1
Q
Methyl Red
A
- Acidic: Red
- Alkaline: Yellow
- Transition range: 4.4-6.4
- Found in: Mixed Acid Fermentaton test
2
Q
Bromcresol Purple
A
- Acidic: Yellow
- Alkaline: Purple
- Transition range: 5.2-6.8
- Found in: Decarboxylase tests
3
Q
Bromthymol Blue
A
- Acidic: Yellow
- Alkaline: Blue
- Transition range: 6.0-7.6
- Found in: OF test and Citrate test
4
Q
Phenol Red
A
- Acidic: Yellow
- Alkaline: Red (breaking down protein)
- Transition range: 6.8-8.4
- Found in: Fermentation broths, MSA
5
Q
Neutral Red
A
- Acidic: Red
- Alkaline: Amber
- Transition range: 6.8-8.0
- Found in: MacConkey
6
Q
Explain the purpose and significance of preforming fermentation and respiration physiological tests to identify bacteria
A
- 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.
7
Q
State the knowledge gained by preforming each test (fermentation and respiration test)
A
8
Q
Why is identifying bacteria important?
A
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.
9
Q
How does on begin to identify bacteria?
A
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.
10
Q
Each test result is used to eliminate _____________ and guide the selection of _______________that rule out all but a single species of bacteria.
A
- a subset of possible species
- further tests
11
Q
What priciple are biochemical tests based on?
A
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.
12
Q
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
- a particular compound
- Color change
- identification
13
Q
Oxidation-Fermentation test (OF test)
A
- 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.
14
Q
Oxidation
A
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).
15
Q
Fermentation
A
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.
16
Q
Which one is Incompletely oxidative
A
1
17
Q
Which on is Strictly Oxidative?
A
2
18
Q
Which one is Facultative?
A
3
19
Q
Which one is Strictly Fermentative?
A
4
20
Q
Phenol Red Broth Sugar Fermentation test
A
- 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.
21
Q
MRVP Test
A
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.
22
Q
Fermentation tests
A
- Oxidation-Fermentation test
- Phenol Red broth sugar fermentation test
- MRVP test
23
Q
Respiration tests
A
- Catalase test
- Oxidase test
- Nitrate reduction test
24
Q
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.
A
- distinguishing characteristic
- a particular enzyme
25
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.
26
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.
27
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.
28
Depending on the test, the results should be read __________ when incubated at 37º C. Interpretation of the result before or after could provide misleading results. Other test results will not change after incubating for 24 hours and can be read later. Write down the inoculation date as well as the interpretation date. When you run these tests on your unknowns, you will need to write down the dates.
* at 24 hours
29
How to preform an oxidation fermentation test
* Obtain four tubes of O-F glucose.
* In two tubes, inoculate using E. coli.
* In the other tubes, inoculate using Pseudomonas.
* Take one tube of E. coli and one tube of Pseudomonas. Set these aside. They will serve as the aerobic environment.
* Take the remaining two tubes (one of E. coli and one of Pseudomonas) and apply a layer of mineral oil. YOU DON’T NEED A LOT, JUST ENOUGH TO COVER THE SURFACE. These will serve as the anaerobic environment.
30
How do you preform a sugar fermentation test?
* \*\*Check at 24 hours\*\*
* Glucose Fermentation \*\*\*Note – this test is performed and interpreted the same for ALL sugar broths.
* Obtain three test tubes of this broth containing this sugar and Phenol Red
* Inoculate the tubes with E. coli, Proteus mirabilis, and Pseudomonas. Aeruginosa. One bacterium per tube.
31
How do you preform a MRVP test?
This broth is tested after the 24-48 hour incubation period, then again several days later if the initial results are negative.
Obtain two MR broth tubes. #Note: the broth may be labeled as MRVP.
Inoculate one tube with E. coli and the other tube with Enterobacter aerogenes
32
How do you preform a catalase test?
Obtain one NA plate, divide in half.
One one half, inoculate with Staphylococcus aureus. and Enterococcus faecaelis on the other half. A simple streak is fine.
After incubation add hydrogen peroxide
33
How do you preform an Oxidase test?
Obtain one NA plate, divide in half.
One one half, inoculate with Pseudomonas. and E. coli on the other half. A simple streak is fine.
Add dimethyl-p-phenylenediamine hydrochloride
34
How do you preform a Nitrate reduction test?
Obtain three tubes of nitrate broth.
Inoculate one tube with Pseudomonas, another tube with Enterobacter aerogenes, and the other tube with Enterococcus faecaelis.
add sulfanilic acid and dimethyl-a-napthylamine to tubes
35
Test type: OF test
* Indicator or substrate: bromthymol blue
* Positive result: yellow
* Negative result: green
* looks for a biochemical pathway
36
Test type: Sugar Fermentation
* Indicator or substrate: Phenol red
* Positive result: yellow
* Negative result: orange
37
Test type: MR
* Indicator or substrate: methyl red
* Positive result: Red
* Negative test result: orange or yellow
38
Test type: Catalase
* Indicator or substrate: Hydrogen peroxide
* Positive: bubbles
* Negative: no bubbles
* Enzyme required for a positive result: catalase
39
Test type: Oxidase
* Indicator or substrate: Oxidase reagent
* Positive result: Borders of colonies are purple
* Negative result: no purple color
* Enzyme required for a positive result: Cytochrome c oxidase
40
Test type: Nitrate reduction
* Indicator or substrate: Sulfanilic acid and dimethyl-a-napthylamine
* Positive result: red and gas, does not turn red after adding sulfanilic acid and dimethyl-a-napthylamine or after adding zinc.
* Negative: No gas, not red after adding sulfanilic acid and dimethyl-a-napthylamine, but turns red with addition of zinc.
* Enzyme needed for positive result: nitratase
41
42
Nutrient Utilization test
* Simmon's Citrate test
43
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.
44
Amino Acid Tests
* Decarboxylation Test
* Phenyalanine Deamination
* Tryptophan (inndole) test
45
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.
46
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.
47
Tryptophan (indole) test
* Some bacteria have an enzyme, **tryptophanase**, **which can hydrolyze the amino acid** t**ryptophan 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.
48
How do you preform a Lysine decarboxylase test?
Lysine Decarboxylase: Note – the test is performed the same for the examination of Ornithine or Arginine as well.
Obtain two tubes of the lysine decarboxylase broth.
Inoculate one tube with Enterobacter aerogenes and the other tube with Proteus mirabilis.
Apply a layer of mineral oil. YOU DON’T NEED A LOT, JUST ENOUGH TO COVER THE SURFACE.
49
How do you preform a simmons citrate stab and streak?
Obtain two tubes of Simmons Citrate slants.
Inoculate using E. coli and Enterobacter aerogenes. \*\*\*\*\*\*\*\*\*\*\*\*This test requires a streak AND a stab.\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*
50
How do you preform a Phenylalanine Deaminase test?
Obtain two phenylalanine agar slants.
Inoculate using Proteus mirabilis and E. coli.
51
How do you preform a Tryptophan hydrolysis (indole) test?
Obtain two tubes of tryptone broth.
Inoculate using E. coli and Enterobacter aerogenes
52
Hydrolysis
a chemical reaction in which a substance is broken down by exoenzymes, and the remaining components are imported for further metabolism by the bacteria. The ability (or lack thereof) to hydrolyze particular molecules can be indicative of the genus and species.
53
Hydrolytic enzyme tests
* Bile Esculin Test
* Starch hydrolysis test
* Urea hydrolysis test
* ONPG Test
* Casein Hydrolysis test
* Gelatin hydrolysis test
54
Combination tests
* Kligler's Iron agar (KIA) test
55
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**
56
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
57
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.
58
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**.
59
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.
60
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.
61
Bile Esculin test set up
Obtain two slants for bile esculin.
In one slant, inoculate using Enterobacter aerogenes and in the other slant, inoculate using E. coli.
62
Starch Hydrolysis Test set up
Obtain one starch plate. Divide the plate in half.
One one half streak using Bacillus sp. (but not Bacillus sphaericus) and on the other half streak using E. coli.
63
Urea Hydrolysis/Urease test set up
Obtain two tubes of sterile water, 3 ml is sufficient.
Inoculate using Proteus mirabilis and Enterobacter aerogenes.
Add to each tube one Urease disk. Found in the north refrigerator, in the door.
64
ONPG test set up
Obtain two tubes of ONPG broth.
Inoculate one tube with Proteus mirabilis and the other tube with Enterobacter aerogenes.
After adding the bacteria, add a single ONPG disk to each tube.
65
Casein Hydrolysis test set up
Obtain two tubes of Litmus Milk broth OR Skim Milk agar.
Inoculate one tube with Bacillus cereus and the other tube with E. coli.
66
Gelatin Hydrolysis test set up
Obtain two tubes of Gelatin.
Inoculate one tube with Bacillus ceres and the other tube with E. coli.
67
KIA (Kligler's Iron agar) test set up
Obtain four KIA slants
Inoculate the tubes with E. coli, Proteus mirabilis, Pseudomonas aeruginosa and Shigella sonnei. One bacterium per tube. \*This test requires a streak AND a stab.\*\*\*\*\*\*\*
