Neisseria spp. For isolation of H. influenzae, chocolate agar with bacitracin can be used. Encapsulated strains appear more mucoidal than non-encapsulated strains, which appear as smaller compact grey colonies. Alternatively, if the blood culture bottle has a screw-cap, open the bottle and remove the fluid using sterile technique (i.e., flaming the bottle mouth upon opening and closing the cap). Differentiating pneumococci from viridans streptococci on a CAP is difficult. Incubate the inoculated T-I medium at 35-37°C with ~5% CO. Examine the blood culture bottle for turbidity at 14-17 hours and then every day for up to 7 days. The small grey, flat colonies surrounded by a greenish zone of alpha-hemolysis are S. pneumoniae and the white colonies with no hemolytic activity are S. epidermidis (black arrow). It is possible to use simple water (filet d’eau de robinet) if distilled water is not available for the entire Gram stain procedure. ... N. gonorrhea. No growth was observed on MacConkey agar. Do not over-heat the slide as over-heating will cause significant distortion or destruction of the cells. Table 1. The Centers for Disease Control and Prevention (CDC) cannot attest to the accuracy of a non-federal website. Performed biannually after initial QC testing. Stab the CTA sugar several times into the upper 10 mm of medium. Incubate the MacConkey agar for 18-24 hours at 35-37°C with ~5% CO, Inspect plate for growth and identify the bacteria (. Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae based on growth on primary culture media and Gram stain results, Figure 10. Depending on the supplier of the Mab, the final dilution may range from 1:10 to 1:500,000. RDTs should be stored at 4°C in a moisture-proof bag until use. Once it has been well-mixed, one or two drops of sediment should be used to prepare the Gram stain and one drop should be used to streak the primary culture media. Apr 7, 2016 - The growth of Neisseria meningitidis colonies on New York City Medium Agar To streak two plates, draw approximately 100-200 µl with the syringe at one time to minimize the possibility of contaminating the T-I medium. Centers for Disease Control and Prevention. Repeat the test directly on the slide using growth from another section of the same plate. No agglutination with any of the antisera or the saline characterizes the strain as non-reactive. If available, mechanical rotation at 100 rpm is recommended. Some serotypes appear more mucoidal than others; the fresher the medium, the more mucoidal the cultures appear. A further modification of these early formulations was developed to improve the isolation of pathogenic Neisseria sp. It has been shown that Neisseria meningitidis can be grouped by coagglutination directly upon growth on sheep blood or chocolate agar plates. Figure 16 shows the two different types of colonies growing on a BAP. Neisseria meningitidis. Add the Mabs to the respective wells. Rinse the strips under running water, align them on a glass plate, and absorb the excess water with filter paper or tilt the plate slightly to dry the strips at room temperature (25°C). Gram-positive organisms will appear dark violet or purple. Carbohydrate utilization by some Neisseria and Moraxella spp. N. meningitidis is a fastidious organism, which grows best at 35-37°C with ~5% CO 2 (or in a candle-jar). You will be subject to the destination website's privacy policy when you follow the link. White (Lac −) colonies from growth on MacConkey agar with 60 μM FAS were selected. The suspension should be moderately opaque (see saline control in. Because the primary purpose of this manual is to aid in the identification of N. meningitidis, S. pneumoniae, and H. influenzae from clinical specimens collected from suspected cases of bacterial meningitis, the methods described here will not allow for identification of other isolates that may be of clinical importance but are less likely encountered. Four species have been reclassified1,2. molecular tools are described in Chapter 10: PCR Methods and Chapter 12: Molecular Methods. Shake off excess water. Streak for isolation and incubate for 18-24 hours at 35-37°C with ~5% CO, Incubate an uninoculated plate for 18-24 hours at 35-37°C with ~5% CO. 103-107 K. kingae, the most common human pathogen of the Kingella species, grows on blood and chocolate agar but fails to grow on MacConkey agar. Carefully remove the antibody solutions from the strips using a pipet tip connected to a suction pump or similar device. If the oxidase test is positive, carbohydrate utilization testing should be performed. Abstract. In the lower portion of each of the sections of the glass slide described in step (2), add 10 µl of the 5% formalinized saline with a micropipettor. Counterstain with safranin for 30 seconds or with carbol-fuchsin for 10-15 seconds. Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae can be made on the basis of a cytological examination of the CSF, specific colony morphology on blood and/or chocolate agar, staining properties on a Gram stain, or by detection of specific antigens in the CSF by a latex agglutination test or using a rapid diagnostic test (RDT). Figure 1. Use a separate disposable loop for inoculating each carbohydrate to be tested. Growth of N. meningitidis on lower left, S. pneumoniae on top, and H. influenzae on lower right of a CAP. For isolation of S. pneumoniae, tryptic soy agar with 5% sheep blood and either gentamicin, neomycin or sulfamethoxazole-trimethoprim (SXT) may be useful. Please refer to Chapter 4: Biosafety in order to follow the guidelines that have been established for laboratorians working in BSL-2 facilities as many of the tests described in this chapter require opening plates with live cultures and are often performed outside of a biosafety cabinet (BSC). When growing Neisseria meningitidis, one usually starts with a normally sterile body fluid (blood or CSF), so a plain chocolate agar is used. Blood agar plate (BAP): trypticase soy agar (TSA) + 5% sheep blood A BAP is used as a general blood agar medium. Alternatively, the reagent could be dispensed into 1 ml aliquots and stored frozen at -20°C. Each isolate will require as many sections on the slide as the individual serogroup-specific antisera that will be tested as well as a saline negative control. Meningitis occurs when the membranes that cover the brain and spinal cord become inflamed. Printer friendly version Cdc-pdf[26 pages]. Laboratories may also receive joint fluid, pleural fluid, or other sterile site specimens from these patients. Prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. For primary isolation of N. meningitidis, a chocolate agar base containing vancomycin, colistin, nystatin, and trimethoprim can be used. Place one drop of each latex reagent on a disposable card provided in the kit or a ringed glass slide. This medium is GCS medium. Follow the manufacturer’s instructions on the package insert. The instructions specify using a micropipettor with sterilized filtered tips to measure the 10 µl of the 5% formalinized saline to suspend the bacteria. General instructions are listed below: Figure 4. Additional health and safety information can be found in Chapter 4: Biosafety. Abstract. Figure 13. N. meningitidis colonies on a CAP, Figure 3. Older cultures (> 24 hours) become more opaquely grey and sometimes cause the underlying agar to turn dark. Neisseria meningitides . The antiserum must give 3+ or 4+ agglutination with homologous antigens within 1-2 minutes. Colonies of N. meningitidis are grey and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. It is used for growth and testing of N. meningitidis and S. pneumoniae. The upper line on the dipstick is the positive control and should always be present. Listed above are the most commonly encountered species. Dot 2 µl of the whole-cell suspension of a relevant reference isolate in the middle of the next marked section and 2 µl of the test isolates successively onto each of the next sections, as shown in. Failing result: no growth or poor growth of the reference strain on appropriate media and growth of organisms on the uninoculated media. Follow the manufacturer’s instructions on the package insert for the specific latex kit being used. Divide a glass slide into two sections using a marker. Let the suspension air dry. N. meningitidis appear as large, colorless-to-grey, opaque colonies on a CAP. Place the two dipsticks (RDT1 and RDT2) into two separate tubes (3 ml disposable plastic tubes are recommended) of 150-200 µl of CSF or a reference strain suspension in PBS, pH 7.2. If the carbohydrate utilization test indicates that the isolate may be N. meningitidis, serological tests to identify the serogroup should be performed. H. influenzae cannot grow on a BAP. Bacterial colonies usually appear after 24–48 hours of growth. Proper streaking and growth of N. meningitidis on a BAP, Figure 7. Oct 14, 2013 - Neisseria meningitidis colonies on agar plate. Catlin, B. Wesley (Marquette University, Milwaukee, Wis.) and Gertrude M. Schloer.A defined agar medium for genetic transformation of Neisseria meningitidis.J. Kovac's oxidase test: a negative and positive reaction on filter paper Plate method, Figure 5. Biochemical characteristics of the isolate included rapidly positive catalase and ace- tate reactions and negative nitrate. Place the tubes in a total volume of 2 minutes, examine the agglutination reactions under a bright light magnification! Immersion objective and Chapter 12: molecular Methods N. gonorrhoeae are 0.5–1 mm in size both the... From overnight growth on chocolate agar plate clean paper towel is working properly the results on! Some serotypes appear more mucoidal than non-encapsulated strains, which consists of a CAP ( Figure 12.... Into 1 ml aliquots and stored frozen at -20°C or another Staphylococcus species the tip of the blood broth. Cut strips ( 0.5 x 10 cm ) of nitrocellulose paper ( pore 0.45 µm ) and kept a... 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