Lab 15 - Biochemical Reactions: Enterobacteriaceae

Where are enterobacteriaceae typically found?

usually inhabit the guts of humans and other animals; usually associated with intestinal infections, but can be found in almost all natural habitats

What is the gram-type and shape of enterobacteriaceae?

large gram-negative rods

What are the characteristics that all members of enterobacteriaceae share?

oxidase negative, glucose fermenters, nitrate reducers

How can you usually determine the pathogenicity of enteric bacterium

by its ability to metabolize lactose; non-fermenters are usually pathogenic, while the fermenters are not

What types of diseases do enterobacteriaceae cause?

meningitis, bacillary dysentery, typhoid, and food poisoning

What are the two types of fermentation reactions?

sugar fermentation, MRVP (methyl red voges-proskauer test)

What are the common end products commonly generated by fermentation?

organic acids and gases such as CO2 and H

What do fermentation test media contain?

1. a single carb for energy production

2. nonfermentable sources of nitrogen/other nutritional req.

3. pH indicator such as phenol red

4. a Durham tube that collects gas

What indicates a positive fermentation reaction?

a change in the color of the indicator from red to yellow (below ph 7) due to the production of acid end products; gas production

Name the 3 sugar fermentation reactions we performed

glucose, lactose, sucrose

What is the purpose of the MRVP test?

used to differentiate between certain intestinal bacteria called coliforms

What is the carb source in the MRVP broth?

dextrose

What is a mixed acid fermentation?

some coliforms will ferment the dextrose to acid products that will cause the pH to drop below pH 5; other coliforms will convert the dextrose to less acidic products such as ethanol or butanediol

What bacteria are positive in the methyl red test?

mixed acid fermenters; after incubation, the addition of methyl red (a dye which turns red below pH 4.4) will indicate whether such fermentation has occurred

What is the purpose of the Voges-proskauer test?

butanediol fermentation; measures the presence of acetion, a precursor to butanediol

What is added to the broth after incubation in the voges-proskauer test?

barritt's reagents, alpha-naphthol, and potassium hydroxide; the tube is shaken to aerate the solution

What is indicates a positive reaction in the voges-proskauer test?

development of a pink or red color after agitation is a positive reaction for the production of acetoin

What are the 3 types of enzyme production tests that we performed in this lab?

catalase production, oxidase production, and citrate utilization

What is catalase?

an enzyme that splits H2O2 into water and oxygen

What indicates a positive catalase reaction?

when H2O2 is added to a colony of catalase-producing bacteria, it is broken down and the oxygen that is produced can be seen as bubbles

What is oxidase?

enzymes that catalyze the reduction of oxygen during respiration

Describe the oxidase test

utilizes a colorless reagent to detect oxidase; this chemical in the presence of oxygen and oxidase will form a colored compound

What indicates a positive oxidase reaction?

a color change within 20 seconds for a strong positive

Describe Citrate agar

a medium containing citrate as the sole source of carbon and ammonium salts as the sole nitrogen source (begins as a green slant); bromo thymol blue is present as the indicator dye

Describe the citrate utilization test

organisms that metabolize citrate utilize the ammonium salts, release ammonia and increasing the pH of the medium; the bromo thymol blue is green at neutral pH, deep blue at a pH above 7.6

What indicates a positive reaction in the citrate utilization test?

citrate medium changes from a green (neutral pH) to a blue (pH above 7.6); indicates the organism metabolizes citrate

What are the 4 types of single media/multiple tests that were performed in the lab?

SIM, TSI (triple sugar iron agar), urease test, nitrate reduction

What are the 3 reactions tested by the SIM?

production of hydrogen sulfide from sulfur-containing amino acids, indole production, and motility

How do you check for motility in the SIM test?

observing the migration of the inoculum from the stab line through the semisolid medium

How do you check for the production of hydrogen sulfide in the SIM test?

indicated by a blackening of the medium

How do you check for the production of indole in the SIM test?

determined after the addition of indole reagent (Kovac's reagent) and the production of a red color

What is the SIM media primarily used for?

primarily used for differentiation of gram-negative enteric bacteria

What is the purpose of the TSI (triple sugar iron) test?

widely used in the identification of gram-negative bacteria, particularly Enterobacteriaceae

What is contained in the TSI medium?

sucrose in addition to the dextrose and lactose; phenol red is present as an indicator

Describe the procedure to the TSI test

the media are poured as slants and are inoculated with a STAB TO THE BUTT followed by a streak of the slant surface (the bacteria therefore are exposed to both an anaerobic environment (butt) ad an aerobic one (slant))

Describe the TSI reaction of nonfermenters

they can grown on the slant by the aerobic degradation of protein components in the medium to alkaline products; the slant and the butt will remain red

Describe the TSI reaction of bacteria that can ferment glucose, but not sucrose or lactose

acid is produced in the slant, and the butt and the medium turn yellow; However, the glucose is used up in 12 hours and bacteria at the surface continue to grow by degrading proteins. By 18 to 24 hours, the alkaline end products cause the medium in the slant to revert to a red color. At 24 hours, the butt is yellow and the slant is red.

Describe the TSI reaction of bacteria that can ferment glucose, but not sucrose or lactose up to 12 hours after incubation

butt and medium are yellow

Describe the TSI reaction of bacteria that can ferment glucose, but not sucrose or lactose at 24 hours after incubation

butt is yellow and slant is red

Describe the TSI reaction of bacteria that can ferment lactose and/or sucrose as well as glucose

the slant and butt will remain yellow after prolonged incubation

In the TSI test, what will yield an acid butt with a black precipitate and an alkaline slant (red)

Salmonella; TSI also contains sodium thiosulfate and ferrous sulfate as indicators of hydrogen sulfide production; this means that the organism only fermented glucose and produced hydrogen sulfide

What does the lifting of agar in the TSI test indicate?

gas production

What is urease?

an enzyme that breaks the carbon-nitrogen bond of amides to form CO2, ammonia, and water

How can urease be detected?

inoculating bacteria in an amide-containing medium, specifically urea

Describe the urease test

When urea is broken down, ammonia is released and the pH of the medium increases (becomes more basic); the pH change is detected by a pH indicator that turns pink in a basic environment

What indicates a positive urease test?

a pink medium

What is the purpose of the nitrate reduction test?

used to determine the ability of an organism to use nitrate as the terminal electron acceptor to carry out nitrate respiration; used in identification of Enterobacteriaceae (usually +)

What does the enzyme nitrate reductase do?

catalyzes the transfer of electrons from cytochrome b to nitrate reducing it to nitrite

What does the nitrate broth contain?

beef extract and potassium nitrate

Describe the nitrate reduction test

Bacteria inoculated in broth and incubated for at least 48 hours. After incubation, reagent A and an equal amount of Reagent B are added.

If a red color develops in the nitrate broth after the reagents are added, what does this mean?

nitrate was reduced to nitrate, which means nitrate reductease was present

What should you do if a red color does not develop after adding the reagents to the nitrate broth?

add a pinch of zinc powder ad shake vigorously

What is the purpose of adding zinc to the nitrate broth if it does not turn red after the reagents are added?

zinc will react with nitrate and reduce it to nitrite

If a red color develops after adding zinc to the nitrate broth, what does this mean?

negative for nitrate reductease; nitrate was still present in the broth (because the enzyme was not there to reduce it), so the zinc reacted with it and turned the broth red (it reduced nitrate to nitrite)

If a red color does not develop after adding zinc to the nitrate broth, what does this mean?

positive for nitrate reductease; the nitrate was reduced to nitrite by the enzyme, and further reduced to nitrogen gas, so zinc was not able to react with the nitrate (it was already reduced!)

What organisms were positive for glucose fermentation?

E. coli (also gas production)

What organisms were negative for glucose fermentation?

Pseudomonas aeruginosa

What organisms were positive for lactose fermentation?

E.coli (also gas production)

What organisms were negative for lactose fermentation?

Pseudomonas aeruginosa

What organisms were positive for sucrose fermentation?

none

What organisms exhibited a mixed acid fermentation in the MRVP (methyl red)

E. coli

What organism did not exhibit a mixed acid fermentation in the MRVP (methyl red)

Enterobacter aerogenes

What organism produced acetoin (precursor to butanediol) in the MRVP (voges-proskauer)

None

What organisms were catalase positive?

E. coli

What organisms were catalase negative?

Streptococcus lactis

What organisms were oxidase positive?

none

What organisms were positive in the citrate utilization test?

Enterobacter aerogenes; media turned blue

What organisms were negative in the citrate utilization test?

E. coli; the media remained green

What organism was positive for hydrogen sulfide in the SIM?

Proteus vulgaris

What organism was positive for indole production in the SIM?

P. vulgaris, E. coli

What organism was positive for motility in the SIM?

E. coli, E. aerogenes, P. aeruginosa, P. vulgaris

What organism fermented lactose and/or sucrose and glucose in the TSI?

P. vulgaris (slant and butt yellow)

What organism was a nonfermenter in the TSI?

P. aeruginosa (slant and butt remained red)

What organism fermented lactose, but not lactose/sucrose?

E. coli, yellow butt, red slant

What organism was urease positive?

P. vulgaris

What organism was positive for nitrate reductease?

E. coli and Streptococcus lactis