Culture media and Bacterial Growth
Bacterial Growth and Culture media are solid, semisolid, and liquid nutrient preparations employed for the cultivation of microorganisms.
For strict autotrophic organisms – Only inorganic compounds.
For strict heterotrophic organisms – Complex organics substances such as vitamins, amino acids, proteins, coenzymes, etc.
The majority of bacteria show characteristics intermediate between the two, which can utilize both organic and inorganic compounds.
The various ingredients employed for the preparation of the common laboratory media and their uses are as follows :
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1. Water –
- Absolutely necessary for the existence of all living cells.
- Distilled water is generally preferable to tap water because it is of definite composition.
- Uniform media cannot always be prepared from tap water.
- The calcium and magnesium in tap water react with the phosphates present in peptone, beef extract, and other ingredients of culture media to give insoluble calcium and magnesium phosphates.
- The formation of precipitate during the sterilization process usually proves to be objectionable.
2. Peptone –
- Are produced by the action of certain proteolytic enzymes (usually trypsin) on native proteins.
- The commercial preparations employed in bacteriology are composed of proteoses or albumoses, peptones, peptides, and amino acids.
- The proportions vary considerably, depending upon the type of peptone desired.
Function of peptone –
- To furnish an available source of nitrogen.
- Since amino acids are amphoteric compounds, peptone is also an excellent buffer.
3. Meat /beef Extract
- Meat extract is prepared by boiling lean beef/beef in water, removing the liquid by filtration, and concentrating in a vacuum.
- It is a dark-colored, thick, pasty mass.
- Serves as a source of carbon and nitrogen.
- Rich source of nitrogenous nutrients such as creatinine, xanthine, urea, glutamine, beta-alanine, etc.
- Rich source of other nutrients such as glucose, hexophosphates, lactic acid, fat, inorganic salts, etc.
4. Yeast extract –
- Obtained from spent yeat from distilleries or breweries.
- To extract the cells, heated in distilled water at 45°C, and the pH is adjusted to 6.5.
- Suspension is stirred up to 14-16 hours.
- Heating is controlled to avoid denaturation of contents on autolysis.
- During stirring, cells autolyze and contents come into the suspension, this extract is dried under the vacuum to a paste or powder.
- Provide nutrients like that of meat extract, especially the B-complex group.
5. Agar –
- Agar is the dried mucilaginous substance extracted from several species of Gelidium and closely related species growing chiefly along the coasts of Japan, China, Ceylon, and Malaya.
- Agar is a complex carbohydrate classified with polysaccharides.
- It dissolves in water at a temperature of about 98°C. and does not solidify until the temperature drops to about 45°C.
- It acts as a solidifying agent.
- There are certain organisms that can attack and liquify agar. e.g. Vibrio, Pseudomonas, and Cytophaga.
6. Sodium chloride –
- Is an essential ingredient required to maintain the isotonicity of the medium?
- It is necessary for the transport of nutrients into the cell.
Other salts –
Media must contain 10 known elements;
Ammonium, sodium, and potassium salts are added as a buffer, ammonium serves as a nitrogen source.Co, Mn, Zn, Cu( trace elements) are required in a small amounts for the metabolism of microorganisms.
Fermentable Compounds –
(1) They furnish readily available sources of energy.
(2) Fermentation reactions are of great help in identifying and classifying organisms.
- Pentoses – Arabinose, xylose, rhamnose.
- Hexoses – Glucose, levulose, mannose, galactose.
- Disaccharides – Sucrose, lactose, maltose, trehalose, melibiose.
- Trisaccharides – Raffinose, melezitose. 4.Polysaccharides: Starch, inulin, dextrin, glycogen, cellulose.
- Trihydric – Glycerol.
- Pentahydric – Adonitol.
- Hexahydric – Mannitol, dulcitol, sorhitol.
- Glucosides – Salicin, amygdalin.
- Noncarbohydrate Compounds – Inositol.
Types of culture media
There are main two types of media. i.e. Synthetic and non-synthetic media.
Synthetic media –
- Composed of compounds of known chemical composition.
- Made up entirely of inorganic salts or most of the ingredients are inorganic and only 1 or 2 complex but chemically defined organic substances.
- The purpose is one can easily duplicate it from batch to batch.
- Used to determine the nutritional requirements of bacteria.
Complex or non-synthetic media –
- Contain ingredients of unknown chemical composition such as yeast/ meat/ beef extract, peptone, blood, serum, etc.
- Not possible to exactly duplicate the composition of the medium.
- Commonly used in Microbiological work.
- To isolate and grow particular organisms
- Simultaneously inhibiting /suppressing the growth of other types of cultural conditions which will selectively promote the growth of the desired organism.
- The liquid medium is used for enrichment purposes.
- A solid medium is used for isolation.
- Brilliant green agar is selective for Salmonella by inhibition of Gram-positive bacteria by brilliant green.
- Sabouraud’s agar is selective for fungi due to its low pH (5.6) and high glucose concentration (3-4%).
Composition of MacConkey’s broth
- Peptone – 20.0 g
- Sodium taurocholate – 5.0 g
- Lactose – 10 g
- Distilled water – 1 liter
- pH – 7.2
- Neutral red (1% sol.) – 4 mL
- The dye should be added only after adjusting the pH.
- Distribute 10 ml in different test tubes.
- Put Durham’s tubes in an inverted position in these tubes.
- Sterilize by autoclaving at 121° c for 15 minutes
Composition of MacConkey’s agar
- The lactose fermenters ( E.coli, Klebsiella spp.) utilize lactose in the medium and produce acid and gas
- The Colour of neutral red to pink and gas production is detected in inverted Durham’s tubes in broth.
- MacConkey’s broth + 2-3 % agar-agar
- In MacConkey’s agar plates color of colonies of lactose fermenting organisms are pink and are opaque as at acidic pH, neutral red precipitates in the colony.
- Lactose non-fermenters (Salmonella spp. and Vibrio spp.)show no pink coloration around the colony.
- Medium changes the color to yellow and the colonies remain translucent.
Differential media is used for growing and differentiating between different types of bacteria on solid media.
- Blood agar can be used to differentiate between hemolytic and non-hemolytic bacteria.
- Selective and Differential media Classical example of such a medium is MacConkey’s broth and MacConkey’s agar.
- Used to grow fastidious organisms ( growth factor requirement is high)
- Such as human pathogens which require blood or serum added into their medium.
- Blood agar is necessary to grow and detect the hemolytic activity of Streptococcus pyogenes.
- Lowenstein – Jensen’s medium contains egg albumin.
- Egg serves a dual purpose –
- Provides rich protein source for the organism
- Helps in gelling of the medium.
- The basic principle involved is that of selection.
- Used when the desired organism is a minority in the sample, say 0.1% of the total.
- The essence of this technique is to provide growth conditions that are very favorable for the organism of interest, and as unfavorable as possible for competing organisms.
Various methods used in enrichment culture technique –
1) Enrichment by modifying the physical conditions. To isolate a bacterium that is a thermophile (prefers to grow at a high temperature such as 55°C),
- incubate the sample at that high temperature.
- Organisms that cannot tolerate that temperature will die or simply fail to grow.
- While thermophiles will grow and increase in number, over time becoming a large and larger proportion of the total bacterial population in the sample.
2) Enrichment by modifying the nutrient content of the culture medium,
- For example, for enriching free-living nitrogen fixers like Azotobacter, the medium used is Ashby’s liquid medium which lacks a nitrogen source.
- When the broth is inoculated with the soil sample, only those species that can fix atmospheric nitrogen can grow.
- These organisms form a pellicle at the surface of the medium and hence can be isolated easily.
3) Manipulating the culture conditions – to isolate bacteria that can break down a xenobiotic (man-made, unnatural compound) such as a pesticide.
- Pesticides are organic compounds (most Carbon and Hydrogen).
- Bacteria generally break down organic compounds as a source of building materials and energy.
- In a growth medium in which the pesticide Is the sole carbon source for growth, only those bacteria that can use the compound will grow appreciably, while others will not
- Eventually, the culture will contain a sufficiently high proportion of pesticide degraders that it would be easy to isolate them using the streak plate technique.
Winogradsky’s column : (Enrichment medium)
- An effective means of enriching photosynthetic bacteria, sulfur bacteria, and anaerobic bacteria in a single shot is Winogradsky’s column.
- The column is simple to prepare.
- Take a glass jar or a measuring cylinder.
- Place a handful of mud along with shredded paper at the bottom.
- Add a few grams of calcium sulfate and calcium carbonate over it.
- Then fill the jar to 3/4th its height with pond water and keep it in a place with appropriate diffused light.
- The contents can be sampled after every 3 to 4 days.
- Samples should be withdrawn from different depths and observed microscopically with and without staining.
- Typically, Algae and cyanobacteria grow at the top.
- Green and purple sulfur bacteria, Beggiatoa.
- Thiothrix and Thiobacillus sp. in the middle and anaerobic bacteria at the bottom.
Minimal medium and Complete medium
- Prototrophs – They are wild-type bacteria that can synthesize all compounds needed for growth from simple ingredients /any microorganism that can synthesize its nutrients from inorganic material.
- Auxotrophs – They are mutant strains that lack one or more enzymes required for metabolizing nutrients and can only grow on supplemented media.
- Minimal Media contains the essentials for bacterial species to grow.
- The media is often used to define if a particular microbial species is a heterotroph, namely an organism that does not have any nutritional requirements beyond core sources of carbon (sugars) and nitrogen (to synthesize amino acids and nucleic acid bases).
- Prototrophs can grow but Auxotrophs or organisms with nutritional requirements will not be able to grow on minimal media.
Important components are –
- A source of carbon, commonly the sugar glucose. A
- A source of nitrogen needed to synthesize amino acids for proteins and bases for nucleic acids. Ammonium chloride is commonly used (NH4Cl), particularly given that the majority of bacterial species cannot fix nitrogen (convert atmospheric N2 into biologically usable ammonia or amine groups).
- Ions/electrolytes – like us, bacterial cells need core nutrients such as sodium, chloride, potassium, calcium, phosphorus, and magnesium. These components are provided in the “M9 salts” solution
A culture medium that is enriched to contain all of the growth requirements of a strain of organisms.
A medium for an in vitro culture that contains the supplemental nutrients as well as the basic nutrients to support fastidious or mutant(auxotrophs) growth requirements. Eg. Nutrient agar, Plate count agar
Nutrient Agar is a general-purpose, nutrient medium used for the cultivation of microbes supporting the growth of a wide range of non-fastidious organisms.
Nutrient agar is popular because it can grow a variety of types of bacteria and fungi, and contains many nutrients needed for bacterial growth.
Nutrient Agar/broth is used for the cultivation and maintenance of non-fastidious organisms as well as enumeration of organisms in water, sewage, dairy products, feces, and other materials.
Plate Count Agar (PCA), also called Standard Methods Agar (SMA), is a microbiological growth medium commonly used to assess or to monitor the “total” or viable bacterial growth of a sample. PCA is not a selective medium.
The composition of plate count agar may vary, but typically it contains (w/v) – 0.5% peptone, 0.25% yeast extract ,0.1% glucose ,1.5% agar pH adjusted to neutral.