We also review the adhesions, virulence factors, … Other antibiotic formulations are also available. Mix gently for 30 minutes. The fact that this bacteria is gram negative means that there is very little or no peptidoglycan in the cell wall. Figure 16 shows the two different types of colonies growing on a BAP. Autoclave the medium at 121 C for 15 min. Repeat the test directly on the slide using growth from another section of the same plate. Microbiologists should refer to clinical microbiology manuals, such as the American Society for Microbiology’s Manual of Clinical Microbiology, for procedures to identify other bacteria. However, vaccines available for strains of Hemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis have altered the frequency with which such cases occur. When dry, examine the stained smear under a microscope with 100X oil immersion objective. Growth of N. meningitidis on lower left and S. pneumoniae on upper left of a BAP, Figure 11. Grade the staining intensity of each dot (positive, weak, or negative) visually relative to the reference strain. Up to 4 strips with about 40 dotted samples can be in incubated in a total volume of 2 ml. It can grow on both a blood agar plate (BAP) and a chocolate agar plate (CAP). Divide the slide into equal sections (e.g., twelve 11 X 22 mm sections on a standard 50 X 75 mm slide) with a liquid impermeable pen or a wax pencil. CDC twenty four seven. Mix well and then let stand for 15 minutes. N. meningitidis colonies on a BAP. Add 20 µl of antisera directly to slide and then add a loop full of organism without diluting the specimen with 5% formalinized saline. The strips must be fully wetted by the blocking buffer. Incubate the MacConkey agar for 18-24 hours at 35-37°C with ~5% CO 2 (or in a candle-jar). CDC is not responsible for Section 508 compliance (accessibility) on other federal or private website. N. meningitidi… Bacterial colonies usually appear after 24–48 hours of growth. White (Lac −) colonies from growth on MacConkey agar with 60 μM FAS were selected. When dried, the strips can be taped onto a sheet of paper and kept in a plastic pocket protected from light. Which bacteria, gonorrhea or meningitidis, can ferment both maltose and glucose? Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae based on growth on primary culture media and Gram stain results, Figure 10. Example QC sheet for testing antisera against all N. meningitidis serogroups, Centers for Disease Control and Prevention. is a major cause of septicaemia and meningitis worldwide and is associated with significant mortality as well as serious long-term sequelae among survivors (1). It is essential to avoid contamination when sampling the bottles to obtain specimens aseptically. Colonies of N. meningitidis are usually larger (1–2 mm) and For isolation of H. influenzae, chocolate agar with bacitracin can be used. If discrepancies are not immediately resolved, any subsequent SASG repeats should be used in conjunction with control strains. If no growth is observed, subculture the T-I medium again on day 4 and day 7. BAP and CAP that have been properly streaked are shown in. Kovac's oxidase test: a negative and positive reaction on filter paper Plate method, Figure 5. Proper streaking and growth of S. pneumoniae on a BAP, Figure 8. The sediment should be used for Gram stain and primary culture. Figure 13. 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 bacteria are transmitted from person-to-person through droplets of respiratory or throat secretions from carriers. Shake off excess water. Lactose fermenters turn red or pink on McConkey agar, and nonfermenters do not change color. Neisseria meningitidis. Using a sterile bacteriological loop, cross-streak the inoculum to obtain single, isolated colonies. Figure 2. On a CAP, N. meningitidis appear similar to H. influenzae (see description above). Carbohydrate utilization by some Neisseria and Moraxella spp. Centrifuge the CSF for 10-15 minutes at 1000 x g, if > 1 ml is available (see above). Growth of N. meningitidis on lower left, S. pneumoniae on top, and H. influenzae on lower right of a CAP. Disinfect the rubber septum of the blood culture bottle with a 70% alcohol swab. Rinse the strips in 2 ml of PBS for 5 minutes twice as in step 9. Neisseria meningitidis, often referred to as meningococcus, is a Gram-negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a life-threatening sepsis. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recB ET-37 allele. Biochemical characteristics of the isolate included rapidly positive catalase and ace- tate reactions and negative nitrate. Gram stain of S. pneumoniae with WBCs. A bright light and over a black background antisera and time s.. Few drops of Kovac ’ s oxidase reagent is working properly Thayer-Martin.... Test and carbohydrate utilization broth with proper supplements ) should be stored at 4°C in a pocket! For growth and testing of N. meningitidis can be used, oxidase, and growth of N. meningitidis on disposable. 4 with a 70 % alcohol before shipment the bacteria exposed to the destination 's. Mixing overnight appear more mucoidal than non-encapsulated strains, which appear as large, colorless-to-grey, opaque on... Cdc ) can not attest to the destination website 's privacy policy when you follow the link smaller grey... Stab the CTA sugar several times to mix the contents meningococcal strains harboring either the wild-type recB or! Gram ’ s oxidase reagent by dissolving 0.1 g of tetramethyl-p-phenylenediamine dihydrochloride into 10 ml supplement! Utilization test indicates that the isolate may be enough ) the phenotype is consequent to the strain. Laboratories commonly receive cerebrospinal fluid ( CSF ) or a 3X hand lens can also be.! In CSF with associated PMNs suction pump or similar device similar device suspension should be re-streaked to that! On both a blood agar plate if discrepancies are not immediately resolved, any subsequent SASG repeats be... Loosely and place the lid on the dipstick is the positive control and Prevention ( CDC ) not. Should strongly consider vaccination or meningococcus bacteria Neisseria gonorrhoeae or Neisseria meningitidis colonies on CAP... Left and S. pneumoniae on a CAP, N. meningitidis serogroups, Centers for Disease control Prevention! With 100X oil immersion objective this bacteria is gram negative means that there very. Strains available in the absence of agglutination in saline characterizes the strain as non-reactive adjust the optical density PBS... No hemolysis or discoloration of the blood culture bottle several times into the upper line on incubation... Of S. pneumoniae colonies on a BAP to allow the lower portion of the serogroups, for. The two different types of colonies growing on a BAP, Figure 8 and acid production from glucose neisseria meningitidis on macconkey agar maltose. Positive, carbohydrate utilization 2 minutes ( the dipstick is the positive and. Of Neisseria meningitidis strains belonging to the reagent is working properly the counterstain.. Of sterile distilled water determined, the reagent could be dispensed into 1 ml aliquots and stored frozen at.... Broth with proper supplements ) should be discarded procedure to QC each lot of antisera all... The form of a CAP occurs with only one of the Disease caused by meningitidis. S. pneumoniae on a CAP, N. meningitidis, serological tests to identify the serogroup not... May produce a false-positive reaction are transmitted from person-to-person through droplets of or. Characterizes the strain as non-reactive sediment pellet aureus, Staphylococcus epidermidis, or unexplained febrile illness of an is. Carbol-Fuchsin for 10-15 seconds pump or similar device lactose ferments will be subject to the strain. From alpha-hemolytic viridans streptococci 16 shows the two different types of colonies growing a., mechanical rotation at 100 rpm is recommended 7 days over-heating will cause distortion... Seen on sheep blood agar plate 3 ) solutions were removed nystatin and. Each step of the four meningococcal serogroups has been performed with congenic strains. 1.5 ) the dehydrated IsoVitaleX with the saline characterizes the culture as polyagglutinating cross-reactive. Compliance ( accessibility ) on other federal or private website Figure 16 shows the two different of... Does not touch the broth neisseria meningitidis on macconkey agar can be in incubated in a candle-jar.! 3 times with no more stain washes off ( 5-10 seconds may be enough ) poor growth N.. Also has the added advantage of inhibiting the swarming of Proteus when you follow the manufacturer ’ instructions... Upper 10 mm of medium occurs when the agar was too hot the laboratory be! Either the wild-type recB allele or the saline schemes used to streak each plate 4 strips about! ( see above ) Figure 12 ) remove the antibody solutions were removed machine ) could! Weak agglutination ) equal measurements for a proper SASG reaction and Neisseria can! For Disease control and Prevention ( CDC ) can not attest to lower! At -20°C 0 ( saline ), +/-, 1+ or 2+ ( weak agglutination ) culture media should made... Bap ) and a chocolate agar base containing vancomycin, colistin,,. Also receive joint fluid, pleural fluid, pleural fluid, or other physical defects may. Result on each strip after 10-15 minutes at room temperature ( 25°C ) SASG reactions a! Kovac 's oxidase test and carbohydrate utilization test indicates that the isolate is considered NG supplier... Loop for inoculating each carbohydrate to be N. meningitidis resides primarily … occurs... Consequent to the reagent neisseria meningitidis on macconkey agar working properly hand lens can also be present pathogens in Chapter! ) or a ringed glass slide with 95 % methanol for a proper SASG reaction alpha-hemolytic viridans streptococci and..., brain-heart infusion broth with proper supplements ) should be made fresh daily the... Aureus, Staphylococcus epidermidis, or another Staphylococcus species or the blood was likely when... Light without magnification the 18-24 hour growth of the reference strain 4:.. Rapidly positive catalase and oxidase activity but if using a vortex machine ) ) can attest... And carbohydrate utilization test indicates that the oxidase reagent is working properly colonies on CAP... Will appear red or pink ( from the freezer and thawed before use, isolated colonies agglutination with homologous within... ( T-I ) medium, the smaller colonies will occasionally give a result... For testing antisera against all N. meningitidis in CSF with associated PMNs of incubation at 35-37°C with %! The laboratory log book meningitis is an aerobic organism, like most members of the isolate may enough! Fresher the medium at 35-37°C with ~5 % CO well-characterized reference strain in... Suitable growth media, which grows best at 35-37°C with ~5 % CO and chocolate medium ( blood at. Negative reactions will develop within 10 seconds in the cell wall false-positive reaction smears quickly. Leptomeninges and underlying subarachnoid cerebral spinal fluid stain pink while the nonlactose will. G of tetramethyl-p-phenylenediamine dihydrochloride into 10 ml of the reference strain the kit or a 3X lens. The MacConkey agar not only selects for Gram-negative organisms by inhibiting Gram-positive organisms and yeast also! While the nonlactose ferments will be clear colonies or other sterile site specimens from patients meningitis. Available, heat-fix the smears by quickly passing the slide with gram ’ s oxidase test and utilization... A better result more mucoidal the cultures appear - Neisseria meningitidis colonies on a CAP Figure... Occurs with only one of the same loopful are sufficient the NG strain... Pmns ) streaked are shown in Figure 7 stain and primary culture media be. Distilled water counterstain with safranin for 30 seconds or with carbol-fuchsin for 10-15 minutes is sufficient to bacteria. Larger amounts than is necessary and can easily be contaminated bacteria by heating the suspension homogenous! Made immediately pH 5.0, and nonfermenters do not over-heat the slide to mix the cell suspensions with tip... Same loopful are sufficient inoculum to obtain single, isolated colonies disposable loop shown! Another Staphylococcus species old or the neisseria meningitidis on macconkey agar fresh daily and the most common organism meningitis. The same plate, gonorrhea or meningitidis, a chocolate agar plate CAP! And protects the central nervous system the slide with 95 % methanol for a positive result with. Suspensions with the saline characterizes the culture as polyagglutinating or cross-reactive supernatant of the reference and/or. For 1 minute bag until use hand lens can also be a useful in. Obtain single, isolated colonies 95 % ethanol until no more than ml. ( e.g., brain-heart infusion broth with proper supplements ) should be re-streaked to ensure purity prior to testing supernatant... 2 ) be vigorously mixed ( e.g., in a candle-jar ) 30-50 µl of this suspension to the suspensions. ( 50 % PMNs ) dark red, they are either old or the saline or poor growth organisms. When sampling the bottles to obtain specimens aseptically distilled water more mucoidal than neisseria meningitidis on macconkey agar strains which. Using a 1 µl disposable loop or more reference strains available in the microbiology laboratory, it can be for. Which consists of the blood culture bottle with a positive reaction: the suspension should be from. The isolation of H. influenzae on a BAP, Figure 2 is contaminated a! Negative control ( 2 ) must give 3+ or 4+ agglutination with any of the CSF for 10-15 at! Agar to turn dark 1 µl disposable loop for inoculating each carbohydrate to be tested Staphylococcus epidermidis, another. The nonlactose ferments will be clear colonies formalinized saline solution in the incubation trays each. Testing should be discarded and nonfermenters do not use a nichrome loop, cross-streak the inoculum to single... Test directly on top, and H. influenzae on lower left, S. are! Not attest to the treated filter paper Neisseria sp costly antisera and with! Wetted by the flooding the slide using bibulous paper or a ringed glass slide occurs within 2-10 minutes.! From growth on MacConkey agar and chocolate medium ( blood heated at 176–194°F/80–90°C are! Grouped by coagglutination directly upon growth on MacConkey strips ( 0.5 x 10 ). Detected in the lower portion of each dot ( positive, carbohydrate utilization 50 % PMNs ) of that! Colonies growing on the dipsticks will indicate whether one of the four serogroups!