Microbiology, Sterile Products and Infection Control
Microbiology
Classification and identification
Bacteria are classified by Gram stain (peptidoglycan thickness), shape, oxygen requirement and biochemical profile. Mycobacteria are acid-fast due to mycolic acids and do not Gram-stain reliably; Pseudomonas aeruginosa is a non-fermenting Gram-negative rod; MRSA is methicillin-resistant Staphylococcus aureus via altered PBP2a. Fungi are eukaryotes (ergosterol-containing membranes), viruses obligate intracellular particles, and prions infectious proteins resistant to standard sterilisation.
Antimicrobial mechanisms and resistance
Targets: cell wall (beta-lactams, glycopeptides), protein synthesis (aminoglycosides, macrolides, tetracyclines, oxazolidinones), nucleic acid (fluoroquinolones, rifampin), folate synthesis (trimethoprim-sulfamethoxazole), and membrane integrity (polymyxins, daptomycin). Resistance arises from enzymatic inactivation (beta-lactamases, ESBLs, carbapenemases), efflux pumps, target modification, and reduced permeability.
Sterilisation and kinetics
Moist-heat autoclaving at 121 °C, 15 psi for 15 minutes kills spores; dry heat and ethylene oxide serve heat-sensitive items; filtration removes but does not kill. The D-value is the time to reduce organisms tenfold, and sterility assurance level 10⁻⁶ means fewer than one survivor in a million units.
Worked example
A bioburden of 10⁴ organisms with D-value 2 minutes at a given condition needs 4 × 2 = 8 minutes for a 4-log reduction, plus the additional 6 logs needed to reach SAL 10⁻⁶: total 10 × 2 = 20 minutes. Trap: forgetting to add the SAL margin.
Sterile Products and Infection Control
Cleanroom logic
Compounded sterile preparations are classified by risk (low, medium, high) and by the environment: an ISO Class 5 critical zone (laminar airflow hood or biological safety cabinet) inside an ISO Class 7 buffer room, with ISO Class 8 for lower-risk ante-areas in older frameworks. First air is the filtered air leaving the HEPA filter before it contacts anything - nothing may be placed between the filter and the critical site, and airflow must not be interrupted. Horizontal hoods protect the product; vertical BSCs protect the operator and are required for hazardous drugs.
Aseptic technique and BUDs
Concepts to master: disinfection of stoppers with 70% isopropyl alcohol and friction, no-touch technique, syringe and needle handling, and beyond-use dates that shorten with risk level and storage temperature. Higher risk, room-temperature storage and preservative-free products give shorter BUDs; refrigerated or frozen storage extends them. Follow your institution's policy and the current USP <797>/<800> framework rather than memorised numbers.
Traps
- Confusing disinfection (chemical, reduces but does not eliminate) with sterilisation (kills all organisms including spores).
- Forgetting hand hygiene moments and correct PPE doffing order - gloves last on, first off.
- Overlooking that multidose vials are a contamination risk and single-dose vials should not be reused.
- Ignoring hazardous-drug handling requirements (closed-system transfer devices, negative-pressure rooms).
Rule of thumb
If any component is exposed to air, contaminated by touch, or prepared from non-sterile starting material, risk escalates and the BUD shortens. When in doubt, choose the shorter BUD and the stricter environment.