1. Unit 4 Seminar
SC246 Microbiology

2. Lab: Unknown Identification
Microorganisms are the most common and the most numerous group of organisms on Earth. The world of Microbiology revolves around the ability to categorize and identify these microorganisms. The identification is especially important in the medical community to determine the cause of disease and effectively treat patients.
To identify unknown bacteria, Microbiologists use a diverse collection of tests including stains, biochemical tests, and selective and differential media. In general, the identification begins with a Gram stain and continues with a scientific identification process, sometimes dependent upon which identification is suspected. For example, if a child goes to the pediatrician with a sore throat, the doctor might look specifically for Streptococci in the child’s throat.

3. Given the massive number of bacterial species on Earth, you can imagine that all of this information can pile up. For decades, Microbiologists have relied upon a collection of books known as Bergey's Manual. This actually refers to a collection of books that provide detailed information on all recognized species of prokaryotes. Each chapter in Bergey's Manual is written by an expert, contains tables, biochemical test results, and further systematic information that is useful for identification purposes.

4. Bergey’s Manual The bible for Microbiology
Resource that provides characteristics of the genus and species of many bacteria

5. Q1
The Gram stain is typically the first test performed in a microbiology laboratory in the process of identifying an unknown. WHY? Give some examples of instances when a Gram stain would NOT be a useful first step.

6. Q2
What are biochemical tests and why are they performed?

7. Give 2 examples of biochemical tests and what the tests are used for.Q3
Give 2 examples of biochemical tests and what the tests are used for.

8. Chapter 5 lab Manual
Biochemical tests

9. Indole SIM medium Tests for: presence of the enzyme tryptophanase
Can this enzyme break down tryptophan into NH3 + pyruvic acid + indole
Kovacs reagent

10. MR-VP test Methyl Red (MR) Voges-Proskauer test (VP)
Same media is used for both
Different reagents are added to do each test

11. Methyl Red
Used to identify enteric bacteria based on glucose metabolism
Checks for mixed acid fermentation
Reagent- methyl red

12. Voges-Proskauer
Used to identify enteric bacteria based on glucose metabolism
Checks for the production of acetoin
Reagent- alpha-napthol and KOH

13. Urea Tests for the presence of urease
If the bacteria have urease, they can break down urea into NH3 and CO2

14. Carbohydrate Utilization
Tests for fermentation of a sugar and gas production
Durham tube inside tube measures gas production
Red/purple= negative for fermentation
yellow = fermentation
pH indicator
Results should indicate fermentation and gas production

15. Citrate Simmons citrate agar Tests for the enzyme citrase
Green = positive
Blue = negative

16. Q4
Imagine that you are working in a microbiology lab and you are given a sample of bacteria on an agar plate. You are told that it is either Staphylococcus aureus or Streptococcus pyogenes. How would you determine which one the sample contains? Are there any tests you could do to differentiate them?

17. Staphylococcus aureus or Streptococcus pyogenes??

18. Catalase Tests for the enzyme catalase
Catalase breaks down hydrogen peroxide
Most aerobic and facultatively anaerobic bacteria make catalase
Some lack this enzyme (Streptococcus and Micrococcus)
Reagent- hydrogen peroxide
Bubbles = positive

19. Q5
WHY must these steps be followed? Wouldn’t it be easier to just observe the organism under a microscope and make an identification based on observation?

20. Unit 5 assignment

21. Case #1
Following a round of water testing in rural Minnesota, contaminated water was discovered in a stream. The source of the contamination is most likely fecal contamination caused by run-off from a 600-acre dairy farm close to the stream. Heavy rains in the area caused localized flooding and the stream measured water levels well above normal.
After isolating a species of bacteria from the water, the following laboratory tests were completed. The lab notes are listed under laboratory observations. You should record in the results column a positive or negative for each test. To complete the Gram stain results, simply interpret whether it is Gram positive or Gram negative and list the gram reaction and shape. For example: Gram + rods in singles, Gram negative cocci in chains.

22. Laboratory observations
Results
Gram stain
Pink rods
Glucose
Yellow media, gas bubble in durham tube
Indole
Red layer of reagents at the top of the test tube
Methyl Red
Red
Urea
Pinkish-red color
Catalase
Bubbles
Table 1.1

24. References and photo credits
Tortora, G., Funke, B., & Case, C. (2007). Microbiology: An Introduction (8th ed.). San Francisco, CA: Pearson.
Alexander, S., & Strete, D. (2001). Microbiology: A Photographic Atlas for the Laboratory . San Francisco, CA: Benjamin-Cummings.