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Pointers on pouring petri plates

Cathy's Lab - a slice of science pie for the mushroom minded

- Cathy Scott is Chief Science Officer at Everything Mushrooms; she handles all the mycelium! Cathy is also a tremendous cook and dedicated fungal fanatic. This little slice of our web space is dedicated to Cathy's experiments, recipies, and mushroom musings. Be sure to check this page regularly for updates on some of the exciting things happening "behind the scenes" in Cathy's Lab at Everything Mushrooms.


 

- posted February 18, 2014 

Pouring media plates is one of the first things I would teach students when they entered the microbiology lab. Pouring plates does not require ultra precision. It a simple task to introduce the students to in the lab and gives them confidence. It also helped supply the never ending need of plates the lab required.

For the Mycophiles interested in culturing mushrooms this skill is a must. If you don't have a laminar flow hood, quick hands can get away with pouring next to a flame on just about any indoor clean area that is protected from wafts of air.

tare-scale.jpg mea-weigh.jpg

LEFT: Tare scale to zero with empty container.          RIGHT: MEA and MYA can be weighed directly in an Erlenmeyer flask

Mix Media - The recipes below are for 20 petri dishes (one sleeve of petri dishes). Shake dry media to mix settled Ingredients and to break up lumps.

MEA - Malt Extract Agar

20g Everything Mushroom Malt Extract Agar-Light

add hot water to a final volume of 500ml

swirl to dissolve before sterilization

MYA - Malt Extract Yeast Agar

20g Everything Mushroom Malt Extract Agar-Light

1g Everything Mushroom Nutritional yeast

add hot water to a final volume of 500ml

swirl to dissolve before sterilization

PDA - Potato Dextrose Agar

15-20g Everything Mushroom Potato Dextrose Agar

add hot water to a final volume of 500ml

heat on low and stir until fully dissolved-sterilize

PDA requires heat and stirring to dissolve potato dextrose before sterilization, it can be done on a magnetic stirrer or on your stove top with a pan and a spoon.

weigh-pda.jpg spinning-pda.jpg

LEFT: PDA should be measured on aluminum foil or wax paper.     RIGHT: Add PDA to heated water while stirring to avoid lumps.

We recommend adding 25mg of Everything Mushroom Media Antibiotic prior to sterilization to prevent bacterial growth. Everything Mushroom Media Antibiotic (Gentamycin Sulfate) is stable enough to withstand the heat and pressure of sterilization. Many antibiotics will breakdown during sterilization or even just heating. Check the information of your antibiotic before adding, many require media to be sterilized first and cooled to 120ºF.

Sterilize at 15psi for 15-30 minutes following the directions provided by the manufacturer of your sterilizer. Be sure to leave caps loose so that hot and cooler air can be liberally exchanged during sterilization. Tightened caps can lead to inadequate sterilization, even implosion or explosion of containers. Cleaning up after an exploded container filled with media is a lesson most only need once.

pour.jpg

 

Pour plates: under aseptic conditions

Move quickly! When agar cools below 110ºF it will solidify!

  1. Open the cover halfway

  2. pour just enough media to cover the bottom of the plate.

  3. repeat for all dishes by pouring in stacks of 5-20

  4. Allow Plates to cool completely before moving them.

Resleeve and store plates upside down in a refrigerator.

resleeve-plates-1.jpg resleeve-and-lable-2.jpg

LEFT: Be sure to save the plastic sleeve     RIGHT: When plates have cooled, resleeve plates from the top down then turn the whole thing over for proper storage (agar up, lid down), tape opening end, label plate type and date.  

Stacking plates as you pour makes whole plate warmer, so it helps keep some of the water from leaving the agar and condensing on the cooler lid. Media will be juicy which is perfect for keeping fungus happy. When I start new cultures I pour plates extra thick knowing I will want to keep some of the low passage cultures for as long as possible. The thick layer of media won't dry out as quickly and you can keep the cultures under refrigeration longer.

These plates can be kept upside down under refrigeration for over a month. The simple extracts, antibiotics and agar do not break down quickly so you can get away with keeping the plates longer in the fridge or even for a while at room temperature. The sleeve will help keep most microbe invaders out but not all. If you are thinking of using older plates it is a good idea to put a few blanks in the incubator to make sure contamination hasn't crept into the edges of the plate.