MacConkey agar is one of the earliest and most common bacteriological media used in clinical microbiology for the isolation and identification of Gram-negative bacteria (1 – 3). Rinse with distilled water. Transfer the strips to be tested with the same Mab to a well (maximum 4 strips per well). However, once the pneumococcal culture ages 24-48 hours, the colonies become flattened, and the central portion becomes depressed, which does not occur with viridans streptococci (Figure 15). Dispose of the tip or loop used to transfer the antisera to the slide in a waste container after each use to avoid contamination of the antisera. S. pneumoniae colonies on a BAP, Figure 15.S. Figure 6. Bacteria colonies Colonies of bacteria in MacConkey agar (culture medium plate) diplococcus stock pictures, royalty-free photos & images. If the T-I medium can be transported to a microbiology laboratory the same day of inoculation, do not vent the T-I bottle until it arrives in the receiving laboratory. Suspend the bacteria in the 5% formalinized saline solution in the lower portion of each of the sections of the slide. Wash the strips for several minutes in 0.05 M sodium acetate buffer, pH 5.0, and then discard the solution. It can grow on both a blood agar plate (BAP) and a chocolate agar plate (CAP). Clean a glass slide with alcohol (optional if slides are pre-cleaned). RDTs should be stored at 4°C in a moisture-proof bag until use. Use a separate disposable loop for inoculating each carbohydrate to be tested. Add 10 ml of supplement per liter of base medium i.e., 1% (v/v); mix thoroughly. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recB ET-37 allele. In this chapter we provide an overview of the disease caused by N. meningitidis and the schemes used to type the meningococcus. Figure 6. If the original plate contains different size colonies, make a subculture for each type of colony and test both cultures the next day. Do not use a circular motion while rocking, as it can cause the sections with different serogroup-specific antisera to run together and contaminate each other. The antiserum must give 3+ or 4+ agglutination with homologous antigens within 1-2 minutes. Rinse the tip with water between each set of strips that were incubated with different Mabs to prevent transfer of the Mabs. Shake off excess water. Young colonies of N. meningitidis are round, smooth, moist, glistening, and convex, with a clearly defined edge on a BAP (Figure 13). Gently blot the slide using bibulous paper or a clean paper towel. Rinse with distilled water. Remove the PBS in the same manner in which the antibody solutions were removed. If only one type of plate is available, a CAP should be used because it contains the hemin and NAD needed for H. influenzae, whereas a BAP does not. Negative reactions will not produce a color change on the treated filter paper. Centrifuge the CSF for 10-15 minutes at 1000 x g, if > 1 ml is available (see above). Prepare a 1.0% Kovac’s oxidase reagent by dissolving 0.1 g of tetramethyl-p-phenylenediamine dihydrochloride into 10 ml of sterile distilled water. Avoid touching the slide with the tip of the reagent bottle or applying liquid directly onto the smear. MacConkey agar is a selective and differential culture medium for bacteria.It is designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and differentiate them based on lactose fermentation. A back-up broth (e.g., brain-heart infusion broth with proper supplements) should be inoculated with some of the sediment pellet. Centrifuge the CSF for 10-15 minutes at 1000 x g and collect the supernatant. Figure 7. The larger colonies usually indicate better capsule production and therefore better reactivity. Young pneumococcal colonies appear raised, similar to viridans streptococci. The aliquots should be removed from the freezer and thawed before use. Performed biannually after initial QC testing. Meningitis occurs when the membranes that cover the brain and spinal cord become inflamed. On a CAP, N. meningitidis appear similar to H. influenzae (see description above). If the CSF is positive for one of the serogroups, a lower red line will also be present. Meningitis. In the upper portion of each of the sections of the glass slide described in step (2), add 10 µl of the serogroup-specific antisera to be tested as well as unformalinized saline or phosphate buffered saline (PBS) for a negative control with a micropipettor. Neisseria meningitides . Label the strips with a permanent pen on the left section. Oct 14, 2013 - Neisseria meningitidis colonies on agar plate. Biochemical characteristics of the isolate included rapidly positive catalase and ace- tate reactions and negative nitrate. If discrepancies are not immediately resolved, any subsequent SASG repeats should be used in conjunction with control strains. BAP and CAP that have been properly streaked are shown in. Thayer-Martin agar was initially developed in … 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. Abstract. Transfer approximately 0.5 ml of the blood culture broth onto MacConkey agar and streak for isolated colonies. Proper streaking and growth of S. pneumoniae on a BAP, Figure 8. Finally, the development and application of PCR-based diagnostic testing, along with an elevated index of suspicion, has been important in recognizing this emerging pathogen in children. The upper line on the dipstick is the positive control and should always be present. Dispense a few drops of Kovac’s oxidase reagent directly on top of a few suspicious colonies growing on the 18-24 hour BAP. The bacterium was positive for catalase, oxidase, and acid production from glucose but not maltose. Before subculture, swirl the blood culture bottle several times to mix the contents. 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). While H. influenzae produce a pungent indol smell that can differentiate it from N. meningitidis, plates should not be opened in order to smell the cultures. Depending on the supplier of the Mab, the final dilution may range from 1:10 to 1:500,000. Add 30-50 µl of the supernatant of the CSF to each latex reagent. Colonies of N. meningitidis are grey and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. Place one drop of each latex reagent on a disposable card provided in the kit or a ringed glass slide. The position of that red line indicates the specific serogroup based on the RDT that was tested. Approximately 8 stabs with the same loopful are sufficient. Lactose ferments will stain pink while the nonlactose ferments will be clear colonies. Subculture and retest fresh growth the following day. Cool to 50 C in a waterbath. Shake off excess water. Be sure that the strips are fully wetted. Rinse the strips 3 times with no more than 2 ml PBS directly in the wells. Inspect plate for growth and identify the bacteria . You will be subject to the destination website's privacy policy when you follow the link. A negative result is designated by a 0 (saline), +/-, 1+ or 2+ (weak agglutination). Four species have been reclassified1,2. For primary isolation of N. meningitidis, a chocolate agar base containing vancomycin, colistin, nystatin, and trimethoprim can be used. If a micropipettor and tips are not available, sterile, disposable 10 µl inoculation loops can be used to transfer 10 µl of the 5% formalinized saline, but often do not deliver accurate amounts (between 5-10 µl). Approximately 50-100 µl is used to streak each plate. The Divide a glass slide into two sections using a marker. Saving Lives, Protecting People, Presumptive identification by Gram stain, latex agglutination, or rapid diagnostic test (RDT), Inoculation of primary culture media from CSF specimens, Primary culture from a blood culture bottle, Chapters 7: Identification and Characterization of, 8: Identification and Characterization of, 9: Identification and Characterization of, National Center for Immunization and Respiratory Diseases, Laboratory Methods for the Diagnosis of Meningitis, U.S. Department of Health & Human Services, Increased opening pressure (>180 mm water), Pleocytosis (usually of polymorphonuclear (PMN) leukocytes); WBC counts > 10 cells/mm, Decreased glucose concentration (<45 mg/dl), Increased protein concentration (>45 mg/dl). Neisseria gonorrhoeae and Neisseria meningitidis are two important pathogens in this group. from specimens, which contained large amounts of mixed microbial flora. Perform steps 1 and 2 with a positive and negative QC strain to ensure that the oxidase reagent is working properly. 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. The degree of mucoidness of S. pneumoniae colonies is strain and serotype dependent and is also influenced by the freshness of the medium and the incubation atmosphere. Colonies of N. meningitidis are usually larger (1–2 mm) and Put the strips in a plastic box and add 10 ml of sodium acetate buffer with 0.4 ml of 3-amino-9-ethylcarbazole (AEC) in dimethylformamide (DMF). Neisseria meningitidis only infects humans; there is no animal reservoir. Figure 2. Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae based on growth on primary culture media and Gram stain results, Figure 10. Positive reaction: agglutination (or visible clumping) of the latex particles and slight clearing of the suspension occurs within 2-10 minutes (. Grade the staining intensity of each dot (positive, weak, or negative) visually relative to the reference strain. Agar plates and broth inoculated with the CSF sediment should be incubated for 18-24 hours at 35-37°C with ~5% CO, If the CSF cannot be transported to a microbiology laboratory immediately (within 1 hour from the time of collection) for culture and analysis, a bottle of T-I medium should be inoculated (. Make a cell suspension in a small tube and vortex if the result from SASG directly on the slide is unclear and repeat the test. The following result combinations are all reported as NG: Agglutination in the saline, regardless of strong reactions with other antisera, characterizes the culture as autoagglutinating. Saving Lives, Protecting People, Preparation of 1% oxidase reagent from oxidase powder, Slide agglutination serogrouping (SASG) test for serogrouping, Rating the intensity of the agglutination reaction, Quality control (QC) of antisera for SASG testing, Performing the dot-blotting serotyping/serosubtyping test, National Center for Immunization and Respiratory Diseases, Laboratory Methods for the Diagnosis of Meningitis, U.S. Department of Health & Human Services. Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website. ... N. gonorrhea. Incubate the inoculated T-I medium at 35-37°C with ~5% CO. If a serogroup is not determined, the isolate is considered NG. It was identified, with 98% probability, as N. gonorrhoeae (bionumber 464001) by the Vitek Neisseria-Haemo-philus Identification (NHI) card (bioMérieux Vitek, Inc., Hazelwood, MO, USA). Tilt the plate and observe colonies for a color change to purple. No hemolysis or discoloration of the medium is apparent. Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website. Autoclave the medium at 121 C for 15 min. Disposable loops are preferred, but if using a wire loop, it must be sterilized prior to each step of the plate-streaking process. The classic gross and microscopic findings are similar to exudative processes elsewhere caused by … Inoculate the media with pure colonies from 18-24 hour growth of a well-characterized reference strain. If the source of antisera is contaminated, a new vial must be used. General instructions are listed below: Figure 5. Flow chart for identification and characterization of a N. meningitidis isolate, Figure 4. Do not flood the entire plate as the bacteria exposed to the reagent are usually not viable for subculture. Fix the smear by the flooding the slide with 95% methanol for a minimum of 2 minutes (3). Shake off excess water. See. It is essential to avoid contamination when sampling the bottles to obtain specimens aseptically. The bacterium has a tendency to resist decolorization and may therefore … Laboratories may also receive joint fluid, pleural fluid, or other sterile site specimens from these patients. Neisseria meningitidis strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. 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. The antiserum must not react with heterologous. The instructions specify using a micropipettor with sterilized filtered tips to measure the 10 µl of the 5% formalinized saline to suspend the bacteria. Additional health and safety information can be found in Chapter 4: Biosafety. Printer friendly version Cdc-pdf[26 pages]. When dried, the strips can be taped onto a sheet of paper and kept in a plastic pocket protected from light. Listed above are the most commonly encountered species. Proper streaking and growth of N. meningitidis on a BAP, Figure 7. Up to 4 strips with about 40 dotted samples can be in incubated in a total volume of 2 ml. CarboFerm™ uses acid production from carbohydrates and the presence of butyrate esterase to differentiate and identify Neisseria species and M. catarrhalis .Phenol red to yellow (Positive) for carbohydrates To prepare a smear using an isolate, add a small drop of sterile water or physiological saline to the slide and create a slightly turbid, uniform suspension of cells from an overnight culture. Blood agar plate (BAP): trypticase soy agar (TSA) + 5% sheep blood A BAP is used as a general blood agar medium. Figure 16. Flood the slide with crystal violet ammonium oxalate for 1 minute to stain. Gram-positive organisms will appear dark violet or purple. The bacteria are transmitted from person-to-person through droplets of respiratory or throat secretions from carriers. The micropipettor will transfer precise and equal measurements for a proper SASG reaction. Flood the slide with Gram’s iodine for 1 minute. Streak the plates for isolation, incubate at 35-37°C with ~5% CO. Once pure bacterial growth has been confirmed by subculture from the blood culture bottle, the bottle should be disposed of according to proper safety procedures. H. influenzae appear as large, colorless-to-grey, opaque colonies on a CAP (Figure 12). Shake off excess water. The Centers for Disease Control and Prevention (CDC) cannot attest to the accuracy of a non-federal website. Colonies of N. gonorrhoeae are 0.5–1 mm in size. Appearance of red lines on the dipsticks will indicate whether one of the four meningococcal serogroups has been detected in the CSF (. Rotate by hand for 2-10 minutes. Inspect the media for any visible microbial contamination, discoloration, drying, deterioration, or other physical defects that may interfere with use. CDC is not responsible for Section 508 compliance (accessibility) on other federal or private website. S. pneumoniae appear as small, grey, moist (sometimes mucoidal), watery colonies with a surrounding green zone of alpha-hemolysis on a BAP (Figure 14) and CAP. If > 1 ml of CSF is available (i.e., if the specimen volume is sufficient for centrifugation), it must be centrifuged at a force sufficient to sediment the bacteria. Figure 14. 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. Repeat the test directly on the slide using growth from another section of the same plate. We also review the adhesions, virulence factors, … Which bacteria, gonorrhea or meningitidis, can ferment both maltose and glucose? 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