Unknown project (microbiology)
sbudha
2420Unknown11.pptxA “Virtual” Bacterial Unknown Identification
Unknown #1
Identifying Unknown Bacteria
It is nearly impossible to ID a bacteria based on physical characteristics alone
Only a few basic shapes
Handful of common features
Biochemical testing can be used to ID bacteria to species level
We will use "Bergey's Manual of Determinative Bacteriology” to help us to determine our unknown
Can you tell which one is Salmonella and which is Escherichia?
No? Me neither!
Family Enterobacteriaceae
Class Gammaproteobacteria
Gram-negative
Enteric bacteria
Use this key to help you determine your Genus
Instructions for the Unknown Project:
You have been given a series of biochemical tests and their results. You will use these results to determine the genus and species of your assigned Unknown.
It is highly encouraged that you work with other classmates to read tests and determine your unknown.
You must turn in the Unknown Results and Biochemical Chart
Using your Unknown Results, you will fill out the Unknown Results and Biochemical Chart
For each test:
What is the purpose of the test?
What is the medium used for that test?
What indicator is in the medium (so it can be interpreted) OR what reagent is added (after the organism is grown in/on the medium) to see the test reaction?
What does a + reaction look like?
What does a – reaction look like?
What reaction does your organism have?
For your Genus and species of bacterium:
What kinds of infections are caused?
What is the normal habitat (human, all animals, soil, air, etc)?
Plate culture and staining
This is a gram stain: What is the gram reaction? The morphology?
Acidfast stain done: negative
Endospore stain done: no spores
Flagella stain: peritrichous
Isolation plate culture
Any pigment?
Describe the individual colonies
Use the chart below
Oxygen Requirement
Look at the exercise on O2 requirements in your lab manual.
Will you call your microbe---strict aerobe, strict anaerobe, or facultative anaerobe?
Thioglycollate broth
uninoculated
after incubation
Oxygen Requirement
Look at the exercise on O2 requirements in your lab manual.
Will you call your microbe---strict aerobe, strict anaerobe, or facultative anaerobe?
TSA plates in 3 oxygen environments
Your organism on 3 different TSA plates in 3 different environments
How can this tell us anything about the oxygen requirements of our unknown?
uninoculated
after incubation
Catalase and Oxidase Test Results
Catalase test
YOUR ORGANISM’S TEST RESULTS
Oxidase test
Biochemical test – Carbohydrate Utilization
Carbohydrate broths
Glucose
Lactose
Sucrose
Mannitol
Name the broth, indicator/reagent
What is the purpose of the little tube in the broth?
uninoculated
after incubation,
phenol red mannitol, sucrose, lactose, and glucose broths look like this
10
Biochemical tests – IMViC tests
SIM agar deep
What tests correspond to this media?
Uninoculated SIM deep
after incubation,
with reagent
Biochemical test results
TTC agar deep
Not technically an IMViC test
But why do we run it?
uninoculated
after incubation
Biochemical tests – IMViC tests
Uninoculated
MRVP broth
MRVP broth
What tests correspond to this media?
after incubation, with Methyl Red reagent
after incubation,
with Barritt’s A and Barritt’s B reagents
Biochemical tests – IMViC tests
Simmon’s Citrate agar slant
What test corresponds to this media?
Uninoculated Simmon’s citrate agar slant
after incubation
Family of Enterobacteriaceae
Using your results, can you narrow down your Genus to either the right or left side of the key or down to maybe 2-3 potential options?
Biochemical test – Nitrate Test
Nitrate Broth
What is the purpose of the test?
Why is there a small upside tube in the broth?
uninoculated
after incubation, with reagents
Biochemical test – Decarboxylation of amino acids
Moeller broths
You will inoculate (4) Moeller broths
No amino acid
+ Arginine
+ Lysine
+ Ornithine
What is the purpose of the test?
What the purpose of adding mineral oil?
Uninoculated Moeller broth
after incubation,
lysine, ornithine, arginine from left to right
Biochemical test – Deamination of amino acids
Phenylalanine slant
What is the purpose of the test?
Uninoculated Phenylalanine slant
after incubation,
with reagent
Biochemical test results
Urea Hydrolysis
YOUR ORGANISM’S TEST RESULTS
uninoculated
after incubation
Biochemical test results
YOUR ORGANISM’S TEST RESULTS
Gelatin Hydrolysis
uninoculated
after incubation
Biochemical test – Casein Hydrolysis
Skim milk agar
What does this test test for?
Does it use an indicator or reagent? Name it.
uninoculated
after incubation
21
Biochemical test – Lipid Hydrolysis
Methylene blue tributyrin agar
What does this test test for?
Does it use an indicator or reagent? Name it.
uninoculated
after incubation
Biochemical test – Starch Hydrolysis
Starch agar
What does this test test for?
Does it use an indicator or reagent? Name it.
uninoculated
after incubation,
with reagent
Family of Enterobacteriaceae
Using your results, you should know your Genus
Now use the next two slides to determine your species
Bergey’s Manual of Bacterial Identification
Biochemical chart for unknown identification
(+) or (-) = usual reaction, d = different reactions
Incubator/ ambient air
GasPak Candle Jar
Incubator/
ambient air
GasPak Candle Jar
