Aseptic techniques, smear preparation and simple staining

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Aseptic techniques, smear preparation and simple
staining

• Lab 2
• Bio 261, Microbiology
• Prof. Santos

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AIM

• Exs 9, 11, and 12

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Aseptic technique

• Aseptic technique refers to the reduction
  of contamination from the environment and other
  areas as the scientist is transferring bacteria
  from one medium to another.

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Aseptic techniques

• 1- wipe your work area before, and after you are
  done working
• 2- If you ever spill something, please call me
  because it needs to be wiped immediately and the
  area needs to disinfected again with Lysol!
• 3- when dealing with microorganisms, you need to
  maintain sterile conditions.

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Transferring cultures

• 1- when transferring from a liquid medium to a
  liquid medium, flame the mouth of the tubes and
  flame the loop until it is red hot. Follow the
  protocol on the book. I will demonstrate in class
  as well. The purpose is to maintain the purity
  and to make sure you have only a pure culture!

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• 2- when transferring from a liquid to a solid,
  flame the mouth of tube, flame the loop and only
  open the plate slightly and gently streak the
  agar surface. DO NOT LEAVE THE PLATE EXPOSED TO
  AIR!
• Label the bottom of a plate and invert in the
  incubator to prevent moisture from condensing on
  the agar surface.

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• 3- When transferring from a solid to liquid,
  flame the mouth of tube, flame the loop, and take
  out only a small sample of the culture.
• I will demonstrate!

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Smear preparation

• The goals of preparing a good smear are
• 1- allow the cells to adhere to the surface of
  the slide so that they are not washed off during
  staining.
• 2- allow the cells to adhere properly so that
  they do not shrink during staining otherwise
  distortions and artifacts can interfere with our
  results

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• 3- prepare a thin smear to allow only one layer
  of cells to adhere otherwise you will get layers
  of cells on top of each other and you will not be
  able to examine the individual cells!

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Procedure for making a bacterial smear

• From liquid medium
• A- draw a target circle on the bottom of the
  slide
• B-Place 2 loopfuls of culture in the center of
  target circle and spread over the entire circle
• C-Let it air dry
• D-Using a clothespin, grab slide and pass it
  several times over the flame to heat kill and fix
  the organisms.

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• From solid medium
• A- Place 2 loopfuls of water in the center of
  target circle
• B- disperse a small amount of cells with
  inoculating loop in water over the entire circle
• C- let it air dry
• D- Using a clothespin, grab slide and pass it
  several times over the flame to heat kill and fix
  the organisms.

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Simple staining

• The use of a single stain to color a bacterial
  cells.
• Common dyes used to stain cells are methylene
  blue, crystal violet, and basic fuchsin.
• Dyes are either negative or positive.
• Since cells are negatively charged, they are best
  stained with positive dyes that contain cationic
  chromophores or coloring bearing ions!

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Procedure

• We will either stain an avirulent strain of
  Corynebacteruim diphtheria or E. coli.
• 1- prepare a good smear of the culture
• 2- stain with methylene blue for one minute, use
  staining tray!
• 3- wash the stain off with water briefly!
• 4- blot dry with bibulous paper
• 5- look under the microscope

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If you stained C. diphtheria, look for

• 1- pleomorphism, several shapes of the cells
• 2- metachromatic granules, reddish granules of
  volutin
• 3-Palisade arrangement of the cells, picket fence
  arrangement of the cells