Transcript ESG 5300- Classroom Activity - Lakehead Science Education (Matt

ESG 5300- Classroom Activity
Fingerprinting of an Unknown
Bacterial Species- “Whodunit”
June 8, 2011
J-P Nadeau
1st year Masters student at the University of
Ottawa
Concept
• Use of various tests to identify an unknown
bacterial species.
Brainstorming Session
• Identify bacterial species, and diseases
they may cause. (or vice versa)
• Ie: Streptococcus – Strep throat
Relevance
• Each small group will have the task of
determining the identity of the bacteria on
the plate that they will be given!
What you will learn today
1. How to perform 2 biochemical tests
2. How to examine colony morphology
3. How to examine cell shape/ aggregation
• Overall, how to identify an unknown
bacterial species.
Background
1,000,000’s of
cells
Grow at 37°C for 16
hours
Inoculate
~1000
cells
Grow at
37°C for 16
hours
Result……
20 minutes = doubling
0 min = 0
20 min = 2 cells
40 min = 4 cells
60 min = 8 cells
2 hr = 64 cells
4 hr = 4096 cells
6 hr = ~250, 000 cells
8 hr = millions…
LB agar plate
1.
Oxidase
test
Tests whether or not the
bacteria has cytochrome
oxidase (electron
acceptor) and thus uses
oxygen to breathe.
If the enzyme is present,
a colourless dye in the
reagent turns purple.
+ result
Beware: An area on the plate that turns purple
where there is no bacteria is a negative result!
Colonies must turn purple http://www.youtube.com/watch?v=7Aa1xO2cC1M&feature=related
2. Catalase test
-Enzyme required
for aerobic
metabolism
(organisms that
use oxygen)
-Used to remove
damaging free
radicals such as
hydrogen
peroxide (H202)
2H202 2H20 +
02
+ result
http://www.youtube.com/watch?v=yFcAORmt6qY
3. Colony Morphology
* You will receive this as a handout to help you decide for your
unknown
4. Cell Shape and Aggregation
• You will use the handout provided to decide
the cell shape and aggregation type of your
unknown.
4a. SHAPE
4B: AGGREGATION
• Single cell:
• Multi cell:
• Mixed:
Other tests
• These tests were already completed for you. You
can use these results to help identify your
unknown.
• Amylase- checks for the presence of amylase
enzyme (the bacteria can breakdown
carbohydrates)
• Lipase- for lipids
• Dnase- breaks down DNA
• Urease- breaks down urea
• Nitrate- breaks down nitrate
Safety
• Don’t do anything silly (i.e eat the colonies
etc…)
• All bacterial unknowns are Biosafety Level
1 agents- are not of concern to normal
individuals.
Schedule
• 15 minutes for each group of 2 to get
results for the 4 activities, and determine
the identity of their unknown.
• Following the activity, some groups will
present their results to the class (for small
prizes).
KEY
Your Tests
Bacterial Species
2.Bacillus Pseudomonas 3.Escherichia Proteus
subtilis
syringae
coli
mirabilis
1.
Bacillus 4.Serratia 1.Lactobacillus
sphericus marcesans plantarum
Oxidase
-
+
-
-
-
-
+
2. Catalase
+
+
+
+
+
+
+
Irregular
Circular
Circular
Irregular
Circular
Circular
Circular
Entire
Entire
Entire
Entire
Entire
Entire
3a. Colony
Morphology
(form)
3b. Colony
Undulate
Morphology (Margin)
4. Cell Shape and
aggregation
Rod
shaped,
chains
Rod shaped,
single cells
Other tests:
Amylase
+
+
-
-
+
+
-
Lipase
+
+
+
+
+
+
+
DNase
+
+
+
+
+
+
+
Urease
-
+
+
+
-
-
+
Nitrate
+
+
+
+
+
+
+
Unknown #2
Your Tests (fill in)
Unknown
#2
1. Oxidase
2. Catalase
3a. Colony
Morphology (form)
3b. Colony Morphology
(Margin)
4. Shape and
aggregation
Other tests:
Amylase
Lipase
DNase
Urease
Nitrate
+
+
+
+
4. Shape and AggregationLook at the stain of your unknown below, determine the shape (rod or cocci) and aggregation (single cell, mixed, multi cell)
of your unknown.
Record above, and use this information to help you identify your unknown!
Activities
1. Oxidase test- bring your plates to me, I will
add a drop of reagent to your unknown
2. Catalase test- perform this test at your
bench by adding a few drops of H202 to
bacteria on your plates
3. Colony morphology- examine your plates
and compare to the handout
4. Cell Shape and Aggregation- data is