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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 . -kc

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 (FeCl 3 ) solution to the slant. After addition of the reagent, rotate the tube to dislodge the surface colonies.

3. Results 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. 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. P rocedure: 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. Place cap loosely on the tube. 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. 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.

8. RESULT: Urea agar test (a) uninoculated , (b)  Proteus mirabilis  (rapidly urease positive), (c)  Klebsiella pneumoniae  (delayed urease positive), (d)  Escherichia coli  (urease negative).

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 (NO 3 – ) to nitrite (NO 2 – ) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH 3)  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 (NO 3 – ) to nitrite (NO 2 – )  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 Nitrate Reduction Positive:  ( Red  after sulfanilic acid + alpha- naphthylamine ; no color after zinc) 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 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. Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to give a milky suspension. Incubate the tube at 35°C in ambient air or in a water bath for 4 hours. Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90 °. 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 Inoculate two tubes of OF test medium with the test organism using a straight wire by stabbing “halfway to the bottom” of the tube. 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. Incubate both tubes at 35°C for 48 hours (slow-growing bacteria may take 3 to 4 days before results can be observed)

16. 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  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   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 Escherichia coli  (MR test positive): the appearance of red color after adding methyl red reagent. 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 20 th  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 Inoculate a tube of MR/VP broth with a pure culture of the test organism. Incubate for 24 hours at 35°C At the end of this time, aliquot 1 mL of broth into a clean test tube. 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.) 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: Klebsiella  species Enterobacter  species