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

Unit 4 Seminar: Unknown Identification

Evelyn I. MilianInstructor

2011

Fundamentals of Microbiology

Microbiology: Unit 4 Seminar – Unknown Identification

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/m

ode/2up

1. 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.

2. What are biochemical tests and why are they performed?

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

4. 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?

5. 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 2


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.



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.



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.



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

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.



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.



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.



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.



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)



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)



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.



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.

2011 14

A clinical microbiology lab report form

Evelyn I. Milian - Instructor


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 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)


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



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 aerogenesPositive = Escherichia coli


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:1)Escherichia coli2)Proteus vulgaris

Examples:1)Enterobacter aerogenes2)Serratia marcescens


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:Escherichia coli

Example:Enterobacter aerogenes


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


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?



Biochemical Tests:Catalase Test

Evelyn I. Milian - Instructor 22

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


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.



Unit 5 Assignment – Example: CASE #1TABLE 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

1. Interpret the tests in Table 1.1 and answer the questions below:a. What is the result of the Gram stain? b. Does this organism ferment glucose? How can you tell?c. Would the catalase test be helpful to identify your unknown? Why or why not.

2. Using the unknown identification chart, compare the results recorded above. What is the genus and species of the unknown organism?

3. If this water was ingested by humans, what type of infection could result? What types of signs and symptoms would an infected individual display?

4. Is the only source of contamination the dairy farm? Give another possible explanation for the water contamination.



Unknown Bacterium Identification Chart

2011 25Evelyn I. Milian - Instructor


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.


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

Documents

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