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How to Make Agar Plates?

How to Make Agar Plates?

Agar plates are an essential and very commonly used tool in microbiology. Their solid surface provides the perfect growing environment for microorganisms. The plates are made of a special growth medium containing nutrients and an energy source, and agar for the growth of microbes. Agar is a substance that keeps the mixture solid and gel-like. When microorganisms grow on agar plate, they form visible spots called colonies. Each colony develops from a single microbe and contains identical cells. Different microbes form colonies with unique shapes, colours, and textures. These help scientists identify them or check if a culture is pure.

How to Make Agar Plates?


The preparation of agar plates involves choosing a recipe and deciding how many plates will be needed. Making agar plates involves several steps:

  1. Sterilize the equipment – Before starting, sterilize all equipment like glassware and tools. This helps avoid contamination. Often, an autoclave is used to properly sterilize the materials in a wet heat sterilization process.
  2. Mix the agar solution – Mix the required amount of agar powder with distilled water and heat until it dissolves. After that, cool the solution to about 45-50 °C so it can be poured without overheating. It is recommended to prepare the agar fresh when starting the experiment rather than preparing stock. This is because the agar solution needs to be stored at a specific temperature. On top of that, it is not recommended to reheat the solution more than a couple of times.
  3. Add nutrients – Nutrient agar contains things like proteins, carbohydrates, and minerals that help microbes grow. Sometimes, special ingredients can be added to help identify or study specific types of microorganisms. Among others, these include glucose, egg yolk or antibiotics. The type and amount of nutrients within an agar solution depends on the type of microorganism.
  4. Pour the agar into plates - Once cooled, pour the agar solution into plates and leave it to solidify. It's important to pour the right amount and avoid bubbles to achieve the best results.
  5. Label the plates – Label each plate with details like the type of agar, the microorganism it’s for, and any specific conditions like pH and temperature.

Materials

Basic Equipment:

  • Agar plates - pre-prepared or freshly poured and set.
  • Inoculation loop - sterile metal or disposable plastic loop for transferring microorganisms.
  • Bunsen burner - to sterilise the metal loops.
  • Sterile pipettes / micropipettes & tips

Additional Sterile Handling Tools

  • Gloves & lab coat - for sterility and personal protection
  • Ethanol (70%) or another disinfectant
  • Tape - to seal plates for incubation

How are Agar Plates Used?


Agar plates are used to grow microorganisms by introducing a sample onto the surface. This is called inoculation, and it must be done in a clean, sterile environment, such as the laminar flow hood, to avoid contamination from airborne particles, microbes, or unsterile equipment. Depending on the goal of the experiment, different inoculation methods can be used. Selecting the correct plating method helps researchers to achieve the goal of their experiment. Whether it be the isolation of pure cultures, counting microbial populations or studying growth patterns.

Streak Plate Method

The streak plate method is primarily used to isolate individual colonies from a mixed sample. This technique allows microbiologists to separate different species or obtain pure cultures for further study by progressively diluting the bacterial load across the agar surface. During this technique, as the sample is spread, fewer and fewer microbes remain in each section, leading to the formation of distinct, isolated colonies that can be studied further.

Procedure:

  1. Sterilization: Sterilize a metal loop using a flame from Bunsen burner until it glows red. This gets rid of any residual microorganisms. Allow it to cool briefly.
  2. Inoculation: Dip the loop into the microbial sample, ensuring it picks up a small amount of bacteria.
  3. First streak: Drag the loop lightly through the first section of agar plate in a zig zag pattern.
  4. Flaming and cooling: Sterilize the loop again, allow it to cool, and streak another section of the plate by dragging microbes from the first section into the second.
  5. Sterilize and repeat: Repeat this process for additional sections to spread the sample and dilute it across the plate.
  6. Incubation: Once streaking is done, close the plate and incubate under appropriate condition to allow the colonies to grow.
Stages of the streak plate method for using agar plates
Streak Plate Procedure

Pour Plate Method

The pour plate method is useful for counting viable microorganisms and analysing microbial populations. Unlike streaking, this method involved suspending microbes within the agar, allowing for the growth of both surface and subsurface colonies. This method is particularly useful for quantitative studies, as colonies within the agar represent embedded bacteria, while those on the surface arise from organisms that prefer aerobic conditions (oxygen is present).

Procedure:

  1. Agar preparation: Prepare a molten agar solution and let it cool to 45°C. This helps avoid killing the microbes.
  2. Dilution (if necessary): Dilute the microbial sample if necessary to ensure colonies will grow without overcrowding.
  3. Mixing: Mix a measured amount of the microbial sample with the agar solution in a sterile container.
  4. Pouring: Pour the mixture, carefully, into an empty sterile plate, covering the bottom evenly.
  5. Solidification: Allow the solution to solidify at room temperature.
  6. Incubation: Incubate under appropriate condition.
Stages of the pour plate method for using agar plates
Pour Plate Procedure

Spread Slate Method

The spread plate method is used for estimating microbial population size and ensuring even colony distribution across an agar plate. This technique is often employed when working with liquid samples that require uniform spreading. The spread plate method is widely used for colony-forming unit (CFU) counting, as each distinct colony represent a single viable microorganism from the original sample.

Procedure:

  1. Sample transfer: Use a micropipette to transfer a small, measured volume of the microbial sample onto the agar surface.
  2. Sterilization: Sterilize a glass or metal spreader by passing it through a flame.
  3. Spreading: Gently spread the liquid across the entire agar surface in a circular motion, being careful not to tear the agar.
  4. Drying: Allow the plate to dry slightly before incubating it under appropriate conditions.
  5. Incubation: Incubate under specific conditions for the colonies to grow.
Steps of the spread plate method for using agar plates
Spread Plate Procedure

Dilution Plate Method

During the dilution plate method, also known as the serial dilution method, the sample is diluted step by step, and small amounts are transferred to plates. This technique is often used when analysing complex microbial communities as it helps count microbes and identify different types in a sample by reducing the concentration in a step by step manner.

Procedure:

  1. Dilution series: Prepare a series of sterile dilution tubes, each containing a set volume of sterile diluent.
  2. Initial dilution: Add a measured amount of the microbial sample to the first tube and mix thoroughly.
  3. Serial dilution: Transfer a small amount from the first tube to the second tube and mix again, repeating this process to create a series of increasingly diluted samples.
  4. Plating: Use a micropipette to transfer a small volume of each dilution onto a separate agar plate.
  5. Spreading: Use the spread plate method to evenly distribute the sample on the agar surface.
  6. Incubation: Incubate the plates under appropriate conditions.
Steps of the dilution plate method for using agar plates
Dilution Plate Procedure

Agar Plate Preparation in a Laminar Flow Hood


A laminar flow hood helps in prevention of contamination thanks to the HEPA-filtered air over the workspace. Preparing agar plates in a laminar flow hood ensure that only the desired microbes grow on the nutrient filled gel. To achieve the aseptic environment, turn the hood on for 10-15 minutes before starting the inoculation. Wipe down the work area including the required materials with 70% ethanol or another disinfectant to remove contaminants. To minimize the exposure to contaminants and protect yourself, wear gloves and a lab coat. Keep the plates closed outside of the laminar flow hood.

Key details to remember:

Check MarkAlways work inside the hood's sterile airflow zone.

Check Mark

Minimize talking and coughing while you work.

Check Mark

To maintain the optimal performance, check the laminar flow hood regularly.

Limitations of Agar Plates


While agar plates are very useful, they have some limits:

  • Limited nutrients – not all microorganisms can grow on agar plates because they might need specific nutrients that aren’t included.
  • Can’t grow some microorganisms – some microbes can’t be cultured on agar because they have very special growth requirements.
  • Small growth space – agar plates have limited surface area, so they can only grow a small number of microorganisms at a time.
  • Not like natural environments – agar plates are many simples than real-world conditions, so they might not show how microorganisms behave in nature.
  • Can't study certain behaviours - some experiments, like studying how microorganisms move in 3D space, can't be done on flat agar plates.

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References


  1. K.N. Kragh, M. Alhede, M. Rybtke, C. Stavnsberg, et al The inoculation method could impact the outcome of microbiological experiments Applied and Environmental Microbiology, ASM Journals, 2018
  2. S.J. Jo, Y.M. Lee, K. Cho, et al Standardization of the agar plate method for bacteriophage production MDPI, vol. 14, issue 1, 2024

Contributors


Written by

Linda Vidova, MSc.

Scientific Writer

 

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