* Flora of the skin*

           ~ Aseptic technique ~

                   - used almost always when transferring organisms from and to different media
                       
                     The technique is simple !!

                     1) Flame sterilize inoculating loop by use of a bunsen burner

                     2) Flame sterilize mouth of tube that culture will be transferred to, and sterilize mouth of  tube that culture is taken from

                     3) Dip sterile loop into culture, then transfer to media

                     4)  Flame sterilize inoculating loop and mouth of both tubes by bunsen burner once transfer is complete

                   *Note- only steps 1 and 4 are required for flora of the skin experiments.

               

              

           

           

            pictures from : http://www.mc.uky.edu/oaa/curriculum/iid98/manual/00labtechniques.htm

              

            ~ Swabbing technique ~

                        - media used would be a Nutrient agar plate and a Malt extract agar plate supplemented with 0.1% erythromycin and 0.1% olive oil

                         The technique :

                         1) Sterilize cotton swab by dipping into sterile water or saline

                         2) Swab area on skin

                         3) Inoculate plate with one streak from cotton swab near the top of plate

                         4) Then using inoculating loop, streak away from main streak

               

               

Image taken from Applications in General Microbiology, A Laboratory Manual; Kerr/McHale; 6th edition;copyright 2003; pg.333

               ~ Additional techniques ~

                          - Gram Staining

                          - TSI agar slant and motility stab

*Flora of the throat and nasal passage*

- Different media are used to differentiate between pathogenic and  non-pathogenic organisms

Media and Techniques used:

    ~ Blood agar plates~

                -aseptic and swab techniques are used the same way as in flora of the skin except Blood Agar plate is used
                -shows different types of hemolysis
                    * alpha hemolysis- partially lysed; greenish or brownish zone around colony


                                   


                    * beta hemolysis- completely lysed; clear zone around colony
                   
                                   



                     * gamma hemolysis- no lyse; no change around colony

               
                                   


                   pictures from:  http://gold.aecom.yu.edu/id/micro/hemolysis.htm

                -shows difference in colony morphology
                    * Streptococcus colonies appear small and translucent on Blood agar plates
                    * Staphylococcus colonies appear large and opaque
               
                *Note---> incubation should be in a candle jar or a carbon dioxide incubator

                -Steps to follow to differentiate S.pneumoniae and S. mitis:

                    1) Take a throat and nasal swab and inoculate on Blood agar plate.
                    2) Pick and transfer an alpha hemolytic organism into a tube of saline solution
                    3) Pipet 0.5 ml of of this solution into tube with Bile solution, and another 0.5 ml into a tube of saline
                    4) If Bile tube has lost turbidity and becomes clear, then organism is bile soluble

    ~ Staphylococcus Medium No. 110~

                -differentiates Staphylococcus aureus from other Gram-positive cocci

                - Steps to follow: 

                    1) Take a throat and nasal swab and inoculate on Blood agar plate
                    2) Plate Staphylococcus colonies (yellow colonies with beta hemolysis) on Staphylococcus medium No. 110, and incubate 48 hours in 37C
                    3) For detecting coagulase activity---> using aseptic technique remove yellow or orange colonies from Staphylococcus medium and mix in tube of BHI                           (brain heart infusion) Broth. Then with a Pasteur pipet add 2 drops of BHI broth to a tube with rabbit coagulase plasma. Any clotting
                        within 3 hours is positive for coagulase.
                    4) Add Brom cresol purple indicator to Staph No.110 plate where the colony was picked. Any change in color means there is
                        fermentation of mannitol.
                    5) Flood plate with saturated ammonium sulfate and incubate. Clear zone around colonies picked--->gelatinase positive.
       

        

           

Image taken from Applications in General Microbiology, A Laboratory Manual; Kerr/McHale; 6th edition;copyright 2003; pg.334

                         

*References*
Applications in General Microbiology, A Laboratory Manual; Kerr/McHale; 6th edition;copyright 2003; pg. 327-335




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