Physiological Tests Flashcards
Methyl Red
- Acidic: Red
- Alkaline: Yellow
- Transition range: 4.4-6.4
- Found in: Mixed Acid Fermentaton test
Bromcresol Purple
- Acidic: Yellow
- Alkaline: Purple
- Transition range: 5.2-6.8
- Found in: Decarboxylase tests
Bromthymol Blue
- Acidic: Yellow
- Alkaline: Blue
- Transition range: 6.0-7.6
- Found in: OF test and Citrate test
Phenol Red
- Acidic: Yellow
- Alkaline: Red (breaking down protein)
- Transition range: 6.8-8.4
- Found in: Fermentation broths, MSA
Neutral Red
- Acidic: Red
- Alkaline: Amber
- Transition range: 6.8-8.0
- Found in: MacConkey
Explain the purpose and significance of preforming fermentation and respiration physiological tests to identify bacteria
- The carbohydrate fermentation test is used to determine whether or not a bacteria can utilize a certain carbohydrate. It tests for the presence of acid or gas produced from carbohydrate fermentation. … This indicates that the bacteria can ferment the carbohydrate in the tube, producing acid.
State the knowledge gained by preforming each test (fermentation and respiration test)
Why is identifying bacteria important?
Accurate identification of the genus and species of the microbe causing an infection is essential for proper treatment of the infection and for reporting of potential and actual outbreaks.
How does on begin to identify bacteria?
Identification starts with determining the morphology and arrangement of cells under a microscope and observing colonies macroscopicly. Stains can assist in determining the genus of a bacteria but often physiological tests are needed to determine genus and species. The identity of a bacterium is determined by collecting data from various biochemical tests.
Each test result is used to eliminate _____________ and guide the selection of _______________that rule out all but a single species of bacteria.
- a subset of possible species
- further tests
What priciple are biochemical tests based on?
Many biochemical tests determine the ability of an isolated (pure) culture to utilize various compounds, produce specific waste products or byproducts, and reveal the presence or absence of specific enzymes.
Physiological test media usually contains _________ and if the bacteria use it they make byproducts, and if the byproducts accumulate they can be detected by changes in media such as the formation of gas bubbles, or __________. If bacteria do not use the compound this can also be helpful with ___________.
- a particular compound
- Color change
- identification
Oxidation-Fermentation test (OF test)
- O-F medium is useful in determining whether a bacterium uses oxidative or fermentative carbohydrate metabolism, or is unable to metabolize the source carbohydrate (inert).
- The medium is generally used in the identification of non-glucose fermenting Gram negative rods.
- Two tubes are always inoculated; one tube is covered with melted Vasoline or mineral oil to maintain an anaerobic environment, and is labeled “F”. The second tube remains exposed to air trapped within the tube and is considered aerobic, as oxygen in the tube diffuses into the medium, and is labeled “O”.
- Incubation is performed over several days because some non-fermenters grow slowly.
- Uninoculated medium contains a particular sugar, such as maltose, and the pH indicator Bromthymol Blue, which is yellow at pH 6.0 and green at pH 7.1.
- A color change to yellow in both tubes, or yellow in the fermentative tube only, means the organism is fermentative.
- A color change to yellow only in the presence of oxygen means that the organism is oxidative only.
- No color change in either tube means the organism cannot utilize the sugar and is considered inert.
Oxidation
refers to the ability of a bacterium to breakdown a sugar molecule to make ATP using oxygen as the final electron acceptor (that is oxygen is required for the metabolism of that particular sugar).
Fermentation
is an anerobic process in which a sugar is partially broken down to pyruvate, and acidic products accumulate from further metabolism of pyruvate to produce ATP in the absence of oxygen.
Which one is Incompletely oxidative
1
Which on is Strictly Oxidative?
2
Which one is Facultative?
3
Which one is Strictly Fermentative?
4
Phenol Red Broth Sugar Fermentation test
- This medium can be prepared using a single sugar in broth containing the pH indicator Phenol Red.
- A variety of different sugars can be tested and the test is performed the same each time. Examples of sugars include glucose, mannitol, lactose, xylose, etc.
- Phenol Red turns yellow at a pH below 7.0 and darker pink above pH 7.0. If bacteria ferment the sugar, the broth will change from red to yellow color, indicating a positive result.
- This test is also prepared with an inverted Durham tube, which is used to trap gas that might be produced during fermentation.
- The final COLOR, not the presence/absence of gas, is recorded for the sugar test. It is advisable to note gas production as secondary to fermentation of the sugar.
- An important issue to consider when using phenol red broth is reversion, in which bacteria incubated for an extended period of time break down protein in the medium after exhausting the available carbohydrate. The ammonia produced from protein metabolism is basic, and increases the pH. As a result, the media will revert back to a red or fuschia color, causing misinterpretation of the result. For this reason, the phenol red test should be interpreted within 24 hours at 37 degrees C, or within 48 hours at 25 degrees C.
MRVP Test
This test is called the Mixed Acid Fermentation (MR) test and is used to detect the presence of multiple stable acid end products from glucose fermentation. Many bacteria, particularly enteric (intestinal) bacteria, produce multiple acids from glucose fermentation such as: lactic, acetic, succinic, formic, etc. If a bacterium produces enough acids to change the medium to a pH <5, they are called mixed acid fermenters. MRVP tubes contain glucose (a fermentable sugar), peptone (a protein source), and dipotassium phosphate (a buffer to resist pH changes and therefore prevent death of the microbes being studied). After incubation for 48 hours, 2 ml of broth is removed and added in a REACTION TUBE. A few drops (3-4) of the indicator methyl red are added to the REACTION TUBE (not the incubation tube). Methyl red is red at pH 4.4 and yellow at pH 6.2. The broth will turn pink-red if positive for mixed acid fermentation; any other color is negative or inconclusive.
Fermentation tests
- Oxidation-Fermentation test
- Phenol Red broth sugar fermentation test
- MRVP test
Respiration tests
- Catalase test
- Oxidase test
- Nitrate reduction test
Biochemical pathways involved in ATP production can serve as _____________________ among different bacterial species. The presence or absence of a ________________or product is determined, and that information is used in the identification of the species.
- distinguishing characteristic
- a particular enzyme
Catalase Test
- The ability of organisms to utilize oxygen is considered to be one of the most significant evolutionary developments in the history of life, as it greatly enhanced the amount of energy obtained from the metabolism of food molecules
- As with most advantageous traits, this ability has an associated disadvantage, namely the production of harmful reactive oxygen species (ROS) that can damage membranes and DNA. Most aerobes and facultative organisms produce the toxic ROS hydrogen peroxide (H2O2). Many of these organisms (including humans) produce catalase to convert hydrogen peroxide into water and oxygen.
- This test can be performed two ways. Option 1: mix a small sample of an isolated colony into a drop of water on a clean glass slide, then add hydrogen peroxide. Visible bubbles will form if the bacteria are catalase positive. Option 2: add a drop of hydrogen peroxide directly to a plate or slant. **NOTE: Hydrogen peroxide will kill cell! Therefore, do not add hydrogen peroxide to your working slant or cultures, unless you do not mind using up the slant or plate.
Oxidase test
This test detects the presence of an ETS system through the activity of an enxyme or electron transport molecule, cytochrome C oxidase, which can be detected by dropping dimethyl-p-phenylenediamine hydrochloride (oxidase reagent) on a colony. Not all microbes may have this particular enzyme, but still depend on ETS systems to produce ATP. If the bacteria have the enzyme, the colony turns a bluish-purple color. If no blue-purple color is observed, the result is negative. **NOTE: Oxidase reagent will kill cells, therefore do not add it to your working slant or cultures, unless you do not mind using up the slant or plate.
Nitrate Reduction test
- Some bacteria use nitrate (NO3-) as an electron acceptor during anaerobic respiration.
- The enzyme nitratase converts nitrate to nitrite (NO2-) in this pathway. Facultative bacteria will reduce oxygen as the final electron acceptor during aerobic respiration before switching to nitrate, so this test should be done in anaerobic conditions. The broth is prepared in test tubes with screw top caps and inverted Durham tubes. The cap must be tightened during incubation to prevent additional oxygen from entering the tube. There should not be any gas (bubbles) trapped in the Durham tube prior to incubation, so inspect the Durham tube and note any existing gas trapped prior to incubation.
- Bacteria are grown for 2-4 days. After incubation, examine the Durham tube and note if gas is present or not. Next, add a few drops of sulfanilic acid and dimethyl – α – napthylamine to the tube. A deep red color indicates a positive reaction for the presence of nitrite.
- If after adding the first two reagents, the broth remains clear, there are two possibilities: (a) the bacteria did not reduce the nitrate, or (b) the bacteria further reduced the nitrite into other compounds, including the possibility of N2 gas. To distinguish between these possibilities, the broth is tested further by adding a few sprinkles of zinc to any tubes that are not positive. If nitrate remains in the broth, zinc can “take” electrons from nitrate, and the broth will turn a pinkish-red, indicating that nitrate has not been reduced and still remains in the broth. This is a negative test result. If after the addition of zinc, the broth remains clear, the test is interpreted as positive because the bacteria utilized the nitrate in the broth and produced metabolic products other than nitrite.
Simmons Citrate test
Some bacteria can utilize citrate as their sole source of carbon if they have the enzyme citratase. The medium also contains ammonium salts. If the bacterium utilizes citrate, it is thought to also use the ammonium salts as a nitrogen source, which results in an alkaline pH shift in the media. The indicator is bromthymol Blue, and the media is initially green, but will turn cobalt blue if the test is positive for citrate utilization. The test is a slant and is inoculated with a surface streak and a stab to the butt of the tube.
Decarboxylation test
- This is a useful test for the determination of Gram negative bacteria through detection of the enzyme decarboxylase.
- Decarboxylation, the removal of the carboxyl group from an amino acid (lysine, arginine or ornithine), yields alkaline products, and can be detected using bromocresol Purple, which is yellow at pH 5.2 and purple at pH 6.8.
- After inoculation, a layer of mineral oil or melted Vasoline is added to prevent oxidative deamination. After 3-4 days a positive reaction will turn the medium a darker purple, while a negative reaction is indicated by no color change or a more yellow color change.
- Some bacteria will first produce a VERY acidic environment which will turn the medium yellow. This acidic environment activates the decarboxylase pathways which will turn the medium purple as the medium shifts from acidic to alkaline.
Phenylalanine test
- Some bacteria, notably Proteus or Morganella, produce the enzyme phenylalanine deaminase, which removes an amino group from the amino acid phenylalanine to produce phenylpyruvic acid and ammonium.
- Ferric chloride is added following incubation, and reacts with phenylpyruvic acid (if present) to form a green precipitate. This indicates a positive result. A negative result is indicated by yellow color after addition of the ferric chloride.
Tryptophan (indole) test
- Some bacteria have an enzyme, tryptophanase, which can hydrolyze the amino acid tryptophan into indole and pyruvic acid.
- The bacteria use the pyruvic acid, but indole accumulates in the medium. Indole can be detected using Kovac’s reagent.
- After 24-48 hours incubation, a few drops of Kovac’s reagent is added, and a positive test appears when a red ring accumulates at the top of the medium.
Bile Esculin test
- Some bacteria tolerate the presence of bile, and can hydrolyze esculin.
- This test can be used for Gram negative enteric bacteria and to separate some species of streptococci and enterococci.
- The medium is prepared as a slant and contains bile (a salt, to select for bacteria that are salt tolerant), esculin (a nutrient whose metabolic products can be detected) and ferric citrate (reacts with the product of esculin hydrolysis, producing a dark brown precipitate). Following incubation, a color change to dark brown is indicative of a positive result
Starch hydrolysis test
- Starch contains a straight chain of amylose (200-300 glucose residues) and a larger, branching amylopectin (glucose containing phosphates).
- Bacteria which have amylases hydrolyze the starch into maltose, glucose and dextrin.
- Starch turns blue in the presence of iodine. Bacteria which produce an exo-enzyme amylase will remove starch from the agar immediately surrounding bacterial growth. The addition of iodine will result in a clear halo surrounding the bacteria, indicating the absence of starch.
- The halo is an indicationof a positive test as the amylases have digested the starch in the media surrounding the bacteria. The addition of iodine will kill bacteria.
- To perform the test, inoculate a plate containing starch agar. Incubate and add iodine following incubation. You may have to wait several minutes for the halo to become visible
Urea Hydrolysis test
- A common test in the differentiation of Gram negative bacteria is whether the bacteria can utilize the carbon in urea.
- Urea is the main nitrogenous compound in mammalian urine, therefore urease positive species are important urinary tract pathogens (Leboffe & Pierce). If a bacterium makes urease, urea will be hydrolyzed to ammonium and carbon dioxide.
- Bacteria are grown in a urea broth that is produced by placing a urea disk into a test tube containing 2-3 ml of sterile water. The disk contains urea and Phenol Red. Following incubation, the presence of ammonium produces an alkaline pH, and can be detected by using the indicator phenol red.
- The indicator will turn pink at approximately pH 8.1, and pink is the positive color. No color change, orange or yellow colors indicate a negative test result.
ONPG test
- ONPG stands for O-Nitrophenyl β-D galactopyranoside. The ONPG test is used to determine if bacteria produce β-galactosidase, one of the many enzymes necessary to utilize lactose (think of the lac operon).
- In order to ferment lactose, bacteria need a permease to transport lactose into the cell, and galactosidase to hydrolyze lactose into glucose and β-galactose. Some bacteria lack permease, but will produce β-galactosidase when induced by the presence of lactose. If produced, β-galactosidase hydrolyzes ONPG resulting in a change of the medium to yellow. The test is prepared using ONPG broth (often a VERY faint yellow) and the addition of an ONPG disk.
Gelatin hydrolysis
- The gelatin test detects the ability of a bacterium to hydrolyze gelatin through the use of gelatinases.
- The medium appears semi-solid, and inoculation is performed as a stab. Following incubation, if the medium appears to be liquid,
- place the tube in a beaker of ice water for 20-30 minutes. The warm temperature of the incubator may liquefy the gelatin, so if the liquid appearance becomes a semi-solid while in the ice water, the test is negative. If the medium remains a liquid after a cooling period, the test is positive.
Kligler’s Iron Agar test (KIA)
- Inoculation of this medium is done with a stab to the butt of the tube, and a smear across the surface of the slant.
- This medium can be used to test for the ability of bacteria to produce hydrogen sulfide (H2S) as it converts the amino acid cysteine into pyruvic acid. The iron in the medium will react with hydrogen sulfide to produce a black precipitate, iron sulfide.
- Kligler’s Iron Agar also contains glucose, lactose, peptone and Phenol red to indicate fermentation of either glucose or both sugars. Following a 24 hour incubation, an acidic (yellow) butt and an alkaline (red) slant indicate the fermentation of glucose.
- The combination of yellow butt/red slant also indicates the bacterium cannot ferment lactose. After glucose fermentation, the bacterium will digest the peptone, producing ammonium, an alkaline product causing the slant to revert to red.
- If the test is not read after 24 hours, the shift from glucose fermentation to peptone utilization may conceal evidence of glucose fermentation.
- If the entire tube (butt and slant) is yellow, the organism can ferment both glucose and lactose. Keep the 24 hour window in mind as a reversion to red can occur even if both sugars are fermented.
- If the tube remains pinkish-orange or red, the bacterium does not ferment either sugar.
- If inoculation is done carefully without disrupting the agar, evidence of gas production can be detected by cracks or bubbles within the agar.
