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Materials Required: Bunsen Burner Inoculating loopMedia Used: Mannitol salt agar MacConkey’s agar Eosin Methylene Blue Agar Phenylethyl Alcohol Agar Hektoen Enteric Agar Blood Agar Chocolate AgarCultures used: Staphylococcus aureus Staphylococcus epidermidis Escherichia coli Pseudomonas aeruginosa Enterobacter aerogenes Proteus vulgaris Shigella flexneri Salmonella typhimurium Streptococcus pyogenes Streptococcus pneumoniae Streptococcus salivarius Haemophilus influenzae
Procedure:Mannitol salt agar, MacConkey’s agar, EMB agar, Phenylethyl Alcohol agar, HektoenEnteric agar, Blood agar and Chocolate agar plates are prepared and sterilized. Staphylococcus aureus and Staphylococcus epidermidis were inoculated into MSA plates. Escherichia coli and Pseudomonas aeruginosa were inoculated into two halves of the MacConkey’s agar plate. Escherichia coli and Enterobacter aerogenes were inoculated into EMB agar plates. Staphylococcus aureus, Staphylococcus epidermidis and Proteus vulgaris were inoculated into PEA agar plate. Shigella flexneri and Salmonella typhimurium were inoculated into HE agar plates. Three species of Streptococci namely Streptococcus pyogenes, Streptococcus pneumoniae and Streptococcus salivarius were inoculated into blood agar plates. Haemophilus influenzae was inoculated into chocolate agar plate.All these plates are incubated at 37oC for 24 hours.Expected Results1. Mannitol Salt Agar (MSA):On MSA, pathogenic Staphylococcus aureus ferments mannitol, thereby changing thecolour of the medium from red to yellow.
Figure 11: Culture of Staphylococcus aureus on Mannitol Salt AgarStaphylococcus epidermidis grows on MSA, but does not ferment mannitol (mediaremains light pink in color & colonies are colorless).Figure 12: Culture of Staphylococcus epidermidis on Mannitol Salt Agar2. MacConkey’s Agar (MAC):
In Macconkey’s agar, E.coli ferment lactose and turn pink in color while P.aeroginosawill be colorless as they are non lactose fermenters.Figure 13: MacConkey’s agar with lactose fermenters (left) and non-lactosefermenters (right)3. Eosin Methylene Blue (EMB) Agar (Levine):On EMB agar, E.coli produces colonies which are very dark, almost black, whenobserved directly against the light. By reflected light, a green sheen can be seen which isdue to the precipitation of methylene blue in the medium from the very high amount ofacid produced from lactose fermentation. Those which form this type of colony aremethyl red-positive lactose-fermenters.
Fig 14: Escherichia coli on EMB agarOn EMB agar, Enterobacter aerogenes produces colonies which are less dark. Often apink, dark center is seen surrounded by a wide, light-colored, mucoid rim – resulting in a"fish-eye" type of colony. Those which form this type of colony are methyl red-negativelactose-fermenters.
Fig 15: Enterobacter aerogenes on EMB agarNon-lactose-fermenting colonies produce no acid from fermentation, so the lighter-colored alkaline reaction is seen.Eg: Proteus vulgarisFig 16: Proteus vulgaris on EMB agar4. Phenylethyl Alcohol Agar:There will be selective growth of Gram positive organisms (S.aureus and S.epidermidis)in PEA agar.
Figure 17: PEA agar plate with selective growth of Gram positive bacteria5.Hektoen Enteric (HE) Agar:In Hektoen Enteric agar plates Shigella flexneri develop into green-colored colonies withdarker blue-green centers while Salmonella typhimurium appear as green colonies withblack centers.
Figure 18: Growth of and Shigella flexneri and Salmonella typhimurium onHektoen Enteric Agar6. Blood Agar:In blood agar, Streptococcus pyogenes cause beta hemolysis resulting in complete lysis of
red blood cells and hemoglobin. This results in complete clearing of the blood around thecolonies.Fig19: Beta hemolysisStreptococcus pneumonia cause alpha hemolysis which results in a greenish-greydiscoloration of the blood around the colonies.
Fig 20: Alpha hemolysisStreptococcus salivarius cause gamma hemolysis resulting in no change in the medium.Fig 21: Gamma hemolysis7.Chocolate Agar:
Haemophilus influenzae uses the enrichment growth factors and grow on chocolate agar.Figure 22:Haemophilus influenzae on Chocolate Agar PlateDifferences Encountered in a RealLaboratory:In an actual laboratory setting, there are certain important steps that are not necessarilyapplicable in a virtual lab: 1. Always wear gloves, and lab coat. 2. Tie your hair properly to prevent any contamination from the culture you are working with. 3. When you enter the lab switch on the exhaust fans. 4. Switch on the lights of the laminar air flow and blower. Prepare your work space
(Laminar Air Flow Cabinet) or lab bench by wiping down the area with disinfectant.5. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone; it is not a large plume, nor is it orange.6. Always label all tubes and plates with: 1. The name of the organism 2. The type of media 3. Your initials 4. The date7. While flaming the inoculation loop be sure that each segment of metal glows orange/red-hot before you move the next segment into the flame.8. Once you have flamed your loop, do not lay it down, blow on it, touch it with your fingers, or touch it to any surface other than your inoculums. If you do touch the tip to another surface or blow on it, you will have to re-flame the loop before you proceed to your experiment.9. Allow your loop to cool before you try to pick up your organism to avoid killing the inoculum.10. When removing the caps from tubes, always keep the caps in your hand. Never set them on the table, as they could pick up contaminants.11. Always handle open tubes at an angle near to the flame of the burner; never let them point directly up, since airborne or other environmental organisms could fall into the tube and cause contamination.12. Open the lid of the plate sufficiently (45 degrees) to introduce an inoculation loop and only for the time it takes to obtain inoculums.13. Rotate the plate counter clockwise 90 degrees and cross the prior streaks to pick up some bacteria and spread them into the next quadrant (Repeat in all the four quadrants;applicable only in the case of quadrant streaking).14. Streak gently; does not gouge the agar.15. As soon as the inoculation is completed, flame your loop or needle. Never place a contaminated tool on your workbench.16. Turn the inoculated petriplate upside down while keeping it in the incubator.17. Discard all contaminated materials properly and return your supplies to the proper storage locations, and clean up your working area.18. Always disinfect your work area when you are finished.19. Turn off Bunsen burner and make sure that the regulator of the gas cylinder is tightened.20. Switch off the laminar air flow and exhaust fans before leaving the laboratory.21. Ensure proper hand washing before you leave from the laboratory.
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