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

Selective and Differential Media Practice

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

Media

What type of microorganism

grows on this agar? (i.e. Gram

positive, Gram negative,

halophile)

What type of microorganism is

inhibited on this agar? (i.e. Gram

positive, 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 Condition

Observation

Conclusions

Colony pigmentation on TSA plate

N/A

Whole colony shape on TSA plate

N/A

Colony elevation on TSA plate

Slightly raised

N/A

Colony margin on TSA plate

N/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:

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

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

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

Test Condition

Observation

Conclusions

Phenylethyl Alcohol Agar

Growth 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 Condition

Observation

Conclusions

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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