Micro Lab Flashcards
What does crystal violet do?
Dyes Gram + bacteria purple
Why is iodine needed after adding crystal violet?
It helps fix the crystal violet to the Gram + cell walls
Why after adding iodine is the slide washed with acetone/alcohol?
This happens in order for the excess crystal violet to be removed from gram negative cells
What does safranin do to bacteria?
It stains Gram - cells pink
MacConkey agar
Has crystal violet and bile salts to prevent the growth of Gr + bacteria
Way to only grow Gr - bacteria
Contains lactose and neural red dye
Therefore lactose fermenting bacteria will produce acids (lactic acid) to lower the pH.
Neural red is the pH indicator for when it drops.
The bacteria will have pink rings around them if they ferment lactose.
The bile salts will precipitate from this pH change which causes the pink halo around the colonies
Mannitol Salt Agar
Selective isolation for Staph species
Contains 7.5% NaCl which prevents many Gr - and Gr + bacteria from growing
Staph aureus can grow!
Contains mannitol as carbon source and phenol red as the pH indicator
If the bacteria can ferment mannitol then the pH will drop from the excess acid produced and the red will turn yellow
Will turn pink if pH is above 8.4
Will remain red if pH is 6.9-8.4
Will turn yellow if pH is below 6.9
What can be accomplished with the use of fermentation tubes in identifying bacteria?
A way to see if a bacteria ferments the molecule (carbohydrate) in the tube
Can do lactose fermentation, glucose fermentation…etc…
We are using phenol red fermentation, turns yellow if acid is produced from active fermentation by the bacteria
What is the purpose of a Durham tube in a fermentation tube?
They collect gas (H2 or CO2) which can be produced during fermentation
EMB media
Selective for Gr - bacteria
The dyes in it prevent the growth of Gr + bacteria
Has lactose in it so if lactose is fermented, the Gr - bacteria colonies will have a green sheen to them
Starch agar media
Tests to see if the bacteria hydrolyses starch
Has protein, starch and agar
Is used on both Gr + and Gr - bacteria
After two days you add Gram’s iodine onto the plate and the starch is stained dark brown by the iodine
If the media gets red and white surrounding it then the test is positive and the bacteria hydrolyses starch
IMViC
Is a set of tests for Gr - bacteria
Indole test for the production of indole from tryptophan
Methyl red for acidic fermentation products
Voges-Proskauer for acetoin production
Citrate to see if citrate is the sole carbon source for the bacteria
What do you use the stab for?
SIM, fluid thioglycollate and other media where you inoculate into the media in the tube
What is SIM medium
Sulfide, indole and motility medium
Use stab for this
Sulfide
Production of hydrogen sulfide from sodium thiosulfate
Media will turn black (H2S reacts with iron in the media
Motility
Diffuse growth from stab line or turbidity of tube
Indole
Test after observing results for first sulfide and motility tests
You add 3-4 drops of Kovac’s reagents
Red ring is a positive test
MR-VP Media
Two media: one for Methyl red test and the other for VP test
Broth tests contains glucose as carbohydrate. Inoculate an MR-VP tube with a colony and grow for 48 hours. Half the culture is added to a new tube
Add methyl red do detect acidic fermentation. 5 drops. red color is a positive test
VP test is done with the other tube and detects acetoin which is a neutral product in pyruvate metabolism
Add 6 drops of reagent A and two drops of reagent B. Vortex gently. Let sit for 10-15 minutes
A positive test yields a red color in the tube
Simmons citrate agar
citrate is the sole carbon source
Brom thymol blue is the indicator dye.
Green is at neutral pH and blue is at alkaline pH
If plate is blue then the test is positive
Urea agar
Urea is hydrolyzed by the enzyme urease into carbon dioxide and ammonia
Media has phenol red which is yellow at pH 6.8 and pink at pH 8.2
Production of ammonia increases the pH of the media
The test is used on Gr - bacteria
The agar is prepared as a slant in test tubes and just streak across the top of the agar
Pink is a rapid urease positive test
Pink and yellow butt is delayed urease positive test
Yellow is negative result
Lysine iron agar
Agar slant in a tube
Used for Gr - bacteria
Has lysine and sodium thiosulfate
Lysine is used to detect lysine decarboxylase and lysine deaminase
The sodium thiosulfate is a substrate for the production of H2S
The dye is brom cresol purple
Yellow at pH 5.2 or lower
Purple at pH 6.8 or higher and is positive for lysine decarboxylation if the butt is purple
Red slant is positive for lysine deamination
Negative reaction for both is a purple slant and a yellow butt
H2S production is black but positive decarboxylation may suppress this so compare to SIM media as well
The decarboxylation of lysine results in the production of amines that raise the pH of the media
Catalase test
Catalase breaks down H2O2
Enzyme is present in Gr + and Gr - species
Level of enzyme tells you if it is pathogenic
Add H2O2 to streak plate
Bubbling from the addition of hydrogen peroxide is a positive test and indicates the release of O2
Gelatin test
Contains just gelatin and no agar. Get gelatin from boiling collagen
Tests to see if bacteria can hydrolyze gelatin
Is used for both Gr - and Gr + bacteria
Some bacteria secrete gelatinases
Inoculate a gelatin stab with your strains
Grow 37C for a week
Each day test to see if gelatin has been hydrolyzed by placing tubes in an ice bath for 15-30 minutes
If liquid then the gelatin has been hydrolyzed
If solid then the gelatin has not been hydrolyzed
After test put them back into incubator
