BIO 351
Identification of Unknown
Bacterial Culture
19.11.2009
Quiz
• What is the first step of identification of
unknown bacteria?
• How do you understand if your bacteria is
catalase positive or negative?
The Aim of the Lab:
• Bergey’s Manual of Determinative bacteriology
• To classify and identify on the basis of
1.
2.
3.
4.
5.
morphology
biochemistry
physiology
growth requirement
evolutionary relationships
There are different tests to differentiate these properties
Steps in identification of unknown bacteria
culture
• The first step is to start with a pure
culture
• The first step should be the broadest
categories:
gram staining group your bacteria as gram(+/-)
 identify the shape (coccus. Rod. Etc.)
Catalase test:
• The catalase test identifies organisms which
produce the catalase enzyme; this enzyme
converts hydrogen peroxide to water and
oxygen gas.
• H2O2 --------------
H2O + O2
Catalase enzyme
• This enzyme helps protect bacterial cells against
hydrogen peroxide. Hydrogen peroxide is a highlyreactive compound which damages cell components
Catalase test:
Procedure:
• On a slide, put 15ul of H2O2 and 15 ul of
your unknown bacteria.
• Observe bubbles.
Catalase Test:
Oxidase Test:
• The oxidase test is for determining the
production of cytochrome c oxidases by a
bacteria.
• Cytochrome oxidase participates in the
electron transport chain by transferring
electrons from a donor molecule to
oxygen.
• You can differentiate bacteria species as
aerobes / unaerobes.
Oxidase Test:
Procedure:
• Put 20ul of bacteria on oxidase test paper redox indicator.
• Observe the color.
• The reagent is a dark blue to purple color
when oxidized and colorless when reduced.
Oxidase Test:
Motility Test:
• To determine the motility, the presence of
flagella
Procedure:
• Take a drop of bacteria culture by pipetting
and put it onto a slide, put a coverslip and
observe under the microscope to see the
motility of the bacterium.
• Truly motile organisms will zip across the
microscope field.
• All bacteria have some vibrational
movement, even nonmotile ones, known as
brownian movement
Today’s experiment:
• On each bench you have 5 unknown
bacteria. They could be ...
1.
2.
3.
4.
5.
6.
Pseudomonas
Staphylococcus
E.Coli
Corynebacterium
Enterobacter
Proteus
• Each group should start with gram
staining of these 5 unknowns (page 36)
• So first classify them into 4 main groups:
Gram (+)
coccus
Staphyloccus
streptococcus
catalase test
Staphylococcus (+)
Streptococcus (-)
Gram (+)
rod
Gram (-)
coccus
neisseria
Bacillus
Corynebacterium
Gram (-)
rod
Pseudomonas
Proteus
E.Coli
Enterobacterium
Motility test
Bacillus (+)
Oxidase test
Pseudomanas (+)
In your reports:
• In your result part, draw a diagram
showing the steps of your procedures that
leads you to the result as the steps in your
manual page 41.