Identifying Unknown Bacteria: Table 1: Brief Description of General Tests and Probable Results

Identifying Unknown Bacteria
Overview
This lab should help give you the background information and techniques
you will need to successfully perform general biochemical tests in order to
help identify unknown bacterial samples. The micro lab website, your
textbook, the web and assorted books available in lab will be the
reference materials necessary for you to successfully complete the next
several weeks of lab work.
 You will perform general biochemical tests on an unknown

organism.
 For each biochemical test you perform, make sure to record the
following in your lab book:
o What does a positive test result look like?
o What is the biochemical basis of the test?
Table 1: Brief Description of general tests and probable results.
Test Brief Instructions Probable Results
TSA/BHI Staphs/Enterics on TSA; Streps on BHI Determine macromorphology
Gram To confirm culture purity Staphs/Streps (Gram+), Enterics (Gram-)

Stain
Motility Stab with a needle straight in and straight Motile organisms have obvious growth
out of the center of the tube half way down. away from inoculation area; Non-motile
Incubate for 24 hours at 37°C. organisms grow only in inoculation area.
Staphs/Enterics in O2; Streps in CO2.
Test Brief Instructions Probable Results
McFarlan Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to a 0.5
d McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard
Standard against the black-lined McFarland reference card to accurately rate the turbidity.
FTM Use a sterile transfer pipette to add 1 mL of Strict aerobes will grow near the top of
your McFarland standard organism in the the media; Facultative anaerobes will
middle of the tube. Cap tightly; do not jostle. grow throughout; Strict anaerobes will
Incubate for 24 hours at 37°C. grow near the bottom.
Catalase Transfer a well-isolated colony to a clean Staphs/Enterics are catalase positive;

slide & add 1 drop of 3% H2O2. Do not bubble formation should occur. Note: do
reverse the order & do not mix. Observe for not take colony from a blood plate.
immediate bubble formation.
Oxidase Add a few drops of oxidase reagent onto Look for appearance of purple color
Whatman filter paper. Smear with a loop-full within 30 seconds.
of organisms. Use colonies from low
glucose, non-selective media.
Motility Test Medium
Intended Use
 Used for detecting motility of microorganisms.
Principle
Motility is apparent by the presence of diffuse growth away from the line
of inoculation.
Test Procedure
1. Inoculate with growth from an 18-24 hour culture by stab
inoculation with a needle.
2. Incubate at a temperature and duration appropriate for the
organism being tested.
3. Examine tubes for growth and signs of motility.
Results
 Motility is apparent by the presence of diffuse growth away from the

line of inoculation.
 Non-motile organisms only grow along the line of inoculation.
Limitations
 The motility of Proteus spp. is temperature dependent.

 Due to the temperature dependency of motility in some organisms,
a negative tube should be incubated an additional 5 days at a lower
temperature of 22-25°C.
Fluid Thioglycolate Medium (FTM)
Tests the oxygen requirements of different microorganisms.
Intended Use
Characterizes microbes according to their oxygen requirements
 Obligate aerobes
 Facultative anaerobes
 Obligate anaerobes
Principle
Various types of bacteria require various oxygen (or oxygen-free)

environments to grow in.
Test Procedure
1. Dilute your organism in a tube of sterile water to obtain a turbidity
equivalent to the 0.5 McFarland test standard. Hold your diluted
tube and the 0.5 McFarland test standard against the black-lined
McFarland reference card to accurately rate the turbidity.
2. Using a sterile 1mL pipette, place 1 mL of organism into the middle
of the tube.
3. Cap tightly; do not jostle.
4. Incubate for 24 hours at 37°C.
Results
 Strict (obligate) aerobes grow at the surface of the medium where

there is a high concentration of oxygen.
 Obligate anaerobes grow near the bottom of the broth tube where
there is no oxygen.
 Facultative anaerobes grow best where more oxygen is present, but
growth will occur throughout the broth tube.
Catalase
Intended Use
 Differentiates Streptococcus (-) from Micrococcus (+)

 Differentiates Staphylococcus (V+) and Bacillus (+) from Clostridium
(-)
Principle
Hydrogen peroxide (H2O2) is the end product of aerobic breakdown of

sugars. Since it is toxic to bacterial cells, most aerobic bacteria produce
catalase or peroxidase to protect themselves. Streptococcus,
Enterococcus, and Lactobacillis are exceptions. Since they do not use the
cytochrome c pathway, they do not produce H2O2 and lack catalase.
Test Procedure
1. Transfer a well isolated colony to a clean glass slide and add 1 drop
of 3% H2O2.
o Do not reverse the order and do not mix.
2. Observe for immediate bubble formation.
3. Use 15% H2O2 for the detection of catalase in anaerobes.
Results
 The formation of bubbles is considered a positive result.
Limitations
 Do not take your colony from a blood agar plate. The catalase
present in the erythrocytes will give a false positive result.
 H2O2 is unstable. You can do a quality control test of the H2O2 reagent
by placing a drop on a blood agar plate. Vigorous bubbling should
result.
Oxidase Biochemical Assay
Tests for the presence of the enzyme indophenol oxidase.
Intended Use
The oxidase test is based on the production of an enzyme called

indophenols oxidase. This enzyme oxidizes a redox dye (present in the
reagent) which results in a color change of yellow to dark purple.
Principle
Indophenol oxidase, in the presence of atmospheric oxygen, oxidizes the

phenylenediamine oxidase reagent to form a dark purple compound,
indophenol.
Test Procedure
1. Have your instructor or IA crush the ampule inside the dropper.
2. Tap bottom on tabletop a few times. Then invert for convenient
drop-by-drop dispensing of reagent
3. Preparation for testing:
o Colonies to be tested must be isolated from other colonies
o The use of fresh isolates (18-24 hr cultures) is recommended
for routine testing.
o If refrigerated, cultures must be allowed to reach room
temperature prior to testing
4. Performing the test – Filter Paper Method
o Add a few drops of oxidase test reagent to a strip of filter
paper (Whatman No. 1 or equivalent).
o Streak a loopful of bacteria onto the reagent-saturated paper
with a platinum loop or wooden applicator stick. Use of steel
of nichrome loops may cause false-positive reactions
Results
Positive reactions turn the bacteria violet to purple immediately, or up to

30 seconds. Negative reactions remain colorless or turn light pink/light
purple after 30 seconds. Delayed reactions should be ignored.
Limitations
 Allow up to 30 seconds for a positive reaction.

 Any delayed reactions should be considered negative.
 Do not add excess reagent, at it may cause the reaction to fade on
oxides-positive organisms.
 Steel loop, nichrome loop, and wire loop containing iron may give a
false-positive reaction. A platinum loop or wooden applicator stick is
recommended.

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Identifying Unknown Bacteria

 You will perform general biochemical tests on an unknown

Table 1: Brief Description of general tests and probable results.

Test Brief Instructions Probable Results

TSA/BHI Staphs/Enterics on TSA; Streps on BHI Determine macromorphology

Gram To confirm culture purity Staphs/Streps (Gram+), Enterics (Gram-)

Catalase Transfer a well-isolated colony to a clean Staphs/Enterics are catalase positive;

Motility Test Medium

 Used for detecting motility of microorganisms.

 Motility is apparent by the presence of diffuse growth away from the

 The motility of Proteus spp. is temperature dependent.

Fluid Thioglycolate Medium (FTM)

Tests the oxygen requirements of different microorganisms.

Characterizes microbes according to their oxygen requirements

Various types of bacteria require various oxygen (or oxygen-free)

 Strict (obligate) aerobes grow at the surface of the medium where

 Differentiates Streptococcus (-) from Micrococcus (+)

Hydrogen peroxide (H2O2) is the end product of aerobic breakdown of

 The formation of bubbles is considered a positive result.

Oxidase Biochemical Assay

Tests for the presence of the enzyme indophenol oxidase.

The oxidase test is based on the production of an enzyme called

Indophenol oxidase, in the presence of atmospheric oxygen, oxidizes the

Positive reactions turn the bacteria violet to purple immediately, or up to

 Allow up to 30 seconds for a positive reaction.

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