I.  MULTIPLE TRUE/FALSE (44 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. 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.)

1.  Regarding the terms cells, colony-forming units and colonies:

  +   A "colony-forming unit" (CFU) is microscopic and can be made up of one or more cells.
  +   A "colony" is visible to the naked eye and arises from a "colony-forming unit" during the incubation of the plate.
  O   The terms "colony-forming unit" and "colony" can always be used interchangeably.
  O   In the quantitation of bacteria, it is important to know the number of cells in the average-sized colony.
  +   Endospores can be "colony-forming units."
  O   A "plaque" is the bacteriophage equivalent of a "colony-forming unit."

2.  A sample of hamburger was diluted to 10^–2 (that is, 1/100). One ml of this dilution was plated, and 300 colonies were counted after incubation.

  O   When one prepares a 1/100 dilution of a sample, there is always a ratio of 1 part of sample to 100 parts of diluent.
  +   To determine the number of CFUs which had been present per gram of the undiluted hamburger sample, one must multiply 300 by 10², rather than by 10^–2.
  +   Theoretically, plating one-tenth ml (instead of one ml) of the 10–2 dilution would give rise to 30 colonies.
  +   Plating one ml of a 10^–2 dilution of the hamburger is theoretically equivalent to plating one-tenth ml of a 10^–1 dilution of the same sample.
  +   Plating one ml of a 10^–2 dilution of the hamburger is theoretically equivalent to plating 10^–2 gram (i.e., 0.01 gram) of the undiluted hamburger.

3.  The following are consistent with good "aseptic technique":

  O   Having one person hold the tubes while another person makes transfers between them.
  O   Leaving culture tubes unplugged and upright in the test tube rack while making inoculations between them.
  O   Placing the top lids of petri dishes on the table top while making transfers or observing colonies.
  O   Touching the wire of a recently-flame-sterilized inoculating loop to your hand to check if it is cool enough to touch the loop to the inoculum.
  O   Pouring plates from a bottle of melted agar medium which was not wiped off after being taken from the water bath, as we expect the water in a 50°C water bath to be sterile.
  O   Doing the slide catalase test on an uncovered slide and then observing it closely for bubbles.

4.  The capsule

  +   can inhibit proper decolorization of a gram-negative cell in the gram-staining procedure.
  O   is an integral layer of the cell envelope of both gram-positive and gram-negative bacteria.
  O   takes up the India ink in the "capsule stain" we did in lab and is thus stained a dark color.

5.  Cells of Mycobacterium are termed "acid-fast" organisms.

  O   "Acid-fast" refers to the rapid production of acid from the fermentation of sugars.
  +   Cells successfully stained with carbol fuchsin hold the stain "fast" and are therefore not decolorized when treated with the acidic decolorizing agent.
  O   The principles underlying the gram staining procedure (why it works the way it does) are the same as those for the acid-fast staining procedure.

6.  A test we did in Experiment 5.1 involved the use of Thioglycollate Medium (shown at right):

  +   This test is designed to determine the "oxygen relationship" of chemoheterotrophic bacteria that can grow well in this medium.
  O   An organism which can grow anaerobically in the medium is one which can obtain energy by the process of anaerobic respiration.
  +   An organism capable of aerobic respiration would be able to grow in the presence of oxygen and also give a catalase-positive reaction.
  +   If the organism shown in Tube #1 can reduce nitrate, and if we were to add nitrate to the medium, we would expect the organism to grow throughout the tube instead of just on top (as shown).

7.  In two experiments involving bacterial quantitation early in the semester, you were provided with a number of tubes of melted Plate Count Agar (PCA) in a 50°C water bath for use in preparing plates by the "pour-plate" method.

  O   Plate Count Agar is an example of a medium that can support the growth of all known kinds and types of bacteria.
  +   We would expect (theoretically) to obtain the same number of colonies on our plates if the tubes each contained twice as much medium.
  O   If you were to keep a tube out at room temperature too long and the medium solidifies, you can put it back into the 50°C water bath and the medium will become liquid again.
  O   After pouring the plates, it is best to keep the petri plate lids off so the medium will cool off and solidify faster.

8.  For growth curves in general:

  O   One plots the CFUs/ml for each time point and then draws a line such that all of the dots are on the line and are thus "connected."
  O   The growth curve will always be the same for any particular species of bacteria, no matter what medium or incubation conditions we use.
  +   If a culture doubles its population every 15 minutes, the growth rate is therefore four generations per hour.
  +   If an actively-growing culture has 1.0 X 10⁶ CFUs/ml at 8:00 AM and 4.0 X 10⁶ CFUs/ml at 9:00 AM, the generation time is 30 minutes.

9.  For the Motility Medium tubes shown at right: Note that Tube #1 is an uninoculated control, #2 and #3 show a visible "stab line," and #3 and #4 are both cloudy throughout the medium. Therefore, the following tubes are considered "positive" for motility:

  O   Tube #2
  +   Tube #3
  +   Tube #4

10.  The genotype possessed by a cell can be changed by

  +   mutation.
  +   recombination.
  O   altering environmental conditions in which the organisms are being incubated (temperature, oxygen availability, etc.).

11.  To isolate a certain kind of organism from a natural source, using the enrichment and isolation principles we learned about in our experiments:

  O   It is best to utilize an all-purpose medium to isolate as many different kinds of bacteria as possible. Then we can study all of the colonies obtained and choose which ones we want to continue with.
  O   We always wish to compare our results to a general key to find out if we isolated the correct strains from the sample and got the correct determination of CFUs per gram.
  O   We always try to duplicate the original habitat of the organisms in the laboratory as much as possible.
  O   We expect each different kind of colony on a plate to represent a different genus.

12.  Selective media include the following:

  O   Blood Agar, as only hemolytic bacteria will grow on Blood Agar.
  +   A medium in which all carbon compounds are excluded.
  O   A medium which allows us to differentiate between glucose fermenters and those which do not ferment glucose.

13.  In the isolation of Streptomyces from soil,

  +   Organisms without extracellular enzymes would find it difficult or impossible to grow and produce colonies on the initial isolation medium we used.
  O   "Penassay Agar" selected specifically for antibiotic-producing bacteria and inhibited others.
  +   When we picked colonies off of the initial isolation medium, we streaked onto an all-purpose medium, as we hoped to obtain pure isolates or (at least) isolates that could be easily distinguished and separated from any contaminants.
  O   Streptomyces is an antibiotic.

14.  Regarding antibiotics:

  O   For an organism to be considered "susceptible" to a particular antibiotic in the antibiotic disc sensitivity test, the zone of inhibition seen around a disc of the antibiotic can be of any size – just as long as some inhibition is seen on the plate.
  O   An antibiotic-resistant organism is always a mutant, as all bacteria are normally antibiotic-sensitive.
  +   Antibiotics can be used as selective agents in bacteriological media.
  O   Antibiotics and antibodies are the same thing; the two terms are interchangeable.
  O   Antibiotics are bacteria that inhibit certain other bacteria.

15.  When looking for purple non-sulfur photosynthetic bacteria, we had to make use of certain medium and incubation requirements in order to isolate them most efficiently, as these organisms

  O   do not utilize sulfur for any purpose.
  +   produce easily-distinguished pigmented colonies when the plates are incubated under anaerobic conditions and in the light.
  +   produce non-pigmented colonies if the plates are incubated under aerobic conditions, and the organisms would unfortunately be virtually indistinguishable from chemotrophs.
  +   may be overrun (crowded out) by respiring chemotrophs if the plates are incubated under aerobic conditions.
  +   may be overrun (crowded out) by fermenting chemotrophs if incubated under anaerobic conditions on a medium containing glucose as a carbon source.

16.  When we heat-shock a suspension of soil, we expect

  O   immediate production of endospores by vegetative cells of Bacillus.
  +   to kill reproductive spores of such soil organisms as Streptomyces and molds.
  +   to kill vegetative cells of non-endosporeforming microorganisms.
  +   to kill vegetative cells of Bacillus.

17.  You are studying a gram stain of a black colony obtained from a plate of Vogel-Johnson Agar, and your partner decided to make a wet mount of it. After finishing observations, you and your partner both discard your slides into the red bucket on the stage (designated for broken glass and stained smears). After awhile, you decide to retrieve your slide and – while looking for it in the red bucket – you cut your hand on the cover slip that came off your partner's wet mount. The next day you have a nasty infection on your hand.

  +   This episode helps to confirm the rule that wet mounts must be discarded only into the disinfectant and not into the red bucket with the stained smears.
  O   Any black colony on Vogel-Johnson Agar must be Staphylococcus aureus, and further steps to identify it would really be unnecessary.
  O   Just doing a catalase test would confirm a black colony on Vogel-Johnson Agar to be Staphylococcus aureus.

18.  In the production of fermented milk products, such as yogurt and the "pseudo-yogurt" we made in lab:

  O   We can use any species of lactic acid bacteria, as they all ferment lactose.
  O   We depend on bacteria already present in the milk to participate in the production.
  O   Slime produced by the organisms was the reason for the solid consistency of the product.
  +   We noticed in our titrations that more base (NaOH) was used to neutralize the milk after incubation than before.

19.  Regarding the group of organisms known as the lactic acid bacteria (the "lactics"):

  O   They are all strict anaerobes.
  +   They are gram-positive rods and cocci that form substantial amounts of lactic acid from fermentation of sugars.
  O   They include the enteric group of bacteria.
  O   All lactic acid bacteria ferment lactose and produce slime from sucrose.

20.  Regarding the group of organisms known as the enteric bacteria (the "enterics"):

  O   They are found only in the intestinal tract.
  +   They are all facultative anaerobes.
  O   They all ferment glucose and lactose.
  O   The group includes Neisseria and Pseudomonas.
  +   The group includes Salmonella and E. coli.

21.  Enrichment and isolation procedures for the "enterics"

  +   are aided by media which contain one or more selective agents that inhibit gram-positive bacteria.
  +   may involve media which contain lactose as the only fermentable sugar, even though many enterics do not ferment lactose.
  +   often results in the isolation of Pseudomonas.

22.  Among the requirements of a good "indicator organism" is/are the following:

  O   the ability to cause the problem being examined.
  +   the ease in which it is detected.
  O   the ability to remain in the contaminated environment indefinitely.

REMINDER: Be sure you indicated + for true and O for false. Graders only look at these answers and do not consider any modifications or additions you may have written.

II.  MATCHING (14 points).

1.  Place the letter of the best, most appropriate item from the second column in each blank by each statement in the first column. Only one letter per blank. Any letter may be used any number of times or not at all. Some statements in the first column have more than one blank. (One-half point for each blank.)

2.  Place the letter of the best, most appropriate organism from the second column in each blank by each organism in the first column. Only one letter per blank. Any letter may be used only once. (One-half point for each blank.)

3.  Place the letter of the best, most appropriate item from the second column in each blank by each statement in the first column. Only one letter per blank. Any letter may be used any number of times or not at all. (One-half point for each blank.)

III.  SHORT ANSWER (25 points).

1.  (2 points) Give one reason why a cell which has a gram-positive type of cell envelope may give a gram-negative staining reaction – other than too much alcohol-acetone was used or some other error in technique. (Saying "gram-variability" is too general; a further explanation is needed.)

The cell wall/envelope can weaken during prolonged incubation of the culture, and this can allow easier decolorization with the alcohol-acetone in the gram-stain procedure.

2.  DEFINITIONS (12 points) Briefly and effectively define four of the following five terms. (If you answer all five, we can only then grade the first four.) Place an X by the term not defined.

• bacillus (the general term; not the genus Bacillus)
  Rod-shaped cell.
  
• antibiotic
  Chemical produced by a microorganism which will kill or inhibit certain other microorganisms.
  
• starter culture
  Culture which is added [usually to a raw food product] to cause a desired fermentation.
  
• chemoorganotroph
  Organism which obtains energy from chemical reactions, oxidizing (deriving electrons from) organic compounds.
  
• any bacteriological term of your choice that you feel has been under-represented in this exam.
  (Splammo may not be an acceptable term. Best to check with your instructor.)
  

3.  (2 points) The same organism was inoculated into these two Succinate Agar tubes. The tube on the left was incubated for several days in the light, and the one on the right was incubated for several days in the dark. Note that there is surface growth in each tube, and the tube on the left has heavy, red-pigmented anaerobic growth.

• The best term to describe this organism would be facultative phototroph. The answer would not be "facultative anaerobe," as oxygen relationship categories do not reflect anaerobic growth due to phototrophy (recall Exp. 5.1).
  
• The red color is due to photosynthetic pigments.
  
  

4.  (2 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 glucose fermentation and catalase reactions.
  4   Determination of the generation time of a typical culture.
  1   Determination of gram reaction and the shape and arrangement of the cells.
  3   Determination of lactose fermentation and amylase reactions.

5.  (2 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   Heart Infusion Agar
  1   Heart Infusion Agar + yeast extract

6.  (2 points) With numbers (1-4; 1 is the first), arrange the following procedures which are involved in the isolation and identification of enterics:

  1   Inoculate source material into the selective enrichment medium.
  2   Streak plate for isolated colonies.
  3   Inoculate Kligler Iron Agar (KIA) tubes with pure cultures.
  4   Eliminate any tubes which show the obvious presence of non-enterics (such as Pseudomonas) and run a number of different biochemical tests on the various cultures.

7.  (3 points) You have cultures of five organisms as listed below. However, the labels of the tubes have come off and you need to re-label the tubes correctly! First, you consider the various reactions you know for the organisms in question:

• The results obtained from what one specific laboratory procedure will differentiate Bacillus and Staphylococcus from each other and also from the remaining three genera?  Gram stain! (This gives the gram reaction and also allows observation of morphology and arrangement.)
  
• The remaining three genera can each be distinguished by the result of one test. Indicate a test (just the test, not the reaction) which will give the distinguishing reaction for each of the organisms:
  Enterobacter:  lactose fermentation
  Morganella:  phenylalanine deamination
  Pseudomonas:  glucose fermentation
  

IV.  PROBLEMS, PROBLEMS, PROBLEMS (17 points).

1.  (2 points) The same dilution can be obtained in each of the following situations:

• The addition of 1 ml of a sample to 4 ml of sterile diluent.
  
  
• The addition of 11 ml of the same sample to 44 ml of diluent.
  
  
• The addition of 10 ml of the same sample to 40 ml of diluent.

These each add one part sample to 4 parts diluent, resulting in a 1/5 dilution.

2.  (1 point) The inoculation of 1 ml of a 10^–3 dilution of hamburger is theoretically equivalent to the inoculation of 10^–3 gram of the undiluted hamburger.  Remember how the so-called "plated dilution" always represents the amount of sample that is plated.

3.  A sample of lake water was diluted and inoculated into plates of Plate Count Agar (PCA) and tubes of Lactose Lauryl Tryptose Broth (with Durham tubes). After incubation, the results were obtained as indicated on the following table. (Note: Each of the three columns of results on this table shows the observations for two plates and three tubes.)

• (3 points) Determine the number of CFUs per ml of the lake water.
  
  

  dilutions made	X	amount inoculated	=	"plated dilution"
  10–2	X	1	=	10–2
  dilution factor	X	no. colonies	=	no. CFUs/ml
  102	X	86	=	8.6 X 103

  
  
• (2 points) Determine the presumptive, most probable no. of coliforms per ml of the lake water.
  

  As growth and gas constitute a positive reaction in this particular test, the results for tubes showing gas – 3,2,1 – corresponds to 1.5 in the MPN table. Therefore, 1.5 X the dilution factor represented by the middle set of tubes will give the most probable number per ml: 1.5 X 10² or 150.

4.  (1 point) Consider this situation: 20 Hfr cells are mixed with 20 F^– cells, and 8 pairs of Hfr and F^– cells undergo conjugation. If 2 F^– cells each incorporate DNA from an Hfr cell into its chromosome, the "recombination frequency" would then be: 10%.

"Frequency" means the number who did divided by the number who can, so the number of F^– cells that actually underwent recombination (2) is divided by the total number of F^– cells (20). The recombination frequency is therefore 2/20 or 10%.

5.  (3 points) Exactly one-half of a 100-gram chunk of cheese was mixed with 450 ml of sterile diluent, and the other half was placed in the refrigerator for future consumption. Two successive 1/100 dilutions were then made. From the last (most dilute) dilution, one-tenth ml was plated onto each of 2 plates of PCA. After incubation, 48 colonies were counted on one plate and 52 were counted on the other.

Calculate the number of CFUs we expect to be present in the entire, other half of the chunk of cheese which is still in the refrigerator and waiting to be eaten.

Note that the initial dilution – 50 grams cheese into 450 ml diluent – is a 1/10 dilution.

6.  You are given two strains of E. coli – strain "A" and "B" – and a 5 ml tube of the phage P1. Given these materials, you proceed to determine the concentration of phage in the tube by the following methods:

First you make eight consecutive dilutions of the original phage stock, adding 1 ml of the previous dilution to 9 ml of sterile diluent. Then, you take cultures of both strains of E. coli, each grown to a density of 1.0 X 10⁹ cfu/ml. You add 0.1ml of your final phage dilution to 0.1ml of each of the two E. coli cultures in separate tubes of melted top agar. Then you plate the top agar tubes, each onto a separate plate of PCA, an all-purpose medium. You incubate both plates overnight at 37°C.

The next day, you discover that there are no plaques on the plate with E. coli A, but there are 79 plaques on the plate with E. coli B.

• (1 point) What is a likely reason there are no plaques on the plate where E. coli A was added? Assume there was no problems with experimental technique. Phage P1 does not infect E. coli A.
  
  
• (2 points) What is the concentration of the original phage stock – i.e., the concentration in the original tube of P1?
  

  dilutions made	X	amount inoculated	=	"plated dilution"
  (10–1)8	X	1/10	=	10–9
  dilution factor	X	no. colonies	=	no. CFUs/ml
  109	X	79	=	79 X 1010

  
  
• (2 points) Predict what a plate would look like if no E. coli cells were added to the top agar, and explain why you predict this. It would basically look like a sterile plate. There would be no growth of E. coli and no resulting plaques, as there would be nothing for Phage P1 to infect.
  
  

V.  EXTRA CREDIT (2 points).

1.  You isolated an enteric from beautiful Lake Splammo, and your Kligler Iron Agar culture of the isolate has a red (alkaline) slant and a yellow (acidic) butt after one day of incubation at 37°C. To begin the identification process, you consider the reactions on the following table:

• From the results of just the pH-related reactions, you know the isolate would not be identified as a coliform or Pseudomonas, and you can also see that the isolate is negative for H₂S production.
  
  
• Your lab partner has been experimenting with differential media and has added a somewhat large amount of mannitol and ornithine to her KIA formulation, and she also got the same result with the same isolate. According to the table, the organism would be therefore identified as this genus: Providencia.