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Selective and differential media

So far you have used general purpose complex media (such as tryptic soy) to culture a broad range of bacterial species. This media was not formulated to support or inhibit growth of any particular species. We mentioned earlier that samples of mixed cultures on plates could result in confluency (wall-to-wall growth) or TNTC (>300 colonies) if the cell density of the sample is too great. In this case, you could plate a smaller sample or dilute the sample before plating. Another way to decrease colony density is to utilize a selective medium. Selective media are formulated to select for a particular group of (ie. inhibit the growth of all but a particular group of) bacteria. This is especially useful if the organism of interest to you exists in low numbers, and is heavily diluted by the other members of a mixed culture (trying to find a needle in a hay stack). This is a frequent situation in clinical samples.

Selective media contain at least one selective agent which inhibits the growth of the unwanted or “contaminating” microorganisms. Selective agents include salts, antibiotics or other inhibitory chemicals at varying concentrations. An example of a selective medium would be TSA with 7.5% added NaCl (table salt). This concentration of salt inhibits the growth of most bacteria, and therefore selects for those that require and/or are capable of growing in the presence of high salt concentrations. The selective media that we will use in this exercise will be Mannitol salt agar and Eosin methylene blue (EMB) agar. Other common selective media include xylose lysine desoxycholate (XLD) agar used for the isolation of Salmonella and Shigella species.

Using general purpose complex media, we are able to enumerate the total number of viable cells in a sample by counting the number of colonies that grow up on that media. This is true of pure cultures and mixed cultures alike. What if you wanted to know how many cells of a particular group of bacteria were present in a mixed culture, and you could not depend upon morphology alone to determine which colonies represented that group? One solution would be to plate the sample on a special type of media on which the colonies arising from a particular group of organisms looked unique. Differential media are designed to distinguish certain species, genera or larger physiological groups from others via differences in appearance of colonies, most often color differences of the colonies or surrounding medium. An example of a differential medium would be one containing a particular carbohydrate that could only be fermented to acid by certain microorganisms, mannitol for example. The medium would also contain a pH indicator which would change color as acid accumulated. Those microbes that do not cause the color change must be respiring mannitol, fermenting mannitol to something other than an acid, or using a different carbon and energy source present at a concentration too low to generate enough acid to cause a color change. Another commonly used differential medium is Triple sugar iron (TSI) slants which contains agar, a pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate. The medium differentiates Gram negative rods in 2 ways. First is by acid production from 1 or more of the carbohydrates. The other is by thiosulfate reduction resulting in the formation H2S, which then interacts with iron to form black FeS.

Some media are BOTH selective and differential. Consider the differential mannitol media above with 7.5% NaCl added. You now have a medium that selects for salt tolerant species, and differentiates between those that ferment mannitol to acid vs those that do not. Such a medium exists and is called Mannitol salt agar. Mannitol salt is used to differentiate mannitol fermenting Staphylococcus species (most of which are opportunistic human pathogens) such as S. aureus from non-fermenting Staphylococcus species (most of which are non-pathogens) such as S. epidermidis. See image “Mannitol salt agar.” Micrococcus and Streptococcus are not salt tolerant, and will either fail to grow on Mannitol salt media, or grow poorly. Other commonly used media that are both selective and differential for Gram negative enteric genera include MacConkey’s, Hektoen enteric (HE) and Salmonella Shigella (SS) agars. The differential basis of these media types involves color changes due to acid production from various carbohydrates in the medium.

EMB agar is a defined medium which contains lactose as a carbon and energy source. It also contains eosin and methylene blue which inhibit the growth of Gram positive bacteria. The basis for the differential property of EMB media is this: Gram negative lactose fermenting bacteria will take up the purplish-emerald green dyes at a rate proportional to the amount of acid produced from lactose fermentation. This medium will be used in our lab for differentiation of the “enteric” Gram negative rods, otherwise known as the oxidase negative, Gram negative rods. Of the organisms that we are working with this semester, this includes E. coli, E. aerogenes, C. freundii, K. pneumoniae, P. vulgaris, S. enteritidis, and S. flexneri. The term “enteric” implies “of the intestines,” or “of fecal origin.”

Appearance of Gram negative, oxidase negative colonies on EMB:

The characteristics described below only apply to isolated colonies. As you will see below, much variation exists among the results for most organisms on EMB, depending on crowding, time and temperature of incubation, and variance in media formulation and pH.

* Species of the genus Salmonella, Shigella and other non-lactose fermentors commonly produce little or no acid resulting in nearly colorless colonies on EMB.

* Enterobacter, Klebsiella and other “Aerogenes-type” organisms commonly produce a moderate amount of acid from lactose fermentation resulting in a characteristic “fish-eye” colony (light with dark center) on EMB agar. Some strains produce more acid resulting in colonies with dark coloration throughout. Alternatively, some cultures of these organisms will form nearly colorless colonies. Although most reports suggest otherwise, certain strains of Klebsiella will produce a transient green metallic-sheen on EMB. See image “EMB agar – fish eye colonies.

* E. coli, Citrobacter, Proteus and other “Coli-type” organisms produce relatively large amounts of acid from lactose fermentation resulting in the formation of colonies that are purplish-black throughout, or have a green metallic-sheen. Although the green sheen is most often attributed only to E. coli, the organism given credit as the most robust lactic acid producer, other genera such as Citrobacter and Klebsiella will sometimes form the green sheen. One distinction here is that the green sheen of E. coli is persistent whereas the sheen formed by Klebsiella fades over a period of 6-24 hours. Proteus colonies on EMB are commonly very small and purple-black throughout, although some Proteus cultures form nearly colorless colonies. See image “EMB agar – green metallic sheen.

Streak these plates for isolation just as you would streak a TSA plate. Having isolated colonies is not critical for proper results on mannitol salt agar, but IT IS on EMB. You will not see clear fish-eyes, etc. if colonies on EMB agar are not isolated.

NOTES

* We use mannitol salt agar for Gram positive cocci.

* The color change for mannitol salt occurs in the media itself, as well as the colony.

* We use EMB agar for Gram negative rods, especially for the oxidase negative variety.

* The media that we have discussed in this lab are both selective and differential. Understand these terms. Know the selective and the differential ingredients of each medium discussed. Understand the selective and differential basis of how these work. Know the expected result of each organism discussed above on mannitol salt and EMB.