Bacteriology/Food Science 324:

Given December, 1996

I.  MULTIPLE TRUE/FALSE (18 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.)


  O    Glass petri dishes are discarded in the same container as plastic petri dishes.
  +    One should not discard stained smears into disinfectant. The cells are already dead, and the stain messes up our attempts to recycle the slides.
  +    Proper aseptic technique includes the flame-sterilization of the entire wire of the inoculating loop or needle, not just the tip.

1.  The following reactions are involved in "stormy fermentation" of Litmus Milk with reduced iron:

  +    Fermentation of lactose resulting in acid and gas production.
  +    Curd formation.
  O    Hydrogen sulfide (H2S) production.
  O    Nitrate reduction.

2.  Extracellular enzymes

  O    are involved in the breakdown of compounds after they have been transported into the cell.
  +    include coagulase and lecithinase.
  +    include gelatinase and amylase.
  O    include catalase and deaminases.

3.  In the test for nitrate reduction,

  O    one should be able to use the results to differentiate between Bacillus and Clostridium.
  +    a bubble in the Durham tube indicates formation of gaseous nitrogen (N2).
  O    formation of a red color – only after the addition of the reagents and the zinc – indicates that you should record that the organism reduces nitrate to nitrite.

4.  Bacillus cereus – when tested for catalase and inoculated into Dextrose Tryptone Agar (DTA) – shows positive reactions for catalase and glucose fermentation.

  +    The catalase reaction is sufficient to distinguish B. cereus from any species of Clostridium.
  O    One cannot tell from these reactions if the organism is a strict aerobe or has some other oxygen relationship.
  O    Any organism which ferments glucose must also be able to ferment lactose.

5.  Preparation of a slide culture (where a "growth chamber" is set up for direct observation on a slide)

  +    is necessary to note the appearance of spores and their structural relationship to vegetative cells of molds.
  O    is necessary to note the appearance of spores and their structural relationship to vegetative cells of Bacillus or Clostridium.
  +    can help us distinguish between various genera of filamentous fungi.

6.  Potato Dextrose Agar

  +    tends to select against bacteria, as the medium lacks sufficient nutrients for the growth of most bacteria.
  O    becomes inhibitory toward the growth of molds when acidified with tartaric acid.
  +    becomes inhibitory toward the growth of molds when supplemented with propionate.

7.  In the isolation of Staphylococcus aureus from food samples,

  +    A count of black colonies on Baird-Parker Agar can give us a "presumptive" determination of S. aureus.
  O    A count of black colonies on Baird-Parker Agar can give us a "confirmed" determination of S. aureus if the black colonies are surrounded by zones of proteolysis and precipitated fatty acids.
  O    The coagulase reaction can be used to differentiate between the genera Staphylococcus and Micrococcus.

8.  You make a 10–2 dilution of a sample of hamburger, and you plate out 0.1 ml from the dilution onto an all-purpose medium.

  +    This procedure is equivalent to plating 1 ml of a 10–3 dilution of the hamburger.
  O    One should multiply the colony count by 10–3 to determine the CFUs per gram of the sample.
  +    The 10–2 dilution could have been made by a variety of procedures. One method would be to mix one-tenth gram of the sample with 9.9 ml of diluent.
  O    If no growth appears on the plate after incubation, it is OK to say that the sample is sterile.
  O    If there is little or no growth on the plate, it would be useful to make an enrichment of the hamburger and then determine the CFUs per ml of the enrichment.
  O    Aerobic incubation of the plate will result in the growth of only strictly aerobic bacteria.
  O    All dilutions and inoculations in the procedure can be made with the use of the same pipette tip.

9.  The following procedures are advisable when making dilution plates of a food sample for the detection of Clostridium perfringens:

  O    cooling of the food sample
  O    vigorously mixing the dilutions prior to plating
  +    utilization of cycloserine as a selective agent

10.  The purpose of the "non-lactose route" in the isolation and identification of Salmonella,

  O    is to restrict the analysis of colonies to those which show no lactose fermentation.
  +    is to give lactose-fermenting strains of Salmonella the same opportunity to be detected as the typical non-lactose-fermenting strains.
  +    is assisted by the use of an isolation medium which does not contain lactose.

II.  MATCHING (11 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.)

  O    A term describing an organism that can tolerate high concentrations of salt and/or sugar.
  H    A term for the filaments of cells formed by a mold.
  C    Yeasts divide by budding or by this method.
  A    Species of Bacillus can be facultatively anaerobic or this.
  J    An old culture of Bacillus cereus will most likely cause one to indicate this regarding the gram reaction of the organism.
  D    What the name Escherichia coli represents as opposed to just Escherichia.
  I    An extremely specific designation given to a group of strains of Salmonella.
  F    Taxonomic group to which both yeasts and molds belong.
  P    Material used in the coagulase test.
  R    Reagent used in the catalase test.
  K    Reagent used in the indole test.
A.  strictly aerobic
B.  bacteria
C.  binary fission
D.  species
E.  gram-positive
F.  fungi
G.  genus
H.  hyphae
I.  serovar
J.  gram-variable
K.  Kovacs reagent
L.  Litmus Milk
M.  motility
N.  strictly anaerobic
O.  osmophilic
P.  plasma
R.  H2O2

III.  SHORT ANSWER (6 points).

1.  (2 points) If samples of foods suspected of containing an active population of either Bacillus cereus or Clostridium perfringens are heated prior to plating, such that only Bacillus and Clostridium endospores can survive the heat treatment, one may find no growth on any plates. Why might this be so?

The food might have sufficient nutrients for active growth of the organisms, and the process of endospore formation may not be triggered yet.

2.  (2 points) A sample of a food suspected of transmitting Salmonella to a large number of consumers is inoculated directly onto various highly-selective plating media, and none of the resulting colonies is identified as Salmonella. However, that same food – when tested correctly for Salmonella – yields numerous confirmed Salmonella colonies. What was the problem with the first method?

One should not plate out directly onto a highly selective medium, unless one is confident the sample contains a large number of healthy, actively-metabolizing cells. In this problem, stressed cells were probably killed off by the highly selective media and were therefore undetected.

3.  (2 points) How can one tell if an organism is able to break down gelatin? Include in your answer the purpose of the ice-water bath.

If the gelatin medium is cooled to where it would normally be solid, and it remains liquid, the organisms must have broken down the gelatin – the only solidifying agent in the medium.

IV.  PROBLEM (5 points).

Fecal contamination was suspected in a batch of ground beef. You obtained a sample and made the two, serial 1/100 dilutions indicated in the table below. From each dilution, 1.0 ml and 0.1 ml amounts were inoculated into plates of Plate Count Agar (PCA, in duplicate) and tubes of Lauryl Sulfate Tryptose (LST) Broth (in triplicate). These plates and tubes were incubated appropriately, and the results are summarized on the table below. Subsequent tubes of Brilliant Green Lactose Bile (BGLB) Broth and EC Broth were inoculated and incubated appropriately; their results are also shown below.

dilutions of ground beef 10–2 10–4
amount inoculated into each of the tubes and plates 1.0 ml 0.1 ml 1.0 ml 0.1 ml
dilution factors are indicated here for convenience 102 103 104 105
colony count on PCA overgrown
no. of positive LST tubes 3 3 2 1
no. of positive BGLB tubes 3 3 1 0
no. of positive EC tubes 0 0 0 0

1. (2 points) Determine the total aerobic plate count in CFUs per gram of ground beef.

dilution factor  X  no. colonies  =  no. CFUs/gram
104  X  55  =  5.5 X 105

2. (2 points) Determine the confirmed, most probable number (MPN) of coliforms per gram of ground beef.

Using the BGLB results: 3,1,0 points to 0.43 on the MPN table. The table tells us that an average of 0.43 organisms (confirmed coliforms, in this case) were in the inoculum of the middle set of tubes (of the 3 sets chosen), which was 1 ml of a 10–4 dilution. Therefore, there were 0.43X104 coliforms per gram of the ground beef, better expressed as 4.3X103/g.

3. (1 point) Why can't one say that there are no fecal coliforms in the ground beef? How might the procedure be improved to answer this question more definitively?

For one thing, the MPN table doesn't translate 0,0,0 as zero any more than it translates 3,3,3 as infinite. There is still a possiblity of fecal coliforms, and one could use increasingly concentrated inocula (such as 0.1 gram, 1.0 gram, etc.) into additional sets of tubes.

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Page last modified on 11/25/97 at noon, CST.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison