Biochemical Tests Flashcards
what does the nitrate reduction test test for?
Nitrate reductase
Can be a sign of anaerobic respiration
What biochemical tests are we responsible for?
- Nitrate reduction test
- litmus milk test
- urease test
- exoenzymes
- carbohydrate fermentation (duram test)
- Gelatin test
- Ames test
what type of organism participates in anaerobic respiration and what enzyme do they contain?
gram -
nitrate reductase
what specific kind of bacteria may participate in anaerobic repiration?
enterobaceriaceae
what is the process of converting nitrate to molecular nitrogen called?
denitrification
what kind of solution is used in the Nitrate reduction test?
Nitrate broth
what is the nitrate broth made of?
- beef extract
- potassium nitrate (KNO3)
how do you know if gas was produced during the nitrate reduction test?
by looking at the inverted Durham tube
is there a color indicator in the nitrate broth? how does it change colors?
no
color change takes place as a result of the reactions between the metabolic products and the reagents added after incubation
how would you test for denitrification in a nitrate reduction test?
look to see is gas collected in the inverted durham tube
if there is no evidence of denitrification, what should you do next? and what are you testing for?
8 drops sulfanilic acid (reagent A) and 8 drops naphthylamin (reagent B) should be added
the reduction of nitrate to nitrite
once reagents A and B are added and there is a presence of nitrite, what will be produced from this reaction and what color will it be?
nitrous acid (HNO2)
red
if there is no color change after you add reagents A and B, what can you assume and what would you do next?
- the nitrate was not reduced or was reduced to one of the other nitrogenous compounds.
- add zinc
why is the zinc added to the solution and what color would it be if specific products are present?
- to catalyze the reduction of nitrate to nitrite
- red
the red color seen after the addition of zinc in a nitrate reduction test indicates what ?
that Nitrate was not reduced by the organism
what does it mean if there still isnt a color change after zinc is added in a nitrate reduction test?
the organism reduced the nitrate to NH3, NO or N2O2 or some other non-gaseous nitrogenous compound.
What are the steps in a nitrate reduction test?
1) inoculate pure bacteria into tube
2) incubate at 37 degrees C for 24 hours
3) examine durham tube (no gas, do next step)
4) add 8 drops of reagent A and B ( no color change, do next step)
5) add zinc (results now shown)
what does the urease test test for?
tests if an organism can metabolize urea
what is urea a product of?
decarboxylation of amino acids
what enzyme hydrolyzes urea?
urease
what is urea hydrolyzed to?
- ammonia
- CO2
what specific organisms are capable of hydrolyzing Urea?
enteric bacteria (proteus, morganella, providencia)
what is the solution called used for the urease test?
urea broth
what is in urea broth?
- urea
- trace of yeast extract
- buffers to inhibit alkalization
- phenol red
what colors are produced by the urease test?
- yellow or orange
- red or pink
what does yellow or orange color of the urease test indicate?
a pH lower than 8.4
is negative for urease
what does the red or pink color of the urease test indicate?
- a pH above 8.4
- is positive for urease
what would the clinical application of the urease test be?
to test for the Urinary Tract pathogen (proteus)
what are the steps in the urease test?
1) inoculate the urea broth with pure organism
2) incubate 24 hours at 37 degrees C
3) observe color changes
What does the litmus milk test test for?
multiple reaction such as….
- lactose fermentation
- reduction of litmus
- casein coagulation
- casein hydrolysis
what does the litmus milk test contain?
Skim milk
litmus
what is the purpose of the Litmus in the solution for the litmus milk test?
it is a pH indicator
if lactose fermentation occurs what are the litmus milk test results?
color change from purple to pink (due to lower pH)
and is an acid reaction
when can you tell that litmus reduction has occurred in the litmus milk test?
when the solution turns white
what color is the litmus milk solution when there is an organism present that do not ferment lactose but instead metabolize it?
blue color due to ammonia production (higher pH)
what does the exoenzymes test test for?
tests for what exoenzymes are present by testing them on different mediums
what are the 4 exoenzyme media and what do they test for?
- fat plate - lipase
- milk agar - casease
- gelatin deeps - gelitinase
- starch agar - amylase
When looking at the exoenzyme tests what determines a positive test?
a clearing zone around the microbe
what determines a positive result in the gelatin deep media?
liquifying of the medium.
what does the carbohydrate fermentation test test for?
testing to see is the organism can grow in that medium
what are the signs that the organism is growing in the carbohydrate medium?
- color change
- production of gas
- turbidity
what proves that fermentations has occurred in the carbohydrate fermentation test?
- color change
- gas production
- along with growth
what proves that the organism can grow in the carbohydrate medium, but does not ferment?
- turbidity (cell growth) but no color change or gas produciton
what special step has to be performed when analyzing the starch plate in the exoenzyme test?
addition of iodine to dye the plate so you can see the clearing zones.
what does the Ames test test for?
mutagenic compounds
what kind of mutation occurs naturally?
spontaneous mutations
what kind of mutations occur unaturally?
induced mutations
what is auxotrophic bacteria?
bacteria that lost its ability to synthesize a specific amino acid.
what happens when you expose an Auxotrophic bacteria to a mutagen in the ames test?
it produces colonies which proves that the agent that you are testing is a carcinogen.
(this is due to the fact that the Auxobacteria are mutated by the agent and can now produce histidine when they weren’t able to before)
how do you test whether something is a mutagenic compound?
exposing auxobacteria to the mutagen on a plate that lacks histidine. if colonies grow then the substance is a mutagen, no colonies grow = the substance wasnt a mutagen)
what would be the point of adding bile salts to the ames test?
to test if the digests of a compound are mutagenic
why would you run a negative and positive control in the ames test?
negative control - introducing an agent that you know isnt mutagenic will cancel out the possibility of having bacteria that were going to grow on the media anyways (proves the results you are seeing are a result of the agent being mutagenic)
Positive control - ^^ same thing just in reverse…. (should see growth to make sure that you arent getting false negative results)
