1.19: Pouring Agar Plates

Learning Objectives

Goals:

• Learn the basics of aseptic technique.
• Learn to prepare sterile agar plates for growing bacteria

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

• Practice aseptic technique.
• Sterilize and pour agar plates by hand

Introduction

Microbes are all around us. In lab 4, our class sampled various surfaces and found that microbes were easily found everywhere in the environment. We used sterile agar plates that provided the nutrients and correct pH for bacteria to grow. In this lab, you will be learning to produce these sterile agar plates.

Agar is a polysaccharide derived from red algae. The agar powder is first dissolved in a boiling liquid, and then cooled to form a gelatinous solid matrix. As microbes cannot digest agar, this material is used commonly in laboratories to hold the nutrients that bacteria need.

The main instructions for pouring agar plates are presented here. But there are many different recipes to prepare growth media for bacteria, as some bacterial species require different combinations of nutrients. Some types of common agar include blood agar, Luria Bertani (LB) agar, MacConkey agar, nutrient agar (NA), and tryptic soy agar (TSA). Follow the specific package instructions regarding the amounts of powder and water to use for the growth media you are making. The recipe to make 1-liter LB agar is 9.1 g tryptone 4.6 g yeast extract, 4.6 g NaCl and 13.7 g agar. If an antibiotic additive is needed in the medium recipe, that is added after the sterilized agar has cooled to 60oC to avoid denaturation.

An autoclave is a high-pressure apparatus that is used by laboratories, dentists, and hospitals to sterilize equipment, instruments, glassware, growth media, liquids and biohazardous waste. The autoclave applies high pressure (15 psi) and saturated steam at 121oC (250oF) for 15-20 minutes to kill microbes and spores. After the media has gone through this cycle, it is sterile. Cool to 60-65oC before adding any antibiotics and pouring into sterile Petri dishes.

Figure 1. Digital Balances and Weigh Boats

Materials

• disinfectant spray
• paper towels
• petri dishes
• gloves
• LB agar powder
• Weigh boat
• Stir bar
• 500 mL autoclavable bottle
• Autoclave tape
• Sharpie marker (colored)
• Deionized or distilled water
• Electronic balance
• Autoclave or pressure cooker
• Metal tray
• Ampicillin or arabinose

Figure 2. Some supplies for preparing plates with media

Procedure

Preparing the media

1. Label a clean glass autoclavable 500 mL autoclavable bottle with media name, date, and initial.
   • Note: only fill bottle halfway, to avoid overflow during the heating process in the autoclave.
2. For a 500 mL bottle, calculate the needed weight of powdered media to make 250 mL. Subtract that from 250 to determine the volume of water to add.
3. Add __ mL of distilled water to the bottle.
4. Add __ g of media with agar powder to the same bottle. (your total should be 250 mL)
5. Add a stir bar (optional). Stir or shake until fully mixed and check that there are no lumps.
6. Add a piece of autoclave tape to the cap or bottle and loosen the cap a half-turn. If using a container with no cap, then cover loosely with aluminum foil

Setting up the autoclave

1. Place the prepared media bottles into a metal tray.
2. Add distilled water until it covers the bottom of the tray; about 1-2 cm deep.
3. Place into the autoclave.
4. Autoclave at 121^oC for 15 minutes at 15 psi.
5. Once the cycle is complete, wear heat-resistant gloves to remove the tray and bottles from the machine.
6. Allow bottles to cool to approximately 60^oC.

Prepping the workspace:

1. To keep as sterile of an environment as possible to avoid contaminating the media and plates, don a lab coat and gloves, and use a disinfecting agent or wipes to wipe down all surfaces.
2. This includes tabletops and edges, gloves, scissors, permanent markers, etc.
3. Make sure to clean your gloves if they have touched another surface that is not disinfected (eg.if you touched face, arm, chair, etc.).
4. If available, use Bunsen burners and carefully pour near the open flame to better prevent airborne contaminants.
5. Once the area is disinfected, bring out the sterile Petri dishes. Keep the sterile dishes closed.
6. Stand a bag up vertically.
7. To conserve the plate bag to reuse for storage, ignore any “Tear Here” markings, and snip a small corner off the top of the bag. Insert half of the scissors into this opening and cut along the crease of the bag.
8. Flip the entire stack of plates upside down, with the cut opening at the very bottom of the stack.
9. Gently apply a small amount of pressure downward while simultaneously rolling the bag up.
10. Fold or roll up the empty bag and put aside to use for re-bagging.

Striping the plates

1. To quickly differentiate between plates that have a similar appearance, a stripe code can be used. A combination of different colored permanent markers can signify different additives to a plate. For example, a green stripe may mean ampicillin, a type of antibiotic, was added to the media in that plate. Striping codes are specific to an individual lab, so always double check the code key.
2. To stripe a plate, the top AND bottom must be labeled with the code.
3. Take the marker with the color in the key, and with the unbagged stack of plates, apply a gentle amount of pressure with your hand onto the top of the stack.
4. Draw a line straight down from the top edge to the base of each plate in the stack.
5. If done quickly, you may need to go back in and redraw the line on the bottom base.
6. If the stripe code has another line or color, repeat the process by adding another line.
7. The spacing of the second line should be within 1 cm from the first line.
8. Repeat the process until the stripe code is complete.

Setting out the plates

1. Begin to unstack the plates. Make sure the plate tops and bottoms do not separate.
2. Place individual plates around the edge of the table (not in stacks), to create a line or chain of plates.

Figure 3: Pouring Plates

Pouring the Plates

1. Once the media has cooled to 60 o C, the liquid solution is ready to be poured.
2. At this time, an antibiotic (ex: ampicillin) may be added to the media and gently swirled or stirred to mix. Note: do not add the antibiotic if the liquid is hotter than 60 o C, as the antibiotic would be denatured.
3. Uncap the media bottle and hold the bottle in your dominant hand. Note: once the bottle is opened, do not talk. Talking will allow bacteria from your mouth to become airborne and may contaminate the media.

4. The cap can be held in the same hand (between fingers) as your bottle or can be placed on a disinfected surface.
5. Grab a plate with your other hand and slide it towards the edge of the table, while keeping it closed.
6. Once the plate is at the edge, open the lid as if there is an imaginary hinge at one end; so the plate opens like a clamshell.
7. Pour the media into the bottom of the plate until it just covers the surface. Do not over fill.
8. Close the lid and allow to cool. The media will be solid.
9. Leave the plates out for a day if possible so the condensation will evaporate from the plate. You may place the plates in a 25C incubator overnight.
10. Stack plates with the same type of media and slide the plastic sleeve over the top.
11. Flip the stack over and seal the plastic sleeve with masking tape.
12. Label the tape with the type of media, date produced, and name of individual that produced the stack.
13. Store sealed stacks in the refrigerator until use.

STUDY QUESTIONS

1. What is the typical amount of agar included in 1 L of media?
2. Why must one take such precautions to disinfect the space and avoid actions that may cause bacteria and mold to become airborne? What is the term applied to these precautions and procedures?
3. Does agar provide nutrients for bacteria?
4. Why can’t you include ampicillin in the media before it is autoclaved?
5. Why must you leave solidified plates out for a day before you seal them and refrigerate them?