Pollution sources and identification of clean rooms in pharmaceutical enterprises

Industrial modular Clean Room time:2022/09/06 17:24:06 click:20

Pharmaceutical enterprises often need to carry out environmental microorganism monitoring in clean rooms to ensure the cleanliness of the production environment and will not cause drug pollution. The common pollution sources in clean rooms of pharmaceutical enterprises are as follows (according to the lecture PPT of the National Drug Microbiological Inspection and Control Technology Elite Training Class in 2018 by China Institute for Food and Drug Control) :

Pollution sources and identification of clean rooms in pharmaceutical enterprises

1, the people. The microorganisms carried mainly include: Staphylococcus aureus, Table staphylococcus, Octococcus, Acinetobacter, alkali-producing bacilli, lipophilic yeast, skin fungus, etc.

2. Air. Carrying microorganisms mainly include: Micrococcus genus, Bacillus genus, Staphylococcus genus, Corynebacterium, Aspergillus, Penicillium.

3, equipment, wall, ground surface. The remaining microorganisms mainly include: Pseudomonas, table Portuguese, Bacillus and so on.

4. Raw materials and packaging materials. Carrying microorganisms are: Lactobacillus, Pseudomonas, Bacillus, Streptococcus, Clostridium, mold (Cladosporium, Penicillium, Aspergillus).

5, and water. The microorganisms in the water mainly include: Pseudomonas, alkali-producing bacilli, oligotrophic mononas, Burkholderia cepacia, Burkholderia pisi, methyl bacilli, Serratia bacteria, flavobacterium and so on.

In order to identify bacterial species, many enterprises often use a large amount of funds for the purchase of automatic identification instruments and related reagents for microorganisms. In fact, some microbial biochemical identification kits (without software and equipment), identification media and PCR kits can complete the identification of common strains.

The species or genera highlighted in red above are known to be identified by biochemical identification kits produced by HiMedia. The biochemical identification kit contains biochemical identification cards and auxiliary reagents. Each biochemical identification card has 12 holes, and the relevant media and biochemical reagents are preset, corresponding to 12 biochemical reactions. After the reaction is complete, the strain identification can be completed manually through the result interpretation table and strain standard table provided in the kit. Its kit covers most common strains, has high accuracy, and is registered in the European Union and the United States FDA.

Among the above common bacterial groups, Staphylococcus aureus and Table Staphylococcus can be identified by the biochemical identification kit (article number KB004), and Acinetobacter, Bacillus, Lactobacillus and Streptococcus all have corresponding biochemical identification kits. Pseudomonas and Burkholderia cepacia could be identified by the non-fermentative bacteria biochemical identification kit (article No. KB019). Serratia belongs to Enterobacteriaceae and can be identified by the biochemical identification kit of Enterobacteriaceae (article number KB003).

For the species in yellow: Oligotrophomonas, also known as Stenotrophomonas maltophilia, formerly "Pseudomonas maltophilia". It, Burkholderia pylori and Flavobacterium can be identified by non-fermentative bacteria kit.

For the species in blue:

1. Micrococcus. Micrococcus luteus was the most common. It is Gram-positive, similar to staphylococcus, with smooth colonies and yellow or red in color. It is an opportunistic pathogen and widely exists in nature. "Berger's Bacterial Identification Manual" points out that it is positive for contact enzyme, has no motility, can use acetate, lactate and propionate, does not produce indole. The identification of Micrococci luteum by biochemical characteristics is mainly based on its growth in complex media containing sugar on aerobic oxygen without producing detectable acid. Its colonies are yellow, yellow-green or orange. For specific identification, the following methods can be used: 1) Gram stain first, and observe the morphology under the microscope, such as spherical, Gram-positive, and suspected Staphylococcus or micrococcus. 2) The Fermentation Medium for Staphylococcus and Micrococcus (item #MV827) can be used to distinguish between Micrococcus and Staphylococcus. The principle is that Staphylococci produce acid anaerobically when the medium contains glucose, while micrococci do not. The medium contains bromocresol violet as a pH indicator, which can indicate the pH change caused by acid production. The liquid medium contains a small amount of AGAR, thus creating an anaerobic environment. After 18-24 hours of incubation, the reaction tubes showed no color change in the case of Micrococcus luteum, whereas the tubes of Staphylococcus turned golden.

2. Octococcus: Belonging to the family Gastrococci, the cells are spherical, showing a cubic pile of eight or more cells. Spores may form and are gram-positive. Fermentable glucose, acid producing, obligate anaerobic, can grow at pH1 to 9.8. No contact enzymes. It shouldn't be hard to make out.

For alkali-producing bacilli, which are very similar to alkali-producing Pseudomonas, its distinguishing feature is the formation of ammonia in broth medium containing peptone, which increases the pH. Faecal alkali-producing bacilli were the most common and showed a strong alkali-producing reaction on carbohydrate medium. Xylose oxidizes alkali-producing bacilli to produce acid in OF glucose medium and OF xylose medium. This bacterium can reduce nitrite, but not nitrate. Therefore, OF Basal HiVegTM Medium (MV395) and corresponding sugar solution were used to identify alkalogenic bacilli.

For Corynebacterium, it belongs to Gram-positive Macrobacter, which is positive for contact enzymes. Tellurite blood AGAR plates could be used for isolation and culture, and corynebacterium colonies were grayish-black.

For methylobacter, 16S rDNA and mxaF gene of methanol-dehydrogenase large subunit could be amplified by PCR. The amplified products were sequenced, compared by BLAST in NCBI database, and analyzed by phylogenetic tree using MEGA4.0 software. Methyl bacilli were gram-negative and positive for oxidase and contactase.

For Clostridium, most are strictly anaerobic bacteria. A table is given in the Berger Handbook of Bacterial Identification, which identifies the individual species within the genus Clostridium mainly on the basis of sugar fermentation. HiMedia offers a 35 sugar fermentation kit (article KB009) that can be used to identify Clostridium SPP.

For fungi, such as lipophilic yeasts and molds (Cladosporium, Penicillium, Aspergillus), first from the morphology of bacteria can be distinguished, also can distinguish yeast and mold, mold often have filamentous hyphae. Different molds, some of which are morphologically distinguishable. According to the characteristic results of PenicilliUM, the genus and even the species level can be identified according to the colony characteristics, conidial stem, broom stem, bottle stem and conidial morphological characteristics. Aspergillus has septum mycelium, conidial pedunculum and special conidial head structure, which is easy to distinguish from other fungi. It can also be further identified according to the speed of growth on SDA or PDA plate, colony morphology, conidial pedunculum, etc. HiMedia also offers a discriminating medium, item number M1127, that can distinguish between Aspergillus brasiliensis, Aspergillus flavus, and Aspergillus parasitica.

In conclusion, for the common bacteria in the clean room of pharmaceutical enterprises, morphological staining, biochemical identification (biochemical identification kits available on the market can be used), PCR product sequencing and other means have been enough to meet the needs of identification, so as to complete the task of traceability and diagnosis.