Bacteriology/Food Science 324:
given December, 1996

I. Multiple True/False (39 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.)

Example: The following are some good things to know:

 +   The following words are always used to denote only the plural – never the singular – when they are used correctly: media, bacteria, genera.
 +   One writes the name of a biological entity as (for example) Escherichia coli or Escherichia coli – never escherichia coli.
 O   Yeasts generally grow well at 37°C and above.

1. Regarding the recovery of injured cells:

     Cells in an older culture (in the stationary phase) are more susceptible to injury than are actively-growing cells in a younger culture.
     Injured cells of enterics should have no problem growing and producing colonies on selective media such as MacConkey Agar and Bismuth Sulfite Agar.
     Once injured, a pathogenic bacterium such as Clostridium perfringens no longer poses a threat.

2. When performing plate counts:

     The use of an all-purpose medium such as Plate Count Agar will allow one to examine and count all species of bacteria present in the sample.
     The use of MacConkey Agar allows one to perform a count of gram-negative colony-forming units (CFUs).
     It is best to perform counts from a plate with about 5 to 25 colonies.
     One can expect that the number of colony-forming units determined per ml or gram of the sample is less than the number of viable cells per ml or gram of the sample.

3. When we quantify bacteria in a given amount of sample – by our usual method where we make dilutions of the sample and analyze plates inoculated from the various dilutions – we should consider the following combination of items:

     The number of cells in an average-sized colony.
     The dilutions made of the sample.
     The amount inoculated onto the plate(s) observed.
     The number of colonies on the plate(s) observed.
     The amount of medium in the plates.
     Very tiny colonies can be ignored, as these must be contaminants or something other than bacteria.
     Colony-forming units and colonies mean exactly the same thing.
     Any given colony can arise from the plating of one or more cells, so we cannot say we have a certain number of cells per gram or ml of the sample.

4. A one-tenth ml inoculum from a 10–2 dilution of a sample of milk

     will contain one-tenth the number of colony-forming units (CFUs) that are in 1 ml of the same, 10–2 dilution.
     is mathematically equivalent to the inoculation of 10–3 ml of the milk sample.
     should give rise to 100 colonies if the original, undiluted sample contains 105 CFUs/ml.

5. Growth of bacteria is always associated with

     an aqueous (water) environment.
     production of gas which can be detected by the hot-loop test.

6. 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.
     flames petri dishes after opening and again after streaking for isolated colonies.
     incubates plates in an inverted fashion (i.e., medium side up) in order to prevent condensation from forming on the medium.
     observes and studies colonies through the bottom lid of a petri plate (i.e., through the layer of medium) rather than looking at the surface of the colonies.
     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.
     considers it OK to drop the caps and plugs of the tubes onto the table while making transfers from one tube to another.
     practices "aseptic technique" when working with pathogenic organisms as it is not necessary to do so for non-pathogenic organisms.

7. The following are tests or observations one can make routinely on cultures which can help in their identification:

     Determination of gram reaction.
     Determination of the shape and arrangement of the cells.
     Determination of the D and Z values associated with the cultures.
     Determination of their abilities to grow in particular food products.

8. The heating of the milk in the production of yogurt can

     reduce the number of microbial contaminants.
     deactivate certain natural inhibitors in the milk.
     alter the milk protein in order to result in a more consistently-smooth product.

9. The following are characteristics of all lactic acid bacteria:

     They are gram-positive organisms.
     They are strict anaerobes.
     They are cocci which can be seen in a variety of arrangements.
     They characteristically ferment lactose.

10. The following are characteristics of all enteric bacteria:

     They are gram-negative organisms.
     Microscopically, they appear as distinct, rod-shaped cells which tend to lengthen during the growth of the culture.
     They are strict aerobes.
     They tend to deaminate amino acids and thereby cause an alkaline product to be formed on the slants of TSI Agar.
     They characteristically ferment lactose.

11. Reactions one may observe and record in Triple Sugar Iron (TSI) Agar include:

     lysine decarboxylation
     glucose fermentation
     lactose and/or sucrose fermentation
     H2S production

12. In the serological testing of Salmonella,

     the reaction seen in the slide agglutination test depends on "O" antigens being on particles (i.e., the cells) and the antibodies being in solution.
     nonmotile strains would not be expected to possess "H" antigens.
     one generally uses cells taken directly from colonies on the isolation plates.

13. Pseudocatalase is indicated by two of the following:

     a positive benzidine reaction
     a negative benzidine reaction
     a positive catalase reaction

14. To isolate microorganisms from food products one might choose the following food products as source material for the successful isolation of the indicated microorganisms.

     A late stage sauerkraut sample can be used as source material for the isolation of a coliform.
     A hamburger sample may be used as source material for the isolation of a fecal coliform.
     One can expect that fermented grapes may be an excellent source material for the isolation of
strictly thermophilic strains of Bacillus.
     A moist food which is rich in nutrients should not be heat-shocked if one is interested in obtaining the highest possible number of colonies of Bacillus cereus or Clostridium perfringens.

15. The following medium ingredients help to make Baird-Parker Agar a differential medium:

     Egg yolk

16. Selective agents in Baird-Parker Agar

     allow the growth of Staphylococcus aureus.
     may allow growth of organisms other than S. aureus.
     include agar and beef extract.

17. Yeasts and molds

     can be isolated efficiently by the use of Baird-Parker Agar.
     are discrete taxonomic groups of fungi.
     are the chief spoilage agents of acidic foods along with lactic acid bacteria.

18. When one observes a plate for mold colonies,

     one finds that they are difficult to distinguish from bacterial colonies.
     one may consider spores as potential CFUs (colony-forming units).
     one may consider fragments of mycelia as potential CFUs.

19. One may take advantage of the following characteristics of yeasts for their selective isolation from food products:

     Yeasts can generally tolerate highly alkaline conditions (pH as high as 10.5).
     Yeasts can generally tolerate highly acidic conditions (pH as low as 3.5).
     As yeasts are very capable biosynthetically, they can be cultivated on a nutritionally poor medium.
     Yeast colonies are generally difficult to distinguish from bacterial colonies.

20. The development of a desirable color of sausage during the curing process is due to a combination of these things:

     production of acid by lactic acid bacteria
     growth of pigmented bacteria

II.  Matching (20 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.)


     Fermentation of glucose with the production of lactic acid as the sole or major end product.
     Fermentation of glucose with the production of equivalent amounts of lactic acid, CO2, and ethanol.
     Group of organisms associated with the above two types of fermentation.
     Test that will distinguish between these types of fermentation.
     A specific organism that indicates fecal contamination.
     A term for an organism that requires a medium supplemented with multiple growth factors.
     During the fermentation of sausage, the addition of nitrite serves to inhibit the growth of this pathogen.
     A certain group of similar organisms within the family Enterobacteriaceae which is more of a physiological group than a formal taxonomic group.
     A designation of an organism that reflects the antigenic uniqueness of that organism.
     An enzyme that detoxifies hydrogen peroxide produced as a by-product of aerobic respiration.
     A group of organisms that are valued by the food industry.
     A highly-selective medium which gives lactose-negative and lactose-positive Salmonella colonies an equal chance to be detected.
     A highly-resistant "alternative cell type" produced by a few genera of bacteria.
     A term for the cellular filaments of a mold.
     A term describing an organism that can tolerate high concentrations of salt and/or sugar.
     An enzyme that causes blood plasma to solidify.
     An old culture of Bacillus cereus will most likely cause one to indicate this regarding the gram reaction of the organism.
     A mechanism by which cells of many species of yeasts reproduce.
     An extracellular enzyme produced by many species of Bacillus that degrades starch.
     The oxygen relationship generally associated with organisms of the genus Clostridium.

A.  amylase
B.  Bismuth Sulfite Agar
C.  catalase
D.  budding
E.  endospore
F.  facultatively anaerobic
G.  gram-positive
H.  hyphae
I.  hot-loop test
J.  gram-variable
K.  coliforms
L.  lactic acid bacteria
M.  MacConkey Agar
N.  homofermentation
O.  osmophilic
P.  protease
Q.  Clostridium botulinum
R.  heterofermentation
S.  serovar
T.  fastidious
U.  Escherichia coli
V.  species
W.  Baird-Parker Agar
X.  coagulase
Y.  methyl red test
Z.  strictly anaerobic

III.  Short Answer (30 points).

1.  (4 points)  Regarding the thermal processing of food, define the following terms:

2.  (3 points)  List three methods of food preservation:

3.  (2 points)  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.  (6 points)  Sauerkraut is produced via a wild fermentation that involves a succession of microorganisms:

5.  (2 points)  List or briefly describe two methods by which manufacturers of cultured dairy products (e.g., yogurt, cheese, etc.) reduce the potential for contamination of their starter cultures by starter-specific bacteriophages?

6.  (2 points)  Regarding sausage:

7.  (2 points)  According to FDA guidelines, coliforms are detected through presumptive and confirmatory tests.

8.  (4 points)

9.  (2 points)  List two ways one can differentiate between molds and yeasts in the laboratory.

10.  (1 point)  How can one can differentiate between yeasts and bacteria in the laboratory?

11.  (2 points)  What two types of canned food spoilage can be caused by facultatively anaerobic species of Bacillus?

IV.  Problems (11 points).

1.  (2 points)  The following results were obtained in an aw experiment for three food products by by the use of our filter-strip technique:

Product aw value of salt on strip
0.99 0.98 0.96 0.95 0.93 0.91 0.85 0.82 0.77 0.71
A dry dry dry dry dry dry dry dry wet wet
B dry dry dry dry dry dry wet wet wet wet
C dry dry wet wet wet wet wet wet wet wet

2.  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 for the Presumptive Test for Coliforms; each tube was inoculated with one ml. After appropriate incubation, the results were recorded as follows:

Dilution made of the original hamburger first second third fourth fifth
For convenience, you can indicate the "plated dilution" or "dilution factor" here          
Colony count on PCA TNTC
No. of broth tubes showing positive reaction 3 2 0 0 0

3.  (3 points)  You dilute a sample of pie and plate it out onto Baird-Parker Agar. After incubation, you find 200 black colonies on a plate inoculated with 0.1 ml of a 10–2 dilution. You then pick 20 black colonies at random, and you find that 18 of them test out to be gram-positive, catalase-positive cocci arranged in clusters. The same 18 isolates turn out to be positive for the coagulase test. How many Staphylococcus aureus CFUs were there per gram of the pie?

4.  (1 point)  One should expect the same number of CFUs in 100 ml of a 10–2 dilution of a hamburger sample as in     gram(s) of the undiluted sample.

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Page last modified on 12/4/97 at 9:45 AM, CST.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison