Today

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Today

• Assignments
• Return MWA 1, Pre-lab 1
• Collect Pre-lab 2, MWA 2
• Exercise 2 Staining
• Simple Stain
• Gram Stain
• Capsule Stain
• Environmental Isolate
• Continue Purification

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Problems From Last Week

• No trash in the Nalgene bucketsonly used pipettes

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Assignment Problems

• Pre-lab
• A little more detail in the methods.
• Good Ex. We will perform a simple stain by
  staining Bacillus with methylene blue.
• Bad Ex. We will do a simple stain.
• Remember Relevance should be to the entire field
  of Microbiologynot this class!

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Assignment Problems

• MWA-1
• Date should be the day you turn the assignment
  in, not the day you did it.
• Plural vs. singular
• Bacteria plural
• Ex. The bacteria found in the wound were
  responsible for the infection.
• Bacterium singular, rarely used
• Ex. An isolated colony originates from a single
  bacterium.

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Assignment Problems

• Important definitions
• Cell biology the study of cells (structure,
  function, etc.), sometimes called cytology
• Histology the study of tissues
• Microbiology the study of microorganisms,
  normally unicellular, including bacteria
  (prokaryotes), and fungi and protists (eukaryotes)

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Staining

• Brightfield illumination is used to visualize
  cells when they have been stained to show
  different features.
• Likewise, when using Brightfield illumination,
  bacterial cells are often stained to provide
  contrast and make them easier to see.

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Staining Types

• Simple simple stains use a single dye to observe
  cellular morphology and arrangement.
• Methylene Blue is a simple stain.
• Differential differential stains use multiple
  dyes to differentiate between two different
  cells, or two different parts of the same cell.
• Gram stain differentiates between cell types.
• Capsule stain differentiates between a cell and
  its capsule.

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Staining Types

• Anionic these stains are repelled by negatively
  charged cell walls, and are used in negative
  staining techniques.
• Negative Stains are used to determine morphology
  and cellular arrangement of cells that are to
  delicate to withstand heat fixing.
• Negative stain is repelled by the negatively
  charged cell wall, so it stains the background
  instead of the cell.
• Eosin, Nigrosin and Methylene Blue are all
  examples of negative stains.

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Staining Types

• Cationic these stains are positively charged, so
  they bind to the negative cell wall.
• Gram Stain an important differential stain that
  reveals differences in the nature of bacterial
  cell walls based on the amount of peptidoglycan.
• Gram-positive cells appear purple.
• Gram-negative cells appear pink.

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Staining Procedure

• Wet Mount Procedure used last week in lab. Used
  to view live organisms.
• Dry mount Used as a first step before staining
  cells. Cells are heat fixed.
• Smear Cells are spread over the whole slide.
  Cells are NOT heat fixed.

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Staining Procedure

• Dry Mount
• Remember to clean all your slides with Ethanol!
• Spread a loop-full of bacteria from liquid
  culture onto a slide.
• OR
• Emulsify (mix) a small amount of bacteria from a
  plate in a drop of water on the slide.
• Allow the slide to air dry. The thinner you
  spread the bacteria, the faster it will dry!!!

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Staining Procedure

• Heat fixing
• After preparing your dry mount, pass the slide
  over the Bunsen burner flame.
• It should be passed over the flame 3 to 4 times.
• The slide should be hot when pressed against the
  skin, but not hot enough to burn!
• Purpose of Heat fixing
• Kills the bacteria
• Helps the bacteria adhere to the slide
• Makes the bacteria more permeable to stains

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Staining Methylene Blue

• Methylene Blue is a negative, simple stain
• uses a single dye (Methylene Blue)
• Used to observe cellular morphology and
  arrangement.
• Organism Bacillus megaterium (large,
  Gram-positive. rod)

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Staining Methylene Blue

• Prepare a dry mount
• Heat fix the cells
• Flood slide with Methylene Blue
• Gently rinse slide with distilled water
• Blot dry with Bibulous paper
• View slide under 100x oil immersion
• (Focus at 10x, proceed to 40x, then to 100x)

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Staining Gram Stain

• Gram Stain a differential stain that is widely
  used to aid in identification of bacteria
• Originally devised by Hans Christian Joachim
  Gram, a Danish doctor, so Gram should always be
  capitalized.
• Gram stains differentiate between two major cell
  wall types

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Staining Gram Stain

• Gram-positive (purple) Bacteria with walls
  containing relatively LARGE amounts of
  peptidoglycan and NO lipopolysaccharide.
• Gram-negative (pink) Bacterial species with
  walls containing SMALL amounts of peptidoglycan
  and, characteristically, lipopolysaccharide (LPS).

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Staining Gram Stain
Streptococcus pneumoniae G cocci, Pathogenic
Proteus vulgaris G- rod, Opportunistic
Staphylococcus epidermidis G cocci, Opportunistic
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Staining Gram Stain

• Gram-negative rods, Gram-positive rods, and
  Gram-positive cocci will be available for
  staining
• Gram negative cocci are often pathogenic, so they
  will not be used.
• All three cultures (e.g. a Gram-negative rod, a
  Gram-positive coccus, and a Gram-positive rod)
  should be prepared on the same slide and stained
  simultaneously

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Staining Gram Stain

• Begin by preparing a dry mount, mixing a
  loop-full (inoculating loop) of each of these
  cultures
• Flood the slide with Crystal Violet and let stand
  1 minute
• Gently rinse with water
• Flood the slide with Gram's Iodine solution and
  let stand 1 minute

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Staining Gram Stain

• Pour off the excess iodine solution and
  decolorize with 70 ethanol
• Gently rinse with water
• Flood the slide with Gram's Safranin and let
  stand for about 2 minutes
• Rinse the Safranin off with water and blot the
  slide dry
• Observe under 100X oil immersion

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Staining Gram Stain (recap)

• Prepare a dry mount
• Flood with Crystal Violet 1 minute
• Rinse with water
• Flood with Gram's Iodine let stand 1 minute
• Pour off iodine and decolorize with 70 ethanol
• Gently rinse with water
• Flood with Gram's Safranin and let stand 1-2
  minute
• Rinse with water
• Blot dry
• Observe under 10x, 40x, 100x dry then oil
  immersion

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Staining Capsule Stain

• A negative, differential stain used to identify
  bacteria that have capsules.
• Capsule
• Protective layer around a bacterium
• Made of glycoprotein or polypeptides
• Impervious to stains
• Many pathogens have a capsule

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Staining Capsule Stain

• Emulsify Klebsiella pneumoniae into 1 drop of
  Congo Red on the end of a slide.
• Do not heat fix!!!
• Spread the emulsion over the entire slide by
  dragging the drop over the slides length with
  another slide. (similar to blood smear)

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Staining Capsule Stain

• 3. Allow to air dry.
• 4. Flood the slide with Maneval's Stain and let
  stand for 1 minute.
• 5. Rinse with water and blot dry.
• 6. Observe under 10x, 40x, 100x dry then oil
  immersion.

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Helpful Hints

• Remember to wear safety glasses when working with
  cultures.
• You are not required to wear gloves today.
• Dont forget to clean all slides with Ethanol.
• The thinner you spread your culture on the slide,
  the faster it will dry.
• If you get stain on the lab bench or microscope,
  clean immediately with Ethanol and paper towels.
• Clean oil off of the 100x lens after each slide.
  It will bake onto the lens if you leave it!

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Helpful Hints

• If you wear contact lens you will see weird
  floaty things when you look in a scope. Do not
  confuse those with your specimen!
• Do NOT lower the stage when you put oil on.
• Move the object lens to the null position, add
  oil, and then move straight back to 100x.

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Helpful Hints

• Empty stain tray after each stain to prevent
  messes.
• Pick up cultures by the tube, not the cap, and be
  sure to vortex them.
• Bunsen Burners
• Make sure your flame isnt to high
• Make sure the gas is all the way off
• Do not set hot loops on the bench place them in
  your tube rack

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Env. Iso. Purification

• Choose an isolated colony.
• Isolated colonies should be found in the third
  section of your plates.
• Re-streak colony on a new plate.
• You will re-streak your organisms until you reach
  purity

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Env. Iso. Purification

• Isolation Streak Pattern

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Purification

• Remember to flame loops between each section.
• Use only light pressure when streaking. Your
  loop should just brush across the top of the
  agar.
• You need to come and re-streak your isolates
  through out the week.
• As soon as there are isolated colonies in the
  third section, re-streak on a new plate.

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Before You Leave

• Your drawer and station materials are all in
  place
• Your staining tray and rack are clean and in the
  sink
• Your microscope is clean, checked in and put away
  correctly (no stain or oil on the scope)
• Your Env. Isolate is labeled and stored on the
  appropriate shelf
• Your lab top is disinfected and your hands washed

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Next Week

• Assignments
• Pre-lab 3.1
• MWA 3
• Exercise 3
• Stains acid fast, endospore
• Sterile Technique
• Environmental Isolate
• Continue purification