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BISC209: Enrichment: Difference between revisions
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→Enrichment media and Isolation protocols (Starting with either SOIL or SOIL EXTRACT)
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Dilute nutrient agar is a solid medium with the same formulation as nutrient agar except that it contains a lower concentration of each component. It also a general purpose medium, but lowering its nutrient concentration further limits fungal growth and favors soil bacterial culture. In broth form, the solidifying agent (agar) is not included. <BR><BR> | Dilute nutrient agar is a solid medium with the same formulation as nutrient agar except that it contains a lower concentration of each component. It also a general purpose medium, but lowering its nutrient concentration further limits fungal growth and favors soil bacterial culture. In broth form, the solidifying agent (agar) is not included. <BR><BR> | ||
==Enrichment media and | <font size="+1">'''Selective / Differential / Enrichment media'''</font size="+1"><BR> | ||
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. Media can be any permutation or combination of selective, differential, and/or enrichment, depending on its ingredients and the microbial growth desired or undesired. <BR> | |||
For more information on the formulations and types of media available in microbiology see: [http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp BD diagnostice Systems Difco catalog of media] http://www.bd.com/ds/technicalCenter/inserts/difcoBblManual.asp<BR> | |||
==Selective for Gram positive Bacteria== | |||
'''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 . <BR> | |||
'''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.<BR><BR> | |||
'''Mannitol Salt Agar''' (an alternative to PEA, not used in 2010)<BR><BR> | |||
Postive control organism: ''Staphylococcus epidermidis'' | |||
==Selective for Gram negative Bacteria== | |||
'''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.<BR><BR> | |||
'''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 <BR><BR> | |||
'''Table 2.''' Colonial appearance on EMB Agar after 18-24 hours at 35°C. A differential medium can be used to differentiate Gram negative enteric organisms based on the colony color.<BR> | |||
{| border="1" | |||
|+ | |||
! '''Organism''' !! '''Colonial Appearance''' | |||
|- | |||
! ''Escherichia coli'' | |||
| purple with black center/ green metallic sheen | |||
|- | |||
! ''Klebsiella pneumoniae'' | |||
| dark centered colonies/ green metallic sheen | |||
|- | |||
! ''Enterobacter aeorogenes'' | |||
| pink colonies/ no metallic sheen | |||
|- | |||
! ''Proteus mirabilis'' | |||
| colorless colonies | |||
|- | |||
! ''Salmonella typhimurium'' | |||
| colorless colonies | |||
|- | |||
|} | |||
<BR><BR> | |||
Reference: Dehydrated Culture Media and Reagents for Microbiology. DIFCO Laboratories, Detroit, MI. 1984. | |||
==Enrichment media to isolate specific soil bacterial community members based on functional roles<BR> | |||
(Starting with either SOIL or SOIL EXTRACT)== | |||
<font color="purple">'''Actinomycetes enrichment <BR> | <font color="purple">'''Actinomycetes enrichment <BR> | ||
Glycerol Yeast Extract Media and/or Arginine-Glycerol-Salt (AGS) medium'''</font color="purple"><BR> | |||
'''It is important that we isolate representatives of this diverse group of organisms so make sure both soil samples from your habitat are enriched for ''Actinomycetes'' using both of the media described, Glycerol Yeast and AGS.''' Most of the | '''It is important that we isolate representatives of this diverse group of organisms so make sure both soil samples from your habitat are enriched for ''Actinomycetes'' using both of the media described, Glycerol Yeast and AGS.''' Most of the | ||
