Question

Illness and food spoilage can result from microbial growth in foods. The sanitary control of food quality is concerned with testing foods for the presence of pathogens. During processing (grinding, washing, and packaging), food may be contaminated with soil microbes and microbiota from animals, food handlers, and machinery. Standard plate counts are routinely performed on food and milk by food-processing companies and public health agencies. The standard plate count is used to determine the total number of viable bacteria in a food sample. The presence of large numbers of bacteria is undesirable in most foods because it increases the likelihood that pathogens will be present, and it increases the potential for food spoilage. In a standard plate count, the number of colony-forming units (CFU) is determined. Foods are the primary vehicle responsible for the transmission of diseases of the digestive system. For this reason, they are examined for the presence of coliforms because the presence of coliforms usually indicates fecal contamination. Eosin Methylene Blue (EMB) agar is used for isolation of coliforms. It contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue. The sugars provide fermentable substrates to encourage growth of coliforms. The dyes inhibit the growth of Gram-positive organisms and, under acidic conditions, also produce a dark purple complex usually accompanied by a green metallic sheen in fecal coliforms (e.g. E. coli). This sheen serves as an indicator of the vigorous lactose or sucrose fermentation typical of fecal coliforms. Smaller amounts of acid production (typical of Enterobacter aerogenes and slow lactose fermenters) result in a dark pink/purple coloration of the colonies (darker than the medium). Non-fermenters (i.e. non-coliforms) retain their normal color or take on the coloration of the medium. Growth on EMB media Scenario Imagine that you are working as a microbiologist for the FDA. Your job requires you to test raw hamburger meat before it is distributed for public consumption. The FDA standards for hamburger are from 2.5×10^5 to 1×10^7 bacteria per gram (total count). The typical number of coliforms is 2-3 orders of magnitude lower than the total count. Your most recent inspection of a ground hamburger sample from the Happy Cow meat purveyors yielded the following results: Total bacterial count: 8.2×10^6 CFU/g (as determined from plating on TSA medium) Coliform bacterial count: 2.24×10^4 CFU/g (as determined from plating on EMB medium) Objectives: To submit your findings you must present the details of the experiment performed that yielded these results. Specifically, a detailed account of your experimental approach and methods, a diagram of your dilution/plating scheme, a table showing dilutions and volumes used for plating and number of colonies counted for each medium, all pertinent calculations. See details below under Reporting. Materials: Ground meat Sterile empty petri dishes and sterile spatulas or tongue depressors (to be used for weighing ground meat subsample) 50-mL sterile centrifuge tube Sterile saline for dilutions 5 sterile microcentrifuge tubes for dilutions Automatic pipettors and sterile tips L-shaped cell spreaders 3 TSA plates 3 EMB plates Hints: Model your experiment design on the experimental design found in the Soil Microbiology Lab as a guide. The suggested approach is as follows: a subsample of hamburger is weighed aseptically, placed in a known volume of sterile saline, mixed thoroughly, serial dilutions are prepared, known volumes of the dilutions are then plated onto suitable media. Spread plate technique is employed. After incubation, the number of colonies is counted. Plates that have between 30 and 300 colonies are suitable for counting. CFU/g of hamburger are calculated for total bacteria and coliforms. Reporting You should explain in writing how you approached the task and your experimental design (~1 page, typed, 12-pt font size). Be sure to address the following. Why did you use both TSA and EMB? Why did you use a serial dilution approach? Why did you choose your selected incubation time and temperature?

Illness and food spoilage can result from microbial growth in foods. The sanitary control of food quality is concerned with testing foods for the presence of pathogens. During processing (grinding, washing, and packaging), food may be contaminated with soil microbes and microbiota from animals, food handlers, and machinery.

Standard plate counts are routinely performed on food and milk by food-processing companies and public health agencies. The standard plate count is used to determine the total number of viable bacteria in a food sample. The presence of large numbers of bacteria is undesirable in most foods because it increases the likelihood that pathogens will be present, and it increases the potential for food spoilage. In a standard plate count, the number of colony-forming units (CFU) is determined.

Foods are the primary vehicle responsible for the transmission of diseases of the digestive system. For this reason, they are examined for the presence of coliforms because the presence of coliforms usually indicates fecal contamination. Eosin Methylene Blue (EMB) agar is used for isolation of coliforms. It contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue. The sugars provide fermentable substrates to encourage growth of coliforms. The dyes inhibit the growth of Gram-positive organisms and, under acidic conditions, also produce a dark purple complex usually accompanied by a green metallic sheen in fecal coliforms (e.g. E. coli). This sheen serves as an indicator of the vigorous lactose or sucrose fermentation typical of fecal coliforms. Smaller amounts of acid production (typical of Enterobacter aerogenes and slow lactose fermenters) result in a dark pink/purple coloration of the colonies (darker than the medium). Non-fermenters (i.e. non-coliforms) retain their normal color or take on the coloration of the medium.

Growth on EMB media

Scenario

Imagine that you are working as a microbiologist for the FDA. Your job requires you to test raw hamburger meat before it is distributed for public consumption.

The FDA standards for hamburger are from 2.5×10^5 to 1×10^7 bacteria per gram (total count). The typical number of coliforms is 2-3 orders of magnitude lower than the total count.

Your most recent inspection of a ground hamburger sample from the Happy Cow meat purveyors yielded the following results:

Total bacterial count: 8.2×10^6 CFU/g (as determined from plating on TSA medium)
Coliform bacterial count: 2.24×10^4 CFU/g (as determined from plating on EMB medium)

Objectives:

To submit your findings you must present the details of the experiment performed that yielded these results. Specifically, a detailed account of your experimental approach and methods, a diagram of your dilution/plating scheme, a table showing dilutions and volumes used for plating and number of colonies counted for each medium, all pertinent calculations. See details below under Reporting.

Materials:

Ground meat
Sterile empty petri dishes and sterile spatulas or tongue depressors (to be used for weighing ground meat subsample)
50-mL sterile centrifuge tube
Sterile saline for dilutions
5 sterile microcentrifuge tubes for dilutions
Automatic pipettors and sterile tips
L-shaped cell spreaders
3 TSA plates
3 EMB plates

Hints:

Model your experiment design on the experimental design found in the Soil Microbiology Lab as a guide. The suggested approach is as follows: a subsample of hamburger is weighed aseptically, placed in a known volume of sterile saline, mixed thoroughly, serial dilutions are prepared, known volumes of the dilutions are then plated onto suitable media. Spread plate technique is employed. After incubation, the number of colonies is counted. Plates that have between 30 and 300 colonies are suitable for counting. CFU/g of hamburger are calculated for total bacteria and coliforms.

Reporting

You should explain in writing how you approached the task and your experimental design (~1 page, typed, 12-pt font size). Be sure to address the following. Why did you use both TSA and EMB? Why did you use a serial dilution approach? Why did you choose your selected incubation time and temperature?

Added by Miguel G.

Observation Result Explanation of Observation/Result Organism or Organisms Indicated Gram reaction & Shape Pink rods scattered around the field Negative Pink (red) rods scattered around the field show Gram-negative Escherichia coli, Salmonella Typhimurium, Enterobacter aerogenes Glucose Durham Tube Fermentation Broth is yellow and there is an air bubble in the Durham tube Positive The pH level of the liquid changes and turns yellow, indicating a positive fermentation reaction. Escherichia coli, Salmonella Typhimurium, Enterobacter aerogenes Lactose Durham Tube Fermentation Broth is yellow and there is an air bubble in the Durham tube Positive The tubes are observed for color changes and/or bubble formation to determine if acid and/or gas is formed. The yellow color of the sample and bubble formation in the Durham tube indicate lactose formation. Escherichia coli, Enterobacter aerogenes Mannitol Durham Tube Fermentation Broth is yellow and there is an air bubble in the Durham tube Positive If mannitol is fermented to produce acid, the pH of the indicator changes color from red to yellow along with the production of gas. Escherichia coli, Salmonella Typhimurium, Enterobacter aerogenes Indole Production After the addition of Kovac's reagent, no red ring is visible Negative The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. We detect indole using Kovac's reagent, which reacts with indole to give a red compound. Salmonella Typhimurium, Enterobacter aerogenes, Staphylococcus aureus, Enterococcus faecalis Methyl Red After the addition of Methyl red, the broth is yellow Negative (no mixed acids are produced) Methyl Red is a less sensitive indicator than Phenol Red (which changes to yellow at pH 6.8 and cannot detect the difference between small and large drops in pH). Enterobacter aerogenes Voges-Proskauer After the addition of Barritt's reagent, the broth is pink Positive test results, alcohol produced The Voges-Proskauer (VP) test is used to determine if an organism produces alcohol from glucose fermentation. This test helps us determine if there is a fermentation intermediate product such as acetoin. For this purpose, we use Barritt's reagent to detect that compound. The reagent and acetoin give us a pink/red color when positive. Enterobacter aerogenes Citrate Utilization Agar is blue Positive Some microbes depend on citrate, which is a pure carbon source for them. The ability to use citrate relies on citrate permease, which is an enzyme. This enzyme helps transport citrate across the cell membrane and into the cell. Once the citrate enters, the Krebs Cycle begins and the production of ATP and CO2. Enterobacter aerogenes, Salmonella Typhimurium H2S Production No black color in agar Negative Some microorganisms remove sulfur and reduce it to hydrogen sulfide in the desulfurase process. H2S reacts with the iron in the medium to form a black precipitate. Since there is no black color, there is a negative test and hence no hydrogen sulfide. Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus Motility Agar is turbid throughout Positive Motility is the ability of bacteria to move via the use of flagella. If the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile). Enterobacter aerogenes, Escherichia coli, Salmonella Typhimurium Catalase Bubbles are seen after the addition of hydrogen peroxide Positive H2O2 is a toxic byproduct of aerobic respiration. Usually, bacteria enzymatically break down the hydrogen peroxide to non-toxic H2O and oxygen gas. In a positive result, oxygen gas is observed as bubbles after dropping H2O2 onto growth on the plate. Enterobacter aerogenes, Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli Lab Report for the Identification of Bacteria Associated with the Gastrointestinal Tract Directions: Your gastrointestinal tract unknown is one of the following bacteria: Escherichia coli, Enterobacter aerogenes, Enterococcus faecalis, Salmonella typhimurium, or Staphylococcus aureus. Your lab report will consist of the following: 1. A Table (30 points) Record your observations in the table for each test performed to identify your unknown. In the column labeled "Observations," write what you saw (for example, a color change, the presence or absence of growth, a particular growth pattern). Record your results in the table for each test performed to identify your unknown. In the column labeled "Result," write whether your unknown is either positive or negative for a particular enzyme or metabolic activity. In the column labeled "Explanation of Observation/Result," write a brief explanation of the reaction observed (meaning what reaction took place resulting in an observable change). In the column labeled "Organism(s) Indicated," write the name(s) of all the organism(s) indicated by the result you wrote on your table. You need to include all of the organisms that give the result you listed in (b). For your report, you will submit a typed version of your table. 2. A Written Report You will attach a discussion to your table. Your report is the most important part of the unknown identification process. In your discussion, focus on the results you obtained and how you identified your unknown based on a process of elimination. The discussion portion of your report is to be typed, double-spaced. You will staple the table to the written report (table first followed by the report). Please do not include information about the procedures you conducted. The steps for writing your discussion are: List all possible GI bacteria used in this laboratory exercise. (5 points) Discuss your Gram staining results; describe if your Gram staining results allowed you to eliminate any of the possible bacteria as your unknown. (10 points) Note: once you have eliminated bacteria based on Gram staining, you do not need to continue to eliminate them. Discuss the results for your Durham tube fermentation tests. Describe how you used the three fermentation tubes to rule out certain bacteria. You need to describe phenol red glucose, lactose, and mannitol results as one series of tests; do not describe each sugar fermentation test separately. Be sure to clearly list which bacteria are included and/or excluded based on the series of Durham tube fermentation results and give clear reasons why. (20 points) Note: once you have eliminated bacteria based on Durham tube fermentation, you do not need to continue to eliminate them. Discuss several tests that clearly allowed you to identify your unknown (these are known as definitive tests). For this part of your report, you cannot include Gram staining and phenol red glucose, lactose, and mannitol since you have already discussed them in parts b and c. Give reasons why certain organisms are excluded and how you reached your specific decision on the identity of your unknown. You should describe how you excluded bacteria that remained as your possible unknown after Gram staining and Durham tube fermentation. Do not include a description of all the tests you performed. Include only the most relevant tests and explain how they allowed you to eliminate the remaining bacteria that were not previously excluded and determine the identity of your unknown. (20 points) State your unknown number and the identity of your unknown. (10 points) Conclude your report with a summary statement of how you were able to identify your unknown. (5 points) Based on the result provided in the table, answer the questions for part 2 of the lab and distinguish which bacteria is the unknown.

Suman K.

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