Culture Media Preparation Tips

Types of Culture Media

Written by MicroDok

GENERAL PURPOSE MEDIA

General purpose media or basic medium is a routine culture media which is used for the cultivation of microorganisms in the microbiology laboratory. General purpose media can also be called simple or basal culture media. They are basically used for the culturing of bacteria that do not need extra growth nutrients, and are not fastidious in nature. General purpose media support the growth of a wide variety of bacteria, and they do not contain any growth inhibitory substance. Commonly used general purpose media in the microbiology laboratory include nutrient agar, nutrient broth, peptone water, tryptic soy broth, tryptic soy agar, Mueller-Hinton broth and Mueller-Hinton agar amongst others. Mueller-Hinton (MH) agar is the best medium for conducting antimicrobial susceptibility testing (AST) in the microbiology laboratory (Figure 1).

ENRICHMENT AND ENRICHED MEDIA

Enrichment media are usually liquid media or broth that supports the growth of a particular bacterium while inhibiting the growth of unwanted bacteria. Typical example of an enrichment medium is the Selenite F broth culture medium which is used for the culture of faecal specimens. Selenite F broth inhibit the growth of commensals or non-clinically relevant bacteria in feacal specimens prior to their subculture onto solid culture media plates. Alkaline peptone water is another example of an enrichment medium; and enrichment media are generally used to recover pathogens from feacal samples. Enriched media are culture media that also contain additional growth nutrients (e.g. blood, serum and egg yolk) like enrichment media for the cultivation and isolation of fastidious bacteria but unlike enrichment media, enriched media are mainly solid culture media ; and they are generally used to cultivate fastidious bacteria e.g. Streptococcus species and Haemophilus species. Blood agar and chocolate agar are examples of enriched culture media.

Figure 1: Illustration of Mueller-Hinton agar plates inoculated with test bacteria and single antibiotic disks prior to incubation for antimicrobial susceptibility testing (AST).

SELECTIVE MEDIA

Selective media are culture media that promote the growth of certain type of bacteria while inhibiting the growth of the undesired organisms. Such culture media contain inhibitory substances such as dyes, salts and antibiotics which prevent the growth of undesired microorganisms by suppressing them so that only the desired microbes will grow. Selective media used in the microbiology laboratory for the culture of microbes include mannitol salt agar (which contain NaCl that inhibit some bacteria), MacConkey agar (which contain bile salts and crystal violet that inhibit the growth of Gram-positive bacteria), Sabouraud dextrose agar (which contain antibiotics that inhibit bacterial growth) and Tellurite media (which contain potassium tellurite that inhibit many bacteria excluding Corynebacterium diphtheria).

Another example of a selective medium used for bacteriological investigation is the Lowenstein-Jensen media (that contains egg yolk) used for the isolation of Mycobacterium tuberculosis from mixed cultures or specimens. Sabouraud dextrose agar (SDA) and Salmonella-Shigella agar (SSA) are selective media used for the cultivation and isolation of fungi and enteric pathogens (e.g. Shigella and Salmonella) respectively. SDA is selective because it contains cycloheximide and chloramphenicol which inhibit the growth of saprophytic fungi and bacteria respectively while allowing only the fungi of interest to grow.    

DIFFERENTIAL MEDIA

Differential media are growth media that allow certain bacteria to have distinct colonies on the culture media. Organisms growing on differential media produce characteristic colonies that differentiate them from other group of microbes. They are also known as indicator media because they contain certain indicators (e.g. neutral red, chemicals and dyes) which changes in colour especially when the definite organism (i.e. the organism of interest) is present in the specimen being cultured. Unlike selective media which only encourage the growth of particular microbes, differential media differentiate between different groups of bacteria; and some culture media can serve as both selective and differential media.

Differential media or indicator media are also used for the presumptive identification of some bacteria. For example, MacConkey agar (MAC) is a differential media that help microbiologists to differentiate lactose fermenting bacteria (e.g. Escherichia coli) which ferments lactose to produce pink colonies on MAC from non-lactose fermenting bacteria (e.g. Salmonella) which does not ferment lactose and thus appear as pale or colourless colonies on the growth medium. Examples of differential media are cystein lactose electrolyte deficient (CLED) media, mannitol salt agar (MSA), MacConkey agar and blood agar amongst others.

TRANSPORT MEDIA

Transport media are used for transporting specimens or microorganisms from one location to another. They are mainly used in cases where the samples collected will not be cultured immediately after collection. Transport media provide all the nutrient and environmental factors necessary to preserve the samples and/or organisms en route to the laboratory where the formal investigation will take place. Of most importance is the fact that transport media prevent the overgrowth of commensals or contaminants in the collected samples Examples of transport media include Amies medium and Cary-Blair media.

STORAGE MEDIA

Storage media which may include a basic or general purpose media such as nutrient agar in slants or tubes are used to preserve and store microbial cultures for long period until they are needed for formal or further laboratory investigations. Chalk cooked meat broth and egg saline medium are typical examples of storage media used to store bacterial cultures in the microbiology laboratory. There are several reasons of storing or preserving microorganisms and maintaining them for many days, weeks, months or years in the laboratory under controlled conditions before reviving them again for futuristic use. Microorganisms including bacteria, fungi, protozoa and viruses can be preserved in different ways and in different types of media in order to study them in the future or to use them for research.

Microbes are preserved for epidemiological, microbiological, educational, and clinical purposes especially for the diagnoses of infectious diseases. Microorganisms can also be preserved for commercial purposes – in which unique strains of microbes are maintained in dormant but viable states to be sold for research purposes and other industrial usages, which are all geared towards enhancing research works in the field of microbiology. There are several organizations that specialize in the storage of microorganisms for commercial purposes; and these companies serve as sources for obtaining important strains of microbes that guide microbiological research in the industry, hospitals and in educational institutions.

The American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC) are typical examples of such companies that maintain microbes in a dormant but viable state for commercial purposes and other research purposes too. One of the simplest method of maintaining the viability of microorganisms especially bacteria in the microbiology laboratory is by a “periodic subculturing of the bacteria onto freshly prepared culture media preferably in nutrient agar slants made in McCartney or Bijou bottles”. In this crude technique of preserving microbes in the microbiology laboratory, bacteria to be preserved is subcultured from a culture media plate onto the slant of a nutrient agar bottle; and the organism is subcultured onto freshly prepared nutrient agar slants on a weekly or monthly basis – while ensuring that other environmental conditions such as temperature and pressure are maintained at optimal levels for the growth of the organism being preserved. However, this method though cheap and less cumbersome, may give room for mutation to occur in the organism being subcultured at intervals.

To counter this problem, there is several commercial maintenance or microbial preserving culture media in the open market that allows microbiologists to preserve their cultures over certain periods of time. These processes as described above only preserve microbes for short-term purposes. For long-term preservation of microbes, the process of lyophilization (freeze drying) is highly recommended for the preservation of microorganisms and other important laboratory working materials for many years. The concept of lyophilization or freeze drying is used for the long-term storage of microorganisms; and it removes volatile substances such as water from the material being stored and the preserved material is held under high vacuum in freeze-drying liquids like liquid nitrogen. Bacterial cultures are maintained at a very low temperature and in a dry state using the technique of lyophilization. When a culture is required for any microbiological purpose after freeze-drying, it is simply reconstituted with nutrient broth or distilled water – which resuscitate the dormant but viable organism to start growing again.

In terms of oxygen utilization, microorganisms (bacteria in particular) can be aerobic, anaerobic, facultative or microaerophilic. Aerobic bacteria grow in the presence of oxygen while anaerobic bacteria grow in the absence of it. Some bacteria can grow either in the presence or absence of oxygen, and thus are called facultative bacteria. Facultative bacteria utilize aerobic respiration in the presence of oxygen for growth; but in the absence of O2, they grow by anaerobic respiration or via the presence of fermentation. Microaerophilic bacteria are organisms that grow in the presence of a very low level of oxygen concentration. Bacteria that are microaerophilic in nature are often called microaerobes because they are capable of growing in the presence of oxygen which is lower than that in the atmosphere. The cultivation of aerobic bacteria in the microbiology laboratory is usually straightforward unlike anaerobic bacteria (e.g. Neisseria species and Streptococcus species) that usually requires anaerobic environment for their propagation. In the microbiology laboratory an anoxic environment (i.e., an environment that is devoid of oxygen) is usually provided for the cultivation of anaerobes using the anaerobic jar (Figure 2).

An anaerobic jar contains chemical substances provided by palladium catalyst or gas packs that remove molecular oxygen which might interfere with the growth of the organism as well as generate CO2 required for the unperturbed growth of the anaerobic bacteria. When gas packs are not available (i.e. when the conventional anaerobic jar with palladium catalyst pellets and methylene blue as anaerobic indicator as shown in Figure 2 is not readily obtainable in the laboratory), an anoxic or anaerobic environment can be provided or improvised in the microbiology laboratory by lighting a candle stick in an empty container that contain the cultured anaerobic bacteria (Figure 3).

As the candle burns, the concentration of oxygen in the jar is reduced until the candle light goes out to provide sufficient CO2 necessary for the organism’s growth. There also exists an anaerobic chamber for the cultivation of anaerobic bacteria in the microbiology laboratory (Figure 4). Anaerobic bacteria are bacterial organisms that exist or thrive in the absence of oxygen. They can also be called anoxic organisms since they do not require oxygen for growth. Anaerobic (anoxic) chamber is mainly used for culturing and handling anaerobic bacteria and/or cultures and oxygen-labile samples in the microbiology laboratory. An anoxic environment is an environment without oxygen present. 

Figure 2: Illustration of anaerobic jar. Anaerobic jar is mainly used for the cultivation of anaerobic bacteria in the microbiology laboratory. The presence of CO2 provides an anoxic environment that allows the anaerobic bacteria to thrive in the absence of O2. The palladium catalyst pellets combines with molecular O2 and hydrogen to remove the O2 so that anaerobic bacteria can grow. CO2 and H2 are produced when sodium bicarbonate and sodium borohydride (contained in the gas pack envelope) are mixed with water; and this is how the cultured organism get its carbon and energy requirements for growth.

Figure 3: An improvised anaerobic jar. It is used for incubating anaerobic bacteria in the microbiology laboratory; and this particular anaerobic container is usually used when the conventional anaerobic jar is not readily accessible. The illuminated candle jar in the container (arrow head) provides an anoxic environment (i.e. a background without oxygen) because the CO2 generated displaces the oxygen content of the jar; and this allows the cultured bacteria to thrive under optimal condition.

Figure 4: An illustration of an anaerobic (anoxic) chamber.

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