Fish disease diagnosis through Microbiological diagnosis method (Biochemical tests)
Aim: To diagnose the fish disease by biochemical tests.
Various biochemical tests are used for the identification of bacteria based on the character of particular bacteria to give its positive and negative result for a particular biochemical test.
1. The Indole Test
The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan. Indole positive bacteria such as Escherichia coli produce tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products. When Kovac's reagent (p-dimethylaminobenzaldehyde) is added to a broth with indole in it, a dark pink color develops. The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits its growth.
2. The Methyl Red and Voges-Proskauer Tests
The methyl red (MR) and Voges-Proskauer (VP) tests are read from a single inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes. One tube is used for the MR test; the other is used for the VP test. Media contains glucose and peptone. All enterics oxidize glucose for energy; however, the end products vary depending on bacterial enzymes. Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose. E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test). Klebsiella and Enterobacter produce more neutral products from glucose (e.g., ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. At this pH, methyl red indicator is a yellow color (a negative MR test).
The reagents used for the VP test are Barritt's A (alpha-naphthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30 minutes to develop.
3. Catalase Test
Catalase is the enzyme that breaks hydrogen peroxide (H2O2) into H2O and O2. Hydrogen peroxide is often used as a topical disinfectant in wounds, and the bubbling that is seen is due to the evolution of O2 gas. H2O2 is a potent oxidizing agent that can wreak havoc in a cell; because of this, any cell that uses O2 or can live in the presence of O2 must have a way to get rid of the peroxide. One of those ways is to make catalase.
4. The Citrate Test
The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's (around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.
5. Oxidase Test
The oxidase test identifies organisms that produce the enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electrons from a donor molecule to oxygen. The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds.
6. Nitrate Reduction Test
Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. Nitrate broth contains nutrients and potassium nitrate as a source of nitrate. After incubating the nitrate broth, add a dropperful of sulfanilic acid and a-naphthylamine. If the organism has reduced nitrate to nitrite, the nitrites in the medium will form nitrous acid. When Sulfanilic acid is added, it will react with the nitrous acid to produce diazotized Sulfanilic acid. This reacts with the a-naphthylamine to form a red-colored compound. Therefore, if the medium turns red after the addition of the nitrate reagents, it is considered a positive result for nitrate reduction. If the medium does not turn red after the addition of the reagents, it can mean that the organism was unable to reduce the nitrate, or it could mean that the organism was able to denitrify the nitrate or nitrite to produce ammonia or molecular nitrogen. Therefore, another step is needed in the test. If the medium does not turn red after the addition of the nitrate reagents, add a small amount of powdered zinc. Be careful, as powdered zinc is hazardous! If the tube turns red after the addition of the zinc, it means that unreduced nitrate was present. Therefore, a red color on the second step is a negative result. The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous acid, which reacted with Sulfanilic acid. The diazotized Sulfanilic acid that was thereby produced reacted with the a-naphthylamine to create the red complex. If the medium does not turn red after the addition of the zinc powder, then the result is called a positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite present in the medium, either that means that denitrification took place and ammonia or molecular nitrogen were formed.
7. Urease Test
Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme, called urease that hydrolyzes urea to ammonia and carbon dioxide. The broth contains two pH buffers, urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red. Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. If the urea in the broth is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink. Many enterics can hydrolyze urea; however, only a few can degrade urea rapidly. These are known as "rapid urease-positive" organisms. Members of the genus Proteus are included among these organisms. Urea broth is formulated to test for rapid urease-positive organisms. The restrictive amount of nutrients coupled with the use of pH buffers prevent all but rapid urease-positive organisms from producing enough ammonia to turn the phenol red, pink.
8. Phenol Red Broth
Phenol Red Broth is a general-purpose differential test medium typically used to differentiate gram negative enteric bacteria. It contains peptone, phenol red (a pH indicator), a Durham tube, and one carbohydrate. We use three different kinds of phenol red broths. One contains glucose; one contains lactose, and the last contains sucrose. The objective of the exercise is to determine which organisms can utilize each sugar. Phenol red is a pH indicator which turns yellow below a pH of 6.8 and fuchsia above a pH of 7.4. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. If the organism is unable to utilize the carbohydrate but does use the peptone, the by-product is ammonia, which raises the pH of the media and turns it fuchsia. When the organism is able to use the carbohydrate, a gas by-product may be produced. If it is, an air bubble will be trapped inside the Durham tube. If the organism is unable to utilize the carbohydrate, gas will not be produced, and no air bubble will be formed.
9. Casease Test
Skim milk agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called casease, that hydrolyzes casein. Casein forms an opaque suspension in milk that makes the milk appear white. Casease allows the organisms that produce it to break down casein into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. When casein is broken down into these component molecules, it is no longer white. If an organism can break down casein, a clear halo will appear around the areas where the organism has grown.
10. Gelatinase Test
Nutrient gelatin is a differential medium that tests the ability of an organism to produce an exoenzyme, called gelatinase, which hydrolyzes gelatin. Gelatin is commonly known as a component of gelled salads and some desserts, but it's actually a protein derived from connective tissue. When gelatin is at a temperature below 32°C (or within a few degrees thereof), it is a semisolid material. At temperatures above 32°C, it is a viscous liquid. Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell membrane and be utilized by the organism. When gelatin is broken down, it can no longer solidify. If an organism can break down gelatin, the areas where the organism has grown will remain liquid even if the gelatin is refrigerated.
11. Lipase Test
Tributyrin agar is a differential medium that tests the ability of an organism to produce an exoenzyme, called lipase, which hydrolyzes tributyrin oil. Lipases break down lipids (fats). Tributyrin oil is a type of lipid called a triglyceride. Other lipase tests use different fat sources such as corn oil, olive oil, peanut oil, egg yolk, and soybean oil. Lipase allows the organisms that produce it to break down lipids into smaller fragments. Triglycerides are composed of glycerol and three fatty acids. These get broken apart and may be converted into a variety of end-products that can be used by the cell in energy production or other processes. Tributyrin oil forms an opaque suspension in the agar. When an organism produces lipase and breaks down the tributyrin, a clear halo surrounds the areas where the lipase-producing organism has grown.
12. Starch Hydrolysis
Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, which hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.
13. Triple Sugar Iron Agar
Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a small amount of glucose (dextrose), ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates. As with the phenol red fermentation broths, if an organism can ferment any of the three sugars present in the medium, the medium will turn yellow. If an organism can only ferment dextrose, the small amount of dextrose in the medium is used by the organism within the first ten hours of incubation. After that time, the reaction that produced acid reverts in the aerobic areas of the slant, and the medium in those areas turns red, indicating alkaline conditions. The anaerobic areas of the slant, such as the butt, will not revert to an alkaline state, and they will remain yellow. This happens with Salmonella and Shigella
14. Decarboxylation Test
Decarboxylase broth tests for the production of the enzyme decarboxylase, which removes the carboxyl group from an amino acid. Decarboxylase broth contains nutrients, dextrose (a fermentable carbohydrate), pyridoxal (an enzyme cofactor for decarboxylase), and the pH indicators bromocresol purple and cresol red. Bromocresol purple turns purple at an alkaline pH and turns yellow at an acidic pH. We also add a single amino acid to each batch of decarboxylase broth. The three amino acids we test in our decarboxylase media are arginine, lysine, and ornithine. The decarboxylase test is useful for differentiating the Enterobacteriaceae.Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three different but very similar media. If the organism is unable to ferment dextrose, there will be no color change in the medium. If an organism is able to ferment the dextrose, acidic byproducts are formed, and the media turns yellow. As the organisms ferment the dextrose, the media initially turns yellow, even when it has been inoculated with a decarboxylase-positive organism. The low pH and the presence of the amino acid will cause the organism to begin decarboxylation. If an organism is able to decarboxylate the amino acid present in the medium, alkaline byproducts are then produced. Arginine is hydrolyzed to ornithine and is then decarboxylated. Ornithine decarboxylation yields putrescine. Lysine decarboxylation results in cadaverine. These byproducts are sufficient to raise the pH of the media so that the broth turns purple. If the inoculated medium is yellow, or if there is no color change, the organism is decarboxylase-negative for that amino acid. If the medium turns purple, the organism is decarboxylase-positive for that amino acid.
15. Coagulase Test
The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents. The coagulase slide test is used to identify the presence of bound coagulase or clumping factor, which is attached to the cell walls of the bacteria. Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate. This causes the cells to agglutinate, or clump together, which creates the "lumpy" look of a positive coagulase slide test. You may need to place the slide over a light box to observe the clumping of cells in the plasma. The coagulase tube test has been set up as a demo for you to observe in class. This version of the coagulase test is used to identify the presence of either bound coagulase or free coagulase, which is an extracellular enzyme. Free coagulase reacts with a component of plasma called coagulase-reacting factor. The result is to cause the plasma to coagulate. In the demo, the coagulase plasma has been inoculated with Staphylococcus aureus and Staphylococcus epidermidis and allowed to incubate at 37°C for 24 hours. Staphylococcus aureus produces free coagulase; Staphylococcus epidermidis does not. The coagulase test is useful for differentiating potentially pathogenic Staphylococci such as Staphylococcus aureus from other Gram positive, catalase-positive cocci.
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