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Microbiology

Microbiology 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 Demonstration

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Microbiology

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  1. Microbiology Science in Motion Summer Biology Teacher Workshop 2004

  2. Aseptic Technique and Staining Techniques in Microbiology • Background Information: • Aseptic Technique • Negative Stain • Smear Preparation • Simple Stain • Gram Stain

  3. Aseptic Technique Demonstration • 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

  4. Negative Stain • Use Nigrosin Stain to stain SLIDE NOT organisms • Background should appear dark gray and organisms should appear opaque or translucent against the background • Used for morphological studies

  5. Simple Stain • Bacteria must be stained in order to be observed • 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

  6. 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.

  7. Bacterial Cell Wall Diagrams

  8. Gram Stain Procedure

  9. Analysis of Hand Washing Techniques • Hand washing is the beginning of infection control. • 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

  10. 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.

  11. Antibiotics (E) Erythromycin (T) Tetracycline (S) Streptomycin (N) Novobiocin (C) Chloramphenicol Antiseptics (Bet) Betadyne (Bio) Biotene (Via) Viadent (Bac) Bactine (Lis) Listerine Antibiotics and Antiseptics for Experiment

  12. Effect of UV Light on Microbial Growth • The effect of UV light negatively affects most bacteria • 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

  13. 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.

  14. 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

  15. 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

  16. 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

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