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1. Biochemical tests?
Biochemical tests are the tests used for the identification of bacteria species based
on the differences in the biochemical activities of different bacteria.
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2. Phenylalanine Deaminase Test or phenylpyruvic acid (PPA) test:
1}Uses of Phenylalaine Deaminase Test
Phenylalaine deaminase test is used to differentiate members of the genera Proteus, Morganella,
and Providencia (+ve) from other members of Enterobacteriaceae which give negative results.
Phenylalanine deaminase test also known as phenylpyruvic acid (PPA) test is used to test the ability of an
organism to produce enzyme deaminase. This enzyme removes the amine group from the amino acid
phenylalanine and produces phenylpyruvic acid (PPA) and ammonia i.e. oxidative deamination of
phenylalanine. Phenylpyruvic acid reacts with ferric iron (10% ferric chloride is added to the medium)
producing a visible green color.
Procedure
Take or prepare a phenylalanine deaminase agar medium
Inoculate the phenylalanine slant (with a loop on the surface) with a test organism.
Note: If you are using the test medium i.e. phenylalanine agar for the first time use positive (Proteus vulgaris)
and negative control (Escherichia coli) to check the efficacy of the test medium.
Incubate the test medium at 37°C for overnight.
Add 4-5 drops of 10% aqueous ferric chloride (FeCl3) solution to the slant. After addition of the reagent,
rotate the tube to dislodge the surface colonies.
3. Results
1.Positive test: Production of green color (Phenylpyruvic
acid thus formed reacts with ferric chloride producing a
green colored compound thus turning the medium dark
green). Proteus sp., Morganella sp., Providenica sp give
positive PPA test.
2.Negative: No color change (medium remains
straw/yellow color; no PPA to react with ferric chloride).
4. 2}Citrate Utilization Test:
The citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red, VP, and
Citrate) tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products.
Citrate utilization can be used to distinguish between coliforms such as Klebsiella aerogenes (formerly Enterobacter
aerogenes) (+ve) which occur naturally in the soil and in aquatic environments from fecal coliforms such as Escherichia
coli (-ve) whose presence would be indicative of fecal contamination.
Citrate utilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and
inorganic ammonium dihydrogen phosphate (NH4H2PO4) as the sole nitrogen source.
Citrate Positive Organisms
Many members of the family Enterobacteriaceae are citrate positive.
Klebsiella pneumoniae
Enterobacter species (minority of strains gives negative result)
Salmonella other than Typhi and Paratyphi A
Proteus mirabilis (a minority of strains gives negative result)
Proteus vulgaris
Vibrio cholerae
Citrate Negative Organisms
Escherichia coli
Shigella spp
Salmonella Typhi
5. Procedure:
1.Inoculate Simmons citrate agar lightly on the slant* by touching the tip of a needle
to a colony that is 18 to 24 hours old.
2.Place cap loosely on the tube.
3.Incubate aerobically at 35°C to 37°C for 18 to 24 hours. Some organisms may
require up to 7 days of incubation due to their limited rate of growth on citrate medium.
4.Observe the development of blue color along the slant; denoting alkalinization.
Citrate utilization test
A: Negative
B: Positive
RESULT:
Citrate positive: growth will be visible on the slant surface and the medium will be
an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-
products of citrate catabolism raise the pH of the medium to above 7.6, causing
the bromothymol blue to change from the original green color to blue.
Citrate negative: trace or no growth will be visible. No color change will occur; the
medium will remain the deep forest green color of the uninoculated agar. Only bacteria
that can utilize citrate as the sole carbon and energy source will be able to grow on the
Simmons citrate medium, thus a citrate-negative test culture will be virtually
indistinguishable from an uninoculated slant. E. coli is citrate negative.
6. Urease test /Chrishtensions urea biochemical test:
Urease is a constitutively expressed enzyme that hydrolyzes urea to carbon dioxide and ammonia. Many
organisms especially those that infect the urinary tract, have a urease enzyme that is able to split urea in the
presence of water to release ammonia and carbon dioxide. The ammonia combines with carbon dioxide and
water to form ammonium carbonate which turns the medium alkaline, turning the indicator phenol red from
its original orange yellow color to bright pink.
Urease test can be used as part of the identification of several genera and species
of Enterobacteriaceae including Proteus and Klebsiella. It is also useful to
identify Cryptococcus species, Brucella, Helicobacter pylori.
Urease test helps for the identification of Proteus species (urease positive) and to differentiate it from other
non-lactose fermenting members of the Enterobacteriaceae family.
7. Medium used for urease test: Any urea medium, agar (Christensen’s urea agar), or broth
(Stuart’s urea broth)
Procedure for Urease test:
A)For Christensen’s urea agar
Streak the entire slant surface with a heavy inoculum from an 18-24 hour pure culture (do
not stab the butt as it will serve as a color control).
Incubate tubes with loosened caps at 35°C.
Observe the slant for a color change at 6 hours and 24 hours unless specified for longer
incubation.
B)For Stuart’s Urea Broth;
Inoculate the broth with a heavy inoculum from an 18-24 hour pure culture
Shake the tube gently to suspend the bacteria
Incubate the tubes with loosened caps at 35°C.
Observe the broth for a color change at 8, 12, 24 hours.
9. Nitrate Reduction Test:
Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis of their
ability to produce nitrate reductase enzyme that hydrolyzes nitrate (NO3
–) to nitrite (NO2
–) which may then
again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia
(NH3) depending on the enzyme system of the organism and the atmosphere in which it is growing.
Procedure:
1} Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the broth is tested for
reduction of nitrate (NO3
–) to nitrite (NO2
–) by adding sulfanilic acid reagent and α- naphthylamine.
If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When sulfanilic
acid is added, it will react with the nitrous acid to produce diazotized sulfanilic acid. This reacts with the α-
naphthylamine to form a red-colored compound. Therefore, if the medium turns red after the addition of the
nitrate reagents, it is considered a positive result for nitrate reduction.
10. 2} If the medium does not turn red after the addition of the reagents, it can mean that the organism
was unable to reduce the nitrate, or the organism was able to denitrify the nitrate or nitrite to
produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. Add a
small amount of powdered zinc. If the tube turns red after the addition of the zinc, it means that
unreduced nitrate was present*. Therefore, the red color on the second step is a negative result.
If the medium does not turn red after the addition of the zinc powder, then the result is called a positive
complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the
medium, either, that means that denitrification took place and ammonia or molecular nitrogen were formed.
Result and Interpretation
1.Nitrate Reduction Positive: (Red after
sulfanilic acid + alpha-naphthylamine; no color
after zinc)
2.Nitrate Reduction Negative: (No color after
sulfanilic acid + alpha-naphthylamine followed
by Red after zinc)
11. Coagulase Test:
The coagulase test helps to identify Staphylococcus aureus which produces the enzyme coagulase
(coagulates) plasma from coagulase-negative staphylococci (CONS). Coagulase is also
the virulence factor of S. aureus, It converts soluble fibrinogen in plasma to insoluble fibrin, and the
fibrin coat the bacterial cells making protecting them from opsonization and phagocytosis.
Tube Coagulase Test Procedure
1.Prepare a 1-in-6 dilution of the plasma in saline (0.85% NaCl) and place 1 ml volumes of the diluted plasma in small
tubes.
2.Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to give a milky suspension.
3.Incubate the tube at 35°C in ambient air or in a water bath for 4 hours.
4.Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90°.
5.Leave negative tubes at room temperature overnight and re-examine.
(This step is essential, for some strains of S. aureus, including many MRSA, which produce a delayed clot that is
rapidly lysed at 37°C by the organism’s staphylokinase.)
12. Result:
Read as positive any degree of clot formation. Often the plasma is converted into a stiff
gel that remains in place when the tube is tilted or inverted, but sometimes clots are
seen floating in the fluid.
Coagulase Positive: Clot of any size eg. Staphylococcus aureus
Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent or ropy
precipitate). eg. Staphylococcus epidermidis
13. Bile-Esculin test:Bile-esculin test is widely used to differentiate Enterococci and non-enterococcus group
D streptococci, which are bile tolerant and can hydrolyze esculin to esculetin, from non-group D viridans
group streptococci, which grow poorly on bile.
Bile-esculin test is based on the ability of certain bacteria, notably the group D
streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile (4%
bile salts or 40% bile).
Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin). The two moieties of the
molecule (glucose and 7-hydroxycoumarin) are linked together by an ester bond through oxygen. For this test,
esculin is incorporated into a medium containing 4% bile salts.
Bacteria that are bile-esculin positive are, first of all, able to grow in the presence of bile salts. Hydrolysis of the
esculin in the medium results in the formation of glucose and a compound called esculetin.
Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black
diffusible complex
Results and Interpretation
Diffuse blackening of more than half of the slant within 24-48 hours indicates esculin hydrolysis. On plates, black haloes will
be observed around isolated colonies and any blackening is considered positive. All group D streptococci will be bile-esculin
positive within 48 hours.
14. Oxidative fermentative (OF) test:
The oxidative-fermentative (OF) test was developed by Hugh and Leifson in 1953. They developed
OF media to differentiate between oxidative bacteria (that produces acid from carbohydrates under
aerobic condition only) and fermentative bacteria (that produces acid both under aerobic and
anaerobic conditions).
Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively. The end products
of fermentation are relatively strong mixed acids that can be detected in a conventional fermentation
test medium. However, the acids formed in oxidative degradation of glucose are extremely weak and
less, and the more sensitive oxidation fermentation medium of Hugh and Leifson’s OF medium is
required for the detection.
The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation
or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a
variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during
fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or
absence of oxygen.
15. Procedure
1.Inoculate two tubes of OF test medium with the test organism using a straight wire by stabbing
“halfway to the bottom” of the tube.
2.Cover one tube of each pair with a 1 cm layer of sterile mineral oil or liquid paraffin (it creates an
anaerobic condition in the tube by preventing diffusion of oxygen), leaving the other tube open to the air.
3.Incubate both tubes at 35°C for 48 hours (slow-growing bacteria may take 3 to 4 days before results
can be observed)
16. 1.Fermentative result: Acid production on both (open and covered) tubes. The acid produced
changes the pH indicator, bromthymol blue, from green to yellow. e.g. Escherichia coli
2.Oxidative result: Acid production in the open tube (aerobic) and not the oil-covered tube
(anaerobic) indicates an oxidative result. Nonfermenting bacteria that metabolize glucose via
oxidative metabolism give an oxidative result. e.g. Pseudomonas aeruginosa
3.Non-saccharolytic (negative OF result): Nonsacchrolytic bacteria give a negative OF result.
The negative result is indicated by no color change in the oil-covered tube and in some cases an
increase in pH (pH 7.6) changing the bromthymol blue from green to blue on the top of the open
tube. The increase in pH is due to amine production by bacteria that break down the peptone
(protein) in the medium.
e.g. Alcaligenes faecalis.
17. Methyl Red (MR) Test:
Methyl Red (MR) test determines whether an organism performs mixed acid fermentation and
produces stable acid end products. Methyl red is the indicator that detects the pH after an enteric
Gram-negative rod has fermented glucose to completion.
In mixed acid fermentation, bacteria form three acids (acetic, lactic, and succinic) in
significant amounts, decreasing the medium’s pH below 4.4. It is visualized by using a
pH indicator, methyl red (2-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid), a pH
indicator which is red at pH ≤ 4.4, and yellow color at pH 5.8.
The pH at which methyl red detects acid is considerably lower than the pH indicators used in bacteriological culture
media. Thus, the test organism must produce large quantities of acid from the supplied carbohydrate source to
produce a color change. Change in the MR-VP broth’s color is observed after adding a pH indicator, and the result
is categorized as;
•MR Positive: The culture medium turns red (because the pH of the medium is at or below 4.4 from the
fermentation of glucose).
•MR Negative: Culture medium remains yellow (less acid is produced from the fermentation of glucose).
18. Expected results of MR Test
1.Escherichia coli (MR test positive): the appearance of red color after adding methyl
red reagent.
2.Klebsiella (formerly Enterobacter) aerogenes (MR test negative): the lack of color
change after adding methyl red.
Methyl Red Test
Left: Negative
Right: Positive
19. Voges Proskauer (VP) Test:
The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol
from glucose fermentation. Voges-Proskauer is a double eponym, named after two microbiologists
working at the beginning of the 20th century. They first observed the red color reaction produced by
appropriate culture media after treatment with potassium hydroxide.
It was later discovered that the active product in the medium formed by bacterial metabolism is acetyl methyl carbinol,
a product of the butylene glycol pathway.
Pyruvic acid, the pivotal compound in the fermentative degradation of glucose, is further metabolized through
various metabolic pathways, depending on the enzyme systems possessed by different bacteria. One such pathway
results in the production of acetoin (acetyl methyl carbinol), a neutral-reacting end product.
If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and
atmospheric oxygen. The diacetyl and guanidine-containing compounds found in the peptones of the broth then
condense to form a pinkish-red polymer.
20. Procedure of Voges Proskauer Test
1.Inoculate a tube of MR/VP broth with a pure culture of the test organism.
2.Incubate for 24 hours at 35°C
3.At the end of this time, aliquot 1 mL of broth into a clean test tube.
4.Add 0.6mL of 5% α-naphthol*, followed by 0.2 mL of 40% KOH.
(Note: It is essential that the reagents be added in this order.)
5.Shake the tube** gently to expose the medium to atmospheric oxygen and allow the
tube to remain undisturbed for 10 to 15 minutes.
Voges-Proskauer (VP) Positive Organisms of Enterobacteriaceae family are:
1.Klebsiella species
2.Enterobacter species
