Clinical Bacteriology Lab: Identification of Gram-Negative Bacilli

CLINICAL BACTERIOLOGY LABORATORY IDENTIFICATION OF SMALL GRAM NEGATIVE NONFERMENTATIVE BACILLI PRINCIPLE • OUTLINE • Introduction • The X and V Factor Test o Principle o Expected Results o Lab Exercise • Oxidase Test o Principle o Purpose/Uses o Result Interpretation o Quality Control o Lab Exercise • Carbohydrate Fermentation Test o Principle o Result Interpretation o Uses o Lab Exercise • Other Tests for Identification of Small Gram-Negative NonFermentative Bacilli o Acetamide Utilization Test o Cetrimide Test o Growth at 42 Degrees Celcius o Oxidation-Fermentation Test • INTRODUCTION • • • • • Nonfermenting gram-negative bacilli (NFGNB), such as Pseudomonas aeruginosa, Acinetobacter baumanii and Steno-trophomonas maltophilia, are important nosocomial pathogens contributing significantly to morbidity and mortality, while others are being increasingly recognized as clinical pathogens. NFGNBs such as A. baumannii or P. aeruginosa have been implicated in nearly all kinds of infections, including bloodstream infections (BSI), pneumonia and meningitis. Clinical presentation of BSI due to A. baumannii or P. aeruginosa varies. It may range from benign transient bacteremia to fulminant septic shock with high mortality and is not different from other gram-negative bacteria. Most NFGNB have a high intrinsic resistance which makes them frequently resistant to the major classes ofantimicrobial agents, often leaving few therapeutic options. Several NFGNB are highly resistant to environmental conditions favoring epidemic spread. Members of the genus Haemophilus require accessory growth factors in vitro. Some Haemophilus spp. require X factor (hemin) alone, V factor (nicotinamide adenine dinucleotide [NAD]) alone, or a combination of the two. Consequently, this significant difference in growth factor requirements of Haemophilus spp. allows for their differentiation. In the laboratory, a lawn of the test organism is streaked onto heart infusion agar, tryptic soy agar, Haemophilus agar, or nutrient agar. The impregnated disks or strips (X, V, or XV) are placed directly on the confluent inoculation, allowing diffusion of the accessory growth factor into the medium. The organisms will grow only around the disk that provides the appropriate factor for growth of the organism ie. the presence of growth around the disc but not elsewhere on the plate indicates a requirement for that particular factor. Differentiation is hence based on the presence or absence of growth around and/or between disks impregnated with factors X, V and XV. EXPECTED RESULTS • • Positive: o Growth around the XV disk only shows a requirement for both factors. o Growth around the V disk, no growth around the X disk, and light growth around the XV disk shows a V factor requirement. Negative: o Growth over the entire surface of the agar indicates no requirement for either X or V factor. L1: THE X AND V FACTOR TEST • • • • • • Haemophilus spp are small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. A clinically important species of the genus, influenzaeis a fastidious organism which grows best at 35-37°C with ~5% CO2 and in the presence of special accessory growth factors called X and V factors. X factor comprises protoporphyrin IX, also called haemin or other iron-containing porphyrins. These are required for growth because X-dependent strains are unable to convert d-aminolaevulinic acid to protoporphyrin. They are heat stable. V factor comprises nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). They are heat labile. However, different species of Haemophilushave varying requirements for X and V growth factors. For example. Haemophilus parainfluenzaerequires V factor only for grow while Haemophilus ducreyi requires only X factor without need of V factor. LAB EXERCISE: X AND V FACTOR TEST • • Materials o PPE o Alcohol lamp o Prepared Chocolate Agar Plate o Gram’s stain o GC Agar Base II o slides o Horse blood o dropper o Taxo X and V o Distilled water o Oxidase reagent o Bacterial cultures Testing Discs o X – and V-Factor Disks are paper disks impregnated with growth factors. Each X-Factor Disk is impregnated with hemin. Each V-Factor Disk is impregnated with NAD (nicotinamide adenine dinucleotide). Each XV-Factor Disk is impregnated with a combination of hemin and NAD. • Procedure 1. Using aseptic technique select isolated colonies of suspect colonies and prepare a suspension equivalent to a 0.5 McFarland standard. 2. Dip a sterile swab into the organism suspension. Roll the swab over the entire surface of a trypticase soy agar plate. Inoculate by streaking in three different directions by rotating the plate in a 60 degree angle after each streaking. Streaking in this manner ensures confluent growth. 3. Allow the surface to dry 3-5 minutes. 4. Place the X, V, and XV factor disks on the agar surface. If using separate disks, place them at least 4 to 5 cm apart. 5. Incubate overnight at 35°-37°C. 6. Observe for growth or no growth around the disks. PURPOSE/USES • • • The oxidase test is used to determine if an organism possesses the cytochrome oxidase enzyme. The test is used as an aid for the differentiation of Neisseria, Moraxella, Campylobacter and Pasteurella species (oxidase positive). Also used to differentiate pseudomonads from related species. L2: OXIDASE TEST • • The oxidase test detects the presence of a cytochrome oxidase system that will catalyse the transport of electrons between electron donors in the bacteria and a redox dyetetramethyl-p-phenylene-diamine. The dye is reduced to deep purple color. This test is used to assist in the identification of Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of which produce the enzyme cytochrome oxidase. A number of reagents can be used for this test. o Kovacs Oxidase Reagent: 1% tetra-methyl-pphenylenediamine dihydrochloride, in water o Gordon and McLeod’s Reagent: 1% dimethyl-pphenylenediamine dihydrochloride, in water o Gaby and Hadley (indophenol oxidase) Reagent: ▪ 1% α-naphthol in 95% ethanol ▪ 1% p-aminodimethylaniline HCL PRINCIPLE • • • RESULT INTERPRETATION OF OXIDASE TEST • • Positive Result o Development of a deep purple-blue/blue colour indicates oxidase production within 5-10 seconds. Negative Result o No purple-blue colour/No colour change. DRY FILTER PAPER METHOD Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple. Organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless within the limits of the test, and are oxidasenegative. Oxidase positive bacteria possess cytochrome oxidase or indophenol oxidase (an iron containing haemoprotein). Both of these catalyse the transport of electrons from donor compounds (NADH) to electron acceptors (usually oxygen). The test reagent, N, N, N’, N’-tetramethyl-pphenylenediamine dihydrochloride acts as an artificial electron acceptor for the enzyme oxidase. The oxidised reagent forms the coloured compound indophenol blue. The cytochrome system is usually only present in aerobic organisms which are capable of utilising oxygen as the final hydrogen receptor. The end product of this metabolism is either water or hydrogen peroxide (broken down by catalase). WET FILTER PAPER METHOD SWAB METHOD EXAMPLES • • Oxidase Positive Organisms: Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella, Helicobacter pylori, Legionella pneumophila, etc. Oxidase Negative Organisms: Enterobacteriaceae (e.g. E. coli) QUALITY CONTROL FOR OXIDASE TEST • • Positive Control: Pseudomonas aeruginosa ATCC 27853 Negative Control: Escherichia coli ATCC 25922 • LAB EXERCISE: OXIDASE TEST MATERIALS • • • • • • • • • • • • • When using Kovac’s oxidase reagent, microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes. 4. When using Gordon and McLeod reagent, microorganisms are oxidase positive when the color changes to red within 10 to 30 minutes or to black within 60 minutes. Microorganisms are oxidase negative if the color does not change. Swab Method 1. Dip swab into reagent and then touch an isolated suspect colony 2. Observe for colour change within 10 seconds. Test Tube Method 1. Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth (or standard media that does not contain a high concentration of sugar). 2. Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% paminodimethylaniline oxalate (Gaby & Hadley reagents). 3. Shake vigorously to ensure mixing and thorough oxygenation of the culture. 4. Observe for color changes. 5. Microorganisms are oxidase positive when the color changes to blue within 15 to 30 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 2 to 3 minutes. Microorganisms are oxidase negative if the color does not change. 3. PPE CTA Medium Prepared Blood agar plate Oxidase test (tetramethyl-p-phenylenediamine dihydrochloride) TSA/Columbia Agar Base Microscope Sheep’s blood Bacterial cultures Prepared Choclate agar plate slides GC Agar Base II Horse blood Inoculating loop • PROCEDURES • • • Dry Filter Paper Method 1. Strip of Whatman’s No. 1 filter paper are soaked in a freshly prepared 1% solution of tertramethyl-pphenylene-diamine dihydrochloride. 2. After draining for about 30 seconds, the strips are freeze dried and stored in a dark bottle tightly sealed with a screw cap. 3. For use, a strip is removed, laid in a petri dish and moistened with distilled water. 4. The colony to be tested is picked up with a platinum loop and smeared over the moist area. 5. A positive reaction is indicated by an intense deeppurple hue, appearing within 5-10 seconds, a “delayed positive” reaction by colouration in 10-60 seconds, and a negative reaction by absence of colouration or by colouration later than 60 seconds. Wet Filter Paper Method 1. A strip of filter paper is soaked with a little freshly made 1% solution of the reagent. 2. A speck of culture is rubbed on it with a platinum loop. 3. A positive reaction is indicated by an intense deeppurple hue, appearing within 5-10 seconds, a “delayed positive” reaction by colouration in 10-60 seconds, and a negative reaction by absence of colouration or by colouration later than 60 seconds. Direct Plate Method 1. Add 2 -3 drops of reagent directly to suspect colonies on an agar plate. Do not flood the plate with reagent. 2. Observe for colour change within 10 seconds. o Note: The Direct Plate method should be carried out on a non-selective agar plate. L3: CARBOHYDRATE FERMENTATION TEST • • The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds is a widely used method for the identification of microorganisms. Different fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. Purple Broth is used for studying carbohydrate fermentation reactions, particularly in the identification of gram-negative enteric bacteria with desired carbohydrates added. Purple media were originally formulated by Vera. This medium is recommended by FDA for fermentation studies of sugars. PRINCIPLE • The principle of carbohydrate fermentation states that the action of organism on a carbohydrate substrate results in acidification of the medium, detected by a pH indicator dye. Carbohydrate fermentation is the process microorganisms use to produce energy. Most microorganisms convert glucose to pyruvate during glycolysis; however, some organisms use alternate pathways. A fermentation medium consists of a basal medium containing a single carbohydrate (glucose, lactose, sucrose, mannitol etc.) for fermentation. However, the medium also contains various pH indicators. In addition to a pH indicator to detect the production of acid from fermentation, a Durham tube is placed in each tube to capture gas produced by metabolism. The carbohydrate fermentation patterns shown by different organisms are useful in differentiating among bacterial groups or species. RESULT INTERPRETATION • • • • Positive: The development of a yellow color in the medium is indicative of a positive carbohydrate fermentation reaction. Negative: Lack of yellow color development is indicative of a negative carbohydrate fermentation reaction. Gas formation is indicated by the appearance of gas bubbles in the Durham tube. Principle o The medium contains acetamide as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for acetamide utilization. When the bacterium metabolizes acetamide by the enzymatic action of an acylamidase, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue, indicative of a positive test. PROCEDURE 1. 2. Inoculate acetamide slant with a needle using growth from an 18- to 24-hour culture. Do not inoculate from a broth culture, because the growth will be too heavy. Incubate aerobically at 35°-37°C for up to 4 days. If equivocal, the slant may be re- incubated for 2 additional days. EXPECTED RESULTS USES • • It is recommended for the determination of fermentation reactions of microorganisms, especially enteric bacilli and Enterococcus They are used primarily for the differentiation and presumptive identification of gram-negative enteric bacilli based on patterns of carbohydrate fermentation. • • USES • LAB EXERCISE: CARBOHYDRATE FERMENTATION TEST • • Materials o PPE o CTA Medium o Microscope o Bacterial cultures o Inoculating loop Procedures 1. Allow medium to warm to room temperature prior to inoculation. 2. Inoculate the Purple Broth (with carbohydrate of choice) with isolated colonies from an 18-24 hour pure culture of the organism. 3. Inoculate a control tube of Purple Broth Base in parallel with the carbohydrate based media. 4. Incubate inoculated media aerobically at 35-37ºC. for 3-5 days. o Note: Increased incubation up to 30 days may be necessary for some microorganisms. 5. Observe daily for development of a yellow color in medium. • • • • Cetrimide is a quaternary ammonium salt, which acts as a selective agent and inhibits most bacteria by acting as a cationic detergent (Cetyltrimethylammonium bromide). Cetrimide when added into a culture medium results into a selective solid medium recommended for use in qualitative procedures for selective isolation and presumptive identification of Pseudomonas aeruginosa and other nonfermentative, gram-negative bacilli. Objective o To isolate and purify Pseudomonas aeruginosa from contaminated specimens. PRINCIPLE • ACETAMIDE UTILIZATION TEST Acetamide test is used to test an organism’s ability to utilize acetamide by deamidation. It is used in the qualitative procedures to differentiate non-fermentative, gram negative bacteria, especially Pseudomonas aeruginosa, on the basis of acetamide utilization. Objective o To differentiate microorganisms based on the ability to use acetamide as the sole source of carbon. The test is recommended for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting, Gramnegative rods. CETRIMIDE TEST L4: OTHER TESTS FOR IDENTIFICATION OF SMALL GRAM NEGATIVE NON-FERMENTATIVE BACILLI • Positive: De-amination of the acetamide, resulting in a blue color. Negative: No color change or development of a yellow color. • The test is used to determine the ability of an organism to grow in the presence of cetrimide, a toxic substance that inhibits the growth of many bacteria by causing the release of nitrogen and phosphorous, which slows or kills the organism. Pseudomonas aeruginosa and very few other organisms are resistant to cetrimide. Pseudomonas aeruginosa produces a number of water soluble iron chelators, including the yellow-green or yellow-brown fluorescent pyoverdin. When pyoverdin combines with the water-soluble pyocyanin, the bright green colour characteristic of Pseudomonas aeruginosa is created. A culture medium containing 0.03% cetrimide is employed for the detection of organisms and especially Pseudomonas aeruginosa. Organisms resistant to cetrimide withstand its germicidal activity resulting in growth which is observed on the inoculated slant of the agar medium. PROCEDURE PRINCIPLE 1. • 2. 3. 4. Inoculate a cetrimide agar slant from an 18-24 hour old pure culture or directly from the specimen. Streak so as to obtain isolated colonies. Incubate at 35°-37°C for up to 7 days. Examine the slant for bacterial growth. EXPECTED RESULTS • • Positive: Growth, variation in color of colonies. Visual examination may also reveal the typical yellow-green to blue color which indicates the production of pyocyanin. Negative: No growth • Pseudomonas aeruginosa is member of the Gamma Proteobacteria class of bacteria. It is a Gram-negative, aerobic rod belonging to the bacterial family Pseudomonadaceae. The bacterium is ubiquitous in soil and water, and on surfaces in contact with soil or water. Its optimum temperature for growth is 37 degrees, but it is uniquely able to grow at temperatures as high as 42 degrees. Growth at 42’C has been considered to be a key differential test for separating non-pigmented P. aeruginosa and the other species of fluorescent pseudomonads. Thus the test is used to determine the ability of an organism to grow at 42°C. For this the organism is cultured on an agar medium and then incubated at a high temperature of 42°C. Only organisms that are capable of enduring the temperature grow while others do not. Several Pseudomonas species have been isolated in the clinical laboratories that are capable of growth at elevated temperatures. The differentiation of apyocyanogenic Pseudomonas aeruginosa from the other fluorescent pseudomonads is clinically important due to the seriousness of infections caused by P. aeruginosa. PROCEDURE 1. • • Pyocyanin Production: o Positive Test – Blue-green pigmentation surrounding growth o Negative Test – No color development Fluorescein Production (requires the use of ultraviolet light): o Positive Test – Yellow-green fluorescence o Negative Test – No fluorescence USES • • • 2. 3. Inoculate two tubes of trypticase soy agar (TSA) with a light inoculum by lightly touching a needle to the top of a single 13- to 24-hour-old colony and streaking the slant. Immediately incubate one tube at 35°C and one at 42°C. Record the presence of growth on each slant after 18 to 24 hours. EXPECTED RESULTS • • Positive: Good growth at both 35°and 42°C Negative: No growth at 42°C, but good growth at 35°C. Used for the isolation and identification of Pseudomonas aeruginosafrom clinical and non-clinical specimens. It may also be used for determining the ability of an organism to produce fluorescein and pyocyanin. The test can also be used for microbial limit testing for non-sterile products. GROWTH AT 42 DEGREES CELCIUS • • Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its minimum growth temperature. The highest temperature at which growth can occur is its maximum growth temperature. However, growth at both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes. Only few organisms grow well in such temperature ranges. Thus the effect of temperature on organisms can be observed to differentiate organisms. Objective o To differentiate species of the fluorescent pseudomonas group o To differentiate other non-fermentative bacteria. Growth at 42°C. A, Positive; good growth. B, Negative; no growth. USES • • Growth at 42oC is advocated to differentiate species of the fluorescent pseudomonas group as well as to differentiate other non-fermentative bacteria. This test is mostly used to differentiate a pyocyanogenic pseudomonads from other Pseudomonas. OF (OXIDATION-FERMENTATION) TEST • • • Carbohydrates are organic molecules that contain carbon, hydrogen and oxygen in the ratio (CH2O)n. Organisms use carbohydrate differently depending upon their enzyme complement. The pattern of fermentation is characteristics of certain species, genera or groups of organisms and for this reason this property has been extensively used as method for biochemical differentiation of microbes. Objective o To detect the oxidation or fermentation of carbohydrates by bacteria. PRINCIPLE USES • • • • Whether an organism is oxidative or fermentative can be determined by using Hugh and Leifson’s medium, commonly called as OF medium which contain tryptone and bromothymol blue (an indicator). One of the sugars, such as glucose, xylose, mannitol, lactose, sucrose, and maltose is added to the medium which serves as the fermentable carbohydrate. An organism is inoculated to two tubes of each OF Medium. Once inoculated, one tube is overlaid with mineral oil or melted paraffin producing an anaerobic environment. The other tube is left open to the air. Growth of microorganisms in this medium is either by utilizing the tryptone which results in an alkaline reaction (dark blue colour) or by utilizing glucose, which results in the production of acid (turning bromothymol blue to yellow). Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of true fermentation, while acidic development in the open tubes are due to oxidative utilization of the carbohydrate present. Asaccharolytic organisms will not produce acid in either tube. PROCEDURE 1. 2. 3. 4. 5. Obtain pure, isolated colonies from an 18-24 hour culture. For each test organism, inoculate tubes in duplicate. Inoculate by stabbing the agar to approximately 1/4 inch from the bottom. Apply sterile mineral oil, sterile melted paraffin, or sterile melted petrolatum to one of each duplicate tubes. Tighten the cap of the overlaid tube, and loosen the cap of the non-overlaid tube. Incubate both tubes aerobically at 35ºC. for up to 14 days. Examine tubes daily for colour change. EXPECTED RESULTS • • Positive: A positive carbohydrate utilization test is indicated by the development of a yellow color in the medium. o Oxidative: Development of a yellow colouration in the open tube only. o Fermentative: Development of a yellow colouration in both open and closed tubes. Negative: A negative carbohydrate utilization test is indicated by the absence of a yellow color (media remains green or turns blue). o Non-oxidizer/Non-fermenter • • • It aids in the identification of gram-negative bacteria on the basis of their ability to oxidize or ferment a specific carbohydrate. It is used to determine whether an organism uses carbohydrate substrates to produce acid byproducts. Non fermentative bacteria are routinely tested for their ability to produce acid from six carbohydrates (glucose, xylose, mannitol, lactose, sucrose, and maltose).