Optochin Susceptbiolity Test: How To Identify Streptococcus Pneumoniae

Optochin Is a chemical, ethylhydrocupreine hydrochloride, and is water-soluble, meaning that in the presence of water it will break down.

Today we are going to explore everything about it, and the opticians suspect an ability test for the identification of streptococcus pneumoniae.

The reason we look at optochin is that it is useful in helping identify streptococcus pneumoniae, and the alpha-hemolytic streptococcus commonly susceptible to this chemical.

Different Types of Phylum Hemichordates and Their Characteristics

What’s interesting is that other Alpha hemolytic streptococcal species are optochin resistant.

Therefore this test will determine whether bacteria are sensitive to oxygen or are impact-resistant to the chemical.

Along with the Differentiation of streptococcus pneumoniae from other forms of viridans, it will depend on demonstrating oxygen suspect ability, as well as bile solubility, reaction with specific DNA probe, as well as the detection of polysaccharides via species-specific capsular.

Although initially used for differentiating pneumococcus optochin susceptibility in 1915, What is well known is that this test was virtually unused by Labs until around the 1950s when it starts to gain traction

How Is The Test Used?

The most common form of Optochin test is done via filter paper discs, in which they are impregnated with ethylhydrocupreine hydrochloride, and applied to inoculate before being given the chance to be incubated.

What we find is that this type of test is generally less time-consuming than other tests such as the bile solubility test.

Fundamentals Of The Test

Identification of pneumoniae is confirmed when a definite zone of inhibition is found around the impregnated disc.

What makes this clear to confirm is that other alpha-hemolytic streptococci will not display a clear zone of inhibition when in the presence of oxygen.

The chemical analyses the sensitivity of the bacterial cell membrane and causes pneumoniae to lyse which is down to alterations in the surface tension.

How To Run An Optochin Susceptibility Test

Here Are the fundamental steps you need to take to run an effective oxygen susceptibility test:

  1. Select three or four well-isolated colonies of an Alpha hemolytic organism which will be your tested samples, done via an inoculating loop. You’ll need to allow for around 18 to 24-hour culture of isolated organisms for testing.
  2. Next, you will need to streak the isolate to ½ of a TSA 5% sheep blood agar plate, which will allow you to obtain confluent growth. Please note That we use sheep blood agar to allow for the best identification, otherwise you risk false identification.
  3. Whilst using sterile forceps as your instrument to navigate the test, you should place an option disc onto the inoculated surface of the sheep blood agar.
  4. Next, press the disc with the sterile forceps, and what will result is that the disc adheres firmly to the sheep blood agar surface.
  5. Incubate the plate at around 35 degrees/- 2.0 degrees celsius for around 24 Hours in an A & E enriched CO2 environment, aiming for around 5-10% CO2.
  6. Finally; if a zone of inhibition is present, then measure the diameter with a ruler or caliper.

Interpreting Your Results

Now The moment you’ve been waiting for; is how to assess your results and what you should be looking for.

Presumptive identification of pneumoniae can be done if the Alpha hemolytic colony creates a zone of inhibition.

You’re looking for around 14 mm or greater which should be located around the disc. It’s not difficult to spot and if you are having trouble finding it, likely, pneumoniae isn’t present.

If you –have organisms that have produced smaller zone sizes, in solubility. Smaller zone sizes around 6 to 14 mm can be assumed as pneumococci but only if it is bile soluble.

In short:

  • A positive test will be identified by a Zone of inhibition that is greater than 14 mm in diameter when using a 6 mm disc.
  • a negative test will show no zone of inhibition or a Zone of inhibition that is less than 14 mm.

The Limitations Of Optochin Susceptibility Test

With this test comes a few common limitations that need to be addressed for us to be as objective as possible.

It has been shown that other strains of Alpha hemolytic streptococcus may result in a slight susceptibility to opticians.

Another thing to note is that susceptibility is a presumptive test only and shouldn’t be seen as gospel on its own. Therefore, further testing is required from a biochemical perspective for a thorough analysis and 100-percent complete identification.

S. pneumoniae isolates must be activated in a CO2 enrichment environment, at least 5 to 10% CO2. This is because someone’s lips grow poorly, whilst others will not grow at all aerobically.

Something that is often overlooked is that isolates will show indeterminate zones on Media other than our recommended sheep blood agar, which we have already alluded to. Isolates that show indeterminate zones will also require further testing to identify them as S. pneumoniae.

Frequently Asked Questions

What Materials Are Required For An Optochin Sensitivity Test?

You will require a number of items to complete an oxygen sensitivity test.

The first item you need is an optochin disk, these can be purchased from vendors and each disc will be impregnated with around 5 ug of optochin.

Make sure to stop them in a climate that is around 2 to 8 degrees Celsius, and ensure they are protected from lights, excessive heat, as well as moisture as this, will potentially damage the disc.

It’s also vital that before you make a purchase check the disc for expiration date and that the disc is used before expiry.

We recommend that you use sheep blood agar as other options can potentially run the risk of false identification.

You will also require a standard microbiological loop, sterile forceps, as well as a CO2 incubator that is around 35 degrees Celsius.

Final Thoughts

Following the steps above will ensure that your test goes smoothly and that you limit any potential limitations that can compromise your test results.

Remember to measure correctly and ensure that your 14 mm or greater diameter is a positive result.

Jennifer Dawkins

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