carbohydrate fermentation quiz

Phenol Red Broth (PRB) - bacteria used

Ec, Psa, Sle, Efl, 2 unks

PRB - purpose

Determine if the bacteria ferments a single carbohydrate and if it produces gas during the fermentation process

PRB - 3 types of carbs

dextrose, sucrose, lactose

defined vs undefined media

Defined media are media with a known composition and concentration. undefined is a medium with an unknown concentration that contains plant or animal byproducts

PRB - composition

undefined liquid media containing peptones, a single carb, and a phenol red indicator

PRB - indicator

Phenol red indicator is a pH indicator that is red at neutral pH, if it stays red it does not ferment its carb. if it turns yellow, it has turned more acidic due to lactic acid released in fermentation. if the broth turns magenta, it had turned more basic due to peptones being broken down

PRB - Durham tube

a small inverted glass tube inside the broth. if gas is released it will get trapped in the tube and show a bubble.

PRB - limitations (reversion)

the first limitation is reversion. PRB is time sensitive and results need to be read no longer than 48 hours. this is b/c the bacteria will start to eat the peptones after 48 hrs when the carbs are finished. this will cause the color to revert to red than magenta

PRB - limitations (DT)

the durham tube may already have a bubble, leading to a false positive

KIA - Bacteri used

Ec, Psa, Sle, Sm, 2 unks

KIA - purpose

3 results are given. does the bacteria ferment glucose, ferment lactose, and reduce sulfur. it also has a deep butt w/ glucose while the remaining slant has lactose, this shows you the location of the carbs being eaten

KIA - composition

undefined, solid media containing peptones, 2 carbs (10 g lactose, 1 g glucose), iron components, and indicator phenol red, which will turn yellow if the carb if fermented (turns acidic)

KIA - sulfur reduction

If the bacteria reduces sulfur, it will release H2S, which mixes with the iron components and turns the media black

PRB - results

Ec (unf) - ferments dextrose and lactose, but not sucrose. produces gas.

Sle - ferments only dextrose and produces gas

Psa - does not ferment any, oxidizes only. does not produce gas

Efl - ferments all. does not produce gas

KIA - results

Ec - ferments both lactose and glucose. does not reduce sulfur

Sle - Because it reduces sulfur, the other results are indeterminate

Psa - does not ferment either, doesnt reduce sulfur but it changes the color of the agar slant

Sm - ferments glucose but not lactose. does not reduce sulfur

KIA - limitations

Reversion after 48 hours

SIM - bacteria used

Ec, Sle, Ml, 2 unks

SIM - purpose

Sulfur, indole, and motility

tests if bacteria reduces sulfur, produces indole, and is motile

SIM - composition

undefined semisolid with a reduced agar concentration. no indicator

SIM - sulfur reduction

If the bacteria reduces sulfur, it will release H2S that reacts with iron components and will produce black parcipitate in media

SIM - indole production

To produce indole, the bacteria has to break down tryptophan, meaning it needs to produce the enzyme tryptophanase that will break down tryptophan into pyruvate, indole, and ammonium. This result is not visible until a reagent is added after incubation

SIM - motility

Determines if the bacteria is moving through the media. This is seen by looking at the fuzziness surrounding the stab. This result is not as conclusive in SIM due to the sufur reduction making motility less visible

Motility - Bacteria used

Sa, Sle, 2 unks

Motility - purpose

determine if bacteria are capable of moving in the media

Motility - composition

undefined, semisolid containing an indicator TTC, which accepts electrons from bacteria when they move making it turn red.

Motility - positive result

We look for red radiating off the stab

Citrate - bacteria used

Kp, Ec, 2 unks

Citrate - purpose

determines if citrate is the only carbon source the bacteria uses

Citrate - process

The bacteria has to bring in citrate and then ferment it. They do this by using the enzyme citrate permease to bring in citrate and then the enzyme citrate lyase is used to breakdown and ferment citrate

Citrate - composition

defined, solid media which contains citrate taken up from the citric acid cycle for carbon, this allows the bacteria to grow. then ammonium for nitrogen which is eaten by the bacteria which causes the media to be basic in pH. this is indicated by the pH indicator Bromothymol Blue with is green when neutral and blue when citrate is used as the sole carbon source and ammonium is eaten to turn the media basic

Citrate - limitations

A light hand is needed for this test. if too much bacteria is used growth can be mistaken.

Citrate - positive results

we are looking for if bacteria grew and if it changed color. it can still be positive if growth occured but color didnt change

SIM - results

The reagent used for indole is Covax, the top layer will turn red if indole is present.

Ec - does not reduce sulfur, it does contain tryptophanase and produces indole, it is motile

Sle - it does reduce sulfur, it does not contain tryptophanse, and therefore does not produce indole, the sulfur reduction makes the motility nonvisible and indeterminate

Ml - no sulfur reduction, no tryptophanse so no indole production, non motile

Motility - results

Sa - non motile

Sle - very motile

Citrate - results

Ec - It did not grow so citrate is not its only carbon source

Kp - grew so citrate is its only carbon source

OF - bacteria used

Ec, Psa, Afl, 2 unks

OF - purpose

Does the bacteria undergo oxidation, fermentaion, or both

OF - composition

undefined, solid media containing glucose and peptones, it has a high ratio of glucose. the indicator bromothymol blue is used. if the bacteria goes for glucose or both glucose and peptones, acids are released and the media turns yellow. if it goes for peptones only it releases bases and turns blue

OF - Aerobic and Anaerobic

Oxidation only occurs in aerobic conditions and fermentation can occur in both. so the tubes are split 5:5 Aerobic to anaerobic. Mineral oil is added after inoculation in a top layer to the anaerobic tubes

MRVP - bacteria used

Ec, Kp, 2 unks

MRVP - purpose

stands for Methyl red and Voges-Proskauer. It determines if bacteria is a mixed acid fermenter for the MV portion and it determines if the bacteria utilizes 2,3 - butanediol pathway for VP portion which releases acetoin to form diacetyl, which turns the media red

MRVP - composition

Defined, liquid media containing glucose, peptones, and a phosphate buffer. 2 reagents need to be added after inoculation

MRVP - VP process

A ml of the media needs to be obtained with a disposable pipette and put into a separate glass tube. To this tube, 15 drops of VP-A and 5 drops of VP-B are added. then it is vortexed for 2 seconds 6 times every 10 mins or until color change is observed

MRVP - MR process

To the remaining media, 2-3 drops of methyl red are added. if the bacteria is a mixed acid fermenter, it will release stable acids and change the color to red

OF - results

Ec - both aerobic and anaerobic tubes yellow meaning it fermented only or fermented and oxidized

Psa - aerobic tube is yellow, anerogic tube is green meaning it oxidizes only

Afl - one or both tubes turned blue meaning Afl is saphrophilic and did not touch the carbs at all instead the peptones were degraded and bases were released.

MRVP - results

Ec - changed color for MR portion so its a mixed fermenter

Kp - did not change color for MR but did change color for VP portion so it utilizes 2,3 - butanediol pathway.

Sucrose vs. lactose vs. dextrose

Sucrose and lactose are glucose plus smth else ( fructose/galactose resp.) so they are disaccharides while dextrose is just glucose and is a monosaccharide.