Bacteriology/Food Science 324:
ANSWERS TO OLD QUIZ NO. 2

Given October, 1996


I.  MULTIPLE TRUE/FALSE (5 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.  Regarding the pseudomonads:

  O    Refrigeration prevents the growth and spoilage of foods by these organisms.
  O    The acidic reaction associated with their growth in Glucose O/F Medium is an indication of glucose fermentation by these organisms.
  +    They can grow on selective media used routinely for the isolation of enterics.
  +    They are like enterics in being gram-negative, catalase-positive rods.

2.  Regarding the enterics:

  O    They are defined by microbiologists as being any organism whose primary habitat is the intestinal canal of warm-blooded animals and humans.
  +    They all ferment glucose.
  +    The enteric group includes fecal coliforms and Salmonella.

3.  Regarding the lactics:

  O    The group name "lactics" refers to the fact that they all ferment lactose.
  +    They are all gram-positive organisms which can be rods or cocci.
  O    As a rule, they are all capable of respiration and fermentation.

II.  MATCHING (14 points).

1.  (4 points) Match the bacteria in column a with the associated category of food-related organisms in column b. Only one letter per blank. Any letter may be used any number of times or not at all. (1 point for each blank.)

a

  B    sauerkraut bacteria
  A    yogurt bacteria
  C    fecal coliforms
  C    Enterococcus
  D    fluorescent pseudomonads
b

A.  starter culture
B.  wild fermentation
C.  indicator organism
D.  spoilage organism (i.e., one which
causes a defect which renders a food
item undesirable)

2.  (10 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.)

a

  D    Nitrite burn is associated with this pigment.
  B      L    Two physiological types of lactics.
  J    Test which differentiates between these two types.
  C      H    Two physiological types of enterics.
  E    Test which differentiates between these two types.
  A    Term associated with a particular organism's need for
lots of growth factors.
  J    The presence of citrate in a medium used for this test
can cause a false-positive reaction.
  K    The benzidine test is needed to tell if a positive catalase
reaction is due to true catalase or this enzyme.
b

A.  fastidious
B.  homofermenters
C.  mixed-acid fermenters
D.  myoglobin
E.  methyl red test
F.  oxidase
G.  Durham tube
H.  butanediol fermenters
I.  fluorescein
J.  hot-loop test
K.  pseudocatalase
L.  heterofermenters

III.  SHORT ANSWER (15 points).

1.  (2 points) The controversy over the addition of nitrite in cured meat products involves what two conflicting effects of this preservative?

Helpful aspects: Helps prevent growth of certain pathogens – e.g., Clostridium botulinum. Also assists in development of good pigmentation of the meat.
Negative aspect: It can contribute to the production of nitrosamines during the cooking process.

2.  (2 points) Name and briefly explain two important factors in the use of indicator organisms.

Remember definition of indicator organisms in that they are not the problem organisms but rather are associated with (found along with) certain problems. Here are several factors:
They should be easy to detect. (That's mainly why they're used. As E. coli is an indicator of fecal pollution, it's easy to test for E. coli and more difficult to test for each possible intestinal pathogen.)
They should not be found in the environment except where the problem is.
They should not be "problems" themselves and pose a health risk for lab workers.

3.  (6 points) What are the three steps in the coliform detection and isolation procedure? Briefly explain the purpose of each step.

Presumptive Test: Initial enrichment to recover as many coliforms as possible.
Confirmatory Test: Another enrichment. It is much more selective for coliforms and should lead to their easy recovery on EMB Agar (the isolation medium).
Completed Test: Isolation and identification of individual coliforms.

4.  (3 points; 1/2 point per item)

a.  A certain amount of salt (NaCl) is included in the formulation of some fermented food products such as sauerkraut and sausage. List two effects of the addition of NaCl.

Here are 3: Affects taste, inhibits spoilage organisms and pathogens, draws water out of tissues (more condusive for microbial growth).

b.  List three ways in which acid produced by the fermentation of organisms in sausage can affect the product.

Taste, preservation, "tightens up" the protein.

c.  What group (genus or group of genera) of organisms is responsible for any desired acid production in sausage?

the lactic acid bacteria

5.  (only 2 points) A little medium thought question: You wish to exploit certain properties of the difficult-to-isolate bacterium Excalibacterium (an enteric) in order to help you detect and isolate it from certain food samples which are highly contaminated with other enterics. You decide to start with MacConkey Agar which you know contains lactose as the only fermentable sugar. Peptone is another medium ingredient which you recall; it contains a mixture of various amino acids – none in any especially high amount. Following is a table showing what has been found out about the organisms associated with this particular food product:

genusfermentation ofdecarboxylation of
glucosemaltoselactosesucrosemannitollysinearginine
Edwardsiella+++
Aquabacterium++++
Excalibacterium+++
other enterics+++ or –+ or –+ or –+ or –+ or –

a.  On MacConkey Agar, what would you expect the net pH reaction would be for any of the three genera specifically listed on the table above?

ALKALINE

b.  As these 3 genera don't ferment or respire lactose, how can they grow (think about generating energy and obtaining carbon) on MacConkey Agar?

The amino acids in the peptone can serve as possible carbon and energy sources.

c.  What would be the best choice for a sugar to add to MacConkey Agar which will assist greatly in the differentiation of Excalibacterium colonies from the other organisms on the table?

MALTOSE. (Excalibacterium colonies will be alkaline; all others will be acidic.)

d.  If lysine were to be included in the medium in a relatively large amount, what effect would this have on the pH reaction associated with Excalibacterium colonies?

MORE ALKALINE


IV.  PROBLEMS (6 points).

1.  (4 points) An one gram sample of hamburger was added to 9 ml of sterile saline and blended vigorously. 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 also inoculated with one ml. After appropriate incubation, the results were recorded as follows:

Dilution made of the original hamburgerfirstsecondthirdfourthfifth
Actual dilution of the hamburger
(Note why the "plated dilution" will be the same.)
10–110–210–310–410–5
Colony count on PCATNTC
TNTC
TNTC
TNTC
82
78
9
12
1
0
No. of broth tubes showing growth31000

a.  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  80  =  8.0 X 104

b.  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: 3,1,0 means there were ("most probably," on the average) 0.43 organisms per inoculum of the middle set of tubes (of the three sets of tubes you used to get the MPN value).
So, 0.43/ml of the 10–2 dilution leads to 43/gram of the undiluted hamburger.

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

a.  The addition of 1 ml of a sample to 9 ml of sterile diluent.

b.  The addition of   3    ml of the same sample to 27 ml of diluent.

c.  The addition of 10 ml of the same sample to   90    ml of diluent.


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