Lab 5-2 (O/F Glucose Test) "Dodger" Test

Facultative Anaerobes

can live with or without oxygen

What is the purpose of lab 5-2?

to identify organisms which are capable of aerobic respiration (oxidation) or fermentation or BOTH using O/F glucose media.

Interpret results for Test Tube A (oil tube) in the picture

Neutral pH (green), Negative for fermentation,

Since the same organism inoculated in those two test tube is negative for fermentation and negative for aerobic respiration, what is the oxygen requirement? Double negative result

No oxygen requirement to report. Organism can't metabolize glucose which is a rare scenario.

Since the same organism inoculated in those two test tube is negative for fermentation and positive for aerobic respiration, what is the oxygen requirement?

Obligate aerobe because it needs oxygen. It couldn't ferment because the bacteria can't not live in a no oxygen environment and it was positive for aerobic respiration meaning it used oxygen.

What do you call an organism (by its oxygen requirements) that can oxidize the sugar but not ferment it?

Obligate aerobes

Why is mineral oil added to one of the test tubes?

Oil is added to give the bacteria an anaerobic condition. For fermentation to occur we need to eliminate the oxygen in the tube.

What bacteria did we inoculate that give those results in the picture?

P. aeruginosa

What two catabolic processes does this experiment differentiate between?

fermentation pathway and cellular/aerobic respiration pathway

Give the gram reaction and morphology (shape) of the two organisms used in this experiment

- Escherichia o are gram negative and rod shape can be single or in pairs. - Pseudomonas o are gram negatives and straight shaped or slightly curved rods.

Write out the name of the section or group in Bergey 's Manual to which each organism belongs. Explain your "expected" results for these two organisms.

- Escherichia is in group 5 (subgroup 1) and we expected them to be part of the E. family, gram negatives, facultative anaerobes and rod shape. - Pseudomonas is in group 4 (subgroup 4a) and we expect our results to be gram negatives, aerobic/ microaerophile, rod and cocci shape.

Why is a high sugar to peptone ratio used in the medium?

We need a lot of sugar for our organisms to use either the fermentation pathway or aerobic respiration pathway. On the other hand, we use a little bit of peptone because we don't want our organism to make a lot of ammomia because then it will interfere with our pathways we are trying to test in this experiment.

Why is glucose the focal point for this lab?

We want to see if the organisms use it for either the fermentation pathway OR aerobic respiration. Sugar is the starting point for each pathway.

Can fermentation occur in the unsealed/no mineral test tubes?

Yes, fermentation can still occur in an unsealed/ no mineral test for microbes that are considered Aerotolerant anaerobe which is not common. They sometimes ignore the oxygen. The fermentation will be seen at the bottom of the test tube NOT top portion. On the other hand, fermentation cannot occur if the microbe is NOT considered aerotolerance anaerobe. For fermentation to occur, no oxygen needs to be present which is why it cannot ferment in an unsealed/no mineral test tube.

What are the ingredients for this lab?

Glucose, peptone, low % of agar, Bromothymol blue

What would be worse for a patient infected with an organism; to be infected with an organism that turns both the sealed and unsealed tubes yellow OR to be infected with an organism that only turns the unsealed tube yellow? Explain why.

It would be worse if we had a person infected with a microbe that give both sealed and unsealed yellow results because they can use both options to make their energy and grow.

Aerotolerant anaerobe

do not utilize oxygen but can survive and grow in its presence

RESULTS: . How would you interpret a yellow result in BOTH the sealed and unsealed tubes? (What pathways were used AND what are the oxygen requirements?)

- Oil/ F tube (sealed): Positive for fermentation, positive for acid and positive for motility. o They carried out the fermentation pathway and they are considered facultative anaerobebecause in this experiment we used E. Coli who had a double yellow result for both sealed and unsealed tube. We know that E. coli who is part of the E. family and they are all considered Facultative anaerobe. - O/F tube (unsealed): Positive for fermentation, Positive for aerobic respiration and positive for motility. o They carried out both Fermentation and Aerobic pathway and they are also considered Facultative anaerobe. They prefer oxygen but can live without it which is why we get both pathways. Another possible oxygen requirement for the bottom of the test tube is Aerotolerant anaerobe.

What color do we want to see for a positive result in fermentation and aerobic respiration?

Bright orange or Bright yellow

What is our pH indicator?

Bromothymol Blue. Bright orange or Bright yellow for Acidic, Green is neutral, Blue is for basic.

What is the difference between cellular respiration and fermentation. What is the purpose of performing these two metabolic processes?

Cellular respiration requires oxygen in its pathway and fermentation cannot have oxygen in its pathway. The purpose of performing the two metabolic pathways is to make energy (ATP).

Since the same organism inoculated in those two test tube is positive for fermentation and positive for aerobic respiration, what is the oxygen requirement?

Facultative Anaerobe because the organism can live with or without oxygen. HOWEVER, for test tube B, it could of possibly fermented at the bottom of the test tube. So we can possibly have a Aerotolerant anaerobe do not utilize oxygen but can survive and grow in its presence of it. Aerotolerant is a possibility ONLY if both test tubes can ferment.

What do you call an organism (by its oxygen requirements) that can ferment AND oxidize the sugar in this experiment?

Facultative Anaerobes

What color does it change when the pH is low? When high?

For an organism with a low pH (acidic) we get a yellow color and for an organism that has a high pH (basic) gives a blue color.

RESULTS: If an organism can grow in the media (with and without the mineral oil) and turns the media blue, is the organism positive or negative for oxidation or fermentation? Why? How were the bacteria able to grow?

If we get a blue media, it means the environment is basic. We are both negative for oxidation and negative for fermentation because to get those two pathways we need an acidic environment. The bacteria were able to grow because it ate the peptone and made ammonia waste.

Which other experiment will we have we performed that tests for motility?

Lab 5-20 (SIMS)

Interpret results for Test tube B (oil tube) in the picture

Negative for Acid, negative for fermentation

Interpret results for Test tube A in the picture

Negative for acid, Basic pH which is positive for ammonia waste, negative for Aerobic respiration

Interpret results for Test Tube A (oil tube) in the picture

Positive for Acid (yellow), Positive for fermentation, positive for motility

Interpret results for Test Tube B in the picture

Positive for acid (yellow) upper portion , Positive for Aerobic/oxidation Respiration, positive for motility

Interpret results for Test Tube B in the picture

Positive for acid, Positive for aerobic/oxidation respiration, OR positive for fermentation (bottom of the tube), positive for motility

What is the purpose of using the low agar concentration in this experiment?

The low % of agar helps promote motility to see if organism spread away from the stabs.

RESULTS: Describe the appearance of your test tubes if you inoculated an obligate aerobe in this experiment.

Tube w/ Oil (sealed): The test tube would be all green. Obligate aerobes are dependent on oxygen to survive. The oil in the tube deprives the oxygen so they can't grow in this tube. Tube without Oil (Unsealed): The test tube would have some orange/yellow on top and green at the bottom.

Obligate aerobes

require oxygen