Bacteriology/Food Science 324:
FINAL LABORATORY EXAM
given December, 1999

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I.  MULTIPLE TRUE/FALSE (19 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. (One-half point each.)

1.  You have identified an isolate from an outbreak as Salmonella, and included in your results is the determination of the antigenic formula as 1,2,4,5:i:1,2.

          You can designate the organism with the formula (i.e., Salmonella1,2,4,5:i:1,2) and probably also with a name such as "Salmonella typhimurium."
          Salmonella isolates with different antigenic formulas would be different species.
          Included in this formula is coded information about certain important biochemical test reactions.

2.  Regarding a_w determinations and related things,

          The a_w value for a moist food such as hamburger or fresh bread which is left out in a dry room, unwrapped, will decrease over time.
          The a_w of a food where all of the water is chemically bonded to the food is 0.
          The a_w value for pure water is 1.
          "Phelps index" can be used to adjust results in MPN and a_w problems.

3.  Injured bacterial cells

          making up the entire population of a broth culture would have a lower D-value than an equal concentration of healthy cells of the same organism in the same medium.
          in a food are dead for all practical purposes as they will have no spoilage or pathogenic potential.
          can recover in all-purpose media and in the human body.
          can recover when the food sample they are in is added to a non-selective broth medium, and a count of the number of CFUs per gram of the sample can be made from the broth after it has incubated overnight.
          can escape detection on some media – even plating media formulated for their isolation.

4.  The following are among the primary tests or observations one would run on a pure culture to identify it.

          Determination of the organism's D-value.
          Determination of the gram reaction and cellular morphology and arrangement.
          Determination of the catalase reaction.
          Determination of lactose fermentation as a higher priority test than glucose fermentation.

5.  The "enterics" (members of the Family Enterobacteriaceae)

          are defined as being any organism whose natural habitat is the enteric (intestinal) tract.
          include Pseudomonas and Enterococcus.
          include Salmonella and the true coliforms.
          all ferment lactose.

6.  The "lactics" (lactic acid bacteria)

          are defined as being any organism which produces lactic acid as a byproduct of fermentation.
          and enterococci are two completely separate groups of bacteria.
          are gram-positive cocci and rods.
          all ferment lactose.

7.  In the "total aerobic plate count,"

          all strictly aerobic organisms in a sample are represented as colonies on the plates.
          an "all-purpose" plating medium is utilized.
          we always expect to wind up with an underestimation of the total number of viable cells per gram or ml of the sample.
          we generally expect to have an equal or higher count that what we would get if we utilized a selective medium.

8.  A one-tenth ml inoculum from a 10^–3 dilution of a sample

          will contain one-tenth the number of colony-forming units (CFUs) that are in 1 ml of the same 10^–3 dilution.
          is equivalent to a 1 ml inoculum from a 10^–4 dilution of the same sample.
          should give rise to 100 colonies if the original, undiluted sample contains 10⁵ CFUs/ml.

9.  The addition of nitrate in the manufacture of sausage

          requires the presence of a population of microorganisms which will reduce it.
          requires the activity of lactic acid bacteria to assist in the ultimate adjustment of the color of the meat.
          always leads to nitrite burn.

10.  Some miscellaneous items:

          Yeasts, molds and lactic acid bacteria can be selectively isolated more appropriately with acidic media than with alkaline media.
          Addition of spices such as cloves and cinnamon to an all-purpose medium will make the medium selective.
          Fermentation, decarboxylation and hydrogen sulfide (H₂S) production are anaerobic processes which may be detected on certain differential plating media which are incubated under aerobic conditions.
          The gram stain is of no practical value in food microbiology, as it is tricky and gives variable reactions.

II.  MATCHING (15 points). In each of the following matching sets, 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 each.)

III.  SHORT ANSWER (51 points).

1.  (1 point) The first thing a food microbiologist should do in the laboratory when examining a food for a microbiological problem is a           . (It takes just a few minutes.)

2.  (3 points) You have determined the D-value and Thermal Death Time (TDT) for a culture of E. coli (1 X 10⁸ CFUs/ml) heated at 55°C. In the blanks below, indicate whether the D-value and TDT would be expected to increase, decrease or stay the same.

3.  (5 points) In this medium, consider that glucose is present to help the growth of the inoculated organism and any acid produced from fermentation of glucose will be too small to be detectable.

1. A coliform would be expected to produce a(n) alkaline / neutral / acid (circle one) reaction.
   
   
2. Salmonella would be expected to produce a(n) alkaline / neutral / acid (circle one) reaction.
   
   
3. Two medium components from which an organism can produce alkaline products:           and          
   
   
4. Medium component from which Salmonella could produce H₂S:          
   

4.  (3 points) With Triple Sugar Iron (TSI) Agar, we can tell if an organism can ferment sucrose and/or lactose.

1. List two other things we can test for with TSI Agar.
   
   
2. What further use can be make of TSI Agar?
   
   
3. An analyst mistakenly poured a deep layer of mineral oil over a freshly-inoculated slant of Triple Sugar Iron Agar. If the organism inoculated were an enteric which did not ferment either lactose or sucrose, how would the medium appear? Why?
   

5.  (14 points) Briefly indicate why each of the following procedures are done in the lab:

1. Stab-inoculating the butt of a Lysine Iron Agar slant. (Think about the primary purpose of the medium.
   
   
2. Incubating petri dishes upside down.
   
   
3. Utilizing Low Glucose Agar when testing lactic acid bacteria for pseudocatalase.
   
   
4. Setting up anaerobic conditions during the sauerkraut fermentation.
   
   
5. Not being required to utilize anaerobic conditions for the yogurt fermentation.
   
   
6. Heating the milk prior to inoculation of the yogurt starter cultures (other than for any pasteurizing effect).
   
   
7. Using a two-stage selective enrichment process prior to plating in the analysis of foods for coliforms. (Why not inoculate the food directly into the confirmatory broth media?)
   

6.  (8 points) Briefly indicate one possible problem associated with each of the following procedures:

1. Testing the methyl red reaction in MR-VP Broth at 24 rather than 48 hours of incubation.
   
   
2. Utilizing a loop instead of a needle when picking colonies off a petri dish.
   
   
3. Gram-staining.
   
   
4. Use of sterile distilled water when diluting cultures for microbial plate counts.
   

7.  (10 points) For each of the following five pairs of organisms, first indicate (in the blank) the test or observation one can make to distinguish between the two organisms. Secondly, circle the organism giving the positive reaction or observation.

8.  (3 points) In the analysis of food for Salmonella, a number of procedures are done in order. With numbers (1, 2, etc.), arrange the following in proper sequence.

                    Inoculate preliminary biochemical test media.

                    Inoculate non-selective broth medium.

                    Perform serological tests.

                    Inoculate selective broth medium/media.

                    Inoculate a wide variety of differential media.

                    Streak selective-differential plating media.

9.  (2 points) An organism is capable of growth in laboratory media containing 4% NaCl. However, when 4% NaCl is added to hamburger, the organism is unable to grow. It is, however, able to grow in unsalted hamburger. Briefly explain.

10.  (2 points) Over the course of sauerkraut fermentation, one can note the appearance and disappearance of certain types of colonies on the various isolation media. In the following tables, the presence (+) or absence (–) of these colonies is noted at four sampling times during the fermentation. In the first column at the left, indicate the order (1-4) in which one would see the results indicated for a typical sauerkraut fermentation.

IV.  PROBLEMS (15 points).

1.  (6 points) An one gram sample of hamburger was added to 9 ml of sterile saline. Two subsequent, 1/100 dilutions were then made. As indicated in the table below, 1 ml and 0.1 ml amounts from the dilutions were plated in duplicate on Plate Count Agar (PCA). From the same dilutions, a 3-tube most probable number analysis was set up with LST Broth. After appropriate incubation, the results were recorded as follows:

1. Calculate the total number of colony-forming units (CFUs) per gram of the hamburger.
   
   
   
2. Calculate the presumptive, most probable no.of coliforms per gram of the hamburger.
   
   

2.  (1 point) If you were to plate 0.1 ml of a sample of undiluted milk, this would theoretically result in the same number of colonies as would plating one ml of a           dilution of the same milk sample.

3.  (2 points) The same dilution can be obtained in each of the following situations:

1. The addition of 1 ml of a sample to 9 ml of sterile diluent.
   
   
2. The addition of           ml of a sample to 18 ml of sterile diluent.
   
   
3. The addition of 10 ml of a sample to           ml of sterile diluent.
   

4.  (2 points) State the indicated number of bacteria per gram of food product in each of the following. (Note: One ml inocula were made from the dilutions.)

5.  (2 points) The following results were obtained in an a_w experiment for a food product by the use of the filter-strip technique we used in lab:

1. What was the approximate a_w range of the food product?
   
   
2. If the food were placed in a room with 81% relative humidity, would it gain or lose water?
   

6.  (2 points) Would an organism with a maximum NaCl tolerance for growth of 10.5% (w/w) be able to grow in a food of the following composition?