Fermentation is the process of using organic molecules

1
Microbiology Lab

• Oct 23 Oct 24

2
Overview

• Review Lab Report Format Graphing
• Count Hamburger plates
• Biochemical tests for Environmental Isolate
• Staining for Environmental Isolate
• Storage and Growth for Environmental Isolate

3
Report Format

• Introduction
• Relevant background information
• Summary of the experiment
• Materials and Methods
• Describe what you did in enough detail so I could
  complete the experiment using your report as
  directions
• Use past tense passive voice
• Results
• This is where you report our findings
• Discussion
• This is where you interpret the results

4
Graphing

• You will put two figures in your report
  containing the numbers we generate today
• In which section do these belong?
• Use the worksheet to help you create the graphs
  in Excel
• You will have two figures-they will be created
  from the same data and will both be labeled as
  Figure 1, have the same title, and the same
  caption
• One will be done on Excel
• The other will be hand drawn on 3-cycle log paper
• This is in your lab manual

5
Tables and Figures

• Weve already established that you will have two
  figures in this report (both your graphs)
• You also need to include a table
• This will be composed of the information we post
  on the board today in class
• Essentially, copy the information from the board
  down, place it in a table and include that in
  your results
• You can do the table in Word or Excel
• See me for help if you cant get it to work
  correctly

6
Technicalities

• No late reports will be accepted
• Reports are due within the first 5 minutes of
  lab!!!
• If you do not turn in a first submission you
  CANNOT turn in a second submission
• Dont forget to attach your rubric
• You will lose points if you do not attach them to
  both submissions
• Use my comments on pre-labs as a guide/a way to
  improve your writing
• You may want to write the materials and methods
  section first
• Label all your sections with the appropriate
  headings

7
Bacterial Enumeration

• Find your plates from last week
• Count all the colonies on each plate with at
  least 30 colonies
• lt30 colonies Not Statistically Significant
• gt300 colonies To Numerous to Count
• Write your plate counts on the board
• Calculate the averages to use in the report

8
Bacterial Enumeration

• Each colony on a plate is assumed to have come
  from one cell
• Colonies will be on the surface (aerobic
  conditions) of the spread plates colonies will
  be embedded into the medium of the pour plates
  (anaerobic conditions)
• Remember lt30 colonies NSS, gt300 TNTC

9
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
• 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 are added when they are
  multiplied
• The units are colony forming units per gram
  (cfu/gm)

10
Bacterial Enumeration

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

11
Bacterial Enumeration
SAMPLE TABLE
12
Bacterial Enumeration
Comparison of average bacterial titer from 3
locations
Figure 1. Enumeration of Bacteria in hamburger
samples from various supermarkets.
13
Creating a Graph in an Excel Worksheet

• Open Excel and enter data in appropriate cells
• Once you have all data entered, select all the
  cells
• Click on the icon that looks like a bar graph on
  the tool bar
• Select column graph and fill in title, axes,
  etc.
• Change the scale to a log-scale
• Again-use the worksheet to help you add/change
  all necessary components

14
Biochemical Tests
15
Biochemical Tests

• The goal of these tests is to provide information
  that will be useful in identifying 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

16
Biochemical TestsControl Organisms

• 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

17
Biochemical Tests

• Use the sheet provided for reference as you are
  completing the procedures
• Reading time for the results varies
• Some need to be read immediately
• some after 24 hours
• some wont need to be read for a week (although
  you may need to put them in the refrigerator)

18
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

19
Review of Metabolism

• All organisms require a carbon source (C), a
  nitrogen source (N) and moisture-many require an
  external energy source as well
• 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 the identity of your organism with
  reasonable accuracy
• Bergeys does not always give us exact,
  indisputable results but we can come pretty close
• rRNA is the molecule of choice in most cases for
  obtaining an irrefutable identification of an
  organism

20
Catalase Test read immediately

• 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 release of oxygen gas is the basis for the
  catalase test
• Catalase is the enzyme responsible for converting
  hydrogen peroxide into water and oxygen gas
• A positive reaction will bubble

21
Catalase Test read immediately
Image taken from http//www.life.umd.edu/cbmg/fac
ulty/asmith/200honors/WEBPAGE/spring2004/paulinaso
ha/catalase.jpg
22
Oxidase Test read within 15-30 seconds

• 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.

23
Oxidase Test Procedure read within 15-30
seconds
1
2
positive control
Group A unknown
Group B unknown
Group C unknown
3
4
24
Oxidase test read within 30 seconds
Image taken from http//www.life.umd.edu/cbmg/fac
ulty/asmith/200honors/WEBPAGE/spring2004/paulinaso
ha/catalase.jpg
25
Carbohydrate (CHO) Fermentation check for
growth, read within 24-48 hours

• 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.

26
Fermentation

• Definition Fermentation is the process of using
  organic molecules as electron donors and an
  organic product acts as an electron acceptor to
  produce ATP (energy)
• Fermentation vs. respiration
• Fermentation is a closed or circular system,
  it regenerates some of the reactants
• Respiration linear system it uses inorganic
  molecules as electron acceptors and does not
  often regenerate its starting products

27
Fermentation contd.

• Fermentation and products
• Acid, hydrogen gas and CO2 are common
  fermentation products
• If your organism is able to ferment the sugars we
  provide you will see a color change due to the
  drop in pH of the medium and you may see a bubble
  in the Durham tube

28
Anaerobic Respiration by Nitrate
Reductioncheck for growth with 24-48 hours,
refrigerate

• 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)

29
Motility Test read within 24-48 hours

• 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
• True motility is based on structures such as
  flagella or cillia that allow the organism to
  navigate through its environment
• 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 read within 24-48
hours
31
Simmons Citrate check for growth, read within
24-48 hours

• 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

32
Urea Hydrolysis check for growth, read within
24-48 hours

• 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
• Hydrolyze essentially means to break apart a
  molecule/substance by adding a molecule of water
  (see p 79 in your photoatlas)
• Urea broth is composed of yeast extract, urea and
  the pH indicator phenol red
• What other test(s) is this indicator used in and
  what does it indicate?

33
Kligler's Iron Agar check for growth, read
within 24-48 hours

• 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
• Deaminate to remove the amine (-NH2) group from
  an amino acid
• This medium contains ferrous sulfate, which
  reacts with H2S to form a dark precipitate of
  iron sulfide
• This test can also help you confirm your
  carbohydrate fermentation results-see p.61-62 in
  photoatlas

34
Kligler's Iron Agar Procedure check for growth,
read within 24-48 hours

• A positive test
• dark precipitate forms 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
• Again, you can check the results of your CHO
  tests with this one
• Note that a yellow color in the tube without a
  dark precipitate is still a negative test for H2S
  production.

35
Gelatinase Test incubate at room temp for 1
week, check for growth, chill for 30 minutes

• Many microorganisms produce an enzyme called
  gelatinase that can degrade or breakdown gelatin
  into smaller polypeptides and amino acids
• Remember one of our reasons for not using gelatin
  as a solidifying agent?
• Gelatin liquefies at temperatures above 30?C but
  solidifies at 4?C
• Remember another of our reasons for not using
  gelatin?
• When hydrolyzed by the enzyme gelatinase,
  however, gelatin does not gel when placed at 4?
  or 5?C
• What has happened to the gelatin if is has been
  hydrolyzed?

36
Gelatinase Test incubate at room temp for 1
week, check for growth, chill for 30 minutes

• 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 re-solidify
• A negative test means the molecule was not broken
  apart by the addition of water molecules and the
  gelatin will re-solodify

37
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.

38
Starch, Casien Lipid Hydrolysis
Fig. 5.5 (Shand)
39
Facultative Anaerobes read after 1 week (well
do this next lab session)

• 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)
• Do you think this is a respiration or
  fermentation process-hint think about the
  nitrate reduction test? What is that based on?

40
Aerotolerance

• Definition the ability or inability to grow in
  the presence of oxygen (photoatlas, p. 9)
• Three basic types of aerotlerance
• Obligate aerobes-require oxygen as the terminal
  electron acceptor
• Facultative anaerobes-can use oxygen or another
  compound as terminal electron acceptor
• Obligate anaerobes-cannot use oxygen and are
  often killed by it

41
Why is aerotolerance important?

• Medical perspective
• The human body has aerobic and anaerobic
  environments
• Diagnostically, if someone tells you they
  cultured an obligate aerobe from the intestine or
  a puncture wound, you should be suspicious!
• Conversely, if they tell you they cultured an
  obligate aerobe from the surface of the skinyou
  should be suspicious

42
Why is aerotolerance important?

• Environmental perspective
• Different environmental systems have different
  oxygen levels
• If you are using microbes to treat sewage waste
  in an anaerobic environment you should not try to
  inoculate your sewage with obligate aerobes

43
Facultative anaerobe procedure

• 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

44
Storage Conditions

• You should have started your storage conditions
  already
• If not, start them today
• Remember-do not disturb these tubes for two weeks
• If you need culture for staining/further
  biochemical testing you should streak a third
  slant and use that
• Again, the purpose of the storage conditions is
  to determine where your organism stores best, NOT
  its optimal growth temperature

45
Maintaining your Environmental Isolate

• From now on (as long as you have purity) you will
  keep you organism on TSA slants rather than
  plates
• Today before your biochemical tests go ahead and
  streak a slant and label it growth
• Keep this slant at room temp until it has grown
  well and either refrigerate or re-streak as
  necessary

46
Next Week

• First submission of Hamburger Report Due
• Ex. 5.2 analysis of the biochemical tests that
  ran for a week
• Continue with staining your Environmental Isolate
• Use controls on your slides
• Make sure to make a stress plate for the
  endospore stain