With the importance of microorganisms in many fields and industries like agriculture, medicine, pharmacy, the dairy industry, clinical research, chemical technology, and nanotechnology, it is easy to understand the attention being given to their growth and study in laboratories.
Many types of growth media have been developed by scientists to grow selected or desired microorganisms.
This article is focused on the growth or culture of E. coli (one of the most important bacteria that are of keen interest in teaching, biotechnology, and clinical laboratories) on nutrient agar, one of the vital culture media in microbiology.
What is Escherichia coli?
Escherichia coli (E. coli) are gram-negative, facultative, anaerobic rod-shaped bacteria with very diverse species.
They are a highly studied, common species of bacteria belonging to the family Enterobacteriaceae – so named because many of its members live in the intestines of humans and warm-blooded animals.
E. coli was named after Theodor Escherich, a German-Austrian pediatrician. Escherich first isolated a variety of bacteria from infant feces by using his own anaerobic culture methods and Hans Christian Gram’s staining technique.
E. coli is mostly commensal, living harmlessly in the intestines; though some are pathogenic. They can grow rapidly on a minimal medium that contains a carbon compound such as glucose (which serves both as a carbon source and an energy source) and salts that supply nitrogen, phosphorus, and trace metals.
E. coli grows more rapidly, however, on a rich medium that provides the cells with amino acids, nucleotide precursors, vitamins, and other metabolites that the cell would otherwise have had to synthesize.
Types of growth media
- General-Purpose Media (Basal media)
- Enriched Media
- Anaerobic Media
- Transport Media
- Differential/ Indicator Media
- Selective and Enrichment Media
Growth media are mediums that provide essential nutrients and minerals to support the growth of microorganisms in the laboratory. It is not possible to culture all microbes with only one type of growth medium. This is because microbes have varying characteristics, habitats, and even nutritional requirements.
There are also some microbes that cannot grow on a culture media at all in any condition – these are called obligate parasites.
Based on their practical use, composition, and application, there are six types of standard culture media in microbiology laboratories. They are general-purpose (basal) media, enriched media, selective or enrichment media, differential or indicator media, transport media, anaerobic media, and assay media.
General-Purpose Media (Basal media)
Basal media, also called general-purpose media, are simple media that support the growth of most non-fastidious bacteria. Peptone water, nutrient broth, and nutrient agar (NA) are basal media. These media are generally used for the primary isolation of microorganisms.
Enriched media are made by adding extra nutrients, such as blood, serum, egg yolk, etc., to a basal medium. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Examples include blood agar, chocolate agar, Loeffler’s, and serum slope.
Blood agar is prepared by adding 5-10% (by volume) blood to a blood agar base while chocolate agar is heated blood agar or lysed blood agar.
Selective and Enrichment Media
These media are designed to inhibit unwanted commensal or contaminating bacteria and help to recover pathogens mixed with bacteria. While selective media are agar-based, enrichment media are liquid.
Both these media serve the same purpose. Any agar media can be selective by adding specific inhibitory agents that don’t affect the pathogen of interest. The addition of antibiotics, dyes, chemicals, alteration of pH, or a combination of these is some of the approaches to preparing a selective medium.
Examples of selective media include Thayer Martin Agar; MacConkey’s Agar; Crystal Violet Blood Agar Mannitol Salt Agar and Salt Milk Agar.
Unlike selective media, enrichment media are typically used as a broth medium. Enrichment media are liquid media that also serves to inhibit commensals in the clinical specimen.
Examples like Selenite F broth, tetrathionate broth, and alkaline peptone water (APW) to recover pathogens from fecal samples.
Differential/ Indicator Media
Certain media are designed to recognize different bacteria based on their colony colors. Various approaches include incorporating dyes, metabolic substrates, etc., so those bacteria that utilize them appear as differently colored colonies. Such media are called differential media or indicator media.
Differential media allow the growth of more than one microorganism of interest but with morphologically distinguishable colonies. Examples include MacConkey agar; Mannitol salts agar and Blood agar.
These are used for transporting clinical specimens to the laboratory immediately after collection to prevent the overgrowth of contaminating organisms or commensals and maintain the viability of the potential pathogens.
Such media prevent the drying (desiccation) of a specimen, maintain the pathogen-to-commensal ratio, and inhibit the overgrowth of unwanted bacteria.
Some of these media (Stuart’s and Amie’s) are semi-solid. The addition of charcoal serves to neutralize inhibitory factors. Examples include: Cary Blair transport medium; Venkatraman Ramakrishnan (VR) medium; Pike’s medium and Sach’s buffered glycerol saline
Anaerobic bacteria need special media for growth because they need low oxygen content, reduced oxidation-reduction potential, and extra nutrients.
The media for anaerobes may have to be supplemented with nutrients like hemin, and vitamin K. Such media may also have to be reduced by physical or chemical means. Examples include cooked meat broth (e.g. Robertson Cooked Meat); anaerobic blood agar; egg-yolk agar (EYA); anaerobic phenyl ethyl alcohol agar and Thioglycollate broth.
Other types of medium include assay media, media for the enumeration of bacteria, media for the characterization of bacteria, maintenance media, etc.
Can Escherichia coli (E. coli) Grow on Nutrient Agar?
Nutrient agar is a general-purpose liquid medium supporting the growth of a variety of non-fastidious organisms, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, among others. It contains nutrients that are suitable to grow a wide range of microorganisms. It is an excellent agar medium to check the purity before any biochemical or serological test.
It is similar to nutrient broth in composition, the only difference being that while nutrient agar has agar, the nutrient broth does not. Besides, the addition of agar solidifies nutrient agar, which makes it suitable for the cultivation of microorganisms.
Nutrient Agar is an ideal medium for demonstration and teaching purposes as it allows more prolonged survival of cultures at ambient temperature without the risk of overgrowth that might occur with more nutritious mediums.
How to prepare nutrient agar
- 28 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water in a beaker
- The suspension is then heated to a boiling point to dissolve the medium completely. – The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.
- When the autoclaving process is complete, the beaker is taken out and cooled to a temperature of about 40-45°C.
- If enrichment is desired, the addition of blood or biological fluids can be done after the autoclaving process.
- The media is then poured into sterile Petri plates under sterile conditions.
- Once the media solidifies, the plates can be placed in the hot air oven at a lower heat setting for a few minutes to remove any moisture present on the plates before use.
Composition of Nutrient Agar (gm/liter)
- 5.0 peptone: this provides organic nitrogen
- 2.0 beef extract/yeast extract: the water-soluble content of these contribute vitamins, carbohydrates, nitrogen, and salts
- Lab-Lemco’ powder 1.0
- 15 agar: this gives the mixture solidity
- 5.0 sodium chloride: this gives the mixture proportions similar to those found in the cytoplasm of most organisms
- distilled water: water serves as a transport medium for the agar’s various substances – pH 7.4 ± 0.2
- Temperature 25°C
Limitations of Nutrient Agar
- Nutrient Agar becomes unreliable during isolation if different organisms differ in their growth requirement which leads to variable growth patterns on the medium.
- In cases where some microorganisms demonstrate similar colony morphologies on nutrient agar, it becomes difficult to distinguish them without microscopic examination.
- It cannot be used as a selective medium for the growth of fastidious organisms that have specific nutrient requirements because nutrient agar promotes the growth of many organisms, and the chances of contamination during isolation are quite high.
- While nutrient agar allows the isolation of bacteria, the same cannot be said of other microorganisms like fungi.
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Last Updated on April 11, 2023 by Our Editorial Team