Background Information

WHY YOU SHOULD CARE ABOUT: Growing Microbes

Making Media

Making media is nowadays as easy as making a cake from a package mix! Premixed media for a vast variety of organisms can be purchased ready made, or already as agar plates with often new and proprietary ingredients. Growing microbes is a sophisticated undertaking, starting about 200 years ago, realizing that different types of Microbes need the right kind of media, specific supplements and the right environment (oxygen, pressure, nutrients, etc) to grow.

The newer approaches to grow unculturable microbes, like microbes that can grow on mars or the myriad of microbes that live in our intestines, are creative and innovative and pushing the envelope compared to traditional methods. We still need to learn a lot more to culture and understand many more of the less explored microbes.

I took this image when I visited “Micropia”, the coolest Microbiology Museum in Amsterdam.

– Dr. Oli

For your reference, download and familiarize yourself with the DIFCO/BBL manual. This is an amazing resource about the huge variety of media available to microbiologists! You may be asked to use this manual when answering questions, so having a copy available will be handy!

A more novel way of growing microbes is by using a specialized agar, chromagar, where specific pathogens grow and develop a unique color, making it super easy to identify a pathogen very quickly. For more information check out the Chromagar website.

Microorganism Nutrient Requirements

• Proper nutrients must be available.
• Moisture is necessary.
• The medium must have an appropriate pH.
• ·The medium must be free of interfering bioburden (competing organisms)
• Contamination must be prevented.
• A satisfactory microbiological culture medium must contain available sources of:
  • Amino-Nitrogen Peptone, protein hydrolysate, infusions and extracts
  • Growth Factors Blood, serum, yeast extract or vitamins, NAD
  • Energy Sources Sugar, alcohols and carbohydrates
  • Buffer Salts Phosphates, acetates and citrates
  • Mineral Salts and Metals Phosphate, sulfate, magnesium, calcium, iron
  • Selective Agents Chemicals, antimicrobials and dyes
  • Indicator Dyes Phenol red, neutral red
  • Gelling agents Agar, gelatin, alginate, silica gel

Microorganism Environment Requirements

• Proper temperature relations must prevail.
  • mesophilic bacteria and fungi have optimal growth at temperatures of 25-40°C.
    • Human pathogenic organisms are generally mesophiles
  • thermophilic (“heat loving”) organisms grow only at temperatures greater than 45°C.
  • psychrophilic (“cold loving”) organisms require temperatures below 20°C.
• Oxygen or other gases must be available, as required.

Functional Types of Culture Media

• Non-Selective Media. General and multipurpose media that incorporate a wide variety of nutrients and growth factors for general cultivation of most bacteria. Examples include Brain heart infusion medium (BHI), Tryptic spy medium (TSA) Nutrient agar/broth (NA, NB)
• Enrichment Media. Microorganisms to be analyzed are often outnumbered or outcompeted by other microbes (especially in food, water, and soil). Enrichment media increase the number of target microorganisms while suppressing the growth of competing microflora. Specific enrichment media exist for listeria, vibrios and other pathogens.
• Selective Media. Generally, support the growth of several species, but also include inhibitory agents, which restrict the growth of undesired microorganisms. Examples include MacConkey agar, which includes bile salts and crystal violet to suppress growth of most Gram-positive organisms or Mannitol salt agar, which has a high salt concentration that prohibits growth of salt intolerant organisms
• Differential Media. Contains reagents (usually dyes) that react with pH changes or other metabolic consequences of growth to produce reactions that are indicative of particular species. Colonies will have species-specific characteristics, which are easily recognized. Examples include blood agar plates, mannitol salt agar and MacConkey agar.
• Media for fastidious organisms. Only about 1% of the bacteria in nature has been cultured, most organisms cannot be grown because their growth requirements are unknown. Media for organisms that are harder to grow and have more sophisticated nutritional requirements (=fastidious organisms). Examples include blood agar and chocolate agar.
• ChromAgar ® is a newer and very popular approach for more rapid diagnosis of pathogens. Proprietary reagents and chromogenic compounds are in the pre-prepared agars, which allows presumptive identification of specific pathogens. Examples include specific Chromagar to identify MRSA, urinary pathogens, pathogenic yeast, toxigenic E. coli and others.
• Defined media and Undefined (complex) media. All media containing complex carbon, nitrogen or sulfite sources derived from yeast, casein, tissues etc. are considered undefined or complex media as the exact composition of the nutrients is not known. In defined media the molar concentration of each component is known. Most media described above are undefined media. Examples of defined media includes Minimal salt medium
• Transport media. After a sample is collected from a patient, the sample must be transported to the laboratory for analysis. Often this process can take up to 24h. The viability of the potential pathogen must be preserved to allow for growth on specific media. Transport media contain preservatives and minimum amount of nutrients and pH stabilizers (buffers) which preserve the viability of the organisms in the sample.

Media Ingredients

• Purified water is recommended for use in the preparation of culture media. As defined by the United States Pharmacopeia, Purified Water is water obtained by a suitable process, complying with the U.S. Environmental Protection Agency National Primary Drinking Water Regulations.
• Peptone, protein hydrolysates, infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material, e.g., meat, milk.
• Carbohydrates are employed in culture media as energy sources and may be used for differentiating genera and identifying species.
• Buffers maintain the pH of culture media.
• Selective Agents include bile salts, dyes and antimicrobial agents. Bile salts and desoxycholate are selective for the isolation of gram-negative microorganisms, inhibiting Gram positive cocci.
• Dyes and indicators are essential in the preparation of differential and selective culture media. In these formulations, dyes act as bacteriostatic agents or indicators of changes in acidity or alkalinity of the substrate.
• Antimicrobial agents are used in media to inhibit the growth of bacteria, yeasts and fungi.
• Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state.
• Chromogens and fluorogens are substrates incorporated into culture media to allow organism differentiation and identification. When these substrates are degraded by specific enzymes, they release differently colored or fluorescent compounds

History Connection

The Petri Dish is named after the German physician and bacteriologist Julius Richard Petri (1852-1921). He was the assistant to Robert Koch (1843-1910, “Koch’s postulates”). It may not seem a big invention to you, but growing microbes on a solid surface were the beginning of isolating organisms and growing them in a pure culture so that they can be studied in detail. Before everything was grown as (mixed) culture in broth. Agar is the solidifying component that makes Petri plates solid, replacing gelatin that was used before. Agar is derived from algae. The American born microbiologist, Fanny Hesse, worked alongside her husband in the lab of Robert Koch in Berlin. Fanny suggested the use of agar over gelatin as agar would not become liquid at 35oC (95oF).

Virtual Lab Simulations

BioMan Biology: Bacterial Growth Virtual Lab

Online Lab

Activity: Making Nutrient Agar Plates

Video Tutorials

Introduction to DIFCO-BBL Manual

Making Nutrient Agar Plates

Procedure

1) Making Nutrient Agar Plates

1. Perform calculations to determine how much powdered media to use:
   1. If each plate holds 25 mL agar, how much agar is needed for 5 plates?  (A: 125 mL agar)
   2. To make 1 L (1000 mL), you would use 40 g of nutrient agar media mixed into 1 L of water.
   3. To make 125 mL agar,  how many g of media would you need? (A: 0.125 L (125 mL) water x 40g agar = 5 g agar media)
2. Use the weigh boat to measure out 5 g of nutrient agar using your scale (make sure to zero it first).
3. Using your scale to measure out  125 mL of water in a microwavable cup.
   1. Set your microwavable cup on your scale and tare it.
   2. Add water to your cup until the scale reads 25 g.
   3. Measure out 25 g 5x so you have 125 mL total (NOTE: the scale has a limit so you need to weigh out 5 amounts of 25mL; 1 g = 1 mL)
4. Heat the water for 30 seconds- a minute in the microwave until boiling. BE CAREFUL NOT TO BURN YOUR HANDS WHEN WORKING WITH HOT WATER AND MEDIA.
5. Immediately mix in the powdered media and swirl the container with the tongue depressor.
6. Once cooled down to almost room temperature, pour roughly 25mL into each plate.
7. Once the media has become solid, invert and refrigerate all five plates to prevent contamination..
8. Keep the plates made for the Isolating Microbes module.

Results

In-Person Lab

Procedure: Growing Microbes

Results

Resources

• Quizlet Link: Take a look at the appropriate module for the vocabulary that is being tested: Microbiology @ UF Lab Terminology Quizlet.
• DIFCO/BBL manual
• ATCC Introduction to Microbiology
• 9.1 How Microbes Grow
• 9.6 Media Used for Bacterial Growth.
• ATCC Bacteriology Culture
• Chromagar website
• Brief explanation how to use our autoclave and also check out how to operate a professional autoclave