Exercise 5 Part I

1
Exercise 5 Part I

• Biochemical Tests
• Data Analyses

2
TODAY- What we are doing

• INTRODUCTION
• WHAT WE ARE DOING TODAY
• Data analysis of hamburger counts.
• Go over lab report format sheets and review the
  requirements for first lab report (Hamburger
  experiment 4) due at the beginning of class next
  week.
• Biochemical tests.
• Environmental isolates.

3
Lab Reports

• Writing report handout
• Citation handout
• Graphing handout
• Rubric
• Sample papers

4
Data Analysis of Hamburger Counts

• Count the number of colonies on each set of
  spread plates and pour plates.
• All of the colonies will be on the surface of the
  spread plates
• Colonies will be both on the surface and embedded
  into the medium for the pour plates. ONLY COUNT
  COLONIES INSIDE OF THE AGAR (anaerobic)
• Note that colonies in the pour plates will
  frequently be very small when embedded in the
  agar and often look like little footballs (or
  lenses, hence, the proper term is "lenticular").

5
Data Analysis of Hamburger Counts

• Plates that have 29 or fewer colonies are not
  statistically significant and although the data
  should be recorded as Not Statistically
  Significant (NSS), they should not be used in
  determining the titer.
• Plates that have much greater than 300 colonies
  are difficult to count. Plates that have too many
  colonies to count accurately should be recorded
  as Too Numerous To Count (TNTC).

6
Data Analysis of Hamburger Counts

• Determine the titer for both spread plates and
  pour plates.
• This is done by dividing the number of colonies
  found on a plate by the dilution.
• For example, if there were 229 colonies on a
  10-5 dilution plate, the formula would be 229
  divided by 10-5. This is equivalent to 229 X
  105 (remember when you divide by an exponent,
  you change the sign of the exponent, so negative
  exponents become positive and positive exponents
  become negative).

7
Data Analysis of Hamburger Counts

• The units here are colony forming units per gram
  (cfu/g). Record the spread plate and pour plate
  titers for your sample on the board. Record all
  of the class data and use the pooled results for
  writing your laboratory report.

8
Report Due Next Week

• You will need the data incorporated into a table
• You will need a hand drawn histogram (bar graph)
  done on 3 cycle log paper of your groups data.
• You will also need a computer generated histogram
  done on excel of the class data. (see me if you
  need help)
• Writing for the Biological Sciences by McMillan
  will help with Tables and Graphs.

9
Table Example of 1 set of Data
10
Enumeration of Bacteria
Average titer of bacteria from hamburger samples
Figure 1. Enumeration of Bacteria in hamburger
samples from various supermarkets.
11
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 uninterruptible results.
• You will need to come in 24 and 48 hours to check
  these
• Record your findings on page 5-9 we will discuss
  the results next week

12
Biochemical Tests

• In this set of experiments, you will subject your
  unknown(s) to a battery of biochemical tests and
  compare the results to known positive and
  negative results produced by control organisms.

13
Biochemical Tests What is a Control ?

• 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.

14
Biochemical Tests 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. The liberation of oxygen gas
  is the basis for the catalase test.

15
Catalase Test Procedure
Biochemical Tests

• To test for catalase activity Remove a small
  amount of your environmental unknown from your
  agar slant, or a loopful 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. Check under the microscope for bubbles.
  Use Bacillus spp. for the positive control and
  Streptococcus lactis for the negative control.

16
Oxidase Test
Biochemical Tests

• 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.
• The oxidase test involves substituting an
  artificial substrate p-phenylenediamine (note!
  this compound is toxic!) for the reduced
  cytochrome that the cytochrome oxidase usually
  acts upon.
• There are very few oxidase positive organisms.
  However, since most pseudomonads are oxidase
  positive, use a pseudomonad for the positive
  control.

17
Oxidase Test Procedure
Biochemical Tests

• 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)

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Oxidase Test Procedure
Biochemical Tests

• 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
20
Carbohydrate Fermentation
Biochemical Tests

• 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.

21
Carbohydrate FermentationProcedure

• 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.

22
Biochemical TestsCarbohydrate Fermentation
23
Biochemical Tests Anaerobic Respiration by
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).

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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 Procedure

• (i) 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.

26
Nitrate Reduction Procedure

• (ii) 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 Procedure

• (iii) 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.

28
Nitrate Reduction Procedure
29
Motility Test
Biochemical Tests

• 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.

30
Motility Test Procedure

• To test 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).

31
Motility Test ProcedureFig. 5.4 (Shand)
32
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.

33
Simmons CitrateProcedure

• To perform at test with Simmons Citrate agar
  Flame sterilize your inoculating needle, allow it
  to cool and transfer some of your test
  microorganism onto the tip.
• 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.

34
Urea Hydrolysis
Biochemical Tests

• 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.
• 1 is uninoculated
• 2 is positive
• 3 is negative

35
Urea Hydrolysis Procedure

• 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.

36
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.

37
Kligler's Iron Agar Procedure

• 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.

38
Kligler's Iron Agar Procedure

• A positive test shows a dark precipitate that has
  formed in the tube. The absence of a precipitate
  is a negative test.
• 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.

39
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.

40
Gelatinase Test Procedure

• To test for gelatinase production Stab two
  tubes of gelatin medium, one with your unknown
  culture and the other with a sterile needle.
  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 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.

41
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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Starch, Casien Lipid Hydrolysis Fig. 5.5 (Shand)
43
Starch Hydrolysis
Biochemical Tests

• 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 protein.

44
Starch Hydrolysis Procedure

• 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.
• 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.

45
Biochemical Tests Casein (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.

46
Casein (milk protein) HydrolysisProcedure

• To test 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.

47
Biochemical Tests Lipid Hydrolysis

• Lipases (or esterases) are enzymes which
  hydrolyze the ester linkages that hold fatty
  acids to glycerol.

48
Lipid Hydrolysis

• To test for lipid hydrolysis Microorganisms
  that excrete enzymes that break down fats
  (lipids) can be identified by growing them on a
  spirit blue agar. 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.

49
Biochemical Tests 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.

50
Facultative AnaerobesProcedure

• 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.

51
Environmental Isolate

• In the time remaining, continue microscopic
  examination of your environmental unknown(s),
  including Gram stains, capsule stain and
  endospore stain.
• Remember your control organisms
• Examine to determine best storage conditions.