sc246 microbiology. lab: unknown identification microorganisms are the most common and the most...
TRANSCRIPT
SC246 Microbiology
• 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.
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.
Bergey’s Manual The bible for
Microbiology
Resource that provides characteristics of the genus and species of many bacteria
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.
Q2
• What are biochemical tests and why are they performed?
Q3
• Give 2 examples of biochemical tests and what the tests are used for.
Chapter 5 lab Manual
Indole SIM medium Tests for: presence
of the enzyme tryptophanase
Can this enzyme break down tryptophan into NH3 + pyruvic acid + indole
Kovacs reagent
MR-VP test
Methyl Red (MR) Voges-Proskauer test (VP)
Same media is used for both
Different reagents are added to do each test
Methyl Red Used to identify enteric bacteria
based on glucose metabolism Checks for mixed acid fermentation
Reagent- methyl red
Voges-Proskauer Used to identify
enteric bacteria based on glucose metabolism
Checks for the production of acetoin
Reagent- alpha-napthol and KOH
Urea Tests for the
presence of urease If the bacteria have
urease, they can break down urea into NH3 and CO2
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
Citrate Simmons citrate
agar Tests for the
enzyme citrase Green = positive Blue = negative
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?
Staphylococcus aureus or Streptococcus pyogenes??
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
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?
Case #1Following 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.
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
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.