Ans. Hektoen Agar is a selective medium that helps in detecting and isolating only the Salmonella and Shigella organisms from other enteric bacteria. Since it can also isolate the growth of salmonella from shigella sps., it can be classified under differentiating media. The bile salts present in the composition of the medium inhibit the growth of gram-positive organisms in the culture and allow only gram negative bacteria to grow. Sucrose, salicin and lactose are added into Hektoen Agar in order to allow the growth of pathogens that are lactose fermentors. Ammonium ferric citrate acts as indicator (reacts with substrate to give rise to a color for visualisation) and Sodium thio-sulfate reacts with hydrogen sulfide (released by salmonella Sps. but not by schigella Sps.) and helps in formation of black precipitate with iron (II) present in ferric citrate. Thus black-centered salmonella colonies represent precipitated black ferrous ammonium citrate while schigella appear as bluish green colonies. The indicators consisting of acid fuchsin and bromothymol blue help in improved recovery of gram negative bacteria. So, the composition of Hektoen Agar is peptone or yeast extract, bile salts, sucrose, lactose, salicin, ferric citrate, sodium chloride, sodium thio-sulfate, acid fuchsin, agar and bromothymol blue (qtd. in King and Metzger 77).
2. Hektoen Agar was designed to differentiate salmonella and shigella from other enteric. Salmonella species sometimes produce black precipitate in their growth shigella species do not. If you were designing a medium to differentiate these two genera, which ingredients from this medium would you include Explain
Ans. Shigella species show negative result for motility test and they cannot ferment lactose in the medium and hence do not produce gas (fermentation leads to gas production). Most of the Salmonella species are lactose fermentors and produce gas. Addition of ammonium ferric citrate and sodium thio-sulfate to the newly designed medium will differentiate Salmonella Sps. from Schigella Sps. as Salmonella appear in black centered colonies due to hydrogen sulfide production and reaction of it with iron, while Schigella appear in bluish green colonies. Ammonium ferric citrate which is a source of iron is used as indicator for hydrogen sulfide (H2S) produced by Salmonella Sps. and sodium thio-sulfate is used as the substrate for building hydrogen sulphide which gives together with iron a black precipitate (Iron(II) sulphide FeS). H2S-positive colonies (salmonella sps.) have black centers and bluish green periphery. So, if there is release of hydrogen sulfide then black centered colonies appear while shigella do not release hydrogen sulfide hence appear in only bluish green (qtd. in King and Metzger 77).
3. All enteric ferment glucose. What would be some consequences of replacing the sugars in Hektoen agar medium with glucose. What color combinations would you expect to see
Ans. Fermentation of glucose gives pink-red color by all other enterobacteriaceae members as they are lactose fermentors. Salmonella Sps. as usual show black centered colonies while Schigella show bluish green colonies as usual as it cannot ferment lactose. Colorless colonies will be shown by those bacteria like pseudomonas (obligate aerobe) that did not get inhibited in their growth in Hektoen agar.
4. Which ingredient in Hektoen Agar contains Carbon Which Ingredient contains nitrogen
Ans. Mixed peptone and yeast extract are rich sources for nitrogenous compounds, vitamins, carbon, sulphur and amino acids.
With respect to the macConkey Agar, what would be the possible consequence of
A. Replacing the lactose with glucose
Ans. As macConkey agar is inhibitory and differential medium used to distinguish lactose-fermenting Gram-negative organisms from those that cannot ferment lactose. By replacing lactose with glucose this medium may not distinguish lactose fermentors from lactose non-fermentors. All the enterobacteriaceae members can grow in the presence of glucose.
B. Replacing the neutral red with phenol red (yellow when acidic red or pink when alkaline)
Ans. Crystal violet, bile salts and neutral red are inhibitory agents. Neutral red is the pH indicator. Neutral red exists in red color normally and changes to yellow at pH 6.8-8.0. While phenol red exists in yellow color normally and changes to red at pH 6.8-8.0. So in the presence of lactose fermentors, lactose get converted into acid and hence the colonies turn pink-red with neutral red indicator but turn into yellow with phenol red indicator. In the case of non-lactose fermenting bacteria no acid production is made and hence colonies remain colorless.