3.3 Surface Microbial Sampling: Contact Plates, Swabs, Incubation Protocols & Action Levels

Purpose and Timing of Surface Microbial Sampling

While air sampling monitors the dilution and filtration efficiency of the ventilation system, surface sampling provides a direct measure of microbial bioburden transferred to cleanroom surfaces by human touch, supply introduction, and surface deposition. Surface sampling verifies the adequacy of personnel work practices, garbing integrity, and facility cleaning and disinfecting procedures.

Operational Timing: End of Compounding

USP <797> mandates that routine surface sampling must be conducted at the conclusion of compounding or at the end of an operating shift, prior to cleaning and disinfecting.

Sampling Devices and Techniques

1. Contact Plates (RODAC Plates)

• Application: Used for flat, accessible surfaces such as PEC work tables, staging countertops, pass-through chambers, equipment housing, and door handles.
• Design: Replicate Organism Detection and Counting (RODAC) plates have a convex meniscus of agar that rises slightly above the rim of the plastic dish.
• Technique: The plate is pressed gently against the target surface with uniform pressure for 3 to 5 seconds without twisting or sliding (which smudges colonies and damages agar).
• Mandatory Post-Sampling Cleaning: After sampling, the sampled area must be thoroughly cleaned and disinfected (USP <797> 6.3.2), followed by sterile 70% IPA if it is inside a PEC. Agar left on a surface is a nutrient bed for microbes.

2. Sterile Swabs

• Application: Used for irregular, grooved, creviced, or confined surfaces where a rigid contact plate cannot make complete physical contact (e.g., inside IV compounding automated pumps, electrical outlet bezels, seams, and glove box glove rings).
• Technique: A sterile swab moistened with sterile diluent (such as sterile peptone water or phosphate buffer with neutralizers) is rubbed firmly across the target area in multiple intersecting directions, then streaked onto an agar plate.

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Mandatory Surface Sampling Frequencies

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Surface Microbial Action Levels

Under USP <797>, action levels for surface sampling are expressed in colony-forming units (CFUs) per contact plate (standard surface area of approximately $24\text{ to }30\,\text{cm}^2$):

Incubation Parameters

Surface devices use the same incubation as air samples: 30°C–35°C for no less than 48 hours, then 20°C–25°C for no less than 5 additional days. The alternative is two devices incubated concurrently at the two temperatures. If two devices are collected at one location, the action level applies to each device separately; the counts are not added.

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Neutralizer Chemistry and Inactivation Kinetics

Cleanroom surfaces are subjected to routine chemical disinfection with quaternary ammonium compounds, phenolics, sodium hypochlorite, and isopropyl alcohol. Residual amounts of these antimicrobial chemicals adhere to cleanroom surfaces and are picked up onto the agar during sampling.

If surface sampling were conducted using standard Trypticase Soy Agar (TSA) without neutralizing agents, residual disinfectants would continue to exert bacteriostatic or fungistatic action inside the Petri dish during incubation. Viable microorganisms present on the surface would be prevented from replicating, producing a false-negative culture report that conceals environmental contamination.

To ensure accurate recovery, USP <797> requires surface sampling media to contain neutralizing additives, naming lecithin and polysorbate 80 as examples. Commercial media may add others:

• Lecithin: Neutralizes quaternary ammonium compounds (QACs) and biguanides (e.g., chlorhexidine).
• Polysorbate 80 (Tween 80): Inactivates phenolic disinfectants, hexachlorophene, and formalin.
• Sodium Thiosulfate: Neutralizes halogenated disinfectants including sodium hypochlorite (bleach), iodine, and chlorine dioxide.

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Personnel Gloved Fingertip and Thumb Sampling (GFT)

Personnel glove testing measures the touch contamination risk introduced directly by compounding operators. A contact plate is held, and the operator presses all five digits (thumb and four fingers) lightly onto the agar surface for 2 to 3 seconds.

USP <797> uses gloved fingertip testing in three settings, with two different action levels:

1. Initial Garbing Competency

• Conducted immediately after garbing, inside a classified area or SCA, before sterile 70% IPA is applied to the gloves.
• The operator must pass three separate, successive evaluations, each after a complete hand hygiene and garbing procedure.
• Action level: > 0 CFU total for both hands. Any growth is a failure.

2. Ongoing Garbing Competency

• Repeated at least every 6 months (Category 1 and 2 compounders) or every 3 months (Category 3), and every 12 months for supervisors who do not compound.
• It is the same test: visual observation plus GFT after garbing, before IPA. The action level stays > 0 CFU.

3. GFT After a Media-Fill Test

• Part of the aseptic manipulation competency, taken inside the ISO Class 5 PEC immediately after the media fill.
• Action level: > 3 CFU total for both hands combined. A media-fill, GFT or direct-compounding-area surface sample failure fails the whole competency.
• USP does not require microbial identification of GFT colonies, although identification can help an investigation.