Today: Exercise 3 Part 1

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Today Exercise 3 (Part 1)

• Assignments
• Collect Pre-lab 3.1, MWA 3
• Return Pre-lab 2, MWA 2
• Exercise 3
• Stains acid fast, endospore
• Sterile Technique
• Environmental Isolate
• Continue purification

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McMillan Ch.3

• Scientific papers are often supplemented with
  tables, graphs, photos, etc.
• Table and figures are NOT essential to all
  scientific papers.
• It is important to determine if your data can be
  summarized in the text, or should be in a table

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McMillan Ch. 3 Tables

• Use a table to present numerical values or to
  summarize or (occasionally) to emphasize verbal
  material.
• Large amounts of quantitative data
• Do not use a table to show an important trend in
  the data
• Tables can also summarize numerous important
  points, summarize literature reviews, or to
  compare and contrast
• Use this type of table sparingly and carefully

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McMillan Ch. 3 Tables
Table 1 Soil Analyses of four farm fields near
Malverne, Vermont
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McMillan Ch. 3 Tables
Table 2 Occurrence of Plantago at five vacant
lots in Morrisville, Ohio
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McMillan Ch. 3 Tables

• Number tables consecutively, and make them
  understandable on their own.
• Number the tables in the order you refer to them
• Even if there is only one table in the paper, it
  should still be numbered
• A table should be able to stand apart from the
  text and still be understandable

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McMillan Ch. 3 Tables

• Writing a title
• Usually a sentence fragment
• Usually lacks a verb
• At the top of the table
• Only the first word is capatilized
• The title of the graph must describe the data
• Bad Field data, Test Results, etc.
• Good Reponses of inexperienced adult blue jays
  to monarch and viceroy butterflies

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McMillan Ch. 3 Tables

• Use a logical format
• Arrange similar elements so they read vertically
• Center data under the column headings
• Put zeros in front of decimal places
• Ex. 0.23 not .23

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McMillan Ch. 3 Tables

• Make the contents concise.
• Include only important info in tables
• Do not include peripheral details, repetitive
  data, or uniform and unvarying values
• Abbreviations may be used in tables
• Any unfamiliar abb. Should be explained in
  footnotes
• Put any units of measurement in the column
  headings
• If the same abbreviations or procedures are used
  in more than one table, refer the reader back to
  the first table

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McMillan Ch. 3 Tables

• Check tables for internal consistency and
  agreement with the rest of the paper.
• Does what youre saying the text make sense with
  the table?
• Do the trends in the table match the trends in
  the graph?
• Proofread!

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McMillan Ch. 3 Figures

• Use a graph to illustrate an important, pattern,
  trend, or relationship.
• A graph is better than a table when you want to
  see the shape of data
• Generally
• Independent variable on the x-axis, and the
  dependent variable on the y-axis
• Axes should meet

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McMillan Ch. 3 Figures

• Graphs are not needed when
• Trends are not statistically significant
• Sparse or repetitive data
• If you can summarize the data in one sententce,
  you dont need the graph

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McMillan Ch. 3 Figures

• Number graphs consecutively, separately from
  tables, and make them understandable on their
  own.
• Number figures in the order they are discussed
• Figure 1, Figure 2etc.
• Write out the word Figure in the title and in
  the text, but not when in appears in parentheses
  (Fig. 1)

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McMillan Ch. 3 Figures

• Writing a title- Just like tables
• Usually a sentence fragment
• Usually lacks a verb
• At the top of the table
• Only the first word is capatilized
• The title of the graph must describe the data
• Legends
• Incomplete sentence, only the first word
  capatilized
• Should be brief and concise

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McMillan Ch. 3 Figures

• Plot data accurately, clearly, and
  economically.
• Make your axes only as long as necessary
• Combine related data sets on the same figure

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McMillan Ch. 3 Figures

• Depict data logically, in a manner consistent
  with your overall hypothesis.
• How you plot your data can change the
  interpretation
• Be sure to understand the purpose of the
  experiment before plotting data
• You dont always need to connect the dots
• Best-fit, statistical, lines
• Trend lines
• No lines

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Safety First!

• Carbol Fuchsin Malachite Green are known
  carcinogens (cancer causing substances)
• Always wear gloves when working with these
  stains!!!

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Staining Acid Fast

• Acid-Fast stain
• used to identify organisms of the genera
  Mycobacterium and Nocardia, including
  Mycobacterium tuberculosis
• contain waxes (mycolic acids) in their cell walls
  
• impervious to dyes such as those used in the Gram
  stain
• dyes can be driven into these organisms with
  heat, and once inside, are very difficult to
  remove (those not acid-fast can be
  counterstained with methylene blue)

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Staining Acid Fast

• Clean Slide
• Prepare a dry mount of your organism
  Mycobacterium smegmatis with a non-acid-fast
  control on the slide (Bacillus megaterium).
• Heat fix
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with carbol fuschin.
• Gently heat the slide for 5 minutes. Add more
  carbol fuschin as the filter paper starts to dry
  out.

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Staining Acid Fast

• 6. Remove paper and gently rinse slide with
  distilled water.
• 7. Decolorize slide with acid alcohol for 5-10
  seconds.
• (decolorizes Bacillus megaterium)
• 8. Rinse with distilled water. Counter-stain with
  methylene blue for 1 minute.
• 9. Rinse with distilled water and blot dry.
• 10. Visualize with 100x oil immersion lens.
• Acid-fast cells are pinkish-purple, non-acid
  fast cells are blue

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Staining Acid Fast
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Help Hints Acid Fast

• Cover base of Bunsen burner with paper towels
• Don't over heat fix
• Don't allow blotting paper to get dry while
  steaming in the carbol fuschin, add stain as
  needed.
• Hold with a clothespin while steaming
• Wear gloves
• Acid fast organisms will be pink, non-acid fast
  organisms will be blue

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Staining Endospore

• Endospore stain (Schaffer and Fulton staining
  method)
• A differential stain used to visualized
  endospores within an organism
• The staining procedure is very similar to the
  acid-fast procedure, with a very important
  exception Do Not Use Acid Alcohol to Decolorize!

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Staining Endospore

• Endospores are formed by some Gram positive
  bacteria.
• Many pathogenic organisms are spore-forming.
• E.g. B. anthracis, C. tetani, C. botulinum
• Endospores are resistant to heat, cold,
  dessication, UV irradiation, chemical
  disinfectants, dyes etc.

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Staining Endospore
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Staining Endospore

• Placement can be
• Terminal
• Subterminal
• Central
• Shapes may vary
• Spherical
• Elliptical (oval)

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Staining Endospore

• The resistance of the endospore is due to a tough
  outer covering made of the protein keratin.
• Keratin resists staining
• Malachite green is forced into the stain by
  steaming the bacterial emulsion.
• Malachite green has a low affinity for cellular
  material, so vegetative cells and spore mother
  cells can be decolorized with water and
  counterstained with Safranin.

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Staining Endospore

• Clean Slide
• Prepare a dry mount of your organism Bacillus
  megaterium by emulsifying a small amount from a
  colony on a plate.
• Heat fix
• Place a piece of filter paper over the slide,
  hold it in place with a clothespin and saturate
  the paper with Malachite Green.

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Staining Endospore

• Gently heat the slide for 5 minutes. Add more
  Malachite Green as the filter paper starts to dry
  out.
• Remove paper and gently rinse slide with
  distilled water.
• Counter-stain with Safranin for 1-2 minute.
• Rinse with distilled water and blot dry.
• Visualize with 100x oil immersion lens.

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Staining Endospore
Endospore (green)
Cell (pink)
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Helpful Hints Endospore

• Don't allow blotting paper to dry while steaming
  in the malachite green, add stain as needed.
• Hold with a clothespin while steaming
• Cover base of Bunsen burner with paper towels
• Wear gloves
• Endospores will appear green while vegetative
  cells will appear pink.
• Take your sample from the outer edges of the
  growth on the plate- otherwise there will be few
  vegetative cells.

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Sterile Technique

• Aseptic Technique
• Aseptically transferring liquids by pipetting
  sterile media from one tube to another.
• This exercise requires (and teaches) dexterity
  and technical skill. Today you will be using
  water instead of sterile media.
• Properly manipulating the equipment required to
  perform sterile transfers is very important, even
  though the medium used is water.

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Sterile Technique

• Transferring liquids (e.g., sterile broth or pure
  cultures) aseptically from one tube to another
  takes practice so that the liquid is not
  contaminated.
• When using sterile disposable pipettes, a Pipette
  Pump is attached to the top of the pipette in
  order to draw the liquid into the pipette. Never
  pipette by mouth.
• Green pipette holder for 5 and 10 ml pipettes
• Blue pipette holder for 1 and 2 ml pipettes.

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Sterile Technique

• 3. Insert the pipette into the Pipette Pump and
  gently but firmly push in and twist (Fig. 3.1).
  Make certain that you do not contaminate the
  pipette by touching the tip with your hands.

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Sterile Technique

• Remove lid, flame the mouth of the bottle or
  tube.
• Holding the vessel at an angle, introduce the
  loop or needle into the liquid and agitate
  gently. If a pipette is used, a measured volume
  of liquid can be released using the pipettor.

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Sterile Technique

• Flame the mouth of the bottle or tube before
  returning the cover.
• Flame the loop or needle after use to incinerate
  any remaining organisms. Dispose of all
  contaminated pipettes or tips in the appropriate
  nalgene containers.

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Sterile Technique

• Practice transferring water from a culture bottle
  to tubes aseptically in various amounts with the
  three different sizes of pipettes (1, 5 and 10
  ml).
• Next week you will use an enriched media to
  transfer and contamination will count.

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Sterile Technique

• You should be able to accurately transfer volumes
  of 0.1, 0.5 and 1.0 ml using a 1.0 ml pipette
• 0.5, 1.0 and 5 ml with a 5.0 ml pipette
• 1.0, 5.0 and 10.0 ml with a 10.0 ml pipette.

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Sterile Technique

• Practice all these transfers until you can
  comfortably transfer liquids aseptically
  (sterilely) and accurately (correct volume).
• Make sure to flame the lips of bottles and
  culture tubes before and after removing or adding
  liquids.
• Do not place bottle caps or culture tube closures
  on your lab bench hold them in your hand.

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Gram Stain Quiz

• You will be given a numbered tube containing E.
  coli, B. megaterium, S. epidermidis, or any
  mixture of the three.
• Spelling and format count!
• You must right the entire name (no abbreviations)
• Genus species (written)
• Genus species (typed)
• You can/should come into open hours to practice
  your gram stain.

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Gram Stain Quiz Format

• Unknown
  Name
  
• 
  
  Date Sec
  
• Gram Stain Quiz
• Directions Perform a Gram Stain and fill out the
  blanks as you go. Be patient and stay calm, as
  you will be given ample time to practice and
  finish. If your technique doesnt work out the
  first time, clean another slide and start again.
  There are a minimum of 2 and a maximum of 3
  genera in each sample.
• Remember Spelling counts and THIS IS A QUIZ,
  therefore no talking or helping your neighbor.
  Raise your hand when you are finished and I will
  come to you. Good Luck!
• 1.The NUMBER of different genera in your unknown
  is, (circle the correct number).
• 1 2 3
• 2. The MORPHOLGY, GRAM STAIN, AND GRAM REACTION
  of the different genera are,
• Morphology Gram
  Stain Gram Reaction
• Cocci or Rod Color
  (pink or purple) G or G-
• A._________________ _________________ ____________
  ____
• B._________________ _________________ ____________
  ____
• C._________________ _________________ ____________
  ____
• 3. The names of the different genera are, (Genus
  and Species).
• A. Please remember you
  have more than one chance at
• B. this quiz before you
  turn it in. Take your time and good luck.

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Next Week

• Assignments
• Pre-lab 3.2
• MWA 4 due (Last one!)
• Exercise 3 (part 2)
• Sterile Technique Quiz
• Gram Stain Quiz
• Environmental Isolate
• Microscopic examination
• Staining
• Storage conditions

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MWA 3

• Objective Describe the data in a results
  paragraph, and then analyze in a discussion
  paragraph.

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MWA 3

• Things Im looking for
• Keep results and discussion separate.
• At least one reference pertaining to whooping
  cough. It does not need to be a journal article.
• Proper grammar, spelling, correct units from
  graph, etc.
• Remember data is plural, datum is singular.