BISC209: Enzyme tests

Enzyme Tests for Differentiation of Microorganisms by Metabolic Processes

Microorganisms vary in their ability to process nutrients and to make energy. Many of these differences stem from variations in metabolic pathways. These pathways use different enzymatically catalyzed reactions. One way to differentiate microorganisms is to test for particular enzymes that are charactistically present or absent in certain groups.

Catalase test

Background
Catalase protects aerobic bacteria from the accumulation of hydrogen peroxide, an extremely toxic form of oxygen, by catalyzing the reaction:

H₂O₂ --------> H₂O + O₂

Procedure
Since the reaction liberates bubbles of oxygen, it is easy to test for the presence of the enzyme.
Place a drop of fresh 3 % hydrogen peroxide (H₂O₂) on a clean glass slide.
Using aseptic technique, take a loopful of bacteria that you want to test and use the loop to mix the cells in the peroxide until you have created a circular emulsion about the size of a quarter.
Remove the loop and look for the formation of bubbles in the emulsion.
A positive test is indicated by the evolution of oxygen bubbles.
Bacillus or Staphylococcus species are good positive controls.

Since hydrogen peroxide is formed by the interaction of molecular oxygen with a variety of cellular reaction systems, most aerobic bacteria are catalase positive while most anaerobes are catalase negative. Lactic acid bacteria are unusual. This group lacks catalase, but can grow in the presence of atmospheric oxygen; however, colonies of these bacteria never get very large on aerobically-incubated plates.

When performing a catalase test, if a colony is taken from a blood-agar plate, you must keep in mind that the enzyme catalase is present in red blood cells; therefore, any carry-over of blood cells with the colony can give a false-positive reaction.

Oxy-Swab Oxidase Test

Background
The Oxidase test is used to detect the presence of the enzyme cytochrome c oxidase. Organisms such as Micrococcus luteus, Neisseria, and Pseudomonas species are positive for this enzyme. If cytochrome c oxidase is produced by the organism, cytochrome c will be present in the oxidized form and can react with either substrate, dimethyl or tetramethyl-p-phenylenediamine dihydrochloride, to produce a purple colored product, indophenol.

Procedure
Using aseptic technique, obtain an Oxy-Swab and use it to pick up a small amount of a pure colony of bacteria to be tested.
Press the swab against the sterile inside wall of the slant or sterile part of the inside wall of the plate to diffuse the inoculum into the fibers of the swab.
Look for the appearance of a purple or black color within 10-30 seconds on the swab in the area containing the bacteria.
Purple or black color is a positive result (+).
Good positive controls are Bacillus spp, Neisseria, Micrococcus or Pseudomonas)
No color change is a negative result (-).
Discard used swabs in an autoclave bag.

Methyl Red/Voges-Proskauer Tests (MR-VP)

Background
The Methyl Red (MR) test is used to identify mixed acid fermenting bacteria that yield a stable acid end product. The Voges-Proskauer (VP) test is used to identify bacteria capable of 2,3 butanediol fermentation following mixed-acid fermentation. MRVP media contains glucose, peptone and phosphate buffer. Many organisms can overcome the buffering capacity of the media by producing large quantities of a stable acid end product, thus lowering the pH. Acid produciton is detected using the pH indicator methyl red (red pH<4.4, yellow pH > 6). In order to insure that the acid is stable this test should be conducted a minimum of 48 hrs. post inoculation. Some organisms do not produce stable acid end products and instead further metabolize acids to more neutral end products like 2,3 butanediol. The reagents used however, don't test for 2,3 butanediol, but rather it's precursor acetoin.

Procedure
a. Methyl red tests for acid production from dextrose: Inoculate a tube of dextrose peptone broth ( 0.7% peptone, 0.5% dextrose, 0.5% dipotassium phosphate) using a small amount of a pure colony of bacteria you want to test. Inoculate another tube with a positive control. Incubate the tubes for 48hours at 37C. Remove 1ml of broth culture into a small glass tube and add 5 drops of methyl red indicator. Read immediately for red color (positive test) or yellow color (negative test). If negative, the test may be rerun after additional incubation of 24-48 hours.

b. Voges-Proskauer tests for the production of acetylmethylcarbinol from dextrose: Inoculate a tube of dextrose peptone broth ( 0.7% peptone, 0.5% dextrose, 0.5% dipotassium phosphate) using a small amount of a pure colony of bacteria you want to test. Inoculate another tube with a positive control. Incubate the tubes for 48hours at 37C. Remove 1 ml of the cultured dextrose peptone broth and place it in a small glass tube. Add 15 drops of 5% alphanaphthol in absolute ethyl alcohol and 10 drops of 40% KOH. Do not mix contents after addition of test reagents! A positive test is indicated by the development of a red color in 15-30 minutes.

Gelatin Liquefaction by Collagenase (a protease)

Background
Organisms that have the ability to liquefy gelatin possess proteases (collagenase) that will hydrolyze gelatin.
Procedure
Using aseptic technique, sterilize an inoculating needle and pick up a small amount of a pure colony of bacteria that you would like to test for collagenase. Stab a tube of sterile gelatin media ( 0.3% beef extract, 0.5% peptone, 1.2% gelatin) with this inoculum by inserting the needle through the center of the media almost to the bottom. Carefully remove the needle in the same path as the entry. Inoculate a positive control organism in the same way. Incubate at 37˚ for at least 48 hours. To observe liquefaction place the 48 hour incubated gelatin culture in the refrigerator. The test is positive (+) if the gelatin remains liquefied after 30 minutes at 4C. If the gelatin is solid after 30 minutes at 4C, the test is negative (-).

Ability to use citrate as a sole source of carbon

Background
The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative. Thus, E.coli is a good negative control for the Citrate test, while Enterobacter and Klebsiella are good positive controls.

Procedure
Using aseptic technique, inoculate a Simmons Citrate agar slant ( 200µg/ml magnesium sulfate, 0.1% ammonium phosphate, 0.1% potassium phosphate, 0.2% sodium citrate, 0.5% NaCl, 1.5% agar, 80 µg/ml bromthymol blue by streaking the slant and stabbing the butt area with a small amount of a pure colony of bacteria to be tested. Inoculate another slant with a positive control organism. Utilization of citrate as a source of carbon is indicated by the development of a blue color (+) in the Simmons citrate agar slant. If the slant remains green, the test is negative (-).

Reagents
Simmons Citrate Agar
Sodium chloride 5g
Magnesium Sulfate 0.2g
Ammonium dihydrogen phosphate 1g
Dipotassium hydrogen phosphate 1g
citric acid 2g
deionized water to 1 liter
Agar agar 15g
pH to 6.8 with 4% (v/v) NaOH
Add 20ml of 0.4% bromothymol indicator to 1 liter of medium

Test for urease and phenylalanine deaminase (PDA): Urea hydrolysis to ammonia and PDA Disk test

Background
The extracellular enzyme urease is produced by some organisms and results in the production of ammonia from urea hydrolysis. Organisms capable of splitting urea release ammonia into the surrounding medium raising the pH.

Organisms containing the enzyme phenylalanine deaminase are capable of deaminating phenyalanine to produce phenylpyruvic acid. Ferric chloride reagent reacts with phenylpyruvic acid to produce a green color.

Procedure
To test for the presence of urease and phenlyalanine deaminase:

a.) add 0.3 ml of sterile saline to a sterile plastic test tube;
b.) using an overnight culture of the organism to be tested, make a heavy suspension of the organism in the sterile saline;
c.) place a Urea – PDA disk into the saline/bacteria suspension;
d.) incubate at 37°C for 2 hours; development of a red color within this time indicates a positive test (+) for urease; no color indicates a negative test (-);
e.) if a red color develops in the tube, add 2 drops of 1N HCl to clear the red color; if no red color has developed, do not add HCl and proceed directly to step f;
f.) add 2 drops of 10% Ferric Chloride Reagent to the tube; immediate development of a green color indicates a positive test (+) for the presence of phenylalanine deaminase; no color change is a negative test (-).

Hydrogen Sulfide Production and Indole Formation and motility using SIM medium (this test is also included as technique 2 within Motility Tests)

Background
The ingredients in SIM (sulfate/ indole/ motility) Medium enable the determination of three activities by which enteric bacteria can be differentiated. Sodium thiosulfate and ferrous ammonium sulfate are indicators of hydrogen sulfide production. The ferrous ammonium sulfate reacts with H₂S gas to produce ferrous sulfide, a black precipitate. The casein peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. Motility detection is possible due to the semisolid nature of the medium. Growth radiating out from the central stab line indicates that the test organism is motile. Organisms possessing the enzyme tryptophanase cleave trytophan, producing three end products. One of these endproducts is indole, produced in aerobic conditions, and skatole, produced in anaerobic conditions. Amyl alcohol in Kovacs reagent acts as a solvent for indole which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. Skatole will also give a positive indole reaction. Organisms which do not produce the enzyme produce no color change in the medium when Kovacs is added.

Procedure
Using aseptic technique, take a small amount of bacteria to be tested from a pure colony with an inoculating needle and make a stab inoculation into SIM agar tubes (also used for motility). Inoculate a positive control tube using the same technique. Incubate at 37C for 24 to 48 hours.

Motile organisms will exhibit growth radiating from the stab inoculation line. Non motile organisms will exhibit growth only along the stab inoculation line.

A blackening of the medium, either along the stab line or throughout the medium, indicates the production of hydrogen sulfide by the microorganisms.

To detect indole production, add three or four drops of Kovacs’ reagent and observe for a red color(positive reaction).

Bactidrop Rapid Spot Indole Test for Tryptophanases Background
The indole test is a qualitative procedure used to determine whether or not microorganisms contain tryptophanases that catalyse the production of indole from the amino acid tryptophan. If indole is present, it reacts with ρ-dimethylaminocinnamaldehyde (DMACA) to produce a blue-green colored complex.
Procedure
Add 2 drops of Bactidrop Spot Indole Reagent to one of the filter paper strips provided. Using a sterile inoculating loop remove a small amount of bacteria to be tested from a nutrient agar slant or an isolated colony from a nutrient agar plate and smear it into the area of the filter paper that has been saturated with the reagent. Do not use colonies from MacConkey or EMB agar.

Blue color within 3 minutes = positive indole test (+)
Pink color = negative indole test (-)

Control Organisms:

Organism	ATCC	Motility	H2S	Indole
Escherichia coli	25922	+	-	+
Salmonella choleraesuis subsp. choleraesuis serotype Typhimurium	13311	+	+	-
Shigella flexneri	9199	-	-	-

SIM agar and Reagent recipes:
SIM agar:
Approximate Formula* Per Liter
Pancreatic Digest of Casein - 20.0 g
Peptic Digest of Animal Tissue - 6.1 g
Ferrous Ammonium Sulfate - 0.2 g
Sodium Thiosulfate - 0.2 g
Agar - 3.5 g

Kovac's reagent: (per liter)
p-Dimethylaminobenzaldehyde 50,0g
Amyl Alcohol 750.0 ml
Hydrochloric acid 250.0 ml

Indole Spot Test Reagent
p-Dimethylaminocinnamaldehyde 10.0 g
Hydrochloric acid 100.0ml
Water, deionized 900.0 ml

Links to Labs

Lab 1
Lab 2
Lab 3
Lab 4
Lab 5
Lab 6
Lab 7
Lab 8
Lab 9
Lab 10
Lab11
Lab 12