A guide to environmental monitoring colony counting

03 August 2022

What is a colony forming unit (CFU)?

Bacteria and fungi grow on agar in colony forming units (CFUs). We can count these to estimate the number of viable bacteria or fungi in a sample.

In your environmental monitoring facility, you can measure the number of CFUs to determine the state of microbial cleanliness in your cleanroom. Incubation of plates gives any microorganisms on your sample time to grow. If there are no CFUs on your plates, you can be assured that your cleanroom is likely to be free from contamination. If there are growths, you may need to carry out further investigation to find out why.

How to count CFUs

Manually counting colonies involves marking each CFU on the back of your petri dish using a pen, so you only count each colony once. It may help to hold the plate up to a light for better visibility, or to set the plate on a white background (such as a piece of paper underneath). Some choose to set the petri dish on a grid, allowing for a methodological approach to counting.

Colony counting technology

In other facilities, particularly those with a high throughput, colony counting technology may be used. Colony counters automate an otherwise error-prone process, ensuring accurate results each time. After all, humans tire as the day progresses – unlike a colony counter.

Whilst some colony counters may struggle to count complex environmental monitoring plates, an innovation by Microgenetics uses cutting-edge Machine Learning technology to overcome this challenge.

Able to integrate with any software you may use in your lab such as a LIMS system or Microgenetics’ own Environmental Monitoring System (EMS), SmartControl EM, SmartControl Colony Counter can help you by:

  • Ensuring repeatable results
  • Removing subjectivity
  • Saving you time
  • Helping you to meet regulation

Learn more about SmartControl Colony Counter here.

Second checking environmental monitoring plates

Environmental monitoring plates tend to be complex, often with overlapping colonies. This can make accurately counting colonies a challenge. That’s why best practice is to second check your plates – especially if you manually count. If the person checking your plate has a different count to you, you will need to check the plate again to ensure accuracy.

Since second checks require additional resource, many environmental monitoring facilities don’t carry them out. In this instance, there are not only implications for your data integrity, but you could have missed a limit breach. If this is the case in your facility, consider using a colony counter to lighten your environmental monitoring load.

Audit trails and image capture

When carrying out your first reading, it may be beneficial to capture an image of each plate, so you have an accurate representation of its state at the time. That way, you can ensure any discrepancies in the count aren’t a result of a delay between your first and second check. Furthermore, an image will support your audit trail, which is an important part of regulation.

Action and alert limits

In your environmental monitoring facility, you’ll have a set alert and action limit for each location. These limits will vary depending on the grade of cleanroom. A grade A isolator, for example, will have lower limits than a grade B, due to the proximity to the final product.

Alert limits are set by your facility, based on the results of performance qualification (PQ) tests and trends in your historical data. Action limits, on the other hand, are generally set through regulatory guidelines such as Annex 1, the USP or ISO standards.

If there has been a breach in your facility, how you react will depend on which of these limits have been breached. For action limits, regulation states that you must carry out a timely and thorough investigation to identify the impact and potential root cause. For alert limits, you must investigate the breach, ensuring there have been no trends

Learn more about setting and analysing your limits here.

Defining trends

Routine review of environmental monitoring data provides confidence in the control of your facility, whilst allowing rapid identification of trends that may be forming. Where a single breach could indicate a temporary issue, multiple breaches, or trends, could indicate a loss of control.

How trends are defined will depend on your facility; however, the 2020 Annex 1 draft suggests trends can include (but are not limited to) the following:

  • Increasing numbers of action or alert limit breaches
  • Consecutive breaches or alert limits
  • Regular but isolated breaches of limits that may have a common cause, for example single excursions that always follow planned preventative maintenance
  • Changes in microbial flora type, as well as numbers and predominance of specific organisms

It is also important to monitor sub-limit trends of data, ensuring control of your environment is maintained. Changes in sub-limit data can indicate a loss of control as well, and if detected early enough, could prevent further issues.

If your trend rules are breached, an out of trend investigation must be performed. It’s therefore important to regularly analyse all data to ensure any potential trends are spotted and investigated where applicable.

How trending is carried out

Trending may be manual, where you create graphs using spreadsheets or a LIMS and look for patterns by eye.

However, this method introduces data integrity challenges if data is moved from one system to another, meaning original records aren’t used. For example, you may initially transcribe data onto paper at the start of your monitoring process, re-transcribe to spreadsheets and then move between spreadsheets or to a LIMS for trending.

Not using original data means there is a higher chance than human error is introduced at some stage in your process, impacting on the accuracy of your data. There may also be missing data which has not been entered into your spreadsheets or LIMS, which could impact your trend detection.

A better way to carry out trending would be to use a system such as SmartControl EM – a dedicated environmental monitoring software solution. Using SmartControl EM, data is recorded straight into the system, so no need for re-transcription. This will boost your data integrity.

When it comes to trending, you can set your own rules in SmartControl EM. If breached, you and your team will receive an alert. This will save you time, as there will be no need for you to manually go through your data to see if a trend has occurred. Additionally, the chances of you missing a trend will be substantially reduced.


  • Measuring the number of CFUs helps determine the state of your cleanroom
  • Manually counting colonies can open your facility up to human error
  • Using an automated colony counter will help you get repeatable, accurate results every time
  • Colony counters can be used as a first or second check, saving resource and helping you to meet regulation
  • Action and alert limits will be set for your cleanroom, depending on its grade. Breaches are analysed for trends, which are subsequently investigated
  • Using environmental monitoring software SmartControl EM can automatically notify you of trends, ensuring none are missed

Learn more about Microgenetics technology

SmartControl EM

SmartControl Colony Counter

SwiftDetect rapid test 

Related reads:

A guide to data integrity in pharmaceutical environmental monitoring

A guide to pharmaceutical trends