1. Purpose

To define a standardized procedure to evaluate and verify the antimicrobial effectiveness of disinfectants and sporicidal agents used for routine cleaning and disinfection of classified and non-classified areas, equipment surfaces, and utilities in the facility.

2. Scope

This SOP applies to Microbiology/QC laboratory personnel performing disinfectant efficacy testing (DET) for:

• Nonporous environmental surfaces (e.g., SS 316L, epoxy, glass, PVC, acrylic)
• Porous/other surfaces where applicable (e.g., vinyl, rubber)
• In-use disinfectant concentrations and contact times
• Routine, rotational, and sporicidal agents used in all production, warehouse, and laboratory areas.

3. References (informational)

• USP <1072> Disinfectants and Antiseptics (latest).
• ASTM E1153 (non-food contact surfaces), ASTM E2197 (quantitative carrier test), ASTM E1054 (neutralizer validation).
• EN 13697 (surface test), EN 1276 (suspension test), AOAC methods for hard-surface sanitizers.
• Company Validation Master Plan (VMP) and Cleaning & Disinfection Program.

4. Definitions

• DET: Disinfectant Efficacy Test.
• Log reduction (LR): log10(N0) − log10(Nt), where N0 is inoculum control CFU and Nt is post-treatment CFU.
• Carrier: A representative test coupon simulating facility surface (e.g., 2×2 cm).
• Neutralizer: A reagent that immediately stops disinfectant action without being toxic to microorganisms (validated for effectiveness and toxicity).
• Organic load/soil: Interfering substance (e.g., 5% serum, 0.3% BSA, 0.03% yeast extract) to simulate worst-case.

5. Responsibilities

• QC Microbiology: Plan, execute, and document DET; trend results; maintain cultures; issue reports.
• Production/Engineering: Provide surface specs and coupons; support selection of representative surfaces and conditions.
• QA: Approve protocols, methods, reports, acceptance criteria; manage change control and deviations.
• EHS: Ensure safe handling of disinfectants and organisms.

6. Safety & Precautions

• Follow institutional biosafety procedures for BSL-2 handling where applicable.
• Use appropriate PPE (lab coat, gloves, goggles/face shield).
• Prepare disinfectants in fume hood where required.
• Handle sporicidal agents (e.g., hypochlorite, peracetic acid) with caution; avoid mixing incompatible chemicals.
• Dispose of biological and chemical waste as per SOPs.

7. Materials & Equipment

• Class II biosafety cabinet, incubators (20–25°C, 30–35°C, 37°C), -20°C/-80°C freezer for stock cultures.
• Sterile test coupons of representative surfaces (SS 316L #4 finish, glass, epoxy-coated panel, vinyl/PVC, acrylic, polycarbonate; optional: vinyl gaskets, rubber).
• Disinfectants/sporicides under evaluation at in-use and worst-case (e.g., lower limit) concentrations.
• Neutralizers (e.g., D/E neutralizing broth, polysorbate 80, lecithin, sodium thiosulfate, histidine, saponin) validated per Section 10.
• Growth media: TSA/TSA-S (bacteria), SDA (yeasts), MEA (moulds), R2A (water isolates as applicable), TSB/BHI (broths).
• Sterile pipettes, vortex, timer, microcentrifuge tubes, spreaders, filtration unit if required.
• Organic soil components (BSA, serum, yeast extract) as per protocol.
• Sterile saline or PBS for suspensions; diluents with neutralizer as applicable.
• Positive displacement pipettes for viscous agents if needed.

8. Test Microorganisms

Select facility-relevant organisms, including compendial strains (ATCC equivalents) and recent environmental isolates (≤12 months) from routine monitoring where available.

Recommended panel:

• Staphylococcus aureus (ATCC 6538 or environmental isolate)
• Pseudomonas aeruginosa (ATCC 15442)
• Escherichia coli (ATCC 8739) or Enterobacter cloacae complex
• Burkholderia cepacia complex (for disinfectants used in non-sterile/aqueous areas)
• Enterococcus faecium (optional for high-level resistance)
• Candida albicans (ATCC 10231)
• Filamentous mould e.g., Aspergillus brasiliensis (ATCC 16404)
• Bacterial spores: Bacillus/Geobacillus (e.g., G. stearothermophilus for sporicides)
• Additional facility isolates of concern (e.g., Micrococcus luteus, Kocuria, Staphylococcus epidermidis, Bacillus spp., Penicillium spp.)

8.1 Culture Preparation

• Revive cultures ≤5 passages from stock.
• Grow to late log phase (18–24 h for bacteria, 48–72 h for yeasts/moulds as per strain).
• Harvest and resuspend in saline/PBS to target ~10^8 CFU/mL for suspension testing or ~10^7 CFU/mL for carrier inoculation; verify by plate count.
• For spores, prepare/obtain standardized spore suspensions; verify purity and titer.

9. Test Design Overview

Perform two complementary methods unless otherwise justified:

1. Quantitative Suspension Test – evaluates intrinsic antimicrobial activity in bulk solution.
2. Quantitative Carrier (Surface) Test – evaluates performance on dried contamination on representative surfaces under soiled and clean conditions.

Include neutralizer effectiveness and neutralizer toxicity controls (Section 10), and the following controls per organism and condition:

• Inoculum control (IC)
• Carrier recovery control (CRC)
• Disinfectant control (DC; disinfectant + neutralizer sterility)
• Neutralizer sterility control (NSC)
• Dilution water control (DWC)

10. Neutralizer Validation (must be completed before DET)

Validate the selected neutralizer for each disinfectant/organism/surface per ASTM E1054 (or equivalent).
10.1 Neutralizer Effectiveness (NE): Demonstrate that neutralizer stops disinfectant action: Disinfectant + Neutralizer + Organism vs. Disinfectant + Organism (without neutralizer). Recovery in NE should be comparable to inoculum control (≤0.5 log difference).

10.2 Neutralizer Toxicity (NT): Demonstrate that neutralizer is non-toxic to test organism: Neutralizer + Organism vs. Organism in diluent. Recovery should be comparable (≤0.5 log difference).

Document acceptance criteria and raw data; failure invalidates DET for that condition.

11. Procedure A: Quantitative Suspension Test (e.g., EN 1276-like)

11.1 Preparation

• Equilibrate disinfectant to test temperature (20±1°C unless otherwise justified).
• Prepare at least two conditions: clean (no soil) and soiled (e.g., 0.3% BSA or 3% BSA/0.3% sheep erythrocytes as justified).
• Prepare organism suspension ~10^8 CFU/mL.

11.2 Execution

1. Mix disinfectant and soil (if used) per validated ratio (e.g., 8 parts disinfectant + 1 part soil + 1 part inoculum).
2. Start timer immediately; maintain specified contact time(s) (e.g., 1, 5, 10 min).
3. At each timepoint, transfer an aliquot into validated neutralizer at a validated neutralization time (≤10 s).
4. Perform serial dilutions in diluent containing neutralizer.
5. Plate appropriate dilutions (duplicate plates per dilution) on TSA/SDA/MEA.
6. Incubate: bacteria 30–35°C/48 h; yeasts 20–25°C/72 h; moulds 20–25°C/5–7 d (or organism-specific).
7. Enumerate colonies (30–300 CFU/plate) and calculate CFU/mL.

11.3 Calculations

• Log reduction = log10(N0) − log10(Nt), where N0 = mean IC titer; Nt = mean survivors after contact.

11.4 Acceptance Criteria (typical)

• Bactericidal: ≥5.0 log10 reduction within claimed contact time.
• Yeasticidal/Fungicidal: ≥4.0 log10 reduction.
• Sporicidal: ≥3.0–4.0 log10 reduction (justify per regulation/claim).
• Meet criteria under soiled condition at lowest in-use concentration and coolest applicable temperature (worst case), unless otherwise justified.

12. Procedure B: Quantitative Carrier (Surface) Test (e.g., ASTM E2197/EN 13697-like)

12.1 Coupon Preparation

• Clean coupons with neutral detergent, rinse with WFI or equivalent, dry, and sterilize (autoclave or depyrogenate if suitable). Document lot IDs.

12.2 Inoculation & Drying

• Deposit known volume (10–20 µL) of organism suspension with/without soil onto each coupon to achieve ~10^5–10^6 CFU per coupon.
• Dry in biosafety cabinet to visible dryness (≤60 min) without desiccation stress beyond method standard.

12.3 Disinfection

• Apply disinfectant to completely cover coupon (spray volume or swab loading defined).
• Maintain contact time(s) (e.g., 1, 5, 10, 20 min) at target temperature. Prevent evaporation by using covered humidity chamber if volatile.
• For wipe/spray methods, define strokes, pressure, and fabric type.

12.4 Neutralization & Recovery

• At end of contact, immediately transfer coupon into neutralizer in a sterile tube.
• Vortex/agitate for defined time (e.g., 60 s) with sterile beads to maximize recovery.
• Perform serial dilutions and plate as in Section 11.2.
• Include carrier recovery controls (CRC) to determine baseline N0 on each surface.

12.5 Calculations & Acceptance Criteria

• Calculate LR per coupon relative to CRC.
• Acceptance as per Section 11.4, applied per surface type and per organism.
• For sporicides, include resistant spores; expect ≥3–4 log10 reduction (justify per claim/regulation).
• No survivors (“complete kill”) may be an additional internal criterion for Grade A/B areas; document LOD.

13. Test Matrix & Worst-Case Justification

Design a matrix covering:

• Disinfectants: all routine, rotational, and sporicidal agents (e.g., QAC, alcohol, hydrogen peroxide, PAA, hypochlorite).
• Concentrations: in-use and lowest allowable (±10%); diluted with worst-case water quality if applicable.
• Surfaces: all critical facility materials.
• Conditions: clean vs. soiled; temperature 18–25°C unless otherwise justified.
• Contact times: label/claim and program times (e.g., 1–10 min).
• Organisms: compendial and recent environmental isolates.

14. Data Recording & Calculations

• Record all raw data on controlled worksheets.
• Use validated spreadsheet or LIMS for CFU counts and LR calculations.
• Report means, SD, and 95% CI where applicable.
• If “no growth,” use half the detection limit for LR calculation and state LOD.

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