The purpose of this lab is to use staining techniques and biochemical testing to identify an unknown bacteria using Bergey’s manual. Bergey’s manual of Systematic Bacteriology is a dichotomous key primarily used to identify a bacterial species. Biochemical tests are used to differentiate different species of bacteria. These tests are effective in determining the characteristics of the microbe being tested. Such characteristics include citrate utilization, gelatin hydrolysis, nitrate reduction, etc. Biochemical testing is very important in the medical field when a patient is infected with an unknown microbe.
Such testing can identify the microbe(s) and types of medications effective in treating the infection. It is also useful in testing cleaning products to determine the most effective method in disinfecting objects and surfaces prone to different types of microbes that are likely present. Materials and Methods: A streak plate is a technique used to isolate a pure culture from a mixture of bacteria. This is the first step in identifying a particular species within a mixture of species. Materials needed include a petri plate containing growth agar, mixture of bacteria, and an inoculating loop.
A gram stain is used to determine the cell shape, arrangement, and size of the bacteria and whether it is gram-positive or gram-negative. Gram-positive bacteria are stained purple, while gram-negative are stained red. Materials for this technique include a slide with a fixed smear of bacteria, crystal violet stain, Gram’s iodine, 95% ethanol, safranin, a staining rack, distilled or deionized water, bibulous paper, and a clothespin. If the bacterium is gram-positive, an endospore stain is preformed to determine if the bacteria produces endospores.
Endospores are able to survive in unfavorable growth conditions. Materials include a slide with a fixed smear of bacteria, malachite green stain, safranin stain, a staining rack, a hot plate, steaming deionized water in a 100 ml beaker, filter paper, distilled or deionized water, bibulous paper, and a clothespin. An oxidase test is used to determine if the bacteria have cytochrome oxidase. Cytochrome oxidase collects electrons at the end of the electron transport chain and facilitates their molecular oxygen.
A positive test results in a dark blue to purple color appearing within seconds. This identifies reduction because the cytochrome oxidase adds electrons to the oxidase reagent. The materials needed include oxidase reagent, a sterile wood stick, and a pure culture of bacteria on an agar surface. A catalase test detects the presence of the enzyme catalase which degrades hydrogen peroxide. Bacteria that require gas for metabolism or tolerate oxygen use catalase to break down hydrogen peroxide. A positive catalase test results in the appearance of oxygen bubbles.
Materials needed are 3% hydrogen peroxide solution, Pasteur pipette and bulb, pure culture of bacteria on an agar surface, an inoculating loop, and a clean microscope slide. The thioglycollate test determines aerotolerance. Thioglycollate broth cultivates obligate anaerobs, bacteria that cannot survive in the presence of oxygen. Thiglycollate is a reducing agent that reacts with oxygen to form water. An indicator dye changes color where oxygen has dissolved into the broth. If the dye is pink or blue oxygen is present and if it is colorless oxygen is absent.
To preform this test a thioglycollate broth tube, and a bacterial culture in broth or on an agar surface is needed. The nitrate reduction test determines if the bacteria are able to reduce nitrate ions to either nitrite ions or to nitrogen gas. In anaerobic respiration the final electron acceptor is the nitrate ion. Nitrate reductase, an enzyme complex, facilitates the reduction of the nitrate ion. Some bacteria use denitrification which uses nitrate reduction during anaerobic conditions of energy production are able to reduce nitrate completely to molecular nitrogen.
Reagent A (sulfanilic acid) and reacgent B (dimethyl-a-naphthylamine) are added to the nitrate broth. A positive result turns pink or red because nitrite is present. The materials needed include a nitrate broth tube, nitrate reagent A, nitrate reagent B, powdered zinc, a wooden stick, disposable gloves, and a pure culture of bacteria. The phenol red test is used to determine the ability of bacteria to ferment a particular carbohydrate. The carbohydrates used included glucose (dextrose), lactose, and mannitol.
A positive test results in the production of acid indicated by the change in color of the phenol red, a pH indicator, from red to yellow. A Durham tube is used to collet gas that is produced. Materials needed include a tube of phenol red-glucose (dextrose), a tube of phenol red-lactose, a tube of phenol red-mannitol, and a pure culture of bacteria. The methyl red test is used to determine the ability of bacteria to ferment glucose by mixed-acid fermentation. Mixed-acid fermentation of glucose produces significant amounts of organic acids.
A positive test is indicated by the pH indicator, methyl red, remains the color red. Materials include methyl redNoges-Proskaur (MR-VP) broth tube, a pure culture of bacteria, methyl red, and a Pasteur pipette and bulb. The Voges-Proskaur test determines the ability of the bacteria to ferment glucose by butanediol fermentation. The butanediol fermentation pathway which produces organic acids, but they are not present in significant amounts after 48 hours of incubation. A positive (VP) test produces a red color when VP reagents react with acetoin in the presence of oxygen.
Citrate utilization determines if bacteria can use citrate as its only source of carbon and energy. The bacteria must posses of the membrane-associated transporter citrate permease to utilize citrate. The byproduct of citrate utilization is alkaline. A positive test results in a color change of the medium from green to blue. Materials needed include simmons citrate agar slant tube, and a pure culture of bacteria. The starch hydrolysis test is used to determine the ability to hydrolyze starch. Bacteria use amylase, an extracellular enzyme to hydrolyze bonds linking the glucose subunits.
Starch is detected by adding iodine to the medium. If the bacteria growing on the starch agar produce amylase, no color change will be seen around the bacterial growth because all of the starch is consumed. The absence of a color change is a positive test. Materials needed to perform this test include a starch agar plate, Gram’s iodine, and a pure culture of bacteria. The casein hydrolysis test determines the ability of bacteria to hydrolyze casein. Casein is the predominant protein in milk and can be used as a substrate to assess the production of proteinases.
If bacteria secrete caseinase, a specific protease, casein molecules are digested in the area around the bacterial growth is clear. A clear zone is a positive test. Materials needed include a skim milk agar plate, and a pure culture of bacteria. The Gelatin hydrolysis test determines whether a bacteria is able to hydrolyze gelatin or not. Gelatinase is an extracellular enzyme that some bacteria can hydrolyze gelatin with. Liquefaction is a positive test. The liquefaction of gelatin is caused by gelatinase cleaving to multiple peptide bonds in gelatin.
Materials needed include a nutrient gelatin deep tube, a pure culture of bacteria, and a refrigerator. The indole hydrolysis test determines the ability of bacteria to split tryptophan, an amino acid, into indole and pyruvic acid. Some bacteria can use tryptophan as a source of energy by degrading the amino acid to get pyruvate and indole is a byproduct that is not used. The presence of indole is indicated by a red layer at the top of the tube and is a positive test. Materials needed are a SIM (sulfide-indole-motility) agar deep, a pure culture of bacteria, Kovac’s reagent, and disposable gloves.
The hydrogen sulfide production test is used to determine if bacteria can dismantle cysteine, an amino acid, or reduce thiosulfate and form hydrogen sulfide as a byproduct. Some bacteria use cysteine desulfurase, an enzyme, to break down cysteine which is a sulfur-containing amino acid to form pyruvate. Pyruvate is used for energy. Hydrogen sulfide is a byproduct which is not used by the bacteria. The blackening of the agar is a positive test and detects hydrogen sulfide production. Materials needed include a SIM deep tube, and a pure culture of bacteria.
The motility test determines if the bacteria are motile. When motile bacteria are stabbed with an inoculating needle into a semisolid agar, they move away from the stab which is indicated by a cloud of growth extending from the stab. This is a positive test. Materials needed are a SIM tube, and inoculating needle, and a pure culture of bacteria. A hanging-drop slide can also be used to determine motility. Materials needed include a clean depression microscope slide, a clean coverslip, petroleum jelly, an inoculating loop, and a bacterial culture.
This method, however, has a higher risk of environmental contamination. The urea hydrolysis test determines if a bacterium can hydrolyze urea. Some bacteria use the enzyme urease to degrade urea into carbon dioxide and ammonia. The appearance of bright pink is a positive test for a ureaseproducing bacteria. Materials include a urea-containing medium, and a pure culture of bacteria. The litmus milk reactions are used to determin whether bacteria have the ability to utilize lactose, protein, and litmus or not. The litmus serves as a pH indicator which turns pink in the presence of acid and blue in alkaline.
If sufficient acid is produced, casein will be denatured and form a firm curd and whey which is a clear, watery fluid at the top of the curd. Some bacteria that form this acid curd will reduce litmus to a colorless compound and appear white, which begins at the bottom of the tube. A soft curd can form when casein is altered by the enzyme renin and the color of the litmus does not change. Medium that is clear results from prteolytic bacteria that completely digest the curd or insoluble casein. Materials needed include a litmus milk tube and a pure culture of bacteria.
