1
Q
(TSI) Test
A
Triple Sugar Iron
- Purpose: test a microorganism’s ability to ferment sugars and to produce hydrogen sulfide.
- Principle:
- Differentiates: members of the Enterobacteriaceae family from other gram-negative rods
- Components: pH-sensitive dye (phenol red), 1% lactose, 1% sucrose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate, is used for carrying out the test.
- Appearance: Yellow during fermentation, gas production, red in alkaline conditions
2
Q
ONPG test
A
- Purpose: To determine the ability of an organism to produce β-galactosidase enzyme. A test for lactose fermentation.
- Differentiates: The test distinguishes late lactose fermenters from non–lactose fermenters of Enterobacteriaceae. Citrobacter from Salmonella.
- Components: O-nitrophenyl-beta-D-galactopyranoside, releasing o-nitrophenol which is a yellow-colored compound.
- Procedure: Incubate for 1 hour @ 35.
- Appearance: Turns yellow if positive. Colorless if negative.
- Note: All organisms tested must be inoculated from a lactose-containing medium (e.g., TSI or MacConkey).
3
Q
Moeller’s Decarboxylases test
A
- Purpose: To test the ability of an organism to produce a decarboxylase enzyme.
- Principle: Arginine, lysine, and ornithine decarboxylase media are used to detect an organism’s ability to decarboxylate or hydrolyze an amino acid, forming an amine that produces an alkaline pH.
- Differentiates: To differentiate the members of the Enterobacteriaceae family on the basis of their ability to produce decarboxylase enzymes. Enterobacteriaceae = POS from klebsiella. Vibrios = POS
- Components: Mineral oil, arginine, lysine, and ornithine, pH indicators are bromcresol purple and cresol red.
- Procedure: 1 drop of culture into each amino acid tube. Incubated 4 days @ 37°C. Checked each day.
- Appearance: Purple = POS, Yellow = NEG
4
Q
Carbon Sources test
A
Lactose fermenters
Sorbital fermenters
Glucose fermenters
Sucrose fermenters
5
Q
Urea test
A
- Purpose: To test the ability of an organism to produce the enzyme urease that hydrolyses urea.
- Principle: Urea is hydrolyzed to ammonia + CO2 raising pH.
- Differentiates: To differentiate urease-positive Proteus from other Enterobacteriaceae. Proteus, Provedencia, morgenella all = POS
- Components: contains urea and the phenol red as a pH indicator. Slant Christianson urea.
- Procedure: Loop a colony. Inoculate onto slant. Incubate @ 35°C for up to 7 days
- Appearance: positive test = magenta to bright pink color in 15 min to 24 hours. A negative test =no color change.
6
Q
PPA or TDA
A
- Purpose: Phenylalanine deaminase medium tests the ability of an organism to produce the enzyme deaminase.
- Principle: This enzyme removes the amine group from the amino acid phenylalanine and releases the amine group as free ammonia. As a result of this reaction, phenylpyruvic acid is also produced.
- Differentiates: to differentiate members of the genera Proteus, Morganella, and Providencia from other Enterobacteriaceae.
- Components: Medium contains nutrients and DL-phenylalanine. Reagent: 10% ferric chloride
- Procedure: Culture in medium then add 4 or 5 drops 10% ferric chloride wait 1-5 mins.
- Appearance: Pos= turn dark green Neg= No color change. Straw colored
7
Q
MRVP
A
- Purpose: This test is used to determine which fermentation pathway is used to utilize glucose.
- Principle: Two Tests. MR portion checks for acid production. Voges-Proskauer test detects the presence of acetoin.
- Differentiates: Between using mixed acid fermentation pathway to produce lactic, acetic, succinic, and formic acids and 2,3 butanediol fermentation pathway to produce acetoin end product.
- Components: (methyl red), 5%a-naphthol 95% ethanol and 40% KOH w/ .03% creatinine
- Procedure: (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear. an aliquot of the MR/VP culture is removed and 0.6 mL a-naphthol and 0.6 mL KOH are added. They are shaken together vigorously and set aside for about one hour until the results can be read
- Appearance: MR: Red = Pos, Yellow = Neg
- Appearance: VP: Red = brownish-red to pink, Yellow = brownish-green to yellow
8
Q
Gelatin
A
- Purpose: This test is used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that liquefy gelatin, a component of vertebrate connective tissue.
- Principle: The presence of gelatinases is detected using a nutrient gelatin medium. When an organism produces gelatinase, the enzyme liquefies the growth medium by hydrolyzing gelatin present in the medium.
- Differentiates: Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli. The ability of an organism that produces gelatinases.
- Components: Nutrient gelatin medium
- Procedure: Inoculate with 4 to 5 drops of 24-hour broth culture. Incubate at 35°-37°C in ambient air for up to 14 days. Place at 4°C to check for liquefaction.
- Appearance: Liquid = Positive, Solid = Negative
9
Q
Nitrate
A
- Purpose: To determine the ability of an organism to reduce nitrate to nitrite.
- Principle: All members of the Enterobacteriaceae family reduce nitrate. Some organisms can reduce nitrate to nitrite. The detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with an α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.
- Differentiates: members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram-negative bacteria that do not produce the enzyme nitrate reductase. Enterobacteriaceae reduce Nitrate
- Components: Reagent A = sulfanilic acid, Reagent B = α-naphthylamine. Durham tube
- Procedure: Inoculate the nitrate broths with bacterial suspension. Incubate @ 30°C or 37°C for 24 hours. look for N2 gas first before adding reagents. 6-8 drops of nitrite reagent A and B. Look for color change about 1 min. If no color develops add zinc powder wait 3 mins.
- Appearance: POS= Red color change after adding reagent A and B if no color change add Zinc if no color change then POS. Neg = red color on the addition of Zn powder
10
Q
Oxidase
A
- Purpose: The oxidase test detects the presence of a cytochrome oxidase system
- Principle: catalyze the transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. The dye is reduced to deep purple color.
- Differentiates: identification of Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella, and Pasteurella, all of which produce the enzyme cytochrome oxidase. Neisseria, Moraxella, Campylobacter and Pasteurella species (oxidase positive).
- Components: Kovacs Oxidase Reagent ( 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water) or Gordon and McLeod’s Reagent or Gaby and Hadley (indophenol oxidase) Reagent
- Procedure: Moisten disk with H20. Rub loop of colony on disk and wait 5-10 seconds. after 60 seconds = neg
- Appearance: Purple = Pos
11
Q
Motility
A
- Purpose: To determine the motility of bacterium.
- Principle: Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility.
- Differentiates: between motile and non-motile bacteria.
- Components: Agar deep.
- Procedure: touch the needle to the colony. Stab agar. Incubate at 35°-37°C and examine daily for up to 7 days.
- Appearance: Pos= growth beyond the stab. Neg = grow only at the stab.
12
Q
Malonate/Mucate
A
- Purpose: To test the ability of the organism to utilize malonate as sole source of carbon and energy for growth.
- Principle: An organism that simultaneously can utilize sodium malonate as its carbon source and ammonium sulfate as its nitrogen source produces an alkalinity due to the formation of sodium hydroxide.
- Differentiates: differentiate organisms on the basis of malonate utilization. differentiate between Escherichia and Enterobacter. Kleb = Pos, E. coli = Neg
- Components: indicator (bromothymol blue) and malonate medium.
-Procedure: light inoculum from an 18-24 hour pure culture, inoculate the tube containing malonate broth.
Incubate 35ºC in an aerobic atmosphere for 24-48 hours.
-Appearance: Pos = Blue, Neg = Green
13
Q
KCN (Potassium Cyanide)
A
-Purpose: This test determines whether the microbe can grow in a medium where potassium cyanide is present as a carbon and nitrogen source.
-Principle:
If the microbe can grow in the presence of potassium cyanide, the broth will become turbid (cloudy) after incubation.
- Differentiates: Potassium cyanide inhibits many bacteria including Salmonella, Shigella, and Escherichia, while members of the Klebsiella, Citrobacter, and Proteus groups grow well.
- Components: 0.75% potassium cyanide
- Procedure: An inoculum from a pure culture is transferred aseptically to a sterile tube of KCN broth. The inoculated tube is incubated at 35-37 C for 24 hours
- Appearance: Pos = Turbid
14
Q
Deoxyribonuclease (DNase) Test
A
- Purpose: To differentiate organisms based on the production of deoxyribonuclease.
- Principle: The test is used to determine the ability of an organism to hydrolyze DNA. DNase agar is a differential medium that tests the ability of an organism to produce an exo-enzyme, called deoxyribonuclease.
- Differentiates: distinguish Serratia (positive) from Enterobacter sp., differentiate Staphylococcus aureus which produces the enzyme, Moraxella catarrhalis (positive) from Neisseria
- Components: contains nutrients for the bacteria, DNA, and mostly methyl green as an indicator.
- Procedure: inoculate the DNase agar, Incubate the plate at 35-37°C for 24 hours.
-Appearance: Positive: Medium is colorless around the test organism.
Negative: No clearing
15
Q
Lipase
A
- Purpose: To determine the ability of microorganisms to produce the enzyme lipase.
- Principle: Microorganisms that possess the enzyme lipase hydrolyze the free fats present in the medium to form glycerol and free fatty acids. Consequently, the release of insoluble free fatty acids results in the formation of an iridescent sheen (oil on water) that can be seen when the plate is held at an angle to a light source.
- Differentiates:
- Components: Egg Yolk Agar
- Procedure: Streak plate with culture, Incubate at 35-37oC for 24-48 hours. Can be anaerobic or not.
- Appearance: Pos = appearance of an iridescent sheen (oil on water) immediately around colonies, Neg = absence of an iridescent sheen.
16
Q
Corn Oil
A
- Purpose: The purpose is to see if the microbe can use corn oil, a lipid, as a source of carbon and energy for growth. Use of corn oil is accomplished by a class of enzymes called lipases.
- Principle: A medium containing lipids (in this case corn oil) and an indicator is used. When lipids are digested, the indicator changes from blue to clear. Halos surrounding colonies is indicative of their ability to digest the lipid in the medium due to the presence of lipase.
- Differentiates:
- Components: Spirit blue agar is a nutrient medium with the dye spirit blue present.
- Procedure: Streak on plate and incubate for 35-37 C for 24 hours.
- Appearance: Pos = Halos around colonies Neg = No change
17
Q
YPA (Polypectate)
A
- Purpose:
- Principle:
- Differentiates: Yersenia will eat into agar
- Components:
- Procedure:
- Appearance:
18
Q
Carbohydrate Fermentation
A
- Purpose: The carbohydrate fermentation test is used to determine whether or not bacteria can ferment a specific carbohydrate. Carbohydrate fermentation patterns are useful in differentiating among bacterial groups or species.
- Principle: tests for the presence of acid and/or gas produced from carbohydrate fermentation. Basal medium containing a single carbohydrate source such as glucose, lactose, sucrose or any other carbohydrate is used for this purpose with a pH indicator.
- Differentiates: All members of Enterobacteriaceae family are glucose fermenters (they can metabolize glucose anaerobically).
- Components: Andrade’s solution, bromcresol purple (BCP), bromothymol blue (BTB) or phenol red
- Procedure: Inoculate each medium and incubate at 35-37°C for 18-24 hours.
- Appearance: Check for color change and gas production. Phenol Red Pos = Yellow and Neg = red.
19
Q
Esculin
A
- Purpose:is a selective and differential medium which is used to presumptively identify enterococci and group D streptococci based on the ability of an organism to hydrolyze esculin.
- Principle: Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe3+ and forms a dark brown to black precipitate.
- Differentiates: differentiates enterococci and group D streptococci from non–group D viridans streptococci.
- Components: esculin, ox bile, ferric ammonium citrate
- Procedure: Inoculate on a slant at 35°-37°C in ambient air for 48 hours.
- Appearance: Pos = Black Neg = Not Black or no growth
20
Q
O129
A
- Purpose: O/129 Disk Susceptibility Testing is useful for the identification of Vibrio and Aeromonas species.
- Principle: Some Vibrio spp. require salt for growth. Therefore, the test is run in duplicate on Mueller-Hinton agar (MHA) with low salt (0.5%) and with added NaCl (4%) to ensure growth on at least one of the plates.
-Differentiates: Vibrio species from Aeromonas species.
Vibio susceptible while aeromonas are resistant
- Components: O/129 disk is the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine phosphate.
- Procedure: Use Mueller-Hinton agar (MHA) to grow a lawn with disks and Incubate MHA plates for 18 to 24 hours at 35°C
- Appearance: Check for Zones of clearing
21
Q
Salt Tolerance
A
- Purpose: To determine the ability of an organism to grow in high concentrations of salt.
- Principle: Salt acts as a selective agent for bacteria and interferes with membrane permeability and osmotic equilibrium. A high salt concentration thus inhibits a range of bacteria but allows salt-tolerant organisms such as enterococci to grow. The broth includes the fermentable carbohydrate, dextrose, and the color indicator, bromcresol purple.
- Differentiates: differentiate enterococci (positive) from non-enterococci (negative). differentiate non-beta-hemolytic strains of catalase-negative, gram-positive cocci (i.e. Enterococcus and Aerococcus)
- Components: broth containing 6.5% NaCl is used as the test medium with bromcresol purple.
- Procedure: Inoculate and incubate @ 35 for 48hrs.
- Appearance: POS = Turbidity Or Purple, NEG = turbidity and no color change after 72 hours
22
Q
Pyrazinamide
A
- Purpose:
- Principle:
- Differentiates:
- Components:
- Procedure:
- Appearance:
23
Q
Indole Test
A
- Purpose: demonstrate the ability of certain bacteria to decompose the amino acid tryptophane to indole
- Principle: Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani, and Providencia species break down the amino acid tryptophan with the release of indole. This is performed by a chain of a number of different intracellular enzymes, a system generally referred to as “tryptophanase.”
- Differentiates: distinguish among members of the family Enterobacteriaceae
- Components: Indole Kovacs Reagent = (p-Dimethylaminobenzaldehyde, Hydrochloric Acid, 37%, Amyl Alcohol), tryptophan broth
- Procedure: Inoculate tryptophan broth then incubate for 18 to 24 hr. Add .05mL of Kovac’s Reagent.
- Appearance: POS= Red top layer, Neg = No color change
24
Q
LIA
A
Lysine Iron Agar
- Purpose: screening test that differentiates lysine utilization and H2S
- Principle:
- Differentiates: proteus turns red due to deamination.
- Components: 1% glucose, lysine, bromcresol purple, sodium thiosulfate, ferrous sulfate, peptone/yeast extract
- Procedure: Inoculate and incubate 18 to 24hrs
- Appearance: Yellow = Negative Purple = Pos glucose fermentation red in the butt. if lysine is decarboxylated then it’s purple throughout. Red is deaminated. Purple if decarboxylated
25
MIO
Motility Indole Ornthinine
- Purpose: differentiate motility indole and ornithine decarboxylation
- Principle:
- Differentiates:
- Components: 1% glucose, ornithine, bromcresol purple, sodium thiosulfate, ferrous sulfate, peptone/yeast extract
- Procedure: Inoculate and incubate 18 to 24hrs
- Appearance: Yellow = Negative Purple = Pos ornithine not decarboxylated it is yellow due to acid production. If ornithine is decarboxylated which is putricin then turns it alkaline turns it back to purple.
26
Indicators
Methyl Red
-pH \< 4.4 = Red
-pH \> 6.2 = Yellow
Neutral Red
-pH \< 6.8 = Red
-pH \> 8.0 = Yellow
Phenol Red
-pH \< 6.4 = yellow
-pH \> 8.0 = Red
Bromocresol Purple
-pH \< 5.2 = yellow
-pH \> 6.2 = Purple
Bromothymol Blue
-pH \< 6.0 = Yellow
-pH \> 7.6 = Blue
Microbiology
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