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Petri Dishes with Agar: How to Make Agar Plates

Reference to this article: ConductScience, Petri Dishes with Agar: How to Make Agar Plates (2022). doi.org/10.55157/CS20220627

Petri dishes and agar are essential components of the culture plate technique developed by Robert Koch. The microbiological technique enabled him and subsequent biologists to isolate, grow, and study microorganisms in laboratories to advance our understanding of bacteriology, mycology, infectious diseases, epidemiology, food science, and biotechnology.[1]

What Are Agar Plates?

Agar plates on a lab's workbench

Agar is a type of natural polysaccharide isolated from red seaweed. It is mixed with a liquid culture medium, which is heated, melted, and solidified into a solid medium. Agar was first used in the 19th century to replace gelatin, the first gelling agent. And soon after, it became the go-to gelling agent for solid culture medium.

Unlike gelatin, agar has a higher melting temperature of about 85°C and starts to solidify when the temperature drops to 42°C. Agar is firm and remains solid if the room temperature has not risen to the melting temperature.

Solidified agar is non-sticky and transparent, allowing users to observe and identify microbial colonies grown on the solid media. Also, microorganisms such as infectious human pathogens, bacteria, yeasts, and fungi, cannot metabolize solidified agar. As a result, the agar remains solid and does not disintegrate when the microbes grow and expand their colonies.[1,2]    

Types of Agar Plates

Agar plates are broadly classified based on the media’s function as follows:[2]

1. Nonselective agar

Nonselective agar is the so-called growth media. Its primary purpose is to allow the inoculated microbes or the specimen of interest to grow without any selection pressure.

Based on the nutrient composition, we can categorize non-selective media into the following groups:[2] 

  • All-purpose media, commonly called nutrient agar, are used to inoculate, culture, and isolate microbes, cells, or specimens of interest. They contain nutrients from sources such as peptones and extracts from beef or yeast.
    • An example of all-purpose media is Lauria-Bertani (LB) agar, also known as a lysogeny medium. LB agar is a nutrient-rich standard media used for cultivating bacteria in most laboratories.
  • Enriched media are nonselective media containing supplemented nutrients from animal blood, serum, and other protein sources. Enriched media are used to culture or identify fastidious microorganisms, which do not grow on all-purpose media like most common microbes.
    • An example of fastidious microbes is Haemophilus species, which dwell in the mucous linings of the human upper respiratory tract. All Haemophilus species require a component of heme-hemoglobin, hemin, and nicotinamide adenine dinucleotide (NAD).
    • They can be grown on, for example, chocolate agar, Brucella agar, and Casman agar, which are enriched media supplemented with animal blood.[3]

2. Selective agar

Selective agar contains one or more selection agents that interfere with the metabolism of the organisms grown on the agar plates.

Selection agents are added to agar plates to prevent unwanted microbes from growing while allowing the target ones to thrive. They are categorized into two:[2]

  • Antibiotics and antiseptics inhibit the growth of unwanted microorganisms. They are added to agar plates to eliminate microbes that cannot metabolize antibiotics or antiseptics.
    • For example, genetically engineered bacteria are selected on nutrient agar plates containing one or more antibiotics. Another example is the combination of colistin and nalidixic acid to select for gram-positive bacteria. Both disrupt different metabolism pathways in gram-negative bacteria, killing them in the presence of antibiotics.[2-3]
  • Organic molecules and minerals can be added to agar plates to inhibit the growth of non-target microorganisms. Examples include specific amino acids, bile salts, sodium chloride, and mannitol.
    • An amino acid species can be excluded from yeast agar plates to select for a specific yeast strain. Bile salts, high concentrations of sodium chloride, and mannitol can be added to the agar plates to terminate microbes that cannot metabolize them.

Selective agar can also be differential or indicator media. This type of selective agar can distinguish and identify a specific group of microorganisms based on the appearance of the colonies or microbes’ reactions to the media. They are frequently used in clinical and non-clinical diagnostic applications.      

To learn more about different media types and their applications, check out our guide on culture media here.

What Are Petri Dishes?

Petri dishes are a set of two plates – a dish and a fitting lid. The dish is a shallow container where liquified agar media is poured and solidified. The lid is shallow and used to cover the dish so that the agar plate is protected from potential contaminations.[1]       

Originally, Petri dishes were cylindrical and made from colorless transparent glass, allowing users to observe the change in the number and appearance of the microbial colonies grown on the plates during the incubation period.

Nowadays, Petri dishes are available in many sizes and forms, including squares and rectangles. Most laboratories use disposable Petri dishes made from plastic, which is colorless, transparent, and hydrophobic. They are pre-sterilized, packaged, and ready to use.

Types of Petri Dishes

  1. Fully stackable Petri dishes have a raised rim. It is designed to prevent the dishes from slipping when liquified agar medium is poured in a stack-pouring manner. This Petri dish is handled manually and is not compatible with most automatic plate-pouring systems.         
  2. Semi-stackable Petri dishes, or slippable, beveled lid Petri dishes, have a slightly raised leveled ridge than fully stackable Petri dishes, which allow them to be used manually and automatically by plate-pouring machines.     
  3. Slippable Petri dishes are Petri dishes with a leveled ridge specifically designed for automatic plate-pouring systems. They are not suitable for the manual stack-pouring of agar plates.  

How to Make Agar Plates

Agar plates can be manually made by the stack-pouring method as follows:

Media Preparation

  1. According to the instruction, prepare the desirable culture media by dissolving the powder in deionized water.
  2. Add an appropriate amount of agar into the dissolved media, and fill up deionized water to the desired volume.
  3. Stir the medium until agar powder is fully incorporated.
  4. Pour medium into a clean autoclavable glass bottle. Leave at least one-fourth of the bottle volume to allow space for air bubbles. Close the cap loosely, label the bottle, and paste a piece of autoclave indicator tape before autoclaving. 
  5. Alternatively, the medium can be sterilized by filtering into an autoclaved glass bottle. 

Plate Preparation

  1. Suppose glass Petri dishes are used; clean and stack the dishes in a metal box or plastic bag. Autoclave and dry the dishes in a hot-air oven. In the case of disposable Petri dishes, remove the packaging. Only remove the lid of each dish under a sterile condition.
  2. Label the side or bottom surface of the dish where the molten media will be poured.
  3. Unstack the labeled Petri dishes, with the dish at the bottom and lid on top.

Stack-pouring

  1. After sterilization, warm/heat the solidified agar medium until it is liquefied using a microwave or water bath.
  2. When the liquified agar medium reaches 50 to 60°C, open the bottle in the laminar flow hood and add the sterilized supplement to the medium. Swirl gently after adding the supplement so that no air bubbles are formed. Be careful not to introduce any contamination.
  3. Hold the bottle containing the liquified agar medium with your dominant hand, and use the other hand to lift the Petri dish’s lid. If a precise media volume is required, use a sterile serological pipette to take up the volume of liquified media.
  4. Dispense the liquified media onto the side of the Petri dish. Gently swirl the media so that it covers the entire surface. If air bubbles are present in the agar plate, quickly pass the plates over the flame while the medium is liquified to get rid of the bubbles.
  5. Leave the lid slightly open in the laminar hood to let the agar plate solidify. Most agar plates will solidify within 30 minutes to one hour.
  6. When the agar plate has completely solidified, close the lid, and restack the agar plates.
  7. Store agar plates upside down in the refrigerator or cold room.

Alternative to hand preparation, agar plates can be prepared by an automated plate pouring system.

Briefly, sterile semi-stackable or slippable Petri dishes are stacked on the machine. Culture medium containing agar is prepared and sterilized before it is pumped and dispensed onto each Petri dish. Depending on the capacity of the automatic system, it can dramatically reduce the time and labor needed to prepare several agar plates.[4]    

Conclusion

Petri dishes and agar plates are the cornerstones of microbiology. They allow microbes to be grown, isolated, and identified in the laboratory.

Agar plates can be made by an automated machine using slippable or semi-stackable Petri dishes. They can also be made by hand using fully stackable or semi-stackable Petri dishes.

Get your quality and durable anaerobic fully stackable Petri dish here!

References:

  1. Das, N., Triparthi, N., Basu, S., Bose, C., Maitra, S., & Khurana, S. (2015). Progress in the development of gelling agents for improved culturability of microorganisms. Frontiers in Microbiology, 6(JUN). https://doi.org/10.3389/fmicb.2015.00698
  2. Lagier, J.C., Edouard, S., Pagnier, I., Mediannikov, O., Drancourt, M., Raoult, D. (2015). Current and past strategies for bacterial culture in clinical microbiology. Clinical Microbiology Reviews, 28(1). https://doi.org/10.1128/CMR.00110-14
  3. Zambro, M.J., Powers, D.A., Miller, S.M., Wilson, G.E., & Johnson. J.A. (2009).  Difco & BBL Manual, Manual of Microbiological Culture Media, 2 edition.
  4. Sharpe, A. N., Biggs, D. R., & Oliver, R. J. (1972). Machine for Automatic Bacteriological Pour Plate Preparation. Applied Microbiology, 24(1). https://doi.org/10.1128/am.24.1.70-76.1972
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