Aqueous Solutions (Water-Based Liquids)
This is the baseline procedure. Most other product types will eventually refer back to these steps once they are dissolved or diluted.
Pre-wetting: Before adding the sample, pour a small amount of sterile diluent (like Fluid A) onto the membrane and filter it. This wets the filter so the sample passes through evenly.
Note: If testing antibiotics, this diluent may contain neutralizing agents to stop the antibiotic from sticking to and destroying the filter.
Filtering the Sample: Pour the required amount of your product (based on the SOP's Tables 1 & 2) through the filter immediately.
Washing (Rinsing): If the product has antimicrobial properties (it naturally kills bacteria/fungi), you must wash the filter membrane to rinse away any leftover product residue that might stop microbes from growing during incubation.
The Rule: Wash at least 3 times using the volume validated in your Method Suitability Test.
The Maximum Limit: Do not exceed 5 washes of 100 mL ($500\text{ mL}$ total) per filter. Even if the antimicrobial activity isn't fully gone, stop at 5 washes to avoid damaging the trapped microbes or tearing the membrane.
🧪 4.8.2 Soluble Solids & 4.8.4 Non-Antibiotic Injectable Solids
The Rule: You cannot filter a solid directly, so you must turn it into an aqueous solution first.
How: Dissolve the solid in a suitable sterile solvent. This can be the solvent provided with the product, Sterile Water for Injection, sterile saline, or Fluid A.
Next Step: Once dissolved, treat it exactly like an Aqueous Solution (Section 4.8.1).
🛢️ 4.8.3 Oils and Oily Solutions
Oils are thick and water-repellent, meaning they require special handling so they don't clog the filter or trap water-loving bacteria inside oil droplets.
Low Viscosity (Thin Oils): Can be filtered directly through a dry membrane without dilution.
High Viscosity (Thick Oils): Must be diluted with a sterile solvent like Isopropyl Myristate (IPM) to thin it out. The IPM must be proven not to kill microbes.
Filtration: Let the oil settle onto the membrane under its own weight first, then gradually apply vacuum suction or pressure.
Washing: Wash at least 3 times with roughly 100 mL of a diluent containing an emulsifying agent (like Fluid K, which contains Polysorbate 80 / Tween 80). The emulsifier breaks up the oil layer so it can be washed off the filter, leaving only the microbes behind.
💊 Antibiotic Preparations (4.8.5 & 4.8.6)
Antibiotics require specific pooling (combining) and sampling rules to ensure a representative sample is tested without overloading the system with antimicrobial powder. They are broken down by packaging size:
1. Pharmacy Bulk Packages $< 5\text{ g}$
Sampling: Take 20 containers.
Preparation: Either take $300\text{ mg}$ of powder from each and dissolve them together in $200\text{ mL}$ of Fluid A inside a sterile $500\text{ mL}$ flask, OR reconstitute all 20 containers as directed on their labels and pull a liquid volume equivalent to $300\text{ mg}$ from each into the $200\text{ mL}$ Fluid A.
Next Step: Filter as an Aqueous Solution.
2. Pharmacy Bulk Packages $> 5\text{ g}$
Sampling: Take 6 containers.
Preparation: Aseptically transfer $1\text{ g}$ of solids (or the liquid equivalent if reconstituted) from each of the 6 containers into $200\text{ mL}$ of Fluid A in a sterile $500\text{ mL}$ flask. Mix well.
Next Step: Filter as an Aqueous Solution.
3. Antibiotic Solids, Bulks, and Blends
Sampling: Take the number of containers required by Table 2.
Preparation: Mix the solids from the containers to create a uniform "composite" mixture. Take an amount of this mix equal to $6\text{ g}$ of solids, and dissolve it in $200\text{ mL}$ of Fluid A in a sterile $500\text{ mL}$ flask.
Next Step: Filter as an Aqueous Solution.
📊 4.9 Sampling Plan of Articles
Quantity of Containers: The number of vials or syringes you need to grab from a batch to test is determined by Table 1 of the master protocol.
Splitting Samples: If a single container has enough volume, you can split its contents equally between the two required culture media (typically Tryptic Soy Broth for fungi/aerobic bacteria and Fluid Thioglycollate Medium for anaerobic bacteria).
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