1 Hygiene procedures for mushroom

cultivation Naomi Diplock

A laboratory cleaning procedure is vital to reducing the introduction of contaminants. The

main sources of contamination should always be kept in mind while entering or working in the
lab. These are: The worker and their clothes, the air and external environment, the media, the
inoculum, the tools and mobile contamination units (flies, rates, cockroaches etc). A few simple
steps can be taken to reduce the risk of introducing contaminants from outside the lab.

1. Cleaning room
A weekly (minimum) cleaning of the facility should be conducted, including all window
ledges, floor edging, bench surfaces and floors. While mopping, 2 buckets should be used to
avoid redistributing dirt throughout the room. Ideally, a 5% bleach or disinfectant solution
should be included in this cleaning procedure.
Cleaning should start from the most critical rooms (i.e., the inoculation room), moving to
the cooling room and followed by all other rooms.
Cleaning should always start from the highest point (e.g., window ledges) and finish with
the floor at the exit door
All work surfaces must be kept clean and cultures protected from aerial and dust
contamination. The work benches and cupboards should be regularly washed down and
wiped with 70% ethanol.
When cleaning and sorting bottles, be sure to place them on a clean bench/trolley rather
than on the floor.
Before beginning work, a light spraying of 70% ethanol should be misted into the air. Start
from the back of the room and move towards the door. Wait 15 minutes before re-entering
for the particles to settle.

2. Shoes and slippers

Staff should take care when removing shoes and stepping into the lab space. When
removing shoes, be sure to step directly into the lab and place slippers on. Avoid walking on
the floor in socks when possible. Changing of shoes into clean slippers should occur in the
buffer room, prior to entering the lab.
Staff should wear clean socks every day to avoid introducing contaminants from outside.
A clean lab coat must be used when entering the inoculation room. These should be kept in
the buffer room and are not to be taken into dirty working areas. Ideally, lab coats should
be washed once per week.
All staff are to avoid entering the inoculation room unless necessary.
Hair must be tied back, and hair nets should be used when possible.
Face masks should be worn when performing critical tasks such as inoculation.

3. Hand washing
Touching of surfaces is a common source of contamination
Scrub hands, nails, wrists, and forearms to elbows for at least 3-5 minutes with a nail
brush, warm water (cold water is OK if warm is not available), and an appropriate
bactericidal soap before carrying out work in the inoculation room.
Gloves - Remember that gloves are only sterile until they touch something that is not
sterile! Gloves can be used to protect your skin; however, their use often leads to a false
sense of sterility. Each time bottles or other non-sterile items are touched or handled, the

2
 cleanliness of gloves or hands is reduced. When entering the lab, hands/gloves should be
sprayed with ethanol, with repeated sprays approximately every 5 minutes when working.

4. Bleach (Sodium hypochlorite) dilution

To clean floors and shelves, a 0.5% sodium hypochlorite solution can be used.

Formula
Concentration1 * Volume1 = Concentration2 * Volume2
E.g., amount of bleach (5.25%) required for 5 L of 0.5% concentration:
C1 V1 = C2 V2
5.25 * V1 = 0.005 * 5000 ml
5.25* V1 = 25 ml
V1 = 25 ml/5.25
V1 = 4.76 ml bleach
Plus Water 4994 ml= 5000 ml

E.g., amount of bleach (30%) required for 5 L of 0.5% concentration:

C1 V1 = C2 V2
30* V1 = 0.005 * 5000 ml
30* V1 = 25 ml
V1 = 25 ml/30
V1 = 0.83 ml

Plus Water 4999.17 ml = 5000 ml

Note: The concentration of bleach decreases over time. Take note of the expiry date,
buy in small quantities and avoid exposure to air.
Avoid contact with skin and clothes. When handling high concentrations, e.g., greater
than 10%, ensure appropriate protective clothing, glasses and gloves are worn.

3
 2 Laminar Flow - an overview of use and
maintenance - Naomi Diplock

1. Laminar Flow Hood

The air surrounding a work space contains many airborne particles, too small to see with
the human eye. These airborne particles contain spores of contaminants, dangerous to the
production of mushroom spawn and other cultures. The use of a laminar flow minimizes
the exposure of these contaminants through the use of a HEPA filter which cleans the air
and creates a clean space for working. The correct use of the laminar flow is vital for the
system to work effectively. It is important to understand how the laminar flow works and
the direction of the airflow (vertical or horizontal) to maximize the effectiveness of your
unit.

1) Cleaning
The laminar flow must be turned on for a minimum of 15 minutes before use. All
surfaces are then swabbed down liberally with 70% ethanol.
Clean the sides of the hood using an up & down motion.
Start at the HEPA filter, working towards the outer edge of the hood.

Order of cleaning:
- walls 1st
- floor of hood 2nd
Frequency:
- beginning of each shift
- before each batch of media to be inoculated
- No longer than 30 minutes following the previous surface disinfection
when activities are ongoing
- after spills
- when surface contamination is known or suspected
Swab any instruments that will be used in the hood with 70% ethanol.

2) Working in the laminar flow hood:

Always minimize clutter!
The work surface inside the laminar flow should be uncluttered and contain only the
items required for a particular procedure. A common mistake is filling the laminar
flow with substrate bottles ready for inoculation. While bottles should be cooled in the
laminar flow after removal from the autoclave, this should not be used as a storage area.
This means that when inoculating substrate, all substrate bottles ready for inoculation
should be removed from the laminar flow after cooling and only a few at a time returned
to the laminar flow hood for inoculation.

The laminar flow works by creating clean air through the use of a HEPA filter. The
HEPA filter works through static electricity, so humidity of 90% or less is required for
effective operation. If required, a dehumidifier may be needed in the inoculation room to
maintain an appropriate humidity.
While the laminar flow does not produce sterilization it prevents contaminants from
settling onto sterile objects, so the correct use and arrangement of equipment is
paramount for effective operation. As the laminar airflow works by creating a flow of

4
 clean air, movement of greater velocity or in a different direction to that produced by
the laminar flow is capable of reducing the effectiveness. Contamination rates in the
laminar flow can be reduced through appropriate and careful techniques. Work at a
smooth, slow and steady pace, at a minimum of 6 inches from any edge and arrange
tools and equipment as to not disrupt the airflow.
A common cause of contamination in the laminar flow is the interruption of airflow by
inappropriately arranged equipment. While arranging equipment and working space, be
sure to maintain a direct and clear path between the filter and the area inside the hood
where the manipulations / inoculations are being performed (Figure 1). Air downstream
from non-sterile objects (such as bottles, hands, etc.) is contaminated by particles blown
off these objects.

Horizontal airflow
In a horizontal style laminar flow, the clean air is produced and moved from the back of
the hood. Avoid placing any large objects near the back of the hood as these will block/
contaminate / change the pattern of the airflow. Arrange objects appropriately, keeping a
clear working space. Critical items should be placed closer to the air source, leaving a gap
of at least 3 inches from the back and sides of the hood. All items (and hands) must be
kept at least 6 inches inside the front of the hood. If required, you may stack some items,
however consider the movement of air. Items should be stacked from lowest to highest
from the back of the hood. This allows for minimum interference to the airflow. Make sure
that materials in use are to the side of your work area, so that airflow from the hood is not
blocked.

Vertical airflow
In a laminar airflow with vertical air movement the placement of objects is less critical,
as the clean air is produced from the top of the hood. Careful consideration must be given
to the position of hands and tools as often these will be placed over critical objects and
contamination is possible. All objects (and hands) must be kept inside the hood at least 6
inches from the front edge. Keep a 3 inch gap between objects and the side and back of the
hood.
Airflow direction

Figure 1: Dead space created around objects in a laminar flow hood (horizontal type)

5
 As little as possible should be kept in the laminar flow hood while working. Tools should
be arranged before starting work in a manner that allows inoculation or other work to be
performed without crossing over of hands while working. E.g. For a right handed person
performing inoculation of substrate, tools and mother spawn should be placed on the right
with sterile jars on the left. All other items should be arranged so that the active work
area is directly in front of the worker. No materials or equipment are to be placed between
the filter and work area (Figure 2). While working in the laminar flow, ensure hands and
equipment are kept at least 6 inches inside from the front edge. The outside air begins to
mix with the air at the front of the laminar flow, resulting in possible contamination.
Don’t talk/cough/sneeze into the laminar flow. If talking is required, direct your face
away from the work area, a face mask may also be worn over your mouth to minimize
contamination. If sneezing/coughing occurs, be sure to spray your hands with ethanol
afterwards.
When removing caps, do not place them down on the surface if possible. If these must be
placed down, keep them ‘open-side’ upright.

Figure 2: Ideal set up of horizontal laminar flow space for inoculation procedure
Note: A side bench could be set up on the left of the laminar flow to store bottles that will be inoculated.

Pouring PDA plates

Prepare PDA according to directions on bottle or use an appropriate recipe.
When removing Petri dishes from the plastic sleeve, cut a slit across the top and squeeze
the required number of dishes from the bottom.
Do not put hands inside the sleeve, as the sleeve can be reused for storage.
Store unused, sealed Petri dishes upside down.
After autoclaving PDA and the temperature has reached 50-60°C, pour 15-20ml of media
per Petri dish.
If pouring in a single layer of plates, pour from the back to the front of the hood to avoid
reaching over the open plates.
If the laminar flow is not big enough to pour in a single layer, stack Petri dishes in the
laminar flow hood in blocks of 4-5 for ease of pouring. Start from the bottom of the pile,
lifting with one hand and pouring with the other.

To minimize the formation of water droplets, leave the plates to set, open in the laminar
flow for 30 minutes. Store in a sealed bag, upside down.

6
Recognizing contamination
Contamination on agar plates or in spawn bottles can be recognized by any growth that is
abnormal to what is expected.
Fungal contaminants are often more easily recognized due to variants in color, and they
normally have a ‘fluffy’ appearance. Bacterial contaminants can be recognized by a wet or
slimy appearance. The location and type of contaminant can often indicate the source.

Checking effectiveness of the laminar flow hood

Most laminar flow hoods in Bhutan are not equipped with a wind speed meter, or working
manometer. It is essential to check the effectiveness of the laminar flow regularly. This
is done simply with the use of PDA/PSA plates. After cleaning, and running the laminar
flow hood for a minimum of 15 minutes place three PDA plates in the center of the laminar
flow. Open the plates and balance the lids on the front side of the plate. Leave the plates
open and time for exactly 30 minutes while the laminar flow is on. Close the plates and seal
appropriately with parafilm or tape. Incubate at approximately 25°C for 5 days. Observe
for any growth. Observations should reveal no growth on the plates, indicating a working
unit. If growth is observed repeat the test. If growth is observed again, replacement of the
HEPA filter or maintenance of other components of the laminar flow is required.

Cleaning of Pre-Filter
The pre-filter of the laminar flow hood should be cleaned approximately every 6 months.
The frequency of cleaning will depend on the cleanliness and humidity of the surrounding
environment. Remove the prefilter by unscrewing it from the unit. Clean gently with soap
and water and allow to dry completely and thoroughly in the sun. Once dry re-attach the
unit.

7
 3 Spawn production method for shiitake and
oyster mushrooms Kazuo Watanabe

I Production of shiitake sawdust spawn

1. Mother culture storage
Mother culture is the most important element of spawn production. Although there are
many ways to store mother culture, the general method involves storage in liquid nitrogen
(-196°C) or a deep freezer (-80~-85°C).
It is also common for subcultures of the mother culture to be maintained at 20 to 23°C.
In this case, subcultures must be restarted once a year. Household freezers (-20°C or
lower) are not suited to long-term storage of mother culture because they do not provide
sufficiently low temperatures, and the survival rate of mother culture is low. To be on the
safe side, the mother culture should be stored using two or more methods simultaneously.
Although liquid nitrogen is difficult to acquire in Bhutan, electricity is readily available;
thus, the use of deep freezers is appropriate. The procedure for storing mother culture in a
deep freezer is provided below.
(1) Inspection of colonies
After checking that the colonies on PDA media are growing normally (i.e., absence of
sectoring, formation of characteristic circular colonies, absence of flat hyphae, etc.),
remove small fungal disks (6 to 8 mm in diameter) from just inside the colony margin
using a sterilized cork borer.
(2) Storage solution and containers
For the storage solution, use a solution containing 10% w/v glycerol and 5% w/v
trehalose. For cryopreservation, add 1 mL of storage solution to tubes (2 mL) or vials
and then sterilize at 121°C for 15 min.
(3) Storage of fungal disks
Place 2 to 5 fungal disks into the sterilized tubes/vials containing storage solution, store
the tubes/vials in a refrigerator for 30 minutes to 1 day and night, and then move the
tubes/vials to the deep freezer (-80~-85°C).
(4) Thawing and culturing
Remove the mother culture from the deep freezer and perform rapid thawing (30°C for
3 minutes). Remove and transfer the fungal disks to PDA media, being careful not to
damage them.

2. Mother spawn preparation

(1) Inoculum inspection and seed culture preparation
Transfer the stored inoculum to PDA media, culture at the specified temperature in an
incubator, and check the colonies for any abnormalities in terms of the speed of hyphal
growth and colony morphology, etc. If no abnormalities are found, the colonies can
be used as an inoculum source for preparing mother spawn. The spawn can then be
used as inoculum for farm-scale spawn production after confirming its ability to form
mushrooms by inducing the development of fruiting bodies on part of the spawn. The
spawn should be prepared so that the mycelia of the mother culture spread rapidly
through the medium and that all areas of the spawn are roughly the same maturity.
For this reason, it is recommended that smaller media than that used for farm-scale
production be used.
(2) Incubation containers
As containers for preparing spawn, use 850-mL polypropylene bottles (PP bottles)

8
 with caps that provide superior dust protection and high breathability. Although
urethane or NK caps are often used in practice, these are somewhat inferior in terms
of dust protection. In such cases, efforts should be made to ensure that the room in
which the spawn is being prepared is clean. ST caps, which do not have filters, are
especially poor in terms of dust protection (although they offer good breathability);
thus, do not use them for spawn preparation. After the mycelia has spread throughout
the medium, the spawn should be allowed to mature for approximately 1 month,
after which it can be used as the inoculum source for farm-scale spawn. Medium
preparation and incubation conditions are the same as for spawn for farm-scale spawn
production.

3. Preparation of spawn for farm-scale production

Spawn for farm-scale production is prepared in Indian-made 1,000-mL plastic bottles and
screw caps that have been drilled (12-mm hole) and fitted with a cotton plug. Although
spawn bags require the use of high-quality PP bags, special containers, or bag necks, from
the farmer’s perspective, bags offer an easy-to-handle alternative to bottled spawn. Thus, it
is anticipated that spawn bags will be used in the future. Here, we discuss the preparation
of spawn for farm-scale production using bottles.
(1) Procedure
1. Prepare medium ingredients 2. weigh medium materials 3. homogenize
medium using a mixer 4. add and mix in nutrients 5. add and mix in water 6. fill
containers (bottles) with medium and create holes for inoculum 7. close containers
8. sterilize 9. allow to cool 10. inoculate medium 11. incubate/store.
(2) Preparation of sawdust medium
① Tree species and sawdust particle size
To ensure uniform mycelial growth and decomposition of the medium, tree species
well suited to decomposition should be selected for preparing sawdust, and the
particle size should be relatively small (3 mm or less). Species well suited for
sawdust preparation include Nepalese alder (Alnus nepalensis ), chinquapin
(Castanopsis spp.), and Himalayan hazel (Corylus ferox ). If the sawdust is dry,
water absorbency should be managed by storing the sawdust outdoors for a certain
period of time while applying water as needed.
② Medium ingredients
Enough nutrients (rice or wheat bran) should be added to the medium so that the
nutrient content is 8 to 10%. As rice bran is readily oxidized, it is not suitable for
long-term storage and should be used as quickly as possible. Adjust the moisture
content to between 59 and 61%. If the initial moisture content is too high, the
moisture content after ripening will be 65% or higher. Further, in addition to
providing little pore space for air, media with high moisture content is prone to
shrinkage after inoculation, reducing the suitability of the spawn as inoculum.
After measuring the moisture content of the sawdust and nutrient material using
a moisture analyzer, the amount of material to be added should be calculated using
the medium recipe calculation software (excel file). The moisture content should be
adjusted as precisely as possible.
③ Mixing and packing medium
After mixing the sawdust and nutrient material for 15 to 20 minutes, add water
and mix for another 30 to 40 minutes. When ambient temperatures are high,
mixing for long periods of time promotes bacterial growth and is not recommended.
If a mixer is used, only add medium material to just above the top of the shaft; care

9
 must be exercised because if too much material is added, it will not mix uniformly.
If the capacity of the mixer is much smaller than the capacity of the sterilizer,
mixing will require a long period of time and promote bacterial growth. Bacterial
growth can change the pH of the medium and, in some cases, lead to the production
of antimicrobial substances, resulting in poor mycelial growth after inoculation.
The medium must be sterilized immediately after it is prepared. Although a
bulk density of 60% (specific weight, 510 g of medium for an 850 mL container)
is recommended for general media, for spawn media, given the fineness of the
sawdust, a bulk density of 50 to 55% (w/v) is required to ensure sufficient void
volume. Medium should be packed into containers in such a way that the medium
near the bottom does not become compacted. If the compactness of the medium is
not uniform, the degree of spawn maturity will also not be uniform.
(3) Medium sterilization
Materials subject to sterilization and microorganisms specified as indicator species
differ by industrial sectors. Accordingly, sterilization requirements also differ by
industrial sector.
① Food processing sector
In the food processing sector, the goal of sterilization is to eliminate or inhibit the
growth of microorganisms that are the most harmful to taste and food texture. In
the case of cow’s milk, the typical sterilization method is ultra-high temperature
processing (2 to 3 seconds at 120 to 130°C). In the case of canned or bottled foods,
the most dangerous contaminant is Clostridium botulinum , which is heat-resistant
and grows under anaerobic conditions. Because C. botulinum does not grow at pH
4.5 or lower, in foods with a pH of 4.5 or lower, sterilization involves pasteurization
at temperatures of 100°C or lower; in foods with a pH of 4.6 or higher, high-
pressure sterilization at 120°C for at least 4 minutes is required. Although some
microorganisms are able to survive such conditions, they do not grow at normal
temperatures after sterilization and are therefore not problematic.
② Mushroom farming sector
In the mushroom farming sector, indicator species for sterilization include
microorganisms capable of growing on the medium used for cultivation, are heat-
resistant and grow in the temperature range required for mycelial growth (20
to 27°C). Even if microorganisms are heat-resistant, they are not targeted for
sterilization as long as they do not grow on the medium used or at temperatures
required for incubation.
In general, molds and yeasts have low heat-tolerance and can be killed by heating at
60°C for 5 to 15 minutes. In contrast, bacteria that produce spores have high heat-
tolerance. Spore-forming bacteria belong to the genera Bacillus and Clostridium .
Clostridium spp. do not grow under aerobic conditions and, thus, are not subject
to sterilization in the mushroom farming sector. On the other hand, Bacillus spp.,
which do grow under aerobic conditions, are important sterilization targets in
the mushroom farming sector. Of the more than 30 species of bacteria in genus
Bacillus , those that exhibit high heat-tolerance are referred to as “thermophilic
bacteria. Two representative thermophilic species are Bacillus stearothermophilus
and Bacillus coagulans . Of these two, B. stearothermophilus does not grow at
temperatures of 28°C or lower (Gordon R. S. and Smith N. R., 1949). Furthermore,
B. stearothermophilus and B. coagulans do not grow on sawdust/rice bran medium,
even at the optimum temperature of 55°C (K. WATANABE, 1997). Meanwhile,
mesophilic Bacillus spp. grow rapidly on media used for mushroom cultivation and

10
 at temperatures required for mycelial growth. From the above, indicator species
for sterilization in mushroom cultivation include heat-resistant mesophilic Bacillus
spp. Examples of sterilization conditions include 8 hours at 99°C (temperature of
the medium itself), 30 minutes at 113°C, 5 minutes at 117°C, and 3 minutes or less
at 122°C. In cases where the autoclave display temperature (not the temperature
of the medium itself) is used, taking into consideration the extra time required for
the medium to reach the display temperature (which depends on the autoclave
configuration), required sterilization conditions are 30 minutes at 121°C for an 850-
mL bottle or 60 minutes at 121°C for 2.5 kg of medium.
③ Air expelling and sterilization efficiency
The thermal conductivity of steam is 26 times greater than that of air. Thus, the
thermal conductivity and sterilization efficiency of an autoclave can be increased by
replacing the air in the sterilization chamber with steam. To replace the air in an
autoclave with steam, after turning on the autoclave heat source (e.g., heater, gas
burner), leave the lid to the chamber slightly ajar for 1 to 2 hours (depending on the
size of the chamber) to expel the air, and then close the lid.
④ Relationship between absolute pressure and temperature
The relationship between absolute pressure and temperature is shown in Fig.
1. This relationship is not affected by external conditions such as elevation or
weather. Theoretically, absolute pressure is the sum of atmospheric pressure and
the pressure inside the autoclave.

Fig. 1 Relationship between absolute pressure and temperature

⑤ Operation of autoclaves used in experiments

・Using a temperature data logger, set up a system for monitoring the internal
temperature of the medium.
・Place the specified number of bottles or bags in the autoclave and turn on
the autoclave. Be careful not to overload the autoclave as this will reduce the
autoclave’s sterilization efficiency.

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 ・After turning on the autoclave, to replace the air in the chamber as well as
in the bottles/bags with steam, leave the hatch slightly open to promote air
purging. Replacement typically requires 1 to 2 hours (depending on the size of the
chamber).
・After replacing the air with steam, maintain the chamber at the specified
pressure (Table 1) As the absolute pressure is the sum of atmospheric pressure
and the pressure measured inside the autoclave, the required chamber pressure
will vary with elevation. The pressures required at different elevations are shown
in Table 1. It should be noted, however, that these pressures are for when the air
in the chamber has been completely replaced by steam. If there is air left in the
chamber, a higher pressure is required to reach the target temperature of 121°C.
If it is possible to monitor the temperature of the medium itself, the autoclave
should be turned off once the temperature reaches 120°C.

Table 1. Pressure required at different elevations to achieve an autoclave

temperature of 121°C.
psi (pound at (technical Mpa (mega
Atm (hpa)
force/inch2 ) atmosphere, kgf/cm2 ) pascal, 10 6 pa)
Absolute pressure
- 29.731 2.090 0.200
at 121°C
Elevation (m) 0 1,013.25 14.689 1.033 0.101
500 950.50 15.949 1.121 0.110
750 919.25 16.402 1.153 0.113
1,000 888.00 16.855 1.185 0.116
1,250 856.75 17.308 1.217 0.119
1,500 825.50 17.761 1.249 0.123
1,750 794.25 18.214 1.281 0.126
2,000 763.00 18.668 1.312 0.129
2,250 731.75 19.121 1.344 0.132
2,500 700.50 19.574 1.376 0.135

・After sterilization is complete, gradually reduce the pressure inside the autoclave.
If the pressure is lowered too quickly, the moisture at the surface of the medium
will be removed, causing the medium to dry.
・After the pressure gauge indicates a return to normal pressure (gauge reads 0),
quickly remove the medium and allow it to cool in a laminar flow hood or other
clean-air environment.
⑥ Contamination by return air
When the temperature inside the autoclave falls below the boiling point of
water, the steam condenses to water, and the volume is reduced by a factor of
approximately 1,700. This reduction in volume creates a vacuum condition inside
the chamber, causing air to rapidly enter the chamber through the pressure release
valve. The return air enters the bags/bottles as well as the chamber. If the return
air contains thermophilic spores and the filtering ability of the caps is low, surface
contamination of the medium may occur. Many large autoclaves are equipped with
a device to filter the return air. However, if no such filter is present, after checking
that the pressure inside the autoclave has returned to zero, open the lid before a
vacuum is created within the chamber (i.e., before the steam starts to condense)
and move the medium to a laminar flow hood or other clean-air environment. Begin
operating the laminar flow hood (or other air cleaning equipment) beforehand so
that the medium can be cooled in a clean air environment.

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(4) Inoculation, incubation, and storage
① Inoculation
After the medium has cooled, move the bags/bottles to the location where
inoculation will be performed (hereinafter “inoculation room”) while preventing
contact with the outside air. Perform inoculation in a laminar flow hood. After
dropping mother spawn into the inoculation holes, inoculate the medium surface.
When using the HEPA filter of a laminar flow hood, the maximum allowable
humidity is RH90%. If the relative humidity of the inoculation room exceeds
this threshold, the laminar flow hood will not function properly. As such, ample
care must be exercised when selecting an inoculation room. Avoid rooms that
remain humid all the time due to evaporation of groundwater through the floor.
Also, because mold can grow on wood, avoid the use of wood in spawn production
facilities as much as possible and, instead, use aluminum or another metal for room
dividers. In the quasi-highlands of Bhutan (such as Chukha and Tsirang districts),
the relative humidity of inoculation rooms during the rainy season is extremely
high and exceeds 90%. When spawn production is performed in such regions, it is
highly recommended that the inoculation rooms be equipped with a dehumidifier.
To ensure that mycelia spread uniformly throughout the bottles, make sure that
the mother spawn falls to the bottom of the inoculation holes before inoculating the
medium surface.
② Incubation environment and spawn storage
The incubation room should be constructed so that it is sealed against
contamination by the outside air. In addition, the floor of the incubation room
should be insulated to ensure efficient temperature control. The incubation
room should be maintained at a temperature that is slightly lower than the
optimum temperature for mycelial growth—typically 21-23°C. Because microbial
contamination occurs more readily when the temperature of the incubation room
fluctuates widely, a temperature controller should be used to maintain a constant
temperature. Incubation rooms are typically ventilated so that the carbon dioxide
(CO2) level does not exceed 3,000 ppm. However, if a CO2 monitor is not available,
a timer should be used to ensure that the incubation room is ventilated for 15
minutes every 3 to 4 hours (depending on the number of incubation bags/bottles).
The exhaust fan should be equipped with a hood to prevent entry of outside air.
Along with the exhaust fan, an air purifier equipped with a HEPA filter should be
installed outside the incubation room and configured to allow clean and fresh air
to be pumped into the room (Fig. 2). When operating the exhaust fan, the outgoing
and incoming air volumes should be checked by measuring the wind speed to
ensure that they are balanced. Air should be moved gently through the incubation
room to promote air exchange inside the bottles through the caps. Relative
humidity inside the incubation room should be maintained at 60-70%. Because
relative humidity can fall below 40% during the dry season, the incubation room
should be equipped with an ultrasonic humidifier. Centrifuge-type misters generate
large droplets of varying size and do not raise the relative humidity effectively.
Fully colonized substrate forms primordia as it nears maturity. Because primordia
require extremely low levels of light (0.01 to 0.0001 lx), incubation rooms do
not need windows and should be kept as dark as possible. Maturation of spawn
requires additional incubation for approximately 1 month after the mycelia have
spread. After the spawn has matured, it is distributed to mushroom farmers. If
distribution will be delayed, the spawn should be stored in a cool room (2 to 4°C) to

13
 prevent over-ripening. In addition, the spawn should be placed in clean plastic bags
or otherwise stored and checked regularly to ensure that it does not dry out. The
maximum storage period for spawn is 2 months. Older spawn should be discarded.

Fig. 2 Diagram of a simple clean air-ventilation system

(5) Production of saw dust plug pawn

① Characteristics of saw dust plug spawn
The term “saw dust plug spawn” refers to plug spawn of a certain size (upper
diameter = 14 mm, lower diameter = 12 mm, length = 20 mm, including a 5-mm
polystyrene stopper) that have been coated with sawdust spawn (same as the
mature spawn distributed to farmers). The materials required include special tools,
molding sheets, and polystyrene stoppers. In addition, a 12.7 mm drill bit is needed
for inoculation. These added requirements notwithstanding, saw dust plug spawn
offers the following advantages.
・Because the mycelia on plug spawn has recovered from any damage incurred
when the spawn is removed from the bottle, they grow as soon as they are
inoculated.
・Because the plug spawn simply needs to be placed in the inoculation holes, no
special inoculation tools or sealing wax are needed. In addition, mycelial growth
is faster because the plugs maintain an air layer in the inoculation hole.
・Inoculation can be performed in a short period of time, reducing labor costs.
・Because the spawn plug spawn is more sensitive to changes in incubation
room temperature and humidity than sawdust spawn, they can be used as
environmental sensors.
・The state of mycelial establishment can be monitored during incubation.
② Procedure for preparing spawn plugs
1. Prepare sawdust spawn 2. empty sawdust spawn onto a sterile tray 3. sieve
using a wire mesh (5 mm or smaller) 4. load into 460-hole molding sheets 5. insert
polystyrene plugs into the molds 6. incubate 7. store
③ Procedure

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 Remove sawdust spawn Sieve using wire mesh Sieved spawn

Disinfect molding sheets Fill molding sheets with spawn Insert polystyrene plugs

Incubation Remove spawn plugs Saw dust plug Spawn

Fig. 3 Procedure for making spawn plug.

④ Precautions when making saw dust plug spawn

・Plug spawn must be manufactured in a clean environment during low-
temperature periods (around 10°C). Risk of contamination increases at
temperatures of 15°C or higher.
・After loading the molding sheets with spawn and inserting the polystyrene plugs,
incubate the molding sheets in a low-temperature environment (around 10°C) for
10 to 14 days. In a low-humidity environment, cover the molding sheets with a
vinyl sheet, etc. to prevent drying.
・After incubation, store the plugs at a low temperature in a refrigerator, etc. The
plug spawn can be stored for around 1 month in unsealed, sterile bags, etc. to
prevent drying.

4. Microbial contamination: inspection and countermeasures

(1) Inspection of the sterilization/cooling processes
① Given potential contamination of the medium surface by return air during the
cooling process, after cooling, samples of the medium surface including the surface
exposed to the inoculation holes should be taken using sterile techniques while the
sample is in the laminar flow hood. Samples should also be taken from inside the
medium (areas that are not in contact with the surface) and transferred to PDA
medium to check for the presence of contaminants. This process is used to check
whether contamination has occurred during the sterilization or cooling process.
② Nutrient agar with high detection capability is typically used to detect bacteria,
however, potato dextrose agar (PDA) is also commonly used. Although incubation

15
 for bacterial detection is typically performed at 35°C, at this temperature,
thermophilic bacteria are also detected. In the mushroom farming sector,
incubation is carried out at the optimal temperature for mycelial growth. After
incubating the samples for 4 to 5 days at 25-28°C, check for the presence/absence
of microbial contamination.
(2) Laminar flow hood management
The most important aspects of laminar flow hood performance are the cleaning
function of the HEPA filter (remove 99.97% of particles that are 0.3 μm or larger) and
the flow rate of clean air.
① The cleaning function of the HEPA filter is checked using PDA media. After
running the laminar flow hood for 15 minutes, place 2 or 3 petri dishes containing
PDA media in the middle of the laminar flow hood with the lids off for 30 minutes.
Incubate the petri dishes in the incubation room for 4 to 5 days. If bacteria are
detected when the laminar flow hood is operated at the maximum allowable
humidity (RH90%) or lower, the HEPA filter must be replaced. The cleaning
performance of the laminar flow hood can also be measured using a particle
counter. Such particle counters are expensive, but they enable instantaneous
checking of cleaning performance. If the laminar flow hood is operating properly,
the particle count for particles 0.3 μm or larger will be zero.
HEPA filter maintenance requires regular cleaning of the prefilter. When the
relative humidity is high, mold grows on dust collected by the prefilter, hindering
the inflow of air and increasing the burden on the Sirocco fan.
② Specifications for the output (flow rate) of clean air after the HEPA filter are
stipulated in the Japanese Industrial Standards (JIS B 9922). For horizontal
laminar flow equipment, the flow rate at 10 cm in front of the HEPA filter should
be 0.3 to 0.6 m/s. For perpendicular flow equipment, the flow rate should be 0.2 to
0.9 m/s. If the flow rate decreases, contaminated air from outside the hood can mix
in. As a general rule, the HEPA filter should be replaced when the flow rate falls
to half the original flow rate. For horizontal laminar flow equipment, HEPA filters
should be replaced when the flow rate falls to 0.2 m/s.

5. Inspection and evaluation of spawn

All spawn should be visually inspected for microbial contamination during incubation and
before shipping to customers. Close observation should be made on the containers (bottles/
bags) while incubated, and containers that are characterized as follows must be discarded.
(1) Containers in which the mycelia do not spread completely through the medium within
1 month.
(2) Containers where the color at the top of the medium and the color at the bottom of the
medium differ substantially (maturity not uniform).
(3) Over-ripe spawn with a moisture content of 65% or higher.

II Manufacturing of grain spawn

1. Adjustment procedure
1. Clean grains 2. boil 3. drain and cool 4. add additives and mix 5. fill containers
6. make cotton plugs 7. cover plugs with a polypropylene sheet 8. Autoclave 9. Cool
10. Inoculate 11. incubate

16
 Fill bags Make cotton plugs Cover with polypropylene sheet

Fill bottles Sterilize Vacuum-sealed bags Cool in laminar flow hood

Inoculate Incubate (bags) Incubate (bottles)

Fig. 4 Procedure for making grain spawn

2. Practical considerations for spawn production

Both wheat and millet are used to make grain spawn, with wheat grains being the most
common. In the case of grain spawn, no nitrogen source is added. The procedure for making
grain spawn using wheat is described below.
(1) Grain preparation
Measure out the specified grain amount and wash with water. At this point, discard
husks and grains damaged by insects.
(2) Moisture content adjustment
Oyster mushroom mycelia grow vigorously at a moisture content of 45-50%, but
growth slows at a moisture content of 40% or less. Although mycelia continue to
grow vigorously at a moisture content approaching 50%, the grains become too soft,
shortening the time that the grains can be used as spawn. Accordingly, grains for
farm-scale production should be adjusted to 43-45%. Because the moisture content
is determined during the boiling step, this step should be performed consistently.
Although moisture content can be measured precisely if a moisture analyzer is
available, if no analyzer is available, the moisture content can be estimated by
calculating the increase in weight after boiling. An increase in grain weight by 1.6
to 1.65 times for fresh grains and by 1.65 to 1.7 times for older grains is optimal
for mycelial growth. The equation used to calculate moisture content and sample
calculations are presented below.

17
 Equation : b = 100(1+c/a)/((100+c)/a)
a = initial grain moisture content before boiling (%)
b = grain moisture content after boiling (%)
c = percent increase in weight before and after boiling (%)

Table 2 Increase in weight and moisture content after boiling

a (Initial MC, %) c (Increase in weight, %) b (MC after boiling, %)
6 60 41.3
8 60 42.5
10 60 43.8
12 60 45.0
6 65 43.0
8 65 44.2
10 65 45.5
12 65 46.7
NOTE: Normally, grain moisture content is 9 to 13%; however, the initial moisture content of old
grains is 8% or lower. MC, moisture content.

(3) Draining, addition of calcium carbonate and calcium sulfate, and filling
After boiling, remove the water on the surface of the grains and allow to dry. If
grains are not sufficiently dried at this stage, water will pool at the bottoms of bags/
bottles during sterilization. After boiling, the grains are removed from the container
and spread on a plastic sheet, etc. and left overnight to dry. However, if there are
time constraints, an electric fan, etc. can be used. Calcium carbonate and/or calcium
sulfate (gypsum) are added to improve the separation of mycelia after inoculation.
The amount of calcium carbonate or calcium sulfate added is 0.3 to 0.6% (w/w of
medium) and 1.2 to 1.6%, respectively. Since oyster mushrooms are saprophytes that
prefer slightly acidic conditions, addition of calcium sulfate (gypsum) by itself is also
acceptable.
When making the mother culture, use bottles and a smaller amount of media. When
making spawn for farm-scale production, used heat-tolerant plastic (polypropylene)
bags filled with 200 to 400 g of medium.
(4) Sterilization of media
Special handling is required for sterilization of plastic bags for farm-scale production
to ensure proper sterilization. The plastic bags containing the grains are pressed
when the pressure inside the autoclave increases, leaving no space between bags for
the steam to flow through uniformly. For this reason, the internal temperature of the
medium does not increase and complete sterilization does not occur. When placing
bags inside an autoclave, form two layers separated by a perforated tray or wire mesh
to create a space between layers (to allow movement of steam). Sterilization is carried
out in the same manner as for sawdust spawn. The procedure can be facilitated by
monitoring the temperature of the medium using a data logger. After sterilization is
completed and the pressure gauge reads zero, gradually open the lid of the autoclave.
Move the bags to a laminar flow hood while they are still under vacuum and allow
them to cool in a clean air environment. After sterilization, grain media (bags/bottles)
must not be left inside the autoclave to cool. When using bagged media, check the
performance of the heat seal of the bags and be sure to use good quality rubber bands
that will not be damaged by heat to secure cotton plugs at the tops of the bags. Also,
wrap the cotton plugs with a polypropylene sheet to keep them from picking up
moisture from the steam and affix the plugs to the bags using a rubber band.

18
Supplementary materials
Spawn production facilities
Although spawn can be produced in many regions of Bhutan, cool, high-elevation regions
with low humidity are the most suitable. Spawn can also be produced in low-elevation
regions; however, in such locations, special equipment for controlling the environment is
needed.
Shiitake spawn production facilities

An ideal spawn production facility

19
 Consumption of
Capacity:
electricity
Room No. Item Size Capacity Quantity Total Origin
1 Exhaust fan with hood 0.025 1 0.03 Malaysia
2 Computer 1
3 Printer 1
Office (line A)
4 Light 0.04kw 6 0.24 Malaysia
5 Heater 0.6kw 2 1.20
6 Control panel 1
7 Electronic balance 3
Microscope 8 Moisture analyzer 2
and precise 9 Exhaust fan with hood 0.025 1 0.03 Malaysia
devices room 10 Microscope 3
11 Light 0.06 3 0.18 Malaysia
12 Autoclave (Big size) 5 KW 1 5.00
Autoclave (Middle size, new
13 1.5 kw 1 1.50
one)
14 Autoclave (Middle size) 1.5 kw 1 1.50
15 Electric cleaner 1.5kw 1 1.50
16 Distillation apparatus 1
17 Desk draft set 1
Supply fan with hood and
18 0.55 1 0.55 Malaysia
HEPA filter
Laboratory
HEPA filter Malaysia or Japan
room (line B)
Exhaust fan with hood 0.55 1 0.55 Malaysia
Exhaust fan with hood 0.3 1 0.30 Malaysia
19 Hot air oven 1
20 pH meter
Lockers for reagent and glass
21 2
wares
Experiment desk with water
22 2
sink
23 Light 0.04 12 0.48 Malaysia
Buffer room 1 24 Light 0.04 2 0.08 Malaysia
25 Clean ventilation unit 1
26 Clean circulation unit 2
27 Supply fan with HEPA filter 0.297 1 0.30 Malaysia
Circulation fan with HEPA
0.35 2 0.70 Malaysia
filter
Cooling room
HEPA filter Malaysia or Japan
Refrigerator 1.9 1 1.90 Malaysia
Oil heater 2 1 2.00 Malaysia
28 Light 0.04 6 0.24 Malaysia
29 UV light 0.015 11 0.165 Malaysia
30 Light 0.04 2 0.08 Malaysia
Buffer room 2
31 Air shower 1.8 1 2.60 Malaysia or Japan
32 Clean ventilation unit 514mm*520mm*150mm(H) 1.20(?) 1 1.20
33 Laminar flower 1
34 UV light (in the room) 0.015 6 0.09 Malaysia
35 Deep freezer 1 Malaysia or Japan
Circulation fan with HEPA
Inoculation 0.35 2 0.70 Malaysia
filter
room 1
HEPA filter Malaysia or Japan
Oil heater 2 1 2.00
37 Hot plate round 1
38 Light 0.04 4 0.16 Malaysia
39 UV light 0.015 6 0.09 Malaysia

20
 40 Laminar flower 1
41 Clean ventilation unit 1
Supply fan with HEPA filter 0.3 1 0.30
Circulation fan with HEPA
Inoculation 0.35 2 0.70
filter
room 2
HEPA filter Malaysia or Japan
Oil heater 2 2 4.00
42 Light 0.04 6 0.24 Malaysia
43 UV light 0.015 6 0.09 Malaysia
44 Clean ventilation unit 1
45 Oil heater 2.5 2 5.00
Supply fan with HEPA filter 0.3 1 0.30 Malaysia
Circulation fan with HEPA
Incubation 0.35 1 0.35 Malaysia
filter
room for
HEPA filter Malaysia or Japan
mother spawn
Ultrasonic Humidifier 2.4L/hur 0.24 2 0.48
Oil heater 2.5 2 5.00
47 Incubator 2
48 Light 0.04 6 0.24 Malaysia
SS-MAC-
49 Clean ventilation unit 0.048 6 0.24
55:514mm*520mm*150mm(H)
50
Supply fan with HEPA filter 0.3 6 1.80 Malaysia
Circulation fan with HEPA
0.35 6 2.10 Malaysia
filter
HEPA filter Malaysia or Japan
Incubation
room (6) 51 Dehumidifier 6
52 Ultrasonic Humidifier 2.4L/hur 0.24 12 2.88 Malaysia or Japan
53 CO2 analyzer 0.05 6 0.30
Device for measurement of
54 0.04 6 0.24
temp. and humidity
55 Oil heater 2.5 12 30.00
56 Light 0.04 36 1.44 Malaysia
SS-MAC-
57 Clean ventilation unit 0.048 2 0.10
55:514mm*520mm*150mm(H)
Spawn
strorage room 58 Refrigerating machine 5.6 2 11.20
(2) Devices for measurement of
59 0.04 2 0.08
temp. and humidity
60 Light 0.04 12 0.48 Malaysia
61 Autoclabe and boiler 1
62 Mixer 2
63 Filling machine 1
Working
(preparation) 64 Expelling machine 1
room 65 Convayer 3
Supply and Exhaust fan with
0.17 6 1.02 Malaysia
hood
66 Light 0.04 18 0.72 Malaysia
Back up and 67 Generator 150KVA 255 1 255.00
air conducting 68 Exhaust fan with hood 0.03 2 0.06 Malaysia
machine room 69 Light 0.04 2 0.08 Malaysia
70 Air shutter with hood
Passage
71 Light 0.04 32 1.08 Malaysia
Entry 72 Light 0.04 3 0.12 Malaysia
Pu insulated sandwitch panel
Thailand
42mm
Total 350.99

21