CLINICAL BACTERIOLOGY LABORATORY IDENTIFICATION OF GRAM POSITIVE BACILLI AND GRAM NEGATIVE COCCI OUTLINE • Introduction • Tests for Identification of Gram Positive Bacilli o MRS Broth Test • Tests for Identification of Gram Negative Cocci o Butyrate Disk Test o Nitrate Reduction Test INTRODUCTION • Gram-positive bacteria retain the color of the primary stain (crystal violet) in the Gram staining procedure and appear as purple/violet under a light microscope. These bacteria have a cell wall containing a thick layer of peptidoglycan. On the basis of cell morphology, Gram-positive bacteria are divided mainly into two groups, “Gram-positive cocci” and “Gram-positive bacilli” • Gram-positive bacteria are bacteria classified by the color they turn in the staining method. Hans Christian Gram developed the staining method in 1884. Staining method uses crystal violet dye, which is retained by the thick peptidoglycan cell wall found in gram-positive organisms. • Neisseria species are non-spore-forming, oxidase-positive, nonmotile, gram-negative cocci (measuring approximately 0.8 μm by 0.6 μm) that usually appear as a biscuit- or kidney-shaped diplococci on smears of infected fluids. Since other organisms may appear similar morphologically, identification rests on biochemical and immunologic techniques. • Gram-Negative Coccobacilli. A coccobacillus is a type of bacterium with a shape intermediate between cocci and bacilli i.e., they are very short rods that may be mistaken for cocci. These small, pleomorphic gram-negative bacteria range in shape from round (cocci) to short, thin rods (bacilli); hence the bacteria are called “coccobacilli.” L1: TEST FOR IDENTIFICATION OF GRAM POSITIVE BACILLI MRS BROTH TEST • The MRS formulation was developed by de Man, Rogosa and Sharpe to replace a variable product (tomato juice) and, at the same time, provide a medium which would support good growth of lactobacilli in general. Hence, MRS agar and broth given to their superiority are commonly employed for culturing and identifying of Lactic acid bacteria especially the Lactobacillus spp. • “Lactic acid bacteria” includes species of the following genera: Lactobacillus, Streptococcus, Pediococcus and Leuconostoc. All these species can produce lactic acid in considerable amounts. • They are Gram-positive, catalase and oxidase negative and are fastidious in their nutritional requirements. Growth is enhanced considerably by microaerobic conditions. • MRS medium is selective for lactobacilli but some growth of leuconostocs and pediococci may occur. Selectivity can be altered by pH adjustment. Lactobacilli will tolerate lower pH levels than streptococci (pH 5.0-6.5) with pediococci and leuconostocs growing best within this range. OBJECTIVE • To determine whether an organism forms gas during glucose fermentation. PRINCIPLE • The MRS broth contains sources of carbon, nitrogen, and vitamins to support the growth of lactobacilli and other organisms. Enzymatic Digest of Animal Tissue, Beef Extract, and Yeast Extract are the carbon, nitrogen, and vitamin sources used to satisfy general growth requirements in Lactobacilli MRS Broth. Dextrose is the fermentable carbohydrate. Sodium Acetate is an inhibitory agent. Sodium Acetate and Ammonium Citrate act as selective agents as well as energy sources. Potassium Phosphate is the buffering agent. Magnesium Sulfate and Manganese Sulfate provide cations used in metabolism. Polysorbate 80 is a surfactant, facilitating uptake of nutrients by lactobacilli. • When an organism is inoculated into the broth medium, those that are able to ferment the dextrose sugar, overcome the selective agents and utilize other nutrients provided show luxuriant growth which is considered a positive result. Some organism may also be able to produce gas for which a Durham tube may be placed to differentiate such gas-producers from the rest of the orgnaisms (eg. Leuconostoc sp. from Lactobacillus spp.). EXPECTED RESULTS • Positive : Growth with marked turbidity of the broth, gas production indicated by a bubble in the Durham tube (Leuconostoc spp.) Growth resulting turbidity, no gas production (Lactobacillus spp) • Negative : No growth (no turbidity) or gas production • MRS broth. A, Positive; gas production by Leuconostoc sp.(Left). • B, Positive: growth, no gas production by Lactobacillus sp.(Right)
USES • It is used for the identification of some Lactobacillus spp. and Leuconostoc sp . which produce gas. • The test is a part of the confirmatory tests performed on organisms isolated on MRS Agar. • Commonly performed to identify Lactobacilli from oral cavity, dairy products, foods, faeces and other sources. L2: TESTS FOR IDENTIFICATION OF GRAM NEGATIVE COCCI BUTYRATE DISK TEST • Butyrate discs are employed in the microbiology lab for demonstrating the enzymatic hydrolysis of bromo-chloro-indolyl butyrate. • The Test Disc is used for rapid detection and qualitative procedures of butyrate esterase for presumptive identification of Moraxella catarrhalis. • Moraxella catarrhalis is recognized as a significant pathogen whose isolation from routine sputum cultures is associated with clinical infection. • The rapid identification of this organism is important, since most strains produce beta-lactamase and are resistant to penicillin and ampicillin. • Conventional methods of identification may take up to 48 hours to perform while the detection of an enzyme, butyrate esterase, is a rapid means of identifying Moraxella catarrhalis , which is performed using butyrate discs. OBJECTIVES • To detect the enzyme butyrate esteraste in microorganisms PRINCIPLE • Organisms capable of producing butyrate esterase hydrolyze bromochlorindolyl butyrate. Disks impregnated with bromo-chloro-indolyl butyrate serve as the substrate for the detection of butyrate esterase. Hydrolysis of the substrate in the presence of butyrate esterase releases indoxyl, which in the presence of oxygen spontaneously forms indigo, a blue to blue-violet color. PROCEDURE 1. Remove a disk from the vial and place on a glass microscope slide. 2. Add 1 drop of reagent-grade water. This should leave a slight excess of water on the disk. 3. Using a wooden applicator stick, rub a small amount of several colonies from an 18- to 24-hour pure culture onto the disk. 4. Incubate at room temperature for up to 5 minutes. EXPECTED RESULTS • Positive : Development of a blue color during the 5-minute incubation period. • Negative : No color change. USES • Aid in the identification of Moraxella (Branhamella) catarrhalis. • Helps to differentiate Neisseria gonorrhoeae(negative) and Moraxella catarrhalis(positive), which are both Oxidase positive, Gram negative diplococci. NITRATE REDUCTION TEST • Anaerobic metabolism requires an electron acceptor other than atmospheric oxygen (O2). Many gram-negative bacteria use nitrate as the final electron acceptor. • Nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate (NO3). • Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases. OBJECTIVES • To determine the ability of an organism to reduce nitrate to nitrite. • Identify the different ways that nitrate can be reduced by bacteria PRINCIPLE • A heavy inoculum of test organism is incubated in a broth containing nitrate. The organisms capable of producing the nitrate reductase enzyme reduce the nitrate, present in the broth, to nitrite which may then be further reduced to nitric oxide, nitrous oxide, or nitrogen. • The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye. • However, only when nitrate is present in the medium, red color will be produced. If there’s no red color in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium.
• There is two explanations for this observation. o The nitrate may not have been reduced; the strain is nitrate-negative. o The nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite; the strain is nitrate-positive. • Thus, when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. This can be done indirectly by adding small amount of Zinc powder to the culture. Zinc powder catalyses the reduction of nitrate to nitrite. The development of the red colour on addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate. If no color change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and thus is a nitrate reducer. • Note: A Durham tube is placed in the nitrogen broth in order to detect deterioration of the broth before inoculation, as evidenced by gas formation in the tube and to identify denitrification by organisms that produce gas by alternate pathways. METHOD • Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is determined by the addition of Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond nitrite is determined by the addition of Nitrate Reagent C (zinc dust). 1. Inoculate the nitrate broths with bacterial suspension. 2. Incubate the tubes at the optimal temperature 30°C or 37°C for 24 hours. 3. After incubation look for N2 gas first before adding reagents. 4. Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B. 5. Observe for the reaction (color development) within a minute or less. 6. If no color develops add zinc powder. 7. Observe for at least 3 minutes for a red color to develop after addition of zinc. EXPECTED RESULTS • Positive Test : o Development of a cherry red colouration on addition of reagent A and B o Absence of red color development on adding Zn powder • Negative Test : o A development of red color on addition of Zn powder USES • All members of the Enterobacteriaceae family reduce nitrate, but some members further metabolize nitrite to other compounds. It is thus used to differentiate members of Enterobacteriaceae that produce enzyme nitrate reductase from Gram negative bacteria that do not produce the enzyme nitrate reductase. • The reduction of nitrate may be coupled to anaerobic respiration in some species. • It is used in differentiating Mycobacterium • Identifying species of Neisseriaand separating them from Moraxella and Kingella The nitrate reduction test is a critical test for differentiating between N. gonorrhoeae and K. denitrificans, particularly when strains of K. denitrificans appear to be gram-negative diplococci in stained smears. • Facilitating species identification of Corynebacterium
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Clinical Bacteriology Lab - 06 Identification of Gram Positive Bacilli and Gram Negative Cocci