Overview

1
Overview

• Quiz
• Return MWA 4
• Go over Lab Report Format Graphing
• Counting plates
• Biochemical tests for Env. Isolate
• Staining for Env. Isolate

2
Bacterial Enumeration Hamburger Plate Data
Collection

• 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
• Calculate the averages (for example if two
  groups used sample A we will compute the average
  for their data and you will use that number)

3
Bacterial Enumeration Hamburger Plate Data
Collection

• 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 TNC

4
Bacterial Enumeration Hamburger Plate Data
Collection

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

5
Bacterial Enumeration Hamburger Plate Data
Collection

• 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.

6
Bacterial Enumeration Hamburger Plate Data
Collection
SAMPLE TABLE
7
Bacterial Enumeration

• You will make 2 graphs for this report
• The first will be hand-done on log paper and will
  be based of YOUR sample
• The second will be computer generated and will be
  composed of the samples from the entire class

8
Bacterial Enumeration Histogram of Hamburger
Plate Data Collection
Comparison of average bacterial titer from 3
locations
Figure 1. Enumeration of Bacteria in hamburger
samples from various supermarkets.
9
Biochemical Tests

• The goal of these tests is to identify your
  organism
• Cell and colony morphology, Gram reaction and
  other differential stains can help, but metabolic
  characteristics are critical
• Do NOT attempt the tests unless you have purity
• Many of these tests require a control organism to
  interpret results accurately

10
Biochemical Test The 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
• Remember, weve used controls for staining
  previously

11
Biochemical Tests

• Use the sheet provided for reference as you are
  completing the procedures
• Some need to be read immediately, some after 24
  hours, and some wont need to be read for a week
  (although you may need to put them in the
  refrigerator)
• Feel free to split the tests between group
  members but make sure each of you takes accurate
  notes so the other knows what happened

12
Review of Metabolism

• Different organisms have different metabolic
  characteristics
• These characteristics help distinguish one
  species from another
• Metabolism A general term for the totality of
  chemical and physical processes occurring in a
  cell
• Catabolism- breaking down complex molecules for
  later use
• Anabolism- building complex molecules to
  incorporate into biomass

13
Review of Metabolism

• All organisms require a carbon source (C), a
  nitrogen source (N) and an energy source
• The form of the C, N, and energy source may be
  different
• Essentially, you will be testing for the presence
  or absence of certain cellular pathways and which
  forms of C, N, and energy can be utilized
• Once you know this, you can use literature to
  determine what your organism is

14
Biochemical TestsCatalase Test

• Obligate aerobes and facultative anaerobes
  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
• Obtain a glass slide and divide into 3 sections
• Remove a small amount of your environmental
  unknown from your agar slant and place on the
  slide
• Do the same for the two control organisms
• Bacillus spp. for the positive control and
  Streptococcus lactis for the negative control
• Place a drop of 3 H2O2 on each of the organisms
  and check for the appearance of gas bubbles
• Bubbles a positive test no bubbles a
  negative test

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)

18
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

19
Biochemical Tests Oxidase Test Procedure
1
2
positive control
Group A unknown
Group B unknown
Group C unknown
3
4
20
Biochemical Tests Carbohydrate Fermentation

• 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
Biochemical TestsCarbohydrate FermentationProced
ure

• 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 read and record the results at
  24 and 48 hours
• 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 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).

23
Biochemical Tests 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.
• Do not mix the tube or do anything to introduce
  oxygen into the culture

24
Motility Test
Biochemical Tests

• True motility (directed movement) is different
  than Brownian movement. Brownian movement is
  caused by invisible molecules striking 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

25
Biochemical Tests Motility Test Procedure

• To test for motility
• Inoculate a tube of motility medium using your
  inoculating needle rather than you inoculating
  loop
• Stab the motility medium to about 2/3rds of its
  depth, then draw 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 red cloudiness around
  the stab pathway (figure 5.4)

26
Biochemical Tests Motility Test Procedure
27
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.

28
Biochemical Tests Simmons Citrate Procedure

• To perform at test with Simmons Citrate agar
• Use your inoculating needle to transfer some of
  your test microorganism into the tube
• 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

29
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
• Urea broth is composed of yeast extract, urea and
  the pH indicator phenol red

30
Biochemical Tests Urea Hydrolysis Procedure

• To test for urea hydrolysis
• 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 a light cherry color (pH
  8.1 or greater)
• No change in the color of the indicator is a
  negative test.

31
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

32
Biochemical Tests 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

33
Biochemical Tests 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.

34
Biochemical Tests Gelatinase Test

• Many microorganisms produce an enzyme called
  gelatinase that can degrade or breakdown gelatin
  into smaller polypeptides and amino acids
• 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.

35
Biochemical Tests 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 (no control organism)
• Incubate both tubes at room temperature for one
  week

36
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.

37
Biochemical Tests Starch, Casien Lipid
Hydrolysis Fig. 5.5 (Shand)
38
Biochemical Tests Starch Hydrolysis Procedure

• To test for starch hydrolysis
• Inoculate a single starch-gelatin agar plate with
  a small amount of your environmental unknown 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)

39
Biochemical Tests Casein (milk
protein)Hydrolysis Procedure

• To test for casein hydrolysis
• Place a small amount of culture from your
  environmental unknown onto the plate
• 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.

40
Biochemical Tests Lipid Hydrolysis

• To test for lipid hydrolysis
• Inoculate a spirit blue agar plate with your
  unknown and control organisms (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

41
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
• The oxygen will be removed chemically and the
  organisms allowed to incubate until the next
  laboratory period