Experiment 11 | Protein catabolism

define deamination and decarboxylation, understand the conditions under which each reaction usually occurs. Interpret a gelatin hydrolysis test, urea broth test, phenylalanine slant test, ornithine decarboxylase broth test, SIM deep test and understand what a positive result means and looks like

What is the structure of a amino acid? (ag,cg,sc)

1-amino group,2- carboxyl group, 3- r side chain

Just like us what is bacteria primary energy source? (think of the 4 main organic molecules)

bacteria perfer carbohydrates as their carbon and energy source of choiceBacteria prefer

If no sugars are available for bacteria, what do they tend to target next as potential sources of energy?(f/p)

They tend to target fats or proteins

Before an amino acid/protein can be used as energy what must happen? (think of the structure of an amino acid, ag must be r_)

Amino (NH2) group must be removed

What else do bacteria pick up to use as energy when the primary source is not available? (most abundant gas in our atmosphere)

Nitrogen as well

What is deamination?(think of what decarboxyaltion is)

Removal of amino group from a molecule,

What does deamination also result in? ( de_ in _think 0-14)

Decrease to pH

What is decarboxylation? (think of what deanimation was)

The removal of the carboxyl group from an amino acid

What does decarboxylation also result in? ( in_ in)

Increase in pH (alkaline environment)

What else can bactera potientally release and what does it look like after incubation?

Hydrogen sulfide (H2S) visualized as a black precipitate

Many enteric bacteria have enzymes to break down the amino acid tryptophan. What is the diagnostic by product of this breakdown?(prod_ oi_

Production of indole

The production of indole can be dectected by the addition of what to the growth medium? (kov_ re_)

Kovacs reagent/dimethylaminobenzaldehyde

How could you tell if indole is present?(py→cr)

Pale yellow turns to cherry red color

What type of macromolecule is gelatin?

Gelatin is a protein that is commonly found in certain kinds of foods.

What is the main purpose of the gelatin hydrolysis test?(exoe_ ge_)

To differentiate between organisms that produce the exoenzyme gelatinase and those that do not

What is gelatinase, and what does it help in?(pat_ ability to in_ hu_ ti_)

A pathogens ability to invade human tissues

How does gelatinase help invade human tissues?

since the gelatin molecule is too large to enter the cell the exoenzyme is secreted by gelatinase + bacteria to cut gelatin into smaller, usable polypeptides

When gelatin is converted to smaller units after the enzyme has done its job what happens to its form?

It changes to a liquid form, loss of solid consistency this is what were analyzing for

Once the media has been liquefied by the enzyme gelatinase, what will not happen again?

It will not re-solidify

What does a positive and negative result look like for a gelatin hydrolysis test?

A positive result will remain a liquid at a temperature below 32 degrees celsius and a negative result will be solid at or below 32 degrees celsius

What is a waste product made by many living organismsn and is the mian component of our urine?

Urea

what can the urea broth determine?(ee u)

If a microbe produces the exoenzyme, urease

What is in the urea broth?(nn, u_, __in_ think 0-14 it is called ph_ r_)

necessary nutrients ,urea, and a pH indicator called phenol red

What color does a phenol red pH indicator turn at a pH of 6 and a pH of 8.

Yellow at a pH of 6 and fuchsia at a pH of 8

What are the two main end products when urease breaks down urea? (a & cd)

Ammonia and carbon dioxide

Since urea is commonly found within the unrinary tract, what is the urease test useful for determining?

The presence of urinary pathogens

What is the phenylalanine agar/slant used for? (dif_ of en_ bac_ based on their ability to produce pa from the amino acid phenylalanine)

differentiation of enteric bacteria based on their ability to produce phenylpyrvic acid by the oxidative deamination of the amino acid

What is the enzyme, and what does it do? (pd_ think of what removal of amino group is called, then what does it leave, pa, en_ prod_)

Phenylalanine deaminase, it can remove the amino group, leaving a phenylpyruvic acid end product

To complete the test after incubation to allow bacteria to grow what is added? (fer_ chl_ think of what they add to pools)

10% Ferric chloride solution is added to the slant

How do you know if a test is + for a phenylalanine slant?

Orange ferric chloride solution will turn olive green

How do you know if a test is - for a phenylalanine slant?

No color change occurs (remains orange) indicating that phenylalanine was not deaminated.

What color does the ornithine decarboxylase broth start off as?

PurpleIt starts off as purple, indicating a neutral pH before fermentation occurs.

How would a + reaction look like?

Gray or purple color

How would a - reaction look like?

It would remain yellow or show no change, indicating no decarboxylation of ornithine.

This reaction also occurs in the absence of oxygen, therefore many will overlay the tube with what? Where do you then observe at?

Mineral oil is added, observing the lower 3/4 anaerobic region of the tube for color change.

SIM agar is a component of what kind of tests used to differenetiate enteric bacteria? (in_ think of previous terms, me_ r_think of pH, vo_-pros_ and cit_ (think of green and blue nice color tube tests)

IMViC - indole, methyl red, Voges-Proskauer, and citrate tests.

The SIM agar deep is used to identify members of what family? (ent_)

Enterobacteriaceae family.

SIM agar allows 3 different results to be visualized what are they? (sr_ common element used in microbio, if_ and mot_ think of tail seen.)

Sulfur reduction, indole formation, and motility.

How can sulfur reduction be visualized in a SIM agar deep?

Sulfur reduction can be visualized in a SIM agar deep by the formation of a black precipitate, indicating the production of hydrogen sulfide gas by the reduction of sodium thiosulfate in the media.

How does indole production occur in a SIM agar deep?

Indole production in a SIM agar deep occurs when the bacterium metabolizes/breakdown tryptophan, producing indole, which can be detected using Kovac's reagent that forms a red ring.

How do you know if indole is present in a SIM agar deep?

reagent will turn red and create a red ring

How do you know if indole is NOT present in a SIM agar deep?

The reagent will not change color and remain pale yellow