Bacteriology/Food Science 324:
1999 QUIZ NO. 1

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I. MULTIPLE TRUE/FALSE (21 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.)

EXAMPLES:

  O     Slides of stained smears must be discarded into the disinfectant.
  +     One always italicizes or underlines genus and species names when writing them in formal communications.

1.  A sample of hamburger was diluted to 10–4 (the same as 1/10,000). One ml of this dilution was plated and 300 colonies were counted after incubation.

        One can only prepare a 1/10,000 dilution of a sample by adding one ml of the sample to 9999 ml of diluent.
        To determine the number of CFUs which had been present per gram of the undiluted hamburger sample, one would multiply 300 by 10–4.
        Theoretically, plating one-tenth ml (instead of one ml) of the 10–4 dilution should give rise to one-tenth as many colonies.
        Plating one ml of a 10–4 dilution of the hamburger is equivalent to plating one-tenth ml of a 10–3 dilution of the same sample.
        Plating one ml of a 10–4 dilution of the hamburger is equivalent to plating 10–4 gram of the undiluted hamburger.

2.  Isolation of lactic acid bacteria from a sauerkraut fermentation will be assisted by a combination of the following factors such that growth of other types of organisms is inhibited as much as possible:

        incubation under aerobic conditions
        incubation under anaerobic conditions
        inclusion of sodium azide in the medium
        the use of MacConkey Agar

3.  In the production of yogurt, which of the following are reasons why one would heat milk prior to the inoculation of yogurt starter cultures?

        to "condition" (partially denature) the milk protein
        to break down the lactose completely
        to reduce the number of contaminating organisms
        to deactivate certain natural inhibitory substances in the milk

4.  In each of the following situations, an increase will occur in the Thermal Death Time (TDT) of a broth culture or other suspension of bacterial cells – when all other factors are held constant:

        an increase in the initial number of colony-forming units (CFUs) per ml
        an increase in the temperature of the heat treatment
        addition of a chemical agent harmful to the cells
        conditions altered such that the D-value is increased by five minutes

5.  The following – when considered together – are factors which can influence the aw of a food.

        amount of water
        amount of solute
        types of microorganisms present

6.  The following media or diluents assist in the recovery of "injured" cells from a food product:

        distilled water
        saline
        an all-purpose medium
        a selective medium

7.  A negative reaction can occur when performing the hot loop test on a culture if

        the organism ferments and produces only acids (as the byproducts of fermentation).
        the organism ferments and produces acids and carbon dioxide.
        the organism ferments and produces acids, hydrogen and carbon dioxide.
        the organism cannot ferment but can respire aerobically.

8.  When performing plate counts:

        The use of an all-purpose medium such as Plate Count Agar (PCA) will allow one to examine and count all species of bacteria present in the sample.
        It is best to perform counts from a plate with less than 30 colonies.
        One can expect that the number of CFUs determined per ml or gram of the sample is always less than (not equal to or greater than) the number of viable cells per ml or gram of the sample.

9.  When going about his/her work, a good microbiologist always

        uses the same pipettor tip when inoculating a series of dilution blanks with decreasing concentrations of inocula.
        understands that it is not necessary to re-flame the "hockey stick" when spreading a series of plates from those with more concentrated inocula to those with more dilute inocula.
        incubates plates in an inverted fashion to prevent condensation from forming on the medium.
        observes colonies from the top rather than looking from the bottom (and through the layer of medium).
        sterilizes just the tip of the loop or needle rather than the entire length of the wire.
        leaves tubes sitting upright and open in the test tube rack while making transfers between them.
        expects a solidified tube or bottle of agar medium will melt when placed in a 50°C water bath.

10.  Regarding gram stains:

        One can skip that step in the identification of bacteria as it is a difficult procedure to do.
        One can usually determine the genus of the organism but not the species.
        The best gram stains are done on "older" cultures such as those incubated over a weekend.
        The best gram stains are made on smears that are so thick that the cells are packed together and one cannot make out the individual shape of the cells – just the color.

II. MATCHING (3 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. (One-half point for each blank.)

a

        Indicates sensitivity of an organism to a particular temperature.
        Indicates sensitivity of an organism to changes in temperatures used in thermal processing.
        Type of organism involved in the first stage of sauerkraut fermentation.
        Type of organism involved in later stages of sauerkraut fermentation.
        Gram reaction associated with a blue or purple color of the cells.
        A bacteriophage is basically this.

b

A. aw
B. D-value
C. gram-negative
D. lactic acid
E. virus
F. Z-value
G. none of the above

III.  SHORT ANSWER (13 points).

1.  (2 points) List two ways one can determine the Thermal Death Time (TDT).

2.  (2 points) Dairy fermentation cultures are often susceptible to bacteriophage contamination. What two things can be done to prevent damage being done by these viruses?

3.  (1 point) How would an increase in a food product's brine strength effect the aw of the food product (with all other factors remaining constant)?

4.  Sauerkraut is produced via a wild fermentation that involves a succession of microorganisms:

5.  (1 point) Suppose you were to add sodium azide to MacConkey Agar and then incubate the inoculated plates under aerobic conditions. What can you suppose about any organism able to grow and produce colonies on this medium? (List at least two probable characteristics.)

6.  (2 points) Lab Technician A performed a "total aerobic plate count" on milk by the usual method – that is, diluting the milk sample and then inoculating plates of all-purpose medium from the dilutions. He came up with a count of 1.1 X 102 CFUs per ml of the milk.

Lab Technician B prepared an enrichment by adding 1 ml from the same milk sample to nine ml of Brain Heart Infusion Broth and letting it incubate at 30°C for 24 hours. He then made more dilutions and platings for his "total aerobic plate count." He came up with a count of 1.6 X 107 CFUs per ml. He reported this result and the dairy plant was closed by reason of excess bacterial contamination.

After a quick review of the results of both technicians, it became obvious that one of the two technicians gave an erroneous and misleading assessment of the condition of the milk. Whose results were the most trustworthy – Technician A or Technician B – and why?

(Note: Both technicians performed specific techniques regarding dilutions, inoculations and incubations correctly. The answer has nothing to do with those techniques.)

IV.  PROBLEM (3 points). A 1 gram sample of tuna was added to 99 ml of sterile saline. Two further 1/100 dilutions were then made. One ml and 0.1 ml amounts from the dilutions were plated on Plate Count Agar (PCA) in duplicate. From the same dilutions and using the same amounts of inocula, a three-tube Most Probable Number analysis was set up with a broth medium formulated to support growth of only gram-negative bacteria. After incubation, the results were recorded as follows:

Dilution made of the original tuna first second third
Amount inoculated 1.0 ml 0.1 ml 1.0 ml 0.1 ml 1.0 ml 0.1 ml
For convenience, you can indicate the
plated dilution or dilution factor here.
           
Colony count on PCA TNTC
TNTC
506
500
71
69
10
8
1
0
0
0
No. of broth tubes showing growth 3 2 1 0 0 0

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John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison