Effect of Incubation Strategy on Recovery of Bacteria and Molds in EM Testing
EM（環境監測）檢測中培養策略對細菌和黴菌回收的影響
One of the most asked questions in microbial EM testing relates to incubation strategy and media choice.
微生物EM（環境監測）檢測中最常遇到的問題之一與培養策略和培養基的選擇有關。
Whether to go single temperature or serial and if serial to go low to high or vice versa with use of one media, TSA, or two, TSA and SDA. Factors that can influence the choice are time to results (single temperature is usually faster than serial), cost (TSA alone is cheaper than TSA and SDA) and quality of microorganism recovery. There are few studies reporting recovery related to incubation choice however in those published a single 30-35°C incubation has been reported to give poorer mold recovery than serial. The data below summarizes a small study performed in our facility.
使用一種培養基（TSA）或者兩種培養基（TSA和SDA），使用單個溫度、兩個溫度，是否從低溫到高溫，還是高溫到低溫。影響微生物回收率的因素有：結果觀察時間(單一溫度通常比兩個溫度早)、成本(隻用TSA比使用TSA+SDA便宜)以及微生物回收率的高低。很少有研究報告回收率相關的培養條件選擇，但是，已有報告單一溫度條件30-35℃培養對於黴菌回收的不如兩個溫度培養的好。下麵的數據總結了在我們實驗室進行的一項小研究。
Three strategies were evaluated using standard TSA LP80 media, single temperature at 30-35°C, and 25-30°C for 5 days and a serial protocol with 72 hours at 20-25°C followed by 48 hours at 30-35°C. A smaller evaluation was performed to review the recovery of molds with SDA media at 20-25°C for 5 days as the control.
采用標準TSA LP80培養基、分別使用30-35℃@5天、25-30℃@5天、20-25℃@72小時+30-35℃@48小時3種方案進行評價。以SDA培養基20-25℃@5天條件為對照，對黴菌的回收情況進行評價。
The samples were taken from close to the manufacturing floor entrances, from un-controlled areas to ensure high enough cell counts to get suitable data sizes. At each site 3 surface contact plates and 1 active air sample (1m3 SAS 180) were taken making 12 points for each day. At each point 3 replicate samples were taken, 1 cassette for each incubation temperature tested. For experiment 2 an SDA sample was also taken at the same time. Samples were taken in Dec-Jan and then 9 months later in Aug-Sept to cover seasonal variation. All samples were incubated on the Growth Direct™. Following incubation, the data series for each cassette was recovered to determine the cfu counts. For experiment 2, three analysts also counted the number of mold colonies present on each cassette and the mean value was calculated for each sample.
這些樣本是從生產車間入口附近的非受控區域采集的，以確保足夠高的計數以獲得合適的數據大小。在每個地點取3個表麵接觸皿和1個浮遊菌(1立方SAS180)，每天取12個點。每個點取3組重複樣本，每個培養溫度測試1組樣品。實驗2中還收集了SDA樣本。樣本分別在12月至1月，以及9個月後的8月至9月采集，以覆蓋季節變化。培養後對每組樣品進行計數，以確定菌落計數。在實驗2中，三位分析員還計算了每個組的黴菌菌落數，並計算了每個樣本的平均值。
Figure 1. Total number of Bacterial colonies detected at each incubation strategy over the 3 days of study 1 in Dec-Jan
圖1。在試驗1的3天內，在每種培養策略下檢測到的細菌菌落總數
Figure 2. Total number of Bacterial colonies detected at each incubation strategy over the 3 days of study 2 in Aug-Sept
圖2。在試驗2的8月至9月的3天內，在每種培養策略下檢測到的細菌菌落總數
Overall the single temperature at 25-30°C appeared to have the least variation by day and may have recovered more colonies than the other 2 methods. There is a fairly high variation in this type of testing, so a larger panel would need to be studied to get a firmer data set.
總的來說，25-30℃的單一溫度日變化最小，可能比其他2種方法回收了更多的菌落。這種類型的測試存在相當大的差異，因此需要研究更大的麵以獲得更可靠的數據集。
Mold detection
黴菌檢測
The frequency of mold colonies occurring at each test point was recorded over the course of the study. Results are shown in Figure 3. As can be seen The SDA recovered the most colonies as expected. Serial incubation recovered the next highest number then single temperature at 25-30°C and finally 30-35°C. The recovery at 30-35°C was less than 1% of that on SDA.
在整個試驗過程中，每個試驗點黴菌菌落的發生頻率都被記錄下來。結果如圖3所示。正如我們所看到的那樣，SDA（22.5℃）回收了大部分的菌落，組合溫度（25-30℃+30-35℃）次之，然後是25-30℃，最後是30-35℃。在30-35℃條件下，回收率小於SDA（22.5℃）的1%。
Figure 3. Total number of mold colonies detected at each incubation strategy over the 3 days of study
圖3.在3天的試驗中，每種培養策略檢測到的黴菌菌落總數
Conclusion
總結
If cost is the driving factor then single media, TSA, with a serial incubation (low to high) is the best option. If speed is the main requirement then the single temperature at 25-30°C is best. If overall recovery is the requirement then 2 media, TSA and SDA is superior.
如果成本是驅動因素，那麽單一培養基，組合溫度培養(從低到高)的TSA是最佳選擇。如果速度是主要的要求，那麽25-30℃的單一溫度是最好的。如果需要全部回收，那麽優選2種培養基，TSA和SDA。
