Sodium Polyanethol Sulfonate (SPS) disk for differentiation of anaerobic cocci

· Bacteriology
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Principle

Sodium polyanethol sulfonate (SPS) is a commonly used anticoagulant. It is also known as Liquoid. It can inhibit the growth of certain anaerobic bacteria such as Peptostreptococcus anaerobius as well as aerobic Gardnerella vaginalis. So, this property of SPS can be used as a tool for differentiating P. anaerobius from other anaerobic cocci. For this test, filter paper disks impregnated with 5% SPS is used.

 

Procedure

 

  1. Prepare a lawn culture of the test organism on a blood agar plate.
  2. Put a SPS disk on the blood agar plate.
  3. If you have more than one organism to test, first streak all the plates and then add the disks to them at the same time. You can use one plate for up to four tests.
  4. Incubate the plate(s) anaerobically for 48 to 72 h at 35 to 37oC.
  5. Examine for a zone of inhibition of growth around the disk.

 

Result and Interpretation

Susceptible: zone of inhibition of ≥12 mm

Resistant: zone of inhibition of <12 mm

Note: P. anaerobius usually gives a very large zone of inhibition (≥16 mm), whereas other anaerobic cocci that appear susceptible to SPS give smaller zones. To presumptively identify P. anaerobius, you must also consider the Gram stain, typical colonial morphology, and odor. Some strains of Peptostreptococcus micros may be susceptible or partially susceptible to SPS. Examine the Gram stain for the small cell size of P. micros and chaining characteristics of P. anaerobius.

 

Controls

Positive contol : P. anaerobius ATCC 27337: Susceptible to SPS

Negative control: P. asaccharolyticus ATCC 29745: Resistant to SPS

 

Preparation of SPS disks

SPS ……………………….   5 g

Distilled water …………100 ml

  1. Dissolve 5g of SPS in 100 ml of distilled water in a clean beaker.
  2. Sterilize the mixture by filtration (0.22- lm-pore-size filter).
  3. Dispense 20µl onto sterile 1/4- inch-diameter filter paper disks that are spread inside an empty sterile petri dish.
  4. Allow these impregnated disks to dry for 72 h at room temperature.
  5. Store the disks at room temperature, and label with an expiration date of 6 months.

 SPS disks are also commercially available

 

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