Cleaning up Confusion; correctly cleaning your room

Confusion over cleaning

I have to admit, I’ve also been confused about the cleaning protocols and procedures for a cleanroom environment. The basic techniques, I’ve got that down (but I’ll talk about that too); there’s a lot of products to clean with and rotating your cleaning agents. It’s confusing to say the least and the internet is vast and for something so important how do you trust the information (since you are reading this online – myself included!)? I’ll share my references and you can see where I’m getting this information on clean room protocol. I’ve found a source that not only knows what they’re talking about and have the proper credentials but also speaks on a practical level that makes sense to me. I’ll try to be as concise as possible in explaining this because you’ll see the basics don’t necessarily warrant an hour long discussion.

The absolute basics

There’s an important concept that I don’t think is explained very well in many CE programs OR they’re so boring I’m missing these points (I’m extremely susceptible to being distracted by shiny objects during powerpoint presentations and tend to zone out). Here it is: there’s cleaning AND disinfecting. These are two distinct actions that must be performed in the correct order to properly clean a cleanroom environment (including your engineering controls and other objects in the room).

Cleaning is the physical removal of “soil” from a surface while disinfection is the application of a chemical germicide to eliminate vegetative organisms. The most important thing here is that cleaning must be done BEFORE disinfection. I try to look at a lot of this stuff very pragmatically and relate it back to things I do on a daily basis to “get” it. SO, something my wife would like me to do more often; cleaning the stovetop. We cook on the stove; drop food and spill liquids. When I’m cleaning I will usually go over the stove first with just a paper towel to remove the debris and maybe even use some soap to scrub off the caked on liquids (spaghetti sauce). Then maybe you’ll follow that up with a clorox wipe to finish things off. Boom! Cleaned (and a happy wife) and disinfected! I’ve just described everything you need to know for how to clean a cleanroom environment.

Cleaning is the physical removal of “soil” from a surface while disinfection is the application of a chemical germicide to eliminate vegetative organisms.

Detergents and Disinfectants

A detergent (soap/surfactant) is used FIRST to physically remove the soil from a cleanroom surface (ceiling, walls, floors and equipment). There’s no point in applying a disinfecting agent to a surface that has a bunch of dirt on it; this just makes sense (SMALL SIDE NOTE: prior to putting materials into pass-throughs it is NOT effective to just spray with alcohol, you should WIPE the materials with IPA sprayed wipes). I just want to add one other step you may want to perform before your detergent though; removing debris. Cleaning your floors should be a daily ritual. They’re the dirtiest part of the clean room by far because we walk on them and the cleanroom is designed to blow air and keep particulate down on the ground. With that being said you should use a DRY microfiber mop (or tacky roller) FIRST to take up the debris before using a detergent. Most likely, you’ll also need to perform a RINSE of the surface after you’ve used the detergent to remove any residue left on the surface; leaving it will actually collect more particulate and microorganisms and may potentially damage the surface (stainless steel). Also, something worth mentioning is that you don’t want a detergent that is going to generate lots of foam (bubbles) and make sure the detergent is compatible with the disinfectant so as to not inactivate it upon application.

A disinfectant is then used AFTER the detergent to kill any microorganisms left on your surfaces. Depending on which disinfectant, you may also need to do a rinse with this as well so as not to degrade and compromise the surface in any way (again stainless steel can be corroded if you leave some of these left sitting).

That’s it! Well, maybe there’s just a bit more to it, but these are the most essential concepts. Let’s go a bit further.

Types of Disinfectants

To make matters just slightly more confusing there are many types of clean room disinfectants but broadly they can be grouped into two categories: oxidizing and non-oxidizing agents. Each of these clean room cleaning agents will kill different organisms: oxidizing agents have a broad spectrum of activity (large bomb; can kill spores) and non-oxiding are more specific (9 mm beretta). Because the oxidizing agents are so non-specific they also present a greater risk to the humans cleaning with them and greater caution should be used.

Non-Oxidizing agents include:

  • alcohols
  • Quaternary Ammonium Compounds (QACs – Quats)
  • Phenolics (update: these have been noted in 483s by the FDA and are not preferred; they’ve also been banned for use in Canada)

Oxidizing agents include:

  • halogens
  • paracetic acid
  • hydrogen peroxide

Tools, Application and Sequence

I’ve already talked about using microfiber wipes for removing some large debris but what about the rest of the clean? You’ll simply use mop heads to apply your detergents and disinfectants. A different mop head should be used for the ceilings and walls than should be used for the floors. There are 2 and 3 mop bucket systems that can be used but to simplify things a bit you need to apply the detergent and disinfectant to the mop head and wipe. I have bottles with spouts that I’m able to pour (a controlled pour) the liquid onto the mop head directly (spray bottles generate aerosolized particles and shouldn’t be used). You’ll apply using unidirectional overlapping (downward in the case of walls) strokes (going back over 25% of the previous stroke) starting with the farthest end of cleanest rooms first (buffer areas) and working your way out of the rooms. If you’re just doing a daily floor and surface clean, again start at the portion of the room opposite the door and mop your way toward the door. If you’re doing an entire room clean on a monthly or weekly basis you’ll start by removing trash from bins, prepare your solutions (according to instructions (may need to be diluted although ready-to-use solutions are available; concentrate may be more economical), clean and disinfect your Primary Engineering Controls, clean and disinfect your room items (tables, carts and cart wheels) followed by the ceiling, walls and floors.

Many of these disinfectants will actually tell you in the directions for use to RINSE them after the contact time to remove residues.

Appropriate rinses for detergents and disinfectant include:

  • sterile IPA (70/30 Isopropyl Alcohol/Water)
  • sterile water

Contact (Kill) times

Each disinfectant may have its own unique contact time that is necessary for it to stay on the surface for a period of time to effectively kill various microorganisms. This may range anywhere from one minute to 10 (or more). You must observe this time to have any chance of your clean being effective and don’t apply your rising agent before that time is up.

To Rotate or Not to Rotate

Rotating two or more disinfectants is common practice in GMP environments as this is a regulatory requirement. However, USP Chater <1072> and prevailing evidence doesn’t support rotation of disinfectants. The practice comes out of the theory of microbial resistance and logically may make sense; but there’s not much proof of this when it comes to cleaning your controlled area. USP <1072> summarizes this:

The development of microbial resistance to antibiotics is a well-described phenomenon. The development of microbial resistance to disinfectants is less likely to occur at significant levels, as disinfectants are more powerful biocidal agents than antibiotics. In addition, they are normally applied in high concentrations against low populations of microorganisms usually not growing actively, so the selective pressure for the development of resistance is less profound. However, the most frequently isolated microorganisms from an environmental monitoring program may be periodically subjected to use-dilution testing with the agents used in the disinfection program to confirm their susceptibility, as there are real differences among different species in resistance to the lethal effects of different sanitizers.

With that said, maybe it’s a decent idea to keep a couple disinfectants in rotation for specific purposes as you are able to target particular bacteria with certain disinfectants. The use of a sporicide is a good example of this as these are tough to kill/deactivate.


While I can’t tell you necessarily how often to clean, this depends on your own environmental monitoring program (more on this below), I’ll give a general guideline. Floors are typically cleaned on a daily basis. Some State Boards of Pharmacy (California) have specific standards that dictate how often you should do an entire room clean. California as far I know at the time of this writing wants sterile compounding pharmacies to do a complete clean at least once a month. However, it may be warranted to clean more frequently than this. Although, if this is the case (based on your environmental data) you may want to look at your practices, procedures and engineering controls for their effectiveness.

Triple Clean

Speaking of sporicides, an effective way to use them is in a worse case scenario event or when you’re bringing a cleanroom online for the first time is a triple clean. It’s simply cleaning your room 3 times; first with a detergent followed by 2 disinfectants then a sporicidal agent (the “triple” being 2 disinfectants then sporicide). Another variation here is doing a triple clean 3 days in a row (a 9 time clean) which is the equivalent of dropping a nuclear weapon on your room. Regular use of a sporicide is also highly recommended meaning once month or quarterly.

Assessing Cleaning Effectiveness

Now we know how to clean, how do we know whether what we’re doing is effective or not? Using environmental monitoring methods to assess whether your cleaning methods and protocols are effective is common practice.

Using contact plates with Tryptic Soy Agar media is typically the media you’ll use for collecting samples. Generally what you’d do is use the contact plates at predesignated places throughout the cleanroom that are fairly critical to production. Typically you’ll sample your ISO 5 PECs, perhaps a stainless steel table or cart you may be using, shelves and pass-throughs.

There’s quite a bit of information that you can attain from doing environmental monitoring and some important conclusions that can help correct or prevent issues going forward. Sampling is usually done PRIOR to cleaning to see whether your cleaning intervals are appropriate. Also, by identifying the organisms to the genus level (which is a requirement) you’re able to see the potential origin of said organism. There are organisms that can generally be seen as from “human,” “environmental” or “materials.” With this information you can figure out a practice or protocol that may need to be changed. Over time you also may be able to trend this data to conclude that “X” occurs as a result of change in season, busier times of production or particular operators not properly gowning.

Not only is it important to have a QUALITATIVE (organism type) approach to collection but monitoring QUANTITATIVELY (number of colony forming units – CFUs) as well. To get the bacteria to grow to a point where you’re able to actually SEE them you’ll need to incubate the media. This varies for media type and you’ll want to be careful not to “cook” the samples as incubating them with temperatures that are too high will kill the sample and not give an accurate assessment. For general purpose media such as TSA an incubation temperature of 30-35 C for at least 2-3 days is appropriate.


I’ve tried to concisely and accurately convey how to clean a controlled environment. However, this is by no means is an exhaustive discussion of all the topics related. I encourage you to look more into this topic as I really haven’t broached vapor decontamination, assessing decontamination (with Total Organic Carbon Analysis) or many other subjects related to the sanitization of a cleanroom environment. Although, I would like to leave you with some of the references I’ve used to write this and have much more detail therein. What I’ve tried to do is condense hundreds of pages into a few paragraphs and inevitably it’s not going to be all-inclusive but this is a good start for cleaning up confusion related to sanitizing your cleanroom environment.


  1. Sandle, T. The CDC Handbook; A Guide to Cleaning and Disinfecting Cleanrooms (2nd Ed.); 2016 – REALLY almost anything with Dr. Tim Sandle as the author is an excellent reference for this topic and anything related to microbiology (it’s what he is – PhD Microbiologist)
  2. USP Chapter <1072>
  3. Sandle, T. Pharmaceutical Microbiology; Essentials for Quality Assurance and Quality Control; 2016
  4. (another Dr. Tim Sandle article)
  5. Updates: credit to Rick Meyer of Superior Laboratory Services Inc.

About the Author:

Seth DiPasquale

Seth DePasquale is a pharmacist and co-owner of BET Pharm, LLC in Lexington, KY; a compounding pharmacy specializing in long-acting injectable hormone formulations for equine reproduction. Seth is a 2002 graduate of Albany College of Pharmacy in Albany, NY and is a Registered Pharmacist in New York, Kentucky, Michigan, Oklahoma, Texas, West Virginia, Virginia, Alabama, Tennessee, Mississippi, Arkansas, Nebraska, Louisiana and Oregon.