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標題: | 以液體方法定量空氣中真菌之效能評估 Evaluation of liquid-based sampling methods to quantify airborne fungi |
作者: | Yun-Tzu Ting 丁韻滋 |
指導教授: | 張靜文(Ching-Wen Chang) |
關鍵字: | 真菌,生物氣膠,收集液,採集效率,儲存效應, fungi,bioaerosol,collection fluid,sampling efficiency,storage effect, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 真菌廣泛存在於各式環境中,人體暴露於空氣中的真菌可引發過敏、呼吸道疾病與其他健康不良效應。為能評估人員的暴露風險,應使用有效的採樣方法搭配適當的樣本儲存方式,以正確定量空氣中真菌。以液體捕集真菌氣膠的採樣方法具有採樣時間較為彈性以及後續分析方法多元等優勢,然現今對於該方法效能評估之研究有限,且尚未被廣泛應用。
為找出針對真菌氣膠以液體為介質之適當定量方法,本研究使用BioSampler與Coriolis μ sampler兩種採樣器搭配8種不同配方之收集液,於室內環境中進行真菌氣膠採樣,同時使用BioStage與前兩種採樣器同步進行採樣,藉以校正不同環境與批次之環境真菌濃度差異,做為基準比較採樣器搭配收集液不同組合間之採樣效能。樣本均推於含氯黴素之麥芽抽出物培養基 (Malt Extract Agar with Chloramphenicol) 上經25℃培養7日後定量真菌濃度 (CFU/m3)。另為評估儲存溫度以及儲存時間對於真菌濃度之影響,採集之環境樣本另亦儲存於4℃與室溫下,並分別於2、4、6日後分析可培養真菌濃度之變化。研究使用之8種收集液其主要基質為磷酸鹽緩衝液 (phosphate buffer saline, PBS),並搭配有無添加非離子型介面活性劑 (Tween 20, Tween 80 或 Triton X-100) 以及消泡劑 (antifoam) 而組成。 本研究共進行10次環境採樣,收集160件環境樣本與160件現場空白樣本;另共分析1120件培養樣本,用於評估採集效率與儲存條件之影響。另外計算RBioStage值 (RBioStage = 液體採樣方法所得濃度/ BioStage採樣所得之濃度),再將RBioStage值取log轉換做為評估採樣效率之指標,若是某一個液體採樣組合之log RBioStage值越大 (即RBioStage值越大),表示採樣效率越高。結果顯示,BioSampler與Coriolis μ sampler相比具有較高之採集效率,其中又以使用Triton X-100之PBS收集液的採集效率最高 (平均log RBioStage = -0.19±0.29)。整體顯著影響採樣效率的因子包含採樣器種類 (P<0.0001)、收集液種類 (P<0.0001)、採樣地點風速 (P=0.0007)、採樣地點濕度 (P=0.02)。在儲存條件方面,將樣本經過不同條件儲存後之濃度 (CFU/mL) 以未儲存之真菌濃度 (CFU/mL) 為基準計算Rstorage值 (Rstorage = 儲存後之樣本濃度 / 未儲存之樣本濃度),再將Rstorage值取log轉換做為評估指標。log RStorage值離0越近 (即RStorage值離1越近),表示樣本經過儲存後濃度越接近採樣後未經儲存之濃度值,代表其較能反映環境中真菌之實態。結果顯示儲存時間增加,樣本濃度與未經儲存時之濃度差異增加,而儲存於4℃時之樣本濃度變異程度較低且較不受時間因素影響 (儲存2-6天平均log RStorage值 = -0.02±0.39),比室溫環境 (儲存2-6天平均log RStorage值 = 0.13±0.67) 適合存放樣本。顯著影響樣本儲存後濃度改變程度之因子包含樣本儲存時間與溫度 (P < 0.0001)、採樣器種類 (P < 0.0001)、收集液種類 (P < 0.0001)、地點 (P < 0.0001)。結合採樣效率與儲存效應進行液體定量真菌方法之評估結果,建議使用BioSampler搭配Triton X-100加PBS之收集液對於空氣中真菌進行採樣,採集回來的樣本若需儲存,則建議置於4℃之冷藏環境並於兩日內分析完畢。 Adverse health effects and degradation of indoor air quality induced by fungi are growing concern. Among various sampling methods for fungi, liquid-based impinger provides multiple choices in sample analysis. However, this method has not been comprehensively assessed for its sampling efficiency (SE) and storage effect after collection. Therefore, this study aims to evaluate various liquid-based sampling methods to quantify airborne fungi and determine the significant factors affecting SE. In addition, effects of storage time and temperature on fungi concentration were also assessed. Airborne fungi were sampled by two widely-used liquid samplers (BioSampler and Coriolis μ sampler) respectively loaded with eight types of collection fluid (contained phosphate buffer saline (PBS), surfactant and antifoam) in three types of location, including storage room, library and animal house. Besides, BioStage was used as reference to compare fungi concentration that collected by each collection fluid (N=160), in order to estimate SE. The present study use log RBioStage as the indicator of SE, which presents a log-transformed ratio of cell concentration in the air collected by liquid methods to that collected by BioStage. The higher the log RBioStage is, the higher the SE is. Effects of storage time (from 0 to 6 days) and temperature (4℃ and room temperature) were estimated by the ratio of fungi concentration in fluids stored for t and 0 day (N=1120). This study use log RStorage as indicator of storage effects, which presents a log-transformed ratio of cell concentration in the analytical sample (CFU/mL) right after sampling to that at various storage times. Sample concentration after storage is close to original concentration when the log RStorage close to 0, which means the results are representive. Generalized linear model (GLM) was used to estimate effects of different factors on SE and storage effect. The GLM results showed that SE was significantly influenced by sampler (P<0.0001), collection fluid type (P<0.0001), wind speed (P=0.0007) and relative humidity (P=0.02). Additionally, sampling by BioSampler with collection fluid which consisted of PBS and Triton X-100 showed the highest efficiency (average log RBioStage = -0.19±0.29). Moreover, sample concentration after storage was significantly affected by sampler (P < 0.0001), collection fluid type (P < 0.0001), sampling location (P < 0.0001), storage time and temperature (P < 0.0001). Fungi concentration tended to be overestimated as storage time increasing when stored at room temperature (average log RStorage = -0.02±0.39, stored for 2-6 days) but remain relatively stable at 4℃ (average log RStorage =0.13±0.67, stored for 2-6 days). Summarizing the results of sampling efficiency and storage effects, sampling by BioSampler with collection fluid consisted of PBS and Triton X-100 followed by sample storage at 4℃ is recommended. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49375 |
DOI: | 10.6342/NTU201602896 |
全文授權: | 有償授權 |
顯示於系所單位: | 環境衛生研究所 |
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