Why is selective media useful

The role of blood is to act as a protective agent against toxic oxygen radicals [ 22 ], but also as a nutritional supplement. For instance, cooked sheep's blood provides the factor X haem necessary for the growth of many pathogenic species, including Haemophilus influenzae [ 23 ]. Serum, such as fetal calf or lamb serum, can also be used as a growth factor by providing a large number of elements such as lipids, vitamins, triglycerides, minerals and others [ 24 ].

This blood has been cleared of cells, platelets and clotting factors. Finally, it is also possible to use the rumen fluid [ 2 , 29 ] to promote the growth of certain bacterial species by mimicking their natural environment.

The rumen fluid used corresponds to the rumen of the sheep. To prepare it, it is first necessary to recover the juice from the fermented plants in the stomach by filtering it through a fine cloth. This juice is then centrifuged 10 rpm for 90 min and the supernatant is collected. Then, it undergoes three successive filtrations at 0. The rumen fluid can promote the growth of certain species of Treponema , such as Treponema hyodysenteriae and Treponema innocens [ 31 ].

Some anaerobic bacteria are fastidious to grow and new culture strategies have been developed to isolate them. Indeed, anaerobic bacteria are most abundant in the human intestinal microbiota, accounting for up to Antioxidants have therefore been added to culture media to allow the culture of strict anaerobic bacteria under aerobic conditions [ 33 , 34 ].

A number of antioxidants have been tested and have shown satisfactory results. This is the case for ascorbic acid and glutathione [ 34 ] or uric acid [ 35 ]. The addition of these antioxidants to the culture medium and its incubation under aerobic conditions allowed the growth of strict anaerobic bacteria, 12 microaerophilic bacteria and 22 strict aerobic bacteria [ 35 ].

A selective culture medium is used to isolate a particular bacterial species or genus. After the addition of a number of inhibitors to the culture medium, the objective of this type of medium is to eliminate unwanted microbial flora. The selective medium is composed of a basic medium to which antibiotics, chemicals, dyes, antiseptics, sodium salts or phages can be added [ 36 ]. Antibiotics are the most commonly used selective agents. Their spectrum of action being well known, it is easier to anticipate their action on bacteria.

There are a large number of antibiotics that can be used in culture media, some of which are called antibacterial because they target bacteria and others antifungal because they eliminate fungi and yeasts [ 37 ]. Some molecules target Gram-positive bacteria, such as penicillin G, bacitracin or vancomycin, whereas others target Gram-negative bacteria, such as colistin or polymixin B. Amphotericin B, cycloheximide or nystatin have an action against fungi and yeasts Table 1 [ 10 , 39 ]. Several antibiotics can be combined to obtain a more selective medium [ 37 ].

Antiseptics are more rarely used in culture media. However, cetrimide [ 43 , 44 ] or acriflavin [ 10 ] can be found in some culture media. Chlorhexidine can be used to select Mycobacterium tuberculosis [ 45 , 46 ] Table 2. Ethanol can also select bacterial species, including sporulated bacteria [ 47 , 48 ] such as Clostridioides difficile [ 47 ]. Cetrimide Agar Base is a culture medium used to selectively isolate and identify Pseudomonas aeruginosa.

Cetrimide is a quaternary ammonium that inhibits a large number of bacteria, including those of the genus Pseudomonas , other than Pseudomonas aeruginosa [ 10 ].

Sodium salts are known for their inhibitory properties. The best known is sodium chloride, used to select halophilic bacteria that resist very high amounts of salts [ 49 ]. In addition, sodium deoxycholate has a strong solvent action on bacteria [ 50 ] Table 3. The Marine Agar E culture medium is used to enumerate marine heterotrophic bacteria. It is composed of a high concentration of salt, which eliminates a large number of bacteria and preserves marine bacteria of interest [ 51 ].

Chemical substances can be added to culture media to inhibit certain bacteria. These inhibiting substances include potassium tellurite and bile salts, which inhibit Gram-positive bacteria [ 10 , 39 , 52 ] or lithium chloride [ 10 , 39 ], which eliminates Gram-negative bacteria Table 4.

Brayton et al. This medium, VV agar, consists, among other things, of potassium tellurite as selective agent for inhibiting Enterobacteriaceae. Dyes can be used as a colour indicator in a culture medium or as a selective agent against certain bacteria.

Crystal violet is one of the most commonly used dyes to inhibit bacteria [ 37 , 54 ]. Malachite green and methylene blue are also used to inhibit Gram-positive and Gram-negative bacteria and Gram-positive bacteria, respectively [ 10 , 55 ] Table 5. A selective medium of Streptococcus pneumoniae has been developed, containing crystal violet. This dye is used to select streptococci and inhibit staphylococci as well as other Gram-positive bacteria [ 54 ].

Bacteriophages are specific viruses of bacteria that can infect and even destroy bacteria, in the case of lytic phages. In order to isolate Mycobacterium tuberculosis , the use of phage lysin decontaminates the sputum of other bacteria present in the pulmonary microbiota [ 56 ]. Sillankorva et al. After 2 hours of treatment, phage T1 reduced the E.

In liquid culture media, also called culture broths, nutrients are dissolved in water. The growth of bacteria in this type of medium can be demonstrated by the appearance of a turbidity in the medium, although this is not always the case. It is difficult to isolate a bacterium specifically in this type of medium. Indeed, the bacteria obtained from a sample inoculated into the culture broth are all mixed with each other. In addition, this type of culture medium does not allow the morphological characteristics of bacterial species to be identified [ 9 ].

However, liquid culture media facilitate access to nutrients for bacteria. These nutrients are all the more accessible as the culture media are incubated under agitation, allowing a renewal of nutrients for bacteria.

Solid culture media are obtained by adding a gelling agent, such as agar, to the culture broth. They make it possible to obtain isolated colonies of different bacterial species, which can be identified.

The different morphological characteristics of the bacterium can be described from these cultures [ 9 , 58 ]. However, in solid culture media, access to nutrients for bacteria may be limited. Media with high gel content, such as agar, will form smaller colonies than low gel content media because nutrient flow and toxin removal are reduced [ 7 ].

In addition, it has been shown that agar, in excessive quantities, can inhibit the growth of certain bacteria, highlighting the need to find other gelling agents [ 9 ]. One of the first gelling agents used in culture media was gelatin. Moreover, the presence of an enzyme in certain bacteria, gelatinase, causes the digestion of gelatin and therefore its degradation.

Agar was then used in culture media. However, over-consumption of agar has led to a reduction in its source, red algae [ 59 ], which has increased costs. In addition, agarase, present in some bacteria, destroys agar, preventing the isolation of these bacteria [ 60 ].

In addition, agar can inhibit the growth of some anaerobic bacteria because inhibitory growth compounds can be produced from autoclaving phosphate with agar [ 61 ]. For all these reasons, new gelling agents have been sought. All these gelling agents have different properties and particular needs to gel Table 6. Gelling agents used in culture media [ 60 , 63 , 67 ].

The first will form a firm gel with a rapid mass build-up when combined with potassium ions. It allows the growth of some bacteria.

This gelling agent resists very alkaline pH values above This gelling agent is produced from brown seaweed extract and forms a flexible gel in the presence of calcium ions. However, this gelling agent does not provide a gel firm enough to grow bacteria [ 63 ]. High-methoxyl pectins require sugar and high acidity to gel. Gellan gum is a polysaccharide produced by a bacterial genus, Sphingomonas spp.

According to Tamaki et al. The use of these different gelling agents could allow the culture of new bacteria, which do not grow on the agar, because of the presence of an agarase for example or because the agar forms a network too dense to allow motility and optimal growth of certain bacteria [ 9 ]. The amount of nutrients available in a culture medium will determine the size of bacterial colonies [ 58 ]. An overly firm culture medium, due to a high concentration of gelling agent, causes a decrease in the flow of nutrients and so a decrease in the access to these nutrients by bacteria [ 7 , 9 ].

On the other hand, in some culture media, the amount of nutrients available is too high and can be toxic for certain bacteria that require a poor culture medium to grow [ 68 ]. To obtain larger colonies, it is sometimes necessary to mimic the bacterium's natural environment by providing it with specific elements.

This is the case, for example, for Phascolarctobacterium faecium and Phascolarctobacterium succinatutens , which form microcolonies. However, when the medium is supplemented with succinate, the colonies have a diameter ranging from 0. After stagnation in the development of new culture techniques, due to the rapid evolution of new microbiological methods such as metagenomics, bacterial culture is experiencing a new boom.

In recent years, culturomics, with the use of new culture media and new culture conditions, has enabled the enrichment of the bacterial repertoire through the isolation of new bacterial species. This shows that, despite the abandonment of culture by a large number of microbiologists, culture media remain a fundamental tool for bacteriologists for the isolation of commensal but also pathogenic bacteria.

Studying the natural environment of bacteria that have remained uncultivated to date would be interesting because it would provide the essential elements for the bacteria to grow.

Indeed, although there are many enriched culture media, each bacterium is unique and has specific requirements. The use of new gelling agents could also allow the isolation of new species for which agar was not suitable for their growth.

Although many gelling agents have been tested, few are still used in commercial culture media and therefore in laboratories. Many developments in bacterial culture are therefore still to come, making it possible to enrich the bacterial repertoire and gain a better understanding of certain diseases.

In addition, intracellular bacteria such as Coxiella burnetii or Tropheryma whipplei require a host cell to survive and multiply. Some of these bacteria cause severe diseases and pose a diagnostic problem because of their fastidious growth or lack of growth on conventional media [ 71 ].

It would be interesting to develop culture media that allow faster and easier detection of these bacteria. In addition, as the microbiota plays an increasingly important role in human health [ 72 , 73 ], the development of probiotics is on the rise [ 74 ]. The use of targeted culture media to select certain bacteria with an important medical role therefore remains a priority.

National Center for Biotechnology Information , U. New Microbes New Infect. Published online Nov Bonnet , 1 J. Lagier , 1, 2 D. Raoult , 1, 2 and S. Author information Article notes Copyright and License information Disclaimer.

Khelaifia: moc. This article has been cited by other articles in PMC. Abstract Microbiology has been largely developed thanks to the discovery and optimization of culture media. Two types of media with similar implying names but very different functions, referred to as selective and differential media, are defined as follows. Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.

Media lacking an amino acid such as proline in conjunction with E. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite.

Much of the study of microorganisms depends on its ability to grow in the laboratory, and this is possible only if suitable culture media are available for the growth of microorganism.

A culture medium is defined as a solid or liquid preparation used for the growth, transport, and storage of microorganisms. The effective culture medium must contain all the nutrients required for the growth of the microorganism. Specialized media are widely employed for the isolation and identification of microorganisms, testing the antibiotic sensitivities, analysis of water and food, industrial microbiology, and other activities.

Although all microorganisms need sources of energy, nitrogen, carbon, phosphorus, sulfur, and various minerals, the exact composition of a satisfactory medium will rely on the species one is trying to identify and cultivate because nutritional requirements vary so greatly among the microorganisms. A medium is used to select and growing specific microorganisms or to help identifying a particular species. In these cases, the function of the medium also depends on its composition. In addition to nutrients necessary for the growth of all bacteria, special-purpose media contain one or more chemical compounds that are essential for their functional specificity.

These include: Selective, Differential and Enriched Media. Selective media allows the growth of certain type of organisms, while inhibiting the growth of other organisms. This selectivity is achieved in several ways. For example, organisms that have the ability to utilize a given sugar are screened easily by making that particular sugar the only carbon source in the medium for the growth of the microorganism.

Like-wise, the selective inhibition of some types of microorganisms can be studied by adding certain dyes, antibiotics, salts or specific inhibitors that will affect the metabolism or enzymatic systems of the organisms.

For example, media containing potassium tellurite, sodium azide or thallium acetate at different concentrations of 0. Tellurite agar, is used to select for Gram-positive organisms, and nutrient agar supplemented with the antibiotic penicillin can be used to select for the growth of Gram negative organisms. Differential media are widely used for differentiating closely related organisms or groups of organisms. Because of the presence of certain dyes or chemicals in the media, the organisms will produce certain characteristic changes or growth patterns that are used for identification or differentiation of microorganism.

Enriched media are media that have been supplemented with highly nutritious materials such as blood, serum or yeast extract for the purpose of cultivating fastidious organisms. Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures. On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this color change is that S.

The growth of other types of bacteria is usually inhibited. This growth differentiates S. The bile salts in the medium precipitate in the immediate neighborhood of the colony, causing the medium surrounding the colony to become hazy appearance.

Non-lactose fermenting bacteria such as , Proteus species , Salmonella , Pseudomonas aeruginosa and Shigella cannot utilize lactose in the medium, and will use peptone instead.

This results in the formation of ammonia, which raises the pH of the agar, and leads to the formation of white or colorless colonies in the plate. But, in some cases, they can also look golden to brown with dark centers. Media that support the growth of many different microorganisms without distinguishing genera or species are nutritive. In contrast, differential media allow several different types of bacteria to grow, but also contain compounds that allow microbial genera or even species to be visually differentiated.

The organisms interact with the added compounds e. This allows for the rapid identification of organisms of interest, which is especially important for heavily mixed cultures, such as stool.