Culture Media: A Detailed Dissection

Microbiology identifies culture medium or growth medium as vital essentials for the growth of different types of cells. Culture medium exists in solid, semi-solid, or liquid, focusing on stimulating the broadening of microorganisms or cells, drawing support from cell proliferation. Culture media context integrates different cell types from plants and animals and microbiological culture that invigorate microorganisms’ growth, specifically the bacteria and fungi.

Research identifies nutrient broths and agar plates as the most common widely used culture medium to grow microorganisms. Cultural medium, irrespective of the growth of any microorganism, operates in a unique principle that mandates the equilibrium mixture of the nutrients to achieve favourable growth. The above goal helps mitigate toxic nutrients that may yield to a high concentration that favours inhibitory growth.

The cultivation technique between parasite and bacterial culture media differs in many aspects, specifically in the epidemiology. There exist some difficulties in the cultivation of parasites since many of them form complex life cycles that continuously undergo evolution in morphological stages. Dominant variables such as the composition of tissues in parasites, host sites, temperature, and immune responses rank some of the barriers that affect culture media in the parasite. Another difference that distinguishes culture media in parasites from bacteria is xenic, monogenic, and axenic cultures that focus on parasites’ growth using the primary growth approach.

The focus in culturing of bacteria is to produce more microorganisms through reproduction techniques at the specified laboratory conditions then they are identified through biochemical tests. The methodology simultaneously tries to isolate the bacteria using their properties and morphology to record possible genetic composition outcomes. Unlike the culturing of parasites, bacteria use streak, lawn, stab, and stroke cultures as the dominant methods. Microbial cultures provide continuity in microbiology as it assists in identifying the organism under study, thus providing crucial information in mitigating infectious diseases.

Culture Media Preparation

Culture media preparation has evolved over the years from the traditional cookbook method that focuses on adding ingredients step by step to a more modern medium preparation known as complex media. The principle in medium preparation first identifies the microorganism as first isolated before integrating different nutrients that form protein structures. The microbiological media undergoes different biochemical reactions that are complimented with pH indicators that act as crucial ingredients in the process. For culture media to be effective and successful, growth factors such as sodium chloride and pH buffers are included to stabilize the medium during the microbes’ metabolizes.

Culture media preparation in the microbiology laboratory first identifies the liquid growth media and culture media that form nutrient agar as the first procedure in the preparation process. Selecting the culture media from different databases ranks as the first procedure in the culture media preparation, hence the need to use cell culture media to give superior and consistent outcomes.

Over the years, research has supported Gibco cell culture media as the preferred mammalian cell as it gives accurate results and support to the cell lines. The next step focuses on the ingredients used with the applied quantities differing depending on the culture media magnitude understudy. The preparation process next follows by adjusting and measuring applied ingredients to the container into which an estimate of 80% deionized water is added to dissolve the ingredients to allow for uniform mixing. For an effective mixture, gentle heating is done, and then pH is recorded using the pH meter.

Regarding the quantity of the culture media, the volume is adjusted to protect cells’ growth since an error in the media volume often results in abnormal behaviour in the cells, hence limitations in their growth. Labelling of the containers as the next step in culture media preparation allows microbiologists to record accurate results, hence generating cells. Sterilization and autoclave is the next procedure as it allows the microbiological media to be free from any contamination that may arise from air hands and glassware.

The sterilization process eliminates any impurities that may affect the medium, making the growing cells sterile after the process. Media sterilization depends on an autoclave procedure that functions as a steam cooker, assisting in maintaining optimum pressure and temperature, making the cells of the organisms viable. Documentation of the results finalizes as the last procedure assisting microbiologists for future research.

Base Components in Culture Media

Development and formulation of culture media depend on the base components of nutrients, energy, minerals, buffering agents, pH factors, selective agents, and gelling agents, which in most cases comprises agar.
Nutrients, as the main components of culture media, are complimented with proteins, peptides, and amino acids, which help the bacteria undergo growth development. Various nutrients components need to be evaluated with the primary aim of capturing the composition of organisms that might be anaerobes. Blood agar in the culture media often contains peptones that generate peptides that assist some of the cells’ biological functions to be stimulated, for example, initiation of hormones. Research advocates for careful analysis of microorganisms’ nutrition to record uniformity and stability in their growth status.

As the other component, energy circulates carbohydrates that are integrated into the culture media to hasten the growth development of organisms. Glucose remains the energy source that is commonly applied as the energy requirement in the culture media process. Metals and minerals as inorganic products also form vital base components in the development of culture media. Examples include calcium, magnesium, iron, phosphates, and sulfates that are further classified into macro and micro components.

Buffering agents support the addition of energy ingredients in the culture media process. Determination of the pH of the culture medium through the buffering agents helps optimize the growth of microorganisms. Phosphates, citrates, acetates, zwitterion elements, and amino acids form the examples of buffering agent dominant in the culture media. As components in culture, buffering agents ensure the equilibrium of pH by stabilizing the acids and bases in the solutions. During culture, media buffering agents need to be analyzed, and correct proportions are initiated to mitigate the growth failure of microorganisms’ cells.

An indicator substance that alters pH change is another component of culture media. This component’s paramount importance is to identify the fermentation of the applied energy mechanisms, in which most cases are carbohydrates. During the procedure, indicator substances alter the colour change at the expected pH, assisting microbiologists to know the practical investigation’s endpoints. Examples of indicators substances that are dominant in culture media include phenol and Bromo-cresol. Selective agents also form parts of the components present in culture media as they assist in combating the growth of unfavourable organisms that may limit the procedure’s results.

Similarly, selective media agents help stimulate and record favourable microorganism growth by maintaining the required concentration. Bile salts, azide, tellurite, and tetrathionate are examples of selective agents applied in culture media. Gelling agents such as silica gel, alginates, and polyacrylamide play a colossal role in culturing agar media. Agar properties such as its ability to maintain unique gel structure and optimum temperature make it popular to be used in the culture media process. Similarly, it provides less toxic results, high and diffusion gel that stimulates microorganisms’ growth process.

Types of Culture Media and Uses

Culture media types are categorized based on the chemical composition of the culture medium and the consistency. Based on consistency the media can either be solid or liquid.

The solid culture medium is prepared by the use of Agar which is obtained from polysaccharide of red seaweed. Solid media are efficient in isolation of bacteria as pure culture and in the studying of bacteria which exist in colonies. Solid media are useful in the study of the morphology of the colonies of bacteria such as yeast and fungi such as actinomycetes. The margins, texture, colour, size and shape of the bacteria are easily studied. Agar’s transparency, inertness and its capability to remain solid at 37 degrees Celsius makes it an efficient media.

Liquid media are useful in the isolation of single organisms which exhibit a high growth rate such as blood culture medium. Peptone water is an example of a liquid medium where organisms are isolated at a temperature of 37 degrees Celsius as a test for their fermentation abilities.

Based on the chemical composition the media are categorized into routine laboratory media and synthetic media. Routine laboratory media are of six types including;

  1. Basal Media,
  2. Enriched Media,
  3. Selective Media,
  4. Indicator Media,
  5. Transport Media, and
  6. Storage Media.

Basal media include peptone water, nutrient agar and nutrient broth which are used for culture of organisms which do not require enrichment of the media such as staphylococci and Enterobacteriaceae. Basal media optimum temperature is between 25-45 degrees Celsius. They are used for primary isolation of organisms.

Enriched media are culture media with additional nutritional supplements such as blood, yolk sac, serum and chocolate e.g. Lowenstein- Jensen media. Optimum incubation temperature for the growth of these bacteria is 37 degrees Celsius.

Enrichment media are used in the growth of non-fastidious bacteria such as streptococci.

Selective media allow the growth of certain bacteria while inhibiting the growth of others by addition of antibiotics, dyes or alteration of pH. These media include Lowenstein-Jensen, MacConkey Agar, Tellurite media and Mannitol Salt which is used in the isolation of S. aureus by addition of 10% sodium chloride.

macconkey agar plate
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MacConkey Agar plate with sorbitol

In indicator media, an indicator is added to the media and the different organisms in the media identify themselves by altering the colour of the media. The organisms exhibit themselves with colonies possessing different colours and morphology. Examples include TCBS media which is used in isolation of Vibrio cholerae by exhibiting yellow colonies due to sugar fermentation. Indicator media are useful in the growth of different types of organisms.

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TCBS culture media plate

Transport media are used in the transportation of specimens to the lab by preventing desiccation, maintaining pathogen to commensal ratio, inhibit the overgrowth of unwanted bacteria and also prevent contamination. These media include Stuart’s and Amie’s media which are maintained at 30 degrees Celsius and are used for Gonococci bacteria Cary-Blair medium is used in the transport of faeces suspected of Salmonella.

Storage media are used for the storage of bacteria for a long time by maintaining their incubation temperatures at 37 degrees Celsius. They include chalk cooked meat broth and saline medium which are used to grow Shigella, Salmonella and vibrio.

Selective media are chemically processed media used in research work.

Best Practices in Organism Culturing

Before culturing of organisms various practices need to be observed in order to ensure the effectiveness of the culture media. The equipment and the culture media need to be prepared in an appropriate time and sterilized effectively. These prevent the contamination of the organisms with unwanted components which will interfere with their growth. Stock cultures of the medium culture need to be prepared in order to ensure future references and advancement in the study. The cultures need to be sterilized by autoclaving and safely disposed in order to limit contamination of other equipment and culture media. Prior to culturing the assessment of risks need to be considered for effective laboratory investigations.

The source of the culture medium need to be selected from reputable suppliers and manufacturers, low volume cultures need to be selected in order to reduce the risk of contamination by reducing the liquid media and availability of lab equipment. Appropriate incubation conditions need to be maintained for the pathogens in under investigation. These involve maintaining an optimum temperature, PH, nutrients and water.

Media preparation needs to be done in an organized manner with the timely acquisition of apparatus and ingredients. During preparation, the ingredients should be properly dissolved and the right amount of media should be used to prevent wastage and spillages that cause contaminations. Organisms should be inoculated at the appropriate time in the culture media to ensure their survival and prevent contamination. The workplace should be consequently cleaned and cleared after the experiment as a preventive measure for human contaminations and infections.

Global Manufacturers of Culture Media

Manufacturing of culture media is based on the types of culture media required by the consumers and the purpose of the media. Various manufacturing companies produce culture media based on the type (lysogeny, broth, stem cell media chemically defined media, classical media), application, research type, user and by region. Major global manufacturers of culture media include;

  1. BioMÉerieux SA,
  2. BD,
  3. Thermo Fisher Scientific Inc.,
  4. HiMedia Laboratories Private Limited,
  5. Fujifilm Holdings Corporation,
  6. Merck & Co.,
  7. Corning Incorporated,
  8. Lonza,
  9. CellGenix GmbH, and
  10. Sartorius AG.

BioMÉerieux SA is a biotechnology company located in France which deals with various scientific researches. It majorly focuses on the manufacture of broth, stem cell and chemically defined media.

Thermo Fisher Scientific Inc is an American company which manufacturer’s laboratory ingredients and regimens including cell culture media. They specialize in the manufacture of dehydrated microbial culture media, prepared culture media and atmosphere generation systems. These products include; MacConkey Agar, buffered peptone water, ESBL Agar and chromomeric coliform Agar.

HiMedia Laboratories is a recognized company in the manufacturing of lab equipment and products. It manufactures culture media ranging from enriched media, indicator media, and transport media to basal media. The company manufactures these media in prepared form, dehydrated form and atmospheric generated media.

Condalab is a leading culture media manufacturer in India. They manufacture dehydrated culture media, chromomeric culture media, and ready to use media, stains and other nutritional supplements for the media. These companies manufacture these media culture for the purpose of research, education and diagnosis purposes in clinical laboratories. They also participate in research strategies in the development of new culture media which will enhance the diagnosis and identification of various organisms.

Branding

Culture media exist in various brands which tend to have different purposes and mechanisms of use. They include the dehydrated culture media, prepared media and the atmospheric generated media. The dehydrated culture media brand is effective as it contains all ingredients required by the organism to grow in a form which is readily soluble. However, the prepared culture media brand is the best as it saves time and labour of preparation of the media.

Prepared media are accurate, consistent and effective in their preparation. They use high-quality media plates and convenience formats in ensuring the effectiveness of the media. Prepared media are effective in labs which run a large number of diagnostic tests a day in order to save time and increase the accuracy of the results. Prepared media in comparison with other brands is readily available, cheap and simple to use as the process of media preparation reduced as it comes in ready to use form.

Conclusion

Culture media exists in different types which serve different purposes. Consistency and composition of the culture media should be considered when selecting a media for culturing organisms. Culture media are set at optimum conditions which allow for optimal growth of the organisms. Sterilization, decontamination, preparation and organization are effective culturing practices which should be applied in the growth of organisms. Manufacturing companies and brands are vital factors in the selection of effective culture media. Competent personnel should be considered when selecting a culture media and the process of culturing organisms.