Unit 4 Seminar: Unknown Identification

1. Fundamentals of Microbiology Unit 4 Seminar: Unknown Identification Evelyn I. Milian Instructor 2011

2. Questions Assigned in Unit 4 Seminar Page • Before seminar, take a look at the introduction in Bergey’s Manual. • http://www.archive.org/stream/bergeysmanualofd1957amer#page/n21/mode/2up • 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. • What are biochemical tests and why are they performed? • Give 2 examples of biochemical tests and what the tests are used for. • 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 aureusor Streptococcus pyogenes. How would you determine which one the sample contains? Are there any tests you could do to differentiatethem? • 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? Evelyn I. Milian - Instructor

3. Identification of Unknown Microorganisms • Microorganisms are the most common and the most numerous group of organisms on Earth. The world of Microbiology revolves around the ability to categorizeand identifythese 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. Evelyn I. Milian - Instructor

4. Identification of Unknown Microorganisms • 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 Manualis written by an expert, contains tables, biochemical test results, and further systematic information that is useful for identification purposes. Evelyn I. Milian - Instructor

5. Classification Schemes for Microorganisms • A classification scheme provides a list of characteristics and a means for comparison to aid in the identification of an organism. Once an organism is identified, it can be placed into a previously devised classification scheme (Tortora, 2010). • Microorganisms are identified for practical purposes—for example, to determine an appropriate treatment for an infection. They are not necessarily identified by the same techniques by which they are classified. Most identification procedures are easily performed in a laboratory and use as few procedures or tests as possible. • Protozoa, parasitic worms, and fungi can usually be identified microscopically. Most prokaryotic organisms do not have distinguishing morphological features or even much variation in size and shape. Consequently, microbiologists have developed a variety of methods to test metabolic reactions and other characteristics to identify prokaryotes. Evelyn I. Milian - Instructor

6. Classification Systems in the Prokaryotes • The original classification system was based on traits such as morphology (shape), cell wall composition, motility, variations in cellular arrangement, growth characteristics, and habitat. • Today, classification schemes are based mainly on genetic and molecular traits (such as comparing sequence of nitrogen bases in ribosomal RNA) and their evolutionary relationships (phylogeny). New groups are being identified and studied. • Prokaryotes are divided into two domains: Archaea and Bacteria. • Archaea share certain traits with bacteria and other traits with eukaryotes. • Bacteria and Archaea were grouped in Kingdom Monera in the past; now they are separate domains because of important molecular differences. Evelyn I. Milian - Instructor

7. Classification Systems in the Prokaryotes • Definitive published source for bacterial classification: • Bergey’s Manual – since 1923. • The basis for early classification was the phenotypic traits of bacteria such as morphology, cell wall composition, motility, growth features such as oxygen use, and biochemical reactions, including staining. • Bergey’s Manual of Systematic Bacteriology: Current version combines phenotypic information with molecular characteristics such as rRNA sequencing. Evelyn I. Milian - Instructor

8. Classification Systems in the Prokaryotes:Diagnostic Scheme • In clinical microbiology it is more useful to use a more informal system to classify bacterial species based on their phenotypic (observable) characteristics. • This system is restricted to bacterial disease agents and is based on readily accessible morphological and physiological tests rather than on phylogenetic (evolutionary) relationships. • It also divides the bacteria into gram-positive, gram-negative, and those without cell walls. • It subgroups bacteria according to cell shape, arrangement, and certain physiological traits such as oxygen usage. • Aerobic bacteria use oxygen, anaerobic bacteria do not use oxygen, and facultative bacteria may or may not use oxygen. Evelyn I. Milian - Instructor

9. Question 1: Gram Stain as a First Test • 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. Evelyn I. Milian - Instructor

10. Question 1: Gram Stain as a First Test • The Gram stain helps differentiate bacteria into two broad groups based on the structure of the bacterial cell wall. Most bacteria possess a cell wall that contains either a thick peptidoglycan layer (Gram-positive; retain crystal violet, a purple stain) or a thin peptidoglycan layer with an additional lipopolysaccharide layer (Gram-negative; retain safranin, a red stain). • The Gram stain is not be useful in identifying bacteria without a cell wall or with unusual cell walls. Evelyn I. Milian - Instructor

11. Questions 2 and 3: Biochemical Tests • What are biochemical tests and why are they performed? • Give 2 examples of biochemical tests and what the tests are used for. (Chapter 5, Lab Manual) Evelyn I. Milian - Instructor

12. Biochemical Tests for the Identification of Bacteria • Simple, differential, and structural stains even if combined with cultivation and observation of colony characteristics, are not sufficient for the identification of bacterial isolates. Results of staining and cultivation must be combined with the results from biochemical tests. • Biochemical testsevaluate the metabolic properties of an isolate, which are unique for each species. • A combination of biochemical tests can be used to determine the biochemical pattern for an isolate. This enables the identification of an isolate using an identification scheme. (Alexander, 2001) Evelyn I. Milian - Instructor

13. Biochemical Tests • Biochemical activities are widely used to differentiate bacteria. • Even closely related bacteria can usually be separated into distinct species by subjecting them to biochemical tests, such as one to determine their ability to ferment an assortment of selected carbohydrates. Evelyn I. Milian - Instructor

14. In health care, morphology and differential staining are important in determining the proper treatment for microbial diseases. • A clinician completes the form to identify the sample and specific tests. In this case, a genitourinary sample will be examined for sexually transmitted infections. Biochemical Tests A clinical microbiology lab report form Evelyn I. Milian - Instructor

15. Biochemical Tests: Carbohydrate Utilization • Examples: phenol red broth; purple broth • Tests used to determine whether an organism has the ability to ferment various carbohydrates (sugars) and produce acidand gas. • Inverted durham tube inside test tube shows gas production. • pH indicator:acid lowers pH causing color change • Yellow= fermentation (acid) • Purple or red = negative for fermentation • Bubbles in durham tube = gas (from fermentation) Evelyn I. Milian - Instructor

16. Biochemical Tests:Citrate Utilization • Tests for the enzyme citrase, produced by some bacteria such as Enterobacter aerogenes and Salmonella typhimurium, but not by others, such as Escherichia coli and Shigella flexneri. • Medium: Simmon’s citrate agar, containing citrate as the only carbon source and pH indicator bromthymol blue (blue if pH increases due to alkaline products from citrate metabolism) • Blue = positive • Green = negative Evelyn I. Milian - Instructor

17. Biochemical Tests: Indole Production • Tests for presence of enzyme tryptophanase, which breaks down amino acid tryptophan to form ammonia, pyruvic acid and indole. • Medium: SIM, also used to detect motility and hydrogen sulfide production. • Reagent: Kovac’s ( 5 drops added to culture after incubation); reacts with indole to form a red color (in alcohol layer of Kovac’s). • Red = positive • No red = negative Examples: Negative = Enterobacter aerogenes Positive = Escherichia coli Evelyn I. Milian - Instructor

18. Biochemical Tests: MR-VP Test • Methyl Red–Voges Proskauer: combination medium used for two tests for enteric bacteria; different reagents are added to do each test. • Methyl Red (MR): To detect enteric bacteria capable of performing a mixed acid fermentation, lowering the pH. • Methyl red indicator added after incubation. • Positive = red (stable acids produced) • Negative = no color change (yellow/orange) (neutral end products) • Examples: • Enterobacter aerogenes • Serratiamarcescens • Examples: • Escherichia coli • Proteus vulgaris Evelyn I. Milian - Instructor

19. Biochemical Tests: MR-VP Test • Voges Proskauer: for organisms able to ferment glucose and convert acid products to acetoin and 2,3-butanediol. • Voges-Proskauer reagents (alpha-naphthol and KOH) added after incubation. • Positive = red (2,3-butanediol fermentation; acetoin produced) • Negative = no color change or copper color Example: Enterobacter aerogenes Example: Escherichia coli Evelyn I. Milian - Instructor

20. Biochemical Tests: Urea Utilization • Tests for presence of urease, an enzyme that breaks down urea (a product of amino acid metabolism) into ammonia (alkaline product) and carbon dioxide. • Distinguish Proteus from other enteric bacteria. • pH indicator: phenol red; changes to pink when pH increases. • Positive = pink • Negative = no color change or yellow (from acid products) Examples: Positive: Proteus, Morganella, Providencia (rapid urease-positive) Negative: Escherichia coli Evelyn I. Milian - Instructor

21. Questions 4 and 5: Biochemical Tests • 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 aureusor Streptococcus pyogenes. How would you determine which one the sample contains? Are there any tests you could do to differentiatethem? • 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? Evelyn I. Milian - Instructor

22. Biochemical Tests:Catalase Test • Detects the enzyme catalase, possessed by most aerobic and facultatively anaerobic bacteria. • Catalase breaks down hydrogen peroxide produced during aerobic respiration. If H2O2 accumulates in the cell, it becomes toxic. • Some bacteria lack this enzyme: Streptococcus , Enterococcus • Reagent: 3% hydrogen peroxide added to 18-24 hour culture on an agar slant or glass slide • Bubbles = positive (formed within seconds from breakdown of hydrogen peroxide into water and oxygen). Example: Staphylococcus Evelyn I. Milian - Instructor

23. Unit 5 Assignment – Example: 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. Evelyn I. Milian - Instructor

24. Unit 5 Assignment – Example: CASE #1 • Interpret the tests in Table 1.1 and answer the questions below: • What is the result of the Gram stain? • Does this organism ferment glucose? How can you tell? • Would the catalase test be helpful to identify your unknown? Why or why not. • Using the unknown identification chart, compare the results recorded above. What is the genus and species of the unknown organism? • If this water was ingested by humans, what type of infection could result? What types of signs and symptoms would an infected individual display? • Is the only source of contamination the dairy farm? Give another possible explanation for the water contamination. Evelyn I. Milian - Instructor

25. Unknown Bacterium Identification Chart Evelyn I. Milian - Instructor

26. References • Alexander, S.K., & Strete, D. (2001). Microbiology: A Photographic Atlas for the Laboratory. Pearson Education, Inc.-Benjamin-Cummings. CA, USA. • Alters, S. & Alters, B.(2006). Biology, Understanding Life. John Wiley & Sons, Inc. NJ, USA. • Audesirk, T.; Audesirk, G. & Byers, B.E. (2005). Biology: Life on Earth. Seventh Edition. Pearson Education, Inc.-Prentice Hall. NJ, USA. • Black, J.G. (2005). Microbiology, Principles and Explorations. Sixth Edition. John Wiley & Sons, Inc. NJ, USA. www.wiley.com/college/black. • Campbell, N.A.; Reece, J.B., et al. (2008). Biology.Eighth Edition. Pearson Education, Inc.-Pearson Benjamin Cummings. CA, USA. • Cowan, M.K.; Talaro, K. P. (2009). Microbiology A Systems Approach.Second Edition. The McGraw-Hill Companies, Inc. NY, USA. www.mhhe.com/cowan2e • Dennis Kunkel Microscopy, Inc.(2010). http://www.denniskunkel.com • Leboffe, M.J. & Pierce, B.E. (2010). Microbiology Laboratory Theory and Application.Third Edition. Morton Publishing. Englewood, CO; USA. • Mader, S.S.(2010). Biology.Tenth Edition. The McGraw-Hill Companies, Inc. NY, USA. • Tortora, G. J.; Funke, B.R.; Case, C.L. (2010). Microbiology An Introduction.Tenth Edition. Pearson Education, Inc.-Benjamin Cummings; CA, USA. www.microbiologyplace.com. Evelyn I. Milian - Instructor