EXOENZYMES
The following tests detect the presence of exoenzymes. Exoenzymes are enzymes that are secreted into the surrounding medium and work on substrates found outside the cell. In general, these exoenzymes are hydrolytic and break down large biomolecules that are too large to be easily transported into the cell.
These biomolecules must be broken down into their smaller building blocks before they can be made available as a nutrient source for the cell. Starch must broken down into glucose, protein into amino acids, and triglycerides into fatty acids and glycerol.
EX. 5-12:
STARCH HYDROLYSIS
TEST – This is a test for the amylase exoenzyme that breaks down starch into glucose
Organisms : E. coli and B. cereus
Media : One starch agar plate per pair
Procedure :
1.
Divide plate in half and label each section with the appropriate organism. Do a straight line inoculation of each organism onto the plate. Incubate, inverted, at 37° C.
2.
After incubation, flood the plate with Gram’s iodine. Observe for the presence of dark color surrounding the inoculation line. Dark color up to the edge of the inoculum indicates the absence of starch hydrolysis. A clear zone surrounding the inoculum indicates a positive reaction, meaning that the starch in the medium has been broken down (hydrolyzed). Discard the plate after it has been reacted with iodine.
EX. 5-14:
CASEASE TEST – This is a test for protease exoenzymes that breakdown the milk protein, casein, into amino acids
Organisms : E. coli, S. marcescens , and Pseudomonas aeruginosa
Media : One Casein agar plate
Procedure :
1.
Divide the plate into thirds and label each section of the plate with one of the three organisms.
Do a straight line inoculation into the correct section of the plate. Incubate, inverted, for 48-72 hours at 37° C.
2.
Observe for a clear zone surrounding the inoculum which is indicative of casein hydrolysis.
EX. 5-15:
GELATINASE TEST – This is a test for the presence of the gelatinase exoenzyme, which breaks down the protein, gelatin, to amino acids
Organisms : E. coli, S. marcescens, and Pseudomonas aeruginosa
Media: Three gelatin deeps
Procedure:
1.
With a needle, inoculate the deeps in a straight line stab inoculation. Incubate the tubes for at least 48 hrs. at 37° C.
2.
After incubation, chill the tubes in an ice bath or the refrigerator and look for the presence or absence of liquefaction after chilling. Remember that any color change you observe is due to the
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pigment produced by that organism, not by any by product of the gelatinase reaction. If the reaction appears to be negative, return the tubes to the 37° C incubator. For some species, liquefaction can take up to a week to observe.
EX. 5-17:
LIPASE TEST for lipase exoenzymes that breakdown lipids to glycerol and fatty acids
Organisms : E. coli, S. marcescens, and Pseudomonas aeruginosa
Media : Tributyrin (lipid) agar plate
Procedure :
1.
Divide plate into thirds. Do a straight line inoculation of each organism into the appropriate section of the plate. Incubate, inverted, for at least 48 hours at 37° C.
2.
A clearing of the agar around the inoculum indicates the breakdown of lipids in the medium.
Lipid hydrolysis may take longer for some lipid + species. If you observe incomplete clearing around an inoculum, return your plate to the 37° C incubator and check it again next period.
CATALASE AND OXIDASE TESTS
After the Gram reaction and cell shape has been determined, the catalase test and the oxidase test are often the first tests performed to identify an unknown organism.
If an organism is Gram negative rod, then a negative oxidase test indicates the organism is a Enteric bacteria; a positive oxidase test indicates a non-enteric organism. We will be using the oxidase to test to determine which rapid ID test should be used to identify the unknown organism you isolated from hamburger.
If an organism is a Gram positive coccus, a negative catalase test indicates it is a Streptococcus ; a positive catalase test indicates is a Staphylococcus . The coagulase test can then be used to further identify the species of the Staphylcoccus .
EX. 5-5: CATALASE TEST
This test is used to indicate whether a microorganism produces the enzyme catalase that breaks down hydrogen peroxide to water and oxygen. This enzyme is important to aerobic organisms because it detoxifies hydrogen peroxide. Hydrogen peroxide forms during aerobic metabolism when components of the respiratory chain donate electrons to molecular oxygen. The other enzyme produced by many microorganisms to detoxify hydrogen peroxide is peroxidase, however, no oxygen is evolved from the breakdown of hydrogen peroxide by peroxidase. This test is used to differentiate between
Streptococcus and Staphylococcus species .
Organisms: S. aureus, S. epidermidis, Streptococcus salivarius and any other organisms used in today’s exercises. Use only cultures grown on agar for the catalase test.
Materials Needed : Microscope slides, hydrogen peroxide in small bottles
Procedure : Place a drop of hydrogen peroxide on a petri dish lid or microscope slide, add a loopful of the organism and observe for immediate bubbling. Catalase positive organisms will exhibit bubbling, catalase negative will not.
EX. 5-6: OXIDASE TEST
This test detects the presence of the enzyme, cytochrome oxidase, which transfers electrons from cytochrome c to molecular oxygen in the electron transport chain. Many aerobic bacteria, such as
Neisseria sp. and Pseudomonas sp., have cytochrome oxidase. On the other hand, many facultative anaerobes such as those in the family Enterobacteriaceae (Gram negative facultative anaerobes), are
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oxidase negative because they lack cytochrome c in their electron transport chain, and therefore, do not have cytochrome oxidase. This test is often used to differentiate between enteric (facultative anaerobic) and non-enteric (aerobic) gram negative bacteria.
Organisms : E. coli, Pseudomonas aeruginosa and Alcaligenes faecalis grown on TSA plates
Materials Needed: Oxistrips (Tetramethyl-p-phenylenediamine dihydrochloride or TPPD)
Plastic sterile disposable transfer loops
Procedure : Place an Oxistrip on a paper towel. Using a PLASTIC loop, smear the test strip (the part with a matte appearance) with a loopful of inoculum. A positive result will produce a dark purple color within 20 to 30 seconds. No color means the organism is negative for cytochrome oxidase.
NUTRITIONAL REQUIREMENTS: DIFFERENTIAL AND SELECTIVE MEDIA
Purpose: To become familiar with various types of commonly used selective and differential media and their uses.
EX. 4-4: MANNITOL SALT AGAR
Organisms : Staphylococcus aureus , Staphylococcus epidermidis , Staphylococcus saprophyticus; E. coli
Media : One MSA (mannitol salt agar) plate per pair
Procedure :
1. Divide plates into four sections. Label each section with the name of a different organism.
2. Using aseptic technique, do a straight line inoculation, with a loop, of each organism into the appropriate section of the plate. Incubate the plate, upside down, at 37° C.
3. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth (scant, medium, or abundant). Growth on the surface indicates salt tolerance. Note any changes in the color of the agar itself. A yellow color indicates the ability to ferment the sugar mannitol.
EX. 4-5: MACCONKEY AGAR
Organisms : Escherichia coli , Enterobacter aerogenes , Staphylococcus aureus , and Proteus vulgaris
Media : One MacConkey agar plate per pair
Procedure :
1 . Divide plate into four sections. Follow same procedure as for Ex. 4-1. Incubate the plate at 37° C.
2. Next lab period: Record your observations on the appropriate pages from the lab manual. Look for the growth on the surface of the agar and make note of the amount of growth (scant, medium, or abundant). Growth indicates that the organism is Gram negative. Note the color of the growth and the surrounding agar. A bright pink/magenta color indicates lactose fermentation. A bright pink
"cloud" in the agar indicates Escherichia coli , produced by the large degree of lactose fermentation.
EX. 5-3: PHENOL RED BROTH
Purpose: test of the capacity of bacteria to oxidize various carbohydrates via a fermentative pathway
Organisms : E. coli, Alcaligenes faecalis, Proteus vulgaris, & S. aureus
Media : Four tubes of each of the following, per pair of students: phenol red glucose (dextrose) broth, phenol red lactose broth, phenol red sucrose broth
Procedure :
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1. These tubes contain an additional glass tube, called a Durham tube, inside the medium. Before inoculating, invert any tubes that may have bubbles to remove the bubbles.
2. Label the tubes and inoculate each tube with the appropriate organism.
3. Incubate tubes at 37° C. Make sure the tops are loosened by 1 /
2
turn when they are incubated.
4. Next lab period: Observe the results and look for a color change (yellow, orange, pink, deeper red), a bubble in the Durham tube (large, medium, small or barely visible), and turbidity (cloudy or not).
NOTE:

Work with your lab partner.


Make sure the cells are suspended before inoculating.
Label the plates carefully. It is easy to inoculate the wrong organism or to inoculate the same organism twice.

Likewise, the plates you are using are red or pink in color and are similar in appearance.
Make certain that you have the correct medium for each part of the exercise.

Be sure to label the plates with your initials, date, and some identifiable mark.

Remember to invert the plates in the incubator. All the plates should be observed when they are removed from the incubator as the results may change after refrigeration.
Follow aseptic technique and flame loops over their entire length until red-hot; failure to do this will result in cross-contamination of the cultures.
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The following tests can be used to differentiate Gram negative rods . In particular, the Indole test,
Methyl Red test, Voges-Proskauer test, and Citrate utilization are sometimes known collectively as the
IMViC test and are used whenever preliminary tests indicate an unknown organism belongs to the family
Enterobacteriaceae . The Enterobacteriaceae include pathogens such as Salmonella and Shigella , occasional pathogens such as Proteus and Klebsiella , and normal intestinal flora such as Escherichia and
Enterobacter . These tests are not entirely inclusive and they may be used for other purposes as well. The table below summarizes the characteristics of a few common species.
Laboratory Tests for Differentiation of Gram Negative Rods
Organism
Enterobacter aerogenes
Escherichia coli
Proteus vulgaris
Salmonella arizoniae
Alcaligenes faecalis
E AG AG AG+/–
– – –
+ + +
– – – – –
E AG AG A+/– – + + – – + – – – – –
E
E
N
AG
AG+/–
–
– AG+/– + + + – +/– + + – + – –
–
–
A+/–
–
+ – + – + + – – – – –
– – – –
+/–
– –
+
– – –
Pseudomonas aeruginosa N – – – – – – – + + – + + – +
A = acid
G = gas
+/– = result can vary between strains of the same species
The tests presented in Chapter 5 of the Lab Manual are all differential. Differential tests are based on the reaction of an indicator reagent with a biochemical product that is produced in the presence of a specific enzyme made by an organism. The differential media must contain the appropriate substrate for the reaction as well as the indicator. It is up to the organism to make the product., which then reacts with the indicator. Selective media are used to enrich for a particular type of organism if you are working with a mixed culture specimen that may have, for instance, been collected from a patient. Remember that some media are both selective and differential, while others are selective only or differential only.
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EX. 5-4: METHYL RED AND VOGES PROSKAUER TEST - The methyl red test is used to identify bacteria that produce mixed acids (lactic, acetic, or formic) as a result of glucose fermentation. The acid end products lower the pH of the growth medium to 5.0 or lower. The Voges Proskauer test identifies bacteria that form non-acidic end products as a result of glucose fermentation. The final fermentation end products of these bacteria are ethanol and acetylmethylcarbinol (acetoin). These tests are frequently used to distinguish between Escherichia coli and Enterobacter aerogenes.
Organisms : E. coli and E. aerogenes - broth cultures
Media: Two tubes of MRVP broth
Procedure :
1.
Inoculate organisms into the appropriate tubes. Incubate at 37° C.
2.
After incubation, aseptically transfer 1 ml of the culture with a sterile bulb pipette to one of the sterile tubes in the hood. You will now have two tubes for each organism. The tube with 4 ml of culture will be used for the methyl red test. The other tube with the l ml of culture will be used for the Voges Proskauer test. Since you have two organisms, you will have a total of four tubes.
3.
To the tubes for the Methyl Red test, add 5 drops of the methyl red indicator. When methyl red is added, the indicator turns the medium red in the presence of acid. This is done under the hood.
Read the tubes immediately and look for color change from pale yellow to red. Red color indicates the presence of acidic end products from glucose fermentation.
4.
To the tubes for the Voges Proskauer test, add 10 drops of Barritt’s solution A (5% alpha naphthol) and then add 10 drops of Barritt’s solution B (40% potassium hydroxide). Allow 15 to
20 minutes to pass and observe for the appearance of a red ring at the top of the culture. This color change is indicative of a positive result. The absence of a red ring is a negative result for the presence of acetylmethylcarbinol.
Note: All chemicals are to be used under the hood and with extreme care!
Ex. 5-7: NITRATE REDUCTION TEST - Some microorganisms are capable of using inorganic molecules other than oxygen as the terminal electron acceptor during an energy yielding metabolic pathway such as respiration. When this occurs, the process is called anaerobic respiration. Carbonate, sulfate, and nitrate are examples of inorganic terminal electron acceptors other than oxygen. The process whereby nitrate is reduced to nitrite, ammonia, or molecular nitrogen, is known as nitrate reduction.
Organisms : E. coli, P. aeruginosa, and Alcaligenes faecalis
Media : Three tubes of nitrate broth per pair
Procedure :
1.
Inoculate and incubate tubes at 37° C.
2.
After the specified incubation time, add reagents A and B (IN THE HOOD) as indicated on the reagent bottles and make observations. Interpretation of the results can be confusing. Read the manual carefully! a.
If nitrate is reduced to nitrite, the nitrite will react with reagents A + B and the medium will turn red. This color change will occur very quickly and can be recorded as a positive result. b.
If the medium remains colorless there are two possibilities: 1) the nitrate was further reduced to nitrogen gas or ammonia or 2) the nitrate was not reduced at all.To differentiate between these two possibilities, zinc is added. Zinc will react with nitrate, the original substrate, and reduce it nitrite. This newly produced nitrite will then react with the reagents A + B that were added previously and turn red. If your medium turns red after adding Zinc, then the reaction is negative because the original substrate, nitrate,
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was not reduced. This reaction may take 5 to 10 minutes to develop.If there is no color change after adding Zinc, then the original substrate nitrate was reduced to a product that cannot be detected by reagents A + B. These products can be either N
2
gas or NH
3
, ammonia. This is a positive result.
Therefore:
If A + B  red, then organism is positive for nitrate reduction
If A + B

no change, then add zinc
If A + B + Zinc

red, then organism is negative for nitrate reduction
If A + B + Zinc  no change, then organism is positive for nitrate reduction
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