Microbiology Lab Review: Chapter 14

Lab Review

Chapter 14: Water Testing 2 Commercial Kits

1. [CH-14-01] What is the pH of un-inoculated phenol red lactose broth? What is the pH after incubation phenol red lactose broth with Escherichia coli, Enterobacter aerogenes, Bacillus subtilis?
   pH of un-inoculated phenol red lactose broth is neutral and therefore appears light red in the tube.
   pH after E. coli incubation: Below 6.8 (yellow, gas)
   pH after E. aerogenes incubation: 6.8-7.4 (red, no change)
   pH after B. subtilis incubation: Below 6.8 (yellow)

2. [CH-14-02] How do you judge the change of pH in phenol red broth? –

Look for the color change in the broth as caused by the indicator, phenol red.
pH below 6.8 = indicator turns yellow.
pH 6.8-7.4 = indicator is red.
pH above 7.4 = indicator is pink/magenta.

3. [CH-14-03] How could you distinguish the growth of Escherichia coli from growth of Enterobacter aerogenes on phenol red lactose broth? –

E. coli is a strong fermenter of lactose and therefore producing BOTH acid and gas changing the tube yellow with gas present.

E. aerogenes also ferments lactose and produces gas, but not to the same degree that E. coli do so.

4. [CH-14-04] You were given two test-tube with cultures Escherichia coli and Bacillus subtilis grown on phenol red lactose broth. Unfortunately, labels came off the tubes. How can you determine which test-tube contains what culture without running any additional tests?
   E. coli is a strong fermenter of lactose, producing gas and acid. Turns the indicator solution yellow.
   
   B. subtilis does not ferment lactose, instead breaks down proteins available in the solution increasing the pH and turning the pH indicator pink.

5. [CH-14-05] Bacillus subtilis is obligate aerobe. What is the reason for pH to go up when this bacterium is grown on phenol red lactose broth? –

Since the bacteria cannot digest lactose, it turns to the digestion of proteins from the pancreatic digest of casein. This protein digestion leads to the amino acids that must be deaminated with ammonia production before it can be used as an energy source. The process increases pH, making the solution basic (color pink/magenta).

6. [CH-14-06] Bacillus subtilis is obligate aerobe. Pink color develops when B. subtilis is grown on phenol red lactose broth. What color of the medium would you expect if this bacterium is grown on phenol red glucose broth? –

Glucose is an easily fermented sugar, which would lead to the production of acid (fermentation product) that would lower the pH turning the color yellow.

7. [CH-14-07] Explain the term coliform. What is the difference between a fecal and non-fecal coliform? –

Coliform: Rod-shaped/cocobacilli, facultative anaerobic/aerobic, gram-negative bacteria capable of fermenting lactose.

            - Fecal: Usually cannot survive long outside of human or animal intestines (non-free living organisms).

            - Non-fecal: Can survive outside of humans or animal intestines (free-living organisms).

8. [CH-14-08] What is meant by the free-living organism? Which of the coliforms are free-living? Which of the coliforms are not free-living? –

Free-living organisms refer to the ability of the bacteria to growth outside of humans and animals in the environment.
Free-living = Citrobacter, Enterobacter, Klebsiella
Non-free living = E. coli

9. [CH-14-09] Why is not water tested for the presence of pathogens but rather for the presence of indicator organisms (Escherichia coli)?
   E. coli commonly present in feces and is a very distinct organism making it the perfect indicator organism of fecal contamination. Testing for E. coli can be done on an assortment of cheap media. (Ex: EMB agar, MacConkey)

10. [CH-14-10] How coliforms and non-coliforms (nonfecal and fecal) are distinguished on ColiScan EasyGel plates? –

Fecal coliforms: Formation of dark blue or purple colonies (has enzyme glucuronidase).

Non-fecal coliforms: Formation of dark red or pink colonies (has enzyme galactosidase).