Please upload and submit your completed Excel spread sheet and Record sheet for your unknown project here. Complete excel sheet by highlighting all your results for each unknown bacteria and every test to show your work process. Complete the Record sheet word document by filling in the “obeservation” and “intepretation” sections of each test outlined in the Record sheet, then fill in your name, unknown bacteria at the bottom of the sheet. For example, if the Urease Test is positive because it changed color from “white” to “black”, you would document your observation column for urease test, as “white to black color change” and intepretation column as “positive”. Samples of these 2 documents are attached below and can also be found in blackboard under the link “Unknown project materials”.  

“Use the second sheet the Gram Negative”

EXERCISE #	TEST	OBSERVATION	INTERPRETATION OF OBSERVATION
COLONY MORPHOLOGY
COLONY PIGMENT
GROWTH IN SLANTS
GROWTH IN BROTH
GRAM STAIN
SHAPE
ARRANGEMENT
FLUID THIOGLYCOLLATE
GELATIN
PHENOL RED- DEXTROSE
PHENOL RED-SUCROSE
PHENOL RED-LACTOSE
GAS
MR
VP
CATALASE
OXIDASE
STARCH AGAR PLATE
UREA
SKIM MILK AGAR PLATE-CASEASE
TRIBUTYRIN AGAR PLATE-LIPASE
CITRATE
PHENYLALANINE
SULFUR
INDOLE
MOTILITY
NAMES OF RESEARCHERS IN YOUR GROUP	UNKNOWN #	UNKNOWN IDENTITY

Biochemical and microscopic
analysis of unknown bacteria

EXPERIMENTAL RESULTS
FOR IDENFICIATION OF THE UNKNOWN

BACTERIUM

Microscopic images:
Gram stain, morphology, arrangement

400X magnification

400X magnification

1000X magnification

Cultural Characteristics:
pigment, colony morphology

Growth in slant

Growth in broth

SIM test results (sulfur, indole, motility)

Catalase test result

On microscope slide

In tube

Oxidase test result

Methyl Red (MR) test result

https://www.bing.com/images/search?view=detailV2&ccid=FUELMhD8&id=4D3B7EBA56F59A72CFAD4A4DCD587018032AB5F7&thid=OIP.FUELMhD8PhSH6gIsQfvqZAEsEs&q=methyl+red+and+voges+proskauer+application&simid=608054868267696835&selectedIndex=1

https://www.bing.com/images/search?view=detailV2&ccid=FUELMhD8&id=4D3B7EBA56F59A72CFAD4A4DCD587018032AB5F7&thid=OIP.FUELMhD8PhSH6gIsQfvqZAEsEs&q=methyl+red+and+voges+proskauer+application&simid=608054868267696835&selectedIndex=1

Voges-Proskauer (VP) test result

https://www.bing.com/images/search?view=detailV2&ccid=3HwOpJNZ&id=AC46E32E3825E673DF38503A6B150A2D69E73AC5&thid=OIP.3HwOpJNZ-vAIogJMYTPDXwEsEs&q=methyl+red+and+voges+proskauer+application&simid=608025585180738723&selectedIndex=0

https://www.bing.com/images/search?view=detailV2&ccid=3HwOpJNZ&id=AC46E32E3825E673DF38503A6B150A2D69E73AC5&thid=OIP.3HwOpJNZ-vAIogJMYTPDXwEsEs&q=methyl+red+and+voges+proskauer+application&simid=608025585180738723&selectedIndex=0

Citrate test result

Urease test result

Gelatinase test result

Phenol red dextrose test result

Phenol red lactose test result

Phenol red sucrose test result

Phenol red Gas formation test result

Aerotolerance test result:
fluid thiolglycollate (FTG)

Obligate Aerobe (OA) or Facultative Anaerobe (FA)??????

Phenylalanine Deaminase test result

Starch Hydrolysis test result

Casein Hydrolysis test result

Lipid Hydrolysis test result

• Biochemical and microscopic analysis of unknown bacteria
• Microscopic images:�Gram stain, morphology, arrangement
• Cultural Characteristics:�pigment, colony morphology

Growth in slant
Growth in broth

• SIM test results (sulfur, indole, motility)�

Catalase test result
Oxidase test result
Methyl Red (MR) test result
Voges-Proskauer (VP) test result

• Citrate test result�

Urease test result
Gelatinase test result
Phenol red dextrose test result
Phenol red lactose test result
Phenol red sucrose test result
Phenol red Gas formation test result

• Aerotolerance test result:�fluid thiolglycollate (FTG)

Phenylalanine Deaminase test result
Starch Hydrolysis test result
Casein Hydrolysis test result

• Lipid Hydrolysis test result�

Sheet1

d

>d

>d

>d

>d

>d

>d

>d

>d

>d

y>

Positive Chart

>3um

rod

coccus coccus coccus rod coccus coccus coccus coccus coccus Morphology

pairs,chains pairs,chains pairs,chains pairs,chains

tetrads/clusters

like

irreg clusters pairs,chains pairs,chains Arrangement

– – – – –

– – – – – – Pigments

+ + – – – – – + – – – – Motility

+ + + – –

– [-] [-] d d Starch

+ + + d – + + + + + + – – Catalase

[+] – – – – + d + – – – – Oxidase

– – – – – – – – – – – – Indole

+ – + + + d d – + + + + VP

[+] + [+] [-] – + – + – d [-] – – Citrate

[-] [+] – – – d – + + + – [+] Urease

+ + + d – [+] [-] – + [+] – – Gelatinase

+ + + + + [-] + – + + + + Glucose

– d [-] + + – – – + + + + Lactose

+ + + + + [-] + – + + + + Sucrose

– – – – – – – – – – – – Gas

– – – – – – – – – – – – H2S

Thioglycollate(o/f)

purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue purple/blue gram stain

Morphology

rod bacillus rod bacillus

rod bacillus spherical coccus spherical coccus spherical coccus spherical coccus spherical coccus spherical coccus spherical coccus spherical coccus Morphology

CHAINS, STREPTO CHAINS, STREPTO

CHAINS, STREPTO

TETRADS TETRADS

clusters, staphylo clusters, staphylo CHAINS, STREPTO CHAINS, STREPTO arrangement

– – – – – Yellow

– – – – – – pigment

SMOOTH, CONVEX, ENTIRE

RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE colony morphology

flat undulate translucent FLAT, IRREGULAR ROUND, SMOOTH CONVEX MUCOID MUCOID, LOBATE ROUND, ENTIRE,SHINY, CONVEX SMOOTH, CONVEX, ENTIRE SMOOTH, CONVEX, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE RAISED, CIRCULAR, ENTIRE growth in slant

SEDIMENT SEDIMENT

UFT SEDIMENT

PELLICLE UFT UFT SEDIMENT SEDIMENT UFT growth in broth

– – – – – – – – – – – – – sulfur (SIM)

– – – – – – – – – – – – – indole (SIM)

+ + + – – + – – + – – – – motility (SIM)

+ + +

– + + + + + + – – catalase

+ – – – – + ± ± + – – – – oxidase

(MR)

+ – – + ± – ± – – – ± – – Methyl Red (MR)

+ – + + + – ± ± – + + + + vogues proskeur (VP)

– + ± ± – + – + – ± ± – – citrate

Urease – ± – – – + – + + + + – ± Urease
Gelatinase – + + ± – – ± – – + ± – – Gelatinase

+ + + + + – + + – + – + + phenyl red dextrose

– – – + + – – – – + – + + phenyl red lactose

+ + + + + – + + – + – + + phenyl red sucrose

– – – – – – – – – – – – – gas

OA OA

FA OA OA OA OA FA FA FA FA fluid thiolglycollate

– – – – – – – + + + – + – Phenylalanine

+ + + + – – ± + + ± ± ± ± starch agar plate

– + + + ± – ± + + + – + – Casein(skim milk)

– ± ± ± ± – ± – – – – – ± Lipase (TBA plate)

FA

OA

+

–

±

UFT

Gram

B Cereus

B megaterium

B subtilis

E faecalis

L lactis

M luteus

M roseus

M phlei

S ureae

S aureus

S epiderm

S pyogenes

S salivarius

Morphology

rod

rod>3um

coccus

Arrangement

pairs,chains

tetrads/clusters

mycelium

–

pairs,tetrads

irreg clusters

Pigments

Yellow

Red

Motility

+

Starch

[+]

[-]

Catalase

Oxidase

Indole

VP

Citrate

Urease

Gelatinase

Glucose

Lactose

Sucrose

Gas

H2S

Thioglycollate(o/f)

gram stain

purple/blue

rod bacillus

spherical coccus

arrangement

CHAINS, STREPTO

clusters, staphylo

TETRADS

TETRADS, sarcinae

pigment

red

colony morphology

flat undulate translucent

FLAT, IRREGULAR

ROUND, SMOOTH

CONVEX MUCOID

MUCOID, LOBATE

ROUND, ENTIRE,SHINY, CONVEX

SMOOTH, CONVEX, ENTIRE

RAISED, CIRCULAR, ENTIRE

growth in slant

growth in broth

SEDIMENT

UFT

PELLICLE

sulfur (SIM)

indole (SIM)

motility (SIM)

catalase

±

oxidase

Methyl Red

vogues proskeur (VP)

citrate

phenyl red dextrose

phenyl red lactose

phenyl red sucrose

gas

fluid thiolglycollate

OA

FA

Phenylalanine

starch agar plate

Casein(skim milk)

Lipase (TBA plate)

facultative anaerobe

obligate aerobe

90% or greater positive

90% or greater negative

50% POSITIVE 50% NEGATIVE

uniform fine turbidity

Sheet2

gram stain

red/pink red/pink red/pink red/pink red/pink red/pink red/pink red/pink red/pink red/pink red/pink red/pink gram stain

Morphology rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus rod bacillus Morphology
arrangement

SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE SINGLE arrangement

pigment – – – – – – – – –

– pigment

colony morphology

FLAT/SMOOTH

MUCOID, convex

MUCOID, SMOOTH MUCOID, SMOOTH MUCOID, SMOOTH FLAT/SMOOTH FLAT/SMOOTH MUCOID, convex MUCOID, SMOOTH colony morphology

growth in slant FLAT/SMOOTH FLAT/SMOOTH MUCOID, SMOOTH MUCOID, convex MUCOID, convex FLAT, irregular, undulate, shiny MUCOID, SMOOTH MUCOID, SMOOTH MUCOID, SMOOTH FLAT/SMOOTH FLAT/SMOOTH MUCOID, convex MUCOID, SMOOTH growth in slant
growth in broth SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT SEDIMENT growth in broth
sulfur (SIM) ± – + – – – – ± + – – – – sulfur (SIM)
indole (SIM) + – – – – + – – + – – – ± indole (SIM)
motility (SIM) + + + + + + – + + + + + – motility (SIM)
catalase + + + + + + + + + + + + + catalase
oxidase + + – + – + – – – + + – – oxidase
Methyl Red ± – + – ± + ± + + – – – + Methyl Red
vogues proskeur (VP) ± – – – ± – – ± – – – + –
citrate ± + + + + – + ± ± + + + – citrate
Urease – – ± – ± – + + + ± ± ± – Urease
Gelatinase + – – – – – – + ± ± ± + – Gelatinase
phenyl red dextrose + – + + + + + + + – ± + + phenyl red dextrose
phenyl red lactose ± – + + ± + + – – – – – – phenyl red lactose
phenyl red sucrose + – ± + + – + – + – ± + – phenyl red sucrose
gas ± – + + + + + + + – – ± – gas
fluid thiolglycollate FA OA FA FA FA FA FA FA FA OA OA FA FA fluid thiolglycollate

– – – – – – – + + – – – – Phenylalaine

starch agar plate + – – – – + + – – – – – – starch agar plate

– – – – – – – + + + + – – casein(skim milk)

– – – + + – + + + + + + – lipids (TBA plate)

FA facultative anaerobe
OA obligate aerobe
+ 90% or greater positive
– 90% or greater negative
± 50% POSITIVE 50% NEGATIVE
UFT uniform fine turbidity

Gram Negative Chart

A hydrophila

A faecalis

C freundii

E aerog

E cloacae

E coli

K pneum

P mirabilis

P vulgaris

P aeruginosa

P fluorescens

S marcescens

S flexneri

red/pink

SINGLE

blue/green

yellow

reddish orange

FLAT/SMOOTH

MUCOID, SMOOTH

MUCOID, convex

FLAT, irregular, undulate, shiny

Phenylalaine

casein(skim milk)

lipids (TBA plate)

Biochemical analysis of unknown bacteria

Aerotolerance Test
Fluid Thioglycollate broth (FTB) is a medium designed to test the aerotolerance of bacteria.
Along with nutrients to support bacterial growth, it contains sodium thioglycollate, thioglycollic acid, L-cystine, methylene blue, and 0.05% agar.
The sodium thioglycollate, thioglycollic acid, and L-cystine reduce the oxygen to water.
Methylene blue is an indicator that is colorless in an anaerobic environment and greenish-blue in the presence of oxygen.
The agar helps retard oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth.
Oxygen is driven out of the broth by autoclaving, but as the broths sit at room temperature, oxygen begins to diffuse back into the tube.
Obligate aerobes will only grow in this oxygen-rich top layer. On another hand, obligate anaerobes will only grow in the lower areas of the tube. Microaerophiles will grow in a thin layer below the richly-oxygenated layer. Facultative or aerotolerant anaerobes can grow throughout the medium but will primarily grow in the middle of the tube, between the oxygen-rich and oxygen-free zones
Reactions typically take up to 1-2 days to develop at 37⁰C
Media is inoculated using an inoculating loop

(A) Escherichia coli and (C) Staphylococcus aureus: both are Facultative Anaerobe, grows both aerobically and anaerobically and growth is seen throughout the tube. Some are capable of growth respiring with oxygen and anaerobically by fermentation.
(B) Clostridium botulinum: Obligate Anaerobe: can not grow in the presence of oxygen, growth is seen approximately 1/4 to 1/2 of the way from the top of the tube.
(D) Neisseria sicca: Microaerophile, requires oxygen but at concentrations below atmosphere, grows just below the surface of the media but not at the top.
(E) Pseudomonas aeruginosa: Obligate Aerobe: oxygen is required for growth and grows at the top of the tube only. The Organism will “settle” and sink into the media if grown longer than 24 hrs.
Aerotolerance Test

Phenol red test
Phenol red broth is a differential test medium prepared as a base to which a carbohydrate such as sucrose, lactose, dextrose or glucose is added.
Included in the base medium are peptone and the pH indicator is phenol red. Phenol red is yellow below pH 6.8, pink to magenta above pH 7.4, and red in between. During preparation, the pH is adjusted to approximately 7.3 so it appears red.
Deamination of peptone amino acids produces ammonia which rises the pH and turns the broth pink.
An inverted Durham tube is added to each tube as an indicator of gas production.
Gas production, also from fermentation, is indicated by a bubble or pocket in the Durham tube where the broth has been displaced.
Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow. Deamination of peptone amino acids produces ammonia which rises the pH and turns the broth pink. Gas production, also from fermentation, is indicated by a bubble or pocket in the Durham tube where the broth has been displaced. Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow. Deamination of peptone amino acids produces ammonia which rises the pH and turns the broth pink. Gas production, also from fermentation, is indicated by a bubble or pocket in the Durham tube where the broth has been displaced.

Phenol red test

Phenol red test
Possible phenol red tube results include: (A) Formation of acid and gas (bubble is indicated by arrow), (B) Formation of acid, (C) uninoculated control, (D) alkaline byproducts, and (E) no acid or gas formation.

Methyl Red (MR) and Voges-Proskauer (VP) Test
The VP test was designated for organism that are able to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol. Adding VP reagents to the medium oxidizes the acetoin to diacetyl, which in turns reacts with guanidine nuclei from peptone to produce a red color. A positive VP result is red. No color change after addition of reagents is negative. The copper color is a result of interactions between reagents and should not be confused with the true red color of a positive result.
MR-VP broth is a combination medium used for both Methyl Red (MR) and Voges-Proskauer (VP) tests. It is simple solution containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein and fermentable carbohydrate, respectively, and the potassium phosphate resists pH changes in the medium.
The MR test is designed to detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the medium and lowers the pH. The acids produced by these organisms tend to be stable, whereas acids produced by other organisms tend to be unstable and subsequently are converted to more neutral products.
Mixed acid fermentation is verified by the addition of methyl red indicator dye following incubation. Methyl red is red at 4.4 and yellow at pH 6.2. Between these two pH values, it is various shades of orange. Red color is the only true indication of a positive result. Orange is negative of inconclusive. Yellow is negative.

Urease Test
Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from orange to pink. The agar must be examined daily during incubation. Rapid urease-positive organisms will turn the entire slant pink within 24 hours. Weak positives may take several days. Urease-negative organisms either produce no color change in the medium or turn it yellow from acid products.
Urea is a product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme urease. Many enteric bacteria (and a few others) possess the ability to metabolize urea, but only members of Proteus, Morganella, and Providencia are considered rapid urease-positive organisms. Urea is a product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme urease..
Urea Agar was formulated to differentiate rapid urease-positive bacteria from slower urease-positive and urease-negative bacteria. It contains urea, peptone, potassium phosphate, glucose, and phenol red. Peptone and glucose provide essential nutrients for a broad range of bacteria. Potassium phosphate is a mild buffer used to resist alkalization of the medium from peptone metabolism. Phenol red, which is yellow or orange below pH 8.4 and red or pink is included as an indicator.

Starch Hydrolysis
Starch is a polysaccharide up of a-D-glucose subunits. It exists in two forms, linear (amylose) and branched (amylopectin), usually as a mixture with the branched being predominant. The a-D-glucose molecules in both amylose and amylopectin are bonded by 1,4-a-glycosidec (acetyl) linkages. The two forms differ in that amylopectin contains polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side chains are identical to the main chain except that the number 1 carbon of the first glucose in the side chain is bonded to carbon number 6 of the main chain glucose. The bond is a 1,6-a-glycosidic linkage.
Starch is too large to pass through the bacterial cell membrane. It splits into smaller fragments. Organisms that produce and secrete the extracellular enzymes a-amylase and oligo-1,6-glucosidase are able to hydrolyze starch.
When organisms that produce a-amylase and oligo-1,6-glucosidase are cultivated on starch agar, they hydrolyze the starch in the area surrounding their growth. Because both starch and its sugar subunits are virtually invisible in the medium, the reagent iodine is used to detect the presence or absence of starch. Iodine reacts with starch and produces blue or dark brawn color, any microbial starch hydrolysis will be revealed as a clear zone surrounding the growth.

Casein Hydrolysis Test
Casein contains a high number of proline residues, which do not interact. There are also no disulfide bridges. As a result, it has relatively little tertiary structure. It is relatively hydrophobic, making it poorly soluble in water. It is found in milk as a suspension of particles, called casein micelles, which show only limited resemblance with surfactant-type micelles in a sense that the hydrophilic parts reside at the surface and they are spherical. However, in sharp contrast to surfactant micelles, the interior of a casein micelle is highly hydrated. The caseins in the micelles are held together by calcium ions and hydrophobic interactions. Any of several molecular models could account for the special conformation of casein in the micelles. One of them proposes the micellar nucleus is formed by several submicelles, the periphery consisting of microvellosities of κ-casein. Another model suggests the nucleus is formed by casein-interlinked fibrils. Finally, the most recent model proposes a double link among the caseins for gelling to take place. All three models consider micelles as colloidal particles formed by casein aggregates wrapped up in soluble κ-casein molecules.
The isoelectric point of casein is 4.6. Since milk’s pH is 6.6, casein has a negative charge in milk. The purified protein is water-insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as aqueous sodium oxalate and sodium acetate. The isoelectric point of casein is 4.6. Since milk’s pH is 6.6, casein has a negative charge in milk. The purified protein is water-insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as aqueous sodium oxalate and sodium acetate.

Lipid Hydrolysis Test
Lipids generally are nonpolar molecules that do not dissolve well in water.  Fats are one type of lipids that are large polymers of fatty acids and glycerol. That are too large to enter the cell membrane. In order to utilize fats, bacteria cells secrete exoenzymes (lipases) outside of the cell that hydrolyze (digestion by the addition of water) the lipid to fatty acids and glycerol. 
The cell can convert glycerol and use it in glycolysis, and the fatty acids can be converted into acetyl-coenzyme A and used in the Citric Acid Cycle.  The cell may also use these precursors to make its own lipids.  Lipid hydrolysis can actually be tasted; it makes food taste rancid.  (However, testing an organism in this manner will not be allowed.)  Lipid hydrolysis (or lipase activity) may be tested by growing an organism on an agar plate providing nutrients and a lipid, and then the plates are checked for hydrolysis of the lipid.  Inclusion of the lipid makes the agar appear opaque.  Plates with hydrolysis will have a clear zone around the growth, whereas those lacking hydrolysis, will have no zones of clearing around the growth. Inclusion of the lipid makes the agar appear opaque. 
Plates with hydrolysis will have a clear zone around the growth, whereas those lacking hydrolysis, will have no zones of clearing around the growth. Inclusion of the lipid makes the agar appear opaque.  Plates with hydrolysis will have a clear zone around the growth, whereas those lacking hydrolysis, will have no zones of clearing around the growth.  Depending on the species being examined, the lipid may be corn oil, tributyrin, egg yolk, or some other lipid.  (Interesting fact: some lipases may be potent cytolytic virulence factors.  Phospholipases can kill cells by dissolving the cell membrane.)

Citrate Test
Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator blue. This test is part of the IMViC tests and is helpful in differentiating the Enterobacteriaceae.
Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromothymol blue turns royal blue. At a neutral pH, blue is green, as evidenced by the uninoculated media.
If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change. Phenylalanine These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium.

Phenylalanine Deaminase test
Organisms that produce phenylalanine deaminase can be identified by their ability to remove the amine group NH2 from the amino acid phenylalanine. This reaction splits a water molecule and produces ammonia NH3 and phenyl pyruvic acid. Deaminase activity is evidenced by the presence of phenyl pyruvic acid.. Deaminase activity is evidenced by the presence of phenyl pyruvic acid.
Phenylalanine agar provides a rich source of phenylalanine. A reagent containing ferric chloride is added to the medium after incubation. The normally colorless phenyl pyruvic acid reacts with the ferric chloride and turns a dark green color almost immediately. Formation of green color indicates the presence of phenylalanine deaminase. Yellow is negative. Phenylalanine agar provides a rich source of phenylalanine. A reagent containing ferric chloride is added to the medium after incubation. The normally colorless phenyl pyruvic acid reacts with the ferric chloride and turns a dark green color almost immediately. Formation of green color indicates the presence of phenylalanine deaminase. Yellow is negative.
This medium is used to differentiate the genera Morganella, Proteus, and Providencia from other members of the Enterobacteriaceae.

Phenylalanine Deaminase test

Sulfur Reduction Indole Production, Motility (SIM) Test
SIM medium is used for determination of three bacterial activities: sulfur reduction, indole production from tryptophan, and motility. The semisolid medium includes casein and animal tissue as sources of amino acids, an iron-containing compound, and sulfur in the form of sodium thiosulfate.
Cysteine desulferase hydrolyzes cysteine to pyruvate and H2S.
Tryptophanase hydrolyzes tryptophan into pyruvate ammonia and indole
Sulfur reduction to H2S can be accomplished by bacteria in two different ways, depending on the enzymes present, Cysteine or thiosulfate reductase. Indole production in the medium is made possible by the presence of tryptophan. Bacteria possessing the enzyme tryptophan can hydrolyze tryptophan to pyruvate, ammonia, and indole. After adding the reagent the formation of red color in the reagent layer indicates a positive reaction and the presence of tryptophan. No red color is indole-negative.
Determination of motility in SIM medium is made possible by the reduced agar concentration and the method of inoculation. The medium is inoculated with a single stab from and inoculating needle. Motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth pattern extending outward in all directions from the central stab line. Growth that radiates in all directions and appears slightly fuzzy is an indication of motility.

Sulfur Reduction Indole Production, Motility (SIM) Test

Gelatin Hydrolysis (Gelatin Test)
Gelatin is a protein derived from collagen, a component of vertebrate connective tissue. 
Gelatin hydrolysis test is a great way to highlight proteolysis by gelatinase secreting species of bacteria.
The presence of gelatinases can be detected using nutrient gelatin, a simple test medium composed of gelatin, peptone, and beef extract.
Nutrient gelatin differs from most other solid media in that the solidifying agent (gelatin) is also the substrate for enzymatic activity.
When a tube of nutrient gelatin is stab inoculated with a gelatinase positive organism, secreted gelatinase will liquefy the medium. Gelatinase negative organisms do not secrete the enzyme and do not liquefy the medium.
Examples: Staphylococcus aureus, which is gelatinase- positive, can be differentiated from S. epidermidis. Serratia and Proteus species are gelatinase positive members of Enterobacteriaceae, whereas most others in the family are negative.
Reactions typically take up to 7 days to develop at 25⁰C
Media is inoculated using an inoculating needle

Gelatin Hydrolysis (Gelatinase Test)
A. Gelatinase positive sample liquefies medium
B. Negative sample, medium remains solid

Catalase Test
The electron transport chains of aerobic and facultatively anaerobic bacteria are composed of molecules capable of accepting and donating electrons as conditions dictate. These molecules alternate between the oxidized and reduced forms, passing electrons down the chain to the final electrons in this sequential transfer is used to perform oxidative phosphorylation. In most cases, electrons in the aerobic electron transport chain follow the stepwise path to oxygen, but other paths can be followed and these result in production of toxic forms of reduced oxygen. For instance, one electron transport chain carrier molecule called flavoprotein can bypass the next carrier in the chain and transfer electrons directly to oxygen, which produces hydrogen peroxide, a highly potent cellular toxin. FADH2 is capable of the same reaction.
Hydrogen peroxide and the superoxide radical are toxic because they oxidize biochemical and make them nonfunctional. However, organisms that produce them also produce enzymes capable of breaking them down. Superoxide dismutase catalyzes conversion of superoxide radicals to hydrogen peroxide. Catalase converts hydrogen peroxide into water and gaseous oxygen. In large parts, the ability to synthesis these protective enzymes accounts for an organism’s ability to live in the presence of oxygen. Bacteria that produce catalase can be detected easily using typical store-grade hydrogen peroxide.
This test is used to identify organisms that produce the enzyme catalase. It is used most commonly to differentiate members of the catalase-positive Micrococci from the catalase-negative Streptococcaceae. Variations on this test also may be used in identification of Mycobacterium species.

Oxidase Test
Considering the life of a glucose molecule entering the cell. It is first split (oxidized) in glycolysis where it is converted to two molecules of pyruvate and reduces two NAD (coenzyme) molecules to NADH (+H+). Then each of the pyruvate molecules is oxidized and converted to a two-carbon molecule called acetyl-CoA and one molecule of CO2, which reduces another NAD to NADH. Then the Krebs cycle finishes the oxidation by producing two more molecules of CO2 (per acetyl-CoA) and reduces three more NADs and one FAD to FADH2.
By this time the cell is becoming quite full of reduced coenzymes. Therefore, in order to continue oxidizing glucose, these coenzymes must be converted back to the oxidized state. This is the job of the electron transport chain.
Many aerobes, microaerophiles, facultative anaerobes, and even some anaerobes have ECTs. The functions of the ECTs are to transport electrons down a chain of molecules with increasing positive reduction potentials to the terminal electron acceptor and generate a proton motive source by pumping H out of the cell thus creating an ionic imbalances that will drive the production of ATP by way of membrane ATPases. Flavoptoteins, iron-sulfur proteins, and cytochromes are important ECT molecules unable to donate protons.
Many bacteria have ECTs resembling mitochondrial ECTs in eukaryotes. These chains contain a series of four large enzymes broadly named Complexes I, II, III, and IV. Complex IV, is called cytochrome c oxidase because it makes the final electron transfer of the chain from cytochrome c, residing in the periplasm, too oxygen inside the cell.
In the oxidase test, the reducing reagent is added directly to bacterial growth on solid media or a bacterial colony is transferred to paper saturated with the reagent. A dramatic color change occurs within seconds if the reducing agent becomes oxidized, thus indicating that cytochrome c oxidase is present.