Contamination Identification Document
Liquid Fungi, LLC.
Contaminant Identification
6.8.23
Author: Alex Hill
Version 1.0
Rhizopus stolonifer: “Black bread mold”, “pin mold”. 4
Bascillus subtilis: “Wet spot”, “Sour rot”. 7
Trichoderma: multiple species, “Green mold”. 8
Background
The production of biological samples---such as liquid culture and inoculated substrates---always carries a risk of contamination. At Liquid Fungi we have processes and controls in place to keep the risk of contamination minimal, but it is important to be aware of common contaminants that could ruin our products and disrupt workflow.
The most important thing to keep in mind as it relates to contamination is to follow our sterile “clean air” techniques. Following proper sterilization procedures, understanding and using the flow hoods properly, and avoiding creating unnecessary turbulent air minimizes the risk of microscopic contaminants entering the sterile substrates and ruining our products.
The following is a list of common contaminants that one may see in the practice of Mycology. If one does suspect contamination of a substrate or liquid culture it is imperative to notify a supervisor immediately, document the suspected contaminant, and follow appropriate incident report procedures.
Rhizopus stolonifer: “Black bread mold”, “pin mold”
One of the most common contaminants that mycologists come across is Rhizopus stolonifer, known as “black bread mold” or “pin mold” (herein “pin mold”). Pin mold is a fast-growing fungus. Once it sporulates, it forms many tall aerial hyphae adorned with black-headed pins. It grows on readily available carbohydrates such as grain, wooden plug substrates, and agar plates. It is worth mentioning that pin mold can not grow on a certain type of agar known as Czapek medium (a.k.a. Czapek’s agar), as this medium does not contain readily available sources of Nitrogen for the pin mold.
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Figure 1 - Notice tall hyphae and black headed pins. |
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Figure 2- Notice tall hyphae and black headed pins. |
Macroscopic Views
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Figure 3- View from outside inoculation jar. |
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Figure 4 - Suspected black pin mold on grain substrate. |
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Figure 5 - Sample with stain or green light filter, notice small dots around sporangia head--those are likely spores |
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Figure 6 - Sample without stain - approx 800x |
Microscopic View
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Figure 7- Spores under microscope, approx 800x. |
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Figure 8 - Sample with lactophenol cotton blue dye stain. |
Bascillus subtilis: “Wet spot”, “Sour rot”
Bascillus subtilis, colloquially known as “wet rot” or “sour rot” (herein “wet rot”), is a bacterium commonly found in soil and the gastrointestinal tract of humans and ruminants; rye berry and plug spawn substrate that is not properly sterilized can be a breeding ground for these bacteria. Although wet rot may not necessarily ruin an inoculated substrate if the mycelium has advanced through the substrate significantly before bacterial colonization has occurred, it is best practice to dispose of contaminated substrate once identified.
Bacterial endospores can survive heat treatment/sterilization procedures; Therefore, at Liquid Fungi, we mitigate this risk by soaking/parboiling our substrate for a minimum of 16 hours before sterilization. This soaking process will encourage endospores to germinate, which can then be killed off during the sterilization process.
Signs of wet rot contamination of substrate will be a dull-gray to mucus-brown colored slime that may carry a sour or rotten smell.
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Figure 9 - Slimy, dull-grey material accumulating in grain jar. |
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Figure 10 - Notice the watery, slimey grey accumulation. |
Macroscopic
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Figure 11 - Wet rot contam in inoculated grain jar |
Trichoderma: multiple species, “Green mold”
Trichoderma, also known as “green mold” (herein “green mold”), is a genus of green-mold fungi that act as biological control agents which can outcompete biological organisms like the fungi we want to grow.
The species that we are most concerned about produce an aggressive white mycelium that leads to masses of emerald-green spores. The white mycelium can look very similar to the mycelium we are attempting to grow in bags, but the Trichoderma variety can be fluffier in structure and extend off the substrate. Green mold can colonize at any point in the cultivation process, so it is important to be mindful of the materials one is working with and look for signs of abnormalities at all times.
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Figure 12 - Trichoderma contamination in rye berry bag, notice the green hue |
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Figure 13- Trichoderma colonizing agar plate |
Macroscopic
Observing substrates for possible contamination before inoculation / transfers
When working on inoculations/transfers of any kind (petri, liquid medium, rye berry, plug spawn), it is important for one to visually inspect the substrate before beginning the inoculation/transfer process. Signs of contamination include:
- Differing colors of mycelial growth
- Texture or growth pattern abnormalities
- Sectoring
Common procedure for contaminated mediums
It is common practice for companies and laboratories working with biological samples, including Liquid Fungi, that samples with any level of contamination be disposed of properly in order to maintain quality of product and samples. Contamination is a common problem faced by companies, laboratories, and home-growers alike and it is not to be alarming.
Step 1: Assess contamination
Follow procedure as detailed in section titled “Observing substrates for possible contamination before inoculation / transfers” in order isolate contaminated specimens and determine the contaminant present.
Step 2: Disposal
- In most circumstances, simply throwing away contaminated items into a trash bag/area is an effective procedure. As contaminants are pervasive within air (unless sufficiently filtered), removing contaminated items from the premises will not protect future samples from risk.
- If one wishes to study or examine the discovered contaminants, do so in areas far away from regions where contamination is not desired, and make sure to follow proper sterilization procedure after examination.
Step 3: Determine and minimize cause of contamination
In order to minimize the possibility / frequency of future contamination, it is important to determine the cause of contamination.
- Frequently, contamination will occur due to exposure of growing substrate (agar, rye, nutrient broth) to bacteria or fungus found in unfiltered air. To verify if this is the problem, one could put an open agar plate into the sterile working area used previously for around 15 minutes. Then, place the lid on the agar plate, seal with parafilm, and leave to incubate for a few days. If there is visible contamination after the allotted time, it is likely that the air/airflow in the working area is at fault. To minimize risk of this happening, attempt to work in areas where the air is either clean (via filtration with HEPA filter in laminar flow hood) or unmoving (via a still air box).
- Another common cause of contamination lies in the tools one uses to work on samples such as scalpels, scissors, gloves, and the working surface. Make sure to completely sterilize each of the tools and surfaces present when working using 70% isopropyl alcohol (remember to sterilize each time a tool exits and enters the working areas thereafter).
- Additionally, contamination could be traveling, even in a sterile environment, from one sample to another. For example, a contaminated Lion’s Mane fungus strain (with no visible contamination) is transferred from a petri dish to a grain spawn bag where contamination becomes visible. Test the strain of fungus in the petri dish again in similar fashion, and if contamination is present, it is likely that the original sample is at fault. If this happens, there is essentially nothing that can be done to decontaminate the sample and it will have to be disposed of.