ISO, Federal, FDA, USP and scientific guidance for microbiological and manufacturing personnel

The identification of live organisms in a controlled environment is a regulatory requirement as set forth in several FDA documents.^{1, 2, 3} The CFR speaks generically of “environmental conditions” that may have an “adverse effect on product quality.” Such conditions include bacteria and fungi as are found in the air or on surfaces. It is the purpose of this paper to present current information as to FDA, ISO, and USP guidance and to provide a basic primer on microbial identification.

QUALITATIVE ANALYSIS OF THE AIR
The settling plate is the standard and simplest technique to obtain a microbial count of the air; an open Petri dish is exposed to air for a stated period of time at designated areas within the controlled environment.

This technique measures microbial content as well as room traffic (difference in settling rate between rooms at rest and air in motion). Furthermore, the entirely passive nature of this simple procedure permits continuous microbial enumeration at points of filling. The exposed Petri dishes are placed at chosen locations throughout the room for specified periods of time, covered and removed to incubation under specified conditions (e.g. 48 to 72 hours or longer on a case-by-case basis at 30 to 35°C with Trypticase Soy Glucose agar for bacteria and perhaps an additional 5 days at 20 to 25°C with the same agar or Sabouraud Dextrose Agar for fungi). If one is trying to correlate environment bioburden with the USP sterility test, additional incubation might be appropriate. The number and types of colonies are recorded and conclusions as to air quality and trends made part of the manufacturing record. A single incubation at either of the above temperatures is called monophasic incubation. An alternate specification calls for the initial incubation at 30 to 35°C for bacteria followed by re-incubation of the same culture at 20 to 25°C for fungi. This is called biphasic incubation. An illustration of a monophasicbiphasic incubation model is shown in Table 1. There can be variations, as to specific SOPs.

QUANTITATIVE ANALYSIS OF THE AIR
For the various centrifugal or impingement air sampling devices, the unit of measurement is the colony-forming unit (CFU) per unit volume. Some typical instruments among others are 1) an Anderson slit sampler, which impinges colonies on to a series of standard Petri dishes; 2) a centrifugal air sampler, which employs bacterial or mold strips; 3) an SAS air sampler which uses contact plates and samples 10 to 1,000 liters. Incubation for growth is accomplished as per Table 1. Typical colonies of bacteria and fungi are shown in Figures 1 (bacteria) and 2 (filamentous fungi).