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Please complete the full lab (all charts) using the attached power point.  

Selective and Differential Media Practice

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Review Biochemicals for Unknown Identification.pptx and your Lab Manual to complete the following table.

MediaWhat type of microorganismgrows on this agar? (i.e. Grampositive, Gram negative,halophile)What type of microorganism isinhibited on this agar? (i.e. Grampositive, Gram negative,halophile)How do fermenters appear? (Remember non-fermenters will appear colorless or media will not change color)
Phenylethyl alcohol agar (PEA)N/A
Mannitol salt agar (MSA)
MacConkey agar
Eosin-Methylene Blue agar (EMB)

Practice Unknown: ___ E. coli ___________

In this lab, you will work independently to record the data for various biochemical tests for your practice unknown. Note that the practice unknown biochemical tests are the same identification tests that will be performed on the graded unknown.

Prior to beginning this lab worksheet, please review the Biochemicals for Unknown Identification.pptx presentation.

Prior to beginning the data interpretation portion of this worksheet, please review the selective and differential media module available through Michigan State University: http://learn.chm.msu.edu/vibl/content/differential.html.

Complete the following data tables with test observations and conclusions from experiments performed on your microbial unknown. Please use complete sentences; don’t just state “positive” or “negative” for the conclusions or you will be marked off. Note: When filling out an observation, describe what you see (i.e. lactose fermentation media has turned yellow and there is air present in the inverted Durham tube). When filling out a conclusion, describe what you can learn from your observation (i.e. the unknown is capable of fermenting lactose to an acid and a gas).

Figure 1a (left). Cultural characteristics of Escherichia coli grown on TSA plate (use this image and p. 57-66 of your lab manual to complete the 4 rows shown on this page). Note that elevation has been filled out for you.

Figure 1b (right). Gram stain of Escherichia coli (use this image to complete the last two rows below on the next page).

Test ConditionObservationConclusions
Colony pigmentation on TSA plateN/A
Whole colony shape on TSA plateN/A
Colony elevation on TSA plateSlightly raisedN/A
Colony margin on TSA plateN/A
Gram Stain Reaction (as viewed under oil immersion lens)(Color?)(Gram-positive or Gram-negative?)
Cell Morphology (as viewed under oil immersion lens)(Shape? Arrangement?) – Remember to list both!N/A

Please use the following results (Figures 3-6) to fill out the table shown on page 8:

Tryptic Soy Agar (TSA): Composition, Preparation and Uses ...

Figure 2. Test result for PEA agar for Escherichia coli.

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Figure 3. Test result of Escherichia coli on Mannitol salt agar

Mannitol salt review: https://youtu.be/kG1_Tf5Vpc0

Figure 4. Test result of Escherichia coli on MacConkey agar.

MacConkey agar review: https://youtu.be/yInQ9jApAlU

6d3a61385ad2d85fcf3d504622d66004.jpg (650×554)

Figure 5. Test result of Escherichia coli on EMB agar.

EMB agar review: https://www.youtube.com/watch?v=y4IJoMPZxk8

Test ConditionObservationConclusions
Phenylethyl Alcohol AgarGrowth on plate?Gram positive or Gram negative?
Mannitol Salt Agar(Growth on plate? Color of colonies? Color of plate?)(Halophile or non-halophile? If you had growth: is the microorganism a mannitol fermenter?)
MacConkey Agar(Growth on plate? Color of colonies? Color of plate)(Gram positive or Gram negative? If you had growth, is the microorganism a lactose fermenter?)
Eosin Methylene Blue (EMB) Agar(Growth on plate? Color of colonies? Color of plate?)(Gram positive or Gram negative? If you had growth, is the microorganism a lactose fermenter?)

Please use the following test results for the table shown below.

For more information on the amylase test watch the following video: https://youtu.be/zFhMbXSgve8

Figure 6. Amylase test result for Escherichia coli

Test ConditionObservationConclusions
Presence of Amylase-Starch plate(Clearing/lightened plate after adding iodine?)(Amylase present?)

Biochemical Tests for Bacterial Identification

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Determination of an Unknown

The first tests on an unknown bacterial organism will allow the researcher to determine the morphology, arrangement, and other basic characteristics of the organism

Selective/differential media will allow the researcher to determine basic colony characteristics, pigmentation, etc.

A Gram stain should be performed to determine if the organism is Gram positive or negative, as well as the cell morphology and arrangement.

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Selective Media

Selective media

Suppress unwanted microbes and encourage desired microbes

Contain inhibitors to suppress growth of unwanted organisms (eg. salts, alcohol)

pH can also be used to select for growth of specific organisms

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Selective media: PEA

PEA plate: Phenylethyl Alcohol Agar plate

Allows for selection of Gram positive organisms by hampering lipids found in Gram negative outer membrane

Typical recipe: (g/liter)

*15 g Pancreatic digest of casein

*15g digest of soybean meal

*5g Sodium Chloride

*2.5 g β-Phenylethyl Alcohol

*15g agar

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Differential Media

Differential media

Allow distinguishing of colonies of different microbes on the same plate

Eg: Uses dyes and pH indicators

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Figure 6.10

Mannitol salt agar (MSA) has both selective and differential characteristics (p. 201)

Mannitol salt agar (7.5% salt)

Selective: Prevents growth of organisms sensitive to salt (selects for growth of Staph)

Differential:

Media turns yellow if mannitol is fermented

(pH change)

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Figure 6.10

Mannitol salt agar (7.5% salt):

Selection of Staphyloccus species

Differentiation of mannitol fermenters and non-fermenters

Typical recipe (per liter):

5.0 g digest of casein 5.0 g digest of animal tissue 1.0 g beef extract 75.0 g sodium chloride 10.0 g mannitol 0.025 g phenol red 15.0 g agar

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Figure 6.10

Mannitol salt agar (7.5% salt):

Inhibits most bacteria other than staphylococci (selective)

Measures ability to ferment mannitol (differential)

Note: phenol red

Pink above pH 8

Yellow below pH 7

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Mannitol fermentation (Note: S aureus can ferment mannitol; S. epidermidis cannot)

Biochemical Tests: Fermentation

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Selective and Differential Media: MacConkey Agar (p. 209)

Allows for selection of Gram negative organisms found in the intestine

Allows for differentiation between different strains of G- organisms based on ability to ferment sugars

Typical Recipe (per 1 liter):  

*17 g peptone 0.03 g neutral red

*10 g lactose (or sorbitol) 0.001g Crystal violet

*1.5 g bile salts 13.5 g agar

*5 g sodium chloride

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MacConkey: Selective and Differential

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MacConkey Agar: Selective and Differential

What should we expect to see on a MacConkey plate?

*Growth of Gram negative, coliform organisms

* Little growth of Gram positive due to inhibition by bile salts and Crystal violet dye

* Pink pigmentation for fermenters of lactose (as media turns acidic, the neutral red should turn pink/red)

*Note: Neutral red is yellow above pH 8, turning red below pH 7***

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EMB: Selective and Differential media

Eosin Methylene Blue Agar Plate (p. 217)

Selection of Gram negative coliforms by

Inhibition of non-coliform organisms.

Differentiation of lactose fermenters and non-fermenters.

Typical contents:

Enzymatic Digest of Gelatin

Lactose

Eosin Y (dye)

Methylene Blue (dye)

Bile salts (inhibits Gram positive and non-coliform organisms)

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EMB: Selective and Differential media

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These can give us a microbial “fingerprint” of the cellular metabolism

They can allow identification of an unknown organism

They can help with development of specific strains of bacteria that can perform specific reactions

What controls the ability of microbes to perform certain chemical reactions or digest certain nutrients?

ENZYMES!!!

What controls what enzymes are in the cell?

Genes/DNA

Biochemical Tests

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The mechanism of enzymatic action.

Substrate

Enzyme

Substrate

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These enzymes act outside the cell, on substances too large to pass through cell membranes

Examples of extracellular enzymes:

Starch hydrolysis (amylase), lipid (lipases), protein hydrolysis (proteases), gelatin hydrolysis (gelatinase)

Biochemical Tests: Extracellular Enzymes (Exoenzymes)

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Starch is a polymer composed of many glucose molecules linked by glycosidic bonds.

It is a form of stored energy in plants.

It is the most common source of carbohydrates in the human diet worldwide.

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Hydrolysis of starch to glucose requires several enzymes (amylase, maltase)

Starch agar plate is a TSA plate with added starch that is flooded with iodine after growth (p. 283).

Biochemical Tests: Extracellular Enzymes (Exoenzymes)

Starch hydrolysis –

Starch hydrolysis +

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Hydrolysis of proteins to peptides and amino acids requires several enzymes (proteases)

Milk agar plate is a TSA plate with added milk that contains the casein protein. Ability to hydrolyze the casein will result in a clear area around the growth.

Biochemical Tests: Extracellular Enzymes (Exoenzymes)

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Protein Catabolism

Protein

Amino acids

Extracellular proteases

Krebs cycle

Deamination, decarboxylation, dehydrogenation, desulfurization

Organic acid

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Gelatin hydrolysis can be a measure of certain exoenzymes (gelatinases)

Nutrient gelatin tubes (TSA tubes with 12% gelatin) can measure whether the microbe has the ability to hydrolyze gelatin.

Biochemical Tests: Extracellular Enzymes (Exoenzymes)

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Ability to ferment specific sugars is a major component of biochemical testing in the microbiological lab

NOTE: Intracellular Enzymes carry out fermentation

(p. 235)

Biochemical Tests: Fermentation (Intracellular Enzymes/Endoenzymes)

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Tubes (containing Durham tubes) can be used to determine ability to ferment various sugars (p. 235).

Dextrose (Glucose)

Biochemical Tests: Fermentation

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Tubes (containing Durham tubes) can be used to determine ability to ferment various sugars.

Sucrose

Biochemical Tests: Fermentation

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Tubes (containing Durham tubes) can be used to determine ability to ferment various sugars.

Lactose

Biochemical Tests: Fermentation

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(Mannitol tubes with protein and pH indicator: Phenol Red)

Uninoculated

S. epidermidis

S. Aureus

E. coli

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Figure 5.19 Types of fermentation.

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MR-VP test (Methyl Red-Voges-Proskauer, p. 243)

These are 2 different tests, but the same media can be used for both.

Methyl Red is red below pH 5, yellow above pH 5.

The MR test will detect organisms that can ferment glucose and produce very acidic (pH below 5) products. For example, E. coli should be MR positive, while Enterobacter aerogenes will be MR negative.

Biochemical Tests: Fermentation

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MR test (p. 243)

Methyl Red is red below pH 5, yellow above pH 5.

Biochemical Tests: Fermentation

Note: Methyl red is added after growth.

A: negative (E. aerogenes)

B: positive (E. coli)

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VP: Voges-Proskauer

The VP test is used to measure organisms that produce a neutral product (acetoin) from fermentation of glucose after a longer incubation time (p. 243). This prevents acid sensitive organisms from being exposed to very low pH.

Biochemical Tests: Fermentation

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VP: Voges-Proskauer

Voges-Proskauer reagents (a-naphthol and KOH) will turn the media pink-red after several minutes if the organism is VP positive. (E. coli should be VP negative, while E. aerogenes is VP positive.

Biochemical Tests: Fermentation

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Some organisms can also ferment citrate but usually only when there is no other carbon source

Citrate Agar Slants are used to determine ability to ferment citrate (p. 269)

Citrate Agar is a defined media,

so no other Carbon source is

available

Biochemical Tests: Fermentation

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Citrate Agar is a defined media, so no

other Carbon source is available.

Typical Recipe:

Sodium Citrate (0.2%)

Sodium Chloride (0.5%)

Ammonium Phosphate (0.1%)

Dipotassium Phosphate (0.1%)

Magnesium sulfate (0.02%)

Agar (1.5%)

Bromthymol Blue (0.0008%)

Biochemical Tests: Fermentation

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Indole production: measures ability of organism to convert tryptophan (amino acid) into indole product.

Biochemical Tests: Amino Acid degradation

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Indole production

This is measured by adding Kovack’s reagent to a culture that has been growing. If indole is present, it will turn pink

Biochemical Tests: Amino acid degradation

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Triple Sugar-Iron Agar Test (TSI) (p. 301)

This media contains 3 sugars: glucose (0.1%), lactose (1%), and sucrose (1%). Thus only organisms that greatly prefer glucose will use it and the acid is oxidized rapidly. There are several different reactions depending on the organism that can help differentiate between different organisms. Phenol red is the pH indicator

Note: This media also contains thiosulfate, so blackening indicates that H2S has been produced.

Biochemical Tests: Fermentation

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Triple Sugar-Iron Agar Test (TSI)

Biochemical Tests: Fermentation

Neutral: no carbohydrate utilized

Alkaline slant/acid butt: only glucose fermentation

3/4. Acid slant/acid butt with or without gas production: lactose and sucrose fermentation has occurred

5. Acid slant/acid butt plus H2S production

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Sulfur-containing amino acids can be metabolized. Sulfur can be reduced to H2S gas by sulfur reducing bacteria. This will form a black precipitate with ferrous sulfate in the media after growth

Biochemical Tests: Amino acid degradation

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Ammonium ion (NH3) causes the pH to increase

Phenol red is the pH indicator

Biochemical Tests: Urease (p. 289)

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Some organisms produce urease. This is particularly useful in identification of Proteus vulgaris

Urease breaks urea down into CO2 and NH3

Biochemical Tests: Urease

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This is a test of whether an organism has cytochrome oxidase that can oxidize cyt c, part of the electron transport chain

Biochemical Tests: Oxidase

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Aerobic, facultative anaerobes have cyt c, whereas anaerobes and Enterobacteria do not.

We will use a BD DrySlide Oxidase test (p. 257)

In this test, cyt c oxidizes the reagent which turns deep purple/blue if positive

Biochemical Tests: Oxidase

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Oxygen

Obligate aerobes—require oxygen

Facultative anaerobes—grow via fermentation or anaerobic respiration when oxygen is not available

Obligate anaerobes—unable to use oxygen and are harmed by it

Aerotolerant anaerobes—tolerate but cannot use oxygen

Microaerophiles—require oxygen concentration lower than air

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Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

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Singlet oxygen: (1O2−) boosted to a higher-energy state and is reactive

Superoxide radicals: O2

Peroxide anion: O22–

Hydroxyl radical (OH•)

Oxygen

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Hydrogen peroxide is produced during aerobic respiration, accumulation of this and superoxide are highly toxic. Hydrogen peroxide can be quickly neutralized by catalase

3% hydrogen peroxide is used to directly test for catalase (p. 251)

Biochemical Tests: Catalase

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Methods of Classifying and Identifying Microorganisms

Classification: placing organisms in groups of related species

Lists of characteristics of known organisms

Identification: matching characteristics of an “unknown” organism to lists of known organisms

Clinical lab identification

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Filled out by one person

Filled out by different person

Figure 10.7 A clinical microbiology lab report form.

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Methods of Classifying and Identifying Microorganisms

Morphological characteristics: useful for identifying eukaryotes; tell little about phylogenetic relationships

Differential staining: Gram staining, acid-fast staining; not useful for bacteria without cell walls

Biochemical tests: determine presence of bacterial enzymes

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Figure 10.8 The use of metabolic characteristics to identify selected genera of enteric bacteria.

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Applications of Microbiology 10.1

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Biochemical Tests

Rapid identification methods perform several biochemical tests simultaneously

Results of each test are assigned a number

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One tube containing media for 15 biochemical tests

is inoculated with an unknown enteric bacterium.

After incubation, the tube is observed for results.

The value for each positive test is circled, and

the numbers from each group of tests are

added to give the code number.

Comparing the resultant code number with a

computerized listing shows that the organism in

the tube is Citrobacter freundii.

Glucose

Gas

Lysine

Ornithine

H2S

Indole

Adonitol

Lactose

Arabinose

Sorbitol

V–P

Dulcitol

Phenylalanine

Urease

Citrate

Code Number

Microorganism

Atypical Test Results

62352

62353

Citrobacter freundii

Citrobacter freundii

Citrate

None

Figure 10.9 One type of rapid identification method for bacteria: EnteroPluri test from BD Diagnostics.

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