lab exam Flashcards
what type of tests are for both gram negative and gram positive bacteria?
the oxygen requirement tests and the catalase test
what are tests that you should conduct on gram negative bacteria?
ONPG test, urease test, Indole test and carbohydrate fermentation on MAC agar and EMB agar + oxidase
lists the tests for gram positive organisms
bile-esculin test, MSA agar, starch hydrolysis, and BAP agar
when should the urease test, bile esculin test actually be conducted
urease should be conducted when if the OXIDASE test is NEGATIVE (test for gram negative, pink bacteria)
bile esculin should only be conducted if the CATALASE test is NEGATIVE
state the procedure for conducting a catalase test
- place 2 drops of hydrogen peroxide on a slide
- use the loop to pick up a colony and mix it with the peroxide
- observe any bubble formations
how does the catalase test work?
the catalase tests if catalase is present, catalase protects the cell from peroxide (h2o2). this enzyme converts peroxide into water and gas
the formation of bubbles is a positive test, because it means that the catalase is able to protect the bacteria from harful oxygen species since it turns hydrogen peroxide into gas
the NO BUBBLES is negative and means that the bacteria has no catalase
how does the oxidase test work?
oxidase tests for cytochrome c, an enzyme for obligate aerobic respiration and in the ETC. uses TMPD as an electron provider
colourless TMPD means that the test is negative and that the organism has no cytochrome C
a blue/purple colour means its POSITIVE, cytochrome c is present
what is the procedure for an oxidase test?
- work near the bunsen burner
- use a sterile applicator swab and collect a single colony from the plate
- tap your swab tip to transfer the colonies on filterpaper
- on a new sterile tip, add one drop of tmpd
- record observations
tests if bacteria can hydrolyze lactose (this is key in gram negative bacteria)
onpg
a colour change to yellow means that Beta galactoside hydrolyzed ONPG into o-nitrophenol
colourless solution means that the test is negative and the bacteria cannot hydrolyze ONPG
how do you conduct the urease and onpg test?
- flame loop and pick a colony from a gram negative sample
- note that urease should be conducted if its oxidase negative
- put colony into yellow capped tube (ONPG) and the red capped tube (urease)
- inoculate cells into the tube (mix the loop in solution)
- do not flame the plastic tube
incubate tubes at 37 degrees celcius and for 60min
what does the urease test assess?
what do the colours mean?
tests for the prescence of the urease enzyme, this enzyme hydrolyzes urea and produces ammonia.
pink colour means that the test is positive and that ammonia was made which increased the pH of the medium to give it that pink colour
colourless solution means that the test is negative and that there is no prescences of a urease enzyme
detects for the action of the trptophanase enzyme . cells with tryptophanase are able to grow and metabolize tryptophanase into indole , which is a waste product of metabolism
indole test
what do the test results of the indole test mean?
a colour change to green or blue/green is positive, meaning that indole is produced form tryptophanase by reacting with DCMA
no colour change=negative
how do i carry out the indole test?
- work near bunsen burner
- use a swab and pick a single colony(gram negative)
- tap the swab on filter paper
- with a NEW SWAB place a drop of DCMACA onto the swab tip
- tap swab tip onto paper and observe any colour changes
what is the carbohydrate fermentation/lactose test about and its incubating procedure?
test involves streaking colonies onto MAC and EMb agar and incubating for 37 degrees for 24 hours
this tests if the end products of carbohydrate femrentation produces lactic acid and pyruvate
what do the results of the carbohydrate fermentation/lactose test mean?
carbohydrate fermentation is for MAC and EMB agar
a colour change in the medium means that the organism produces beta galactosidase that is used to ferment carbohydrates such as lactose
colonies in medium will change colours dependinf on the type of fermentation that happens
what are we testing for the bile esculin test?
tests to see if organisms can grow in bile and hydrolze esculin
test is used to distinguish streptococci bacteria
how do i interpret the results of a bile esculin test?
if the colour changers into a black-brown colour , that means results are positive and means that esculin is present
how do i conduct the bile esculin test?
- flame loop, pick a colony from a gram positive cocci culture only, and streak the bile slant
- incubate for 37 degrees for 60 minutes, record observations at the beginning, 30 minute mark, and 60 minute mark
what does starch hydrolysis test test?
tests if starch can be hydrolyzed by amylase into glucose and maltose
test is used for gram positive bacillus
state interpretations of the starch hydrolysis test
to interpret results of the test, you have to pick an isolated colony, inoculate the bacteria and streak a single line on the plate then incubate at 37 for 24 hours
A blue-black of dark brown colour (this is the iodine) means that starch is present and that iodine reacts with the starch to produce a colour change
what indicates that bacteria are gamma hemolytic on BAP agar?
means that there is no discolouration of the medium surrounding colonies, and white colonies
what indicates alpha hemolytic bacteria?
a greenish opaque halo around the colonies, this means that bacteria are partially hemolytic
what indicates beta hemolytic bacteria?
a thick clear zone (kinda yellow) around the colonies, this means that bacteria are an;e tp cpnduct complete hemolysis (can rupture a lot of rbc’s)
