Microbiology

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

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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

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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

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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.

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Bacterial Cell Wall Diagrams
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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.

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Antibiotics and Antiseptics for Experiment

• Antibiotics
• (E) Erythromycin
• (T) Tetracycline
• (S) Streptomycin
• (N) Novobiocin
• (C) Chloramphenicol

• Antiseptics
• (Bet) Betadyne
• (Bio) Biotene
• (Via) Viadent
• (Bac) Bactine
• (Lis) Listerine

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

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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.

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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

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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,
  youve messed up ALL of them.
• Demonstrate proper shaking technique
• Elbow remains on lab table