Today: Exercise 5'1

1
Today Exercise 5.1

• Assignments
• Collect Pre-lab 5.1
• Return Quiz, Pre-lab 4
• Report Format
• Count Hamburger Plates
• Compile Data
• Exercise 5
• Biochemical Tests
• Environmental Isolate
• anaerobe jars for facultative anaerobes

2
McMillan Ch.7 Drafting and Revising

• The First Draft
• Focus on content
• Get down basic information
• Tinker with commas and prose later when you are
  editing

3
McMillan Ch.7 First Draft

• Devise a working title
• Reflect your most important findings
• What is the main point of your research?
• What does your study contribute?
• Pick a title early in the process to help you
  focus your thoughts
• You can always go back and edit it

4
McMillan Ch.7 First Draft

• Make an outline or rough plan
• Make an outline
• Can be a detailed plan of action
• Can be a rough flow chart
• Some sections are easier to outline than others
• An outline is especially useful for
  Materials/Methods, but may not be as useful for
  your discussion
• Find a chart that works for you!

5
McMillan Ch.7 First Draft

• Start the easiest writing first
• You dont have to begin at the beginning
• It may be easier to do the Materials/Methods
  first
• Results may be your next choice
• Introduction/Discussion sections tend to be the
  hardest to write

6
McMillan Ch.7 First Draft

• Talk to others
• Brainstorm with other people
• Gives you an audience to test out your ideas
• Keeps you on track
• Other people may give you new ideas about what
  your data means

7
McMillan Ch.7 Practical Suggestions for Revising

• Break Revising down into steps
• Consider the paper as a whole
• Check overall content, organization, coherence
  and consistency
• Check grammar, etc. after you are sure your
  arguments are solid

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McMillan Ch.7 Practical Suggestions for Revising

• Polish your style
• Examine paragraphs and sentences for clarity
  accuracy and conciseness
• Look for awkward sentences, improper word usage,
  etc.
• Start with big changes and work your way towards
  the more detailed changes

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McMillan Ch.7 Practical Suggestions for Revising

• Keep older drafts
• You might accidentally delete your file
• An older draft might actually sound better
• Print out your paper and revise by hand
• You might notice formatting errors, etc.
• Dont depend on spell check to much
• Misses things like no or on, to or too, etc.

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McMillan Ch.7 Practical Suggestions for Revising

• Allow plenty of time for editing
• Dont try to write your paper and then edit it
  right away
• Give yourself hours or days to edit
• Read your paper out loud
• Easier to spot awkward sentences
• Ask other people to edit you paper
• Always back up your files!

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McMillan Ch.7 Checking Content and Structure

• Improve logic, continuity, and balance
• Keep track of your argument
• What are your questions?
• What are your hypotheses?
• Do your data relate to your hypotheses and your
  conclusions?
• Try to visualize you paper as a unit
• Is its structure clear?
• Do you lead readers through your line of
  reasoning?
• Is there a consistent style?
• Did you follow through on the promises you made
  in your introduction?

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McMillan Ch.7 Checking Content and Structure

• Omit unnecessary material
• Check for places you may have strayed off topic
• Materials/Methods may contain procedures that you
  didnt need
• Results can be a summary of every detail of your
  data
• Discussion may have predictions that are
  impossible to test
• Check to make sure that every point you make is
  related!

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McMillan Ch.7 Checking Content and Structure

• Check for completeness and consistency
• Great checklist of things to make sure are in
  your paper!

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McMillan Ch.7 Improving Paragraphs

• Present coherent units of thought.
• Paragraphs are logically constructed passages
  organized around a central idea
• Central idea is given in the topic sentence of
  the paragraph
• Topic sentence is supported by material that
  further develops / illustrates the point
• Dont clutter the paragraph with irrelevant
  details
• Confuses the reader

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McMillan Ch.7 Improving Paragraphs

• How long should a paragraph be?
• Aim for four to six sentences and then
  shorten/lengthen as needed
• Avoid one sentence paragraphs
• Dont simply group related sentences together
• You should link them together so the reader can
  clearly see the point
• Include transitional elements thus, however, etc.

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McMillan Ch.7 Improving Paragraphs

• Make paragraphs work as integrated parts of the
  text
• Paragraphs should mesh
• The beginning of the paragraph should fit with
  the end of the previous paragraph
• Problem in Materials/Methods and Results
• Use the same transitional elements to link
  paragraphs

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McMillan Ch.7 Improving Paragraphs

• Vary your sentences
• Pay attention to structure, length, and rhythm of
  sentences
• Keeps the reader interested
• Helps your paper flow

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McMillan Ch.7 Writing Clear, Accurate Sentences

• Use words that say precisely what you mean
• Dont use a word just because you think it sounds
  right
• Use a dictionary
• There are online dictionaries for normal and
  technical vocabulary

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McMillan Ch.7 Writing Clear, Accurate Sentences

• Commonly misused words
• Affect vs. effect
• Comprise
• Correlated
• Significant
• That/which
• Unique

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McMillan Ch.7 Writing Clear, Accurate Sentences

• Avoid slang.
• Slang is informal vocabulary used by particular
  people
• Slang is NOT appropriate in scientific writing!
• Ex.
• Cutting edge of research
• Cop out

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McMillan Ch.7 Writing Clear, Accurate Sentences

• Revise misplaced modifiers
• Avoid vague use of this, that, it and which
• Make comparisons complete
• Make each verb agree with its subject
• Put related elements in parallel form
• Write in a direct, straightforward manner
• Avoid jargon

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McMillan Ch.7 Avoiding Wordiness

• Omits unneeded words
• Shorten wordy phrases
• Ex.
• There is now a method, which was developed by
  Jones (1973), for analyzing the growth of rotifer
  populations
• Jones (1973) developed a method to analyze the
  growth of rotifer populations.

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McMillan Ch.7 Avoiding Wordiness

• Avoid repetition
• Some sentences/paragraphs are wordy because the
  same information is included twice
• Use the passive voice sparingly
• Help for shifting the focus to the materials
  studied
• Can be monotonous
• You can use the active voice as long as your not
  using first person

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McMillan Ch.7 Verb Tense

• Past Tense
• Describing your own findings
• Materials/Methods, Results
• Present Tense
• Describing the published findings of others
• Most of the Introduction and Discussion

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McMillan Ch.7 Punctuation

• Punctuation can make or break your paper
• Can make it clear or confusing for the reader
• Buy a writing handbook and use it
• McMillan goes over common punctuation
  mistakes/uses

26
Bacterial Enumeration

• First find your pour and spread plates from last
  week
• Then count all the colonies on each plate with at
  least 30 and no more than 300 colonies and record
  your data
• lt 30 Not Statistically significant (NSS)
• gt300 To Numerous To Count (TNTC)
• Write your significant plate counts in the table
  on the board
• We will calculate the averages to be used on your
  reports

27
Bacterial Enumeration

• Each colony on a plate is assumed to have come
  from one cell
• Colonies will be on the surface of the spread
  plates and on the surface and embedded into the
  medium for the pour plates.
• Colonies embedded in the agar might be small and
  can look like little footballs or lens.
• These colonies morphology is called lenticular
• Remember lt30 colonies NSS, gt300 TNTC

28
Bacterial Enumeration

• Determine the titer for both spread plates and
  pour plates by dividing the number of colonies
  found on a plate by the dilution.
• If you have 250 colonies on a 10-4 dilution plate
  the formula is 250 divided by 10-4 or 250/10-4
  which is equivalent to 250 X 104

29
Bacterial Enumeration

• It helps to put everything into scientific
  notation making your final formula.
• 2.5 x 102 X 104 2.5 X 106
• remember exponents add when they are multiplied
• The units are colony forming units per gram
  (cfu/gm).

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Bacterial Enumeration

• Record your spread plate and pour plate titers in
  the table on the board
• We will calculate the class pooled averages
• Record all of the class data and use the pooled
  results for writing your laboratory report.

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Bacterial Enumeration
Sample Table
32
Bacterial Enumeration

• Reports are individual and are not to be done as
  a team!
• You will pool data but do not assist each other
  with the report
• This report is the first of two that you can
  re-submit twice to get the total possible 40 pts.
• This report will need to have both hand drawn and
  Excel generated graphs and tables.
• Generate a graph in Excel using the class
  averages.
• Hand draw a graph of your groups average on the 3
  cycle log paper in your lab manual.
• Some helpful hints can be found in your writing
  handbook

33
Bacterial Enumeration
Example graph only graph the class averages.
Figure 1. Enumeration of Bacteria in hamburger
samples from various supermarkets.
34
Biochemical Tests

• Microorganisms can not be identified with any
  precision solely on the basis of cell and colony
  morphology, Gram reaction and source of inoculum.
  
• Pure cultures are critical for biochemical
  analyses as biochemical tests using mixed
  cultures containing two or more organisms will
  generate un-interpretable results.
• To obtain more conclusive results you will
  subject your purified unknown to a group of
  biochemical tests and compare the results to
  known positive and negative control organisms.

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Biochemical Tests

• Control Identical conditions without the
  variable being tested.
• Positive Control Identical conditions using a
  variable with a known positive response.
• Negative Control Identical conditions using a
  variable with a known negative response.
• Control Organism An organism with a known
  reaction to a specific test that is used in
  comparative analysis.

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Biochemical Tests Catalase

• The liberation of oxygen gas is the basis for the
  catalase test.
• Obligate aerobes and facultative anaerobes that
  utilize oxygen frequently produce toxic
  by-products like hydrogen peroxide (H2O2) and/or
  superoxide radicals (O2-) as part of their
  aerobic respiration.

37
Biochemical Tests Catalase

• To test for catalase activity
• Remove a small amount of your environmental
  unknown from your agar slant, or a loop-full of
  control test organisms from a broth culture and
  place it on a glass slide.
• Mix the organisms with a drop of 3 H2O2 and
  check for the appearance of gas bubbles (a
  positive test). No bubbles is a negative test.
• Use Bacillus spp. for the positive control and
  Streptococcus lactis for the negative control.

38
Biochemical Tests Oxidase

• There are very few oxidase positive organisms.
  However, since most pseudomonads are oxidase
  positive, use a pseudomonad for the positive
  control.
• Cytochrome oxidase catalyzes the oxidation of a
  reduced cytochrome.
• This enzyme plays a vital role in the electron
  transport chain.
• In the cell, the reduced cytochrome donates
  electrons to the oxidase and becomes oxidized.
• The oxidase test involves substituting an
  artificial substrate p-phenylenediamine for the
  reduced cytochrome that the cytochrome oxidase
  usually acts upon.
• This substrate is toxic! Wear gloves!

39
Biochemical Tests Oxidase

• To test for cytochrome oxidase
• For the test, you will use a commercially
  prepared test called a "dry slide" oxidase test.
  Squares of filter paper have been impregnated
  with p-phenylenediamine then sandwiched between
  two pieces of plastic (figure 5.1)
• Using a plastic "Steri-loop" rub the cells from a
  plate or slant directly onto the filter paper in
  one of the windows of the dry slide and record
  the color change within 20 seconds.
• If oxidase positive, the reaction area will turn
  dark purple. If oxidase negative, there will be
  either no color change or a change from colorless
  pink to gray

40
Biochemical Tests Oxidase
1
2
Group A positive control
Group A unknown
Group B unknown
Group B positive control
3
4
41
Biochemical Tests Fermentation

• The ability to ferment carbohydrates and the
  types of fermentation end products that are
  formed (e.g., acid or gas) are very useful in
  bacterial identification.
• These tests are set up so that a number of
  different sugars can be tested easily.
• Broth tubes containing the individual sugars also
  contain a pH indicator (phenol red) to
  demonstrate changes in pH and a small tube called
  a Durham tube which is inserted upside down to
  trap any gas that may be produced as a result of
  the fermentation.

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Biochemical Tests Fermentation

• You will test your environmental isolate for the
  ability to ferment
• Glucose (also called dextrose)
• Sucrose (also called saccharose)
• Lactose
• Mannose

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Biochemical Tests Fermentation

• To test for Carbohydrate Fermentation
• Inoculate a tube containing one each of the four
  sugars from your TSA slant of your purified
  environmental isolate.
• Incubate the tubes at room temperature.
• It is critical that you score the tubes at 24 and
  48 hours and record your results.
• Make sure that the broth is turbid and that the
  organism has actually grown before scoring the
  tube.
• A yellow color is a positive test orange is
  still negative after 24-48 hours. Tubes that
  have incubated for greater than 48 hours should
  not be scored.

44
Biochemical Tests Fermentation
45
Biochemical Tests Nitrate Reduction

• Some microorganisms that usually use molecular
  oxygen as a terminal electron acceptor can
  substitute nitrate (NO3-) for this purpose under
  anaerobic conditions (e.g., Pseudomonas).
• Nitrate can be reduced to nitrite (NO2-) and some
  microorganisms can reduce the nitrite further to
  ammonia (NH3) or even to nitrogen gas (N2).

46
Biochemical Tests Nitrate Reduction

• To test for nitrate reduction
• Inoculate a tube of nitrate broth containing a
  Durham tube with your culture.
• Incubate the culture tube until growth appears
  (24-48 hrs), then refrigerate until next lab.
• Since nitrate reduction occurs under anaerobic
  conditions at the bottom of the tube, do not mix
  the tube or do anything to introduce oxygen into
  the culture.
• Do all of the following tests in the same culture
  tube during the next lab period (refer to figure
  5.3).

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Biochemical Tests Nitrate Reduction

• First, check for the presence of gas in the
  Durham tube.
• If there is gas in the Durham tube, it is
  nitrogen and this observation alone is a positive
  test for nitrate reduction.
• You do not need to do any of the following if
  your organism is already positive.

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Biochemical Tests Nitrate Reduction

• Second, to determine if nitrite is present, add
  10 to 15 drops of Nitrite A reagent
• Note that dimethyl-alpha-naphthylamine is
  closely related to compounds that are
  carcinogenic. If any of this reagent contacts
  your hands, wash them immediately.
• If the culture turns red within 15 min. it is
  positive for the presence of nitrite and positive
  for nitrate reduction, and you do not need to
  proceed any further with this test.
• If after 15 min. there is no color change, then
  one of two events have occurred either the
  nitrate has not been reduced or nitrate has been
  reduced beyond nitrite to ammonia or nitrogen gas.

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Biochemical Tests Nitrate Reduction

• Finally, if there was no color change after the
  addition of Nitrite A reagent and Nitrite B
  reagent, test for the presence of nitrate by
  adding a small amount of zinc powder.
• If nitrate is present, it will be reduced to
  nitrite by the zinc and since the Nitrite A
  reagent and Nitrite B reagent are already
  present, the culture will turn red.
• If the culture turns red within 15 min., then
  nitrate was present and the test is negative for
  nitrate reduction.
• If the culture does not turn red upon the
  addition of zinc, this means that the nitrate has
  been reduced to either ammonia or nitrogen gas
  and is positive for nitrate reduction.

50
Biochemical Tests Nitrate Reduction
51
Biochemical Tests Motility

• True motility (directed movement) is different
  than Brownian movement.
• Brownian movement is caused by invisible
  molecules striking the the bacteria making them
  appear to vibrate rather than the bacteria
  actually moving from one place to another.
• Motility can be observed in a wet mount or
  hanging drop preparation of the organism.
  However, wet mounts tend to dry out quickly
  rendering the organisms immotile.

52
Biochemical Tests Motility

• To test for motility
• Inoculate a tube of motility medium using your
  inoculating needle rather than your inoculating
  loop.
• Stab the motility medium to about 2/3rds of its
  depth, then withdraw the needle straight out
  using the same path that was used going in.
• Incubate for 24 to 48 hrs. The test is positive
  for motility if there is cloudiness around the
  stab pathway (figure 5.4).

53
Biochemical Tests Simmons Citrate

• This test determines if an organism can transport
  citrate and use it as the sole carbon source.
• In addition, the sole nitrogen source in Simmons
  Citrate agar is ammonium ions (instead of amino
  acids).
• A third important ingredient is the pH indicator
  brom thymol blue. This indicator is green at
  neutral pH but turns blue above pH 7.6.

54
Biochemical Tests Simmons Citrate

• To perform at test with Simmons Citrate agar
• Using your inoculating needle, transfer some of
  your culture.
• Stab the agar about 2/3rds of the way down and
  then streak the surface of the slant in a zigzag
  fashion before removing the needle from the tube.
  
• Incubate at room temperature for 24 to 48 hrs.
• A positive test is indicated by a change in the
  medium from green to blue. No color change is a
  negative test.

55
Biochemical Tests Urea Hydrolysis

• Urea is a common metabolic waste product that is
  toxic to most living organisms.
• Urease is an enzyme that hydrolyzes urea into
  ammonia and carbon dioxide.

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Biochemical Tests Urea Hydrolysis

• To test for urea hydrolysis
• Urea broth is composed of yeast extract, urea and
  the pH indicator phenol red.
• Inoculate a tube of urea broth with your test
  organism and incubate at room temperature for 24
  to 48 hrs.
• If urease is present, ammonia will be released
  and the pH will rise.
• A positive urease test is a change from yellow to
  cerise (a light cherry color pH 8.1 or greater).
  No change in the color of the indicator is a
  negative test.

57
Biochemical Tests Kliglers Iron Agar

• Kligler's iron agar is used to test for the
  production of hydrogen sulfide (H2S) gas.
• The production of H2S often results from the
  deamination of the sulfur containing amino acid
  cysteine.
• This medium contains ferrous sulfate, which
  reacts with H2S to form a dark precipitate of
  iron sulfide.

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Biochemical Tests Kliglers Iron Agar

• To test for H2S production
• Inoculate a tube of Kligler's iron agar with some
  of your test organism using your inoculating
  needle.
• Make your stab about 2/3rds of the way into the
  agar.
• Incubate at room temperature for 24 hours.
• A positive test shows a dark precipitate that has
  formed in the tube. The absence of a precipitate
  is a negative test.

59
Biochemical Tests Kliglers Iron Agar

• Since this medium also contains glucose, lactose
  and phenol red, the medium might also turn yellow
  due to the fermentation of these carbohydrates.
• Note that a yellow color in the tube without a
  dark precipitate is still a negative test for H2S
  production.

60
Biochemical Tests Gelatinase

• Gelatin is a heterogeneous mixture of very large,
  water-soluble proteins and is prepared from
  collagen by boiling skin, tendons, ligaments,
  bones etc., with water.
• Many microorganisms produce an enzyme called
  gelatinase that can degrade or breakdown the
  gelatin into smaller polypeptides and amino acids
  that can be taken up and used by the cell.
• Gelatin liquefies at temperatures above 30?C but
  solidifies at 4?C. When hydrolyzed by the enzyme
  gelatinase, however, gelatin does not gel when
  placed at 4? or 5?C.
• Thus a positive test for hydrolysis of gelatin is
  the inability of the medium to gel when placed in
  a refrigerator for 30 minutes as compared with a
  control that does gel.

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Biochemical Tests Gelatinase

• To test for gelatinase production
• Stab a tubes of gelatin medium with your unknown
  culture.
• Incubate both tubes at room temperature for one
  week.
• At the end of the one week incubation, test for
  gelatinase production by chilling the tubes in
  the refrigerator.
• Do not shake the tubes when transferring them to
  the ice bath as this medium is already a bit
  "loose."
• If your unknown organism produced gelatinase and
  hydrolyzed the gelatin, the gelatin will remain
  liquid.
• If your unknown organism did not hydrolyze the
  gelatin after one week incubation, continue
  incubating both the control tube and your unknown
  for another week.

62
Biochemical Tests

• Starch, Casien Lipid Hydrolysis
• NOTE For the following biochemical tests that
  are done on plates, the plates should be divided
  into thirds by drawing lines on the back of the
  plates with your Sharpie marker and the
  microorganisms spotted onto the plates as shown
  in Figure 5.5 below.

63
Biochemical Tests Starch Hydrolysis

• Starch is a complex polysaccharide that can be
  hydrolyzed by a variety of microorganisms via
  extracellular enzymes called a-amylases.
• Starch molecules are much too large to be taken
  into the cell, and must be broken down into their
  constituent parts just like large proteins are.

64
Biochemical Tests Starch Hydrolysis

• To test for starch hydrolysis
• Inoculate a single starch-gelatin agar plate with
  a small amount of your environmental unknown(s)
  and use B. subtilis for the positive control and
  E. coli for the negative control.
• Incubate the plate at room temperature for 24 to
  48 hours and then refrigerate it. D
• During the next lab period, add a few drops of
  Gram's iodine (i.e., use just enough to cover the
  surface of the plate).
• Areas on the plate that contain starch will form
  a dark blue or purple complex. Areas around
  colonies in which the starch has been hydrolyzed
  will appear as clear zones.
• A clear zone around your test organism after
  treatment with Gram's Iodine is a positive test.

65
Biochemical Tests Casein Hydrolysis

• In order for microorganisms to take advantage of
  the carbon and nitrogen in large proteins found
  in their environment, the proteins first have to
  be broken down into individual amino acids or
  small peptides (chains of a few amino acids) in
  preparation for transport into the cell.
• The cell accomplishes this by excreting
  extracellular enzymes called proteases which
  break down proteins in the environment.

66
Biochemical Tests Casein Hydrolysis

• To test for casein hydrolysis
• We will use casein in Skim milk plates to
  determine if a microorganism excretes
  extracellular proteolytic enzymes.
• Place a small amount of culture from your
  environmental unknown onto the plate. Use one
  plate for all of your test organisms. Use B.
  subtilis for a positive control.
• The plate will be incubated at room temperature
  for 24 to 48 hours and examined for zones of
  clearing.
• A clear zone will appear around the colony where
  the protein has been hydrolyzed.

67
Biochemical Tests Lipid Hydrolysis

• Lipases (or esterases) are enzymes which
  hydrolyze the ester linkages that hold fatty
  acids to glycerol.
• Microorganisms that excrete enzymes that break
  down fats (lipids) can be identified by growing
  them on a spirit blue agar.
• To test for lipid hydrolysis
• In addition to your unknown, inoculate a plate of
  spirit blue agar with Pseudomonas spp. for the
  positive control and E. coli for the negative
  control.
• Incubate the plate at room temperature for 24 to
  48 hours (may take longer).
• If lipases are produced, a clear zone will
  develop around where the organism has grown. If
  no lipases are produced, then the area will
  retain the original color of the medium.
• TA must check before discarding.

68
Environmental Isolate

• Facultative Anaerobes
• Many bacteria can grow both aerobically and
  anaerobically.
• Organisms that can grow in the presence or
  absence of oxygen are call "facultative
  anaerobes" (E. coli is an example).
• To determine if your unknown organism is a
  facultative anaerobe, inoculate a TSA plate with
  your unknown and place it into the anaerobic jar
  that your instructor has prepared.
• The oxygen will be removed chemically and the
  organisms allowed to incubate until the next
  laboratory period.

69
Environmental Isolate

• If you have extra time today, continue staining
  your unknown.
• Examine your storage slants if they have been
  incubating for two weeks.
• Re-streak each slant onto new slants.
• Be sure to label which is which!
• Examine daily to see which grows faster.

70
Next Week

• Assignments
• 1st submission Hamburger Report
• Pre-lab 5.2
• Exercise 5.2
• Complete Biochemical Tests
• Description of Bergey's Manual