Nutrient Agar

Principle

Nutrient agar allow the growth of a variety of microorganisms that do not usually require specific nutrients or supplements. The primary constituents are peptone, beef extract, and agar. The peptone is the source of nitrogen or protein that acts as a source of amino acids. The beef extract is the primary source of carbon which is essential for the formation of carbohydrates.

It also contains other components like some vitamins, trace minerals, organic compounds, and salts, which enhance the growth of different organisms. sodium chloride is added to maintain the osmotic equilibrium of the medium and prevent the change in pH during growth. The distilled water dissolve the nutrients.

Agar is the solidifying agent that provides a stable surface for the organism to grow on, which allows for the observation of colony morphology and enumeration of organism.

Procedure

Dissolve all ingredients (except sugar) by heating and adjust to pH 7.4. Add sugar powder and digest it by boiling in water bath. Recheck the pH and sterilize it by autoclaving. Nutrient broth has the same composition except that it does not contain the solidifying agent agar it is prepared in the similar manner.

MacConkey’s Agar

Principle

MacConkey Sorbitol Agar is only slightly selective, the concentration of bile salts, which inhibits gram-positive microorganisms. Peptone and proteose peptone supply nutrients like nitrogenous and carbonaceous compounds, amino acids, minerals, vitamins and trace ingredients for the growth of organisms.

Crystal violet inhibits the growth of grampositive bacteria, especially enterococci and staphylococci. Sodium chloride maintains osmotic equilibrium. Neutral red is an indicator. D-Sorbitol is the fermentable carbohydrate.

Differentiation of enteric microorganisms is achieved by the combination of sorbitol and the neutral red indicator.The growth of E.coli O157:H7 on MacConkey agar shows colorless colonies and most of the fecal flora ferment sorbitol and appear pink. Colorless or pink to red colonies are produced depending upon the ability of the isolate to ferment the carbohydrate sorbitol.

Procedure

Dissolve by heating, adjust pH to 7.4 and sterilize by autoclaving. 

Eosin Methylene Blue Agar

Principle

EMB agar is a differential medium used for the isolation of coliforms. Aniline dyes (eosin and methylene blue) inhibit Gram-positive and fastidious Gram-negative bacteria. Both dyes combine to form a neutral compound which precipitates at acidic pH, thus serving as an indicator of acid production. Typical colonies of strong lactose fermenter E.coli are small nucleate with a greenish black metallic sheen. While atypical colonies of weak lactose fermenter En.aerogenes appear as opaque, non-nucleated and mucoid. lactose non-fermenters produce transparent colonies.

Procedure

Dissolve ingredients (except lactose, eosin and methylene blue) in distilled water by heat and sterilize by autoclaving. Add lactose, eosin and methylene blue after autoclaving. Mix well and steam it for 5 minutes.

Wilson & Blair’s Agar for salmonella spp.

Principle

WB agar is highly selective medium for Salmonella. Due to the presence of brilliant green dye and the heavy metal bismuth, most species of Gram-positive bacteria, lactose fermenting coliforms and the shigella are completely inhibited.

As the typhoid organism grow in presence of glucose it reduces sodium sulphite to sulphide which together with bismuth forms bismuth sulphide. The organism in presence of bismuth Sulphide and glucose forms black colonies provided the medium is not too acidic. To prevent excess of acidity (due to fermentation of glucose) sodium phosphate is present the buffer. Ferric citrate is added to neutralize the toxicity of bismuth. Colonies of Salmonella typhi appear black, while Salmonella paratyphi B appear black with a metallic sheen. Salmonella paratyphi A produce greenish colonies.

Procedure

Dissolve separately bismuth ammonium citrate & sodium sulphite, in little amount of distilled water. Mix both solution. Add Na2HPO4 & glucose. Adjust final volume to 200 ml with distilled water.

CLED Medium

Cysteine Lactose Electrolyte Deficient Agar for Urinary Tract Infection

Principle

The CLED comprises of enzymatic digest of Casein & gelatin, and beef extract which provide the nitrogen, vitamins, mineral and amino acids essential for growth. L-cystine supports the growth of cysteine-dependent dwarf coliform colony. Lactose is the fermentable carbohydrate which provides carbon and energy. Organisms capable of fermenting lactose will lower the pH and change the color of the medium from green to yellow. The color change is indicated by the presence of pH indicator Bromothymol Blue. Agar present in the medium is the solidifying agent.

Procedure

Add component to distilled / deionized water and bring volume to 1 liter.Mix throughly . Gently heat while stirring & bring to boiling autoclave for 15 min. at 15 psi pressure and 121`C. Cool to 55`C, pour into sterile petridish or distribute into sterile tubes.

King’s Medium for Pseudomonas sp.

Principle

Pseudomonas aeruginosa is known to produce two types of pigments pyocyanin and fluorescein. Pyocyanin is green while fluorescein is fluorescent yellow Kings Medium B Base is particularly suited for fluorescein. It is used for the non-selective isolation and pigment production of Pseudomonas species.

These media contain proteose peptone, which provides carbonaceous and nitrogenous compounds for the growth of bacteria. Glycerol serves as a source of energy and also enhances pigment production. Magnesium sulphate also enhances pigment production.

The production of pigments especially nonfluorescent blue pigment, pyocyanin is readily demonstrated by culturing on Kings Medium B. The addition of dipotassium phosphate increases the phosphorus content of the medium thereby enhancing production of fluorescent pigment. 

Procedure

Add component to distilled water and bring volume to 1 liter. Mix thoroughly, gently heat & bring to boiling. Distribute into tubes or flask. Autoclave for 15 minute at 15 psi 121`C. Pour into sterile petridish or tubes.

Mannitol Salt Agar for Staphylococcus spp. 

Principle

Mannitol Salt Agar contains peptones and beef extract, which supply nitrogen, vitamins, minerals and amino acids essential for growth. The 7.5% concentration of sodium chloride results in the partial or complete inhibition of bacterial organisms.

Sodium chloride supplies electrolytes for transport and osmotic balance. Mannitol is the fermentable carbohydrate, fermentation leads to acid production, detected by phenol red indicator, Coagulase positive staphylococci (e.g., Staphylococcus aureus) produce yellow colonies and a surrounding yellow medium while coagulase negative staphylococci produce red colonies and no color change of the phenol red indicator.

Agar is the solidifying agent.

Procedure

Dissolve all ingredients into distilled water by heating, pH should be adjusted to 7.3 to 7.5. autoclave at 121°C for 15 minutes at 15 PSI pressure. Cool the medium to 50-55°C and dispense in sterile petridish. 

Advantages of Bacteriological Media

Each of these bacterial culture media has specific advantages for the isolation and identification of different types of bacteria. Here's a brief overview of the advantages of each:

1. Nutrient Agar

It provides a general-purpose medium suitable for the growth of most non-fastidious bacteria. It contains all the necessary nutrients for bacterial growth, making it suitable for primary isolation and cultivation of a wide range of microorganisms.

2. MacConkey’s Agar

Selective and differential medium used for the isolation and differentiation of Gram-negative bacteria, especially enteric bacilli such as Escherichia coli and Salmonella spp. It contains crystal violet and bile salts that inhibit the growth of Gram-positive bacteria and allow for the selection of Gram-negative organisms. It also contains lactose and pH indicators, allowing for the differentiation of lactose fermenters (pink colonies) from non-fermenters (colorless colonies).

3. EMB Agar (Eosin Methylene Blue Agar)

Selective and differential medium primarily used for the isolation and differentiation of fecal coliforms such as Escherichia coli. It contains eosin and methylene blue dyes that inhibit the growth of Gram-positive bacteria and allow for the selection of Gram-negative organisms. It also contains lactose and sucrose as fermentable carbohydrates, allowing for the differentiation of lactose fermenters (dark colonies with a green metallic sheen) from non-fermenters.

4. King’s Medium

It is a selective and differential medium used for the isolation and differentiation of Gram-negative bacteria, particularly Salmonella spp. It contains peptone, lactose, bile salts, and indicators such as neutral red, which facilitate the detection of lactose fermentation and the differentiation of Salmonella spp. from other enteric bacteria.

5. Wilson & Blair’s Agar

This medium is used specifically for the isolation and identification of Salmonella spp. from clinical samples. It contains selective agents such as bile salts and brilliant green that inhibit the growth of other bacteria and favor the growth of Salmonella spp.

6. CLED Medium (Cysteine Lactose Electrolyte Deficient Agar)

It is a differential medium used for the isolation and presumptive identification of urinary tract pathogens, particularly Escherichia coli and other lactose-fermenting bacteria. It contains lactose and indicators such as bromothymol blue, which allow for the differentiation of lactose fermenters (yellow colonies) from non-fermenters (colorless colonies). Additionally, it lacks electrolytes, making it suitable for urine culture, as excessive electrolytes in urine can inhibit bacterial growth.

7. Mannitol Salt Agar

Selective and differential medium primarily used for the isolation and differentiation of Staphylococcus aureus from other staphylococci and non-staphylococcal bacteria. It contains high concentrations of salt (7.5-10%), which inhibit the growth of most bacteria except for staphylococci. It also contains mannitol and a pH indicator, allowing for the differentiation of mannitol fermenters (yellow colonies) from non-fermenters (pink to red colonies).

Disadvantage of Bacteriological Media

Here are some potential disadvantages for each of the mentioned bacterial media:

1. Nutrient Agar:

•    - Not selective: It supports the growth of a wide range of bacteria, making it difficult to isolate specific organisms.
•    - Lack of differentiation: It does not provide any differentiation between different types of bacteria.

2. MacConkey’s Agar:

•    - Limited use: It is primarily used for the isolation and differentiation of Gram-negative enteric bacteria, so it may not be suitable for other types of bacteria.
•    - Selective but not differential: While it inhibits the growth of Gram-positive bacteria, it only differentiates between lactose fermenters and non-fermenters among Gram-negatives.

3. EMB Agar (Eosin Methylene Blue Agar):

•    - Toxicity: EMB agar contains eosin and methylene blue dyes, which can inhibit the growth of some bacteria.
•    - Overgrowth of non-target organisms: Some non-target bacteria may still grow on EMB agar, potentially obscuring the presence of target organisms.

4. King’s Medium:

•    - Complexity: King's medium is relatively complex and may require more preparation time and expertise compared to simpler media.
•    - Specificity: It is designed for specific types of bacteria and may not be suitable for other organisms.

5. Wilson & Blair’s Agar for Salmonella spp:

•    - Limited selectivity: While it is selective for Salmonella spp., it may still allow the growth of other bacteria, potentially leading to false positives or contamination.
•    - Time-consuming: Identifying Salmonella spp. colonies on Wilson & Blair’s agar may require additional biochemical tests, prolonging the overall identification process.

6. CLED Medium (Cysteine Lactose Electrolyte Deficient Agar) for Urinary Tract Infection:

•    - Limited differentiation: CLED agar primarily distinguishes between lactose fermenters and non-fermenters, which may not provide enough information for comprehensive bacterial identification.
•    - Selectivity concerns: While it inhibits the growth of some organisms, it may not completely prevent the growth of all contaminants, leading to potential false positives.

7. Mannitol Salt Agar:

•    - Limited application: It is primarily used for the isolation and differentiation of Staphylococcus species, so it may not be suitable for other types of bacteria.
•    - High salt concentration: The high concentration of salt in Mannitol Salt Agar may inhibit the growth of some organisms while allowing others to grow, potentially leading to inaccurate results.