Laboratory Media

Microbiology media are vital in cultivating, isolating, and studying microorganisms. These media provide the necessary nutrients, moisture, and environmental conditions that microorganisms require to grow. Broadly, there are two main types of microbiology media: broth and agar, each serving unique purposes in the laboratory. Broth media are liquid nutrient solutions without a solidifying agent, like agar. They are primarily used to propagate large quantities of microorganisms quickly and allow for turbidity measurements, where the cloudiness of the broth indicates microbial growth.

History of Agar

The history of agar in microbiology is closely tied to the development of modern microbiological techniques and the study of microorganisms. Agar, a gelatinous substance derived from red algae (particularly from the genera Gelidium and Gracilaria), became a crucial component in microbiological media due to its unique properties.

Before agar, other substances like gelatin were used as solidifying agents in culture media. Gelatin, however, had several drawbacks. It melts at around 37°C (body temperature), which is a problem when cultivating human pathogens that need to be incubated at this temperature. Additionally, many bacteria produce enzymes that degrade gelatin, leading to liquefaction of the media and making it unsuitable for microbial growth studies.

Agar was introduced to the world of microbiology in the late 19th century. The use of agar in microbiology is credited to Fanny Hesse, the wife of Walther Hesse, a German physician and one of Robert Koch's assistants. Fanny Hesse suggested the use of agar as a solidifying agent in culture media around 1881. She had previously used agar in her kitchen for making jellies and desserts, and she realized that it remained solid at higher temperatures and wasn’t degraded by bacteria. Walther Hesse adopted this suggestion, and it quickly proved to be far superior to gelatin for cultivating microorganisms.

Media Types

Since broth media distribute nutrients evenly, microorganisms can grow throughout the medium, making them ideal for producing large cell populations. On the other hand, agar media are solid or semi-solid and contain agar, a gelatinous substance derived from seaweed, as a solidifying agent. These media are particularly useful for isolating individual colonies of microorganisms from mixed cultures, studying colony morphology, and performing biochemical tests. The solid surface provided by agar allows for easy observation of microbial colonies, making it an essential tool in many assays. Agar media come in various forms, such as plates, slants, and deeps, each with specific applications.

Agar plates are used to isolate pure colonies and conduct tests like antibiotic sensitivity. Slants, where the agar is solidified at an angle in a test tube, are commonly used for long-term culture storage and to observe aerobic growth at the surface and anaerobic growth at the bottom. Deeps, which are upright test tubes filled with agar, are used to assess the oxygen requirements and motility of microorganisms. The versatility of agar media in these different forms makes them indispensable in the microbiology lab.

Examples of common media include nutrient broth, a general-purpose medium for growing a wide range of microorganisms, and tryptic soy broth, another versatile medium. Agar-based media include nutrient agar for general growth. There are various media types that provide microbiologists with the tools needed to study microorganisms in great detail, from their growth patterns to their biochemical characteristics.

·         General-purpose media are designed to support the growth of a wide range of microorganisms, especially non-fastidious organisms that do not have specific nutritional requirements. These media are commonly used for routine cultivation and maintenance of bacterial cultures in the laboratory.

o    Nutrient Agar (NA) and Tryptic Soy Agar (TSA), which provide the essential nutrients for a variety of bacteria and fungi.

·         Selective media contain specific agents that inhibit the growth of certain microorganisms while allowing others to thrive, making them ideal for isolating particular groups or species from mixed cultures. They are widely used in clinical and environmental microbiology to detect and isolate specific pathogens.

o    MacConkey Agar, for instance, selectively allows Gram-negative, lactose fermenting, bacteria to grow by inhibiting Gram-positive bacteria.

o    Mannitol Salt Agar (MSA) is selective for Staphylococcus species due to its high salt concentration.

·         Differential media are formulated to distinguish between different types of microorganisms based on their biological characteristics, such as their ability to ferment certain sugars or produce specific enzymes. These media are crucial for differentiating and identifying microorganisms based on their metabolic activities or colony characteristics.

o    Blood Agar (BA) is a prime example, differentiating bacteria based on their hemolytic activity.

o    Eosin Methylene Blue Agar (EMB) differentiates lactose fermenters from non-fermenters through color changes in the colonies.

·         Enriched media are supplemented with additional nutrients like blood, serum, or specific growth factors to support the growth of fastidious organisms that require more demanding nutritional conditions. These media are particularly useful in clinical settings for isolating pathogenic bacteria from patient samples.

o    Chocolate Agar, enriched with lysed red blood cells, is used to grow organisms like Haemophilus influenzae and Neisseria species.

o    Thayer-Martin Agar is specialized for isolating Neisseria gonorrhoeae and Neisseria meningitidis.

·         Anaerobic media are designed to support the growth of anaerobic organisms, which require environments free of oxygen. These media often contain reducing agents to lower oxygen levels, making them essential for growing microorganisms that thrive in oxygen-deprived environments, such as those found in deep tissues or the gastrointestinal tract.

o    Thioglycollate Broth, for example, creates an oxygen gradient that allows for the growth of both aerobes and anaerobes depending on their oxygen needs.

o    Bacteroides Bile Esculin Agar (BBE) is selective for anaerobic Bacteroides species in clinical diagnostics.

·         Specialized media are tailored for specific purposes, such as isolating particular pathogens, detecting specific biochemical traits, or cultivating specific groups of microorganisms. These media are commonly used in research and diagnostic settings.

o    Sabouraud Dextrose Agar (SDA) is formulated with a low pH to selectively grow fungi and yeasts while inhibiting bacterial growth.

o    Lowenstein-Jensen Medium is used for cultivating Mycobacterium tuberculosis due to its enriched formulation.

o    Campylobacter Agar (Skirrow’s Medium) is another specialized medium used to isolate Campylobacter species from stool samples, thanks to its antibiotic content that suppresses other bacteria.

Lab Exercise

Objective

To help prepare for future labs you are going to make the media that everyone will be using.  Making broths, agar plates, deeps, and slants involves preparing the media, sterilizing it, and then pouring or inoculating it into the appropriate containers.  Follow the procedures below to produce various types of media, check with your instructor as to which medias will be made.

Materials

·         Select Media

·         Test Tubes with lids

·         Petri Plates (various sizes)

·         Hot Plates

·         Stir Bars

·         Autoclave

·         Pipettes

Bacteriological Culture Methods - Media Prep

·         Quantity

o    Broths 48

o    Agar

o    Plates 48

o    Slants 24

o    Deeps 24

Procedure for Tryptic Soy

Broths

·         Suspend 7.5g of Tryptic Soy broth powder in 250ml of (hot 80 degrees Celsius) RO water into a 500ml Erlenmeyer Flask.

·         Mix and dissolve the agar in the water completely until homogeneous (consistent throughout).

·         Pipet 5ml of the liquid into the test tubes, put on the caps loosely, and place in test tubes in an upright rack.

·         Sterilize by autoclaving at 121°C for 15 minutes.

Agar Plates

·         Suspend 30g of Tryptic Soy agar powder in 750ml of (hot 80 degrees Celsius) RO water into a 1L Erlenmeyer Flask.

·         Mix and dissolve the agar in the water completely until homogeneous (consistent throughout).

·         Sterilize by autoclaving at 121°C for 15 minutes.

·         When cool enough to handle, pour the liquid into the petri dish (about half full) replace lid and wait for the medium to solidify. Be sure that you are preparing the agar in the clean environment to prevent any contamination.

Agar Slants

·         Suspend 4g of Tryptic Soy agar powder in 100ml of (hot 80 degrees Celsius) RO water into a 250ml Erlenmeyer Flask.

·         Mix and dissolve the agar in the water completely until homogeneous (consistent throughout).

·         Pipet 4ml of the liquid into the test tubes, put on the caps loosely, and place the test tubes in the slanted rack.

·         Sterilize by autoclaving at 121°C for 15 minutes.

Agar Deeps

·         Suspend 6g of Tryptic Soy agar powder in 150ml of (hot 80 degrees Celsius) RO water into a 250ml Erlenmeyer Flask.

·         Mix and dissolve the agar in the water completely until homogeneous (consistent throughout).

·         Pipet 5ml of the liquid into the test tubes, put on the caps loosely, and place in test tubes in an upright rack.

·         Sterilize by autoclaving at 121°C for 15 minutes.