M465:Selective Media

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Selective / Differential / Enrichment media
Selective media helps select for growth of certain organisms in a mixed population by using a ingredient that inhibits the growth of other microorganisms, but not the desired species or group. Enrichment media selects for certain microorganisms by including a nutrient that the desired microorganism or group can use and its competitors can not. (Sometimes enrichment media also limits alternate sources of nutrition). Differential media does not select for any particular group by inhibiting or enhancing their growth over competitors, but it does show a visible difference between or among groups of microorganisms. Some media can be both selective and differential.
For more information on the formulations and types of media available in microbiology see: BD diagnostice Systems Difco catalog of media http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp

Selective for Gram positive Organisms Phenylethyl Alcohol Agar (PEA) PEA selects for the growth of Gram positive organisms by inhibiting the growth of Gram negative bacilli. The phenylethyl alcohol interfers with DNA synthesis in Gram negative organisms. This medium is particularly useful at inhibiting the overgrowth of Gram negative Proteus species that tend to swarm (they are highly motile) and, thus, make isolation of Gram positive organisms difficult in a mixed population .
Recipe: 10g tryptose, 3g Beef extract, 5g Sodium Chloride, 2.5g Phenylethyl alcohol, 15g Agar to 1 liter distilled or deionized water pH 7.1-7.5.

Positive control organism: Bacillus subtilis

Selective for Gram negative Organisms Eosin–Methylene Blue (EMB) Agar is a differential medium for the detection of Gram negative enteric bacteria. The medium contains peptone, lactose, sucrose, dipotassium phosphate, eosin and methylene blue dyes. Eosin and methylene blue act as indicators to differentiate between Gram negative organisms that ferment lactose and those that do not ferment lactose. Most bacteria that ferment lactose form colonies on EMB agar that are dark blue to black with a metallic sheen due to precipitation of the dyes by the acid by-products of fermentation. Colonies produced by lactose non-fermentors are not dark blue or black. The growth of Gram positive bacteria is generally inhibited on EMB agar because of the toxicity of methlyene blue dye. In low concentration, the protective lipid outer membrane of Gram negative bacteria prevents entry of the toxic water soluble dye while the more porous cell wall of Gram positive bacteria without the protective outer membrane makes them more sensitive to the toxicity of methyene blue.

Recipe:10g peptone, 10g Lactose, 2g dipotassium phosphate, 0.4g eosin Y, 0.065 g methylene blue 15 g Agar. final pH 6.9-7.3

Positive control organism: E. coli

Lab Activities

Activity 1: Using selective media to confirm your gram stain results from last meeting.
You will be testing the growth of each of your isolates, and a positive control for each medium.

1. As always, label your plates with your initials and the date.
2. You will inoculate four isolates onto one plate. Therefore, mark out quadrants to be used for each isolate on the back of the agar using a sharpie marker. Label each quadrant. You will need two plates of each media (5 isolates + 2 control strains = 7 quadrants needed)
3. Bring your isolates and your labeled plates into the hood and inoculate the surface of your selective medium with the appropriate isolate.
4. Inoculate the positive and negative controls as well.
4. Place your plates in the 30C incubator when done.

Activity 2: Preserving your bacteria using slants.

You will continue to work with these 5 isolates for many laboratory sessions to come. In order to preserve them we will be growing them in slants. A useful link with pictures of what these look like can be found here: [1].

0. In the hood, sterilize your loop. Bring your patches and plates with colonies into the hood.
1. Label your tubes with your colored tape using your name, the lab (M465) and the isolate number. Using your patches (if you have 5) or a well separated colony from your plates (if you have less than 5 patches), touch the loop to the surface of the bacterial culture. This will form your inoculum for the slants.
2. Uncap the tube with the slanted agar and, using a zig-zag motion, distribute your bacterial culture across the entire surface of the slanted agar. Be careful not to dig or gouge the surface of the slant
3. Recap the tube and resterilize your loop.

4. Put your slants in the 30°C incubator overnight. Remove the following day and store in drawer.