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Kamal, Gray, and Cathy4/13/2004
Production of 30 TSA Plates
Gather the following materials:
- TSA Dehydrated Media
- Weigh Boat
- Top Loading Balance
- Glass Stir Rod
- 1L Fleaker (or a beaker that can hold 1L of liquid)
- Magnetic Stirrer
- Teflon-coated magnetic stir bar
- 1L distilled water
- Autoclave
- Spoon or Scoop
- 20% Bleach Solution
- 30 Sterile Petri Dishes
- 50ºC Water Bath (with Thermometer)
Set up the 50ºC Water Bath
- visually check for instrument validation sticker or paper work, note the date of the last validation
- put enough distilled water to fill the water bath container approximately 20-30 centimeters deep
- flip the power button on the water bath so that the heating element turns on (there should be a lit LED for confirmation)
- place a thermometer in the water bath
- adjust dials or knobs so that the thermostat is set to approximately 50ºC
- the water bath should be ready to use in approximately 30 minutes
Media appearance validation SOP
- open the container of TSA dehydrated media
- make sure the media is a beige-colored powder
- if there are any hard clumps or the color is not beige, then obtain a fresh container of TSA dehydrated media and throw that one out
Measure out 40 grams of TSA dehydrated media
- using a top loading balance SOP (does not need to be quantitative, a top loading balance capable of 2-3 decimal places is fine)
- visually check for instrument validation sticker or paper work, note the date of the last validation
- zero the top loading balance
- place a clean dry weigh boat on the top loading balance
- record the mass of the weigh boat then add 40 grams to that mass
- using a clean dry spoon or scoop, transfer TSA hydrated media from the container into the weigh boat until the mass reading is equal to the calculated mass of the weigh boat and 40 grams of TSA
Carefully place a magnetic stir bar into a 1L Fleaker
Add 700mL of distilled deionized water to a 1L Fleaker
Transfer the measured 40 grams of TSA dehydrated media into the 1L Fleaker
Stir the powder and water mixture until the mixture is uniformly stirred and if necessary use a glass rod to break up clumps
- using magnetic stirrer SOP
- visually check for instrument validation sticker or paper work, note the date of the last validation
- place 1L Fleaker onto magnetic stirrer
- turn dial to the lowest stetting (ie. 1)
- slowly increase the speed of the turning stir bar until there is a visible whirl pool forms and there is uniform stirring
- Stir mixture for approximately 5 minutes
if the magnetic stir bar is not turning uniformly, then turn off the magnetic stirrer for a few seconds and repeat the steps for using a magnetic stirrer
- Turn off magnetic stirrer, then remove Fleaker
Fill Fleaker with distilled deionized water until the liquid volume is 1L (this volume does not need to be quantitative, the graduated markings on the Fleaker will suffice)
Repeat steps for stirring the mixture until the mixture is uniformly stirred
Heat beaker with mixture in a double boiler with boiling water at medium heat for approximately 15 minutes
- using double boiler SOP
- if using a stove top, take a pot (where the pot diameter is larger than the diameter of the Fleaker) and fill it with approximately 20 centimeters (deep) of water and heat until it is boiling
- if using a hot plate, take a heat-resistant container (that is wider than the Fleaker's diameter) and fill it with approximately 20 centimeters (deep) of water and heat until it is boiling
- reduce heat to a medium setting and place Fleaker into the boiling water
- heat the Fleaker with mixture for approximately 15 minutes
- when finished, turn off heating element and remove Fleaker from the boiling water in the double boiler
- use a towel to dry off the bottom of the Fleaker
- pour the boiling water down the drain and allow the double boiling container to dry
Place aluminum foil over the opening of the Fleaker, make sure shiny side is down facing the mixture (ie. down)
Attach autoclave to tape the top of a Fleaker
Put the Fleaker with mixture into autoclave and autoclave for at least 15 minutes
- using autoclave SOP
- transport the Fleaker into the room with the autoclave
- visually check for instrument validation sticker or paper work, note the date of the last validation
- look at the autoclave, make sure the pressure is at zero and temperature is below 80ºC before opening the autoclave, otherwise wait until these conditions are met
- if the autoclave is warm (ie. you can feel heat coming from the autoclave or the temperature gauge reads above 50ºC), wear heat resistant gloves when using the autoclave
- open the autoclave door
- fill the autoclave with water up to the indicated line
- place the Fleaker into the autoclave
- shut the autoclave door and make sure it is closed tightly
- maker sure autoclave is set on slow pressure release
- turn the numbered dial on the autoclave to 17 minutes
- check the autoclave in approximately 10 minutes, make sure the pressure has risen beyond zero and the temperature is above 80ºC, if not, the autoclave is broken, find another autoclave to use
- when the autoclave is done, it needs to cool off and unpressurized, this takes approximately 30 minutes, please be patient
- when the autoclave pressure gauge reads zero and temperature gauge reads below 80ºC, first put on heat resistant gloves, then open the autoclave door
- with the heat resistant gloves on, transport the Fleaker out of the autoclave
Visual Inspection (Validation) of Liquid TSA
- look at the Fleaker of TSA media, make sure the liquid has a beige or tan translucent color, if there are solid particles in the liquid media, then properly discard the media and prepare a fresh batch
With heat resistant gloves on, place Fleaker into 50ºC water bath and leave the Fleaker in the water bath for approximately 30 minutes
Stir the mixture in the Fleaker before pouring plates
- Using magnetic stirrer SOP
- visually check for instrument validation sticker or paper work, note the date of the last validation
- Place Fleaker on the magnetic stirrer
- turn dial on to the lowest setting
- increase the speed until there is uniform but gentle stirring
Wipe down counter (where plates are to be poured) with 20% bleach
Put latex gloves on, bleach latex gloves, and wipe dry with paper towel
Take 30 sterile petri dishes out of the plastic sleeves and place them on the counter with the smaller dish on the bottom
Turn off the magnetic stirrer and Remove the mixture from the magnetic stirrer
Pouring plates SOP
- Please note that the following steps should be performed as aseptically as possible, ie. Don't touch the spout of the Fleaker, try not to spill anything
- After removing petri dishes from the plastic sleeves place into stacks with 3-4 petri dishes per stack (smaller side on the bottom).
- Aseptically fold back the aluminum foil from the pouring spout until there is an opening approximately the size of a quarter.
- With the cooled off media in one hand use one's off-hand to grasp the lid of the bottommost petri dish first lifting the entire stack with the cover of the bottom plate.
- Pour approximately 12 ml of media into the small side of the bottom petri dish, and then replace cover (media thickness should be ½ to 2/3 of the depth of plate). Be careful not to accidentally drop the magnetic stir bar into the petri dish. It's OK if you drip agar on the counter, it can always be wiped up. But try not to drip agar down the sides of the petri dishes. That encourages contaminations.
- Next, use the same procedures for the next to bottommost dish. Moving up the the stack one dish at a time continue to fill the smaller sides of the entire stack, and then move on to the next stack and repeat.
When there is not enough agar for one more plate, place unused petri dishes back into a plastic sleeve and tape up the sleeve
Throw away the aluminum foil
Rinse out the Fleaker with warm water
Leave plates until agar solidifies (6-18 hours)
Once the agar have solidified, the plates should be inverted
Validation
Single sterile plate incubation SOP
- A plate should be taken and put into an incubator
- Leave for 5-7 day at a temperature of 35ºC or 20-25ºC
- After 5-7 days remove plate from incubator and check for sterility then discard the plate
- If plate is contaminated, growth will be visible and all the other plates prepared with this prepared medium should also be checked and a fresh batch prepared
Single plate innoculation SOP
- using a 48 hour old plate, innoculate one plate with the experimental microbe
- incubation this plate for 18-48 hours, it takes longer for some microbes to grow
- if there is no growth after the standard incubation period for that microbe, then throw out the whole batch of plates and prepare a fresh batch
Immediate visual inspection
- Make sure the color and clarity are correct according to product description prior to using the medium. In other words, maker sure the agar has a uniform beige or tan color and is slightly opaque.
- Make sure the media thickness is ½ to 2/3 of the depth of plate
Visual inspection after 48 hours at room temperature SOP
- Making sure medium is gel-like
- Make sure the media thickness is ½ to 2/3 of the depth of plate
- No air bubbles are present
- No growth is present (it takes cultures about 18-24 hours to grow)
- If any of these is present the plate should be discarded and a new one should be obtained and the visual inspection must be repeated
Visual inspection prior to use of the plate
- Check inventory to make sure the plate was pour 48 hours ago or more
- Make sure the media thickness is ½ to 2/3 of the depth of plate
- Making sure medium is gel-like
- No air bubbles are present
- No growth is present
- If any of these is present the plate should be discarded and a new one should be obtained and the visual inspection must be repeated
Visual inspection of instrument validation records (to assure recent validation)
This chart summarizes the importance of using proper techniques to prepare the medium so as to avoid any quality in the prepared product (Difco Laboratory Manual):
Storage
Area in which plates are to be stored should meet the following conditions:
- Recently wiped down with 20% bleach solution, this date should be noted by a sticker in the storage container or area
- The storage area should be a closed off, so that there is no active air currents are going pass the sterile TSA plates
Plates can be stored in the following conditions:
- inverted (larger lids on the bottom and the smaller lid with agar on the top)
- stacked on a tray (which also should have been wiped down with 20% bleach solution)
- at room temperature
- using a sticker, label the tray with the date in which the TSA plates were poured
Inventory Control
A log book should be kept with the following:
- instrument validation dates and records
- lot numbers of all dehydrated media received and used
- a log of TSA media production
Each time TSA media is made, the following should be recorded in a spreadsheet:
- date in which the plates were poured
- the lot number of the dehydrated media used
- the number of plates poured
- the validation dates of instrumentation used
- the name of the lab technician who poured the plates
References
Difco Laboratory Manual
Bacterial Analytical Manual online M152 Trypticase (Tryptic) Soy Agar.. 04 Apr. 2000. U.S. Food & Drug Administration, Center for Food Safety and Applied Nutrition. 03 Apr. 2004. <
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EPA/OPP Microbiology Laboratory, ESC, Ft. Meade, MD, Standard Operating Procedure for Autoplate 4000. 29 March 2004. <
K.C. Pharmaceuticals, Inc. Warning Letter. Nov. 19 2002. Oen, Pramuditya. 01 Apr. 2004. <
Methods Manual Applied Microbiology. 15 Oct. 2002. Carprette, David R. 29 March 2004. <
Microbiology Media Preparation. 02 Jan. 2004. Reynolds, Jackie. 29 March 2004. <
Science Stuff™ Agar Powder - Preparation & Equipment Use. 2004. 29 March 2004.
Soil Fungi Colony Analysis Procedures. 29 March 2004.
Teaching in the Genome Age Artificial Environments for Growing Bacteria. 2002. Pamela Mott et. al. 29 March 2004. <