Question

1. Someone new is preparing media for lab and adds buffers to the EMB agar that raise the pH to 7.8. The starting pH of EMB plates should be 6.9 – 7.3. No one is aware of the change. The EMB plates are inoculated for identification of unknown bacteria. After the appropriate incubation time, the results are interpreted. A. Discuss the consequences of the change in starting pH to the interpretation of the results. (The answer is not that no organisms could grow.) B. Is that a false negative, or a false positive?

2.  Helpful information to answer question #2:

Diagnostic tests of any kind are only as reliable as their sensitivity and specificity allow. A perfect test would be positive 100% of the time only for a positive condition, and negative 100% of the time only for a negative condition. In other words, it would never falsely identify a positive as a negative (called a "false negative") and would never falsely identify a negative as a positive (called a "false positive"). It would never miss a positive and never indicate that a negative condition is positive when it isn't. Unfortunately, tests are normally not perfect. Sensitivity and specificity are probabilities about the results obtained with tests.

Sensitivity refers to the ability of tests to identify a "positive" as "positive". A highly sensitive test gives the True Positive Rate. Highly sensitive tests rarely miss a positive, because they do not incorrectly identify a positive as negative (that would be a "false negative"). Therefore, they produce few false negative results. In medicine, a highly sensitive test is a test that identifies the number of people with a disease who will test positive for the disease.  So a test with 90% sensitivity will identify 90% of the patients with the disease. It will miss 10%. To remember, consider that the "n" in sensitivity refers to giving no (or few) "false negative" results.

Specificity refers to the ability of a test to identify a "negative" as "negative". A highly specific test gives the True Negative Rate. Highly specific media produce few false positive results; the test will not incorrectly identify a negative as a positive (that would be a false positive). In medicine, a test that is 90% specific will identify 90% of patients who do not have the disease and miss 10% of patients who do not have the disease (it will falsely indicate that they are positive). Remember that the "p" in specificity refers to giving no (or few) "false positive" results.

For a diagnostic test checking patients for a disease, the highly sensitive test would be positive for every person with the disease. It would not miss any positive cases (no false negatives). The highly specific test would not be positive for a patient who does not have the disease. It would never indicate that a patient had a disease when they did not (no false positives).

Unfortunately, the two are frequently inversely proportional. When tests have high sensitivity, they often have low specificity. In other words, if a test does not miss any positive cases, it often reports a number of cases that are false positive.

Now for the question:

Would the change in starting pH of EMB agar discussed in question #1 affect the sensitivity of those EMB plates, or would it affect their specificity? Explain why.

3. Your interpretation of an MSA plate is +/-. A. What have you learned about the organism?

4. Jonelle’s TSI tube has a yellow slant and a black butt with several cracks. Jonathan’s TSI tube has a pink slant and a yellow butt.   

A. Which carbohydrate(s) can Jonelle’s organism ferment? How do you know that?

B. Which carbohydrate(s) can Jonathan’s ferment? How do you know that?

C. What do the cracks in Jonelle’s tube indicate? Why do they form?

D. For an organism to produce H2S, what must occur first in the butt of the TSI tube?  

5. After inoculating them with her unknown, Letitia’s Phenyl Red Glucose, Lactose, and Sucrose tubes are all yellow. The Durham tubes are filled with liquid. Her TSI tube results are K/H2S. The EMB plate result is +/+. Do all results agree? Explain why, or why not.   Be thorough!

Answer 1

1.- A) This agar has a pH mechanism to indicate lactose fermentation. Normally, lactose fermentation brings pH into acidic values, this ends up staining the colonies with a dark color, if lactose is not fermented then acidic values are not reached and the colonies stay transparent. But in this case the pH is just initially too alkaline, this means that even a positive fermentation will not be able to reach acidic values and will be presented as a negative when it was actually a positive

B) This is a false negative

2.- In our example in question one we affected sensitivity, that is because we affected the ability of the test to show a positive when it was positive, and it showed a false negative instead

Your interpretation of an MSA plate is +/-. A. What have you learned about the organism?

MSA agar is selective media that allows only cocci to grow, and its mannitol and phenol red allow to differentiate Sthaphylococci since theya re the only ones that ferment mannitol. So if this strain is +/- then it was able to grow in salty condition but was not able to ferment mannitol

A) The bacteria ferments glucose and lactose and produces H2S

B) The bacteria ferments only glucose

C) The cracks indicate gas production, when gas is formed they increase pressure and the agar cracks

D) The but must be acidic since sulfur reduction requires acidic conditions, to obtain acidic conditions you require for a sugar to be fermented

- Do all results agree?

The first phenol red tubes indicate the strain ferments all glucose, lactose and sucrose. The durham tubes indicate no gas production. The TSI shows different results because it is indicating the strain only ferments glucose and produces H2S, but not sucrose or lactose. The EMB agar is telling us the strain can ferment lactose.

The results do not agree between different tests