Biochemical Test performed in routine laboratory work

1. Catalase Test

All of the Enterobacteriaceae are catalase positive except Shigella dysenteriae.

2. Oxidase Test

i) Used to demonstrate the ability of a bacterium to produce the enzyme cytochrome- C oxidase, capable of reducing oxygen.
ii) Only aerobic bacteria have this enzyme. This test will distinguish aerobic vs. anaerobic metabolism.
iii) A positive test will show a color change to blue, then to dark purple or black, within 10 to 30 seconds.
iv) Assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase.
v) In the presence of an organism that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product.
vi) The Family Enterobacteriaceae are oxidase negative.
vii) The lists of Enterobacteriaceae are given below.
Salmonella
Shigella
Enterobacter
Proteus
Serrati
Erwinia
Yersinia
viii) The members of the Family Vibrionaceae are usually oxidase positive.
Vibrio and Aeromonas.
ix) The members of Family Pastuerellaceae are oxidase-positive and non-motile.
Pasteurella, Haemophilus, Actinobacillus
x) The members of Family Pseudomanadaceae are motile, catalase-positive and usually oxidase positive.

3. IMVic Test

IMViC are used in the clinical laboratory to distinguish between enteric microorganisms.

3.1 Indole Test/Typtone broth

*Used to demonstrate the ability of a bacterium to produce the enzyme tryptophanase. This enzyme acts on the amino acid to produce “indole”.

*Description: Tryptophan hydrolysis Some bacteria split tryptophan into indole and pyruvic acid using the hydrolase called tryptophanase. Indole can be detected with Kovac's reagent (Indole reagent). This test is very important in differentiating E. coli (indole positive) from some closely related enteric bacteria. It also differentiates Proteus mirabilis (indole negative) from all other Proteus species (indole positive). Tryptone broth is used for this test as it contains a large amount of tryptophan.

*Interpretation: After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Add a few drops of Indole reagent to the broth culture (tryptone broth). DO NOT SHAKE THE TUBE. A positive result has a red layer at the top. A negative result has a yellow or brown layer.

3.2 Methyl Red Test

i) An indicator of low pH (red below pH of 4.4) – used to show the mixed acid fermentation ability of bacteria.

ii) Description: Mixed acid fermentation – Many gram-negative intestinal bacteria can be differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, and Proteus ferment glucose to produce lactic, acetic, succinic, and formic acids and CO₂, H₂, and ethanol. The large amounts of acids produced lowers the pH of the medium - Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas.

iii) Interpretation: After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test if you are going to use the same broth for the VP test. Add 3-4 drops of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer.

3.3 Voges-Proskauer Test

i) Used to show bacterial production of acetoin, also known as 2,3-butanediol.

ii) Description: Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do. Enterobacter, Serratia and some species of Bacillus produce these substances. There is no satisfactory test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.

iii) Interpretation: After incubation: Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and cannot be tested. Barritt's reagent A (VP A) contains α- naphthol and Barritt's B (VP B) contains 40% KOH. Test 1 ml of your culture from the MRVP broth. If you have already conducted the methyl red test, you should have already placed 1 ml of untested broth in a sterile tube. If you haven’t done this, do so now. Add the entire contents of the VP A reagent (15 drops) and 5 drops of the VP B reagent to the 1 ml of your broth culture. SHAKE WELL. This reaction will take a few minutes before you will see a color change. SHAKE the tube every few minutes for best results. With a positive reaction the medium will change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change color or will be copper colored. Wait at least 15-30 minutes for color to develop before calling the test negative.

NOTE: The ratio of Barrit’s Reagent A and Baritt’s Reagent B should be 3:1.

3.4 Citrate test

i) Simmons citrate agar tests for the ability of a gram-negative organism to import citrate for use as the sole carbon and energy source. Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium.

ii) Description: Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH.

iii) Interpretation: After incubation, a positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green.Citrate is converted to Oxalo acetic acid by citrate lyase and oxaloacetate decarboxylase activity will convert oxaloacetate to pyruvate with the release of carbondioxide. The other products of the reaction are acetate, Lactic acid, formic cid etc. The carbondioxide reacts with sodium and water to form sodium carbonate.

4. Triple Sugar Iron Agar (TSIA) Test

The triple sugar iron agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish them from other gram negative intestinal bacilli. This differentiation is based on the differences in carbohydrate fermentation patterns and hydrogen sulphide production by the various groups of intestinal organisms. Carbohydrate fermentation is detected by the presence of gas and a visible colour change (from red to yellow) of the pH indicator, phenol red. The production of hydrogen sulfide is indicated by the presence of a precipitate that blackens the medium in the butt of the tube. TSI Agar contains three fermentative sugars, lactose and sucrose in 1% concentrations and glucose in a concentration of 0.1%. Due to the building of acid during fermentation, the pH falls. The acid base indicator Phenol red is incorporated for detecting carbohydrate fermentation that is indicated by the change in colour of the medium from orange red to yellow in the presence of acids. In case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This is indicated by the change in colour of the medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide (indicated by blackening in the butt of the tube). To facilitate the detection of organisms that only ferment glucose, the glucose concentration is one-tenth the concentration of lactose or sucrose. The small amount of acid produced in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain orange red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube since it is under lower oxygen tension. After depletion of the limited glucose, organisms able to do so will begin to utilize the lactose or sucrose. To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely. If the tube is tightly closed, an acid reaction (caused solely by glucose fermentation) will also involve the slant.

5. Urease Test

Demonstrates the ability of a bacterium to produce the enzyme urease, capable of hydrolyzing urea. Phenol red indicator is added (fuchsia above pH 8.4) to show rise in pH due to accumulation of ammonia.Urease test is performed by growing the test organism on urea broth or agar medium containing the pH indicator phenol red (pH 6.8). During incubation, microorganisms possessing urease will produce ammonia that raises the pH of the medium/broth. As the pH becomes higher, the phenol red changes from a yellow colour (pH 6.8) to a red or deep pink (cerise) colour. Failure of the development of a pink colour due to no ammonia production is evidence of a lack of urease production by the microorganisms. Urea is a nitrogen containing compound that is produced during the decarboxylation process of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is thus it is an efficient way for the human body to excess nitrogen. This excess urea is then taken away from the body with the help of the kidneys as a part of urine. Certain bacteria produce the enzyme urease during its metabolism process and that will break down the urea in the medium to ammonia and carbon dioxide. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. The urease test is useful in identifying the genera Proteus, and Morganella, which liberate this enzyme. Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia. The presence of urease is detectable when the organisms are grown in a urea broth medium containing the pH indicator phenol red. As the substrate urea is split into its products, the presence of ammonia creates an alkaline environment that causes the phenol red to turn to deep pink. This is a positive reaction for the presence of urease. Failure of deep pink colour to develop is evidence of a negative reaction.

6. Motility

The ability of an organism to move by itself is called motility. Motility is closely linked with chemotaxis, the ability to orientate along certain chemical gradients. Eukaryotic cells can move by means of different locomotor organelles such as cilia, flagella, or pseudopods. Prokaryotes move by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. Almost all spiral bacteria and about half of the bacilli are motile, whereas essentially none of the cocci are motile. The medium mainly used for this purpose is SIM medium ( Sulphide Indole Motility medium) which is a combination differential medium that tests three different parameters, sulphur reduction, indole production and motility. This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness. In soft agar tubes non-motile bacteria will only grow on the inoculated region. Motile bacteria will grow along the stab line and will tend to swim out away from the stabbed area. Therefore, a negative result is represented by growth in a distinct zone directly along the stab. A positive result is indicated by diffuse or cloudy growth mainly at the top and bottom of the stabbed region. SIM agar may also be used to detect the presence of H2S production. The SIM medium contains peptones and sodium thiosulfate as substrates, and ferrous ammonium sulfate, Fe(NH4)SO4, as the H2S indicator. Cysteine is a component of the peptones used in SIM medium. Sufficient agar is present to make the medium semisolid. Once H2S is produced, it combines with the ferrous ammonium sulfate, forming an insoluble, black ferrous sulfide precipitate that can be seen along the line of the stab inoculation. If the organism is also motile, the entire tube may turn black. This black line or tube indicates a positive H2S reaction; absence of a black precipitate indicates a negative reaction.

7. H₂S Production

H₂S Producer: Proteus vulgaris, Citrobacter freundii and Salmonella
Non-H₂S Producer: Proteus rettgerii, Klebsiella ozaenae and Escherichia
Different reaction: Proteus

8. Coagulase

The coagulase test differentiates strains of Staphylococcus aureus from other coagulase-negative species.

Coagulase Positive: Staphylococcus aureus, Staphylococcus delphini, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lutrae and Staphylococcus pseudointermedius

Coagulase Negative: S. epidermidis, S. saprophyticus and Streptococcus pneumoniae

9. Lactose Fermentation

Lactose fermented rapidly
i) Escherichia coli: metallic sheen on differential media; motile; flat, non-viscous colonies
ii) Enterobacter aerogenes: raised colonies, no metallic sheen; often motile; more viscous growth
iii) Enterobacter cloacae: similar to Enterobacter aerogenes
iv) Klebsiella pneumoniae: very viscous, mucoid growth; nonmotile

Lactose fermented slowly
Edwardsiella, Serratia, Citrobacter, Arizona, Providencia and Erwinia

Lactose not fermented
i) Shigella species : nonmotile; no gas from dextrose
ii) Salmonella species: motile; acid and usually gas from dextrose
iii) Proteus species: "swarming" on agar; urea rapidly hydrolyzed (smell of ammonia)
iv) Pseudomonas species: soluble pigments, blue-green and fluorescing; sweetish smell

10. Glucose Fermentation

i) Determines the ability of a bacterium to ferment the sugar glucose as well as its ability to convert end products (pyruvic acid) into gaseous byproducts.
ii) Phenol red indicator is used to show acid fermentation (yellow below pH 6.8) or alkaline fermentation (red above pH 8.4).
iii)Durham tubes collect CO2 gas produced from fermentation process.
Gas From Glucose
Escherichia, Salmonella except S. Typhi, Enterobacter and Proteus

Absence of Gas
Shigella and Yersinia

Different reaction
Serratia

11. Mannitol Fermentation

Mannitol Fermenter
Bacillus megatherium (VP -ve)
Stapylococcus spp.

Mannitol Non-Fermenter
S. saprophyticus and S. epidermidis
Micrococcus luteus

12. Nitrate Reduction

Demonstrates the ability of a bacterium to produce the enzyme nitratase, capable of converting nitrate to nitrite.

Nitrate Reducer Positive
Corynebacterium xerosis

Nitrate Reducer Negative
C. kutscheri

13. ONPG (Ortho-nitrophenyl beta-D-galactopyranoside) Test

ONPG (Ortho-nitrophenyl beta-D-galactopyranoside) is a synthetic colourless compound (galactoside) structurally similar to lactose [1]. The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase [2]. The glucose and galactose can then be metabolized by the bacteria. However, some organisms lack permease and appear as late or non-lactose-fermenters. The ONPG test is considered to be a very sensitive test for lactose-fermentation. O-nitrophenyl-beta-D-galactopyranoside (ONPG) acts an artificial substrate for the beta-galactosidase. This substrate is capable of penetrating the bacterial cell without the presence of permease. If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond, releasing o-nitrophenol which is a yellow-colored compound. Thus, beta-galactosidase cleaves ONPG to galactose and o-nitrophenyl, a yellow compound. The ONPG test is very useful in the rapid identification of cryptic lactose fermenters (late fermenters). Since members of family Enterobacteriaceae are routinely grouped according to their lactose fermenting ability, the ONPG test becomes significant here.

Limitation: All organisms tested must be inoculated from a lactose-containing medium (e.g., TSI or Mac Conkey).

REFERENCES

1.Lowe G.H., 1962., J. Med. Lab. Technol., 19:21
2.Gottschalk, G. 1986; p. 97-98, 178-179. Bacterial Metabolism, 2nd ed. Springer-Verlag, New York.
3. Cheesbrough M.; 2009, “District Laboratory Practice in Tropical Countries”, Cambridge University Press, (Page 40-44)
4. Pelczar M.J., Chan E.C.S., Krieg N.R.; 2011, “Microbiology”, Fifth Edition, Tata Mc Graw Hill Education Pvt. Ltd., New Delhi

Please go to the following link here below for summary of biochemical test for identification of bacteria.

Biochemical Test for Identification of Bacteria

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