Biochemical tests

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biochemical tests

Carbohydrate fermentation Broth What are we testing for?

testing to see if the organism can ferment
Glucose, Lactose, Mannitol

How do you know if carbohydrate fermentation broth is positive or negative?

1 carb + pH indicator (Phenol red)
if carb is fermented, acid end products will be released causing pH to drop. Red---> Yellow if acidic
If there is gas in Durham tube, then non water soluble gases were given off, sign of some sort of reaction.

Why is a positive result in a carbohydrate fermentation Broth help/hurt organism?

if the carb can be fermented, then the organism can use the carb as an energy source.

O/F Glucose Test, What are we testing for?

testing to see does the organism do Aerobic respiration, Fermentation, or Both? (stab)

whats a positive/negative result look like for an O/F Glucose test?

green = negative
Positive = yellow
if yellow in sealed tube= fermentation
if yellow in open tube = aerobic respiration.

Why does this benefit the organism with O/F?

if its fermentation, then can still metabolize with out oxygen present.
if its aerobic respiration then it can metabolize with glucose present.

What are we testing for in the Simmons Citrate Media?

Testing to see if the organism has the ability to use citrate as sole carbon source and energy source

What does a positive/negative result look like for Simmons citrate slant

Negative= remains dark green (slightly acidic)
Positive = turns electric Blue (pH goes up) (alkaline)

Changes color because uses up acids, so pH increases.

Why does this benefit an organism to be citrate positive?

benefit because it gets to use citrate as sole energy source.

What is MR-VP testing for?

Two types of fermentation detected:
--Mixed Acid Fermentation (Methyl Red test)
Ferments carb, produces mixed acid end products
--2, 3 Butanediol Fermentation (Voges-Proskayer Test)

What does a positive result look like in a MR-VP broth?

Mixed acid fermentation (methyl red test)
Add methyl red reagent. Positive result =red
2,3 butanediol Fermentation
Add alpha-haphthol and KOH (Potassium hydroxide)
Positive result= red

What is the benefit to the organism in the MR-VP broth?

if it is able to ferment the carb, then it has another energy source.

What is catalase test testing for?

testing for cytochrome oxidase activity, at the time of sampling. TSA plate

what does a positive result look like for the catalase test?

collect all of mass. Add it to oxidase reagent card.
Positive= Paper turns purple

what is the benefit of the organism for a positive result with the Catalse test?

Positive means its going under aerobic respiration, benefit is the organism can use oxygen as its terminal electron acceptor, which is the most energy efficient acceptor.

What are we testing for with the Nitrate broth test?

We are testing for the ability to use nitrate as a Terminal electron acceptor.
NO3--> NO2
or
NO3--> NO2-->N2O--> N2

what does a Positive result for a Nitrate broth test looks?

First add NO3 A and NO3 B
if pink= positive result (NO3--> NO2) If clear...
then add Zinc dust
if pink= negative. If colorless still= Positive result ( NO3-->N2)

what is the benefit to the organism with a positive result to the nitrate broth test?

If it can use Nitrate as its terminal electron carrier then it can do anaerobic respiration. which is the second most energy efficient electron acceptor.

What are we looking for with the Starch test?

Testing for the ability to degrade starch using Amylase

What does a positive result look like?

Pour iodine over plate
Iodine + starch = black purple color
If amylase is in organism, clearing will be seen around streak.

what is the benefit to the organism with a positive result to the starch test.

If the organism has amylase present then it is able to break down starch molecules (anything more than two glucose molecules, two glucose molecules= malose.) and bring them inside the cell for energy.
amylase breaks down starch moecules down one by one.

What are we testing for with the Milk Agar?

Testing for caseinase?
Caseinase is an enzyme that breaks down mile protein.

What does a positive test result look like for Milk Agar?

If organism has caseinase present, agar goes from white/ opaque--> clearing around growth.
Clearing = Positive result.

What is the benefit to the organism with a positive result to the milk test?

If the organism has Casinase present, then it has the ability to break down milk protein.

What are we testing for with Spirit Blue agar?

Spirit Blue= dye that binds to lipids.
Testing for lipase --> enzyme that degrades fats.

What does a positive result look like in the Spirit blue agar?

Media begins blue, dye binds to lipids in the agar
As bacteria grow, if they can use that fat, the break down and transport it into the cell leaving dye behind, Dark blue line (Dye build up) around growth if positive.

Benefit to organism if they have a positive result to spirit blue agar?

If lipase is present, it is the enzyme that will break down the fats to bring is into the cell.

what are we testing for in the Sulfide Indole Motility stab (SIM)

Sulfide= SO4 is in media as source of sulfer, Fe is also present.
Motility= If cloudy everywhere, cells were motile. If entire tube is black then assumed motile because its not just black on stab line.
Indole= tests for trytophan hydrolysis via trytophanase
****(KNOW STRUCTURE!!!!!!!!!!!)****

What does a positive test result look like in the SIM stab?

If sulfide is produced by the organism then S will react with FE in media to form FeS, a black ppt.
If motile, then it will be cloudy everywhere or black everywhere.
For Indole, add Kovac's reagent, Positive = kovac's layer will turn red (means indole is present)

What is the benefit to a positive result in the SIM stab?

The benefit to being a sulfide producer, is it helps determine between enterobacteria
The benefit to being motile is it can move around to get the nutrients it needs, or leave a unfavorable environment.
The benefit to Indole producer, is it helps determine between enterobacteria.

What are we lookinf for with the Urea broth test?

tests for urea hydorlysis via urease.
****( KNOW EQUATION)****

what does a positive result look like in the Urea broth test?

Positive result= Peach turns to Hot pink color.

What is the benefit to the organism that has a positive result for the Urea Broth test?

the ability to break down Urea via Urease is used to test the the differences between different bacteria.

What are we testing for with the Phenylalanine Slant?

Tests for Phenylalanine deaminase
****( KNOW REACTION/DIAGRAM)****

What does a positive result look like in the Phenylalanine slant?

Add reagent FeCl3 to slant
Positive result= growth turns green

What is the benefit to the organism that has a positive result to a phenylalanine slant

If the organism contains Phenylalanine deaminase then it can degrade Phenylalanine into phenylpyruvic acid and further to pyruvate and benzene (waste product)

what are we testing for in the Litmus Milk test?

testing for lactose fermentation
Litmus= pH indicator

What does a positive reaction look like in the Litmus milk test

Acid end products will be released if lactose is fermented, causing pH to drop. Vigorous lactose fermentation could cause casin to precipitate.
Purple(nuetral)-->Pink(slightly acidic)-->yellow(very acidic)

what is the benefit to the organism to have a positive result to the litmus milk test.

A positive result would indicate that the organism can degrade lactose, which is beneficial to the organism because it can use it as an energy source.

What are we Testing for in the Eosin methylene Blue (EMB) test?

Fermenting carbs, Lactose and sucrose. If either carb is fermented, acid end products are released, lowering pH.

What is the positive result of the EMB test look like?

Lactose fermentation= pink
Vigerous lactose fermentation= metallic Green sheen
Any other color= other fermentation

What is the benefit to the organism to have a positive result to the EMB

having a positive result means that the organism can ferment lactose or sucrose, being able to ferment those carbs is beneficial because it gives the organism an another energy source.

what are we testing for in teh MacConkey Agar (MAC)

selective agents
Bile salts
Crystal Violet
Testing for ability to ferment Lactose
pH indicator= neutral red

What does a positive result look like for the MAC test.

Lactose fermentors =Pink colonies
Vigerous lactose fermentors = Opaque pink cloud in media. indicates bile ppt.
colorless = above 6.8 pH
Red = Below 6.8 pH

what is the benefit to the organism that has a positive result for MAC

Having a positive result means the organism can ferment lactose, which is beneficial because it gives it another source of energy.

What are we testing for in the Salmonella Shigella Agar (SS).

Selective Agent
Sodium Citrate
Brilliant green
bile salts
Testing for the ability to ferment lactose
pH Indicator= nuetral red

what is the positive result look like in a SS agar?

Thiosulfate is in media as sulfer media also contains irons.
If thiosulfate is used, sulfur is given off and sulfide reacts with iron in media which forms FeS, black ppt.
Lactose fermented= pink colonies
Sulfide produced = black colonies.

What is the benefit to the organism to have a positive result to the SS agar.

The benefit is it can ferment lactose and use it as an energy source.

What are we testing for in the triple sugar iron slant (TSI)

Fermentation of Lactose, Sucrose, and glucose.
Lactose an sucrose at 1 % and glucose and 0.1%
Stab and streak.
Phenol red = pH indicator

What does a positive result look like in a TSI slant

Butt = yellow (acidic) = glucose fermented (should always happen)
Slant = yellow (acidic) = Sucrose and lactose ( if yellow one or the other fermented, or both)
Thiosulfate added as sulfur, media contains Fe. If thiosulfate is used sulfate is given off, it reacts with Fe, making FeS, black ppt.
Sulferate is only active at acidic pH, meaning carb had to be fermented.

what is the benefit to the organism who has a positive result to the TSI

the benefit to the organism is if it can ferment the carbs, then it has another energy source.

What are we testing for in the Ornithine decarboxylase, Arginine Decarboxylase, Lysine decarboxylase test.

Tests to see if it can decarboxyate the carb.
four tubes, first one is just a glucose decarb broth. then Orn, Lys, and Arg.
pH indicator = bromresol Purple. (nuetral)
Decarboxylase enzymes require a acidic pH
****(KNOW DIAGRAM)****

What does a positive result look like for the Ornithine decarb, Arginine decarb, and Lysine decarb test.

Decarb broth= should ALWAYS be yellow, if its not, then cant trust any of the results for that amino acid.
Purple --> yellow (glucose fermented) then Yellow--> Purple (amino acid decarboxylated).
Sealed with mineral oil, forces anerobic respiration.

What is the benefit to the organism that has a positive result for Orn decarb, Arg Decarb, and Lys decarb test.

the benefit is the organism can ferment glucose in an anerobic environment, and has Amino acids that help it survive.

bacteriophage

To eat bacteria. A bacterial virus.
Virus attaches to bacteria
Takes over cell, turns it into a virus producing factory
bacterial cell dies.

Plating and counts for Bacteriophages

PFU/mL 30 -300 (too little or too many = invalid test)
1 virus gives rise to 1 plaque
Plaque = clearing because bacteria didnt grow. (cover entire plate.. count clearings.)
#PFC
----------------- x Dilution factor
PFC/mL = Amt plated

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