Announcements

1
Announcements

• Linda will be checking the classes today
• No coming into labs during class time.
• 2 points will be taken off

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What we are doing today

• ENUMERATION OF BACTERIA
• DATA ANALYSIS OF HAMBURGER LAB DATA
• BIOCHEMICAL TESTING OF YOUR UNKNOWN
• STORAGE CONDITIONS OF YOUR UNKNOWN
• DIFFERENTIAL STAINING

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Now, we will find out how much bacteria is in
your hamburger!

• 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
• 300 To Numerous To Count (TNTC)
• Write your significant plate counts in the table
  on the board
• Make sure that you write down all of the other
  plate counts from the board
• We will calculate the averages to be used on your
  reports

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Things to Remember about your 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. Colonies will be embedded into the
  medium for the pour plates. (Dont count the ones
  on the surface)
• Colonies embedded in the agar might be small and
  can look like little footballs or lens. These
  colonies morphology is called lenticular.
• Remember 300 TNTC

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Calculations for Enumerating Bacteria

• Determine the titer for both spread plates and
  pour plates by dividing the number of colonies
  found on a plate by the dilution.
• For example, if you have 250 colonies on a 10-4
  dilution plate, then the formula is 250 divided
  by 10-4 or 250/10-4
• 250/10-4 2500000. 2.50 x 106
• It helps to put everything into scientific
  notation making your final formula.
• The units are colony forming units per gram
  (cfu/gm).

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Titer Recording Information

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

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Bacterial Enumeration

• REMEMBER TO RECORD ALL DATA ON THE BOARD FOR YOUR
  REPORT
• You will use the data in the completed table to
  construct a bar graph on 3 cycle log paper.
• Some helpful hints can be found in your writing
  handbook
• MAKE SURE THAT YOU LABEL EVERYTHING PROPERLY

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Sample Table for Enumerating Bacteria
9
Sample Histogram for Hamburger Data

• Comparison of average bacterial titer from 3
  locations

Figure 1. Enumeration of Bacteria in hamburger
samples from various supermarkets.
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LAB REPORT

• Lab report should be double spaced and follow the
  format from Mini Writing Assignment 1
• Your report should be 5 to 8 pages, preferably
  not more than 6 pages, and will have a title page
  format found on page 70 of McMillan.
• YOUR REPORT TITLE CANNOT BE HAMBURGER REPORT

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LAB REPORT

• Your paper will not have an abstract.
• Each section of your lab report will be labeled.
  (Introduction, Materials and Methods, Results,
  Discussion)
• I suggest you read McMillan, because any lab
  reports that are not written in lab report
  format, or are missing sections, may be
  considered incomplete, and will be returned
  without being graded

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LAB REPORT SECTIONS

• Introduction - should include citations from your
  sources. It should explain the reason for the
  study you did, and its purpose.
• Hypothesis- A tentative explanation for an
  observation, phenomenon, or scientific problem
  that can be tested by further investigation.
• Materials and Methods should describe what was
  done in your experiment in a way that would allow
  the experiment to be duplicated, but should not
  include any detailed descriptions of standard
  techniques.
• Be sure to cite the lab manual as a reference in
  this section.
• Write in past tense and third person impersonal.

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LAB REPORT SECTIONS

• Results a report of the observations you have
  made. The class data should be summarized in
  tables, correctly labeled, as well as summarized
  in brief paragraphs. This section will summarize
  and illustrate the findings, integrating
  quantitative data with the text (explain texts
  and figures), and will NOT interpret the data or
  draw major conclusions.
• Discussion restates and summarizes the
  objectives of the experiment, and interprets the
  results of the experiment, supported by evidence
  (references). Do not present every conceivable
  explanation for the results include only the
  major explanations.

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LAB REPORT SECTIONS

• Literature Cited List all the references you
  cited in your report. Do not list any other
  sources, even if they were helpful to you as
  background reading.
• Use the format found in Chapter 6 of McMillan, or
  the format used in the sample report found in
  your lab manual.
• Your references should be in alphabetical order.

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Writing Rules to Remember

• Its/Its
• Its going to snow.
• The classes average grade is an A. Its average
  is an A.
• Effect/Affect
• Poor grammar will affect your grade.
• The effect of poor grammar will catch up with you
  later in life.
• Than/Then
• I am cooler than my office mate.
• I came into class today and then I lectured you
  on grammar.

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More Writing Rules to Remember

• Whether/Weather
• Lets talk about whether you want to go to the
  party or not
• The weather is nice today, wish it would snow
• Straight/Strait
• Go straight ahead to the stop sign
• This is the Strait of Juan de Fuca
• Also,
• Please do not use this to start every sentence

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Biochemical Tests

• Biochemical tests are used to more precisely
  identify your environmental isolate.
• Pure cultures are critical to correctly
  identifying your isolate. Mixed, impure cultures
  will generate weird results, such as all tests
  turning out positive.
• You will subject your unknown to a series of
  biochemical tests and compare those results to a
  control organisms that is known to be positive or
  negative.

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Why use a control in Biochemical Tests?

• CONTROL Identical conditions without the
  variable.
• CONTROL ORGANISM An organism with a known
  reaction to a specific test that is used in
  comparative analysis.
• POSITIVE CONTROL Identical conditions using a
  variable with a known positive response

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BIOCHEMICAL TESTSCatalase Test

• Tests for the presence of catalase and
  peroxidase, enzymes that convert H2O2 to water
  and free O2 gas.
• This test will activate the enzymes, and they
  will hydrolyze the H2O2, producing gas bubbles.
• A small amount of the unknown is placed on a
  glass slide, and mixed with a drop of H2O2. If
  gas bubbles appear, the test is positive, if they
  do not, the test is negative.
• INTERESTING FACT Found in plant, animal cells
  within the peroxisomes. Has one of highest
  turnover numbers for all known enzymes
  (40,000,000 molecules/second

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BIOCHEMICAL TESTSOxidase Test

• Cytochrome oxidase catalyzes the oxidation of a
  reduced cytochrome by molecular oxygen (O2)
  resulting in the formation of H2O or H2O2. 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.
• There are very few oxidase positive organisms.
  However, since most pseudomonades are oxidase
  positive, use a pseudomonad for the positive
  control.

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Procedure for Oxidase Test

• 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

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Biochemical Tests Oxidase Test Procedure
1
2
Group A positive control
Group A unknown
Group B unknown
Group B positive control
3
4
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BIOCHEMICAL TESTSCarbohydrate 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. You will
  test your environmental isolate for the ability
  to ferment glucose (also called dextrose),
  sucrose (also called saccharose), lactose and
  mannose.
• 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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Procedure 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.

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Chart for Carbohydrate Fermentation
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Chart for Carbohydrate Fermentation
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BIOCHEMICAL TESTSNitrate Reduction

• Tests your organism for anaerobic respiration
  using nitrate instead of molecular oxygen.
• Inoculate tube with unknown, DO NOT MIX TUBE.
  When growth has occurred, place tube in
  refrigerator until the next lab period.
• 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.

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Nitrate Reduction Procedure

• 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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Nitrate Reduction for Next Lab

• 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.
• 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. 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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Nitrate Reduction for Next Lab

• 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.

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Biochemical Tests Nitrate Reduction Procedure
32
BIOCHEMICAL TESTSMotility Test

• To test if the unknown is motile or not
• 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

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Procedure for Motility

• Inoculate a tube of motility medium using your
  inoculating needle rather than you inoculating
  loop. Flame sterilize your needle and once it is
  cool, transfer some cells onto the very tip.
  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.
  Sterilize the needle.
• Incubate for 24 to 48 hrs. The test is positive
  for motility if there is red cloudiness around
  the stab pathway (figure 5.4).
• This test may remain at room temperature for as
  long as the student wishes, as some organism are
  motile but move slowly.

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Positive and Negative View of Motility
35
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.

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BIOCHEMICAL TESTSSimmons Citrate

• Stab the needle with your organism to about 2/3s
  of the depth of the media, and after the needle
  has been withdrawn along the same pathway, streak
  the needle, in a zigzag pattern, onto the media
  slant.
• This test should be read within 24 to 48 hours.
• A positive test is indicated by a change in the
  medium from green to blue no color change
  indicates a negative result.

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BIOCHEMICAL TESTSUrea 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.
• 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.
• This test should be read within 24 to 48 hours.
• A positive result is indicated by a color change
  to yellow or pink a negative result is indicated
  by no color change.

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Biochemical Tests Kligler's 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 TESTSKliglers Iron Agar

• Inoculate tube with needle, stabbing 2/3s into
  the agar, withdraw using same pathway, then
  streak the slant.
• This test should be read within 24 to 48 hours.
• A positive result is indicated by a dark
  precipitate forming in the tube a negative
  result is indicated by lack of precipitate. SEE
  PHOTOATLAS, and record results even if negative.
• Note that a yellow color in the tube without a
  dark precipitate is still a negative test for H2S
  production.

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Biochemical Tests Gelatinase Test

• 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 TESTSGelatinase Test

• Stab needle into tube about ½ or 1 inch.
• At the end of the one week incubation, test for
  gelatinase production by chilling the tubes in an
  ice-water bath. Do not shake the tubes when
  transferring them to the ice bath as this medium
  is already a bit "loose." The control
  (uninoculated) gelatin tube should "firm up" when
  chilled. 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.

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BIOCHEMICAL TESTSFacultative 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).
• We will check the results next lab period.

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Procedure for Facultative Anaerobes

• 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.

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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.

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Biochemical Tests Starch, Casein and Lipid
Hydrolysis Fig. 5.5 (Shand)
46
BIOCHEMICAL TESTSStarch 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.

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Procedure 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.
• The plate will be incubated at room temperature
  for 24 to 48 hours and then refrigerated. 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.

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BIOCHEMICAL TESTSCasein (Milk Protein) 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.

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Procedure for Casein Hydrolysis

• Use one plate for all of your test organisms.
  Use B. subtilis for a positive control. 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.
• 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.

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BIOCHEMICAL TESTSLipid 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.

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Procedure 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.

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Storage Conditions with your Unknown

• Make sure that you have started your storage
  conditions
• IF it has been two weeks
• Streak one new TSA slant with the culture from
  the slant that was stored in the refrigerator,
  and streak a second new TSA slant with the
  culture from the slant that was stored at room
  temperature. Label the slants appropriately.

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Storage Conditions with your Unknown

• The growth on the new slant tubes should be
  checked as frequently as possible, and the two
  cultures should be compared to determine if one
  is regenerating faster or better then the other
• The culture that grows the best has been stored
  at the optimal storage conditions. This slant
  tube should be stored according to these
  conditions, and will need to be sub-cultured
  every two weeks.

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What to do if you are finished

• Make sure TA has checked you off for all of your
  differential stains. If you still need to do
  some of your stains, do them now.
• If you do not complete all of the stains, you
  will lose technique points, so make sure you are
  checked off on my list.