Unknown project (microbiology)
sbudha
VirtualUnknownNotes2.pptxIdentifying Unknowns
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
"Bergey's Manual of Determinative Bacteriology"
"The Prokaryotes"
Identifying Unknown Bacteria
Pure culture is critical to proper identification
Use proper aseptic technique
Absolutely vital!
You must keep a "reserve" plate at all times in case of a suspected contamination
You will not be provided another original stock plate
You must check your tests in a timely manner
Sometimes you may have to come on a non-lab day for the best results!
Family Enterobacteriaceae
Gammaproteobacteria
Gram-negative
Enteric bacteria
Objectives:
Maintain a pure culture throughout the experiment
This is your Table's responsibility!
Determine the physical characteristics of your bacteria
Determine the biochemical characteristics of your bacteria
5
Identifying Unknowns
Identification of Unknown Session 1:
Gram Stain
Streak Plate
TSB and TSA slants
Oxygen Requirements
Catalase
This breakdown follows the Lab Manual and what you normally would have done in an in-person lab
Oxygen Requirements
Culturing Anaerobic Bacteria
Candle Jar
For growing microaerophiles
3-5% carbon dioxide; 8-10% oxygen
Atmospheric is 22% oxygen
GasPak
Growing anaerobic bacteria
Removes oxygen
Thioglycollate broth
Thioglycollate reduces oxygen
Cannot remove all the oxygen in the tube
Resazurin – indicator – indicates areas of higher oxygen
Will appear pink
Unknown Session 1
Each table must have their own labelled rack to keep the unknowns separated!
Store original plate in 2420 10º
Keep your culture going
(1) TSB
(1) TSA slant
(1) streak plate
Gram stain
Incubation Temperature:
M/T = 37℃
W/R = 25℃
Testing oxygen requirements:
(1) thioglycollate broth incubated
Ambient vs Candle Jar vs Anaerobic Conditions
Each plate will the streaked with your unknown and the provided strict aerobe and strict anaerobe
(1) TSA plate at ambient air (incubator)
(1) TSA plate in GasPak (the entire class will be put in one GasPak!)
(1) TSA plate in Candle Jar (the entire class in one candle jar)
Pages like this show you what you would have done in-person
Identifying Unknowns
Identification of Unknown Session 2:
Check results
IMViC
TTC test
Carbohydrate usage
Oxidase
Catalase
Quick Results
What oxygen requirement does your bacteria have? Yours looks like #2
Compare thioglycollate with ambient air, candle jar and Gas Pak results
Catalase test
Perform a catalase test
Family Enterobacteriaceae are all positive
Catalase test
Catalase is an enzyme that breaks down hydrogen peroxide to water and bubbles (oxygen)
Perform an oxidase test
Use of cytochrome oxidase
Reduction of oxygen in the ETC
Colorless reagent reactions with oxygen very obvious blue/purple
Oxidase Reagent
Read immediately
Family Enterobacteriaceae are all negative
Oxidase test
IMViC Tests
Indole, Methyl Red, Voges-Proskauer, Citrate
After Gram Staining, these are the most important tests to determine your unknown!!
Consists of a series of tests using:
SIM agar deep
Indole
Hydrogen sulfide gas
Motility
Additional motility test – TTC motility deep
MRVP broth
Methyl Red
Voges-Proskauer
Simmon's citrate agar slant
Citrate utilization
You must know what media and reagent goes with each test!
You must be able to read the results!
IMViC Tests
Indole, Methyl Red, Voges-Proskauer, Citrate, Hydrogen sulfide, motility
SIM agar deep:
Hydrogen sulfide gas production
Sodium thiosulfate in medium can be utilized to produce H2S gas
H2S gas is colorless
Ferric ammonium sulfite + H2S --> black ferrous sulfide
Indole production
Tryptophan can be converted by tryptophanase to indole
Identified when it reacts with Kovac's reagent
Positive for indole = red
Motility
Not as good as TTC motility
Motile organisms will diffuse out from the stab line (black)
Problem: what happens if the organism doesn’t produce H2S?
http://iws2.collin.edu/dcain/CCCCD%20Micro/SIMdeep.jpg
1. H2S +, Indole -, motile
2. H2S -, Indole +
TTC Motility Deep Review
Used to confirm motility
Semi-solid containing TTC
TTC is tetrazolium chloride, a colorless salt
TTC when reduced by bacterial metabolism becomes red
How to read:
Nonmotile – straight line that may be red
Motile – original line will diffuse out as the bacteria grow
Note: Those bacteria with strict oxygen requirements (strict aerobes) may not grow well in this medium
From the BIOL 2420 lab manual
IMViC Tests
Indole, Methyl Red, Voges-Proskauer, Citrate, Hydrogen sulfide and motility
After Gram Staining, these are the most important!!
MRVP broth:
Methyl red test
Determines the use of glucose with an acidic end product
Acid production shown by the pH indicator methyl red
Voges-Proskauer test
Determines the use of glucose
Tests for acetoin (acetylmethylcarbinol) as an end product
Important for the identification of many bacteria
VERY picky test
You must add the reagents in the proper order for the test to be successful
The test must then sit for up to 45 minutes
You need to know the indicators for the IMViC tests!
16
IMViC Tests
Indole, Methyl Red, Voges-Proskauer, Citrate, Hydrogen sulfide and motility
After Gram Staining, these are the most important!!
Simmon's Citrate agar Slant – Citrate test:
Identifies citrate as the sole carbon source
No other nutrients in this medium
Produces end products: carbonates, bicarbonates, ammonium hydroxide
Uses bromothymol blue indicator
Positive turns the medium from green to royal blue
You need to know the indicators for the IMViC tests!
Carbohydrate Utilization
Sugars are very important test for many microbes!!
Sugar tests can be very finicky
Due to other nutrients available in the media that can be used
Additional byproducts can cause a shift in the indicator color
Note: Best to read within 24 hours
Phenol Red sugar broth with Durham tube
Lactose
Mannitol
Glucose (Dextrose)
Sucrose
Sugar breakdown leads to acids and carbon dioxide gas
Basic sugars to test
You need to know the indicators for the Phenol Red tests!
Unknown Session 2
Make sure your keep your culture growing!
If you want to store results week to week, keep them in the 10°
Perform the catalase test
Perform the oxidase test
(1) SIM deep
(1) TTC motility deep
(1) MRVP broth
(1) Simmon’s Citrate Agar slant
(1) Phenol red sucrose
(1) Phenol red lactose
(1) Phenol red dextrose
(1) Phenol red mannitol
Incubation Temperature:
M/T = 37℃
W/R = 25℃
Identifying Unknowns
Identification of Unknown Session 3:
Check results
Nitrate test
Decarboxylase test
Deaminase test
Gelatin hydrolysis
Quick Results
Indole Production, Hydrogen sulfide, motility
Acidic product of glucose or acetoin production
Add Kovac’s == indole
Black precipitate?
Movement away from the stab line? (TTC?)
Start VP test at beginning of lab
Citrate as a sole carbon source?
How is your sugar used?
Acid or acid and gas?
Nitrogen Fixation vs Nitrification vs Denitrification
Nitrogen Fixation
N2 to ammonium (NH4+) or Nitrogen dioxide (NO2)
Necessary to synthesize amino acids or nucleic acids
Done by bacteria
Nitrification
NH4+/ammonia to nitrate (NO3-) to nitrite (NO2-)
Aerobic process done by prokaryotes
Denitrification
Nitrate to nitrite to nitric oxide to nitrous oxide to N2
Done by facultative anaerobic bacteria
These bacteria can use nitrogen compounds as their final electron acceptor
Our unknowns can be classified by their ability to produce nitrate reductase
http://aem.asm.org/content/80/1/19/F1.large.jpg
Nitrate Reduction Test
Tests for denitrification
Tests for the production of nitrate reductase
Reduces nitrate (NO3) to nitrite (NO2)
How it works: the nitrate is reduced to nitrite and nitrites form nitrous acid. Add the sulfanilic acid (Reagent A) to create an intermediate that reacts with naphthylamine (Reagent B) and turns the broth red
Options:
NO3 to N2 = gas bubble in Durham tube
NO3 to NO2 = red broth
NO3 to NO2 to NH3 = no color + Zn = no color
No reduction of NO3 = no color + Zn = pink
You need to know the broth, indicator, and how to read the test!
Amino acid use by bacteria
20 amino acids
Many biochemical tests based on protein and amino acid used
IMViC tests for indole production
Tryptophanase breaks down tryptophan to indole
Wikipedia
Decarboxylation
Decarboxylase break the bond of carboxylic acid from the amino acid
Must take place in anaerobic conditions
Use mineral oil to exclude oxygen
Produces a basic chemical (increases pH)
Increase in pH causes indicator (brom cresol purple) to go from yellow to purple
Arginine decarboxylase
Ornithine decarboxylase
Lysine decarboxylase
You need to know the broth, indicator, and how to read the test!
Deamination
Deaminases remove an amino group from an amino acid
Produces an acidic chemical (decreases pH)
Phenylalanine deaminates to phenylpyruvic acid
Phenylpyruvic acid reacts with ferric chloride
Phenylalanine deaminase
http://image.slidesharecdn.com/biochem21-150201182649-conversion-gate01/95/biochem2-1-26-638.jpg
You need to know the broth, indicator, and how to read the test!
This is a “perfect world” example. They usually have a much smaller green zone
Gelatin Hydrolysis
Gelatin is solid at room temperature (25º)
Gelatinase hydrolyzes gelatin to a liquid
Aids in the determination of gelatin as a carbon/energy source
Produced optimally at 25º
Gelatin deep is inoculated for up to a week at 25º
To determine liquefaction, place on ice for 30 minutes
Aids in distinguishing some Enterobacteriaceae from each other
wikipedia
Unknown Session 3
Make sure your keep your culture growing!
If you want to store results week to week, keep them in the 10°
(1) Nitrate broth
(1) Phenylalanine slant
(4) Moeller broths + mineral oil
No amino acid, arginine, lysine, ornithine
(1) Gelatin deep
Incubation Temperature:
M/T = 37℃
W/R = 25℃
Identifying Unknowns
Identification of Unknown Session 4:
Check results
Casein hydrolysis
Lipid hydrolysis
Starch hydrolysis
Urea hydrolysis
Quick Results
Is the gelatin firm after 10min?
What color are your Moeller broths? Results?
1 control
2 (-)
3 (+)
4 (-)
5 (+)
6 (+)
Add ferric chloride to your phenylalanine
Add nitrate reagents to your nitrate broth
Use dichotomous key to help determine:
You should know your Genus by now.
Starch hydrolysis
Starch – complex polysaccharide
Amylase
Secreted enzyme (exoenzyme)
Hydrolyzes (breaks down) starch into smaller sugars that can utilized by the cell
To test:
Starch agar plate and Gram's iodine
As the starch is converted to smaller sugars, less starch is available to react with iodine
Positive = yellow/clear zone around bacteria
Negative = purple (no reactions)
http://www.julianbakery.com/wp-content/uploads/2011/12/starch.jpg
http://www.chemistryland.com/CHM107Lab/Exp03_DetectOzone/OzoneLab/GlucoseMakesStarch.jpg
Lipid hydrolysis
Lipid - fats
Lipase
Secreted enzyme (exoenzyme)
Hydrolyzes (breaks down) tributyrin into fatty acids
Tributyrin is a type of vegetable oil (butter)
Smaller fatty acids can be taken up and used by the cell
To test:
Methylene blue tributyrin agar plate
Methylene blue is the indicator
Allows to see the zone of clearing around the streak
Casein hydrolysis
Casein – milk protein
Caseinase
Secreted enzyme (exoenzyme)
Hydrolyzes (breaks down) casein into peptides/amino acids
Taken into bacteria for anabolism
To test:
Skim milk agar
If the agar goes from white and opaque to clear (zone of clearing) = positive
Urea Hydrolysis
Urease breaks down urea to ammonia and carbon dioxide
Causes an alkaline product to be produced
Sometimes the reaction is weakly positive
Causes the phenol red indicator to turn pink
Help to determine if urea can be used as a carbon and energy source
Tube 1 – salmon = uninoculated
Tube 2 – hot pink = positive
Tube 3 – yellow = negative
https://data.epo.org/publication-server/image?imageName=imgb0001&docId=5156467
http://www.blinn.edu/natscience/phillips/Micro%20Pictures_files/image102.jpg
Additional tests available to you:
Unknown Session 4
Make sure your keep your culture growing!
If you want to store results week to week, keep them in the 10°
(1) skim milk agar
(1) methylene blue tributyrin agar
(1) starch agar
(1) urea broth
Do you need to set up any additional tests?
You have one more class to decide.
Incubation Temperature:
M/T = 37℃
W/R = 25℃
Identifying Unknowns
Identification of Unknown Session 5:
Check results
API 20E
Last day for additional tests
Quick Results
Add the iodine to test for amylase
You should know your Genus by now.
If you need additional tests, they are included in your Unknown
Do you have lipase?
Does your bacteria hydrolyze casein?
Do you have urease?
No reaction (L)
Positive (M)
Negative (R )
20 tests to determine Gram-negative, enteric rods
Each compartment is one dehydrated test
Read the results to receive a code
Code will tell you the name of the bacteria
Make sure to follow the directions on how to fill each well/cupule
Some will require mineral oil
API 20E
Unknown Session 5
If you want to store results week to week, keep them in the 10°
At minimum, you should know your genus!
(2) API 20E strips per class
Tables 1, 4, 5
Table 2, 3, 6, 7
Do you need to set up any additional tests?
Your unknown is due next class period.
Incubation Temperature:
M/T = 37℃
W/R = 25℃
