Bacteriology/Food Science 324:

Given October, 1996

I.  MULTIPLE TRUE/FALSE (17 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 true or false 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.)

Example: The following are plural terms which should never (not ever) be used as singular terms:

  +    bacteria
  O    coccus
  +    media

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

  +    Wiping down the entire work area with disinfectant before and after work.
  O    Leaving dilution tubes upright and uncapped throughout the dilution process.
  O    Making a smear with several loopsful of the culture and not bothering to resterilize the loop after touching the slide and before re-entering the tube.
  +    Heating the entire wire of the inoculating loop or needle in the Bunsen burner flame – not just the tip.

2.  A sample of milk was diluted to 10–3 (that is, 1/1000). One-tenth ml was plated, giving rise to 40 colonies after incubation.

  O    To determine the number of CFUs that had been present per ml of the undiluted milk sample, one would multiply 40 by 10–4.
  +    Plating one-tenth ml of a dilution is like making another dilution without the fuss and bother of inoculating another dilution blank. In our example here, when we plated one-tenth ml of the 10–3 dilution, it was equivalent to plating one ml of a 10–4 dilution.
  +    Theoretically, plating one ml (instead of 0.1 ml) of the 10–3 dilution would give rise to 400 colonies.
  +    Directly plating 1 ml or 0.1 ml of the undiluted milk sample would obviously result in an over-grown plate. If we had the technology to plate out directly a 10–4 ml amount of the undiluted milk, we would theoretically get 40 colonies on the plate (provided we were able to get a representative sample).

3.  You are provided with a bottle of melted Plate Count Agar (PCA) in a 50°C water bath for use in pouring plates.

  O    If you were to keep the bottle out at room temperature too long, and the medium solidifies, you can heat it back up to 50°C and it will be 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.
  O    As PCA is a non-selective medium, we expect to be able to recover all possible species of bacteria from whatever we sample.

4.  Regarding the D-value and thermal death time (TDT) associated with a given culture of bacteria, the following is/are true:

  +    Increasing the temperature of the heat treatment will decrease both the D-value and the TDT.
  +    Increasing the number of cells per ml of the culture would have no effect on the D-value (for a particular temperature of heat treatment).
  O    Increasing the number of cells per ml of the culture would have no effect on the TDT (for a particular temperature of heat treatment).

5.  The following is/are important in the proper conversion of cabbage into sauerkraut:

  +    salt (NaCl)
  +    acid produced by the fermentation of bacteria
  O    acid added by the manufacturer at the beginning of the process
  O    a starter culture added by the manufacturer at the beginning of the process
  O    the use of aerobic incubation of the preparation

6.  "Phelp's Index" can be used in problems involving

  +    indicated number determinations.
  O    aw determinations.
  O    plate counts.

7.  Injured bacterial cells can recover in/on

  +    an all-purpose medium like PCA or Brain Heart Infusion Agar.
  O    a selective medium formulated for selective isolation of the type of organism to which these particular bacterial cells belong (e.g., gram-negative bacteria).
  +    the human body if that's where their non-injured counterparts can grow.
  O    distilled water.

8.  As a moist food product (hamburger, bread, etc.) is allowed to dry out,

  O    its aw is expected to increase.
  +    it becomes less susceptible to bacterial spoilage
  +    it can reach a point where spoilage by molds is more likely to happen than spoilage by bacteria.
  +    the solute concentration of the water portion of the food increases.

9.  Things important in the identification of an unknown bacterium include the following:

  +    gram reaction
  +    shape and arrangement of the cells
  O    the Z value of a typical broth culture
  +    the catalase reaction

II.  MATCHING (7 points). Place the letter of the correct item from column b in the blank by each statement in column a. Only one letter per blank. Any letter may be used any number of times or not at all. (1 point for each blank.)


  B      I    Two examples of intrinsic factors associated with microbial growth in a particular food product.
  E      H    Two examples of extrinsic factors associated with microbial growth in a particular food product.
  D    The hot-loop test detects the presence of this highly-soluble gas which is produced during fermentation by certain organisms.
  F    Bacteriophages use this as their "growth medium."
  A    The catalase test involves an enzyme which is important in the breakdown of this compound.

A.  H2O2
B.  natural inhibitory compounds
C.  Top Agar
D.  CO2
E.  temperature
F.  bacterial cells
G.  H2
H.  relative humidity
I.  pH

III.  SHORT ANSWER (12 points).

1.  (2 points) List two reasons why the addition of NaCl is important in the production of sauerkraut.

Taste, preservation (inhibitory effect on spoilage microorganisms), and it helps draw water out of the cabbage tissue.

2.  (3 points) You have three flasks of milk (X, Y and Z) as shown in the following table:

flaskstarter culture
phosphate added
(with heat)

a.  Which flask would be least susceptible to spoilage by certain organisms? (Note that no culture was added to Flask A.) Should the flasks be left open to the environment, one would expect flask Z to "spoil" the least. Here we have the culture achieving the desired fermentation to bring the pH down to a more inhibitory level.

b.  Which flask would contain the higher concentration of bacteriophages after incubation of the flasks? Y

c.  What would be the texture of the milk in flask Z after incubation? solid

3.  (1 point) The first thing a food microbiologist should do in the laboratory when examining a food for a microbiological problem is a gram stain!

4.  DEFINITIONS (6 points) Briefly define each of the following. Do not give an example as the only definition.

a.  wild fermentation: food fermentation achieved with the use of organisms indigenous to the raw food product

b.  Z value: the difference in temperature when decreasing the TDT 90% (one log cycle)

c.  microbial succession: the successive dominance of different microbial groups during the course of time

IV.  PROBLEM (4 points). An ten gram sample of hamburger was added to 90 ml of sterile saline. Four, subsequent 1/10 dilutions were then made. One ml amounts from the dilutions were plated in duplicate with Plate Count Agar (PCA). From the same dilutions, a 3-tube most probable number analysis was set up with a broth medium formulated to support the growth of only gram-negative bacteria; each tube was inoculated with one ml . After appropriate incubation, the results were recorded as follows:

Dilution made of the original hamburgerfirstsecondthirdfourthfifth
Actual dilution of the hamburger10–110–210–310–410–5
Colony count on PCATNTC
No. of broth tubes showing growth32000

1.  (2 points) Calculate the total number of colony-forming units per gram of the hamburger.

dilutions made  X  amount inoculated  =  "plated dilution"
1/10 X 1/10 X 1/10  X  1  =  1/1000 or 10–3

dilution factor  X  no. colonies  =  no. CFUs/gram
103  X  125  =  1.25 X 105
best rounded to 1.3 X 105

2.  (2 points) Calculate the most probable number of gram-negative organisms per gram of the hamburger.

According to the MPN table as it applies to this problem: 3,2,0 means there were ("most probably," on the average) 0.93 organisms per inoculum of the middle set of tubes (of the three sets of tubes you used to get the MPN value).

So, 0.93/ml of the 10–2 dilution leads to 93/gram of the undiluted hamburger.

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Page last modified on 9/8/98 at 7:15 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison