IDENTIFICATION OF ENTERIC BACTERIA Flashcards
Enteric bacteria may be isolated by cultivating_____ samples on differential media containing_____.
fecal
lactose
MacConkey agar is a differential medium containing ______to inhibit______
bile salts
non-enteric bacteria
MacConkey
In addition, it has two dyes,______, which are taken up by_____-fermenting bacteria.
neutral red and crystal violet
lactose
MacConkey
_______is inhibitory to the growth of gram-positive bacteria.
The_____ bacteria form pigmented colonies while the ______fermenters from colorless colonies.
Crystal violet
enteric
non-lactose
In_____ agar, pigmented colonies also are formed by lactose fermenters, but the type of pigmentation is distinctive to various genera.
EMB
_____agar is another differential medium and does not support the growth of gram-positive bacteria.
EMB
Triple sugar iron (TSI) agar is a valuable medium for differentiating enteric bacteria.
The medium contains three carbohydrates -(3)- as well as the pH indicator_____.
Bacteria are inoculated into the _____of the medium and into the the__, where anaerobic conditions prevail.
glucose, lactose, and sucrose
phenol red
butt
Several reactions are possible in TSI agar: First, if the organisin ferments only the glucose, both the slant and the butt will turn yellow after several hours;
Butt:?
Sant:?
…as alkaline conditions reappear from the digestion of_____ and the production of_____ compounds.
the butt will remain yellow
the slant will soon revert to red
peptones
ammonium
Several reactions are possible in TSI agar: Second, if lactose or sucrose or both carbohydrates are fermented
sufficient_____ will be produced to cause both the slant and butt will remain a_____ color.
acid
red alkaline
Several reactions are possible in TSI agar: * Third, gas (COz) production from the carbohydrates also may be determined in TSI agar* by noting the presence of________ in the medium after the incubation period.
If large amounts of gas are produced, portions of the medium may be pushed up in the tube.
cracks and fissures
Several reactions are possible in TSI agar: Fourth, hydrogen sulfide production may be tested because TSI agar contains (2)
Certain bacteria use_____ in their metabolism and release_______. This then reacts with____, yielding_____, which is a ______. The medium becomes black at this point.
iron ions and sodiurn thiosulfate
thiosulfate
hydrogen sulfide (HS)
iron
iron sulfide
black precipitate
The results from the IMViC series of tests provide additional evidence for the
identification of an enteric bacterium.
Though primarily used to distinguish
_____ from ______, the results from the test series may also be used for other organisms.
Escherichia coli from Enterobacter aerogenes
The IMViC series consists of four tests:
indole production (l)
methyl red test (M),
Voges-Proskauer test (V)
citrate test (C)
The_____ identifies species that can digest tryptophan to indole and other products.
indole test
The indole test identifies species that can digest______ to indole and other products.
tryptophan
The______ test depends on an organism’s ability to ferment glucose and produce large amounts of acid.
______indicator, added at the end of the incubation period, remains red in acid solution but becomes yellow in alkaline or neutral solution.
methyl red
Methyl red
The________ uses the digestion of glucose
to acetyimethylcarbinol to differentiate between enteric species.
If present in the culture tube, acetylmethylcarbinol will react with_____ and _____ to form a red chemical compound
Voges-Proskauer test
alpha naphthol and potassium hydroxide
The Voges-Proskauer test
uses the digestion of glucose
to________ to differentiate between enteric species.
If present in the culture tube,______ will react with alpha naphthol and potassium hydroxide to form a_____ chemical compound
acetylmethylcarbinol
red
The______ is based on the ability of certain bacteria to use citrate, a salt of_____, as a sole carbon source in growth.
When this occurs, the ph of the medium rises and the indicator,______, becomes deep blue.
If citrate use has not occurred, the original_____ color of the medium remains.
citrate test
citric acid
bromthymol blue
green
In recent years, several miniaturized systems have been made available to microbiologists for the rapid identification of enteric bacteria.
These products include
API 20E (bioMerieux Vitek, Inc.), Enterotube I| (Becton-Dickinson Microbiology Systems, Inc.), Micro-ID (Organon Teknika), and many others.
NOTE: If TSI reaction shows non-fermentative Gram-negative rods, perform________.
If it is positive, perform_______.
oxidase test
oxidative-fermentative test
Spot Oxidase Test
1. Prepare a solution of__________________ in sterile distilled water each day.
- Place a_____ into a sterile, plastic, disposable Petri dish, and moisten the filter paper with several drops of the fresh reagent.
- Remove a small portion of the colony to be tested with a platinum wire or wooden stick, and rub the growth on the moistened filter paper.
- Observe for a color change to_____ or _____ within____ seconds.
1% tetramethyl-p-phenylenediamine dihydrochloride
filter paper circle
blue or purple
10
Triple sugar iron (TSI) agar is a versatile medium for differentiating enteric bacteria based on their (3).
It contains three carbohydrates—(3)—as well as_____ as a pH indicator.
carbohydrate fermentation patterns, gas production, and hydrogen sulfide (H₂S) production
glucose, lactose, and sucrose
phenol red
TSI
Inoculation involves…
slant (where aerobic conditions prevail)
(butt) of the medium (where anaerobic conditions exist)
TSI
Carbohydrates:
________ : A monosaccharide that, if fermented, will produce acid in both the slant and butt of the TSI agar, initially turning both sections yellow.
_________: Disaccharides that, if fermented, produce larger amounts of acid than glucose alone, keeping both the slant and butt yellow even after prolonged incubation.
Glucose
Lactose and Sucrose
TSI
pH Indicator:
________ : it is a pH-sensitive dye that changes color in response to acidity or alkalinity.
It turns______ in acidic conditions (indicating________) and_____ in alkaline conditions (indicating______ utilization rather than carbohydrate fermentation)
Phenol Red
yellow; carbohydrate fermentation
red; peptone
TSI
______ : Present to detect H₂S production. When H₂S is released as a byproduct of bacterial metabolism, it combines with iron to form a_______, signaling H₂S production.
________: Acts as a source of sulfur, allowing certain bacteria to produce H₂S during their metabolism.
Iron Ions
black precipitate (iron sulfide)
Sodium Thiosulfate
TSI
Initial Reaction: Both slant and butt turn yellow due to acid production.
Final Reaction: The butt remains yellow (indicating acid production and glucose fermentation in anaerobic conditions), but the slant reverts to red as bacteria in the aerobic slant area exhaust the glucose, then metabolize peptones, producing alkaline byproducts.
Interpretation: This reaction indicates________, typical of many enteric bacteria.
It is commonly seen in enteric bacteria such as (2)
glucose fermentation only
Salmonella or Shigella
TSI
Reaction: The large amount of acid produced from fermentation keeps both the slant and butt yellow.
Interpretation: Acid in both the slant and butt suggests fermentation of either_______.
This reaction produces a stronger acidification than glucose fermentation alone, commonly observed in (3)
lactose, sucrose, or both
Escherichia coli or Enterobacter species
TSI
Gas (CO₂) Production
Reaction: Gas production from fermentation is detected by the presence of (3) in the medium. Sometimes, gas can push parts of the medium up in the tube.
Interpretation: The presence of gas (CO₂) confirms that fermentation of at least one carbohydrate (glucose, lactose, or sucrose) has occurred, resulting in the production of gaseous byproducts.
_______ often show gas production during fermentation.
cracks, bubbles, or fissures
Enterobacter species and Klebsiella
TSI
Hydrogen Sulfide (H₂S) Production
Reaction: Hydrogen sulfide (H₂S) reacts with_____ present in the TSI agar to form_______, indicating that the bacteria can reduce sulfur compounds.
Interpretation: The formation of a black precipitate indicates H₂S production, which is commonly seen in sulfur-reducing bacteria, such as certain strains of_________. This is an important characteristic for differentiating these bacteria from others.
iron ions; iron sulfide (black precipitate)
SPC: Salmonella, Proteus, or Citrobacter
The_______ is a set of biochemical tests used to differentiate and identify enteric bacteria. Although it is primarily employed to distinguish(2), the series can also be useful for identifying other enteric organisms.
IMViC series
Escherichia coli from Enterobacter aerogenes
The IMViC acronym stands for the following four tests:
Indole production (I)
Methyl red test (M)
Voges-Proskauer test (V)
Citrate test (C)
IMViC
Purpose: This test identifies bacteria that can metabolize the amino acid tryptophan, producing indole and other metabolites. The presence of indole is determined by adding Kovac’s reagent after incubation.
Indole Production Test (I)
IMViC
Indole Production Test
Procedure: Bacteria are grown in a medium that contains_______. After incubation,______ is added, which reacts with_____to form a red compound.
Result Interpretation:
Positive: A_____ color at the top of the medium (indicating indole production).
Negative:______ (indicating no indole production)
tryptophan; Kovac’s reagent; indole
red
No color change or a yellow coloration
IMViC
Purpose: This test detects the ability of bacteria to ferment glucose and produce large amounts of acid.
The indicator is used to detect the pH change, where the solution remains red in acidic conditions and turns yellow in neutral or alkaline conditions.
Methyl Red Test (M)
IMViC
Methyl Red Test
Procedure: After the organism has been grown in a glucose-containing broth,______ is added. If the bacteria have produced enough acid, the medium will remain____.
Result Interpretation:
Positive:_____ color after adding methyl red (indicating a high acid production, typically from mixed acid fermentation).
Negative:_____ color (indicating either neutral or alkaline pH, suggesting less acid production).
methyl red; red
Red
Yellow
IMViC
Purpose: This test identifies organisms that ferment glucose to produce acetoin (a precursor to 2,3-butanediol), a product that reacts with reagents to form a red compound. The Voges-Proskauer test is used to distinguish between bacteria that ferment glucose via different pathways.
Voges-Proskauer Test (V)
IMViC
Voges-Proskauer Test (V)
Procedure: After inoculation and incubation, reagents (2) are added. If acetoin is present, a red color forms.
Result Interpretation:
Positive:_____ color (indicating the presence of acetoin or the production of 2,3-butanediol).
Negative:_____ (indicating no acetoin production).
alpha-naphthol and potassium hydroxide
Red
No color change
IMViC
Purpose: The test determines whether bacteria can utilize citrate as their sole carbon source for growth. If it is utilized, the pH of the medium will rise, turning the bromthymol blue indicator from green to blue.
Citrate Test (C)
IMViC
Citrate Test (C)
Procedure: Bacteria are inoculated onto a slant of________, which contains citrate as the sole carbon source. If the bacteria can utilize citrate, they will grow, causing the medium to become more______.
Result Interpretation:
Positive:______ color (indicating successful citrate utilization and alkalinization of the medium).
Negative:______ (green remains, indicating that the bacteria cannot use citrate as a carbon source).
Simmons citrate agar; alkaline
Blue
No color change
The Indole test is used to identify bacteria that can break down tryptophan, an important characteristic in the differentiation of enteric bacteria like_______ (which produces indole) from other species that do not.
E. coli
The Methyl Red test is particularly useful for distinguishing organisms that ferment glucose to produce acidic byproducts, such as_____, from those that produce neutral or alkaline byproducts, such as______.
E. coli
Enterobacter
The Voges-Proskauer test is complementary to the methyl red test and helps differentiate bacteria that produce neutral fermentation products, such as_______, from those that produce acids
Enterobacter
The Citrate test is valuable for identifying bacteria that can metabolize citrate, which is often a distinguishing feature for genera like (2)
Klebsiella and Enterobacter
The Oxidase Test is used to determine whether an organism produces ______, an enzyme involved in the electron transport chain of certain bacteria.
The test is important for differentiating between groups of Gram-negative bacteria, particularly those that are non-fermentative, such as______ species, and those that are fermentative, like_____.
cytochrome c oxidase
Pseudomonas
Enterobacteriaceae
Procedure for Spot Oxidase Test:
- Preparation of Reagent:
Prepare a__________ in sterile distilled water. This solution acts as the reagent for detecting cytochrome c oxidase activity. The solution should be prepared fresh every day to ensure accuracy.
- Preparation of Test Surface:
Place a filter paper circle into a sterile, plastic Petri dish. The filter paper provides a surface on which the bacterial sample will be tested.
Moisten the filter paper with several drops of the freshly prepared reagent. This ensures that the test area is adequately saturated for the reaction.
- Sample Application:
Use a platinum wire or a wooden stick to remove a small portion of the bacterial colony to be tested. The choice of tool is crucial as metal may interfere with the reaction if it’s not a platinum wire.
Rub the bacterial growth directly onto the_________. This allows the bacteria to come into contact with the reagent and facilitates the reaction.
- Observation of Results:
Observe for a color change within_____. A positive reaction is indicated by a______ color appearing at the site where the bacterial sample was applied. This color change occurs because the cytochrome c oxidase enzyme catalyzes the oxidation of the reagent, leading to a visible color change.
If no color change occurs within 10 seconds, the result is negative, indicating that the bacteria do not produce cytochrome c oxidase.
1% solution of tetramethyl-p-phenylenediamine dihydrochloride
moistened filter paper
10 seconds
blue or purple
Follow-up Test (if Oxidase Test is Positive):
If the oxidase test is positive (i.e., color change occurs), further tests may be conducted, such as the________.
This test is used to determine whether the organism uses oxygen (oxidatively) or ferments carbohydrates, further distinguishing between bacterial species.
Oxidative-Fermentative Test (OF test)
The _____________ test helps differentiate between oxidative and fermentative bacteria based on their metabolic pathways for carbohydrate utilization.
This test uses a medium that contains glucose, and it helps identify organisms based on their ability to utilize glucose under aerobic or anaerobic conditions.
Oxidative-Fermentative (OF) Test
TSI inoculation
Stab
Streak
LIA inoculation
Stab stab
Streak
Citrate inoculation
Streak surface lightly
SIM inoculation
Stab butt (for H₂S and motility)
Urea inoculation
Streak surface of the slant
