Bile Solubility Test Principle, Procedure, Result

• In qualitative techniques for the presumed identification and discrimination of Streptococcus pneumoniae from other alpha-hemolytic streptococci, the 2% and 10% Bile Solubility Reagents are recommended.
• The bile solubility test can be conducted using a cell suspension or by directly applying the Bile Spot Reagent to the colony.
• The bile solubility test is a qualitative method for assessing the capacity of bacterial cells to lyse in the presence of bile salts under certain time and temperature conditions.
• The primary purpose of the test is to distinguish between bile-soluble Streptococcus pneumoniae and bile-insoluble alpha-hemolytic streptococci.
• S. pneumoniae possesses an autolytic enzyme that breaks down the organism’s own cell wall, resulting in cell lysis.
• The autolytic process is accelerated in the presence of bile salt sodium deoxycholate.
• On a solid medium, colonies of S. pneumoniae dissolve and vanish, and if growth is suspended in saline, the murky solution clears.
• The reactions are characterised as soluble in both instances. Bile (Oxgall) and other bile salts, such as sodium taurocholate, can also be used to do the test, however the findings are frequently varied.
• Other -hemolytic streptococci lack active autolytic enzymes, are insoluble in bile, and are therefore categorised as bile-insoluble.
• Neufeld utilised sodium deoxycholate, a bile salt, in 1900 to detect autolysis, which distinguished bile-soluble S. pneumoniae from bileinsoluble alpha-hemolytic streptococci.
• Downie et al. utilised 10% bile salt solutions to lyse pneumococci broth cultures. Greey later reported that immediately applying bile to a blood agar plate culture of pneumococci was beneficial.
• In 1965, Hawn and Beebe devised a fast method for measuring the solubility of bile on blood agar by employing 2% sodium deoxycholate.
• The 10% reagent may produce more quick reactions; nonetheless, both doses have been observed to yield accurate results using both the tube and spot test procedures.
• It should be noted that only 80% of Streptococcus pneumoniae strains will lyse completely, and additional tests may be necessary to distinguish bacteria that demonstrate only partial or incomplete lysis.

Bile Solubility Test Purpose

• This test tells the difference between alpha-hemolytic streptococci and Streptococcus pneumoniae, which is positive and soluble (negative; insoluble).

Principle of Bile Solubility Test

• When added to actively growing bacteria in agar or broth media, bile salts, specifically sodium deoxycholate and sodium taurocholate, can selectively kill S. pneumoniae.
• S. pneumoniae makes enzymes that break down its own cells. This is why older pneumococcal colonies on agar media have a central depression or umbilication.
• When bile salts are added to a culture of pneumococci, they activate the autolysins and speed up the natural lytic reactions.
• If the organism is soluble, the turbidity of a broth suspension that has been mixed with bile salts also clears up.

Requirements for Bile Solubility Test

• Test organism (α -haemolytic streptococci)
• A pure culture of the test organism grown at 35°C for 18–24 hours in Todd–Hewitt broth (or equivalent)
• Sheep blood agar plate
• Sodium deoxycholate (10% for tube test, 2% for plate test)
• Phenol red solution (1% aqueous)
• Sodium hydroxide (NaOH) solution, 0.10 N

How to make 2% and 10% solutions of sodium deoxycholate (bile salt)?

• 2 g and 10 g of sodium deoxycholate should each be dissolved in 100 ml of sterile distilled water.
• Dispense in small amounts to minimise contamination. It can be kept for 270 days.
• Keep between 15 and 30°C. If the reagent is kept in a cool place, it can get thicker.

Test Organism

• Any alpha-hemolytic, catalase-negative, Gram-positive cocci in chains with a flattened centre or a mucoid colony shape that looks like it could be S. pneumoniae.
• Any Gram-positive cocci in pairs that look like lancets from a successful blood culture. Grow the isolate(s) to be tested on a blood agar plate (BAP) for 18–24 hours at 35–37°C with less than 5% CO2 (or in a candle-jar).

Procedure of Bile Solubility Test

Do some preliminary tests, like a Gram stain and a catalase, to make sure that the test isolate is a Streptococcus.

Spot Test/Plate Method of Bile Solubility Test

1. To obtain well-isolated colonies, do a four-quadrant streak of the test organism onto a blood agar plate. (Before doing the test, it should be determined that the test organism is a -hemolytic, catalase-negative, gram-positive cocci organised in chains)
2. Plates should be inverted for 18 to 24 hours at 35 degrees Celsius.
3. Select a distinctive, well-isolated S. pneumoniae colony. Mark the location of the colony with a wax pencil or permanent felt marker on the bottom of the petri dish.
4. Add 1 drop of 2% or 10% Bile Solubility Reagent to a colony of the test isolate grown on sheep blood agar for 18 to 24 hours.
5. Incubate the plate aerobically at 35-37°C in an upright position for up to 30 minutes, observing intermittently. Leave the lid slightly ajar to facilitate reagent evaporation.
6. After thirty minutes, examine the dish for the presence of the suspect colony.

Tube Test Method of Bile Solubility Test

1. Obtain a pure overnight culture of the desired streptococci on blood agar or Todd-Hewitt Broth.
2. Make a dense suspension of the organism in 1.0 mL of physiological saline solution.
3. Add 1 drop of phenol red indicator and, if necessary, use 0.1 N sodium hydroxide to set the pH to 7.0. The solution should be pink in colour. (This step is optional, but the pH must be greater than 6.8 for appropriate results)
4. Equally divide the saline suspension into two tubes (0.5-mL per tube). Label the test tube and the control tube.
5. Add 0.5 ml of either 2% or 10% Bile Solubility Reagent to the designated tube.
6. Add 0.5 ml of sterile physiologic saline with a pH of 7.0 to the tube labelled control.
7. To ensure the suspensions are homogeneous, agitate both tubes gently.
8. Incubate tubes between 35 and 37 degrees Celsius.
9. Check tubes hourly and form a conclusion after three hours. Observe for the broth to clear.

Interpretation of Results

Spot Test

• Positive test: Colony disintegration and/or the appearance of a -hemolytic zone on the plate containing the colony within thirty minutes.
• Negative Test: Colonies on the plate remain intact after 30 minutes, with no change in colony integrity.

Tube Test

• Positive Test: Clearing or loss of turbidity of the test suspension within three hours indicates a positive result. The suspension of Control remains turbid.
• Negative test: Test and Control suspensions remain turbid after three hours. 

Reporting results

Bile Soluble

• Plate Test – The reagent causes bile-soluble colonies to dissolve and vanish. At the place of the drop, a flattened imprint of the lysed colony may remain, as well as a region of hemolysis.
• Tube Test– When comparing the test sample to the control sample, a reduction in turbidity is noticed. Additionally, a rise in suspension viscosity may be seen.

Bile Insoluble

• Plate Test – Colonies are still visible and intact.
• Tube Test– After 3 hours of incubation, the test aliquot stays turbid and is comparable to the control aliquot. 

• 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 organism is not bile-soluble, it is likely a viridans group streptococcus or still may be S. pneumoniae; further testing is indicated from typical pneumococcal colonies. Bile insoluble colonies resembling S. pneumoniae should be further identified using another method, such as optochin susceptibility and/or DNA Probe and/or matrix-assisted laser desorption ionization time-of-flight spectrometry (MALDI-TOF).

Quality Control

Using the following quality control organisms, all lot numbers of Bile Solubility Reagent 2% and 10% have been evaluated and verified to be acceptable. The examination of control organisms should adhere to recognised laboratory quality control protocols. Patient outcomes should not be published if abnormal quality control results are observed.

Streptococcus pneumoniae (TubeTest)	Positive
Streptococcus sanguinis (TubeTest)	Negative
Streptococcus pneumoniae (Spot Test)	Positive
Streptococcus sanguinis (Spot Test)	Negative

Precautions

• For the plate process, several sources offer a 2% deoxycholate solution as opposed to our 10%. The 10% solution can be further diluted with sterile water to achieve the appropriate 2% concentration, if desired.
• Avoid shaking and undue movement of plates after the reagent has been introduced, as alpha-hemolytic colonies occasionally do not disintegrate during the plate technique but rather lift off and float.
• Since sodium deoxycholate can form a precipitate in an acid suspension (pH 6.5 and below) and produce false-negative findings, the pH for the tube test must be above 6.8.
• Downie reported that the absence of the capsule may affect S. pneumoniae’s sensitivity to lysis by bile salts.
• The reagent can be used to differentiate between -hemolytic Haemophilus species (H.. influenzae and H. aegypticus) and bile-insoluble Haemophilus species.

Limitations

• Only alpha-hemolytic streptococci are tested.
• Keep the plate level when doing the spot test to avoid the reagent from running, as non-pneumococcal colonies may wash away and falsely appear to have dissolved.
• Colonies older than 24 hours may have lost their enzyme activity, resulting in a false-negative test result.
• In the bile solubility test, only 85 percent of pneumococcal strains will lyse completely; other S. pneumoniae will not lyse in the presence of bile, presumably because to the absence of virulence factor or capsule. If lysis is absent, the isolate may still be S. pneumoniae, necessitating additional testing.
• Some strains of Streptococcus pneumoniae are resistant to lysis in the presence of bile salt solution, most likely due to the absence of virulence factor or capsule. In the absence of lysis, the isolate may still be S. pneumonia. Therefore, non-bile-soluble colonies mimicking S. pneumoniae should be further investigated using an other technique, such as optochin sensitivity and/or DNA probe.
• Bile salts will not induce clearance of a dead or overly acidic culture. Thus, suspensions of young cultures in salt are utilised.
• Before introducing the reagent to the bile solubility tube test using saline or unbuffered broth, the pH must be adjusted to neutral in order to avoid false negative results.
• Care must be taken not to dislodge the colony being tested when using the plate method, as this could result in false-positive findings. If the direct plate is difficult to understand, the test should be repeated using a tube or slide.

Uses

• The only use of bile solubility is to distinguish Streptococcus pneumoniae from other alpha-hemolytic Streptococci.
• Used to distinguish between bile-soluble and insoluble organisms.

References

• Bailey & Scott’s Diagnostic Microbiology
• https://microbiologynotes.com/bile-solubility-test-principle-procedure-result-interpretation-examples-and-limitation/
• https://universe84a.com/bile-solubility-test/
• https://www.dalynn.com/dyn/ck_assets/files/tech/RB60.pdf
• https://www.thermofisher.com/order/catalog/product/R21206
• https://biologyreader.com/bile-solubility-test.html
• https://laboratoryhub.com/bile-solubility-test-principle-procedure-results/
• https://us.vwr.com/store/product/20287624/bile-spot-test-hardy-diagnostics
• http://www.keydiagnostics.com.au/clinical-testing-2/products-by-organism/bile-spot-reagent-detail
• https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU21206.pdf
• https://catalog.hardydiagnostics.com/cp_prod/content/hugo/BileSpotRgnt.htm
• https://www.nature.com/articles/s41598-017-07772-x