Disinfectants & Sanitizer Efficacy Testing Protocol

This protocol/SOP is designed to establish the scientific evidence and demonstrate the efficacy of disinfectants and sanitizing agents against various microorganisms by “Use Dilution or Concentration Method” and “Surface Challenge Test.”

Efficacy Evaluation of Disinfectants & Sanitizing Agents

Table of Content – Disinfectants Efficacy Protocol:

Sr. No Topics
1.0 Protocol Approval
2.0 Purpose
3.0 Scope
4.0 Responsibility
5.0 References
6.0 Methodology
7.0 Validation Procedure
8.0 Summary of Deviations
9.0 Abbreviations
10.0 Documentation and Archival
11.0 Annexures

1.0   PROTOCOL – APPROVAL:

    • This is a protocol to demonstrate the efficacy evaluation of disinfectants and sanitizing agents used at the Microbiology Laboratory and Pharmaceutical plant.
    • The protocol has been prepared, reviewed, and approved for execution by personnel from the following departments:
    • Prepared By – Microbiologist
    • Reviewed By – Department Head 
    • Reviewed By – Head Regulatory Affairs 
    • Approved By -Head Production
    • Approved By -Head Quality Assurance 

2.0   PURPOSE – Disinfectants Efficacy Protocol:

    • This protocol is designed to establish the scientific evidence and demonstrate the efficacy of disinfectants and sanitizing agents against various microorganisms by “Use Dilution or Concentration Method” and “Surface Challenge Test.”
    • To establish the scientific evidence and demonstrate the contact time of the disinfectant and sanitizing agents for its application.
    • To establish the scientific evidence and demonstrate the validity of the storage period for in use disinfectants and sanitizing agents.

3.0.   SCOPE – Disinfectants Efficacy Protocol:

    • This protocol is applicable for the efficacy evaluation of –
      • Disinfectants and sanitizing agents,
      • Neutralization study of disinfectants,
      • Contact time establishment and
      • Hold time study for expiry date when used as per supplier recommended dilutions for the routine sanitization and disinfection applications to control the microorganism.

4.0   RESPONSIBILITY – Disinfectants Efficacy Protocol:

    • Microbiology Executive/Designee-
    • Preparation of validation protocol,
    • Execution of the validation studies and
    • Completion of the validation report.
    • Head Q.C./Designee
    • Responsible for review of the protocol and its summary report for the execution of experimental validation study
    • Arranging resources for the validation program and review of validation results and summary report.
    • Head Production/Designee
    • Responsible for review of the protocol and its summary report for any technical aspects of the evaluation study.
    • Head Q.A./Designee
    • Responsible for the final approval of the protocol and summary report,
    • After completion of the qualification, the summary report shall be checked, reviewed, and approved.

5.0   REFERENCES:

    • USP Chapter <1072> Disinfectants and Antiseptics.

6.0   METHODOLOGY – Four Level of Disinfectants Efficacy:

  • Pre-Requisites for Disinfectant Efficacy Testing

    • Media Required:
    • Soyabean Casein Digest Agar
    • Sabouraud Dextrose Agar
    • Tryptone Soya Agar with Lecithin and Tween-80 or Dey/Engley (D/E) agar
    • 1% Peptone Water
    • Dey/Engley (D/E) Broth
    • 9% Saline
    • Accessories Required:
    • Sterilized Membrane 0.45µm
    • Filter Assembly
    • Filter Cups
    • Tubings
    • Micropipette
    • Microtips
    • Forceps
    • Petri plates
    • Surface Test Coupons
  • Challenge Microorganisms – Disinfectants Efficacy Measurement:

    • Selection of ATCC and Environmental isolates is done to cover the entire microorganism based on the gram character and cell morphology.
    • For the studies on disinfectants used for spraying and surface disinfection isolates selected must involve spore bearing prokaryotic and eukaryotic microorganisms.
    • The typical challenge microorganisms that can be employed are listed in Table-01.

Table-01

Sr. No.

Name of the challenge Microorganism (Standard Test Organism) Disinfectant/Sanitizing Agent Used

Cell Morphology

1. Escherichia coli (ATCC 8739) Bactericide Vegetative Bacteria and Gram –ve small bacilli
2. Staphylococcus aureus (ATCC 6538) Bactericide Vegetative Bacteria and Gram +ve cocci in clusters.
3. Pseudomonas aeruginosa (ATCC 9027) Bactericide Vegetative Bacteria and Gram –ve bacilli
4. Bacillus subtilis (ATCC 6633) Sporicide Spore forming bacteria and Gram +ve bacilli.
5. Candida albicans (ATCC 10231) Fungicide Yeast, prokaryotic budding cells.
6. Aspergillus brasiliensis (ATCC 16404) Fungicide/Sporicide Mold, Spore forming mycelium.
7. Environment Isolates Bactericide Gram +ve cocci
    • However, all the environmental isolates, which were isolated from an environmental monitoring program shall be challenged to the efficacy evaluation of disinfectants/sanitizing agents to confirm their susceptibility, other wise most frequently isolated microorganisms is also acceptable.
  • Classification of Disinfectant and Sanitizing Agents:

    • Chemically disinfectants are classified by their chemical type.
    • These include aldehydes, alcohols, halogens, peroxides, quaternary compound (QAC) and phenolic compounds (see Table-02).

Table-02

Chemically Entity Classification Examples
Aldehydes Sporicidal agents 2% Glutaraldehyde.
Alcohols General purpose disinfectant (Bactericide), antiseptic, antiviral agent 70% Isopropyl Alcohol (IPA)
Chlorine and Sodium hypochlorite Sporicidal agent 0.5% Sodium hypochlorite.
Phenolics General purpose disinfectant 500 µg per g chlorocresol, 500 µg per g chloroxylenol.
Ozone Sporicidal agent 8% gas by weight.
Hydrogen peroxide Vapour phase sterilant, liquid sporicidal agent, antiseptic 4 µg per g H2O2 vapour, 10%-25% solution, 3% solution.
Substituted diguanides Antiseptic agent 0.5% Chlorhexidine gluconate
Peracetic acid Liquid sterilant, vapour phase sterilant 0.2% Peracetic acid, 1 µg per g Peracetic acid
Ethylene oxide Vapour phase sterilant 600 µg per g Ethylene oxide.
Quaternary ammonium compounds General purpose disinfectant (bactericide), antiseptic 200 µg per g Benzylkonium chloride.
Β-Propiolactone Sporicidal agent 100 µg per g Β-Propiolactone
  • Selection Criteria of Disinfectants and Sanitizing Agents foe Efficacy Testing:

    • The Following points to be consider for selection of disinfectants and sanitizing agents for efficacy testing.
      • Number and types of microorganisms to be controlled.
      • The spectrum of activity of commercially available disinfectants and sanitizing agents.
      • The claims as a sterilant.
      • The disinfectant or sanitizer supporters by the EPA registrations.
      • The concentration, application method and contact time with the disinfectant or sanitizing agent.
      • Nature of the surface material and its compatibility with the disinfectant or sanitizing agents.
      • The amount of organic compounds on the surface that may inactivate a disinfectant or sanitizing agent.
      • The possible need to maintain a residual bactericidal activity of the disinfectant on the surface.
      • The corrosiveness of the disinfectant to equipment with repeated application.
      • Safety consideration to the operators applying the disinfectant or sanitizing agents.
  • Neutralizing agents for Disinfectants and Sanitizing agents (Efficacy Testing):

    • Neutralizers that inactivate the disinfectants shall be included either in the diluent or microbiological media used for microbial enumeration.
    • Refer Table-03 for information on the disinfectant neutralization.

Table-03

Disinfectant Neutralizing Agent
Alcohols, Dilution or Polysorbate 80
Glutaraldehyde Glycine and Sodium bisulfate
Sodium hypochlorite Sodium thiosulphate
Chlorhexidine Polysorbate 80 and Lecithin
Mercuric chlorides and other mercurials Thioglycolic acid
Quartnery ammonium compounds Polysorbate 80 and Lecithin
Phenolic compounds Dilution or Polysorbate 80 and Lecithin
A universal neutralizing broth which contain a range of neutralizing agents can also be used for example Dey/Engley (D/E) broth which contains 0.5% Polysorbate 80, 0.7% Lecithin, 0.1% Sodium thioglycolate, 0.6% Sodium thiosulphate, 0.25% Sodium bisulfate, 0.5% tryptone, 0.25% yeast extract and 1.0% dextrose.

7.0   Validation Procedure – Disinfectants Efficacy Testing:

    • This validation methodology demonstrated for the following parameters:
      • Preparation of challenge inoculum culture.
      • Neutralization study of disinfectants and sanitizing agents.
      • In vitro “Use dilution” test and contact time Establishment (screening disinfectants and sanitizing agents for their efficacy at various concentrations and contact times against a wide range of standard test organisms and environmental isolates).
      • In vitro “Surface Challenge Test” on test coupons.
  • Preparation of challenge inoculum:

    • Prepare SCDA and SDA slants as per the current version of SOP for “Preparation of Microbial Culture Suspension”.
    • Incubation conditions for the slants is as follows:
      • Bacillus species incubate at 30 to 35 ºC for NLT 2 Days.
      • Vegetative growth of bacteria incubates at 30 to 35 ºC for NMT 3 Days.
      • Yeast incubate at 20 to 25 ºC for NLT 5 Days.
      • Molds incubate at 20 to 25 ºC for NLT 5 Days.
    • After completion of incubation period wash the slants with 0.9% saline to get uniform inoculum suspension.
    • This shall be used to prepare the 106 cfu/ml and to obtain the NMT 100 cfu by serial dilution techniques.
    • Check the suspension for the cell population by culture suspension preparation method as per SOP for “Preparation of Microbial Culture Suspension”.
    • Record the observations and results in the Annexure-1
  • Neutralization study of Disinfectants and Sanitizing Agents (Efficacy Testing):

    • This test method is used to establish and demonstrate the elimination or minimizing the chemical effect of the disinfectant on the microbial population when performing the recovery test.
    • Sample Preparation:
    • Prepare Culture suspension as per the current version of SOP for “Preparation of Microbial Culture Suspension” to determine NMT 100 cfu concentration of the following microorganisms selected:
      • Escherichia coli
      • Staphylococcus aureus
      • Candida albicans
      • Aspergillus brasiliensis
      • Pseudomonas aeruginosa
      • Bacillus subtilis
      • Environment Isolates
    • Prepare “Use Dilution” in a separate tube of each disinfectant and sanitizing agent used for the study.
    • Prepare and sterilize the required quantity of neutralizing media in separate container, dispense 50ml of the media into 100ml test tubes for each challenge microorganisms.
  • List of Disinfectants and Sanitizing Agents for Efficacy Test.

S. N. Name of Disinfectant Use Concentration Category
1. Bacillocid 2.0 % Bactericidal,  Sporicidal
2. Taski Combaton DS 1.0 % Bactericidal,   Sporicidal
3. Virosil 20 % Sporicidal
4. Sterillium Undiluted Bactericidal,   Sporicidal
5. Isopropyl alcohol 70 % Bactericidal,   Sporicidal  
6. Divosan (Clearklens Activ) 1 % Sporicidal
7. Virex II 256 0.5% Bactericidal, Fungicidal, Virucidal
8. Oxivir 516 1% Bactericidal, Fungicidal, Virucidal
  •  Use Dilution Test and its contact time Establishment:

    • This method involves the screening disinfectants for their efficacy at various concentrations and contact times against a wide range of standard microorganisms and environmental isolates.
    • Sample Preparation:
    • Dilute the sanitizers with sterile purified water at ambient temperature for the stock according to the recommendation of the manufacturer.
    • This is termed as “Use Dilution”
    • Prepare the “Use Dilution” of each disinfectant.
    • Dispense 20ml of the dilution into four sterile test tubes and label them as A, B, C and D. A for 0 Min., B for 5 min., C for 10 min. and D for 15 min.
    • Note:   Label on Sterillum or IPA 70% (Hand sanitizer) study tubes as A for 10 Seconds, B for 20 Seconds, C for 30 Seconds and D for 60 Seconds and note down the observations in Annexure-5 and calculate the log reduction.
    • Add 0.1 ml of culture containing 108 cfu/ml (for Bacteria & Yeast) or 105 cfu/ml (for Mold) of specified microorganism into tubes to get the concentration of 1×108 cfu/ml (for Bacteria & Yeast) and 1×105 cfu/ml (for Mold) sample A, B, C and D.
    • Recovery Study from Disinfectant Control Test (Efficacy):

    • Within 1 minute (0 minute) contact time immediately transfer the sample from A (20ml) into the 50 ml of diluents containing neutralizer.
    • Filter the solution through 0.45µm sterile membrane and give three rinses of 100 ml each with 0.1% peptone or suitable diluents.
    • Place a membrane aseptically on pre-incubated agar plates (TSA with Lecithin & Polysorbate 80) or plates containing neutralizer (DE agar) according to the nature of the disinfectants.
    • Repeat the above exercise to establish the contact time for 5 minutes, 10 minutes and 15 minutes for tubes B, C & D respectively for each sanitizing agent used.
    • Recovery Study from Positive Control Test:

    • Add 0.1 ml of the challenge microorganism containing 108 cfu/ml (for Bacteria & Yeast) or 105 cfu/ml (for Mold) to 20 ml sterile normal saline,
    • Further transfer to 50 ml neutralizing media, filter the solution through 0.45 µm sterile membrane and place the membrane on the pre-incubated TSA or DE agar plate aseptically.
    • Repeat the procedure for all specified microorganisms.
    • Incubation Conditions:   Bacteria: 30 to 35 ºC for 48 to 72 hrs.

Yeast & Mold: 20 to 25 ºC for 5days.

    • Interpretation of Results: After the incubation period, count the number of colonies on the membrane from all plates and note down the observation in Annexure-2 and calculate the log reduction.
    • Negative Control:

    • Filter 20 ml sterile normal saline in 50 ml neutralizing media, filter the solution through 0.45 µm sterile membrane filter and place the membrane on the pre-incubated TSA or DE agar plate aseptically..
    • Incubate the plates at 20-25 ºC for 72 hrs. Followed by 30-35 ºC for 48 hrs.
    • Log Reduction Calculation:

                                                      ( X-Y )

            Where, X = Log of population taken for test.

                         Y = Log of observed count after contact time.

  • Description – Disinfectants Efficacy Testing:

    • This method involves using standard test microorganisms and microorganisms that are typical environmental isolates, applying disinfectants to the selected surface at the “Use Dilution” concentration with a specified contact time, and determined the log reduction of the challenge microorganisms.
    • This is considered necessary because critical process steps like disinfection of aseptic processing area, as required by GMP regulation, needed to be validated, based on surface application.
    • Selection Criteria of Surface:

    • All the surface base on the disinfection application criteria.
    • Because a wide range of different material of construction are used in the clean rooms and  other controlled areas each material need to be evaluated separately to validate the efficacy of the given disinfectant and sanitizing agents.
    • Also contains the common materials used in the clean room construction as per Table-04 given below

Table-04

Material Application
Stainless Steel Work Surface, Filling Equipment and Tank
Glass View Panels
Epoxy Floor
Kota Stone Floor
Clestra  Panels Wall Panels
    • Sample Preparation:
    • Surface coupons like S.S., Glass & Kota Stone shall be wrap by aluminum foil and sterilized in steam sterilizer and Epoxy shall be surface sanitized with 20% Virosil.
    • After the sterilization carry the S.S., Glass and other surface coupons into the LAF area and unwrap coupons carefully before analysis.
    • Recovery from Test Surface:

    • Select Three areas of 2 inch x 2 inch square on one coupon surface. 1st area shall be used for test sample surface recovery, 2nd shall be used for positive test surface recovery and 3rd shall be used as a negative control.
    • Add 0.1ml of the cell suspension containing approximately 108 cfu/ml (for Bacteria & Yeast) or 105 cfu/ml (for Mold) of any one selected microorganism on the template surface area (1st and 2nd) and spread equally with an L-spreader.
    • Hold the coupon in vertical position and apply selected disinfectant by fine spray on the spike surface area of the template surface.
    • Take precaution not to over spill the applied disinfectant to other marked surface.
    • Allow the disinfectant as per established contact time on the template surface and recover by challenge inoculums by swab method on the three surfaces with individual swab sticks.
    • Rotate and spread the swab throughout the selected surface (1st area of the template surface) in zigzag fashion.
    • Place the swab immediately in to a tube containing 10 ml Neutralizing broth.
    • Repeat the procedure for all specified microorganisms and with each selected disinfectant on each mentioned surface coupons respectively (0 Minute, 5 Minutes, 10 Minutes & 15 Minutes).

Note:   In case of Hand sanitizer (Sterillum or IPA 70%) the study will be performed on 10, 20, 30 & 60 Seconds and note down the observations in Annexure-6 and calculate the log reduction.

    • Recovery from Control Surface:

    • For control surface specimen spike the 0.1ml of the cell suspension containing approximately 108 cfu/ml of 1×108 concentration culture spread equally for the 2×2 inch area.
    • Take the swab from 2nd area of the template surface similar to the specimen; place the swab into the neutralizing media culture.
    • Recovery from control surface:
    • 3rd part of the selected coupon surface area of 2×2 inch area which is not spike with microorganism and disinfectant treated as test negative control.
    • Swab Test Procedure:
    • Vortex the tube containing swab for about 30 second and proceed by filtration method.
    • Arrange filter assembly, attach the vacuum pump and filter the Neutralizing broth tube through 0.45µm x 47mm membrane and aseptically transfer the membrane on pre-incubated TSA or DE agar plate for bacterial cultures and yeast and mold culture.
    • Incubation Conditions:
    • Bacteria: 30 to 35 ºC for 48 to 72 hrs.
    • Yeast & Mold: 20 to 25 ºC for 5days.
    • Interpretation of Results:
    • After the incubation period count the number of colonies on the membrane from all plates and note down the observations in Annexure-4 and calculate the log reduction.
  • Hold Time Establishment Study (Disinfectants Efficacy Testing) :   
    • To determine the validity of the disinfectant for the certain period of storage time in use container for the regular application shall be demonstrated for its effectiveness when compared to initial day of preparation.
    • Following parameters are established to demonstrate the effectiveness.
      • Efficacy study after 2nd day.
      • Bio burden level of the disinfectant.
    • Sample Preparation:

    • Dilute all sanitizing agents which were used in the contact time establishment study with sterile purified water at ambient temperature from the stock according to the recommendations of the manufacturer.
    • This is termed as “Use Dilution”
    • Use the same dilution which was established in the “Use Dilution Test” (In contact time establishment study).
    • Prepare the “Use Dilution” of all the disinfectants and sanitizing agents used of 20 ml quantity.
    • Prepare the culture suspension to have 1×108 (for Bacteria & Yeast) or 1×105 cfu/ml (for Mold) populations.
    • Refer preparation of challenged inoculum procedure of this protocol.
    • Prepare 20ml of neutralizing media solution in the test tubes for all the challenged microorganisms.
    • Recovery from Test Sample by Use Dilution Method:

    • Add 0.1ml culture suspension of   challenged microorganisms in each 20 ml disinfectant.
    • Hold the diluted disinfectant for 2 days in the aseptic area or in the controlled area.
    • Arrange the filter assembly in the LAF, connect to the vacuum pump and filter the content of each selected dilution through separate 0.45 µm x 47mm membrane filter.
    • Aseptically transfer the membrane filter on pre-incubated media plates of sterile TSA for bacterial cultures and yeast & mold cultures.
    • Recovery study shall be performed at 2nd day hold time.
    • Recovery from Positive Control Sample:

    • Add 0.1ml of   challenged microorganisms in each 20ml neutralizing media solution.
    • Arrange the filter assembly on the LAF, connect to the vacuum pump and filter the content of each prepared above solution through 0.45 µm x 47mm membrane filter.
    • Aseptically transfer the membrane filter on pre-incubated media plates of sterile TSA for bacterial culture and yeast & mold cultures.
    • Positive control recovery is performed along with the test control.
    • Negative Control:

    • Only neutralizing media shall be used for negative control test.
    • Filter the whole 20 ml content of the neutralizing media through 0.45 µm membrane.
    • Aseptically transfer the membrane on the pre-incubated TSA plate and incubate.
    • Incubation Condition:
    • Bacteria- 30-35 °C for 48 to 72 hrs.
    • Yeast & Mold – 20-25 °C for 48 to 5 days.
    • Negative Control- 20-25 °C for 72 hrs. Followed by 30-35 °C for 48 to 72 hrs.
    • Interpretation of Results:
    • After the incubation period count the number of colonies on the membrane from all plates and note down the observation in Annexure– 4 and 5.
  • Bio burden Test (Disinfectants Efficacy Test):

    • Sample Preparation:
    • Take aseptically 1 ml of manufacturer recommendation concentration of the disinfectant and transfer in sterile petri plate in duplicate for Total bacterial count and Total fungal count.
    • Pour approximately 15-20 ml of sterile DE agar in each plate and mix properly by rotating the plate clockwise and anti-clockwise direction and allow them for solidification.
    • Negative Control:
    • Only neutralizing media shall be used for negative control.
    • Incubation Condition:
    • Bacteria- 30-35 °C for 48 to 72 hrs.
    • Yeast & Mold – 20-25 °C for 48 to 5 days.
    • Negative Control- 20-25 °C for 72 hrs. Followed by 30-35 °C for 48 to 72 hrs.
  • Acceptance Criteria (Disinfectants Efficacy Test):

    • For contact time establishment there should be minimum 5 log reduction for vegetative bacteria/ yeast and 3 log reduction for bacteria spore/fungi (mold) with a control disinfectant and sanitizing agents.
    • For surface coupons studies there should be minimum 3 log reduction for vegetative bacteria/ yeast and 2 log reduction for bacteria spore/fungi (mold) with a control disinfectant application.
    • The 2 day hold time study disinfectant in use studies for established contact time should show 5 log reduction for vegetative bacteria/ yeast and 3 log reduction for bacterial spore/fungi (mold) with a control disinfectant and sanitizing agents stored.
    • There should not be any microbial growth in store disinfectant to establish with selected hold time period.

8.0   SUMMARY OF DEVIATIONS (Disinfectants Efficacy Test):

    • Any deviation(s) from the protocol while performing the methodology shall be investigated and documented in the report.

9.0   ABBREVIATIONS:

    • QC – Quality ControlDisinfectants Efficacy Testing
    • QA- Quality Assurance
    • SCDA- Soyabean Casein Digest Agar
    • SDA- Sabouraud Dextrose Agar
    • TSA- Tryptic Soya Agar
    • ATCC- American Type Culture Collection
    • CFU- Colony Forming Units
    • NMT- Not More Than
    • NLT- Not Less Than
    • LAF- Laminar Air Flow

10.0   DOCUMENTATION AND ARCHIVAL

    • Report: At the end of the study a report shall be prepared.
    • Archival: The original and executed document shall be hand it over to QA for archival.

11.0    ANNEXURES – (Disinfectants Efficacy Test):

Annexure-1  : Inoculum Preparation Record.

Name Organism ATCC No.
Date of Preparation Date of Reporting
Media Used Media Lot. No.
Incubation Temperature Incubator ID
From To
Reference: Protocol No.:
Dilution Volume Tested Count /Plate Final Inoculum Population Observed By (Sign / Date)
Plate 1 Plate 2 Average

Annexure-2  : Growth Observation Record for Direct Contact Method.      

Name of Disinfectant Disinfectant Batch No.
Concentration Date of Analysis
Name of Media Media Lot. No.
Neutralizing Diluents Used Lot No. of Diluents
Incubation Details: –  20-25 ºC for 5 Days  and 30-35ºC for 3 Days
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC
From   From
To To
Name of Organism Initial Population CFU/ml Volume Tested Population taken for Test (X) Observed count after contact time(Y) Log reduction  observed (X-Y) Observed by (Sign /Date)
0 Min 5 Min 10 Min 15 Min  0 Min 5 Min 10 Min 15 Min
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Candida albicans
Aspergillus brasiliensis
Pseudomonas aeruginosa
Environmental Isolate-1
Isolate-2
Isolate-3

Annexure-3  : Surface Challenge Test.

Name of Disinfectant Disinfectant Batch No.
Concentration Date of Analysis
Name of Media Media Lot. No.
Neutralizing Diluents Used Lot No. of Diluents
Incubation Details: –  20-25 ºC for 5 Days  and 30-35ºC for 3 Days
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC
From From
To To

 

Name of Organism Surface Initial Population cfu/ml Volume Tested Population Taken for Test cfu/ml (X) Observed count after contact time (Y) – min Log reduction Observed (X-Y)- min Observed by (Sign)
10  15 0 10 15
SS
Epoxy
Glass
Kota Stone
Clestra Panels

Annexure-4  : Hold Time Establishment by Use Dilution Method.

Name of Disinfectant Disinfectant Batch No.
Concentration Date of Analysis
Name of Media Media Lot. No.
Neutralizing Diluents Used Lot No. of Diluents
Incubation Details:  20-25 ºC for 5 Days  and 30-35ºC for 3 Days
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC
From                                      From
To To

 

Name of Organism Initial Population CFU/ml Volume Tested Population taken for Test (X) Observed count After 2 Days(Y) Log reduction observed after 2 days(X-Y) Observed by (Sign /Date)
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Candida albicans
Aspergillus brasiliensis
Environmental Isolate -1
Isolate -2
Isolate-3

Annexure-5  : Growth observation Record of Hand Sanitizer For Direct Contact Method

Name of Hand Sanitizer Hand Sanitizer Batch No.
Concentration Date of Analysis
Name of Media Media Lot. No.
Neutralizing Diluents Used Lot No. of Diluents
Incubation Details: –  20-25 ºC for 5 Days  and 30-35ºC for 3 Days
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC
From                                     From
To To

 

Name of Organism Initial Population CFU/ml Volume Tested Population taken for Test (X) Observed count after contact time(Y) Log reduction  observed (X-Y) Observed by (Sign /Date)
10 Sec. 20 Sec. 30 Sec. 60 Sec. 10 Sec. 20 Sec. 30 Sec. 60 Sec.
Bacillus subtilis
Escherichia coli
Staphylococcus aureus
Candida albicans
Aspergillus brasiliensis
Pseudomonas aeruginosa
Environmental Isolate-1
Isolate-2
Isolate-3

Remarks:

Annexure-6  : Growth observation Record of Hand Sanitizer For Surface Challenge Test

Name of Hand Sanitizer Hand Sanitizer Batch No.
Concentration Date of Analysis
Name of Media Media Lot. No.
Neutralizing Diluents Used Lot No. of Diluents
Incubation Details: –  20-25 ºC for 5 Days  and 30-35ºC for 3 Days
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC
From From
To To

 

Name of Organism Surface Initial Population cfu/ml Volume Tested Population Taken for Test cfu/ml (X) Observed count after contact time (Y) – in second Log reduction Observed (X-Y)- in second Observed by (Sign./Date)
10 20 30 60 10 20 30 60
Bacillus subtilis SS
Epoxy
Glass
Kota Stone
Clestra Panels

Annexure-7  : Bio burden Test Report of Disinfectant and Sanitizing Agents.

Name of Disinfectant Lot No./Batch No.
Analysis Start on Analysis Completion On
Name of Media Media Lot. No.
Incubator ID Incubator ID
Incubation Temperature 22.5±2.5 ºC Incubation Temperature 32.5±2.5 ºC

Observations     

0 Day 7 Days 15 Days 30 Days
TBC TFC TBC TFC TBC TFC TBC TFC
1 2 Avg. 1 2 Avg. 1 2 Avg. 1 2 Avg. 1 2 Avg. 1 2 Avg. 1 2 Avg. 1 2 Avg.

Negative Control:                                                                                          Positive Control:

Remarks:

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pharmabeginers

Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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