Optimal Use of Glass Bottles for Cultivating

Glass bottles or jars are the most frequently utilized culture vessels for the cultivation of Cordyceps militaris mushrooms. If you want to achieve the best yield, consider the following points.

Importance of Glass Bottles or Jars in Cordyceps militaris Cultivation

The primary reason for using transparent glass bottles or jars is to provide adequate illumination to the phototropic Cordyceps militaris mushroom. The following aspects are crucial when selecting these bottles or jars:

Size of Bottle or Jar

The maximum height for bottles or jars should be approximately 10 cm. Most strains produce fruiting bodies that reach a length of 6-9 cm, making a 10 cm deep bottle sufficient for growth. The yield per bottle is directly related to its diameter. A larger diameter increases the functional surface area for cultivation, resulting in a higher yield. Ideally, the diameter should be at least 7 cm, balancing the cost of the bottle, yield, and labor involved in handling these bottles throughout the cultivation process.

Wall Thickness

Glass is not an efficient heat conductor. Therefore, thinner walls promote better heating and cooling during autoclaving and growth phases. While wall thickness has minor negative effects during autoclaving, thicker walls can hinder cooling over the 40-60 day incubation period. Hence, it is advisable to select bottles with thinner walls that are sturdy enough to endure handling and stacking in the autoclave.

Neck to Bottom Diameter Ratio

Opt for wide-mouth bottles with a neck-to-bottom diameter ratio of 1. This design facilitates easier removal of the fruiting media and fruiting body. A narrower mouth may obstruct the efficient removal of the fruiting body.

Depth of Bottle

The depth of the bottle should be around 4-6 inches. This depth allows for proper gaseous exchange of oxygen and carbon dioxide between the sealed bottle and the external environment. It also gives adequate height for the vertical growth of the fruiting body. A shallower depth might lead to curling of the fruiting body and excessive moisture loss in later growth stages. Conversely, a deeper depth could impede effective gaseous diffusion.

Note: Virtually any container that can be autoclaved and facilitates illumination can be used for cultivating Cordyceps militaris.

Gas Exchange Port Design

The gas exchange port is crucial for enabling the diffusion of primarily O2 and CO2 gases while preventing contamination during cultivation. Consider these points when designing the gas exchange port:

Material Selection

• The gas exchange port must be covered with a hydrophobic or non-absorbent material.

• Using hydrophilic or absorbent materials, such as hydrophilic cotton or medical dressing tape, can lead to saturation with water vapor. This saturation negatively impacts growth and yield due to poor gas permeability. Such conditions can promote aerial mycelia dominance, resulting in stunted and uneven growth of fruiting bodies.

  
  
  
  

Using Hydrophobic Materials

Choose hydrophobic materials such as non-absorbent cotton or hydrophobic felt cloth. These materials enhance gaseous exchange while preventing moisture loss from the bottle.

Port Creation

To create an aeration port, use a drill, punch, or red-hot tube to make a hole approximately 10-12 mm in diameter in the cap. Seal this port with hydrophobic cotton or a similar material before autoclaving. This port can also be used for injecting or pipetting the liquid spawn into the bottle during inoculation.

Identifying Non-Absorbent Cotton

Not all commercially available non-absorbent cotton products are truly non-absorbent. For making cotton plugs, non-absorbent cotton must be hydrophobic, meaning it should not absorb water. The "float-test" is practically inadequate for selecting high-quality non-absorbent cotton.

Investigating Absorbency

1. Float Test Irrelevance: The float test is not suitable for our application. Whether the cotton sinks or floats does not guarantee that it is absorbent or non-absorbent due to air bubbles.

   
   

2. Wettability Test: Immerse a small cotton sample (1 inch x 1 inch x 1 inch) in water for 10 seconds. Shake it vigorously to remove water. Absorbent cotton will remain wet, while non-absorbent will appear dry.

   
   

3. Form Retention Test: Submerge a small cotton sample in water for 10 seconds, then press it between your palms. Absorbent cotton retains a thin, sheet-like shape, while non-absorbent cotton returns to its fluffy form.

   
   

Inoculum Injection Port

If you decide to create injection ports in the bottle cap, follow these steps:

1. Drill a 5-7 mm hole in the cap.

2. Apply a 3-5 mm thick layer of Dowsil TRV sealant 736 or 733, or any food-grade heat-resistant silicone. Let it cure at room temperature for 24-48 hours. These sealants are heat-resistant, withstand autoclaving, and provide a self-sealing function post-inoculation.

   
   

Creating Holes or Ports in the Cap

If the cap is made of metal, use a drill or punch to create the hole. For plastic caps, use a red-hot metal tube to create holes. This method is more effective and simplifies the process compared to drilling.

Water Quality Matters

It is vital to use RO water for preparing liquid supplements and for humidification. This method is cost-effective and produces satisfactory results. Avoid tap water unless absolutely necessary, as it may lead to mineral precipitation.

Composition of Seed Culture Media (SCM) and Liquid Supplement for Basal Media (LSBM)

The composition of SCM and LSBM may differ from lab to lab. Check the Unveiling the Secrets of Cultivating *Cordyceps militaris* for detailed information. You can also modify the compositions to suit your needs.

Choosing Rice as Basal Media

Any parboiled or brown rice can be used as the base medium for cultivating Cordyceps militaris. Avoid polished rice, as it tends to overcook during autoclaving. Brown rice can increase production costs unnecessarily. Ensure to wash, soak for 1-2 hours, drain, and dry the rice grains before placing them in the bottle.

Relative Quantities of Basal Media, LSBM, and Inoculum

These values can be adjusted based on individual lab requirements.

Basal Media Quantity

Use 0.40 – 0.52 g per cm² of the bottom surface area of the bottle/jar. For example, if the diameter of the bottle is 10 cm, the bottom surface area equals 78.54 cm². Therefore, the necessary quantity of basal media is 31.4 – 40.8 g.

LSBM Quantity

Employ 1.28 to 1.60 mL of LSBM per gram of the basal media used.

Inoculum Quantity

Utilize a small quantity just to cover the top surface of the autoclaved basal media. Ideally, use 0.15 mL of inoculum per cm² of the bottom surface area. For the bottle mentioned above, around 11.8 mL of inoculum will suffice.

For comprehensive calculations, refer to the Unveiling the Secrets of Cultivating *Cordyceps militaris*.

Temperature, Humidification, and Air Exchange

Use a regular air conditioner to control temperature throughout different cultivation stages. Use RO water for humidification if feasible. Additionally, ensure the humidification process is non-condensing. Water droplets that condense on fruiting bodies can cause carotenoid leaching, leading to gradual whitening.

Make 2-3 air changes in the culture room daily using a fan filter unit or smaller HEPA filters. A speed controller for the fan filter unit is preferable to regulate the air change rate.

Effective Illumination

A normal 6500K (white to bluish light) LED light is sufficient and works well for the needs of cultivating Cordyceps militaris.

By following these recommendations, you can optimize the cultivation process and maximize your yield of Cordyceps militaris. The right choices in equipment and methods directly influence the success of your mushroom cultivation efforts.