Culture Media 2025 PDF

Culture Media  What are we going to study? What is Culture Media Nutritional Requirements of Bacteria Components of media Simple Media Complex Media Synthetic Media Special Media  What is Culture Media ▪ Microbiology has been largely developed thanks to t...

Culture Media  What are we going to study? What is Culture Media Nutritional Requirements of Bacteria Components of media Simple Media Complex Media Synthetic Media Special Media  What is Culture Media ▪ Microbiology has been largely developed thanks to the discovery and optimization of culture media. ▪ The discovery of culture media allowed the development of microbiology in the nineteenth century. ▪ Bacterial culture was the first method developed to study the human microbiota using an artificial medium that allows growth and isolation of bacteria. ▪ The first to have cultured a bacterium in a reproducible way was Louis Pasteur in 1860 thanks to the development of the first so-called artificial culture medium. ▪ The evolution of bacterial culture through the media used for their culture began with the development of the first solid culture medium by Robert Koch, allowing not only the production of bacterial colonies, but also the possibility of purifying a bacterial clone.  Introduction ▪ The first to have cultured bacteria reproducibly in a liquid culture medium was Louis Pasteur. In 1860, he developed a culture medium containing ‘yeast soup’, ashes, sugar and ammonium salts. ▪ His objective was to create a fermentation medium to demonstrate that each fermentation (alcoholic, acetic, lactic) was associated with the development of a particular microorganism. ▪ The presence of these different elements in the medium allowed him to observe that some of these components could promote or inhibit the growth of certain bacteria and that they could also allow the emergence of certain bacteria compared with others. ▪ Indeed, in the course of the study of fermentation of beer, he was confronted with a problem: when the beer was healthy, the microscope only showed brewer's yeast, but when the beer was acid, Pasteur noticed that the tanks contained ‘tiny rod-shaped objects’ producing lactic acid. This fermentation medium therefore made it possible to highlight the multiplication of this bacterium.  Introduction ▪ In 1881, Robert Koch demonstrated optimal growth of bacteria when they were incubated in a broth composed of fresh beef serum or meat extract. However, the use of a liquid culture medium did not produce pure bacterial cultures. Koch therefore sought a way to solidify the medium ▪ First, he tested coagulated egg albumin, starch paste or an aseptically cut slice of potato. However, not all of these techniques allowed him to isolate colonies. Koch then added gelatin to his broth and poured it all over a flat glass plate. ▪ However, gelatin had disadvantages as it liquefied at temperatures above 25°C and could be consumed by gelatinase, an enzyme produced by certain bacteria. Thanks to the wife of one of his assistants, Fannie Hesse, who used agar to solidify her jams, he replaced gelatin with agar, which allowed him to obtain firm agars and isolate bacteria. ▪ In 1887, Julius Richard Petri replaced the glass plate with a circular culture box, the Petri box was created, and is still used today. This allowed him to obtain and observe isolated colonies and to limit contamination. Evolution of culture media: from the first bacterial culture (1673) to culturomics. Culturomics is a high-throughput culture technique consisting in the multiplication of the culture conditions along with a quick bacterial identification using MALDI-TOF MS What is Culture Media? ▪ Culture media is a gel or liquid that contains nutrients and is used to grow bacteria or microorganisms in laboratory for the purpose of identifying and studying them. ▪ They are also termed growth media. ▪ Different cell types are grown in various types of medium. ▪ Nutrient broths and agar plates are the most typical growth media for microorganisms. ▪ Some microorganisms or bacteria need special media for their growth. ▪ Culturing on solid media is another convenient way of separating bacteria in mixture Significance – ▪ Culture media is used in order to identify the causative agent from infected material. ▪ Subsequent clinical diagnosis -studying its morphology and its identification. ▪ Bacteria have to be cultured in order to obtain antigens from developing serological assay for vaccines. ▪ Certain genetic studies and manipulations of the cells also need that bacteria be cultured in vitro. Significance Used to grow bacteria ▪ Can be used to- Enrich the numbers of bacteria. ▪ Select for certain bacteria and suppress others. ▪ Differentiate among different kinds of bacteria. ▪ It is usually essential to obtain a culture by growing the organism in an artificial medium. ▪ If more than one species or type of organism are present each requires to be carefully separated or isolated in pure culture Components Culture Media The nutrients: In order to grow, bacteria need a minimum of nutrients: 1. Water 2. Carbon source 3. Nitrogen source 4. Mineral salts 5. Growth factors Water: Water plays a fundamental role in solubilizing nutrients, transporting them and ensuring hydrolysis reactions. Some bacteria need free water for their growth. If evaporation occurs during the incubation of the agar, there may be a loss of this water, resulting in a decrease in colony size and inhibition of bacterial growth. Carbon sources: ▪ Carbon is the most abundant constituent element in bacteria. ▪ It is essential for bacteria to produce carbon molecules, such as fats, carbohydrates, proteins and nucleic acids. ▪ Bacteria can use inorganic carbon sources, such as carbon dioxide, or organic sources such as sugars and alcohols Nitrogen sources: ▪ As for nitrogen sources, they are numerous and can be found in a large number of compounds used in the composition of a culture medium. ▪ It is found in the organic form, corresponding to protein hydrolysates, particularly in case of hydrolysate, proteose- peptone or tryptone, but also in an inorganic form, nitrates. ▪ Nitrogen allows bacteria to synthesize their proteins. Among the common mineral salts, phosphate, sulphate, magnesium or calcium are needed. Energy sources: There are two types of bacteria, phototrophic bacteria, such as Thiocapsa roseopersicina, which uses light as an energy source by transforming it into an electrochemical gradient of protons, and chemotrophic bacteria, which use the energy of oxidation of mineral or organic compounds as energy sources. Among these bacteria, we can find Listeria monocytogenes Growth factors ▪ The use of a minimal medium does not allow the growth of certain bacteria that need specific elements to grow. ▪ It is sometimes necessary to add growth factors to culture media to boost the multiplication of bacteria. ▪ Growth factors are elements that bacteria are unable to synthesize from the nutrients available in the environment. ▪ Growth factors are required in small quantities in the culture medium and their need is justified by the absence or blocking of a metabolic pathway in the bacterium. Purine and pyrimidine bases ▪ There are different categories of growth factors among which we find purine and pyrimidine bases. ▪ They are necessary for synthesis of nucleic acids. ▪ Indeed, some lactic acid bacteria need adenine, guanine, thymine or uracil for growth. ▪ This is the case in particular for the bacterium Leuconostoc mesenteroides, for which guanine is essential for its growth  Common ingredients of culture media Peptone- source of carbon and nitrogen. Beef extract- source of amino acid, vitamins, minerals. Yeast extract- source of vitamin, carbon, nitrogen. Distilled water-solvent Agar- solidifying agent. Types of Culture Media The culture media are classified in many different ways: Based on the physical state ▪ Liquid media ▪ Nutrient broths or lysogeny broth ▪ Solid media (LB) medium are the two most ▪ Semisolid media popular growth media for bacteria. Based on the presence or absence of oxygen ▪ Agar is frequently added to liquid ▪ Anaerobic media media before being placed into a petri dish to solidify. ▪ Aerobic media ▪ These agar plates offer a stable Based on nutritional factors medium for the cultivation of ▪ Simple media bacteria. ▪ Synthetic media ▪ It stays solid because relatively few ▪ Complex media bacteria can break down agar. ▪ Special media Based on the physical state  LIQUID MEDIA Definition: ▪ Liquid media, also known as broth media, are culture media without a solidifying agent like agar, allowing microorganisms to grow uniformly throughout the liquid. ▪ They are essential for cultivating large quantities of bacteria and studying their physiological properties. ▪ Diffused growth ▪ No characteristics for identification ▪ Difficult to isolate ▪ Earliest liquid medium: urine or meat broth used by Louis Pasteur  Based on the physical state SOLID MEDIA ▪ Distinct colony morphology ▪ Characteristics → easy to identify ▪ Colony – macroscopically visible collection of millions of bacteria originating from a single bacterial cell  SOLID MEDIA ▪ Earliest solid medium: ▪ Cooked cut potato by Robert Koch ▪ Gelatin - not satisfactory - liquefy at 24oC Agar ▪ Frau Hesse ▪ Universally used for preparing solid medium ▪ Obtained from seaweed: Gelidium ▪ No nutritive value  SOLID MEDIA Definition: ▪ Solid media are culture media that contain a solidifying agent, usually agar, to create a firm surface for the growth and isolation of microorganisms. ▪ Solid media are essential for studying colony morphology, isolating pure cultures, and differentiating species.  AGAR ▪ Agar, is an indispensable substance in microbiology. ▪ Agar: A gelatinous substance derived from the cell walls of red algae, primarily species of Gelidium and Gracilaria. It is a mixture of polysaccharides, mainly agarose and agaropectin, enabling the study of microorganisms in a controlled environment. ▪ It serves as a solidifying agent in microbiological culture media.  CHEMICAL STRUCTURE ▪ In the natural state, agar occurs as structural carbohydrate in the cell walls of agarophytes algae. ▪ It is a complex mixture of polysaccharides composed of two major fractions – agarose, a neutral polymer, and agaropectin, a charged, sulfated polymer. ▪ Agarose, the gelling fraction, is a neutral linear molecule essentially free of sulfates, consisting of chains of repeating alternate units of β-1,3-linked- D-galactose and α- 1,4- linked 3,6 anhydro-L-galactose units. ▪ Agaropectin, the non gelling fraction, is a sulfated polysaccharide (3% to 10% sulfate), composed of agarose and varying percentages of ester sulfate, D-glucuronic acid, and small amounts of pyruvic acid. The proportion of these two polymers varies according to the species of seaweed.  KEY PROPERTIES OF AGAR IN MICROBIOLOGY 1.Solidifying Agent: ▪ Agar is used at concentrations between 1-2% to solidify media. ▪ It liquefies at about 85°C and solidifies around 40°C, making it easy to handle and pour into culture plates or tubes. 2.Inertness: ▪ Agar is chemically neutral and does not interfere with the nutrients or the metabolic activities of microorganisms. 3.Transparency: ▪ Solidified agar is clear, allowing easy visualization of microbial colonies. 4.Non-Nutritive: ▪ Most microorganisms cannot degrade agar, ensuring that the nutrients in the medium dictate growth.  SEMI-SOLID MEDIA Definition: Semi-solid media are types of culture media with a gel-like consistency that is softer than solid media but firmer than liquid media. This is achieved by adding a lower concentration of a solidifying agent like agar (typically 0.5% or less, compared to 1.5% in solid media). Composition: Agar: The primary solidifying agent, used in a lower concentration (0.2–0.5%). Nutrients: Carbon, nitrogen, vitamins, minerals, and other growth factors to support bacterial growth. Indicators (optional): For detecting motility or metabolic changes, such as dyes or pH indicators. Based on the physical state Types of culture media based on oxygen requirement Microorganisms have different requirements for growth depending on oxygen requirements. 1. Aerobic media In this media, it is easy to cultivate microbes, on solid media, the growth occurs by keeping the culture in the incubator. It shows the growth; of non-fastidious microorganisms. Examples of aerobic media are- liquid media, solid media Peptone water- 1%peptone + 0.5% Nacl +100ml water. Nutrient agar- nutrient broth +2% agar. 2. Anaerobic media The media cultivates anaerobic bacteria at low oxygen, reducing oxidation-reduction potential. Anaerobic media contains extra nutrients like vitamin K, hemin, and oxygen that get reduced by a physical or chemical process. The addition of glucose (1%), thioglycollate(0.1%), ascorbic acid (0.1%), cysteine (0.05%), or iron fillings added to cause the medium to reduce. The medium is boiled in a water bath to force out dissolved oxygen and packed with sterile paraffin. Examples of Anaerobic media RCM (Robertson cooked meat) isolation for Clostridium sp. Thioglycolate broth– It has sodium glycolate that maintains low oxygen. Based on nutritional factors/ingredients Simple Media ▪ Simple culture media includes nutrient broth. ▪ One percent meat extract and peptone water makes up such broth. Nutrient broth becomes glucose broth when glucose is added to it. ▪ Likewise, it becomes nutrient agar when 2-3% agar is added. ▪ For purposes of diagnosis, this is the simplest and most common media used in laboratories. ▪ A semisolid medium that permits the propagation of motile bacteria can be produced if the concentration of agar is decreased. Examples – peptone water, nutrient agar and glucose broth. BASED ON NUTRITIONAL FACTORS/INGREDIENTS Peptone is primarily obtained from bovine or porcine origin, such as meat, internal organs, gelatin, and milk, as well as from plants and yeasts. It is a partially digested protein and is prepared by the enzymatic or acidic hydrolysis of proteinaceous material. Beef extract powder is a dehydrated extract of bovine tissue for use in preparing microbiological culture media. It is prepared from selected bovine tissues which are free from fat and sinew. Diverse media utilize beef extract, for example, yeast dextrose broth, Herrold′s egg yolk medium. This nutritional supplement provides a mixture of peptides, individual amino acids, nucleotides, organic acids, minerals, and vitamins. Sinew-a piece of tough fibrous tissue uniting muscle to bone; a tendon or ligament Application: Beef Extract Powder is used in general purpose culture media and is recommended for use in the bacteriological examination of water, milk, and other materials, where the uniform composition of media is important. Beef Extract Powder is also relied upon for biochemical studies like fermentation reactions. BASED ON NUTRITIONAL FACTORS/INGREDIENTS Complex Media ▪ Other than simple media, all forms of media are termed complex media. ▪ Complex media contain additional components for bringing out specific qualities or providing the unique nutrients needed for the bacterium’s growth. Here, the source of the amino acids contains a variety of chemicals whose precise composition is unknown, thus it is an undefinable media. Examples – MacConkey agar medium and chocolate agar. BASED ON NUTRITIONAL FACTORS/INGREDIENTS Streptococcus pneumoniae BASED ON NUTRITIONAL FACTORS/INGREDIENTS Synthetic Media (Chemically defined medium) ▪ A synthetic medium is a defined medium. ▪ A defined medium (chemically defined medium) is a medium in which there is no yeast, plant or animal tissue present, and all the chemicals employed are known. ▪ These are made from only pure ingredients whose exact composition is known. ▪ These are employed in specialised investigations like those on metabolic needs. Example – Dubo’s culture medium with tween 80.  SPECIAL MEDIA Special media are of seven different types: 1. Enriched media 2. Selective media 3. Differential media 4. Enrichment media 5. Transport media 6. Indicator media 7. Sugar media Special Media Enriched media – ▪ It is created when a basic medium is supplemented with nutrients like eggs, blood or serum. ▪ As a result of this function, enriched media contains high nutrition in form of egg yolk or blood serum so on. Its basal medium also contains nutrients. ▪ For example, a blood agar medium is used for the growth of bacteria like Streptococcus which specifically requires blood for its proliferation. Special Media Selective media – ▪ Selective media are used to select for the growth of a particular "selected" microorganism. ▪ Selective media contain components that prevent the growth of all but a small number of bacterial species and make it easier to isolate a specific species. ▪ When mixed bacterial flora is anticipated in specimens, these media are utilised to isolate specific bacteria from those specimens. For example, bile salt acts as a selective agent in BSA or bile salt agar. ▪ While preventing the growth of other intestinal organisms, it favours the growth of Vibrio cholerae. ▪ For example, if a certain microbe is resistant to a particular antibiotic (e.g., novobiocin), then that antibiotic can be added to the medium in order to prevent other organisms, which are not resistant, from growing. Special Media Special Media Differential media – ▪ It is a term used to describe a medium that has components that aid in identifying the various properties of bacteria. Peptone, agar, lactose, neutral red and sodium taurocholate are all ingredients in MacConkey’s medium. Here, the colonies made by lactose fermenters are pink, but those made by non-lactose fermenters are pale or colourless. ▪ Differential medium. a medium which is used to differentiate different types of microorganisms based on their different colours or colony shapes. Examples of differential media are: Macconkey's agar Special Media Cystine-Lactose-Electrolyte-Deficient Thiosulfate–citrate–bile salts–sucrose agar Xylose Lysine Deoxycholate Agar  Special Media Enrichment media ▪ This media contains several ingredients that either stimulate the bacteria being grown or suppress their competitors. ▪ Enrichment media are a form of extremely selective medium that only permits a specific species of a microbe to grow in it. These mediums prevent undesirable, commensal, or contaminating microbes from growing. ▪ Examples – Alkaline peptone water and tetrathionate broth.  Special Media Transport media – ▪ These are employed when dealing with delicate organisms that might not make it through the transit period or might become covered with non-pathogenic germs. ▪ Transport media for microorganisms are essentially buffer solutions containing carbohydrates, peptones and other nutrients (excluding growth factors) designed to preserve the viability of bacteria during transport without allowing them to multiply. ▪ Special media are developed for the transportation of such bacteria to laboratories and these are known as transport media. Example – Stuart’s transport medium. Special Media  Special Media Indicator media – ▪ Differential media (or indicator media) in microbiology contain substances that lead colonies of certain organisms to take on a distinctive appearance. ▪ These media thus allow the microbiologist to differentiate various kinds of microorganisms on the same agar plate. An example of differential media is blood agar. If blood agar is inoculated with a mixed culture of bacteria, some of these may produce enzymes that hemolyze the contained red blood cells, forming a hemolytic zone around colonies. This helps to distinguish between hemolytic and nonhemolytic bacteria. There are also differential media that contain pH indicators. These change color if, for example, a bacterial species produces acid during growth that lowers the pH of the medium. ▪ When bacteria multiply in these media containing an indicator, they tend to change their colour. MacConkey’s medium is also an example of an indicator medium. ▪ Another classic example is the black colonies of Salmonella typhi that develop on sulphite-containing Wilson and Blair media. Special Media Sugar media – Media containing any fermentable substance Eg: glucose, arabinose, lactose, starch etc. Media consists: 1% of the sugar in peptone water + Indicator Contain a small tube (Durham's tube) for the detection of gas by the bacteria. ▪ It contains 1% sugar, which can be any fermentable substance like glucose, mannitol, sucrose and lactose. ▪ The generation of acid following the fermentation of sugar transforms the medium into pink due to the presence of an indicator. Also to show that gas is produced, Durham’s tube is kept inverted inside the sugar tube and gas bubbles are observed. Application of culture media To culture microbes. To identify the cause of infection. To identify characteristics of microorganisms. To isolate pure culture. To store the culture stock. To observe biochemical reactions. To test microbial contamination in any sample. To check antimicrobial agents and preservatives effect. To observe microbe colony type, its color, shape, cause. To differentiate between different colonies. To create antigens for laboratory use. To estimate viable count. To test antibiotic sensitivity. Limitations of culture media Viable but nonculturable Limitations of culture media Limitations of culture media Limitations of culture media