antibiotics needed to allow bacterial resistance

C

Which of the following is an antibiotic? a) Ethanol b) Zidovudine (medication used to treat HIV and other retroviruses) c) Penicillin d) Bleach e) All of the above

C

Which of the following is responsible for the differential nature of Columbia CNA agar? a) agar b) colistin and nalidixic acid c) sheep blood d) carbon dioxide e) tryptone

C

A colony picked directly from ____ media should never be considered a pure culture. a) blood agar b) nutrient agar c) selective media d) differential media

C

A positive result in the gelatin hydrolysis assay a) is solidification of the nutrient gelatin only if the negative control is liquid b) is the solidification of the nutrient gelatin only if the positive control is solid c) is the liquefaction of the nutrient gelatin only if the negative control is solid. d) Is liquefaction of the nutrient gelatin even if the negative control is liquid.

C

In order for an organism to be considered "sensitive" to a particular antibiotic, it must: a) Exhibit confluent growth on the plate b) Display a zone of inhibition c) Display a zone of inhibition of a certain size d) Have all its cells die in a certain amount of time

C

You discover a bacteria that will only grow at 55 degrees C. What type of bacteria is this? a) psychrophile b) falcultative thermophile c) obligate thermophile d) mesophile

C

cCNA Test. In your own words, what is the application (purpose) of Columbia CNA with 5% sheep blood agar?

Columbia CNA agar is a selective and differential medium. It inhibits the growth of Gram negatives and allows isolation of Gram positives. Hemolytic Gram positives, such as Staphylococcus, Streptococcus, or Enterococcus species can frequently be differentiated from other Gram positives because of their hemolytic reactions

cCNA Test Compare the recipes of nutrient agar and Columbia CNA agar. If an organism can grow on both media, on which would you expect it to grow better? Explain your answer ??

Columbia CNA agar is much more nutritious than nutrient agar. Both have the same amount of beef extract, so there is no difference there. The only additional nutrient source in NA is peptone, but Columbia CNA agar has casein, animal tissue, yeast extract and corn starch. Columbia CNA agar inhibits Gram-negative organisms, but those that aren't inhibited are treated to a diverse menu.

2. Which of the microscope images could be consistent with Gram-stained S. aureus or S. epidermidis? (a) L (b) M (c) O (d) P (e) Q

D

3. Which microscope image appears to show endospores? (a) J (b) K (c) L (d) N (e) Q

D

EMB Agar is selective and differential medium. It selects for ____ while differentiating between those that can and cannot ____. a) Gram-positive; ferment mannitol b) Gram-positive; ferment lactose c) Gram-negative; ferment mannitol d) Gram-negative; ferment lactose

D

In the Kirby-Bauer antibiotic disk sensitivity testing procedure, antibiotic-resistant colonies of bacteria are most likely to be observed near the ____ of the zone of inhibition. a) Center b) Intermediate/middle area c) Selective range d) Periphery (outside edge) e) Underlayment

D

The peptone in the Eosin Methylene Blue Agar serves as a) a pH indicator b) a selective agent c) an inhibitor d) a nutrient source

D

EMB Test. In your own words, what is the role of lactose in EMB agar?

EMB agar is a differential medium based on the ability of an organism to ferment lactose to acid end products; lactose is the substrate of the fermentation. Coliforms perform this fermentation, whereas noncoliforms do not.

Urea. Did your results from the broth and solid media agree for each organism?

Due to its limited nutrients and strong buffer, urease broth yields a positive result only for rapid urease-positive organisms, so there should have been a difference between the results given by K. pneumoniae if the urea broth was incubated for the suggested 24 hours.

5. Which microscope image appears consistent with gram-negative cocci? (a) J (b) M (c) O (d) P (e) Q

E

How might bacteria become resistant to an antibiotic? a) After the target of the drug b) Alter how the drug gets into the cell c) Break down the drug d) Get rid of the drug by creating an "efflux pump" e) All of the above

E

EMB Test. In your own words, what is the application (purpose) of EMB agar?

EMB agar is used to isolate Gram negatives from clinical samples and to subsequently differentiate between lactose-fermenting coliform and lactose-nonfermenting coliforms

cCNA Test. Is Columbia CNA agar a defined or an indefine medium? Provide the reasoning behind your choice and explain why this formulation is desirable.

It is undefined due to the casein, animal tissue, yeast extract, beef extract, and corn starch in the medium. The exact compositions of casein and corn starch are unknown, and the animal tissue, yeast extract and beef extract contain the array of biochemicals found in the cells of each. ANy one of these would make the medium undefined. The goal of the medium is to isolate desired organims and once the undesired organisms have been inhibited you want the desired ones to grow well. The variety of biochemicals in animal tissue, yeast extract and beef extract make the medium more likely to satisfy the varied nutritional needs of the organisms being selected for.

cCNA Test. Would removing colistin and nalidixic acid from CNA alter the medium's sensitivity or specificity? ??

It would alter specificity because organisms that shouldn't grow on it would. It wouldn't likely alter sensitivity because you would probably still be able to detect growth of organisms that should grow on it.

EMB Test Compare the recipes of nutrient agar and EMB agar. If an organism can grow on both media, on which would you expect it to grow better?

It would probably grow better on NA, which has beef extract (a rich mix of biochemicals) and peptone. Levine EMB supplies one carbohydrate and one protein derivative (gelatin) for carbon and nitrogen sources.

HE Agar. Suppose a mistake is made in preparing a batch of HE agar and the starting pH is 7.0 instead of 7.4-7.8. Would that affect the medium's sensitivity or specificity?

If a bunch of HE were made at a higher than normal pH, it would take more acid production from lactose/salicin/sucrose fermentation to produce the orange-pink color of a positive. It is possible then, that is a true positive for lactose fermentation might not change the medium's color in the allotted incubation time and would be incorrectly recorded as a negative. This would affect the medium's sensitivity.

Gelatin. Some microbiologists recommend incubating this medium at 37 C, along with an uninoculated control, and then transferring all tubes to the refrigerator prior to reading them. Why might this be the preferred technique in some situations? What potential problems can you see with this method?

Incubation at 37 C would favor growth of the organisms that prefer this temperature - such as those associated with humans. Some possible problems might be nonenzymatic alteration of the gelatin or gelatinase denaturation at the higher temperature

Gelatin. In the Theory, we mention that some organisms are slow gelatin liquefiers. What about an organism might make it a slow gelatin liquefier?

Low affinity for gelatin by gelatinase or limited production of the enzyme are possible reasons for slow gelatin liquefaction.

UV Lab This is not a quantitative exercise. Keeping this in mind, can you see a general trend between bacterial death and UV exposure time?

More exposure results in more cell deaths

Gelatin. If the control is solid and an uninoculated tube is also solid, is it acceptable to read the result before the complete incubation time has elapsed?

No the interpretation at this point would have to be "gelatinase negative' since the tube is solid. However, it is not acceptable to read a negative result early.

HE Agar. Would removing bile salts from HE agar alter the medium's sensitivity or specificity?

Removal of bile salts would alter specificity because organisms that "shouldn't" grow on HE agar would, and additionally some of those might be able to ferment lactose/salicin/sucrose to acid end products. It probably wouldn't affect sensitivity, because you would still be able to detect growth of organisms that "should" grow on HE agar as well as detect the orange-pink color indicative of lactose fermentation

EMB Test. Would removing eosin Y and/or methylene blue from EMB agar alter the medium's sensitivity or specificity? Explain your answer.

Removal of one or both of these would alter specificity because organisms that "shouldn't" grow on EMB would, and additionally some of those might be able to ferment lactose to acid end products. It also would alter sensitivity. Although you would still probably be able to detect growth of organisms that "should" grow on EMB, your ability to detect color changes associated with lactose fermentation would either be lost or hampered, depending on whether both or only one are removed.

HE Agar. All enterics ferment glucose. What would be some consequences of replacing the sugars in HE agar with glucose? What color combinations would you expect to see?

Since all enterics ferment glucose, the only difference in growth would be seen in the enterics that do not ferment lactose, salicin, and/or sucrose - Shigella, Salmonella, and Proteus colonies would appear salmon-pink instead of the expected blue green color. The Salmonella and Proteus colonies might also have a black center, due to sulfur reduction; Shigella colonies would not.

Thermal Death Lab. How much did your plotted and calculated values differ? How do you explain any differences?

The calculated value is more concise (answers vary). Sources of error could be pipetting incorrect volumes, not mixing prior to pipetting, and imprecise timing of samples

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 0.5 McFarland standard may be considerably more dense after 30 minutes.

UV Lab The purpose of this exercise is to demonstrate the comparitive effect of UV on two (or three) bacterial populations. This could have been accomplished without the cardboard cover. Why was the cover used?

The cover was used to show how there can be protection from the harmful UV rays of the sun

HE Agar. In your own words, what are the roles of acid fuchsin and bromothymol blue in HE agar?

They are pH indicators that indicate pH changes

Urea. Typically, urea broth is filter-sterilized because autoclaving breaks down the urea. However, even unsterilized broth produces false-positive results. Why do you think this is true?

This medium is nutrient poor (a low concentration of yeast extract and urea are the carbon and nitrogen sources) Not much can grow in it.

Kirby-Bauer Test. What might be the consequence of pouring the plates 2 mm deep instead of 4 mm 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 diffuses outward. Thus, the zone would be smaller.

Gelatin. Suppose that after 7 days a tube inoculated with a slow liquefier shows no evidence of liquefaction. Is this due to poor sensitivity or poor specificity of the medium? Explain.

This would be due to inadequate sensitivity since the organism is hydrolyzing gelatin, but the test system is incapable of detecting it.

8. Based on the phenol red broth results shown (see image on separate page), which sample(s) would be considered "negative" for fermentation? a. sample on the left b. sample on the right c. sample in the middle d. middle and right samples e. all 3 samples

a

9. In the urease assay results shown (see image on separate page), which sample(s) appears to show a positive result? a. sample on the left b. sample on the right c. sample in the middle d. middle and right samples e. none of the above

c

29. The tests we use in this class for differentiation of species are: A) Qualitative B) Infallible / invariant C) Quantitative D) Recently developed, cutting-edge techniques E) All of the answers show are correct

A

EMB Test Growth on the EMB agar and NA plates was recorded as "good growth,' "poor growth," or "no growth." These are qualitative and at least for the first two, subjective terms. What did you use to establish what constituted "good growth?"

The nutrient agar plate inoculated with the same organisms provided examples of what "good growth" for each organism looks like on a nonselective media. Without these for comparison, it wouldn't be possible to tell if sparse growth on EMB was due to inhibition or was just normal growth for that species.

15. A student performed a Gram-stain, but washed excessively during the "decolorization" step (too much ethanol applied); as a result, after completing the stain procedure, both Gram(+) and Gram(-) cells would likely appear: a. safranin-colored b. clear (un-colored) c. cyan-colored d. violet-colored e. iodine-colored

a

24. What type of material is agar? a. carbohydrates, b. nucleotides, c. proteins, d. lipids, e. synthetic polymer

a

31. The tests we use in this class for differentiation of species directly examine A) phenotype (expressed biochemical activities) B) genotype (contents of DNA) C) colony morphologies D) bacterial pathogenicity E) none of the above

A

Kirby-Bauer Test. Suppose you do this test on a hypothetical Staphylococcus species with the antibiotics penicillin (P 10) and chloramphenicol (C 30). You record zone diameters of 25 mm for the chloramphenicol penicillin disks. Which antibiotic would be more effective against this organism? What does this tell you about comparing zone diameters to each other and the importance of the zone diameter interpretive table?

A zone of 25 mm around penicillin disk indicates resistance to the antibiotic. A 25 mm zone around a chloramphenicol disk indicates susceptibility to the antibiotic. Therefore, chloramphenicol would be the antibiotic of choice. This example demonstrates that the zone diameter sizes are only meaningful when compared to the interpretive chart, not to eachother.

Kirby-Bauer Test. In the optional exercise, what was the purpose of inocultation TSA plates with samples taken from the zones of inhibition?

The purpose was to determine if the antibiotic is bacteriostatic (which should result in growth on the TSA plate) or bacteriocidal (which should show no growth on the TSA plate)

Thermal Death Lab. Is the D value of either organism affected by the size of the population? Explain.

It shouldn't be. D value represents the time it takes to produce a 90% reduction in the population size regardless of the starting size.

Kirby-Bauer Test. The Mueller-Hinton II plates are supposed to be used within a specific time after their preparation 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 effect the ability of the antibiotic to diffuse through the agar. Moisture will cause the antibiotic to spread more rapidly

17. Which of the following can cause false negatives on a diagnostic test? a. Low test sensitivity b. excessive amount of sample applied c. Low test specificity d. reading test results after longer-than-specified incubation e. answers "A" or "B"

a

20. Cornyobacterium xerosis is a Gram-positive, spore-negative bacillus. What color would you expect it to appear, following completion of the spore stain procedure? a. Pink b. Green c. Purple d. Pink and Green e. None of the answers shown

a

24. What is the fundamental, underlying reason why species of bacteria vary with respect to their abilities to ferment different carbon sources? a. DNA - fermentation processes are catalyzed by genetically-encoded enzymes b. None of the answers shown c. the oxygen requirements of the microbe (i.e., anaerobes vs. aerobes, etc.) d. variation of the cellular respiration rate and redox potential e. the abundance of a particular carbohydrate (i.e., ferment lactose if environment is lactose-rich)

a

28. What is the fundamental, underlying reason why species of bacteria vary with respect to their abilities to ferment different carbon sources? a. DNA - fermentation processes are catalyzed by genetically-encoded enzymes b. None of the answers shown c. the oxygen requirements of the microbe (i.e., anaerobes vs. aerobes, etc.) d. variation of the cellular respiration rate and redox potential e. the abundance of a particular carbohydrate (i.e., ferment lactose if environment is lactose-rich)

a

33. Historically, some species were grown in candle jars - in this class, we used a closed tub with a CO2-generator to produce such an environment for incubation of the Columbia CNA plate. Which of the following is NOT accurate regarding "candle- jar" type environments? a. they have no oxygen present (anaerobic) b. they are typically used for culturing species of Streptococci c. they mimic the atmosphere inside respiring mammals d. they can be simulated using "Gas-Pak" containers with reagent packets e. they have increased levels of carbon dioxide

a

34. The tests we use in this class for differentiation of species directly examine: a. phenotype (expressed biochemical activities) b. genotype (contents of DNA) c. colony morphologies d. bacterial pathogenicity e. none of the above

a

35. The tests we use in this class for differentiation of species are: a. Qualitative b. Infallible / invariant c. Quantitative d. Recently developed, cutting-edge techniques e. both highly sensitive and highly specific

a

36. The more potent chemotherapeutic agent would be effective at (micromolar / nanomolar / millimolar ) concentrations; the more toxic chemotherapeutic agent would be deadly at (micromolar / nanomolar / millimolar ) concentrations. a. nanomolar, nanomolar b. micromolar, millimolar c. millimolar, micromolar d. nanomolar, millimolar e. millimolar, millimolar

a

38. In the Kirby-Bauer antibiotic disk sensitivity testing procedure, antibiotic-resistant colonies of bacteria are most likely to be observed near the __________ of the zone of inhibition. a. periphery (outside edge) b.intermediate/middle area c. selective range d. center e. underlayment

a

39. In the Kirby-Bauer antibiotic disk sensitivity testing procedure, what (approximately) does the drug concentration at the edge of the zone of inhibition correspond to? a. Minimum inhibitory concentration b. Lethal dose c. Resistance concentration curve d. Maximum inhibitory concentration e. All of the answers shown

a

13. In the image of the blood agar plate (see image on separate page), which sample appears to have alpha- hemolytic activity? (a) A (b) B (c) C (d) All of the above (e) none of the answers shown are correct

b

19. Smears of bacteria are heat-fixed onto microscope slides; Gram's iodine mordant fixes crystal violet stain; some bacteria perform nitrogen fixation... In these scientific contexts, to "fix" means: a. Neutralize b. Immobilize c. Inoculate d. Manipulate e. Sterilize

b

23. A colony picked directly from ________ media should never be considered a pure culture. a. Blood agar b. Selective media c. Differential media d. All-Purpose media e. Chemically undefined media

b

27. Which of the following is true regarding Selective Media? a. It is used to aid in isolation of less-abundant or slower-growing species of bacteria b. Any/all of these answers can be correct c. It can be produced by adding bacteriostatic agents to all-purpose media d. It can be produced by excluding certain nutrient sources from chemically-defined media e. Colonies that appear isolated could have unseen contaminants and should not be considered pure cultures

b

30. What does it mean when a media is differential? a. It only allows for growth of certain species b. It helps distinguish between different species of bacteria c. It allows growth of all bacterial species d. It prevents the growth of contaminants (i.e. fungal spores, etc) e. None of the answers shown

b

37. In the Kirby-Bauer antibiotic disk sensitivity testing procedure (described & used in exercise 7-3), which of the following processes creates a gradient of antibiotic concentration? a. electrical current applied to the media (external mechanical processes) b. passive diffusion (random movement of substances through semi-solid media) c. bacterial flagellar motion (biological processes within the media) d. active transport (flow of substances across membrane, up (or against) the concentration gradient) e. osmosis (flow of substances across membrane, down the concentration gradient)

b

12. Which of the following statements does not belong in the results section of a scientific paper, or a formal lab report? a. All of these choices actually are appropriate b. The pH of the control sample was 6, while the pH of the fermentation test sample was 2.5. c. Bubbles were present in the mild inoculated with starter yogurt, which proved fermentation occured. d. A Gram stain of the unknown bacterial species revealed purple-colored cocci arranged in pairs. e. There were no differences in turbidity among the control and the experimental samples.

c

25. Which of the following would be most likely to cause false positives on a diagnostic test? a. low test sensitivity b. insufficient amount of sample applied c. low test specificity d. proteins in test reagent were destroyed e. answers "a" and "b"

c

26. Suppose a student testing an unknown culture performs the urease test according to the protocol; after the specified incubation, the media has not changed color... Which of the following conclusions would be most proper / justified? a. This result is sufficient to allow species identification - the unknown must be P. vulgaris b. No urea hydrolysis occurred in this test; there was zero urease activity c. No urease activity was observed d. The bacteria tested does not have a urease gene e. This result is positive, indicative of urease activity

c

26. Suppose a student testing an unknown culture performs the urease test according to the protocol; after the specified incubation, the media has not changed color... Which of the following conclusions would be most proper / justified? a. This result is sufficient to allow species identification - the unknown must be S. saprophyticus b. No urea hydrolysis occurred in this test; there was zero urease activity c. No urease activity was observed d. The bacteria tested does not have a urease gene e. This result is positive, indicative of urease activity

c

29. Which of the following experimental results should be considered most conclusive (and/or, most reliable)? a. Urease negative b. no gelatinase activty c. Beta hemolytic d. Gamma hemolytic e. unable to ferment glucose (PR-Glucose negative)

c

31. Which of the following is responsible for the differential nature of Columbia CNA agar? a. agar b. colistin and nalidixic acid c. Sheep blood d. carbon dioxide e. tryptone

c

9. Which of the following would be most likely to cause false positives on a diagnostic test? a. low test sensitivity b. insufficient amount of sample applied c. low test specificity d. proteins in test reagent were destroyed e. answers "a" or "b"

c

10. Based on the antibiotic disc diffusion test results shown (plate "Z" - separate page): Which disc has the largest zone of inhibition (which shows the most microbial inhibition)? a. top left b. top right c. bottom left d. bottom right e. not enough information to determine

d

16. Insufficient decolorization (too little washing with ethanol) while gram-staining a specimen could result in both Gram(+) and Gram(-) organisms appearing what color, upon completion of the Gram stain? a. safranin-colored b. clear (un-colored) c. cyan-colored d. violet-colored e. iodine-colored

d

11. Based on the antibiotic disc diffusion test results shown (plate "Z" - separate page): The drug on which disc would be the most appropriate drug for treating the infection in humans? a. top left b. top right c. bottom left d. bottom right e. not enough information to determine

e

21. Which of the following might cause a bacterial smear to appear spore-free when stained according to the spore stain procedure (Schaeffer-Fulton protocol) we used in class? a. primary stain was omitted b. no spore-forming bacterial species present c. no steam treatment (heating) step performed d. no sporogenesis has occurred e. any/all of the answers shown could be true

e

22. Bacterial spores are typically resistant to: a. typical cell-staining procedures b. desiccation c. very cold conditions d. heat e. all of the answers shown

e

32. Historically, some species were grown in candle jars - in this class, we used a closed tub with a CO2-generator to produce such an environment for incubation of the Columbia CNA plate. Which of the following types of bacteria likely could survive in "candle-jar" type environments? a. aerobes b. microaerophiles c. capnophiles d. facultative aerobes e. All of the types of bacteria listed could likely survive in a candle-jar-type environment

e

40. Which of the following could potentially cause a false species identification when using a dichotomous key? a. genetic changes (mutation / transformation / conjugation / transduction event(s)) previously occurred b. the test specimen / culture contained multiple species c. improper dichotomous key used d. a test result was misread e. any of the answers shown

e