Science and Technology

An Overview Of Microbial Culturing Techniques


Culturing techniques of microbes allow isolating samples from the field to detect the presence of microorganisms by culturing them. Several culture media for growth of different microbes have been established. The most important growth media for microbes are nutrient broths and agar plates. In this article we will talk about culture media and its classification. Let’s get started!

What is Culture Media?

Agar as solidifying agent is used as a culture media. It is a complex polysaccharide function as a solidifying agent for culture media in petri plates, slants, and deeps. Generally, it is not metabolized by microbes, liquefies at 100C and solidifies at ~40C

How Culture Media can be Classified?

 Bacterial culture media are often classified on the basis of at least 3 ways:

 1. Consistency

 2. Nutritional component

 3. Functional use

Classification depending on consistency

  • Liquid media: These are available to be used in test-tubes, bottles or flasks. Liquid media are sometimes called as “broths” (such as nutrient broth). In liquid medium, bacteria may reproduce evenly producing smooth turbidity. No agar is added and are mostly used for inoculums preparation.
  • Solid media: An agar plate may be a Petri dish that contains a growth medium (typically agar plus nutrients) accustomed culture microorganisms. 2% of agar is added. Agar is the most ordinarily used solidifying agent, as a result colony morphology, pigmentation, hemolysis can be appreciated. Examples include agar and nutrient agar.
  • Semi-solid media: They are fairly soft and are important for the demonstration of bacterial movements and differentiating motile from non-motile strains. Example of Semi-solid media includes Hugh & Leifson’s oxidation fermentation in which 0.5% agar is added.

 Classification depending on Nutritional Components

  • Simple media: Simple media like peptone water, culture medium can support most non-fastidious bacteria. It’s also called as basal media. Eg: NB, NA. Nutrient Broth includes yeast extract, peptone, and NaCl. When 2% of agar is added to nutrient broth it forms agar.
  • Complex media: Media aside from basal media are called complex media. They have special components in them that are essential for the growth of microorganisms. These special components such as casein hydrolysate, yeast extracts contain a mixture of a large number of chemicals in an unknown proportion.
  • Synthetic media/Chemically defined media: Specially prepared media for research purposes where the composition of each component is standard. It’s prepared from pure chemical substances. Eg: peptone water (1% peptone + 0.5% NaCl in water).

Classification depending on Functional Use or Application

Enriched media:

Form by the addition of more nutrients within the blood, serum, or egg to basal medium. It is a media accustomed isolate pathogens from a mixed culture which stimulate the growth of desired bacterium and inhibit growth of unwanted bacterium. Enriched media can be incorporated with inhibitory materials to suppress the growth of unwanted organism leading to the increase in numbers of desired bacteria. Examples of enriched media include:

  • Selenite F Broth – for the isolation of Salmonella, Shigella, Tetrathionate Broth – inhibit coliforms, and alkaline Peptone Water – for Vibrio cholera.
  • Chocolate Agar that can be a non-selective, enriched growth medium used for growing fastidious bacteria, like Haemophilus influenza, blood agar.
  • Blood agar plate that may contain mammalian blood usually of sheep or horse, typically at a level of 5– 10 percent. BAP are differential or enriched media that is used to isolate fastidious organisms. They are also involved in the detection of hemolytic activity.

Selective media:

The inhibitory substance is added to a solid media thus causing a rise in number of colonies of desired bacterium. Selective media and enrichment media are prepared to inhibit the undesired commensal or contaminating bacteria. They also help recovering pathogen from a mixture of bacteria. Any agar media can be designed as a selective media by adding specific inhibitory agents that don’t disturb the pathogen. To design a medium as selective, it includes addition of dyes, antibiotics, chemicals, and alteration of pH.

Examples of Selective media are:

  • Thayer Martin works as a selective medium for Neisseria gonorrhoeae.
  • EMB agar acts as a selective media for gram-negative bacteria. The dye methylene blue within the medium inhibits the expansion of gram-positive bacteria; small amounts of this dye effectively inhibit the expansion of most gram-positive bacteria.
  • Campylobacter Agar (CAMPY) is involved in the selective isolation of Campylobacter jejuni.
  • EMB agar Campylobacter agar

Differential Media:

Certain media are designed in such a way that different bacteria are often recognized on the type of their colony color. Several approaches can be involved such as metabolic substrates, incorporation of dyes, in order that those bacteria that use them do not look as the same colored colonies. Substances incorporated in it enable it to differentiate between bacteria. Example of differential media: MacConkey’s agar, CLED agar, Salmonella and Shigella species, XLD agar, nutrient agar, and xylose lysine deoxycholate agar.

Transport Media:

Clinical microorganisms must be transported to the lab instantly after collection to overcome the additional growth of contaminating organisms and commensals. Small organisms with delicate morphology may not survive the time taken for moving the specimen without a transport media. This can be performed by using a transport media. Transport media should fulfill the subsequent criteria:

  • Temporary storage of the organisms being moved to the lab for cultivation.
  • Sustain the viability of all microbes within the specimen without inducing any affect on their concentration.
  • Contain only buffers and salt.
  • Lack of carbon, nitrogen, and organic growth factors so for the prevention of prevent microbial multiplication.
  • Transport media utilized in the isolation of anaerobes must be freed from molecular oxygen.

Indicator Media:

It contains an indicator which changes its color when a bacterium grows in them.  Examples are Wilson-Blair medium – S. typhi forms black colonies, McLeod’s medium (Potassium tellurite)– Diphtheria bacilli, and urease media: urea- CO2 + NH3.  NH3- medium turns pink.

Anaerobic media:

Special media is needed for the growth of anaerobic bacteria because they require additional nutrients, low oxygen content, and reduced oxidation – reduction potential. This media is for anaerobes that may need to be supplemented with nutrients like hemin and vitamin K. Boiling the medium can remove any dissolved oxygen present. Example of Anaerobic media includes thioglycollate medium.

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