Optochin, also known as ethylhydrocupreine hydrochloride, is a derivative of quinine, which is an antimalarial agent.
Introduced by Morgenroth and Levy in 1911, optochin was initially used as an antibiotic for treating pneumococci infections (infections caused by Streptococcus pneumoniae).
Although optochin can inhibit or kill S. pneumoniae, it is not used as a treatment. Instead, it is employed to differentiate S. pneumoniae from other α-hemolytic Streptococci (viridans Streptococci).
Streptococcus pneumoniae is a Gram-positive, catalase-negative, α-hemolytic, anaerobic but aerotolerant diplococcus, belonging to the genus Streptococcus within the family Streptococcaceae, in the class Bacilli.
Commonly found in the upper human respiratory tract, S. pneumoniae can cause pneumonia, meningitis, otitis media, arthritis, and other infections in immunocompromised and susceptible individuals. Due to its significant role in pneumonia, it is referred to as ‘pneumococcus.’
S. pneumoniae is typically located in the respiratory tract and is often isolated from respiratory samples containing diverse groups of α-hemolytic Streptococci, making identification challenging.
In 1955, Bowen and Jeff proposed a method to differentiate S. pneumoniae from other viridans Streptococci by impregnating a disk with optochin. This method is known as the optochin susceptibility test and is used to identify pneumococcus bacteria.
Objectives
To distinguish S. pneumoniae from other α-hemolytic (viridans) Streptococci.
Principle
α-Hemolytic Streptococci produce colonies with a green-colored zone of incomplete hemolysis in a blood agar medium, making it challenging to identify S. pneumoniae among other viridans Streptococci based on hemolysis and common biochemical characteristics.
S. pneumoniae is the only known optochin-sensitive viridans Streptococcus. Therefore, S. pneumoniae exhibits a clear zone of inhibition around the optochin-impregnated disk, whereas other viridans Streptococci do not display a zone of inhibition around the optochin-impregnated disk.
This distinctive feature allows for the easy differentiation of S. pneumoniae from other viridans Streptococci.
Requirements
a. Culture Media
A 5% Sheep Blood Agar Plate is used as the culture medium for performing the optochin sensitivity test.
This medium is prepared by adding 5% v/v defibrinated sheep blood to a molten blood agar base medium.
Measure the appropriate amount of blood agar base powder (or the media components) and mix with the required volume of water in a conical flask (or glass bottle) according to the manufacturer's instructions.
Stir well using a magnetic stirrer or manually, and heat to boiling to ensure all components and agar dissolve completely in water.
Autoclave the flask or bottle at 121°C and 15 lbs pressure for 15 minutes, then let it cool to around 40 – 45°C.
Slowly pour 5% (5 to 10%) v/v sterile defibrinated sheep blood into the flask containing the blood agar base while stirring constantly. Mix properly to ensure the blood is uniformly distributed in the medium, forming the blood agar.
Pour approximately 25 mL of the blood agar into a sterile Petri plate (glass plate with a 10 cm diameter) and allow it to solidify at room temperature.
Store the blood agar plates (BAPs) in a refrigerator at 4°C for use up to a maximum of 2 to 4 weeks.
b. Reagents
Optochin-impregnated disk: 6 mm disk with 5 μg of optochin
Defibrinated Sheep Blood: for BAP preparation
McFarland Standard number 0.5
Composition
1% anhydrous barium chloride (BaCl2) solution
1% sulfuric acid (H2SO4) solution
Preparation of number 0.5 McFarland Standard Suspension
In a clean and clear test tube, add 9.95 mL of 1% H2SO4 solution and 0.05 mL of 1% BaCl2 solution.
c. Equipment
Petri Plates
Forceps
Weighing Machine
Autoclave
Bunsen burner
CO2 Incubator
Inoculating loop/(Cotton Swab)
PPE
Other laboratory materials
d. Test organism (sample bacteria)
Streptococcus pneumoniae ATCC 49619: Positive control
Streptococcus mitis ATCC 49456 or Enterococcus faecalis ATCC 29212 or Streptococcus pyogenes ATCC 12384: Negative control
Procedure
In a sterile test tube, prepare a bacterial suspension using well-isolated α-hemolytic colonies of the sample organism. Adjust the turbidity of the suspension to match the 0.5 McFarland standards.
Using a sterile inoculating loop or cotton swab, streak or spread the bacterial suspension in at least two directions over the 5% Sheep Blood Agar Plate. If S. pneumoniae culture is being used for purposes other than identification, Mueller-Hinton Agar (MHA) can be utilized.
Allow the suspension to adhere to the plate and dry by leaving it in an upright position for approximately 5 to 10 minutes.
Using sterile forceps, place an optochin antibiotic disk in the area of inoculation and gently press the disk to ensure its adherence.
Incubate the plate anaerobically in a 5 to 10% CO2 environment at 35±2°C overnight, which typically ranges from 18 to 24 hours.
After the incubation period, observe the formation of a zone of inhibition around the optochin antibiotic disk and measure the diameter of the zone.
Result and Interpretation
If the bacterium displays a zone size of ≥14 mm, classify it as Optochin Sensitive.
If the zone size measures <14 mm, categorize it as Optochin Intermediate.
If no zone of inhibition is observed, designate it as Optochin Resistant.
Regarding identification:
If the bacterium is Gram-positive cocci in pairs, catalase-negative, and α-hemolytic, and exhibits Optochin Sensitivity, report it as Streptococcus pneumoniae.
If the bacterium meets the above criteria but shows Optochin Intermediate results, conduct the spot bile solubility test. If the bacterium is bile soluble, classify it as Streptococcus pneumoniae.
If the bacterium is Gram-positive cocci, α-hemolytic, and Optochin Resistant, report it as Viridans Streptococci.
Optochin Susceptible Bacteria:
Streptococcus pneumoniae
Optochin Resistant Bacteria:
S. mitis group
S. anginosus group
S. sanguinis group
S. mutans group
S. salivarius group
Quality Control
Streptococcus pneumoniae ATCC 49619 exhibits a distinct zone of inhibition measuring ≥14 mm around the optochin disk.
Streptococcus mitis ATCC 49456 or Enterococcus faecalis ATCC 29212 demonstrate no zone of inhibition surrounding the optochin disk.
Precautions
Avoid pouring blood into molten blood agar base if its temperature exceeds 45°C.
Pour the media into petri plates within a sterile environment before the temperature of the molten media falls below 40°C to prevent clumping.
Always utilize 5% sheep blood agar for bacterial identification purposes.
Position the disk approximately 25 mm away from the edge of the plate. When using multiple antibiotic disks, ensure each disk is at least 25 mm apart from one another.
Applications
Utilized for the identification and differentiation of Streptococcus pneumoniae from other viridans Streptococci.
Limitations
If the test bacteria exhibit α-hemolysis and demonstrate intermediate susceptibility to the optochin, a bile solubility test is necessary.
Occasionally, reports of Streptococcus pneumoniae resistant to optochin have been documented.
For culture and incubation, a 5% sheep blood agar medium and a CO2 incubator are essential.
Verification is crucial for colonies demonstrating optochin resistance or intermediate susceptibility, as they may or may not be S. pneumoniae.
Due to its reliance on culture, results and interpretation require a minimum of 24 hours.
~1.webp "Optochin Susceptibility Test - Principle, Procedure, Results - Basic Cultural Media For Microbiology By Microbiologist Doctor_dr2021")
