There are number of products that are recalled from the market just because of the contamination issue. Now, here are more Question come into mind;
Where did it contamination came from ?
How it can be avoided?
 Do you know a human is the main source of contamination and apart from Human there are other factors that contribute contamination to the products; e.g. poor facility design, poor disinfection of manufacturing area, poor sanitization of equipment so now you can understand why disinfection is necessary. Disinfection is necessary to reduce the risk of contamination to the products and it can be achieved by using a suitable disinfectant. Only suitable disinfectant is not sufficient to kill the microorganism, a validated concentration of disinfectant is also required otherwise it will not work so now here is a question.
How did you know that this is valid concentration of disinfectant? 
It can only be confirmed by doing some validation activities, now you will be thinking HOW then read the complete article. The following approach can be followed to qualify/ validate the disinfectant solution.

Neutralizer Efficacy test:
This neutralizer efficacy test is required to check the recovery of microbial culture and neutralization is required to remove the antimicrobial effect of the disinfectant solution. For this test, Dey-Engley Neutralizing agar can be used. To perform this test first you need to apply 1 ml known concentration of the disinfectant solution on the surface of 90 mm Dey-Engley’s Neutralizing agar place and it should be homogeneously spread on the surface of media plate and allow it to dry. After dry spread 0.1 ml know microbial culture suspension; having 50 to 100 CFU on a plate and spread it gently to all surface and incubate all these plates for a defined period and incubation temperature as per organism requirement; like E.coli required less than or equal to 72 hours at 30 to 35 degree C and Candida albicans required less than or equal to 5 days at 20 – 25 degree C. Count all the CFU’s. A negative and positive control is also required with these tests. Count CFU in positive control. Now you need to calculate % Recovery of an organism by using below formula:
%Recovery = Positive control with disinfectant / Positive control X 100


RECOVERY TEST AND DIFFERENT CONCENTRATION OF DISINFECTANT SOLUTION: 

As different surfaces and materials of construction are used in the pharmaceutical manufacturing environment so you need to identify all the surface and their MOC (Material of construction) which are directly or indirectly into contact of a product and finally coupons of all types of material/surface should be available. The following organism can be used during study along with one or two environmental isolates that you have isolated during the environmental monitoring program of the manufacturing area.
Organism  ATCC Incubating Details
  • Staphylococcus aureus  ATCC 6538 30 – 25°C for 3 – 5 days
  • E.coli ATCC 11229 30 – 25°C for 3 – 5 days
  • Bacillus subtilis ATCC 19659 30 – 25°C for 3 – 5 days
  • Aspergillus niger  ATCC 16404 20 – 25°C for 3 – 5 days
  • Candida albicans  ATCC 10231 20 – 25°C for 5 – 7 days
  • Pseudomonas aeruginosa  ATCC 15442 30 – 25°C for 3 – 5 days

Environmental bacterial isolate Not applicable 30 – 25°C for 3 – 5 days
1. Recovery study can be done by two methods;

(i) Contact plate method and (ii) swab method
1.1. Recovery study by contact plate method: Following culture media are required to demonstrate this test; Dey- Engley’s neutralizing agar plate (pre-incubated), 0.1 % peptone water & sterile normal saline All the coupons should be sterilized by using 70% IPA solution and after applying the IPA solution rinse the coupons by sterile purified water or can be sterilized by autoclave or moist heat sterilization. After drying all the coupons spread 0.1 ml culture suspension of the above microorganism on all coupons surface and allow to dry again under Laminar airflow. Take the contact plate from the inoculated surface and incubate the plates into incubator organism-wise as per the above table at the defined temperature for a defined period make the Positive and Negative control also.

1.2 By the Swab method this test instead of using the contact plates take the swab of all coupon which you are going to challenge.    – Wet the filter paper with 10 ml of 0.1 % peptone water before filtering, extract the organism in 10 ml 0.1% peptone water and filter it.- Rinse the membrane filter with 100 ml of 0.1% peptone water and place this filter paper on  Dey- Engley’s neutralizing agar plate for positive control incubate the 0.1 ml of above-mentioned culture suspension in 10 ml of 0.1% peptone water and filter it followed wash with 100 ml 0.1% peptone water, then place the filter on the pre-incubated Dey- Engley’s neutralizing agar plate.

  • Note: Before filtration wet the filter paper with Peptone water. For negative control, take the swab from coupons before inoculating the culture. Incubate all these samples at defined incubation temperature for a defined period as mentioned in the above table. Recovery should be 50 to 200% in comparison to positive control for both the recovery methods.

    2.0 DETERMINATION OF CONTACT TIME:     Take three sets of 10 tubes for each disinfectant and add 9 ml diluted disinfectant into these test tubes and finally add 1 ml of 10^2 or 10^3 concentration culture suspension or desired concentration, keep aside and after define time interval like after 5 minutes, 10 minutes and 15 minutes filter the solution through 0.45-micron filter and wash the filter three times with 100 ml of 0.1% concentration peptone water. For the product, positive control adds 1 ml of normal saline in place of culture suspension and wash three times with 100 ml of 0.1% concentration peptone water. For a positive control filter, 10 ml peptone water contains 10 to 100 CFU of culture. Place all the filters on the surface of Dey-Engley’s neutralizing agar plate and incubate as per the above table. After incubation calculate the colony and log reduction and select the time where more than 2 log reduction observed

    3.0 SIMULATION STUDY BY CONTACT PLATE:
    All the coupons should be sterilized by using a 70% IPA solution and after applying the IPA solution rinse the coupons by sterile purified water or can be sterilized by autoclave or moist heat sterilization. Allow drying under laminar airflow. Spot the 100µl of culture suspension of each organism at the desired concentration in duplicate for all type of disinfectant allows to dry for 10 to 15 minutes under Laminar airflow and then spread the disinfectant dilution on pre-incubated coupons after allowing the contact time which is validation under above section 2 and after that take sample using contact plate in duplicate for all disinfectant. Positive control prepared by serially dilutes the culture concentration to obtain the 10 t0 100 CFU/plate. Finally, incubate the plates as per the above table. Calculate the colony and log reduction after completion of the incubation period

    4.0 SIMULATION STUDY BY SWAB METHOD:

    All the coupons should be sterilized by using a 70% IPA solution and after applying the IPA solution rinse the coupons by sterile purified water or can be sterilized by autoclave or moist heat sterilization. Allow drying under laminar airflow. Spot the 100µl of culture suspension of each organism at the desired concentration in duplicate for all types of disinfectants. Allow to dry for 10 to 15 minutes under Laminar airflow and then spread the disinfectant dilution on pre-incubated coupons after allowing the contact time which is validation under above section 2 and after that take sample using sterile swab in duplicate for all disinfectant. Wet the filter paper with 10ml of 0.1 % peptone water before filtration. Extract the organism in 10 ml of 0.1% peptone water and filter and finally wash the filter paper by 100 ml0.1% peptone water and place the filter on Dey-Engley’s neutralizing agar plate and incubate as per above table. Positive control filter 10 ml peptone water contains 10 to 100 CFU of the culture and prepared negative control by taking a swab from a sterile saline solution. Finally, incubate the plates as per the above table. Calculate the colony and log reduction after completion of the incubation period.

Recovery should not be less than 70% on both swab and contact plate and minimum 3 log reduction is required to qualify the disinfectant.

“So now you can do the disinfectant validation and select the appropriate disinfectant and its concentration with contact time and make you product contamination-free. I strongly recommend using at-least one sporicidal inside the manufacturing area to avoid the germination of spore as well.”