Growth medium

Micro organisms do exist in natural environment as mixed population growing on suitable substrates. But in laboratories micro organisms are maintained in form of cultures.

CULTURE:

The population of any organisms grown in laboratory by using suitable nutrient substances is called culture.

CULTURE/GROWTH MEDIUM:

An agar plate -- an example of a bacterial growth medium. Specifically, it is a streak plate; the orange lines and dots are formed by bacterial colonies.

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens There are different types of media for growing different types of cells.
There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

TYPES OF CULTURE MEDIUM:

                             Depending on the consistance and nature culture media is divided into

·         Liquid medium

·         Semi-solid medium

·         Solid medium

·                                                       LIQUID MEDIUM:

·                                                              The culture media which occurs in liquid state or in the form of solution is called LIQUID MEDIUM.A liquid medium is known as broth. The liquid medium consists only the nutrients to support the growth of organisms but not any gelling solidifying substances like agar- agar .Liquid medium is mainly used to carryout the different biochemical tests in characterization of bacterial species.

·                                                                               Eg: nutrient broth.

·                                                                               

Ingredients for Nutrient broth:

Ingredient	1 L	500 mL
Distilled water	1 L	500 mL
beef extract	1 g	0.5 g
Yeast extract	2 g	1 g
Peptone	5 g	2.5 g
Sodium chloride (NaCl)	5 g	2.5 g

Instructions

1. Mix and autoclave

Notes:

• A good all-purpose isolation and maintenance medium.
• This medium should be used for Dickeya rather than Kings medium B. Dickeya will die if cultured on KB. Use the broth for shake-culture.

SEMI-SOLID MEDIUM:

 A liquid culture medium is converted into a semi-solid medium with the addition of a gelly or solidifying agent like agar-agar at less than 0.5 % to the liquid medium.

It exhibits a soft , custard like consistancy. This medium is useful for cultivation of microaerophillic bacteria and also for the determination of bacterial motility.

SOLID MEDIUM;

Solid medium is that culture medium which exist in a solid state. Along with the required nutrients,a solidifying agent namely agar-agar is added at 1-2% level to the broth to prepare solid  medium .

              Eg:nutrient agar

Nutrient agar

Nutrient agar is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. It is useful because it remains solid even at relatively high temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly visible as small colonies. In nutrient broth, the bacteria grows in the liquid, and is seen as a soupy substance, not as clearly distinguishable clumps. Nutrient agar typically contains (w/v): 0.5 % peptone

• 0.3 % beef extract/yeast extract
• 1.5 % agar
• 0.5% NaCl and distilled water
• pH adjusted to neutral (6.8)at 25 °C.

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TYPES OF MEDIA BASED ON COMPOSITION;
Based on chemical composition culture media is frequently classified into2 broad categories:
1)Complex medium or chemically undefined medium
2)Synthetic medium or chemically defined medium.
The difference between these 2 media is not in the number of ingrients present but in the knowledge of their exact chemistry.
COMPLEX MEDIUM:
This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown
An undefined medium (also known as a basal or complex medium) is a medium that contains:

• a carbon source such as glucose for bacterial growth
• water

·         various salts needed for bacterial growth

The complex medium is widely used in cultivation of heterotrophic micro organisms.

SYNTHETIC MEDIUM:

The synthetic medium is also called as chemically defined medium and simply defined medium

It is prepared by using pure organic and inorganic chemicals of this medium is known.

Different synthetic medium are widely used in research field.

The synthetic medium may be simple or complex depending on the chemical ingredients used in construction of the medium. The simple synthetic medium contains a carbon source and different inorganic chemicals as nutrients.

eg: Psedomonas medium .This medium does not contain any yeast, animal or plant tissue.

The complex synthetic medium consists of inorganic substances such as amino acids, vitamins, nucleic acids, bases in addition to the inorganic chemicals.

eg: Methnobacterium medium.

Depending on the microorgamisms to be cultivated in the medium they are classified into:

BACTERIAL MEDIUM:

It is used for the cultivation and isolation of bacteria.

It is most predominantly used medium.

Eg: nutrient agar, nutrient broth.

FUNGAL MEDIUM:

It is used for the cultivation and isolation of fungi.

Eg: potato dextrose agar medium

Potato dextrose agar

1. niger growing in potato dextrose agar

Potato dextrose agar (abbreviated "PDA") and potato dextrose broth (abbreviated "PDB") are common microbiological media made from potato infusion, and dextrose (corn sugar). Potato dextrose agar is the most widely used medium for growing fungi and bacteria which attack living plants or decay dead plant matter.
 Potato infusion can be made by boiling 300g of sliced (washed but unpeeled) potatoes in water for 30 minutes and then decanting or straining the broth through cheesecloth. Distilled water is added such that the total volume of the suspension is one litre. 20g dextrose and 20g agar agar powder is then added and the medium is sterilized by autoclaving at 15psi for 15 minutes.^\
Common organisms that can be cultured on PDA are yeasts such as Candida albicans and Saccharomyces cerevisiae and molds such as Aspergillus niger.

Sabouraud agar

view of a Sabouraud agar plate with a colony of Trichophyton rubrum var. rodhaini.

Sabouraud agar is a type of agar containing peptones. It is used to cultivate dermatophytes and other types of fungi. It was created by, and is named after, Raymond Sabouraud in 1892. Later adjusted by Emmons, its pH level was brought closer to the neutral range to support the growth of other subcultures of fungi.

Typical composition

Sabouraud agar typically contains

• 40 g/L dextrose
• 10 g/L peptone
• 20 g/L agar
• pH 5.6

2)czapecks dox medium:

Mostly used for the cultivation and isolation of Aspergillus, Mucor, Rhizopus.

Yeast extract peptone dextrose(YEPD):

·         It is used for the cultivation of yeast cells.

·         Hay infusion agar :

Specific for the culturing of slime moulds (which are not fungi

ALGAL MEDIUM:

It is used for the cultivation of autotrophic algae eg: Bristols medium-used for the cultivation of Nostoc,  Oscillatoria, Anabena, Scytonema belonging to cyanophyceae mambers and green algae like Chlamydomonas, chlorella belonging to chlorophyceae

Virus and phage culture

Virus or phage cultures require host cells for the virus or phage to multiply in. For bacteriophages, cultures are grown by infecting bacterial cells. The phage can then be isolated from the resulting plaques in a lawn of bacteria on a plate. Virus cultures are obtained from their appropriate eukaryotic host cells.

Eukaryotic cell culture

Isolation of pure cultures

For single-celled eukaryotes, such as yeast, the isolation of pure cultures uses the same techniques as for bacterial cultures. Pure cultures of multicellular organisms are often more easily isolated by simply picking out a single individual to initiate a culture. This is a useful technique for pure culture of fungi, multicellular algae, and small metazoa, for example.
Developing pure culture techniques is crucial to the observation of the specimen in question. The most common method to isolate individual cells and produce a pure culture, is to prepare a streak plate. The streak plate method is a way to physically separate the microbial population, and is done by spreading the inoculate back and forth with an inoculating loop over the solid agar plate. Upon incubation, colonies will arise and, hopefully, single cells will have been isolated from the biomass.

Based on the application of media culture, media is divided into:

Enriched medium:
 Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.

Blood agar:

it is enriched media used for the cultivation of fastidious organisms which fail to grow on nutrient agar eg: Streptococcus (Haemolytic  streptococci)

Chocolate agar(heated blood agar):

It is used for the cultivation of fastidious organisms like Neisseria gonorrhoeae, N.meningitis

Selective media:

Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter.

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus culture.

Selective media are used for the growth of only select 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. coli unable to synthesize it were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes.
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. Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker.
Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker. Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase (HSV TK)

some examples of selective media include:

• eosin-methylene blue agar (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria
• YM (yeast and mold) which has a low pH, deterring bacterial growth
• blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolytic Streptococcus
• MacConkey agar for Gram-negative bacteria
• Hektoen enteric agar (HE) which is selective for Gram-negative bacteria
• mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol
• Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.
• xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria
• buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila

Differential media:

Differential media or indicator media distinguish one microorganism type from another growing on the same media.^[5] This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
Examples of differential media include:

• eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation
• MacConkey (MCK), which is differential for lactose fermentation
• mannitol salt agar (MSA), which is differential for mannitol fermentation

X-gal plates, which are differential for lac operon mutants


MICROBIAL CULTURE:

 A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined media. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a media to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the caustive agent of strep throat.^[1] Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.

 A culture of Bacillus anthracis
For the purpose of gelling the microbial culture, the medium of agarose gel (Agar) is used. Agar is a gelatinous substance that is derived from seaweed. A cheap substitute for agar is Guar gum, which can be used for the isolation and maintenance of thermophiles.                                                                                                                                                                                                                                                                                                                                                                                                                
                                                                                                            V.JAHNAVI.