Document Sample
Microbiology Powered By Docstoc
     Science in Motion
Summer Biology Teacher
       Workshop 2004
Aseptic Technique and Staining
Techniques in Microbiology
   Background Information:
       Aseptic Technique
       Negative Stain
       Smear Preparation
       Simple Stain
       Gram Stain
Aseptic Technique

   Will demonstrate smear preparations
       Bacteria are smeared on a clean slide and allowed to
        air dry
       Slide is then passed through a flame which kills the
        bacteria and fixes them to the slide
           If done properly, bacteria will remain on slide throughout
            staining processes
Negative Stain
   Use Nigrosin Stain to stain SLIDE NOT
   Background should appear dark gray and
    organisms should appear opaque or
    translucent against the background
   Used for morphological studies
Simple Stain
   Bacteria must be stained in order to be
   Many different dyes are available to stain:
       Carbol Fushion    (Raspberry Red)   10 sec
       Crystal Violet    (Violet)          1 min
       Methylene Blue    (Blue)            1 min
       Safranin          (Pink)            1 min
       Malachite Green   (Green)           1 min
Gram Stain
   Used to identify unknown species of bacteria
   Gram reaction is based on the structure of
    the bacterial cell wall
       Gram + Crystal violet stain is trapped under
        peptidoglycan layer
       Gram – The outer membrane prevents crystal
        violet stain from reaching peptioglycan layer. The
        outer membrane is then permeabilized by alcohol
        (acetone) treatment, and the pink safranin stain is
        trapped in the peptidoglycan layer.
Bacterial Cell Wall Diagrams
Gram Stain Procedure
Analysis of Hand Washing
   Hand washing is the beginning of infection
   Use Aseptic Technique to wash hands at ALL
    TIMES to reduce infections
   Each group has a different soap sample so
    that we may compare results
   Use the same soap for both parts of the lab
Inhibition of Bacteria –
Antibiotics and Antiseptics
   Alexander Fleming first discovered penicillin (by
    accident) and thus lead to the understanding of
    bacterial inhibition
   Antibiotics inhibit or destroy bacterial cells in
    different ways
       Some inhibit the cell from producing peptidoglycan to
        protect cell walls. Results in a weak cell wall and cell will
        eventually rupture.
       Others inhibit the production of proteins at the ribosomes
        within the cell.
       Still others affect the way bacterial ribosomes read mRNA
        which leads to errors in protein structure.
Antibiotics and Antiseptics for
   Antibiotics                  Antiseptics
       (E) Erythromycin             (Bet) Betadyne
       (T) Tetracycline             (Bio) Biotene
       (S) Streptomycin             (Via) Viadent
       (N) Novobiocin               (Bac) Bactine
       (C) Chloramphenicol          (Lis) Listerine
    Effect of UV Light on Microbial
   The effect of UV light negatively affects most
       Occurs most readily between 260 and 270nm.
   The absorption of UV light causes the
    production of thymine-thymine dimers
       Nucleotides fail to form bonds with dimers, thus
        causing DNA replication to stop.
           Most damage is caused by the cell trying to repair itself
               Heavily damaged DNA attempt SOS repair
                 Eventually results in the production of new DNA strands with
                  a greater number of misplaced bases
                 This mutation leads to faulty protein synthesis and
                  eventually death
Hand Washing Lab Tips
   Make sure to label plates with group number
   Expose Plate A and Plate B at the same time
    in appropriate light boxes. All groups will
    expose under the same UV light
   After Plate A is finished remove plate and
    expose Plate C.
Microbes in the Environment
   This lab demonstrates approximate numbers
    of microorganisms growing in different
    locations in the environment
       It does not attempt to identify particular species
   Each group is assigned 3 locations and 1 of
    their choosing. Be Creative!!!
   Make sure to follow proper aseptic technique
   Document sample source in Data Table 1
The “Sizzler” – Bacterial
Population Counts
   Use SPC (standard plate count) to determine
    the number of bacteria in a culture sample
       Procedure
           Diluting organisms from your source using sterile
            water blanks
           Incubation of plates
           Analyze a plate with 30-300 colonies
           Conduct a calculation to determine the number of
            organisms per milliliter of sample
The “Sizzler” – Helpful Hints
   Tiffany will dispense out pipettes. When you
    need a new pipette see her.
   Read the directions CAREFULLY there are a
    number of dilutions. If you mess up the first
    one, you’ve messed up ALL of them.
   Demonstrate proper shaking technique
       Elbow remains on lab table

Shared By: