Mobile Contain nation Units MCUs

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The over-riding coefficients affecting each vector are the number of contaminants and the exposure time. The more of each, the worse the infestation. This book does not go into detail as to the identity of the common contaminants. However, my previous book, The Mushroom Cultivator (1983), co-authored with Jeff Chilton, has extensive chapters on the identity of the molds, bacteria, and insects. The reader is encouraged to refer to that manual for the identification of contaminants. All contaminants are preventable by eliminating the Six Vectors of Contamination. If you have difficulty determining the vector of contamination, or a solution to a problem, please refer to Chapter 25: Cultivation Problems and Their Solutions: A Trouble-Shoe ting Guide.

1. You, The Cultivator: The human body teems with populations of micro-organisms Diverse species of fungi (including yeast), bacteria and viruses call your body their home. When you are healthy, the populations of these microorganisms achieve an equilibrium. When you are ill, one or more of these groups proliferate out of control. Hence, unhealthy people should not be in a sterile laboratory, lest their disease organisms escape and proliferate to dangerous proportions.

Most frequently, contaminants are spread into the sterile laboratory via touch or breath. Also, the flaking of the skin is a direct cause. Many cultivators wear gloves to minimize the threat of skin-borne contaminants. I, personally, find laboratory gloves uncomfortable and prefer to wash my hands every 20 or 30 minutes with antibacterial soap. Additionally my hands are disinfected with 80% isopropyl alcohol immediately before inoculations, and every few minutes throughout the procedure.

2. The Air: Air can be described as a sea of microorganisms, hosting invisible contami nants that directly contaminate sterilized media once exposed. Many particulates remain suspended. When a person walks into the laboratory, he not only brings in contaminants that will become airborne, but his movement disturbs the contaminant-laden floor, re-releasing contaminants into the lab's atmosphere.

Several steps can prevent this vector of contamination. One rule-of-thumb is to always have at least three doors prior to entry into the sterile laboratory from the outside. Each room or chamber shall, by default, have fewer airborne particulates the nearer they are to the laboratory. Secondly, by positive-pressurizing the laboratory with an influx of air through micron filters, the airstream will naturally be directed against the entering personnel. (For the design of the air system for a laboratory, see Appendix I).

For those not installing micron filters, several alternative remedies can be employed. Unfortunately, none of these satisfactorily compare with the efficiency of micron filters. "Still-air" laboratories make use of aerosol sprays—either commercial disinfectants like Pinesol® or a dilute solution of isopropanol or bleach. The cultivator enters the work area and sprays a mist high up in the laboratory, walking backwards as he retreats. As the disinfecting mist descends, airborne particulates are trapped, carrying the contaminants to the

Floor. After a minute or two, the cultivator, reenters the lab and begins his routine. (Note that you should not n 'x 'iisinfectants—especially bleach and ammonia. Furthermore, this method can potentially damage your lungs or exposed mucous membranes. Appropriate precautions are strongly recommended.)

Without the exchange of fresh air, carbon dioxide levels will naturally lise from out-gassing by the mushroom mycelium. As carbon dioxide levels elevate, contaminants are trig gereri into growth. An additional problem with heavily packed spawn rooms is that with the rise of carbon cioxide, oxygen levels proportionately decrease, eventually asphyxiating the laboratory personnel. Unless the air is ex changed, the lab becomes stifling and contamination-prone Since the only way to exchange air without introducing contaminants ;s by filtering, the combination of fans and micron filters is the only recourse

Other cultivators use ultraviolet lights which interfere with the DNA replication of all living organisms. UV lamps are effective when the contair 'nants are directly exposed. However, since shadowed areas are fully protected from UV exposure, contaminants in those regions remain unaffected. I disdain the use of UV in favor of the micron filter alternative. However, many others prefer their use. Note that the lab door should be electrically switched to the UV light so that the lamp turns off at entry. Obviously, exposure to UV 'ight is health-threatening to humans, potentiating skin cancer and damage to the cornea of the eye.

Frequently, the vector of airborne contain: nation is easy to detect because of the way it forms on petri uishes. Airborne contaniinants enter a petri dish either at the time the lid is opened (during pouring or inocuiation) or dur

Figure 58. Using an elastic film to seal ¿he top and bottom of petri dishes. Thr eliminates the chance of airborne contamination entering during incubation.

Figure 58. Using an elastic film to seal ¿he top and bottom of petri dishes. Thr eliminates the chance of airborne contamination entering during incubation.

ing incubation. When the dish is opened, airborne contamination can spread evenly across the face of nutrient media. Turing incubation, contaminants creep in and form along the inside periphery of the petri dish. This latter occurrence is most common wi h laboratories with marginal clean';ness. A simple solution is to tape together the top and bottom of the the petri dish directly after pouring and/or inoculation using elastic wax film (Parafilm® is one brand. See Figure 58.) Plastic, stretchable kitchen wraps available in most grocery stores also can be used. These films prevent entry of contaminant spores that can occur from the fluctuation of barometric pressure due to natural changes in weather patterns.

One helpful tool in e,;minating each vector of contamination as the source is to leave con tainers of media uninoculated. For instance, the cultivator should always leave some culture dishes uninoculated and unopened. These "blanks" as I like to call them, give the cultivator valuable insights as to which vector of contamination is operating. At every step in the cultivation process, "blanks" should be used as controls.

The air in the growing rooms does not require the degree of filtration needed for the laboratory. For mushroom cultivators, cleaning the air by water misting is practical and effective. (Rain is nature's best method of cleansing the air.) This cultivator's regimen calls for the spraying down of each growing room twice a day. Starting from the ceiling and broadcasting a spray of water back and forth, the floor is eventually washed towards the center gutter. The room feels clean after each session. Each wash-down of a 1000 sq. ft. grow ing room takes about 15 minutes This regimen is a significant factor in maintaining the quality of the growing room environment.

3. The Media: Often the medium upon which a culture is grown becomes the source of contamination. Insufficient sterilization is usually the cause. Standard sterilization time for most liquid media is only 15-20 minutes at 15 psi or 250° F. (121° C.). However, this exposure time is far too brief for many of the endospore forming bacteria prevalent in the additives currently employed by cultivators. I recommend at least 40 minutes @ 15 psi for malt extract or potato dextrose agars. If creating soil extracts, the soil must be soaked for at least 24 hours, and then the extracted water be subjected to a minimum of 1 hour of sterilization. Indeed, soil extracts are resplendent with enormous numbers of contaminants. Because of the large initial populations, do not be surprised if some contaminants survive this prolonged sterilization period. Should they persist, then sterilizing the extracted water first, and then re-sterilizing it with standard malt sugar additives is recommended. Cleariy, sterilization is best achieved when the media has a naturally low contamination content, (See Preparation of Media in Chapter 12.)

A good practice for all laboratory managers is to leave a few samples from each sterilization cycle uninoculated. Not inoculating a few petri dishes, grain jars, and sawdust/bran bags and observing them for a period of two weeks can provide valuable information about the vectors of contamination These quality control tests can easily determine whether or not the media is at fault or there has been a failure in the inoculation process. Under ideal conditions, the uninoculated units should remain contamination-free. If they contaminate within 48-72 hours, this is usually an indication that the media or containers were insufficiently sterilized. If the containers are not hermetically sealed, and contaminants occur near to the end of two weeks, then the contamination is probably endemic to the laboratory, particularly where these units are being stored. Under ideal conditions, in a perfect universe, no contamination should occur no matter how long the uninoculated media is stored.

Many researchers have reported that saw dust needs only to be sterilized for two hours at 15 psi to achieve sterilization. (See Royse et al. (1990), Stamets and Chilton (1983)). However, this treatment schedule works only for small batches. When loading an autoclave with hundreds of tightly packed bags of supplemented sawdust, sterilization for this short a period will certainly lead to failure.

In the heat treatment of bulk substrates, absolute sterilization is impractical. Here, sterilization is more conceptual than achievable.

Figure 59. Heat-sensitive sterilization indicator strips showing no sterilization, partial sterilization, and complete sterilization.

The best one can hope is that contaminants in the sawdust have been reduced to a level as to not be a problem, i. e. within the normal time frame needed for the mushroom mycelium to achieve thorough color-nation. Again, the time peiiod needed is approximately two weeks. Should colonization not be complete in two weeks, the development of contaminants elsewhere in the substrate is not unusual Of course, by increasing the spawn rate, coloniza-:ion is accelerated, and the window of opportunity favors the mushroom mycelium. The recommended sterilization times for var ous media are described in Chapters 15-17. Badham (1988) found that sterilization of supplemented sawdust under pressure for 4 hours at 19 psi was functionally similar (in terms of contamiaa^on reduction, growth rate and yield of Shi' ake) to high temperature pasteurization (190-194° F. or 88-90° C.) for 14 hours at atmospheric pressure (1 ps'). Remote sensing thermometers, placed at a variety of depths, are used to determine a temperature profile. When the coolest probe reads 190° F. (88° C.), steam is continuously supplied for a minimum of 12 hours, preferably 14-16 hours depending on substrate mass.

Since heat penetration varies with each substrate material's density, and is co-dependent on the moisture content, the use of sterilization indicator strips is recommended to confirm that ste ;lization has actually occurred.

Yet another limiting factor is that media biochemically changes, potentially generating toxins to myce,;al growth. Should malt agar be cooked for 2-3 hours at 18 psi, the resulting media changes into a clear, amber liquid as sugars have been reduced. Under these cond: tions, cultivators say the meca has "caramel" ed" and generally discard the media and make up a new batch. Contaminants won't

Figure 59. Heat-sensitive sterilization indicator strips showing no sterilization, partial sterilization, and complete sterilization.

grow on this media; nor does most mushroom mycelia. The cultivator is constantly faced with such dilemmas. What makes a good cultivator is one who seeks the compromises which lead most quickly to colonization and fruitbody production.

4. The Tools: In this category all tools of the trade are included from the scalpel to the pressure cooker to the media vessels. Insufficient sterilization of the tools can be a direct vector since contact with the media is immediate. Flame-sterilizing scalpels is the preferred method over topical disinfection with alcohol or bleach. However, the latter is used widely by the plant tissue culture industry with few problems

If you are using a pressure cooker for sterilizing media and other tools, many forget that although the interior of the vessel has been sterilized, for all practical purposes, the outside of the vessel has not been. Contaminants can be easily picked up by the hands of the person handling the pressure cooker and re-distributed to the immediate workstation. All the more the reason one should disinfect before beginning transfers.

5. The Inoculum: The inoculum is the tissue that is being transferred, whether this tissue is a part of a living mushroom, mycelium from another petri dish, or spores. Bacteria and molds can infect the mushroom tissue and be carried with it every time a transfer is made. Isolation of the inoculum from the mushroom mycelium can be frustrating, for many of these contaminant organisms grow faster than the newly emerging mushroom mycelium. Cultivators must constantly "run" or transfer their mycelium away from these rapidly developing competitors. Several techniques can purify contaminated mycelium.

6. MCU's, Mobile Contamination Units: Mobile Contamination Units are organisms that carry and spread contaminants within the laboratory. These living macro-organisms act as vehicles spreading contaminants from one site to another. They are especially damaging to the laboratory environment as they are difficult to isolate. Ants, flies, mites, and in this author's case, small bipedal offspring (i. e. children) all qualify as potential MCU's. Typically, a MCU carries not one contaminant, but several.

Mites are the most difficult of these MCU's to control. Their minute size, their preference for fungi (both molds and mushroom mycelium) as food, and their penchant for travel, make them a spawn manager's worst nightmare come true. Once mite contamination levels exceed 10%, the demise of the labora^

tory is only one generation away. The only solution, after the fact, is to totally shut down the laboratory. All cultures must be removed, including petri dishes, spawn jars, etc. The laboratory should then be thoroughly cleansed several times. I use a 10% household bleach solution. The floors, walls, and ceiling are washed. Two buckets of bleach solution are used—the first being the primary reservoir, the second for rinsing out the debris collected in the first wipe-down. The lab is locked tight for each day after wash-down. By thoroughly cleansing the lab three times in succession, the problem of mites can be eliminated or subdued to manageable levels. Mycelia are regenerated from carefully selected stock cultures.

I have discovered "decontamination mats", those that labs use at door entrances to remove debris from footwear, are ideal for preventing cross-contamination from mites and similarly pernicious MCU's. Stacks of petri dishes are placed on newly exposed sticky mats on a laboratory shelf with several inches ojf space separating them. These zones -oA||lation, with culture dishes incubatindiMi^highly adhesive surface, make the mites and other insects a most difficult endeavor. The upper sheet is removed every few weeks to expose a fresh, clean storage plane for new cultures.

All of these vectors are universally affected by one other variable: Tiwe of Exposure. The longer the exposure of any of the aforementioned vectors of contamination, the more significant their impact. Good laboratory technicians are characterized not only by their speed and care, but by their rhythm. Transfers are done in a systematically repetitive fashion. Controlling the time of exposure can have a drastic impact on the quality of laboratory technique.

Laboratory Sawdust Column
Figure 60. Storing petri dish cultures on "sticky mats" limits cross-contamination from mites and other mobile creatures.
Laboratory Sawdust Column

Isolation of Mushroom Mycelium from Contaminants

Sterilization and Pouring of Agar Medium

Piopagation of Pure Culture

Inoculation of Grain

Sterilisation of Grain Media

Inoculation of Sawdust/Dowels

Plugging Logs

Inoculation of Spawn

Log Culture

Stump Culture inoculation of Bulk Substrate

Tray Culture

Mound Culture

Wall Culture

Bag Culture

Column Culture

Figure 61. Overview of Techniques for Growing Mushrooms


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