((필독2. site)) Answers to the Old Final Exam in the Manual (Appendix Z)

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Bacteriology 102: Answers to the Old Final Exam in the Manual (Appendix Z)

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Page Last Modified: 5/11/02

Also see the "Things to Think About."

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I. Multiple True/False (53 points). In the blank by each statement, place a + for a true statement or a O for a false statement. There can be any number of + or O statements. Please do not change or qualify the wording of any statement in any way. Each is either true or false as stated. (1/2 point for each blank.)

Example: Without exception, these terms – as they apply to microbiology – are always plural terms, not to be used in the singular:

  +   media
  +   bacteria
  O   Bacillus

1. The following procedures are consistent with what we observe to be proper aseptic technique:

  O   Making loop transfers of a broth culture to slides, flaming the loop at the very beginning of the procedure and only again after the last smear is prepared.
  O   Leaving tubes open and vertical in a test tube rack while performing transfers between them.
  +   Wiping the entire work area with disinfectant before and after working with cultures.
  O   Flaming only the tip (end) of the needle or loop rather than the entire wire.

2. In the preparation of smears from any bacterial culture,

  O   a drop of water is always placed on the slide first.
  O   either thick or thin smears may be prepared for a gram stain; neither will have any effect on how effectively the smear is stained or decolorized.
  O   a cover slip should be placed on the completed smear.

3. Things which are considered differential include

  +   the acid-fast staining procedure.
  +   the endospore staining procedure.
  O   the inclusion of an antibiotic in a medium.
  +   the inclusion of a Durham tube in a sugar-containing medium.

4. The following may be achieved with the use of the microscopes we used in lab:

  O   observing colonial characteristics
  +   noting the difference between Brownian motion and true motility
  O   observing individual bacterial flagella in a wet mount
  O   observing individual bacteriophages in a wet mount

5. When considering the magnifying capabilities of our microscopes, one should realize that

  +   magnification means little without suitable resolution.
  O   the total magnification is calculated by adding the magnification of the ocular lens and the magnification of the objective lens.
  O   one must decrease the light intensity when going from one magnification to a higher one.

6. Tests or observations which should be made on young cultures (18-24 hours old at the most) include determination of

  +   the gram reaction and shape of the cells.
  +   the fermentation reactions in Kligler's Iron Agar (KIA).
  O   the methyl red reaction.
  +   motility by means of a wet mount of a broth culture.
  O   whether or not an organism has produced endospores.

7. Organisms which utilize organic compounds as their sole sources of carbon and energy may be therefore termed:

  +   chemotrophs
  O   autotrophs
  O   auxotrophs
  +   heterotrophs

8. Facultative anaerobes and aerotolerant anaerobes

  +   obtain energy by fermentation when growing under anaerobic conditions.
  +   can be differentiated by the catalase test.
  O   cannot be differentiated from each other in the standard test for oxygen relationship as each shows the same growth pattern throughout the tube of Thioglycollate Medium.

9. Petri dishes are incubated in an inverted position in order to prevent

  O   condensed moisture from dropping onto the colonies, thus contaminating them directly.
  +   condensed moisture from dropping onto the colonies, thus causing them to run together.
  O   the colonies from sinking into the medium.

10. You have broth cultures of the two mating types of Escherichia coli we used in Experiment 8: The Hfr strain is thr⁺met^– and the F^– strain is thr^–met⁺. You also have a Minimal Medium (MM) formulated for growth of prototrophic strains of E. coli.

  O   After undergoing conjugation and recombination, an F^– cell becomes an Hfr cell.
  O   During conjugation, each strain donates DNA to the other.
  O   An Hfr cell will form a colony on MM if the medium has been supplemented with threonine.
  +   An F^– cell will form a colony on MM if it has received the threonine gene from an Hfr cell.
  +   A "back-mutation" for either strain should allow growth on MM.
  O   Hfr and F^– cells always possess one or more mutations which make them auxotrophs.

11. A change in genotype

  +   can be accomplished by mutation.
  +   can be accomplished by recombination.
  O   will not consequently change the phenotype, as the latter is changed only by altering environmental conditions.

12. The following characteristics are possessed by all "enterics" (i.e., members of the family Enterobacteriaceae):

  +   gram-negative rods
  +   glucose-fermenting and catalase-positive
  O   lactose-fermenting
  O   associated only with an intestinal habitat

13. The following characteristics are possessed by all "lactics" (i.e., the lactic acid bacteria):

  O   gram-positive rods
  +   glucose-fermenting and catalase-negative
  O   lactose-fermenting
  +   resistant to chemicals which halt respiration

14. The following apply to the production of sauerkraut:

  +   "floral succession"
  +   "wild fermentation"
  +   lactic acid bacteria
  O   lactose fermentation

15. Extracellular enzymes produced by microorganisms

  +   are needed for an organism to overcome the selective nature of the medium we used to isolate Streptomyces.
  +   include amylase and coagulase.
  O   are a type of antibiotic.

16. The antibiotic disc sensitivity test

  O   shows a zone of inhibition only for those organisms which are sensitive to an antibiotic.
  +   may be used in medicine to choose a particular antibiotic to use in order to stop an infection in a patient.
  O   induces antibiotic-sensitive bacterial cells to mutate to become resistant to the antibiotic.
  O   was used in our lab to test an organism's ability to produce an antibiotic.

17. The tests for antibiotic production and starch hydrolysis are similar in that

  +   the tests are concerned with extracellular products of the organism.
  O   the organisms to be tested are streaked on the plates to get isolated colonies.
  +   the organisms to be tested are inoculated and then allowed to incubate for two or more days before anything further is done to the plate.
  O   an iodine solution is added to the plate after incubation.
  +   a positive result is where nothing is seen immediately adjacent to the culture being tested.

18. In the bacteriological examination of water, use is made of indicator organisms. An indicator organism is

  O   one which indicates the normal, uncontaminated condition of a particular environment.
  +   one which should not pose a health risk for laboratory workers.
  +   not difficult to detect in the laboratory; the enrichment and isolation procedures are designed to exploit certain properties of these organisms.
  +   likely to be E. coli if one suspects fecal contamination of food or water.

19. When enumerating organisms by means of the MPN method, a positive result

  +   for coliforms would be indicated by growth and gas in the appropriate selective-differential broth medium.
  O   for coliforms means that we have a pure culture of a particular coliform in the tube.
  +   for a "total count" would be indicated simply by growth in an all-purpose broth medium.
  +   for bacteriophages would be indicated by no growth in an all-purpose broth medium inoculated with the appropriate host organism.

20. The following is/are true of procedures used to isolate bacteria from natural sources:

  O   One can expect to find any organism anywhere, as these bacteria really get around!
  O   The media utilized are always selective media.
  +   A selective broth enrichment may not be necessary if the desired organisms are in a high enough number for the sample to be plated directly.
  O   Plate counts which are made of an enrichment of a natural sample can be used to estimate the number of CFUs per gram or ml of the sample.

21. You have a lake water sample which contains 4 X 10⁴ CFUs/ml. Which of the following plate inoculations, if any, should result in 40 colonies?

  O   one ml of a 10^–2 dilution of the sample
  +   one-tenth ml of a 10^–2 dilution of the sample
  +   one ml of a 10^–3 dilution of the sample
  O   one-tenth ml of a 10^–3 dilution of the sample

22. A 1/100 dilution of a soil sample can be achieved

  +   by making two 1/10 serial (consecutive) dilutions of the soil.
  +   by taking one gram of soil and adding it to 99 ml of diluent.
  O   by taking one-tenth gram of soil and adding it to 9 ml of diluent.

23. Hemolysis

  +   refers to a change observed in the blood around colonies growing on Blood Agar.
  +   is called "gamma hemolysis" if it is not seen; in this situation, "gamma" simply means "negative."
  O   is a useful characteristic which can be used to differentiate Streptococcus from other organisms.

24. When one puts a layer of mineral oil on an inoculated tube of Lysine Decarboxylase Broth, or when one stoppers a filled bottle of an enrichment for purple non-sulfur bacteria,

  O   One expects anaerobic conditions to be achieved by the activities of only strictly aerobic bacteria in the inoculum.
  O   One expects anaerobic conditions to be achieved by bacteria performing anaerobic respiration.
  +   The mineral oil or stopper is simply meant to prevent any more air from getting into the medium and allowing bacterial activities not associated with anaerobic metabolism.

25. A candle jar

  +   provides an elevated level of carbon dioxide which is helpful to the growth of certain organisms.
  O   provides a lighted environment suitable for the growth of photosynthetic bacteria.
  O   can be used in the cultivation of strictly anaerobic bacteria.

26. The following types of soil organisms become potential colony-forming units (CFUs) when a dilution of non-heat-shocked soil is inoculated onto an all-purpose plating medium:

  +   endospores of Bacillus and Clostridium
  +   vegetative cells of Bacillus and Clostridium
  +   vegetative cells of Streptomyces and molds
  +   reproductive spores of Streptomyces and molds
  +   vegetative cells of a wide variety of other organisms

27. When endospores give rise to colonies:

  +   An endospore inoculated onto the plate must undergo germination and outgrowth to become a vegetative cell.
  O   As the cells divide and the colony expands outwardly, endospores which have formed on the edge of the colony migrate toward the center of the colony.
  O   An endospore stain of a colony reveals endospore(s) originally inoculated onto the plate.
  O   One can expect the endospores to be able to divide, forming colonies of endospores.

28. Nitrogen-fixing bacteria

  O   are those bacteria which reduce nitrate to nitrite and eventually to nitrogen gas.
  O   have air as their main habitat; these are the organisms which are found on air-exposure plates.
  +   can help to support the growth of non-nitrogen-fixing bacteria when both are present in a medium which was not formulated with any nitrogen compound.

II. MATCHING (15 points). Place the letter of the correct item from column b in the blank by each statement in column a. (1 point for each blank).

a   G N   Two extracellular products associated with Streptomyces   I L   Two items which will assist in the isolation of purple non-sulfur photosynthetic bacteria   H J   Two items which will assist in the isolation of Bacillus   H I   Two items which will assist in the isolation of Clostridium   F J   Two items which will assist in the isolation of the "lactics"   D   Decolorizing agent in the gram stain   C   Decolorizing agent in the acid-fast stain   E   Used to help in the specific identification of a Salmonella isolate   A   Produced by "lactics"; helps to preserve certain foods   K   pH-related reaction associated with deamination or decarboxylation of amino acids

b A. acid B. bacteriophages C. acid alcohol D. alcohol acetone E. antibodies F. sodium azide G. geosmins H. heat-shocking I. anaerobic incubation J. aerobic incubation K. alkaline L. light M. bloom N. none of the above

III. SHORT ANSWER (39 points).

1. (4 points) When one is preparing media, one can usually count on trace elements being present in the various medium components such that one usually does not have to add them specifically. A growth factor must be included for the growth of any organism which cannot synthesize it from the other components of the medium. As an example, one which is involved in the transport of iron into a bacterial cell is a siderophore. An undefined mixture of nutrients is conveniently provided with the addition of peptone.

2. (3 points) List three reasons why one would wish to streak a plate from a culture for isolated colonies.

See Experiment 7 (first page). Quantitation is definitely not a reason!

3. (6 points) You have been asked to isolate the following type of bacterium from a soil sample:

• endospore-producing
  
• strictly anaerobic
  
• resistant to streptomycin
  
• ferments the sugar sucrose
  
• grows at normal incubation temperatures
  (25-37°C)

So you say, "No problem. I took Bacteriology 102 at UW-Madison." You then decide to plate a suspension of soil directly rather than first utilize a broth enrichment.

a. What pre-treatment of the soil suspension may you wish to perform?

Heating in hot water bath (over 70 degrees C)

b. What three significant selective and/or differential medium components would you include in your plating medium which will assist (with proper incubation conditions) in the efficient isolation of your organism?

streptomycin, sucrose and a pH indicator

c. Under what conditions would you incubate your plates?

anaerobically!

d. Once you have obtained isolated colonies of the desired type, how would you prove whether or not you have strict anaerobes? Or, have you shown that already?

Run oxygen relationship test or catalase test or try to grow aerobically.

4. (4 points) With numbers (1-4; 1 is the most important), list the priorities of the following procedures with regard to the identification of bacterial cultures:

  2   Determination of oxygen relationship.
  4   Determination of whether or not the organism can produce a siderophore which other bacteria can utilize.
  1   Determination of gram reaction and cellular morphology and arrangement.
  3   Determination of whether or not lactose is fermented.

5. (4 points) With numbers (1-4; 1 is the "best"), arrange the following media according to how you think they would support the growth of a variety of different bacteria.

  3   MacConkey Agar
  4   MacConkey Agar + sodium azide
  2   Nutrient Agar
  1   Nutrient Agar + yeast extract

6. DEFINITIONS (18 points). Briefly, yet effectively, define SIX of the following seven terms, placing an X by the term not defined.

a. acid-fast
ability of a cell to retain the primary stain (in the acid-fast staining procedure) when treated with an acidic decolorizer

b. phototaxis
movement of a cell in response to light

c. phage-typing
use of phages to help characterize a bacterial culture – i.e., whether or not certain known phages will cause lysis of the culture

d. bacteriostatic agent
agent which inhibits (rather than kills) bacteria

e. starter culture
culture added to the raw food product to effect a fermentation leading to the final food product

f. streptococcus (as opposed to Streptococcus)
cocci (spherical cells) arranged in a chain

IV. PROBLEMS (18 points).

1. (2 points) Determine the generation time (in hours or minutes) for this culture. (By careful observation of the graph, you will see that formulas are unnecessary!)

During the exponential phase, one can see that the culture doubles every 15 minutes.

2. You added 10 ml of a lake water sample to 90 ml of sterile diluent. After mixing thoroughly, two, serial 1/100 dilutions were made. From each of these three dilutions, plates of Plate Count Agar and tubes of Lactose Lauryl Tryptose Broth (LLTB) were inoculated as shown in the table below. After appropriate incubation, the plates and tubes were checked, and the results are tabulated below. Subsequent inoculations into BGLB and EC Broth were appropriately made and incubated; their results are also shown below. (TNTC=too numerous to count.)

Dilution of the water sample	1st (=10–1)		2nd (=10–3)		3rd (=10–5)
Amount inoculated into each of 2 plates of PCA and 3 tubes of LLTB	1 ml	0.1 ml	1 ml	0.1 ml	1 ml	0.1 ml
"plated dilution"	10–1	10–2	10–3	10–4	10–5	10–6
"dilution factor"	101	102	103	104	105	106
colony counts on plates	TNTC	TNTC	417/425	55 / 59	8 / 5	0 / 1
no. of positive LLTB tubes	3	3	3	1	0	0
no. of positive BGLB tubes	3	3	2	1	0	0
no. of positive EC Broth tubes	3	2	2	0	0	0

a. (4 points) Determine the number of colony-forming units per ml of the lake water.    5.7 X 10⁵/ml

b. (3 points) Determine the confirmed, most probable number of total coliforms per ml of the lake water.    Using BGLB Broth: 1.5 X 10³/ml

3. You are given a 10^–3 dilution of a suspension of bacteriophage able to lyse E. coli strain B. You then make two, successive 1/100 dilutions of this dilution. From the last dilution you make, 0.1 ml is added to a tube containing 4.8 ml of melted top agar. One-tenth ml of a young culture of host organism is also added to the same tube of top agar. After mixing the tube of top agar is poured onto a plate of bottom agar. After incubation, 44 plaques are counted.

a. (0 points) To help you organize your data, the following can be filled out:

Initial dilution of phage:    10^–3
Subsequent dilutions of phage:    10^–2 and 10^–2
Amount inoculated from last dilution:    0.1 ml
The above give us a plated dilution of 10^–8; therefore dilution factor = 10⁸.
Does the amount of top agar matter in the calculations?    NO
Does the amount of host culture matter in the calculations?    NO

b. (3 points) Determine the number of plaque-forming units (PFUs) per ml of the original, undiluted bacteriophage suspension.    44 X 10⁸ = 4.4 X 10⁹ PFUs/ml

4. (3 points) You dilute a sample of cheese and plate it out onto Vogel-Johnson Agar. After incubation, you find 120 black colonies on a plate inoculated with 0.1 ml of a 10^–1 dilution. NOW, you pick 30 black colonies at random, and you find that 20 of them test out to be gram-positive, catalase-positive cocci arranged in clusters, the typical "staphylococcus" arrangement. Of these 20 isolates, you run the coagulase test, and you find that 15 of them are positive. How many Staphylococcus aureus CFUs were there per gram of the cheese?
120 X 10² = 1.2 X 10⁴ black CFUs/gram.
Therefore, as half of the sampled colonies tested out to be S. aureus, we have 6 X 10³ S. aureus CFUs/gram.

5. (3 points) You plate out 0.2 ml of tap water onto a plate of Plate Count Agar, and 46 colonies arise after incubation. How many CFUs were there per ml of the tap water?
If 0.2 ml (=1/5 ml) yields 46 colonies, then there were (46X5=) 230 CFUs in one ml of the water. (If we use the standard Appendix C formulas, then the dilution factor would be 5.)

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Page last modified on 5/11/02 at 3:30 PM, CDT. John Lindquist, Department of Bacteriology University of Wisconsin – Madison

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