1. Unit 4 Seminar: Unknown Identification
Fundamentals of Microbiology
Microbiology
4/16/2017
Unit 4 Seminar: Unknown Identification
Hi everyone! Welcome to seminar! It is great to have you all here today. We will be starting up in a few minutes.
While you are waiting to get started this evening, please take this opportunity to chat and adjust your speaker volumes. Think about the topic for today and the questions assigned in the seminar page.
If you encounter any technical issues accessing a KHE Seminar, please call KU ACE Help You may also KU ACE Help at
Evelyn I. Milian
Instructor
2011
Evelyn I. Milian - Instructor

2. Questions Assigned in Unit 4 Seminar Page
Microbiology
4/16/2017
Questions Assigned in Unit 4 Seminar Page
Before seminar, take a look at the introduction in Bergey’s Manual.
ode/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 aureus or Streptococcus pyogenes. How would you determine which one the sample contains? Are there any tests you could do to differentiate them?
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?
Hi everyone! Welcome to seminar! It is great to have you all here today. We will be starting up in a few minutes.
While you are waiting to get started this evening, please take this opportunity to chat and adjust your speaker volumes. Think about the topic for today and the questions assigned in the seminar page.
If you encounter any technical issues accessing a KHE Seminar, please call KU ACE Help You may also KU ACE Help at
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

3. Identification of Unknown Microorganisms
Microbiology
4/16/2017
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 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.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

4. Identification of Unknown Microorganisms
Microbiology
4/16/2017
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 Manual is written by an expert, contains tables, biochemical test results, and further systematic information that is useful for identification purposes.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

5. Classification Schemes for Microorganisms
Microbiology
4/16/2017
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.
TAXONOMIC SCHEME
Bergey’s Manual of Determinative Bacteriology, is based entirely on phenotypic characteristics; it categorizes bacteria by traits commonly assayed in clinical, teaching, and research labs. It organizes prokaryotes into four major groups based upon the nature of the cell wall:
Gracilicutes: Bacteria with gram-negative cell walls; thin-skinned.
Firmicutes: Bacteria with gram-positive cell walls; thick and strong.
Tenericutes: Bacteria without cell walls; soft.
Mendosicutes: Archaea (archaebacteria); primitive prokaryotes with unusual cell walls and nutritional habits.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

6. Classification Systems in the Prokaryotes
Microbiology
4/16/2017
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.
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.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

7. Classification Systems in the Prokaryotes
Microbiology
4/16/2017
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.
CHAPTER 10 – TORTORA
Bergey’s Manual of Determinative Bacteriology has been a widely used reference since the first edition was published in 1923.
Bergey’s Manual of Determinative Bacteriology has been a widely used reference since the first edition was published in The American bacteriologist David Bergey was chairman of the group who compiled information on the known bacteria from articles published in scientific journals. Bergey’s Manual of Determinative Bacteriology (9th ed., 1994) does not classify bacteria according to evolutionary relatedness but instead provides identification (determinative) schemes based on such criteria as cell wall composition, morphology, differential staining, oxygen requirements, and biochemical testing.* The majority of Bacteria and Archaea have not been cultured, and scientists estimate that only 1% of these microbes have been discovered.Medical microbiology (the branch of microbiology dealing with human pathogens) has dominated the interest in microbes, and this interest is reflected in many identification schemes. However, to put the pathogenic properties of bacteria in perspective, of the more than 2600 species listed in the Approved Lists of Bacterial Names, fewer than 10% are human pathogens.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

8. Classification Systems in the Prokaryotes: Diagnostic Scheme
Microbiology
4/16/2017
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.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

9. Question 1: Gram Stain as a First Test
Microbiology
4/16/2017
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.
THE GRAM STAIN (Tortora, 10 Ed., Chapter 3)
Differential Stains
Unlike simple stains, differential stains react differently with different kinds of bacteria and thus can be used to distinguish them. The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain.
Gram Stain
The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is one of the most useful staining procedures because it classifies bacteria into two large groups: gram-positive and gram-negative.
In this procedure (Figure 3.12a),
A heat-fixed smear is covered with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain.
After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant.When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.
Next, the slide is washed with alcohol or an alcohol-acetone solution. This solution is a decolorizing agent, which removes the purple from the cells of some species but not from others.
The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye. The smear is washed again, blotted dry, and examined microscopically.
Counterstain: Stains that have a contrasting color to the primary stain; for example: safranin.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

10. Question 1: Gram Stain as a First Test
Microbiology
4/16/2017
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.
CELL WALL
It surrounds the underlying plasma (cytoplasmic) membrane (the plasma membrane is involved in passage of materials into and out of the cell, among other functions).
Differential Staining (Chapter 10, Tortora)
Recall from Chapter 3 that one of the first steps in identifying bacteria is differential staining.Most bacteria are either gram-positive or gram-negative. Other differential stains, such as the acid-fast stain, can be useful for a more limited group of microorganisms. Recall that these stains are based on the chemical composition of cell walls and therefore are not useful in identifying either the wallless bacteria or the archaea with unusual walls. Microscopic examination of a Gram stain or an acid-fast stain is used to obtain information quickly in the clinical environment.
2011
Evelyn I. Milian - Instructor
Evelyn I. Milian - Instructor

11. Questions 2 and 3: Biochemical Tests
Microbiology
4/16/2017
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)
2011
Evelyn I. Milian - Instructor
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 tests evaluate 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)
2011
Evelyn I. Milian - Instructor

13. Evelyn I. Milian - Instructor
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.
2011
Evelyn I. Milian - Instructor

14. A clinical microbiology lab report form
Biochemical Tests
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.
A clinical microbiology lab report form
2011
Evelyn I. Milian - Instructor

15. Biochemical Tests: Carbohydrate Utilization
Microbiology
4/16/2017
Biochemical Tests: Carbohydrate Utilization
Tests used to determine whether an organism has the ability to ferment various carbohydrates (sugars) and produce acid and 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)
CARBOHYDRATE UTILIZATION (From Prof. Annissa Furr’s presentation – Sept. 2011)
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
Examples: phenol red broth; purple broth
2011
Evelyn I. Milian - Instructor
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
2011
Evelyn I. Milian - Instructor

17. Biochemical Tests: Indole Production
Microbiology
4/16/2017
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
2011
Evelyn I. Milian - Instructor
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
Serratia marcescens
Examples:
Escherichia coli
Proteus vulgaris
2011
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
2011
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
2011
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 aureus or Streptococcus pyogenes. How would you determine which one the sample contains? Are there any tests you could do to differentiate them?
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?
2011
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 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.
2011
Evelyn I. Milian - Instructor

24. Unit 5 Assignment – Example: CASE #1
TABLE 1.1
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
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.
2011
Evelyn I. Milian - Instructor

25. Unknown Bacterium Identification Chart
2011
Evelyn I. Milian - Instructor

26. Evelyn I. Milian - Instructor
Microbiology
2010
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.
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.
Dennis Kunkel Microscopy, Inc. (2010).
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.
OTHER REFERENCES:
Alters, Sandra & Alters, Brian. (2006). Biology, Understanding Life. John Wiley & Sons, Inc. NJ, USA.
Belk, Colleen; Borden, Virginia. (2007). Biology, Science for Life. Second Edition. Pearson Education, Inc- Prentice Hall. NJ, USA.
Brooker, Robert J.; Widmaier, Eric P.; Graham, Linda E.; Stiling, Peter D. (2008). Biology. The McGraw-Hill Companies, Inc. NY, USA.
Dennis Kunkel Microscopy, Inc. (2010).
Mader, Sylvia S. (2007). Essentials of Biology. The McGraw-Hill Companies, Inc. NY, USA.
Marieb, Elaine N. (2006). Essentials of Human Anatomy & Physiology. Eighth Edition. Pearson Education, Inc., publishing as Benjamin Cummings. CA, USA.
Presson, Joelle & Jenner, Jan. (2008). Biology, Dimensions of Life. The McGraw-Hill Companies, Inc. NY, USA.
Solomon, Eldra; Berg, Linda; Martin, Diana W. (2008). Biology. Eighth Edition. Cengage Learning. OH, USA.
Tortora, Gerard J.; Derrickson, Bryan. (2006). Principles of Anatomy and Physiology. Eleventh Edition. John Wiley & Sons, Inc. NJ, USA.
Tortora, Gerard J.; Funke, Berdell R.; Case, Christine L. (2007). Microbiology An Introduction. Ninth Edition. Pearson Education, Inc.-Benjamin Cummings; CA, USA.
OLD REFERENCES:
Atlas, Ronald M. & Bartha, Richard. (1998). Microbial Ecology – Fundamentals and Applications. Fourth Edition. Benjamin/Cummings Publishing Company, Inc. CA, USA.
Benson, Harold J. (2002). Microbiological Applications: Laboratory Manual in General Microbiology. Eighth Edition. The McGraw-Hill Companies; NY, USA.
Maier, Raina M.; Pepper, Ian L. & Gerba, Charles P. (2000). Environmental Microbiology. Academic Press- Elsevier. CA, USA.
Prescott, Lansing M.; Harley, John P.; Klein, Donald A. (1996). Microbiology. Third Edition. Wm. C. Brown Publishers; IA, USA.
2011
Evelyn I. Milian - Instructor
Prof. Evelyn I. Milian