ONPG Test- Principle, Procedure, Results, Uses

The ONPG test is a biochemical test that can be used to determine the ability of bacteria to produce the enzyme β-galactosidase. This enzyme is responsible for breaking down lactose, a sugar found in milk and dairy products, into glucose and galactose. The ONPG test can help to differentiate between lactose fermenters and non-lactose fermenters among Gram-negative bacteria, especially the members of the Enterobacteriaceae family and some Neisseria species.

Lactose fermenters are bacteria that can utilize lactose as a source of energy and produce acid and gas as by-products. Non-lactose fermenters are bacteria that cannot metabolize lactose and instead use other sugars or organic compounds. The ability to ferment lactose is an important characteristic for the identification and classification of many bacterial pathogens, such as Escherichia coli, Salmonella, Shigella, Klebsiella, Vibrio and others.

The ONPG test is based on the use of a synthetic substrate called o-nitrophenyl-β-D-galactopyranoside (ONPG), which mimics the structure of lactose but can enter the bacterial cell without the need of a transport protein called permease. Permease is another enzyme that facilitates the uptake of lactose into the cell. Some bacteria can produce β-galactosidase but not permease, which makes them slow lactose fermenters. The ONPG test can detect these bacteria by measuring their β-galactosidase activity independently of permease.

The ONPG test is simple, rapid and inexpensive to perform. It can be done by using either a liquid broth or a paper disk impregnated with ONPG. The test is positive when the bacteria produce β-galactosidase and cleave ONPG into galactose and o-nitrophenol, which is a yellow compound that changes the color of the medium or the disk. The test is negative when the bacteria do not produce β-galactosidase and no color change occurs.

The ONPG test has several applications in microbiology, such as:

• Differentiating lactose fermenters and non-lactose fermenters among Gram-negative bacteria
• Determining the presence or absence of permease in lactose fermenting bacteria
• Characterizing and identifying bacterial isolates based on their biochemical profiles
• Screening for potential mutants or recombinants that have altered β-galactosidase activity

The ONPG test is based on the ability of bacteria to produce an enzyme called β-galactosidase, which can break down lactose and other similar compounds into simpler sugars. Lactose is a disaccharide composed of glucose and galactose, and it requires two enzymes to be fermented by bacteria: permease and β-galactosidase. Permease is a transport protein that allows lactose to enter the bacterial cell, where β-galactosidase can cleave the bond between glucose and galactose. The resulting sugars can then be used as energy sources by the bacteria.

However, some bacteria lack permease or have low permease activity, and thus appear as non-lactose fermenters or late lactose fermenters on conventional media. These bacteria may still possess β-galactosidase, but they cannot utilize lactose efficiently. To detect the presence of β-galactosidase in these bacteria, an artificial substrate called o-nitrophenyl-β-D-galactopyranoside (ONPG) is used instead of lactose. ONPG is structurally similar to lactose, except that the glucose moiety is replaced by an o-nitrophenyl group. Unlike lactose, ONPG can penetrate the bacterial cell without the need for permease. Once inside the cell, β-galactosidase can hydrolyze ONPG into galactose and o-nitrophenol. The latter is a yellow-colored compound that can be detected visually or spectrophotometrically.

The ONPG test can thus differentiate between bacteria that produce β-galactosidase (ONPG positive) and those that do not (ONPG negative), regardless of their permease status. The test can also indicate the permease activity of bacteria by comparing the results with the lactose fermentation test. Bacteria that are ONPG positive and lactose positive produce both enzymes and can ferment lactose rapidly. Bacteria that are ONPG positive and lactose negative produce only β-galactosidase and ferment lactose slowly or not at all. Bacteria that are ONPG negative and lactose negative produce neither enzyme and cannot ferment lactose .

To perform the ONPG test, you will need the following:

• Culture media: You can use either ONPG broth or ONPG disk as the culture medium for the test. The ONPG broth contains casein peptone, o-nitrophenyl-beta-D-galactopyranoside (ONPG), disodium hydrogen phosphate, and sodium chloride. The ONPG disk is a filter paper disk impregnated with ONPG .
• Reagents: You will need sterile physiological saline to prepare the bacterial suspension and McFarland standard no. 2 to adjust the turbidity of the suspension. The McFarland standard no. 2 is a solution of 1% anhydrous barium chloride and 1% sulfuric acid.
• Equipment: You will need sterile test tubes, inoculating loops, an incubator, and a long-wavelength ultraviolet light source .
• Test organism: You will need a pure and fresh (18 to 24 hours old) culture of the bacteria that you want to test for beta-galactosidase production. You can use any suitable culture medium to grow the bacteria, but it is recommended to use a medium containing a high concentration of lactose.
• Positive and negative controls: You will need known beta-galactosidase positive and negative bacteria to verify the accuracy of the test. For example, you can use E. coli ATCC 25922 or Neisseria lactamica ATCC 23971 as positive controls, and Proteus mirabilis ATCC 12453 or Neisseria gonorrhoeae ATCC 43069 as negative controls .

The ONPG test can be performed by using two different types of culture media: ONPG broth or ONPG disk.

ONPG broth

ONPG broth is a liquid medium that contains o-nitrophenyl-beta-D-galactopyranoside (ONPG) as the substrate for beta-galactosidase enzyme. It also contains casein peptone, sodium chloride and disodium hydrogen phosphate as the sources of nitrogen, salt and buffer respectively .

The composition of ONPG broth per 1000 mL is as follows:

• Casein Peptone (or casitose): 7.50 grams
• o -Nitrophenyl -β- D- galactopyranoside: 1.50 grams
• Disodium hydrogen phosphate: 0.35 grams
• Sodium Chloride: 3.75 grams
• Final pH 7.5±0.2 at 25°C

The preparation of ONPG broth is as follows:

• Measure the appropriate amount of ONPG broth medium as directed by the manufacturing company and dissolve it with the sterile water of the required volume in a conical flask (or glass bottle).
• Heat to dissolve the medium, but do not autoclave the medium (The broth can be stored at or below – 10°C away from light.)
• Dispense about 5 mL (or the required quantity) of the broth in sterile test tubes.

ONPG disk

ONPG disk is a filter paper disk that is impregnated with a controlled concentration of ONPG. It can be used to test bacteria that are grown in any suitable culture media .

The preparation of ONPG disk is as follows:

• ONPG Differentiation Disk is prepared by impregnating carefully controlled concentrations of ONPG onto a 0.25 inch diameter filter paper disk.

ONPG broth is a liquid medium that contains o-nitrophenyl-β-D-galactopyranoside (ONPG) as the sole source of carbon and energy for the bacteria. ONPG is a synthetic substrate that mimics lactose and can be hydrolyzed by the enzyme β-galactosidase to produce galactose and o-nitrophenol. The o-nitrophenol is a yellow compound that can be detected visually or spectrophotometrically.

The other ingredients of ONPG broth are:

• Casein peptone (or casitose): It is a source of nitrogen, amino acids, and vitamins for the bacterial growth.
• Disodium hydrogen phosphate: It is a buffering agent that maintains the pH of the medium at 7.5±0.2.
• Sodium chloride: It provides essential ions and maintains the osmotic balance of the medium.

The composition of ONPG broth per 1000 mL is as follows:

The ONPG broth can be prepared by dissolving the ingredients in distilled water and heating to dissolve completely. The medium should not be autoclaved as it may degrade the ONPG substrate. The broth can be stored at or below -10°C away from light.

• Measure the appropriate amount of ONPG broth medium as directed by the manufacturing company and dissolve it with the sterile water of the required volume in a conical flask (or glass bottle).
• Heat to dissolve the medium, but do not autoclave the medium (The broth can be stored at or below – 10°C away from light.)
• Dispense about 5 mL (or the required quantity) of the broth in sterile test tubes.

The preparation of ONPG broth is simple and straightforward. The main thing to remember is not to autoclave the medium, as this can degrade the ONPG substrate and affect the test results. The broth should also be protected from light exposure, as ONPG is photosensitive and can change color over time. The broth can be prepared in advance and stored in a freezer for later use. The amount of broth needed depends on the number of samples to be tested. Each sample should have enough broth to cover the bottom of the test tube.

The ONPG test requires two main reagents: the ONPG substrate and the McFarland standard.

• The ONPG substrate is o-nitrophenyl-β-D-galactopyranoside, a synthetic compound that mimics lactose and can be hydrolyzed by β-galactosidase. The ONPG substrate can be added to the broth medium or impregnated on a paper disk. The ONPG substrate is sensitive to light and heat, so it should be stored in a dark and cool place. The ONPG substrate can be purchased from commercial suppliers or prepared in the laboratory by dissolving 1.5 g of ONPG in 100 mL of distilled water and sterilizing it by filtration.
• The McFarland standard is a turbidity reference that is used to adjust the bacterial suspension to a uniform density. The McFarland standard number 2 corresponds to approximately 1.5 x 10^8 cells/mL, which is suitable for the ONPG test. The McFarland standard can be prepared by mixing 0.2 mL of 1% anhydrous barium chloride (BaCl2) solution with 9.8 mL of 1% sulfuric acid (H2SO4) solution in a clear glass tube. The resulting suspension should have a milky white appearance and should be shaken well before use. Alternatively, the McFarland standard can be purchased from commercial suppliers or measured by a spectrophotometer.

These reagents are essential for performing the ONPG test and should be used with caution and accuracy.

The equipment required for the ONPG test are:

• Sterile test tubes
• Sterile inoculating loop
• Sterile pipettes
• Incubator
• Water bath (optional)
• Colorimeter (optional)

The sterile test tubes are used to prepare the bacterial suspension and to inoculate the ONPG broth. The sterile inoculating loop is used to transfer the bacteria from the culture medium to the test tube. The sterile pipettes are used to measure and dispense the saline and the ONPG broth.

The incubator is used to provide optimal temperature and aerobic conditions for the growth and metabolism of the bacteria. The water bath is optional and can be used to speed up the color development in the ONPG broth by heating it to 45°C for 15 minutes. The colorimeter is optional and can be used to measure the intensity of the yellow color in the ONPG broth by using a wavelength of 420 nm.

The equipment should be clean, dry, and properly labeled before use. The equipment should also be handled with care and disposed of appropriately after use.

The ONPG test can be performed on any Gram-negative bacteria that are suspected to be lactose fermenters or non-fermenters. The test organism should be pure and fresh (18 to 24 hours old) and grown on a suitable culture medium. The test organism can be isolated from clinical specimens, such as urine, stool, blood, sputum, etc., or from environmental sources, such as water, soil, food, etc.

Some examples of bacteria that can be tested by the ONPG test are:

• Escherichia coli: A common Gram-negative bacterium that is found in the intestinal tract of humans and animals. It is usually a lactose fermenter and produces both β-galactosidase and permease enzymes. It is positive for the ONPG test.
• Proteus mirabilis: A Gram-negative bacterium that is associated with urinary tract infections and wound infections. It is usually a non-lactose fermenter and does not produce β-galactosidase or permease enzymes. It is negative for the ONPG test.
• Salmonella Typhimurium: A Gram-negative bacterium that causes gastroenteritis and typhoid fever. It is usually a non-lactose fermenter and does not produce β-galactosidase or permease enzymes. It is negative for the ONPG test.
• Salmonella Choleraesuis: A Gram-negative bacterium that causes septicemia and enteric fever. It is usually a slow lactose fermenter and produces β-galactosidase but not permease enzymes. It is positive for the ONPG test.
• Shigella dysenteriae: A Gram-negative bacterium that causes dysentery and bloody diarrhea. It is usually a non-lactose fermenter and does not produce β-galactosidase or permease enzymes. It is negative for the ONPG test.
• Klebsiella pneumoniae: A Gram-negative bacterium that causes pneumonia and urinary tract infections. It is usually a lactose fermenter and produces both β-galactosidase and permease enzymes. It is positive for the ONPG test.
• Vibrio cholerae: A Gram-negative bacterium that causes cholera and severe dehydration. It is usually a lactose fermenter and produces both β-galactosidase and permease enzymes. It is positive for the ONPG test.
• Neisseria gonorrhoeae: A Gram-negative bacterium that causes gonorrhea and pelvic inflammatory disease. It is usually a non-lactose fermenter and does not produce β-galactosidase or permease enzymes. It is negative for the ONPG test.
• Neisseria lactamica: A Gram-negative bacterium that is part of the normal flora of the upper respiratory tract. It is usually a lactose fermenter and produces both β-galactosidase and permease enzymes. It is positive for the ONPG test.

These are some of the common bacteria that can be tested by the ONPG test, but there are many other bacteria that can also be tested by this method. The results of the ONPG test should be interpreted along with other biochemical tests and phenotypic characteristics to identify the bacteria accurately.

Positive and negative controls are essential for the validity and accuracy of the ONPG test. They help to verify that the test medium, reagents, equipment and procedure are working properly and that the results are reliable.

A positive control is an organism that is known to produce β-galactosidase enzyme and give a positive ONPG test result. A positive control should develop a yellow color in the broth or on the disk within 24 hours of incubation, indicating the hydrolysis of ONPG by β-galactosidase.

A negative control is an organism that is known to lack β-galactosidase enzyme and give a negative ONPG test result. A negative control should show no color change in the broth or on the disk after 24 hours of incubation, indicating the absence of ONPG hydrolysis by β-galactosidase.

Some examples of positive and negative controls for the ONPG test are :

• Positive control: Escherichia coli ATCC 25922 or Neisseria lactamica ATCC 23971
• Negative control: Proteus mirabilis ATCC 12453 or Neisseria gonorrhoeae ATCC 43069

It is important to use fresh and pure cultures of the control organisms and to follow the same procedure as for the test organisms. The control results should be recorded and compared with the test results to ensure their consistency and validity. If the control results are not as expected, the test should be repeated with a new batch of medium, reagents, equipment and organisms.

The ONPG test can be performed by using either the tube method or the disk method. The tube method uses ONPG broth as the medium, while the disk method uses ONPG disk as the substrate. Both methods require a heavy inoculum of pure and fresh (18 to 24 hours old) sample bacteria that have been grown in a medium containing lactose, such as Kligler iron agar (KIA) or Triple Sugar Iron (TSI) agar . This is to induce the production of β-galactosidase enzyme, which is necessary for the ONPG test.

• Prepare a suspension of sample bacteria with turbidity equal to McFarland Standard number 2 .
• Heavily inoculate a tube of ONPG broth with the bacterial suspension using a sterile inoculating loop .
• Incubate the tube at 35±2°C aerobically .
• After one hour, observe for color change (development of yellow color) in the broth . If no color change is observed, continue incubating for up to 24 hours and look for color change.

The disk method is an alternative way of performing the ONPG test using ONPG disks instead of ONPG broth. The ONPG disks are sterile filter paper disks impregnated with ONPG. The advantage of this method is that it requires less time and materials than the broth method. The procedure of the disk method is as follows:

• In a sterile test tube, add 0.5 mL of sterile saline and prepare a heavy bacterial suspension (turbidity equivalent to about number 2 McFarland standards).
• Place an ONPG disk in the suspension.
• Incubate aerobically at 35±2°C for about 4 to 6 hours.
• Observe for color change (development of yellow color) in the ONPG disk.

The result and interpretation of the disk method are the same as the broth method. A positive test indicates the production of β-galactosidase and a negative test indicates the lack of β-galactosidase production. The disk method can be used to differentiate lactose fermenters and non-lactose fermenters among Enterobacteriaceae and Neisseria spp.

The result of the ONPG test is based on the development of yellow color in the broth or on the disk, which indicates the production of o-nitrophenol from the ONPG substrate by the action of β-galactosidase enzyme. The intensity of the yellow color may vary depending on the amount of enzyme produced by the organism.

• Positive ONPG test: Development of yellow color in the broth or on the disk within 24 hours of incubation. This means that the organism can produce β-galactosidase and ferment lactose, regardless of the presence or absence of permease enzyme.
• Negative ONPG test: No color change in the broth or on the disk after 24 hours of incubation. This means that the organism cannot produce β-galactosidase and cannot ferment lactose.

Some examples of ONPG positive bacteria are: Escherichia coli, Citrobacter freundii, Salmonella Choleraesuis, Shigella dysenteriae, Klebsiella pneumoniae, Vibrio cholerae, Serratia marcescens, Neisseria lactamica, etc.

Some examples of ONPG negative bacteria are: Proteus vulgaris, Proteus mirabilis, Salmonella Typhimurium, Vibrio parahaemolyticus, Providencia spp., Pseudomonas aeruginosa, Neisseria gonorrhoeae, etc.

For quality control, it is recommended to use known positive and negative controls along with the test organism. Some suggested controls are:

• Positive control: Escherichia coli ATCC 25922 or Neisseria lactamica ATCC 23971
• Negative control: Proteus mirabilis ATCC 12453 or Neisseria gonorrhoeae ATCC 43069

The positive control should show a distinct yellow color in the broth or on the disk, while the negative control should show no color change. If the controls do not give the expected results, the test is invalid and should be repeated with fresh reagents and media.

Quality control is an essential step in any laboratory test to ensure the accuracy and reliability of the results. For the ONPG test, quality control involves using known positive and negative control strains of bacteria to verify the performance of the ONPG broth or disk and the interpretation of the results.

A positive control strain is a bacterium that is known to produce β-galactosidase and hence can hydrolyze ONPG, resulting in a yellow color development. A negative control strain is a bacterium that is known to lack β-galactosidase and hence cannot hydrolyze ONPG, resulting in no color change. The positive and negative control strains should be tested along with the sample bacteria in each batch of testing.

Some examples of positive and negative control strains for the ONPG test are:

• Positive control: E. coli ATCC 25922 or Neisseria lactamica ATCC 23971
• Negative control: Proteus mirabilis ATCC 12453 or Neisseria gonorrhoeae ATCC 43069

The positive control strain should show a clear yellow color in the ONPG broth or disk within 24 hours (or preferably within 4 to 6 hours) of incubation. The negative control strain should show no color change in the ONPG broth or disk after 24 hours of incubation. If the control strains do not show the expected results, the test should be repeated with a fresh batch of ONPG broth or disk and reagents.

The quality control also involves checking the purity and freshness of the sample bacteria, the turbidity and pH of the bacterial suspension, the storage and handling of the ONPG broth or disk, and the incubation conditions. Any deviation from the standard protocol or contamination of the materials can affect the outcome of the test.

The quality control results should be recorded and reported along with the sample results. Any discrepancy or inconsistency in the results should be investigated and resolved before reporting. The quality control ensures that the ONPG test is performed correctly and provides valid information for the differentiation and identification of bacteria.

• ONPG is highly unstable and it must be purchased or made by skilled personnel in a controlled environment .
• ONPG broth and disk must be stored in a dark area because ONPG is highly photosensitive .
• ONPG broth should not be autoclaved; hence every piece of equipment and saline must be pre-sterilized .
• Use heavy inoculum for testing .
• Bacteria that naturally produce yellow pigment are not suitable for the ONPG test.
• For the rapid ONPG test, the bacterial colonies cultured on the lactose-containing medium are required.
• The bacterial colonies from the glucose-containing medium show lower activity against ONPG.

• The ONPG test is used to differentiate the members of Enterobacteriaceae and Neisseria spp. based on the ability to synthesize β-galactosidase . These bacteria are important pathogens that cause various infections in humans and animals.
• The ONPG test helps in distinguishing Enterobacteriaceae that are late lactose fermenters from non-lactose fermenters . Late lactose fermenters are very difficult to distinguish from non-lactose fermenters because both appear as colorless colonies on MacConkey agar. The ONPG test can detect the presence of β-galactosidase even in the absence of permease, which is required for lactose uptake.
• Coupled with the lactose fermentation test, the ONPG test can also determine the ability of bacteria to synthesize the permease enzyme . Permease facilitates the rapid intake of lactose molecules so that the bacteria can quickly ferment the lactose. Bacteria that synthesize both β-galactosidase and permease are rapid lactose fermenters, while bacteria that synthesize only β-galactosidase are slow or late lactose fermenters.
• The ONPG test is used in the biochemical characterization and identification of Gram-negative bacteria . It is one of the tests included in the API-20E system, which is a standardized identification system for Enterobacteriaceae and other Gram-negative rods. The ONPG test can also be used to identify some Staphylococcus species.

• Bacteria that naturally produce yellow pigment are not suitable for the ONPG test, as they can interfere with the color interpretation of the test results. Examples of such bacteria are Flavobacterium spp., Sphingomonas spp., and some strains of Pseudomonas aeruginosa.
• For the rapid ONPG test, the bacterial colonies cultured on the lactose-containing medium are required, as they induce the expression of β-galactosidase enzyme. The bacterial colonies from the glucose-containing medium show lower activity against ONPG, as glucose represses the synthesis of β-galactosidase.
• The ONPG test is not enough for bacterial identification, as it only detects the presence or absence of β-galactosidase enzyme. It does not provide information about other biochemical or phenotypic characteristics of the bacteria. Therefore, it should be used in conjunction with other tests for accurate identification.
• The ONPG test may give false-positive results for some bacteria that can hydrolyze ONPG but do not ferment lactose. For example, some strains of Vibrio cholerae and Vibrio alginolyticus can produce β-galactosidase but are unable to utilize lactose as a carbon source. Similarly, some strains of Enterococcus faecalis and Enterococcus faecium can degrade ONPG but are not members of the Enterobacteriaceae family.
• The ONPG test may give false-negative results for some bacteria that can ferment lactose but do not produce β-galactosidase. For example, some strains of Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A can utilize lactose but lack β-galactosidase activity. Similarly, some strains of Escherichia coli and Klebsiella pneumoniae can ferment lactose but have low levels of β-galactosidase expression.

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