轉載自Cleanroom Technology（2025.01.02發布）。
Incubating settle plates: Understanding growth patterns and when things are going wrong
培養沉降皿：了解生長模式以及何時出現問題

Published: 2-Jan-2025

Steven Brimble from Cherwell Laboratories discusses why it is important to your environmental monitoring regime to understand growth characteristics of common cleanroom contaminants and how morphology shifts over time on various media.
Cherwell Laboratories 的 Steven Brimble 討論了為什么了解常見潔凈室污染物的生長特性以及形態在各種培養基上隨時間的變化對于環境監測制度很重要。

Environmental monitoring (EM) is essential for ensuring that cleanroom environments maintain the highest standards of cleanliness and are suitable for manufacturing sensitive products, especially in industries such as pharmaceuticals, biotechnology, and electronics.
環境監測 (EM) 對于確保潔凈室環境保持最高清潔度標準并適合制造敏感產品至關重要，尤其是在制藥、生物技術和電子等行業。

Effective monitoring not only ensures product quality, but also serves as a critical data source when deviations or non-conformances occur.
有效的監控不僅可以確保產品質量，而且在出現偏差或不合格時還可以作為關鍵的數據來源。

A robust EM programme should provide insights into the types of microorganisms present in a cleanroom, their behaviour, and how these factors impact operations, such as the selection of disinfectants. So, the ability to identify viable bacteria and mould is crucial for preventing contamination and ensuring the effectiveness of cleaning regimes.
完善的 EM 程序應提供有關潔凈室中微生物類型、其行為以及這些因素如何影響操作（例如消毒劑的選擇）的見解。因此，識別活細菌和霉菌的能力對于防止污染和確保清潔方案的有效性至關重要。

Both species showing similar growth patterns at five days, but with sufficient characteristic differences for accurate identification
兩種物種在五天時都表現出相似的生長模式，但具有足夠的特征差異以實現準確識別

This article delves into the practicalities of EM, focusing on the growth patterns of different organisms, culture media types, and the impact of incubation conditions. We'll explore the growth characteristics of common cleanroom contaminants and discuss how morphology shifts over time on various media.
本文深入探討了 EM 的實用性，重點關注不同生物的生長模式、培養基類型以及培養條件的影響。我們將探討常見潔凈室污染物的生長特性，并討論形態在各種培養基上如何隨時間變化。

Understanding these principles is essential for optimising microbial recovery rates and ensuring that cleanroom environments remain under control.
了解這些原則對于優化微生物回收率和確保潔凈室環境得到控制至關重要。

The role of environmental monitoring in cleanrooms
環境監測在潔凈室中的作用 

Environmental monitoring in cleanrooms primarily focuses on viable bacteria and fungi, ensuring that the microbial load remains within acceptable limits
潔凈室的環境監測主要關注活的細菌和真菌，確保微生物負荷保持在可接受的限度內

The data collected play a pivotal role in releasing products, as cleanrooms must remain free of contamination in order to avoid product quality being compromised.
收集的數據在產品放行過程中起著關鍵作用，因為潔凈室必須保持無污染，以避免產品質量受到影響。

When deviations or non-conformances arise, EM data serve as reference points, allowing microbiologists and quality control (QC) staff to investigate the source of contamination, the effectiveness of disinfectants, and the overall hygiene of the environment.
當出現偏差或不符合情況時，EM數據可作為參考點，讓微生物學家和質量控制 (QC) 人員調查污染源、消毒劑的有效性以及環境的整體衛生狀況。

Dual incubation regimes are often implemented to reduce the need for additional EM plates
通常會實施雙重培養方法，以減少對額外EM皿的需求

Each of the methods used in cleanroom monitoring – the collection of air samples, surface swabs, and settling plates – helps build a comprehensive profile of the cleanroom environment, by highlighting trends and identifying potential sources of contamination.
潔凈室監測中使用的每一種方法-收集空氣樣本、表面拭子擦拭、沉降皿——都有助于通過強調趨勢和識別潛在污染源來建立潔凈室環境的全面概況。

These microbial profiles are often specific to an individual cleanroom, taking into account factors such as the activities carried out, the materials present, and even the operators working within the space. It's therefore critical to regularly review and update monitoring strategies in order to reflect any changes in cleanroom operations or layout.
這些微生物概況通常特定于單個潔凈室，考慮到所進行的活動、存在的物料，甚至在空間內工作的操作員等因素。因此，定期審查和更新監測策略以反映潔凈室操作或布局的任何變化至關重要。

Understanding the types of organisms isolated through EM is key. For example, common contaminants such as Staphylococcus aureus, Staphylococcus epidermidis, and Aspergillus species are often encountered. By studying the growth patterns of such organisms and how they change based on incubation conditions and media type, QC staff can make more informed decisions regarding contamination control and root cause analysis.
了解通過 EM 分離的生物類型是關鍵。例如，經常遇到金黃色葡萄球菌、表皮葡萄球菌和曲霉菌等常見污染物。通過研究這些生物的生長模式以及它們如何根據培養條件和培養基類型發生變化，QC人員可以就污染控制和根本原因分析做出更明智的決策。

Morphology and incubation times: A case study 
形態和培養時間：案例研究    

Let’s take Staphylococcus epidermidis, a Gram-positive cocci, as an example.
我們以革蘭氏陽性球菌表皮葡萄球菌為例。

Following incubation on tryptic soy agar (TSA) at 24 hours incubation at 30-35°C, its colonies appear small, smooth, and regular in shape. This can make it challenging to distinguish between other bacteria that might be present on the plate. However, after an additional 24 hours of incubation, the colonies become more distinct, exhibiting typical characteristics, such as larger size and classic morphology (Figure 1). 
在 30-35°C 的溫度下在胰蛋白酶大豆瓊脂 (TSA) 上培養24小時后，其菌落看起來小而光滑，形狀規則。這使得區分可能存在于平皿上的其他細菌變得困難。然而，再次培養24小時后，菌落變得更加明顯，表現出典型特征，例如尺寸更大和形態典型（圖 1）。

Figure 1: Incubating plates - staph epidermidis and aureus
圖 1：培養皿 - 表皮葡萄球菌和金黃色葡萄球菌

Therefore, incubation time can significantly influence the appearance of colonies. Another example is Staphylococcus epidermidis and Staphylococcus aureus – though belonging to the same genus – can be distinguished by their pigmentation after 48 hours of incubation at 30–35°C. 
因此，培養時間對菌落外觀有顯著影響。另一個例子是表皮葡萄球菌和金黃色葡萄球菌——雖然屬于同一屬——但在 30–35°C 下培養 48 小時后，可以通過它們的色素沉著來區分。

Over time, this visual differentiation becomes more apparent, with both species showing similar growth patterns at five days, but with sufficient characteristic differences for accurate identification. The morphology is a crucial part of microbial identification, whether this is used in conjunction with traditional phenotypic tests or alongside identification platforms. 
隨著時間的推移，這種視覺差異變得更加明顯，兩種物種在五天時表現出相似的生長模式，但具有足夠的特征差異以實現準確識別。形態學是微生物鑒定的關鍵部分，無論是與傳統表型測試結合使用還是與鑒定平臺一起使用。

Moulds spreading across plates will make the ability to record the number and identification of bacteria more challenging
霉菌遍布培養皿，使記錄細菌數量和鑒定細菌變得更加困難

This demonstrates how crucial incubation time and temperature is to effective bacterial identification. It must be noted that if plates are needing to be inspected frequently due to an investigation, reading plates at 24 hours might lead to misidentification or inaccurate results. Knowing those organisms which present better at greater than 48 hours provides clearer and more accurate results – a practice that QC staff should understand as part of their monitoring protocols.
這表明培養時間和溫度對于有效細菌鑒定至關重要。必須注意的是，如果由于調查需要頻繁檢查培養皿，則讀取 24 小時培養皿可能會導致錯誤鑒定或不準確的結果。了解哪些微生物在48小時以上表現更好，可以提供更清晰、更準確的結果——這是 QC 人員應將其作為監測方案的一部分來理解的做法。 

The impact of media on morphology 
培養基對形態的影響

In cleanrooms, the choice of media plays a critical role in the recovery of contaminants. TSA is a commonly-used medium, but when additional components such as Lecithin and Tween are introduced, they can impact on the morphology of recovered bacteria.
在潔凈室中，培養基的選擇對于污染物的回收起著至關重要的作用。TSA 是一種常用的培養基，但當加入卵磷脂和吐溫等其他成分時，它們會影響回收細菌的形態。

For instance, Staphylococcus aureus grown on TSA with lecithin and Tween at 24 and 48 hours exhibits similar morphology to TSA without additions. However, by day five, the media additives can cause morphological changes, which can complicate phenotypic identification (Figure 2). This underscores the importance of understanding the impact of media on microbial growth. 
例如，在含有卵磷脂和吐溫的TSA上培養 24 小時和 48 小時的金黃色葡萄球菌，其形態與未添加任何添加劑的TSA相似。然而，到第五天，培養基添加劑會引起形態變化，從而使表型鑒定變得復雜（圖2）。這強調了了解培養基對微生物生長的影響的重要性。

Figure 2a - Incubating plates - staph aureus TSA L + T 24hour
圖 2a - 培養皿 - 金黃色葡萄球菌 TSA L + T 24 小時

Figure 2b Incubating plates - staph aureus TSA L + T 5 days
圖 2b 培養皿 - 金黃色葡萄球菌 TSA L + T 5 天  

Dual incubation regimes and their effects 
雙重培養方法及其影響

Dual incubation regimes are often implemented to reduce the need for additional EM plates. One set of plates is first placed at a temperature of 20-25°C and following five day incubation, these plates are read and moved to a 30-35°C incubation for 48 hours, or variations of this. This practice ensures the detection of both environmental bacteria and fungi. This may play a role in influencing colony morphology. Again, QC staff should understand how incubation conditions affect the organisms they are monitoring. This knowledge equips them to make better decisions, when choosing media and incubation protocols, to optimise the recovery of viable microorganisms.
通常會實施雙重培養方案，以減少對額外EM皿的需求。首先將一組培養皿置于20-25°C的溫度下，培養5天后，讀取這些培養皿并移至30-35°C的培養箱中培養48小時，或以此為基準進行變化。這種做法可確保同時檢測環境細菌和真菌。這可能對菌落形態產生影響。同樣，QC人員應了解培養條件如何影響他們所監測的生物。這些知識使他們能夠在選擇培養基和培養方案時做出更好的決策，以優化活性微生物的回收率。

Fungal growth will present differently on TSA and SDA at 20-25°C and the ability to see colonies can be more challenging on TSA.
在20-25°C下，真菌生長在TSA和SDA上呈現不同情況，并且在TSA上觀察菌落更困難。

Effective monitoring not only ensures product quality, but also serves as a critical data source when deviations or non-conformances occur
有效的監控不僅可以確保產品質量，而且在出現偏差或不合格時還可以作為關鍵的數據來源

The length of incubation time is important to ensure the visibility of all viable fungal organisms. A risk that may pose a challenge when fungi are present is more rapid growth when TSA plates are moved to the 30-35°C incubation. 
培養時間的長短對于確保所有活體真菌生物的可見性非常重要。當真菌存在時，可能帶來挑戰的風險是，當 TSA皿移至30-35°C培養時，真菌生長速度會更快。

Moulds spreading across plates will make the ability to record the number of bacteria on the plate, along with the identification of these, more challenging. This challenge isn’t restricted to plates exposed to dual incubation, fungal growth can be rapid and present the same challenge on TSA when single incubation is used, where plates are incubated at 30-35°C.    
霉菌在培養皿中蔓延，使得記錄培養皿中細菌數量以及識別這些細菌的能力更加困難。這種挑戰不僅限于暴露于雙重培養的培養皿，真菌生長速度很快；當使用單次培養時，在30-35°C下培養培養培養皿，TSA也面臨同樣的挑戰。

Monitoring mould growth in cleanrooms 
監測潔凈室中的霉菌生長

Mould contamination is a significant concern in cleanroom environments. When these are present on EM plates this can impact on the ability to read the plates effectively.
霉菌污染是潔凈室環境中的一大問題。當EM皿上出現霉菌時，會影響有效讀取皿的能力。

For example, when plates are incubated for 48 hours, Aspergillus may occupy only a portion of the agar plate, but by day five, its growth can cover the entire surface; this makes it almost impossible to identify any bacterial growth that might also be present (Figure 3).
例如，當培養皿培養 48 小時后，曲霉菌可能僅占據瓊脂皿的一部分，但到第五天，它的生長就可以覆蓋整個表面；這使得幾乎不可能識別可能存在的任何的細菌生長（圖 3）。

Figure 3a -Incubating plates - Aspergillus
圖 3a -培養皿 - 曲霉菌

Figure 3b - Incubating plates - Aspergillus 4 days
圖 3b - 培養皿 - 曲霉菌4天

As with bacteria, the choice of media can influence mould morphology. On TSA with lecithin and Tween, Aspergillus exhibits even more rapid growth than on standard TSA, quickly covering the plate and potentially masking the presence of other organisms. It's therefore crucial to evaluate environmental plates regularly during incubation to avoid missing critical contamination events. Another important consideration is the environmental conditions that promote mould growth.    
與細菌一樣，培養基的選擇會影響霉菌的形態。在含有卵磷脂和吐溫的TSA上，曲霉菌的生長速度比在標準 TSA上更快，迅速覆蓋培養皿并可能掩蓋其他生物的存在。因此，在培養期間定期評估環境培養皿至關重要，以避免錯過關鍵的污染事件。另一個重要的考慮因素是促進霉菌生長的環境條件。

A robust EM programme should provide insights into the types of microorganisms present in a cleanroom
強大的環境監測程序應能洞悉潔凈室中存在的微生物類型

Cleanrooms must be monitored for humidity and temperature, both of which can influence fungal contamination. If humidity levels are too high, mould growth can become uncontrollable, even with stringent cleaning and disinfection protocols. Monitoring and controlling these environmental parameters are essential to keeping mould contamination under control.
必須監測潔凈室的濕度和溫度，這兩者都會影響真菌污染。如果濕度過高，即使采用嚴格的清潔和消毒規程，霉菌的生長也難以控制。監測和控制這些環境參數對于控制霉菌污染至關重要。

Recognising atypical growth patterns 
識別非典型生長模式    

QC staff must also be able to recognise atypical growth patterns that may result from accidents or deviations during EM.
QC人員還必須能夠識別由于 EM 期間的事故或偏差而導致的非典型生長模式。

For example, plates that have been inadvertently dropped or touched by operators will exhibit unusual growth patterns, and present organisms that may have not been witnessed before. Recognising these outliers is important for correctly interpreting and trending EM data to enable risk to product to be correctly evaluated. 
例如，被操作員無意中掉落或觸碰的盤子會呈現出不尋常的生長模式，并出現以前可能從未見過的生物。識別這些異常值對于正確解釋和趨勢分析 EM 數據非常重要，這樣才能正確評估產品風險。

For instance, a plate contaminated by accidental contact may show irregular, clustered growth that is unlike the smooth, even distribution typical of cleanroom contaminants (Figure 4). These insights are valuable during investigations of out-of-trend results and help ensure that corrective actions are targeted and effective.
例如，一塊因意外接觸而受到污染的皿可能會出現不規則的聚集增長，這與潔凈室污染物典型的平滑均勻分布不同（圖 4）。這些見解在調查超出趨勢的結果時很有價值，有助于確保糾正措施有針對性和有效性。

Figure 4 - Incubating plates - irreg clustered growth
圖 4-培養皿-不規則簇狀生長 

By understanding the typical growth patterns expected from environmental isolates, microbiologists can more readily identify when something is out of trend, and gain assurance nothing has been missed.
通過了解環境分離物的典型生長模式，微生物學家可以更容易地識別出何時出現趨勢以外的情況，并確保沒有遺漏任何內容。

The choice of media is important, along with the incubation time and temperature. This is to ensure that when a plate presents no growth, this is a true representation of no environmental risk to product and not a result of a false negative due to the environment which the organism requires to grow being compromised. 
培養基的選擇以及培養時間和溫度都很重要。這是為了確保當培養皿中沒有細菌生長時，這真正表明產品沒有環境風險，而不是由于生物生長所需的環境受到損害而導致的假陰性結果。

Optimised environmental monitoring 
優化環境監測

Effective EM is fundamental to maintaining cleanroom control and ensuring product quality.
有效的 EM 對于維持潔凈室控制和確保產品質量至關重要。

The ability to understand how colony morphology changes over time, under different incubation conditions, and on various media types is crucial for identifying contaminants and preventing contamination. QC staff must be equipped with the knowledge and skills to interpret EM data accurately, considering factors such as incubation time, media composition, and the potential for dual incubation regimes to affect microbial recovery.
了解菌落形態在不同培養條件下以及在各種培養基類型下隨時間變化的能力對于識別污染物和防止污染至關重要。QC人員必須具備準確解釋 EM 數據的知識和技能，并考慮培養時間、培養基成分以及雙重培養方案影響微生物恢復的可能性等因素。

In cleanrooms, the choice of media plays a critical role in the recovery of contaminants
在潔凈室中，培養基的選擇對于污染物的回收起著至關重要的作用

By optimising EM practices for microbial contamination, cleanrooms can remain under control, ensuring that final products meet the highest standards of quality and safety. The insights gained from careful observation of growth patterns and colony morphology provide greater confidence in the recovery of viable bacteria, support investigations into root cause analyses, and guide the implementation of corrective actions to prevent future deviations.    
通過優化微生物污染環境監測實踐，潔凈室可以得到控制，確保最終產品符合最高的質量和安全標準。通過仔細觀察生長模式和菌落形態，可以更有信心地恢復活菌，支持調查根本原因分析，并指導實施糾正措施以防止將來出現偏差。

In industries where contamination can have significant consequences, these practices are essential for maintaining the integrity of cleanroom environments and ensuring the consistent delivery of safe, high-quality products.
在污染可能造成嚴重后果的行業中，這些實踐對于維護潔凈室環境的完整性以及確保持續交付安全、高質量的產品至關重要。

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