Cultural characteristics of microorganisms on special media- Differential/ selective media

Differentiation of microorganisms which are morphologically and biochemically related is quite difficult.  The use of special media such as differential, selective, enriched and enrichment media is found to make the identification process much easier.  Selective media are used to select specific groups of bacteria.  They incorporate chemical substances that inhibit the growth of one type of bacteria while permitting growth of another, thus facilitating bacterial isolation.  Differential media incorporate chemical compounds that, following inoculation and incubation, produce a characteristic change in the appearance of bacterial growth and/or the medium surrounding the colonies, which permit differentiation. Eg: Mannitol salt agar (MSA), MacConkey agar, Eosin-methylene blue (EMB) agar.  

 Aim

To differentiate and identify microorganisms on the basis of the cultural characteristics on differential/ selective media.

 Principle

Special purpose media usually contain the necessary nutrients for growth as well as one or more chemical substances that are essential for the functional specificity of the microorganism.  Mannitol Salt Agar (MSA) contains a high salt concentration (7.5 %) which is inhibitory to most of the bacteria except Staphylococci. This medium also helps to differentiate between mannitol fermenting and non fermenting Staphylococci.  The indicator phenol red, in the medium detects the presence of acid produced by the mannitol fermenting Staphylococci which produce a yellow zone surrounding its growth.  Non fermenting Staphylococci will not show change in coloration. 

Mac Conkey agar contains crystal violet which is inhibitory to gram positive bacteria.  The medium also contains the carbohydrate lactose, bile salts and pH indicator neutral red. This medium is useful to differentiate enteric bacteria on the basis of their ability to ferment lactose.  On this basis, enteric bacteria can be separated into Coliform bacilli and Dysentery, typhoid and paratyphoid bacilli.  Coliform bacilli ferment lactose and the acid produced will change the color of indicator.  E.coli produce large amount of acid than other coliforms.  Hence, the medium surrounding the growth also becomes red as the acid precipitates bile salts, followed by absorption of the neutral red.

Eosin-Methylene Blue (Levine) Agar (EMB Agar) contains lactose and the dyes eosin and methylene blue that permit differentiation between  enteric lactose fermenters and non-fermenters as well as identification of the colon bacillus, E.coli.  The E.coli colonies are blue black with a metallic green sheen caused by the large quantity of acid produced and that precipitates the dyes on to the growth surface.  Other coliforms such as Enterobacter aerogenes, produce thick mucoid, pink colonies on this medium.  Enteric bacteria that do not ferment lactose produce colorless colonies, which, because of their transparency, appear to take on the purple color of the medium.  This medium is also partially inhibitory to the gram positive organisms.

         Endo Agar is a medium used primarily to isolate and distinguish coliform bacteria, especially                 lactose-fermenting enterics like Escherichia coli from non- fermenters in water, food, and clinical          samples. It contains lactose as the fermentable carbohydrate, peptone for nitrogen, dipotassium            phosphate as a buffer, and agar as the solidifying agent; its selectivity and differential properties              come from the combination of sodium sulfite and basic fuchsin dye. The sodium sulfite basic                  fuchsin system partially inhibits Gram-positive organisms, allowing Gram-negative enterics to               grow more readily. Lactose fermenters produce acid (and intermediates like acetaldehyde) that                react with the sulfited fuchsin, releasing the dye and causing colonies to turn red/pink; strong                fermenters such as E. coli often develop a characteristic metallic green sheen (“fuchsin sheen”) due         to intense acid production and dye precipitation on the colony surface. Organisms that do not                  ferment lactose form colorless, translucent colonies that may appear to take on the pale                            pink/background color of the medium. 

          MSA, MacConkey agar, EMB agar and Endo agar can be considered as the selective as well as              differential media.

 Requirements 

24 to 48 hr broth culture of species Escherichia coli, Proteus, Pseudomonas, Staphylococcus and Bacillus.

Mannitol salt agar, MacConkey agar and Eosin-methylene blue agar plates, Bunsen burner, inoculating loop, glass marking pencil etc.

Procedure

The given bacterial cultures are inoculated into the special media as follows:

1.  Appropriately label each of the plates.  Divide each of the petridishes into three sections (one section for each different organism) by marking the bottom of the plate.

2.  Inoculate each of the different media with the following:

    Mannitol salt agar:  Bacillus, E coli and Staphylococcus sp.

    MacConkey agar:   Staphylococcus, Pseudomonas and E.coli

    Eosin-methylene blue agar: Bacillus, Proteus and E.coli

              Endo Agar : Bacillus, Proteus and E.coli

                                                                                                                                                           Using sterile technique, inoculate all plates with the designated organisms by making a single line of inoculation of each organism in its appropriate section.  Flame inoculating loop between inoculations of different organisms.

3.  Incubate the plates in an inverted position for 24 to 48 hrs at 37^o C.

 Observation

On Mannitol salt agar:  Bacillus and E coli produced no growth.  Staphylococcus sp. produced characteristic colonies on the medium.  The organism exhibited yellow zone surrounding their growth indicating acid production as a part of mannitol fermentation.

On MacConkey agar:   Staphylococcuss sp. produced no growth. Pseudomonas sp. produced colorless colonies on the medium. E.coli showed pink colored colonies due to lactose fermentation.

EMB agar : Bacillus sp. showed no growth on the medium. Proteus sp., produced colorless colonies. E.coli produced blue black colonies with a metallic sheen.

         Endo Agar: Bacillus sp. showed no growth on the medium. Proteus sp. produced colorless to pale          pink colonies. E. coli produced red colonies with a metallic green (fuchsin) sheen. 

Results

Mannitol salt agar:  Bacillus and E coli are non- halophiles and Staphylococcus sp. is halophile.

MacConkey agar:   Streptococcus and Pseudomonas sp. are non lactose fermenters and E.coli is lactose fermenter.

EMB agar: Bacillus and Pseudomonas sp. are non lactose fermenters and E.coli is an enteric lactose fermenter.

          Endo Agar: Bacillus and Pseudomonas sp. are non lactose fermenters and E.coli is an enteric              lactose fermenter.