Cornell Mushroom Blog

A post by Alex Lando, a doctoral candidate in mycology at Cornell

Some fungi make good lab pets. They’re agreeable, easy to grow fungi that eat simple, ordinary food: like potato or cornmeal extract with a dash of sugar. But my research focuses on fungi in the order Entomophthorales (featured in these previous posts). Most are obligate pathogens of insects— they can only complete their lifecycle on their host insect. Infection requires a spore shot from its host cadaver to fall onto an unsuspecting victim. Spores that fail can form a smaller, secondary version of themselves and shoot again if they don’t hit their bug the first time. Otherwise they die. Other spores form thick walls, and hunker down as resting spores designed to survive over the winter. These fungi are fascinating to look at, but make pretty poor pets.

While some fungi happily grow on wood shavings, or cigarette butts, or your leftovers, many have never been grown in culture. Examples include rust fungi, lichens, and Labouls. And consider mycorrhizal fungi, some of which need a whole tree to grow. When describing or experimenting on a species, researchers aim to do so in axenic culture, meaning the fungus is alone, without other living organisms. An axenic culture helps us analyze fungal biology without confounding factors. But due to the complexity introduced by obligate pathogenicity, some fungi remain impossible to culture axenically. Generations of mycologists have tried all kinds of crazy ingredients to entice all kinds of fungi to grow in the lab; we can’t always guess what they need. Only five years ago we learned arbuscular mycorrhizal fungi receive lipids from their plant hosts—perhaps this clue will help us culture them.⁴

Some fungi in the Entomophthorales prefer liquid media, like Grace’s Medium, in which they’ll grow in their protoplast stage. Protoplasts have shed their chitinous cell wall, usually after they break through the exoskeleton and arrive inside an insect. This allows them to evade the insect’s chitin receptors that recognize fungi and launch insect defenses. Under a microscope, in Grace’s medium, the living protoplasts gently twist and dance, movements induced by their internal cell skeleton.

Grace’s Medium mimics insect hemolymph, the bug equivalent of blood. This liquid medium is named after Thomas D.C. Grace, a pioneer of insect cell culturing.² Grace worked for over ten years, in labs across the world, to develop the first insect cell culture lines in 1962.³They are populations of cells isolated from an organism and grown in lab conditions, to facilitate research on, for example, ovarian or fat cells. The chemical composition of Grace’s Medium is similar to the nutrients insect-infecting fungi get from their hosts, and includes sugars such as trehalose, the primary sugar in hemolymph. Dr. Shang yin Gao developed a similar formula three years before Grace’s publication, but China’s cultural revolution delayed dissemination of his work and its recognition.⁶ Later in life, Grace used this worldwide renown to begin a peaceful life of farming in Australia.

Solid media for entomopathogenic fungi have proved equally challenging to develop. Some of best recipes, including one that involved Kellogg’s Special K cereal, are published in the “Manual of Techniques in Insect Pathology.”⁵ Development of nutrient-rich media to grow these fungi began in the 1950s and continues today.¹ Our favorite solid medium is SDAEYM (Sabourad’s Dextrose Agar Egg Yolk Medium). The main components are found at your local grocery store: yeast, whole milk, and egg yolks. It doesn’t mimic insect hemolymph as Grace and Gao’s media do. We call it Breakfast Medium, from the inevitable French toast smell that wafts across the lab once you cook it. On Breakfast medium, our favorite fungi spread over the dish as hyphae instead of protoplasts, which they do not do inside an insect

Grace called his culturing method “organized neglect,” as he simply switched out half the liquid medium for fresh medium each week or so, when the fungus was looking limp. A fungus needs new food after a while, same as you or I would after we finish a meal and go looking for the next one.  For fungi growing on solid media in Petri dishes, we cut out a small piece of the culture once it’s grown a while, and move it onto a new Petri dish of fresh media, where it grows some more (see our Taming the Fungus post). One problem with the Entomophthorales is that after some generations in culture they become quite unhappy, and will stop growing entirely. How and why they grow in such different forms under such varying conditions, we do not know.

It goes to show that even some things that seem most basic to research, like what your organism can eat, are still a great challenge in the world of fungi. But we do know that these bug-eating fungi enjoy “breakfast” in the morning, just like us.

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