LAB 6

Question 1

What is standard plate count?

Answer 1

• Used to determine the number of mesophilic aerobes (organisms that grow best in moderate temperature).
• It involves taking a sample, serially diluting it aseptically, and then plating out the resulting suspensions to get a countable number of isolated colonies per Petri plate to determine CFU.
• The quantity of these bacteria will affect shelf life and quality of a product and may indicate if a food has been prepared under unsanitary conditions or if the food was transported or stored incorrectly.

Question 2

What are sewage indicator or fecal organisms?

Answer 2

• A common indirect method to predict whether intestinal pathogens are likely to be present is to determine whether the food has been contaminated with fecal matter.
• These are common organisms that are present in very large numbers in the normal gut flora, are restricted to this environment, and are easy to detect.
• If E. coli and E. faecalis are absent, it can be concluded that recent fecal contamination did not take place and that pathogens transmitted by this route are most likely absent.
• Intestinal organisms and pathogens can be tested for directly or after enrichment culture, using the appropriate media.

Question 3

What where the goals of the Lab 6 experiment?

Answer 3

1. Analyze the number of bacteria present per gram of ground beef.
2. Whether fecal indicator E. coli is present and in what quantity.
3. Whether fecal indicator E. faecalis is present after enrichment culture.
4. Whether the pathogen S. aureus is present after enrichment culture.

• In this exercise, the presence of different pathogens and fecal indicator organisms in your ground meat sample be will examined by a series of enrichments followed by a quick series of tests to confirm and complete the identification of the organisms.

Question 4

Describe the steps of Lab 6 for analyzing the number of bacteria present per gram of ground beef.

Answer 4

1. 1g of meat sample is transfered to 99mL of PBS and shaken. This is the 10-1 solution.
2. Solution is diluted to up to 10-5 using serial dilutions.
3. Pour 1mL of each of 10-2, 10-3, 10-4 and 10-5 into separate petri dishes.
4. Pour ~20mL of plate count agar (PCA) onto plates.
5. Allow to solidify for 20 minutes, then incubate at 37 degrees for 24 hours.
6. The following week, calculate the CFU/capsule for each dilution factor. This is the number of bacteria present per gram of ground beef.

Question 5

What are enrichment cultures?

Answer 5

• Provide conducive conditions for specific organisms to grow and inhospitable conditions for others, allowing for the selection of specific bacteria and growth of a small amount of organism into detectable levels.

Question 6

Describe the steps of Lab 6 for analyzing whether fecal indicator E. coli is present and in what quantity.

Answer 6

1. Transfer 200uL of 10-2 solution and spread evenly across an EMB plate using the spread plating technique.
2. Let plates dry for 10 minutes covered upright.
3. Incubate at 37 degrees for 24 hours.
4. Transfer 5mL of 10-2 solution into 5mL of 2X lactose broth.

Question 7

What is the 2X Lactose Broth test?

Answer 7

• This differential medium demonstrates the fermentation of lactose. Acid produced by fermentation pathways reacts with the indicator phenol red to give a yellow colour, and fermentation gases are trapped in the inverted Durham vial.
• E. coli would have acid + gas.

Question 8

Describe the steps of Lab 6 for analyzing whether fecal indicator E. faecalis is present after enrichment culture.

Answer 8

1. Add 5mL of 10-2 solution to 5mL 2X Aside Dextrose Broth.
2. Incubate at 37 degrees for 24 hours.
3. The following week, take a loop full of broth from the azide dextrose broth tube with the most turbidity and streak onto a Kenner Fecal (KF) plate.
4. Incubate at 37 degrees for 24 hours.
5. The following week record plate observations.
6. Aseptically transfer 3 red/pink colonies onto labelled Brain Heart Infusion (BHI) broth and vortex.
7. Incubate in water bath for 2 hours. E. faecalis should be able to survive this.
8. Gram stain an isolated red/pink colony from the KF plate and perform a catalase test.

Question 9

What is the 2X Azide Dextrose Broth test?

Answer 9

• This selective medium contains the respiratory poison azide (N3-) which blocks electron transport along the cytochrome chain and thus inhibits most respiring organisms.
• Streptococci, including Enterococcus spp. grow in this medium because they are aerotolerant anaerobes that never respire, even in the presence of 02.

Question 10

What is a Kenner Fecal (KF) agar?

Answer 10

• Selective medium is designed for the growth of fecal streptococci, including Enterococcus faecalis.
• It contains azide to inhibit aerobic organisms as well as the pH indicator bromcresol purple.
• Fermentation of the sugars, maltose, and lactose, produce acid, causing the pH indicator in the agar to turn yellow.
• Reduction of triphenyl tetrazolium chloride (TIC) dye in the medium gives a deep red colour to the colonies.

Question 11

What is Brain Heart Infusion (BHI) broth?

Answer 11

• Enterococcus faecalis will grow at elevated temperature (45°C) in rich medium such as BHI (Brain Heart Infusion) broth.
• The result will be a turbid yellow solution, from an originally clear yellow solution.

Question 12

Describe the steps of Lab 6 for analyzing whether the pathogen S. aureus is present after enrichment culture.

Answer 12

1. Transfer 5mL of 10-2 solution into Trypticase Soy Broth (TSB) with 7.5%NaCl.
2. Gently vortex and incubate at 37 degrees for 24 hours.
3. The following week streak a loop full of broth onto a BAP plate.
4. Seal with parafilm and incubate at 37 degrees for 24 hours.
5. The following week, recod observations.
6. Transfer an isolated colony into a test tube 0.5mL of citrated plasma.
7. Mix and incubate at 37 degrees for 1 hour.

Question 13

What is Trypticase Soy Broth (TSB)?

Answer 13

• This selective medium contains a high salt concentration, which inhibits most bacteria through dehydration of the cytoplasm and plasmolysis of the cell.
• Staphylococcus spp. are salt tolerant and can maintain cytoplasmic water activity and grow in the presence of up to 10% NaCl.
• Staphylococcus spp. are common, normal inhabitants of human skin where salt concentrations may be high due to perspiration.

Question 14

What is a coagulase test?

Answer 14

• Coagulase is an exotoxin, an extracellular protein released into the environment by pathogenic Staphylococcus aureus. Coagulase is a virulence factor, enabling the bacterium to turn the naturally occurring fibrinogen in blood fluids into fibrin (a clot) thereby immobilizing and protecting the bacteria in a localized infection (e.g. a pimple or boil).
• In this test, the cells are mixed with citrated plasma and, if they the produce coagulase, a precipitate or gel-like fibrin clot is observed.
• Citrate (a chelator of cations such as Ca2+) is added to prevent the normal clotting reaction, which requires Ca2+.