Bile Solubility Test- Principle, Procedure, Types, Results, Uses

Bile solubility test is a biochemical test used for the differentiation and confirmation of Streptococcus pneumoniae from other alpha-hemolytic Streptococci .

Streptococcus pneumoniae is a Gram-positive, catalase-negative, lancet-shaped coccus that causes pneumonia, meningitis, otitis media, sinusitis, and other infections. It is also known as pneumococcus and is one of the most common causes of bacterial meningitis in adults and children.

The bile solubility test has been used as an essential test for the differentiation of S. pneumoniae as it allows the distinction between the two species S. pneumoniae and Streptococcus pseudopneumoniae, which is a challenging task.

The test is based on the lysis of the bacterial cells in the presence of certain bile salts like sodium desoxycholate under certain conditions. The organisms that lyse are considered positive, whereas those that do not are considered negative .

The exact working mechanism of the test is still not clearly understood, but it has been hypothesized that the lysis is brought about by the induction of autolytic enzymes .

The bile solubility test is generally considered to be an accurate test for differentiating S. pneumoniae from other mitis group streptococci, including S. pseudopneumoniae.

The test results from the method are, however, difficult to interpret, particularly because it is based on subjective human evaluation.

The bile solubility test can be performed using a cell suspension on a slide or in a tube or by adding the reagent directly to the colony .

The following sections will describe the objectives, principle, microorganisms tested, media, reagents, and supplies used, procedure, quality control, result interpretation, reporting results, uses, and limitations of the bile solubility test.

The main objective of the bile solubility test is to identify and differentiate S. pneumoniae from other alpha-hemolytic Streptococci. S. pneumoniae is an important human pathogen that causes various infections such as pneumonia, meningitis, otitis media, and sinusitis. It is also one of the leading causes of morbidity and mortality worldwide, especially in children and the elderly. Therefore, accurate and rapid identification of S. pneumoniae is essential for appropriate diagnosis and treatment.

Another objective of the bile solubility test is to detect the ability of an organism to undergo lysis in the presence of bile salts. Bile salts are natural detergents that are produced by the liver and stored in the gallbladder. They help in the digestion and absorption of fats and other lipids in the small intestine. Bile salts also have antimicrobial properties and can disrupt the cell membranes of some bacteria. The bile solubility test exploits this property to distinguish between bile-soluble and bile-insoluble organisms.

The bile solubility test is a simple, inexpensive, and reliable test that can be performed in any laboratory setting. It can be used as a confirmatory test for S. pneumoniae after preliminary screening tests such as Gram stain, catalase test, and alpha-hemolysis on blood agar. It can also be used as a direct test on blood culture broth or colonies from other media. The test results are usually obtained within 15 to 30 minutes for the direct plate or slide method, or within 3 hours for the tube method.

The bile solubility test has some limitations as well, such as false-negative results due to bile-resistant strains of S. pneumoniae or false-positive results due to dislodgement of colonies or evaporation of reagent. Therefore, it is recommended to use the bile solubility test in conjunction with other tests such as optochin susceptibility or molecular methods for definitive identification of S. pneumoniae.

The bile solubility test is based on the ability of S. pneumoniae to lyse in the presence of bile salts like sodium desoxycholate, due to an autolytic enzyme that breaks down its own cell wall . The test can also help distinguish S. pneumoniae from S. pseudopneumoniae, which is a challenging task .

The exact mechanism of the bile solubility test is not fully known, but it has been speculated that the bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism`s natural autolytic process . The addition of bile salts also activates the autolytic enzyme, amidase, which induces clearing of the culture .

The bile solubility test can be performed either with a broth culture of the organism or with colonies growing on agar media. The turbidity of a broth suspension visibly clears with the addition of bile salts if the organism is soluble. On agar medium, bile-soluble colonies dissolve when drops of the reagent are placed on them .

Because sodium desoxycholate may precipitate at a pH of 6.5 or less, the broth culture medium must be adjusted to pH 7.0 to prevent false-negative reactions . The test should also be performed with fresh cultures that have not undergone autolysis .

The bile solubility test is mainly used to test for the presence of Streptococcus pneumoniae, which is a common cause of pneumonia, meningitis, and other infections. S. pneumoniae is a Gram-positive, catalase-negative, alpha-hemolytic coccus that usually appears in pairs or short chains. It can be distinguished from other alpha-hemolytic streptococci by its characteristic flattened or mucoid colony morphology and its susceptibility to optochin.

However, not all S. pneumoniae strains are optochin-sensitive, and some other streptococci may show partial or false-positive reactions to optochin. Therefore, the bile solubility test is used as a confirmatory test for S. pneumoniae identification. The bile solubility test can also differentiate S. pneumoniae from Streptococcus pseudopneumoniae, which is a recently described species of alpha-hemolytic streptococcus that can cause respiratory infections and mimic S. pneumoniae in some biochemical tests.

Other alpha-hemolytic streptococci that may be tested by the bile solubility test include Streptococcus mitis, Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii. These are collectively known as the viridans group streptococci, and they are part of the normal flora of the oral cavity and upper respiratory tract. They are usually bile-insoluble and can cause opportunistic infections such as endocarditis and dental caries.

The bile solubility test can also be used to test for the presence of Neisseria gonorrhoeae, which is a Gram-negative, oxidase-positive, diplococcus that causes gonorrhea and other sexually transmitted infections. N. gonorrhoeae is bile-soluble and can be differentiated from other Neisseria species by its ability to ferment glucose but not maltose.

The bile solubility test should not be used to test for other Gram-positive or Gram-negative bacteria, as they may show variable or nonspecific reactions to bile salts. The test should also not be used to test for fungi or parasites, as they are not affected by bile salts.

Media

The bile solubility test does not require any specific culture media. The test can be performed on any alpha-hemolytic, catalase-negative, Gram-positive cocci in chains that grow on blood agar or other suitable media. The test can also be performed on blood culture broth that contains Gram-positive cocci in lancet-shaped pairs.

Reagents

The main reagent used for the bile solubility test is bile salts, which are usually sodium desoxycholate or sodium taurocholate. These are bile acids that are produced by the liver and stored in the gallbladder. They help in the digestion and absorption of fats and cholesterol in the intestine.

A 10% bile salt solution can be either purchased or prepared by dissolving 10 grams of sodium desoxycholate or sodium taurocholate in 100 ml of distilled water. The solution should be dispensed in small amounts to minimize contamination and stored at 15 to 30°C. The shelf life of the solution is usually 270 days.

Another reagent that is used for the bile solubility test is 0.85% NaCl (sterilized). This is used to prepare a cell suspension of the organism to be tested. Alternatively, a suitable broth medium (e.g. BHI) can be used instead of saline.

Supplies

The supplies that are needed for the bile solubility test are:

• Loops
• Test tubes or slides
• Pipettes
• Incubator or heat block
• Microscope
• Gram stain reagents or methylene blue stain

Bile solubility test can be performed either via the test tube method or direct plate method or direct slide blood culture method.

Test tube method

• About 0.5 ml of sterile saline or suitable broth is dispensed into a small test tube.
• A heavy suspension of the organism is prepared in the saline (equivalent to no.1 McFarland standard). The suspension is then shaken by hand or on a vortex to form a uniform suspension.
• The suspension is divided into two tubes, one labeled “TEST” and the other labeled “CONTROL.”
• Two to five drops of bile reagent are dispensed into both the tubes marked “TEST” and “CONTROL.” Both the tubes are gently mixed.
• The tubes are then incubated for three hours at 35°C, checking hourly for clearing, or each tube can be examined by Gram stain or methylene blue wet mount for lysis of cells at an interval of 15 minutes.

Direct plate method

• A drop of bile reagent is placed near a suspected 18- to 24 hr colony.
• The drop is then gently rolled over several representative colonies by tilting the plate. The colonies should not be dislodged with the bile reagent.
• Note: Do not touch the agar surface with the tip of the dropper of bile reagent.
• The plate is kept right side up and is incubated at 35°C for 15-30 minutes or until the drop has evaporated. The plate can also be placed on a heat block as a substitute for the use of an incubator.
• The flattening of the colony is observed. Proper observation should be made to ensure that the colony does not simply float away.

Direct slide blood culture test

• One drop of blood culture broth is added to 1 drop of bile reagent on a glass slide and allowed to dry.
• As a control, one drop of broth blood culture is added to 1 drop of water and allowed to dry.
• The resulting suspension is Gram stained and examined for cocci.

To ensure the accuracy and reliability of the bile solubility test, it is important to perform quality control procedures before and during the test. Quality control involves the use of positive and negative control organisms, proper preparation and storage of reagents, and adherence to the standard operating procedures.

Control Organisms

The following organisms can be used as control strains for the bile solubility test:

• Positive control: S. pneumoniae ATCC 49619 or any known bile-soluble strain of S. pneumoniae.
• Negative control: S. mitis ATCC 49456 or any known bile-insoluble strain of alpha-hemolytic Streptococcus.

The control strains should be tested along with the unknown isolates using the same method (tube, plate, or slide) and reagents. The expected results are:

• Positive control: Clearing of turbidity in the tube method, disintegration or flattening of colonies in the plate method, or lysis of cells in the slide method.
• Negative control: No change in turbidity in the tube method, no change in colony morphology in the plate method, or no lysis of cells in the slide method.

If the control strains do not give the expected results, the test should be repeated with fresh reagents and cultures. If the problem persists, the reagents and cultures should be checked for contamination, expiration, or deterioration.

Reagents

The reagents used for the bile solubility test are:

• Bile salts: A 10% solution of sodium desoxycholate or sodium taurocholate. The solution can be purchased commercially or prepared in-house by dissolving 10 g of bile salt in 100 ml of distilled water. The solution should be dispensed in small aliquots to avoid contamination and stored at room temperature (15 to 30°C) for up to 270 days. The solution should not be refrigerated as it may thicken and become unusable. The solution should be clear and colorless; if it becomes cloudy, discolored, or precipitated, it should be discarded and replaced with a fresh one.
• 0.85% NaCl: A sterile solution of sodium chloride that can be purchased commercially or prepared in-house by dissolving 0.85 g of sodium chloride in 100 ml of distilled water. The solution should be autoclaved at 121°C for 15 minutes and stored at room temperature for up to one year. The solution should be clear and colorless; if it becomes cloudy, discolored, or contaminated, it should be discarded and replaced with a fresh one.
• Broth culture medium: A suitable broth medium that supports the growth of Streptococcus spp., such as brain heart infusion (BHI) broth or tryptic soy broth (TSB). The broth medium can be purchased commercially or prepared in-house according to the manufacturer`s instructions. The broth medium should be sterilized by autoclaving at 121°C for 15 minutes and stored at 2 to 8°C for up to one month. The broth medium should be clear and free of particulate matter; if it becomes cloudy, discolored, or contaminated, it should be discarded and replaced with a fresh one.

The reagents should be labeled with the name, concentration, date of preparation, expiration date, and initials of the preparer. The reagents should be checked for quality before use by testing with control strains.

Procedures

The procedures for performing the bile solubility test are:

• Test tube method: This method involves preparing a heavy suspension of the organism in saline or broth, dividing it into two tubes (test and control), adding bile reagent to both tubes, incubating at 35°C for up to three hours, and observing for clearing or loss of turbidity in the test tube compared to the control tube.
• Direct plate method: This method involves placing a drop of bile reagent near a suspected colony on a blood agar plate, rolling the drop over several colonies without dislodging them, incubating at 35°C for up to 30 minutes or until the drop evaporates, and observing for disintegration or flattening of colonies in the presence of bile reagent compared to those without it.
• Direct slide blood culture method: This method involves adding a drop of blood culture broth to a drop of bile reagent on a glass slide, allowing it to dry, Gram staining the resulting smear, and examining for lysis of cocci in the presence of bile reagent compared to those without it.

The procedures should be followed strictly according to the standard operating protocols. Any deviations or modifications should be documented and justified. The procedures should be performed by trained and competent personnel using appropriate personal protective equipment and biosafety practices. The procedures should be performed in a timely manner using fresh cultures and reagents. The results should be recorded and reported accurately and clearly. Any discrepancies or unexpected results should be investigated and resolved.

The result of the bile solubility test depends on the method used and the observation of the change in the appearance of the bacterial cells or colonies after exposure to bile salts.

Test tube method

In the test tube method, bile solubility is demonstrated as a clearing or loss of turbidity in the tube labeled "TEST" when compared to the tube labeled "CONTROL" within 3 hours. This indicates that the bacterial cells have been lysed by the bile salts and are no longer visible in the suspension. The result can also be confirmed by observing the lysis of cells under a microscope .

A positive result for bile solubility is obtained when the suspension clears in the test tube, which means that the organism is S. pneumoniae.

A negative result for bile solubility is obtained when the suspension remains turbid in the test tube, which means that the organism is not S. pneumoniae.

Direct plate method

In the direct plate method, bile solubility is demonstrated as disintegration or flattening of the colony within 30 minutes, leaving an area of alpha-hemolysis where the colonies were located. This indicates that the bacterial cells have been lysed by the bile salts and are no longer intact on the agar surface .

A positive result for bile solubility is obtained when the colony dissolves or flattens on the plate, which means that the organism is S. pneumoniae.

A negative result for bile solubility is obtained when there is no change in the integrity of the colony on the plate, which means that the organism is not S. pneumoniae.

Direct slide blood culture method

In the direct slide blood culture method, bile solubility is demonstrated as complete lysis of all the cocci in the smear when Gram stained and examined under a microscope. The control smear should show intact bacteria .

A positive result for bile solubility is obtained when all the cocci in the smear are entirely lysed, which means that the organism is S. pneumoniae.

A negative result for bile solubility is obtained when some or all of the cocci in the smear are not lysed, which means that the organism is not S. pneumoniae.

The results of the bile solubility test should be reported based on the method used and the appearance of the colonies or broth after adding the bile reagent.

• For the test tube method, a positive result is indicated by a clearing or loss of turbidity in the test tube containing bile reagent, compared to the control tube containing water, within 3 hours. This means that the organism is bile soluble and likely to be Streptococcus pneumoniae. A negative result is indicated by no change in turbidity in either tube, meaning that the organism is bile resistant and likely to be another alpha-hemolytic streptococcus .
• For the plate method, a positive result is indicated by the disintegration or flattening of the colony within 30 minutes after adding bile reagent, leaving an area of alpha-hemolysis where the colony was located. This means that the colony is bile soluble and likely to be Streptococcus pneumoniae. A negative result is indicated by no change in the integrity of the colony within 30 minutes, meaning that the colony is bile resistant and likely to be another alpha-hemolytic streptococcus .
• For the direct slide blood culture method, a positive result is indicated by the complete lysis of all the cocci in the smear containing bile reagent, compared to the control smear containing water. This means that the organism is bile soluble and likely to be Streptococcus pneumoniae. A negative result is indicated by no lysis of any cocci in either smear, meaning that the organism is bile resistant and likely to be another alpha-hemolytic streptococcus.

If either the spot or tube test demonstrates bile solubility of an alpha-hemolytic colony from a catalase-negative, lancet-shaped, Gram-positive coccus, definitively report as Streptococcus pneumoniae . If the test does not demonstrate bile solubility, the organism may still be S. pneumoniae or another streptococcus; further testing is advised from typical pneumococcal colonies .

Some possible additional sentences to conclude point 9 are:

• The bile solubility test is a simple and rapid test that can help in identifying S. pneumoniae from other alpha-hemolytic streptococci in clinical specimens.
• The bile solubility test should be performed along with other tests, such as Gram stain, catalase test, optochin susceptibility test, or molecular methods, to confirm the identification of S. pneumoniae.
• The bile solubility test should be interpreted with caution and according to standard protocols, as some factors may affect its accuracy and reliability.

• The bile solubility test is used to determine the ability of an organism to undergo lysis in the presence of bile salts .
• This test can be used for the differentiation and identification of S. pneumoniae from other alpha-hemolytic Streptococci .
• This test is a qualitative test for the differentiation of bile-soluble and bile-insoluble organisms .
• This test can also help distinguish S. pneumoniae from S. pseudopneumoniae, which is a challenging task .
• This test can be used as a confirmatory test for S. pneumoniae after optochin susceptibility test.
• This test can be used to detect pneumococcal infections from blood cultures.
  
  
  Limitations of Bile Solubility Test
  
  The bile solubility test has some limitations that should be considered before interpreting the results. Some of these limitations are:
• The bile solubility test should only be performed on alpha-hemolytic streptococci that are catalase-negative and Gram-positive . Other bacteria may not be affected by the bile salts or may have different mechanisms of lysis.
• Some S. pneumoniae strains may not lyse in the presence of bile salts, possibly due to the loss of virulence factors or capsule . If lysis is not observed, the isolate may still be S. pneumoniae and should be further confirmed by other methods, such as optochin susceptibility, DNA probe, or MALDI-TOF.
• The bile solubility test may not be reliable with old cultures that have autolyzed. The test should be performed on fresh cultures that are 18 to 24 hours old.
• The pH of the broth or saline used for the test should be adjusted to neutral (pH 7.0) before adding the bile reagent, as low pH may cause precipitation of the bile salts and false-negative results.
• The concentration and volume of the bile reagent should be appropriate for the test method used. Too much or too little bile reagent may affect the lysis of the bacteria.
• The incubation time and temperature should be optimal for the test method used. Too long or too short incubation may result in false-positive or false-negative results, respectively.
• The interpretation of the test results should be done carefully and objectively. The clearing of turbidity in the tube method or the flattening of the colony in the plate method should be compared with the control tube or plate, respectively. The Gram stain or wet mount examination of the cells can also help to confirm lysis.
• The storage and handling of the bile reagent should be done properly to avoid contamination or degradation. The reagent should be stored at 15 to 30°C and warmed to liquefy if thickened. The tip of the dropper should not touch the agar surface or the bacterial cells when dispensing the reagent.

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