Biochemical Testing Handout

As you guys know, the purpose of a medical microbiology laboratory is to mainly isolate and identify organisms to provide proper treatment. For this week we will focus on five biochemical tests.

1) Kligler iron agar- Please do not confuse this test with the TSI
when you are looking online (very similar, but one carbohydrate difference). This test is used to differentiate bacteria based on glucose and lactose fermentation, sulfur reduction, and gas production.

The media contains peptones, so if the organism runs out of carbohydrates to ferment (.1% glucose and 1% lactose), it can switch to deamination of peptones raising the pH. This makes it a very time sensitive test (observe at 18-24 hours). So how does it work? Since you have a very small amount of carbohydrate in the medium, the organism will keep on fermenting, producing acidic end products, which in turn will lower the pH, which will cause the pH indicator to turn yellow, but eventually the organism will run out of its preferred food. Some organisms have the ability of switching to peptones as their food source. Peptones are digested proteins. This process of utilizing peptones is known as deamination. This means the organism will use the protein, but first it will remove the amino group, which is basic. This accumulation of amino groups will cause the pH to rise and in turn, make the pH indicator turn pink or light red. This will give you a false-negative result. This is known as reversion. The pH indicator is phenol red. The hydrogen sulfide indicator is ferrous ammonium sulfate. The design of this tube provides both an aerobic and anaerobic environment (one of which is necessary for sulfur reduction). The other property of this test is that it contains sodium thiosulfate as a source of sulfur for organisms. If an organism is able to reduce sodium thiosulfate it will produce hydrogen sulfide

(H2S), which is a gas. This is that rotten egg smell that you recognize. The problem here is that H2S is colorless so we need an indicator to be able to see if this reaction occurred. This is where ferrous ammonium sulfate (some can use ferrous ammonium citrate) comes in. This indicator will immediately react with the gas produced and form a black precipitate. Since sulfur reduction requires a fully anaerobic environment it can only happen in the butt of the tube. So if you see black on the slant it is not H2S. Also, when you see a black butt in the tube you automatically assume glucose fermentation. The reason for that is because apart from the anaerobic environment, sulfur reduction also requires an acidic environment. So acid from glucose fermentation sets the stage for sulfur reduction. The last property of this test is the ability to detect gas production. When organisms ferment a carbohydrate they will sometimes produce gas (I say sometimes, because sometimes the gas produced is minimal and no change is observed and other times no gas is produced. This phenomenon is something to keep in mind when trying to identify or eliminate an organism based on gas production alone). They will produce enough gas to cause cracks and bubbles in the medium. Things to remember about this test: 1. 2. 3. 4. 5. We always read this test from top to bottom. K stands for alkaline and A stands for acidic. Black covers yellow H2S does NOT cause the gaps (H2S only in butt) Gas from carbohydrate fermentation is NOT black

2) Catalase test- This test is known as the great divider of Gram

positive cocci. This is because organisms from the genus Staphylococcus are catalase positive, while organisms from the genus Streptococcus are catalase negative. This test looks at the ability of organisms to respire, which is the conversion of glucose to ATP using glycolysis, the Krebs cycle, and oxidative phosphorylation. Respiration can be: Aerobic- reduces oxygen to water (oxygen is the final electron acceptor during the ETC). Anaerobic- reduces inorganic molecules like NO3 or SO4 to N2, NO2 and H2S. Catalase is an enzyme used to detoxify the cell by converting harmful by-products like H2O2 from respiration to water and oxygen. This is because not only is hydrogen peroxide a common by-product of respiration, but also the chemical used by phagocytic cells to destroy microbes once they are engulfed. Some organisms also carry the enzyme superoxide dismutase, which is similar to catalase, but it catalyzes the conversion of superoxide (another harmful by-product) into oxygen and hydrogen peroxide. We cant use a broth culture because it will provide skewed results. Solid culture MUST be used for this test.

3) SIM (Sulfide, Indole, and motility)- This is probably one of

the most informative tests, especially when dealing with Gram negatives. The test belongs to a group of tests known as IMViC. This stands for Indole, Methyl red, Voges Proskauer, and Citrate and combined these tests are used in the identification of organisms belonging to the Enterobacteriaceae. This test is only differential and semi-solid. It takes advantage of the ability of some organisms to be able to reduce sulfur compounds into sulfide, catalyze the conversion of tryptophan into indole and pyruvate, and be able to move throughout the medium (motility). So how does it work? Some organisms are able to reduce sodium thiosulfate (in the medium) into hydrogen sulfide (H2S (g)) using either thiosulfate reductase or cysteine desulfurase. After the reduction a similar reaction as the one seen in the KIA slant occurs. Hydrogen sulfide gas will immediately react with ferrous ammonium sulfate to create FeS (ferric sulfide), a black precipitate. Tryptophan is an amino acid added to the medium. This is helpful because some organisms have the ability to hydrolyse or break down the amino acid into indole, ammonia, and pyruvate using the enzyme tryptophanase. We can detect if ther eaction occurred by the addition of Kovacs reagent (a carcinogen so wear gloves!) which will react with indole turning a bright pink color at the top of the tube. NOTE: add the Kovacs once you have checked for H2S production and motility. Motility is a very important ability in organisms. It is able to enhance the pathogenicity of an organism, but it is not necessary for it (EX: Klebsiella pneumoniae and Shigella sonnei). The fact that the test is semi-solid allows us to test for motility. If the tube is completely cloudy (growth away from the line of inoculation) then you can assume motility. It is tricky to read, but it can go a long way if you do it correctly. If the tube is completely black (due to Ferric sulfide in the medium) then motility is very easy to read because if FeS is everywhere then the organism is moving around the medium and reducing sodium thiosulfate as it goes along.

4) Urea hydrolysis test- Urea is a waste molecule produced from

the urea cycle. In this cycle, the molecule is made by the decarboxylation of arginine (and sometimes other amino acids). Decarboxylation is just a fancy way of saying that the carboxyl group has been removed from an amino acid. If urea is not eliminated (different organisms have different ways of removing it. Urine is one way. Birds use uric acid crystals), then it can become toxic and damage the or ganism. Below is the structure of urea:

It is helpful to remember the structure because it can help you remember the products of the hydrolysis reaction. Some organisms carry the enzyme urease, which will catalyze the following reaction:

Reaction is hydrolize - products of hydrolysis learn chart above

Notice that each amino group will get one hydrogen from the water molecule used to split it (giving you 2NH3) and the oxygen will go to the carbon (giving you CO2). We can use this reaction to our advantage because in the tube we have the pH indicator phenol red. If you recall from the KIA explanation, phenol red will be yellow under acidic conditions and pink or bright pink under alkaline conditions. In this case the ammonia being produced will rise the pH and turn the phenol red bright pink (hot pink). This is a positive reaction. Yellow is negative and orange is no reaction. The test should be observed at 18-24 hours if you have a Gram positive cocci. If the test is orange you may keep it and check it every 24 hours until you reach 96 hours, but once the test turns either yellow or bright pink you are done. On top of the ability to hydrolyze urea, some organisms can rapidly hydrolyze urea. Rapid urea hydrolysis is commonly a characteristic of

organisms from the genus Helicobacter, Morganella, Proteus, and Providencia. If your tube turns pink at 24 hours (P. vulgaris does it in about 6 hours) then you can conclude rapid hydrolysis. If this happens at 48 hours or above it is slow. The fact that Helicobacter pylori is a rapid urea hydrolyzer is a very important ability because we use the urea breath test to check for stomach ulcers. The carbon in the urea molecule is radiolabeled and the patient drinks it (dont worry, this is pure urea, not urine). In about 24 hours the patient comes back and breathes into a machine that will be able to detect CO2. If the radiolabeled CO2 is detected then something hydrolyzed urea in the stomach. This is an accurate diagnosis of an H. pylori infection.

5) Phenol red broth- Also each tube only has one type of carbohydrate).
Looking for fermentation reaction and gas production. Sugars fermentation makes acid makes phenol red turn yellow. After 24 hours the organism will run out of the carbohydrate. It will switch to peptones cause the breakdown of amino acids known as deamination which will then raise the PH causing it to turn red. When all the sugars have been used the organisms This test is differential only. This process usually involves the following steps: Step One: Carbohydrate Glucose Step Two: Glucose Pyruvate Step Three: Pyruvate Fermentation By-products Phenol-Red Carbohydrate Fermentation Broths are useful for helping to characterize bacteria based on their fermentation abilities. Each PR-Carb broth contains the following: Carbohydrate Each broth contains a single fermentable carbohydrate. There are other organic nutrients (such as amino acids) that are not fermentable but can be used for growth. Phenol-Red is a pH indicator that is RED at pH 7 or higher (alkaline) but turns YELLOW at low pH (acidic). The broth is initially pH neutral. So, if fermentation occurs, and acid by-products are formed, the solution will turn yellow. Durhamn Tube This is a small test tube that is inverted (upside-down) in the broth. If gases are produced, they will be trapped in the Durham Tube and a gas bubble will form. Thus, two things can be determined from a PR-Carb broth. First, can the species ferment the carbohydrate or not? Second, if the species can ferment the carb, is it a gas-producing kind of fermentation? One word of caution: if PR-Carb broths are incubated for too long (more than 24hrs ), acids may be further metabolized and converted back to neutrality or even alkalinity. In these situations, a yellow broth will turn back to red. This test is time sensitive 18-24 hr due to reversion. Change back to yellow. Remember what that is? Ammonia is basic so when they eat the amino acid making the medium basic making the phenol red broth pink knows as deamination. This test is used to differentiate members of the Enterobacteriaceae and to distinguish them from other Gram negative rods.

1. Why does the formation of yellow color indicate fermentation? Yellow means the organism can ferment the sugar increasing the acidity. 2. What can go wrong during the culturing process to lead to erroneous results? You can leave it sit too long causing it to revert showing a false negative result. 3. Even if everything is performed correctly, sometimes observed results do not match expectation. Give some reasons why this might happen. 4. Suppose you have a culture of unknown bacteria. Describe how you would use a PRCarb broth (or several) to help identify your unknown.

1) Citrate Utilization test- This test is part of the IMViC group of

tests used for members of the Enterobacteriaceae. It has the molecule citrate as the sole source of carbon and ammonium phosphate as the sole source of nitrogen. Since we know that citrate is the only source of carbon, this medium is known as a highly defined medium. The pH indicator is Bromthymol blue. The tube itself is a slant and its called Simmons citrate agar slant. If the organism is able to utilize citrate, it will also use the ammonium phosphate, which will alkalinize the medium and turn it blue. The enzymes of interest here are citrate-permease, which allows the organism to transport the molecules into the cell and convert them into pyruvate using citrase. When inoculating this test you must use a needle. The reason for that is that since this is a defined medium, we only want the nutrients in the slant. By using a loop sometimes you can introduce broth from another tube along with the organism and that can allow the organism to grow using the broth nutrients not the citrate. This test may be incubated up to 72 hours. Once it turns blue you can read it and you are done, but if you see growth and no blue color then you can incubate it longer or call it positive.

2) Coagulase- This test is used to identify Staphylococcus aureus

from other Gram positive staph. Although this is one of the enzymes that some members of the genus Streptococcus carry (several enzymes are similar in staph and strep organisms). S. aureus is an opportunistic pathogen that can be highly resistant to the immune system of the host and some antibiotics (MRSA and VRSA). It is resistant because it produces the enzyme coagulase. This causes normal plasma components to form a protective fibrin shield around an individual bacterium or a group of bacteria by catalyzing the conversion of fibrinogen to fibrin, which will then crosslink and form a polymer. This shield protects them from phagocytosis and other types of attack (i.e. antibiotics). The enzyme coagulase can be: 1. Bound- this is also called clumping factor. It is attached to the bacterial cell wall and reacts directly with fibrinogen in plasma. Causes cells to clump together 2. Free- this is an extracellular enzyme that reacts with a plasma component called coagulase-reacting factor. This is similar to the clotting mechanism in the body There are two tests designed for this. One is a tube test and the other one is a slide test. The tube tests for both bound and free coagulase and the slide only for bound. In reality, you should observe this test every 4 hours to make it more accurate because sometimes S. aureus produces the enzyme staphylokinase (streptokinase in Streptococci), which cause the clot to dissolve giving you a false negative.

3) Gelatin hydrolysis test- Gelatin is derived from collagen. It is

normally used to provide texture (gelling agent) to foods. Some organisms have the ability of hydrolyzing gelatin into amino acids using enzymes from the gelatinase family (not gelatinase twice as your book shows). Uses water molecule to break down the gelatin. If the organism carries the enzymes the gelatin will become liquid since gelatin is the only solidifying agent. Remember, gelatin will liquefy on its own at a few degrees above room temperature so be careful when reading it. Final product is amino acids Liquid is positive Solid is negative Incubate at 25 C Will melt at 28C

4) Methyl Red-Voges Proskauer test- This test is used as a

component of the IMViC battery of tests (Indole, Methyl red, VogesProskauer, and Citrate) used to distinguish members of the Enterobacteriaceae and differentiate them from other Gram negative rods. One tube incubate 5-7 days, then separate, MR and one tube VP These are two test performed in one tube with one type of broth (MRVP). This is a simple solution of peptone, glucose and a phosphate buffer to prevent harsh changes in pH. For the MR part of the test we want to see if the organism can have mixed acid fermentation. If it does, then the acidic produces will overcome the buffer and lower the pH. Methyl red is added as an indicator dye after incubation. Only red is an indication of a positive test. Orange is negative or inconclusive. Yellow is negative. For the VP part of the test we want to see if the organism can ferment the carbohydrate (glucose) releasing acid, but quickly convert it to acetoin and 2,3-butanediol (less acidic products). The VP reagents added react with these products and cause a change to the color red. This is positive. No change or copper color is a negative result.

5) Nitrate Reduction test- Many Gram negative organisms have

the enzyme nitrate reductase and perform a single step reduction of nitrate to nitrite (NO3 NO2). Other organisms use denitrification, which is a multi-step process that uses several enzymes to reduce nitrate into molecular nitrogen (NO3NO2NON2ON2). No color indicators are included in the medium itself and it is only a differential test. The color change is due to the reactions between metabolic products and reagents added after incubation The medium is tryptic nitrate broth. It is a semi-solid medium, which contains .1% Agar and KNO3. The semi-solid medium prevents oxygen from diffusing into the media providing an anaerobic environment, which is necessary for optimal nitrate reduction. Reagents: A = Sulfanilic acid (suspected carcinogen) (wear gloves) B = alpha naphtylamine (corrosive and poisonous) (wear gloves) C= zinc dust If you add: A+B red = positive for reduction by one step NO3NO2 using nitrate reductase A+B no color change. Add zinc = red. Negative. No NO3 reduction. Zinc reduces the NO3, not the organism A+B+zinc no color change. Positive for denitrification. End products are NH3 and N2. Zinc didnt react with NH3 or N2