With the malonate test, what happens in the oxidation of succinate dehydorgenase?
- Requires the enzyme succinate dehydrogenase
- Coenzyme FAD is reduced to FADH2
What replaces succinate as the substrate in the succinate to fumerate reaction, thus competatively inhibiting the succinate enzyme
- Malonate (malonic acid) b/c its similar enough to succinate to replace it as the substrate in the reaction.
- This competative inhibition of succinate along with the build up of succinate in the cell shuts down the Kreb cycle and kills the organism unless it can ferment or utilize malonate as its remaining carbon source
Between what 2 mediums does the malonate test differentiate
The malonate test differentiates between escherchia, which grows in the medium, and enterobacter
In the malonate test, what colors indicate the utilization of malonate?
- Dark blue: malonate is utilized, +
- No color change or slightly yellow: malonate not utilized, -
Of what is urea the product and to what can it be hydrolyzed?
- Urea is the product of decarboxylation of cerain amino acids
- It can be hydrolyzed to ammonia nad carbon dioxide bybacteria containing the enzyme urease
Waht does urea agar do?
Urea agar was formulated to differentiate between urease positive bacteria and urease negative bacteria
Waht are the functions of the contents of the urea agar?
- Peptone and glucose: Provide essential nutrients for a blood range of bacteria
- Potassium and phosphate: Mild buffer used to resist alkalinization of medium from peptone metabolism
- Phenol red: An indicator, yellow or orange below pH of 8.4, red or pink above pH of 8.4
What will be the results of urea hydrolysis by different mediums?
- In general, urease positive organisms overcome the buffer in the medium and change it from orange to pink.
- Rapid urease + organisms: Turn entire slant pink within 24 hours (rapid urea hydrolysis; strong urea production, +)
- Weak urease + organisms: May take several days, color can be partially pink in 24 hours and all pink or partially pink in 24 hrs-6 days or just orange/yellow within 24 hours and all pink/partially pink within24 hrs-6 days (slow urea hydrolysis; weak urease production, W+)
- Urease -: orange/yellow in 24 hours, same in 24 hrs-6 days. (No urea hydrolysis; urea absent, -)
Application of urease hydrolysis test
- Used to differentiate organisms based on their ability to hyrdolyze urea iwth the enzyme urease.
- Urinary tract pathogens from teh genus Proteus may be distinguisehd from other enteric bateria by their rapid urease activity
What is the SIM medium used for?
- Determination of three bavcterial activites-
- Sulfer reduction, indhole production from tryptophan, and motilitiy
What does the SIM medium include?
- Casein and animal tissue as ources of amino acids
- An iron containing compound
- Sulfur int he form of sodium thiosulfate
What are the two ways that sulfer reduction to H2S can be accomplished by bacteria?
- It depends on the enzymes present.
- The enzyme cysteine desulfurase catalyzed the putrefaction of the amino acid cysteine to pyruvate
- The enzyme thisulfacte reductase catalyzes the reduction of sulfur (in the for of sulfate) as at the end of the anaerobic respiratory electron transport.
- Both of hese systems produce hydrogen sulfied gas.
- When either reaction occurs in the SIM medium, the hydrogen sulfide that is produced combines with iron, in the form of ferrous ammonium sulfate to form ferric sulifde which is a black precipitate
What makes indole production in the SIM medium possible?
- The presence of tryptophan (contained in casein and animal protien)
- Bacteria possessing hte enzyme tryptophanbase can hydrolyze tryptophan to pyruvate, ammonia (by deamination) and indole
How is the hydrolysis of tryptophan in the SIM medium detected?
- By the addition of Kovac's reagent after a perdiod of incubation
- Kovacs' eagent contains dimethylaminobenzaldehyde (DMABA) and HCL dissolveed in amyl alchohol.
- A few drops of Kovac's reagent are added to the tube and DMABA reats with any indole present and produces a quinoidal compound the turns the reagent layer red.
In the SIM medium test, what indicates a positive reaction and the presence of tryptophanase?
- the formation of a red color.
- No red color: indole negative
What determines motility in the SIM medium?
- Reduced agar concentration and the method of inoculation.
- The medium is inoculated with a single stab from an incoulating needle
- Motile organisms are able to move about in the semisolid medium and can be detected by the radiating growth pattern extending outward in all directions from the central stab line.
- This fuzzy growth in all directions indicates motility
Application of SIM medium
SIM medium is used to identify bacteria that are capable of proucing indole, using the enzyme tryptophanase.
SIM Medium indole production results and interpretation
- Red in the alchohol layer of KOvac's reagent: Tryptophan is broken down into indole and pyruvate, +
- Reagent color is unchanged: Tryptophan not broken down into indole and pyruvate, -
SIM medium sulfur reduction results and interpretations
- Black in the medium: sulfur reduction (H2S production), +
- No black in the medium: Sulfur not reduced, -
SIM medium motility results and interpretations
- Growth radiating outward from stab line: motility, +
- No radiating growth: Nonmotile, -
What does the bile esculin azide agar (BEAA) do?
- It is both selective and differential
- Contains esculin, peptone, bile, sodium, azide, and ferric citrate
What do the contents of the BEAA do?
- Esculin: a glycoside composed of glucos and esculetin)
- peptone and esculin: provide nutrients
- Bile: inhibits grwoth of gram positive organisms (except group D which are streptococci and enterococci)
- Sodium azide: inhibits growht of gram negative organisms
- Ferric acid: an indicator
What happens when esculin molecules are split?
Esculin reacts wtih ferric citrate and forms a dark brown phenolic iron complex
Application of BEAA
Commonly sed for the presuptive ID of enterococci and members of the strep bovis group all of which are +
Results of BEAA
- More than 1/2 the medium is darkened within 48 hours: presumptive ID as a member of Group D strep or enterococcus, +
- No color change or less than 1/2 the medium is darkened within 48 hours: presumptive determinatino as not a member of group D strep or enterococcus