The Use of a Single Growth Medium for Environmental Monitoring Of

The use of a single growth medium for environmental monitoring of pharmacy aseptic units using tryptone soya agar with 1% glucose John Rhodes1*, Jennifer Feasby1, Wayne Goddard1, Alison Beaney2 and Mike Baker3 1 North Tees and Hartlepool NHS Foundation Trust, Stockton-on-Tees, UK 2 Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, UK 3 Pharma Quality Consulting, Cheshire, UK

The suitability of tryptone soya agar, Sabouraud dextrose agar and tryptone soya agar with 1% glucose plates for general environmental monitoring was compared. Plates were incubated at three different temperatures to assess an optimal temperature for growth. Results indicated that there are benefits from using tryptone soya agar with 1% glucose incubated at 25°C as an all-purpose medium for environmental monitoring.

Key words: Tryptone soya agar, Sabouraud dextrose agar, tryptone soya agar with 1% glucose, environmental monitoring of aseptic rooms, settle plates, finger dabs.

Introduction any test methods or suitable media. For cleanroom monitoring of UK National Health Service (NHS) aseptic services, more The choice of a microbiological growth medium is not simple. detailed guidance has been provided4 for environmental settle The Difco Manual has proven to be a comprehensive source of plate agars. It indicates that the media recommended is: information since the first edition appeared in 1927. The tenth edition was published in 1984 and found its way into most 1 “standardised on tryptone soya for bacterial count (this microbiology laboratories . The guide for the selection of will also detect yeasts and moulds to an extent) and culture media formed a 9-page table and contained many agars Sabouraud dextrose medium for the selective and broths for isolation, differentiation and propagation of determination of yeasts and moulds.” 4 different classes of micro-organisms. The most ubiquitous propagation agars were tryptone soya agar (TSA) for aerobic This advice was based on the experience of the authors over bacteria and Sabouraud dextrose agar (SDA)/Sabouraud several years in NHS pharmaceutical quality control, maltose agar (SMA) for yeasts and moulds. however, the extent that fungi grow on TSA had not been The media supplied by Difco and similar companies were published. formulated for specific applications, namely clinical, Sabouraud agar medium was developed by a French serology, water, foods, beverages and pharmaceutical dermatologist Raymond JA Sabouraud in the late 1890s to microbiology. Pharmaceutical microbiology was based on 5,6 2 support the growth of fungi . J Hare provides a history and formulations provided by Pharmacopoeias for testing the theory of Sabouraud agar7: products. Little was published for environmental microbiology. “The available means of inhibiting bacterial growth in European Union Guidelines to Good Manufacturing Sabouraud’s pre antibiotic era was an acidic medium Practice Annex 1 describes the manufacture of sterile (pH 5.6)…Glucose is present at the high level of 4% in medicinal products and contains recommended limits for 3 Sabouraud’s formulation to assist in vigorous environmental microbial contamination . It provides a fermentation and acid production of any bacteria maximum number of colonies for settle plates, contact plates, present, inhibiting future bacterial growth. In 1977, airborne viable counts and finger dabs, but does not reference Emmens formulated an alternative version of Sabouraud’s agar which contains half the amount of glucose (2%) and a neutral pH of 6.8–7.0. The neutral *Corresponding author: John Rhodes, Stockton Quality Control pH seems to enhance the growth of some pathogenic Laboratory, University Hospital of North Tees, Hardwick, Stockton-on-Tees, 7,8 UK, TS19 8PE. Email: [email protected]. Tel: +44(0)1642 614194 fungi.”

50 USE OF TSA + 1% GLUCOSE FOR THE ENVIRONMENTAL MONITORING OF PHARMACY ASEPTIC UNITS 51

The ideal medium for environmental monitoring is broad Table 1. Agar plates prepared by Oxoid UK and Cherwell spectrum and encourages the growth of sub-lethally Laboratories UK used in the tests. damaged organisms and spores. In the 1930s, Bowers and 9 Medium Oxoid catalogue Cherwell catalogue Hucker developed tryptone glucose skim milk agar, a number number medium for detecting bacteria in milk and other dairy TSA+G PO0330B 104466 products. TSA PO0241B 104131 In 1932, Norton recommended the incorporation of 0.5 to 1% dextrose10 and the Becton Dickinson updated Difco SDA PO0410B 104066 manual11 indicates the following for dextrose agar:

“Dextrose is an energy source used by many of nine plates, three of each type of agar on different organisms. The high concentration of this surfaces in the laboratory in order to collect environmental ingredient makes dextrose agar a suitable medium organisms. The plates were incubated at 22.5°C, 25°C and for the production of early, abundant organism 32.5°C in such a way that each type of media was growth and shortening the lag periods of older incubated at each temperature per location. The plates cultures.” were read over 5 days. Oxoid plates were exposed in a laboratory with no air conditioning. Cherwell plates were Tryptone glucose extract medium, used as a standard plate exposed in a laboratory supplied with air filtered through count agar for dairy products and water, and plate count an EU8 filter. agar, used for obtaining microbial plate counts from milk and dairy products, foods, water and other materials of sanitary importance, were developed from the Bowers and Results Hucker formulation11. These media were formulated using The plates for the ten locations were read and the number 1% glucose and a pH of 7.0 ± 0.2 in contrast with SDA of bacteria and fungi recorded. The total number of formulated with 4% dextrose and pH 5.6 ± 0.22. organisms identified as bacteria for the ten locations were The optimal pH range for growth for the majority of presented as a bar chart for each manufacturer of plates in bacteria is between 6.5 and 7.5 which is in line with Figures 2 and 3. The total number of organisms identified Emmons philosophy8. A plate incorporating the general as fungi (moulds and yeasts) are presented as a separate purpose formulation of TSA and 1% glucose (TSA+G) bar chart for each manufacturer of plates in Figures 4 and has been formulated at pH 7.0 ± 0.2 for general use in 5. Oxoid plates were read daily over the 5 days of cleanrooms as indicated in Figure 1. Stockton Quality incubation. Control Laboratory has been using TSA+G since 1973 for plates used for routine environmental monitoring. The aim of the study was to validate the use of TSA+G Discussion by comparing the effect of typical environmental The tests were carried out using pre-poured and irradiated organism growth on TSA, SDA and TSA+G settle plates plates prepared by Oxoid and Cherwell Laboratories. from two different suppliers when incubated at three Oxoid plates were being purchased at the time of the different temperatures, 22.5°C, 25°C and 32.5°C. initial validation. The repeat tests were carried out as part of a revalidation study when the laboratory was located in an air conditioned building and at that time plates were Pancreatic digest of casein 15 g purchased from Cherwell Laboratories. Both sets of plates Enzymatic digest of soya bean 5 g were exposed in a general laboratory environment under turbulent air. Staff wore laboratory coats over personal Sodium Chloride 5 g clothing and, therefore, a challenge by human Agar 15 g commensals was expected. The laboratory did not control the cleanliness of items brought into the test areas and, Glucose 10 g therefore, a range of environmental organism challenge is Water 1000 mL not unusual. It is known that the chance of particles carrying organisms landing on a plate is poor and pH at 25°C 7.0 ± 0.2 12 determined by particle size, air flows and eddies . Figure 1. Formulation of TSA+G plates. Therefore, the total number of organisms for the ten locations were added together to reduce this variation. The total number of bacteria recovered on the Oxoid and Cherwell plates were similar as shown when comparing Materials and methods Figures 2 and 3, however, the total number of fungi Agar plates prepared by Oxoid UK and Cherwell obtained for the Cherwell plates were significantly lower Laboratories UK were used in the tests and detailed as in as shown when comparing Figures 3 and 4. It is believed Table 1. that the difference arises because the Cherwell plates were Plates were exposed for between 1 and 4 hours to exposed in an air conditioned laboratory and not a simulate different challenges. They were placed in groups variation in quality of the product. 52 JOHN RHODES, JENNIFER FEASBY, WAYNE GODDARD, ALISON BEANEY, MIKE BAKER

Figure 2. Total number of bacteria recovered from the three formulations of Oxoid agars incubated at three temperatures after 5 days.

It was not possible to control the challenge size as the 219 bacteria which corresponded to 74% of the total count number of organisms landing on the plate depended on the for the study. The corresponding plates incubated at activity in the room and the airflow around the different 22.5°C and 32.5°C gave counts of 96 and 100, group sites. Plates were grouped closely together to respectively. A Grubbs outlier test13 indicated that the 219 minimise variations due to location and exposed for up to result was an outlier (p < 0.01). This suggests that this 4 hours to average out the number of particles landing on a result introduced a bias into the statistics. When all the plate in the group. Figure 2 implies that TSA+G plate results for this location were removed from the plots, incubated at 25°C recovered twice as many organisms as Figure 6 replaces Figure 2 and Figure 7 replaces Figure TSA plates or the TSA+G incubated at a higher or lower 4. The removal of entrance area plates did not change the temperature. However, when the individual plate counts shape of the fungi plot but did alter the TSA+G profile in were examined, one Oxoid plate exposed on the floor in line with the TSA and SDA profiles. the laboratory entrance area for 3 hours gave a count of Clearly, the SDA plates from Oxoid or Cherwell did

Figure 3. Total number of bacteria recovered from the three formulations of Cherwell agars incubated at three temperatures after 5 days. USE OF TSA + 1% GLUCOSE FOR THE ENVIRONMENTAL MONITORING OF PHARMACY ASEPTIC UNITS 53

Figure 4. Total number of fungi recovered from the three formulations of Oxoid agar incubated at three temperatures after 5 days.

Figure 5. Total number of fungi recovered from the three formulations of Cherwell agar incubated at three temperatures after 5 days.

Figure 6. Total number of bacteria recovered from the three formulations of Oxoid agar incubated at three temperatures after removal of the entrance set of results after 5 days. 54 JOHN RHODES, JENNIFER FEASBY, WAYNE GODDARD, ALISON BEANEY, MIKE BAKER

Figure 7. Total number of fungi recovered from the three formulations of Oxoid agar incubated at three temperatures after removal of the entrance set of plates after 5 days.

not support the growth of bacteria (Figures 6 and 3) to any TSA plates especially when growth is observed in SDA great extent which was not unexpected as the pH of the plates. 8 formulation is low . The bacteria that settled on all the The comparative data for each of the three media Oxoid plates grew better at the higher temperature (see indicates that either 22.5°C or 25°C would be an Figure 6), however, the bacteria that settled on the appropriate incubation temperature for fungi, but not Cherwell plates appeared to grow equally well at any of 32.5°C. TSA+G gave similar total fungal counts to SDA the incubation temperatures. and a greater recovery than TSA for both Oxoid and Incubating at 32.5°C reduced the recovery of fungi for Cherwell plates. Either 25°C or 32.5°C would be an all three media from both suppliers as shown in Figures 5 appropriate incubation temperature for bacteria on TSA or and 7. It is well established that lower temperatures 14 TSA+G plates. The TSA gave slightly better recovery than encourage the growth of filamentous fungi . However, TSA+G for both bacteria and fungi but this was not dermatophytes or dimorphic fungi prefer the higher 7 significant (p > 0.1) temperature but take 2 to 4 weeks for growth . As test Dual temperature incubation has not been considered14 results are required to establish whether a cleanroom in this study, however, 25°C does appear to be a good remains microbiologically in control, timely results are compromise for single temperature incubation of TSA+G. essential in the event of a high count exceeding an alert or It gave the best recovery of bacteria on Cherwell plates action level to ensure an investigation is undertaken and and the best recovery of fungi on Oxoid plates. Slightly appropriate action is taken in order to minimise the risk of better results were obtained for fungi on Cherwell plates at preparing a microbially contaminated product. Extending 22.5°C and for the recovery of bacteria on Oxoid plates at the incubation time to permit the growth of slow-growing 32.5°C. The authors believe that there would be little organisms will delay the commencement of an benefit from changing incubation temperatures. investigation and potentially allow faster growing The use of a single media to monitor clean air devices organisms to overgrow a plate. The British Pharmacopoeia total viable count test for has the advantage of taking up less space in critical areas, non-sterile preparations2 has separate limits for bacterial such as isolators or laminar flow cabinets. Finger dabs can counts and fungal counts. It utilises TSA incubated at only be carried out once at the end of a work session and a 32.5°C for 3 days and SDA incubated at 22.5°C for 5 days single general purpose agar will allow the culture of a for mould and yeast count. In order to allow fungi to be broad range of organisms. It is a requirement that environmental monitoring results are trended on a counted independently from bacteria, SDA inhibits the 15 growth of bacteria. When a total viable count is not monthly basis . Results from a single plate at a given performed by filtration, molten agar is poured over the position will be easier to record and manipulate than from sample and growth appears as small discrete colonies two plates. There will be no possibility of mix up if the embedded in the agar. The agar is incubated at 22.5°C to wrong media is exposed, especially if SDA plates are encourage mould growth and the incubation time is 5 days exposed less frequently. If different temperatures are used as growth is slow. Bacteria are grown on TSA as it is not for the TSA and SDA plates, there is a possibility of inhibitive of bacterial growth. Bacteria tends to grow incubating agar at the wrong temperature if they are sufficiently well for the visual counting of colonies after 3 wrapped together, mislabelled in error or simply placed in days at 32.5°C. However, experience has shown that the wrong incubator, organism numbers could then be occasionally yeast or mould contamination is observed in underreported. USE OF TSA + 1% GLUCOSE FOR THE ENVIRONMENTAL MONITORING OF PHARMACY ASEPTIC UNITS 55

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