General Procedures for Preparing Media

Agar Petri Plates

Calculate the amount of media that needs to be made.
o Each plate requires 25 30 mL of agar.
o If 100 plates are needed, 2500-3000mL of agar is needed.
o Always add 200mL to the amount required in case of spills or miscalculation.
Follow package instructions for preparation.
o Instructions are typically written for 1L (1000mL) of media. If less is desired calculate the amount
needed as shown:
For example: If the instructions state 23g for 1L and 600mL is desired, use a ratio to calculate the
amount needed (in this example 13.8 g is needed for preparing 600mL):
23g/1000mL = Xg/600mL
23 * 600= 1000X
13800 = 1000X
13800/1000 =X
13.8 g = X
o Always prepare media in a beaker with 1/3 of empty space. (i.e. prepare 600mL of media in a 1000mL
beaker). If the amount of media to be prepared is greater than 1L, prepare it in 500mL aliquots or use a
2000mL beaker.
o Label the beaker with autoclave tape and state what media is being prepared, the date, and your initials
(i.e. Nutrient Agar 8-18-08 KH).
o Add powder to beaker first, and then fill with necessary amount of water.
o Stir with a glass stirring rod to mix.
o Place in microwave and heat at 3-5 minute intervals.
o Stir between intervals, using caution and allowing the media to sit for 30 seconds in the microwave
before stirring.
o Heat for approximately 10 minutes or until boiling has been achieved.
o Test the pH of the media to insure that it is within the acceptable range as stated on the package. If the
pH needs to be adjusted, add drops of 1N Hydrochloric Acid (HCl) (to make more acidic) or 1N Sodium
Hydroxide (NaOH) (to make more basic) as necessary until desired pH is achieved.
o Cover the beaker with foil and secure with autoclave tape.
o Autoclave for 20 minutes. Refer to the Standard Operating Procedure for Autoclave Use.
o While the media is in the autoclave, arrange Petri plates on the counter top.
Marywood University Standard Operating Procedure Microbiological Media Preparation

o Once sterilization is complete, open the autoclave and remove the beaker of media. You must wear the
autoclave gloves when removing anything from the autoclave.
o Allow media to cool slightly, but not for longer than 10 minutes as the agar may solidify.
o Poke a small hole in the foil covering the top in order to pour the agar.
o Pour approximately 10-15mL of agar into the plates. The bottom of the plate needs to be covered. If
necessary, swirl the plate slightly in order to evenly disperse agar.
o Allow plates to cool on the countertop overnight to reduce condensation.
o When cooled, store upside-down in plastic bags in the refrigerator to prevent the agar from drying out.
Be sure to seal the plastic bag with masking tape.
Be sure the bag is labeled with its contents, the date it was prepared, and your initials (i.e. Nutrient Agar
8-18-08 KH).
Prosedur umum untuk Mempersiapkan Media
Agar Petri Pelat
Hitung jumlah media yang perlu dibuat.
o Setiap lempeng membutuhkan 25 - 30 mL agar.
o Jika 100 piring diperlukan, 2500-3000mL agar-agar yang dibutuhkan.
o Selalu tambahkan 200ml untuk jumlah yang diperlukan jika terjadi tumpahan atau salah perhitungan.
Instruksi paket Ikuti untuk persiapan.
o Instruksi biasanya ditulis untuk 1L (1000ml) media. Jika kurang diinginkan menghitung jumlah yang
dibutuhkan seperti yang ditunjukkan:
For contoh: Jika 23 g petunjuk negara untuk 1L dan 600 ml yang diinginkan, menggunakan rasio untuk
menghitung jumlah yang dibutuhkan (dalam contoh ini 13,8 g diperlukan untuk mempersiapkan 600ml):
23 g / 1000ml = Xg / 600ml
23 * 600 = 1000X
13.800 = 1000X
13800/1000 = X
13,8 g = X
o Selalu menyiapkan media dalam gelas dengan 1/3 dari ruang kosong. (yakni mempersiapkan 600ml
media dalam gelas 1000ml). Jika jumlah media yang akan disiapkan lebih besar dari 1L, mempersiapkan
dalam 500ml aliquots atau menggunakan gelas 2000ml.
o Label gelas dengan pita autoclave dan negara media apa yang sedang dipersiapkan, tanggal, dan inisial
Anda (yaitu Nutrient Agar KH 8-18-08).
o Tambahkan bubuk ke Beaker pertama, dan kemudian isi dengan jumlah yang diperlukan air.
o Aduk dengan batang pengaduk kaca untuk campuran.
o Tempat di microwave dan panas pada interval 3-5 menit.
o Aduk antara interval, menggunakan hati-hati dan memungkinkan media untuk duduk selama 30 detik
dalam microwave sebelum aduk.
o Panas untuk sekitar 10 menit atau sampai mendidih telah dicapai.
o Uji pH media untuk memastikan bahwa itu adalah dalam kisaran yang dapat diterima sebagaimana
tercantum pada paket. Jika pH perlu disesuaikan, tambahkan tetes 1N Asam klorida (HCl) (untuk
membuat lebih asam) atau 1N Sodium Hidroksida (NaOH) (untuk membuat lebih banyak dasar) yang
diperlukan sampai pH yang diinginkan tercapai.
o Tutup gelas dengan foil dan aman dengan pita autoclave.
o Autoclave selama 20 menit. Mengacu pada Standard Operating Procedure untuk Autoclave Gunakan.
o Sementara media di autoclave, mengatur Petri piring di atas meja.
Marywood Universitas Standard Operating Procedure Mikrobiologi Media Persiapan
Halaman 2 2015/02/18

o Setelah sterilisasi selesai, buka autoclave dan lepaskan gelas media. Anda harus memakai sarung tangan
autoclave saat melepas sesuatu dari autoklaf.
o Biarkan media untuk mendinginkan sedikit, tetapi tidak lebih lama dari 10 menit sebagai agar dapat
memperkuat.
o Poke sebuah lubang kecil di foil meliputi atas untuk menuangkan agar-agar.
o Tuangkan sekitar 10-15mL dari agar-agar ke dalam piring. Bagian bawah piring perlu ditutupi. Jika
perlu, aduk piring sedikit untuk merata membubarkan agar.
o Biarkan piring dingin di meja semalam untuk mengurangi kondensasi.
o Ketika didinginkan, menyimpan terbalik dalam kantong plastik di dalam lemari es untuk mencegah agaragar kering.
Be yakin untuk menutup kantong plastik dengan selotip.
Be yakin tas yang diberi label dengan isinya, tanggal itu disiapkan, dan inisial Anda (yaitu Nutrient
Agar KH 8-18-08).
pH is adjusted to neutral (7.4) at 25 C.
Preparation of Nutrient Agar
1. Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
5. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.
6. Replace the lid of each Petri dish and store the plates in a refrigerator.
Broth Preparation
Calculate the amount of media that needs to be made.
o Each broth tube requires 5-7mL of broth.
o If 100 tubes are needed, 500-700mL of broth is needed.
o Always add 200mL to the amount required in case of spills or miscalculation.
Follow package instructions for preparation.
o Instructions are typically written for 1L (1000mL) of media. If less is desired calculate the amount
needed as shown:
For example: If the instructions state 23g for 1L and 600mL is desired, use a ratio to calculate the
amount needed (in this example 13.8 g is needed for preparing 600mL):
23g/1000mL = Xg/600mL
23 * 600= 1000X
13800 = 1000X
13800/1000 =X
13.8 g = X
o Always prepare media in a beaker with 1/3 of empty space. (i.e. prepare 600mL of media in a 1000mL
beaker). If the amount of media to be prepared is greater than 1L, prepare it in 500mL aliquots or use a
2000mL beaker.

o Label the beaker with autoclave tape and state what media is being prepared, the date, and your initials
(i.e. Nutrient Broth 8-18-08 KH).
o Add powder to beaker first, and then fill with necessary amount of water.
o Stir with a glass stirring rod to mix.
o Place in microwave and heat at 3-5 minute intervals.
o Stir between intervals, using caution and allowing the media to sit for 30 seconds in the microwave
before stirring.
o Heat for approximately 10 minutes or until boiling has been achieved.
o While heating, place test tubes in racks and label with autoclave tape (i.e. Nutrient broth 8-18-08 KH).
o Test the pH of the media to insure that it is within the acceptable range as stated on the package. If the
pH needs to be adjusted, add drops of 1N Hydrochloric Acid (HCl) (to make more acidic) or 1N Sodium
Hydroxide (NaOH) (to make more basic) as necessary until desired pH is achieved.
o Pour the broth into an appropriately sized glass bottle for pipette dispenser use.
o Before attaching the pipette dispenser, set it to the proper setting for the volume of media required for
each tube.
o Attach the dispenser to the bottle top.
o While over the sink, test the dispenser to ensure that the liquid media is filling the pipette dispenser.
o Fill each tube.
o Place loose caps on filled tubes.
For screw caps, leave the caps partially unscrewed to allow steam to enter and escape.
Marywood University Standard Operating Procedure Microbiological Media Preparation
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o Autoclave for 20 minutes. Refer to the Standard Operating Procedure for Autoclave Use.
o After sterilization is complete, remove the test tube rack, tighten all test tube caps and allow the tubes to
cool at room temperature.
o Once cooled, place in the refrigerator for storage.
Agar Slant Tube Preparation
Calculate the amount of media that needs to be made.
o Each broth tube requires 7-10mL of broth.
o If 100 tubes are needed, 700-1000mL of broth is needed.
o Always add 200mL to the amount required in case of spills or miscalculation.
Follow package instructions for preparation.
o Instructions are typically written for 1L (1000mL) of media. If less is desired calculate the amount
needed as shown:
For example: If the instructions state 23g for 1L and 600mL is desired, use a ratio to calculate the
amount needed (in this example 13.8 g is needed for preparing 600mL):
23g/1000mL = Xg/600mL
23 * 600= 1000X
13800 = 1000X
13800/1000 =X
13.8 g = X
o Always prepare media in a beaker with 1/3 of empty space. (i.e. prepare 600mL of media in a 1000mL
beaker). If the amount of media to be prepared is greater than 1L, prepare it in 500mL aliquots or use a
2000mL beaker.
o Label the beaker with autoclave tape and state what media is being prepared, the date, and your initials
(i.e. Nutrient Broth 8-18-08 KH).
o Add powder to beaker first, and then fill with necessary amount of water.
o Stir with a glass stirring rod to mix.
o Place in microwave and heat at 3-5 minute intervals.
o Stir between intervals, using caution and allowing the media to sit for 30 seconds in the microwave
before stirring.
o Heat for approximately 10 minutes or until boiling has been achieved.
o While heating, place test tubes in racks and label with autoclave tape (i.e. Nutrient Agar Slants 8-18-08
KH).
o Test the pH of the media to insure that it is within the acceptable range as stated on the package. If the
pH needs to be adjusted, add drops of 1N Hydrochloric Acid (HCl) (to make more acidic) or 1N Sodium
Hydroxide (NaOH) (to make more basic) as necessary until desired pH is achieved.
o Pour the broth into an appropriately sized glass bottle for pipette dispenser use.
o Before attaching the pipette dispenser, set it to the proper setting for the volume of media required for
each tube.
o Attach the dispenser to the bottle top.
o While over the sink, test the dispenser to ensure that the liquid media is filling the pipette dispenser.
o Fill each tube.
o Place caps on filled tubes.
For screw caps, leave the caps partially unscrewed to allow steam to enter and escape.
o Autoclave for 20 minutes. Refer to the Standard Operating Procedure for Autoclave Use.
o After sterilization is complete, remove the test tube rack, tighten all test tube caps, and tilt the tubes of
liquid agar on a support that is about inch thick (Plastic weighing dishes work well) or use special white
slant racks.
o Allow the agar to solidify (about 25 minutes) and store in the refrigerator.
Marywood University Standard Operating Procedure Microbiological Media Preparation

First Aid
In case of contact with eyes, immediately wash the eyes with large amounts of water for 15 minutes,
while holding eyelids open. Get medical attention immediately. If contact lenses are worn remove them
immediately.
In the event of skin contact, wash the area thoroughly.
In the event of skin contact with a hot beaker, seek medical attention.
Report any incident to the laboratory manager.
Disposal Requirements
Spills can be cleaned up with a paper towel and disposed of in the trash. Use caution with hot media.
Unused plates can be disposed of in the waste to be autoclaved trash cans.
See the microbiological waste SOP for further instruction on waste disposal.
References:
1. Techniques in Microbiology: A Student Handbook, Lammert