#321 STS Lab Note: 3 Proven Methods to Increase Aseptic Technique

Aseptic Technique is a direct application of Pasteur’s “Germ theory of disease”
In this Lab Note we will look at 3 methods we always try to enforce in the STS, consisting of Clean work area, you as the major contaminant and learning the difference between sterilization and disinfection.

Introduction

Aseptic technique is the use of practices, methodologies or procedures to prevent contamination of your cultures and/or spawn. It involves applying the strictest rules to minimize the risk of contamination. Contamination can be moulds/fungi, bacteria/yeasts or spores among others.

Aseptic technique is a set of routine measures that are taken to prevent cultures, spawn, substrate, or solutions from being contaminated by unwanted microorganisms. Since the goal of a mycologist is to grow mycelia without the introduction of extraneous organisms, aseptic techniques are crucial for accurate and meaningful experimentation.

Sometimes misnamed as ‘Sterile Technique’, that is limited to operating theaters, etc. One should always remember that a completely sterile working environment does not exist for Urban Farmer. However, there are a number of simple, common sense procedures that will reduce the risk of culture contaminations.

Germ Theory of disease:

Louis Pasteur introduced the ‘Germ theory of disease’ in the 1860’s and many of his theories are used and crucial fundamentals for Aseptic techniques. Not going to do a thesis on this, but think important to have idea of it, and would advise reading up WikiPedia’s entry.

To state the importance of Aseptic technique in mycology has to be reiterated. Contamination vectors [albeit, spores, bacteria, or other fungi/Trichoderma] can be one miniscule spore – that ruins a whole batch of spawn/substrate/inoculant and weeks of work down drain.

General rules to follow for any aseptic technique.

• Close windows and doors to reduce draughts
• Prevent sudden movements which might disturb the air.
• Make transfers over a disinfected surface.
• Ethanol disinfection is recommended because of its rapid action.
• If the bench surface is difficult to clean, cover the bench with a sheet of tough material, which is more easily disinfected.
• Good initial disinfection of work area can be done with 1:9 concentration of household bleach to water and liberally dousing area.
• “The wetter, the better…”, to steal pun – but the wet surface will entrap spores and prevent them blowing around.
• Start the operations only when all apparatus and materials are within immediate reach.
• Complete all operations as quickly as possible, but with methodical movements
• Vessels must be open for the minimum amount of time possible.
• If working in open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards.
• On opening a test tube or bottle, the neck must be immediately warmed by flaming with the vessel held as near to horizontal as possible and so that any movement of air is outwards from the vessel.
• During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to contamination from the air.
• The parts of sterile pipettes/scalpels which will be put into cultures or sterile vessels must not be touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the surface of the working area, or the outside of bottles/ test tubes.
• Scalpel blades which come into contact with microorganisms must be sterilized before and after each such exposure.
• Please take care when working near Bunsen burner as the alcohol based sterilizer you use on hand is HIGHLY flammable.

Now that you have a basic grasp of Aseptic Technique, can maybe explain better the heading. Three proven methods over the years that have employed that vastly increased my Aseptic Technique.

1. Aseptic Technique work area

This is the most important, to do ‘clean work’ you need a ‘clean area’. It is advised to make an dedicated enclosed area as your Aseptic workspace.

1. Using a SAB [still air box] is a prerequisite and can easily be made from clear plastic tote with holes cut in for arms & hands. This allows you to properly sanitize a restricted workspace, and will prevent air movement with smooth movements. Also prevents breath on plates/work area.
2. For more intense workload it is advised to get a HEPA [high-efficiency particulate air] flow hood. A certified HEPA filter must capture a minimum of 99.97% of dust, pollen, mold, bacteria, and any airborne particles with a size of >0.3 μm at 85 liters/min. They are costly – but give you freedom of movement and blow a ‘sterile’ air, so ensure that you work ‘downwind’ from air flow.

To the top over the glass I installed a 15cm plank and behind this is a UVC germicidal lamp to bath interior. Top of the box can also install a 2 foot LED T8 fluorescent tube light.
NOTE: The UVC light you only put on when not working in box. This is the minimum would advise.

But for only a fraction of point 2 [HEPA Flowhood] you can get yourself a HEPA air purifier. In this original box as above I mounted the HEPA purifier at back blowing forward, but subsequently have mounted it at the top to blow down and then out in front to you. This has several benefits:

• More efficient clearing of air from box, and prevention of ‘dead spaces’, the flow of air out of the access slot in front will be less turbulent.
• Keep the interior sanitized with bleach/alcohol and the germs will be entrapped by both gravity and the air flow down.
• More practical, and lower footprint of SAC [Still Air Cabinet], as you do have the HEPA filter suction protruding out the back.
• Very easy to make a pre-filter ‘sock’ and put over whole HEPA unit if mounted top of cabinet blowing down. I used felt under-carpet and stitched a “sock” and placed over HEPA Unit. This minimises the work load on the HEPA unit itself, especially if a smoker like me [😷].

This way you can make a clean space, or Aseptic Work Area, for less than R4000 [$200]. Compared to a HEPA Flowhood that costs over R20000 [$1000].

2. YOU and Aseptic Technique

Remember that 90% of the time the major contamination vector is YOU! And as John Wesley said: “Cleanliness is indeed next to Godliness…”.

• Advised to take a shower before work, tie up hair, and avoid loose clothing.
• Always wipe your hands and work area with 70% ethanol. Can use 99% isopropyl but the 70% rubbing alcohol is better as a longer ‘drying time’ so can sterilize before evaporates.
• It is recommended to wear gloves. This will prevent any foreign contaminants from coming in contact with you or sample during testing. If gloves are not used, it is necessary to sanitize hands before and after testing, or when leaving work area.
• Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the work area.
• Whenever you leave the sterile work area then sterilize hands, and objects, with alcohol before returning.
• Always cap the bottles and flasks after use. Seal Petri plates with tape or place them in resealable bags. To prevent microorganisms and airborne contaminants from gaining entry.
• Never uncover a sterile flask, bottle, Petri dish, etc. until the instant you are ready to use it. Never leave it open to the environment, return the cover as soon as you are finished.
• Before work advised to sterilize area with 9:1 bleach spray, can easily be made by adding 50 ml household 5% bleach to 450 ml water in spray bottle. If taking a break then spray work area again, as the sanitizer will entrap stray spores.

This is especially true for the Urban farmer, where you can be planting next crops – or harvesting the worm bin. Remember that Trichoderma is all over, especially in the Garden, they part of the microbial life in soil. So bear in mind that you will be full of the minuscule spores – Trichoderma spp spores are 3-5μm in size. How many times haven’t I cut the corner as just want to quickly make some spawn, or transfer a plate, just to contaminate Green Monster!

So advise plan your day and prepare for the Aseptic work window by taking a shower, refreshing, and putting on clean clothes before playing with your Agar.

3. Sterilizing your workspace

The saying: “Failing to plan, is planning to fail…” is quite literal when working in the microbial world. So make a time slot for ‘Clean Work’, a quick rundown would be:

1. Switch on HEPA fan and UVC light.
2. Spray inside of SAC liberally with 1:9 Bleach. Will dicuss more below on disinfectant.
3. Go have your shower, put on clean clothes, meditate a bit and relax.
4. Come back to do work, spray inside of SAC again with sterilant.
5. Remember to switch off the UVC lamp. May give you a tan at best, or “Arc-eyes” at worst. Most probably will only happen once (😜)
6. Wear short sleeves, and properly sanitize hands [70% rubbing alcohol]
7. Meditate again, do the “Ohmmmm” excercise, and methodically do the work you planned.
8. Everytime your arms leave the SAC then sanitize your hands. And sanitize anything you bring in – containers, etc.
9. The working surface of SAC must be wet with disinfectant. If dry then give a quick blitz again to moisten. The idea here is that if any errant spores are introduced then the HEPA unit will blow them down entrapping in the disinfectant. This is reason to keep moist.
10. When finish work then spray down inside of SAC again with Sterilant, switch off electrical equipment.

Sounds like a Schlep? But if you go through the motions, remember that you are literally the biggest contaminant in the room, it becomes like second-nature after a while. And there is a kind of Bliss in mindless repetition…

Antiseptic Disinfectant

Just a quick word here, what’s the difference between Disinfectant and Sterilant?

DIsinfectant (Noun):

a chemical agent used on inanimate objects (i.e., nonliving) (e.g., floors, walls, sinks) to destroy virtually all recognized pathogenic microorganisms. aka Sanitizer.

cdc.gov

Sterilant: 

a liquid chemical germicide that destroys all forms of microbiological life, including high numbers of resistant bacterial spores.

cdc.gov

Alcohol is a disinfectant, after COVID aka Sanitizer, and kills microbial life. In this mean living bacteria, viruses, fungi, etc. BUT does not kill endospores. There is also a time differential to do this, not instant, think its a couple of minutes. This is why we add water to the alcohol to make 70% “Rubbing Alcohol”. The water slows down the evaporation of the alcohol so all the beasties can be killed.

Household bleach [typically 5%] is a sterilant, and will kill the spores as well. Because clean bleach is ‘snotty’ you thin it down in a 1:9 ratio with water. So in a 750ml spray bottle put in 75ml household 5% bleach and fill up. This is also not instantaneous, if memory serves me right, it takes 1 hour to sterilize with 0.1% Bleach. This is reason to keep the SAC work space damp, this will entrap errant spores, and also gives the time exposure to kill them.

As a Sterilant I use a combination of 1:9 bleach and Methylated Spirits, in separate bottles. First give the SAC work area a liberal dousing with the Bleach spray, then give a quick spray with Methylated spirits bottle.

---

Socratic Questions (5 Search Terms):

1. How to improve aseptic technique in a home lab?
2. Best sterilization methods for mushroom cultivation?
3. How to use a canner for mycology sterilization?
4. Preventing contamination in mushroom grain spawn?
5. Proper use of alcohol and bleach in a still air box?