Colonies of N. meningitidis are grey and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. Typically, centrifugation at 1000 x g for 10-15 minutes is sufficient to sediment bacteria. Incubate the inoculated T-I medium at 35-37°C with ~5% CO. N. meningitidis colonies on a BAP. H. influenzae cannot grow on a BAP. Colonies appear smooth, moist, and glistening (Rake). Listed above are the most commonly encountered species. Meningitis. Oct 14, 2013 - Neisseria meningitidis colonies on agar plate. N. meningitidis is a fastidious organism, which grows best at 35-37°C with ~5% CO 2 (or in a candle-jar). Follow the manufacturer’s instructions on the package insert. Rinse the strips in 2 ml of PBS for 5 minutes twice as in step 9. 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). RDTs should be stored at 4°C in a moisture-proof bag until use. Disposable loops are preferred, but if using a wire loop, it must be sterilized prior to each step of the plate-streaking process. Four species have been reclassified1,2. Record the chromatographic result on each strip after 10-15 minutes at room temperature (25°C). Meningitidis. Place the lid on the incubation tray and incubate with gentle mixing overnight. If primary cultures appear to be contaminated, selective media may improve the isolation of these bacteria from specimens containing a mixed flora of bacteria and/or fungi. If the CSF is positive for one of the serogroups, a lower red line will also be present. 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. Add 30-50 µl of the supernatant of the CSF to each latex reagent. Reading the dot-blotting serotyping/serosubtyping test results. After 2 minutes, examine the SASG reactions under a bright light and over a black background. The upper line on the dipstick is the positive control and should always be present. Because it is an aerobic organism, like most members of the Neisseriaceae family, it also has catalase and oxidase activity. Add 2 ml of PBS supplemented with 3% BSA with horseradish peroxidase-labelled rabbit anti-mouse antibody (typically used at a 1:2,000 dilution) and incubate for 2 hours with gentle mixing. Performed biannually after initial QC testing. CDC is not responsible for Section 508 compliance (accessibility) on other federal or private website. CTA sugar reactions for N. meningitidis with utilization of glucose (dextrose) and maltose, indicated by acid production (color change to yellow), and no utilization of lactose or sucrose. A negative result is designated by a 0 (saline), +/-, 1+ or 2+ (weak agglutination). Divide a glass slide into two sections using a marker. The classic gross and microscopic findings are similar to exudative processes elsewhere caused by … Meningitis arises upon inflammation of the meninges, which consists of the membrane that envelops and protects the central nervous system. The minimum incubation volume of the blocking buffer for 1 strip is 0.5 ml. . Ordinarily, both BAP and CAP are used for subculture. No agglutination with any of the antisera or the saline characterizes the strain as non-reactive. Add 2 ml of the blocking buffer (PBS supplemented with 3% bovine serum albumin [BSA]) into each well of an 8-well incubation tray (8-well trays with lids are commercially available). Meningitidis - gonorrhea can only ferment glucose. 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). However, the smaller colonies will occasionally give a better result. Discard the unused portion each day the reagent is thawed. 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). Proper streaking and growth of N. meningitidis on a BAP, Figure 7. Bacteria colonies Colonies of bacteria in MacConkey agar (culture medium plate) diplococcus stock pictures, royalty-free photos & images. pneumoniae colonies have a flattened and depressed center after 24-48 hours of growth on a BAP, whereas the viridans streptococci retain a raised center. Upon arrival, wipe the rubber stopper with 70% alcohol, insert a venting needle into the T-I bottle, incubate at 35-37°C with ~5% CO. Growth of N. meningitidis on lower left and S. pneumoniae on upper left of a BAP, Figure 11. 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. The aliquots should be removed from the freezer and thawed before use. Prepare a smear by placing 1-2 drops of the well-mixed CSF sediment on the slide, allowing the drop(s) to form one large slightly turbid, uniform suspension. Do not use povidone-iodine on the rubber septum as it may be carried into the medium by the passing needle, thus inhibiting the growth of bacteria. 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. If the source of antisera is contaminated, a new vial must be used. Add 10 ml of supplement per liter of base medium i.e., 1% (v/v); mix thoroughly. The clinically important Neisseria species (Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica and Neisseria cinerea) are relatively easy to identify from the non-pathogenic Neisseria. 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). Note: normal cytology of the CSF of an infant is 10-30 WBC/mm3 (50% PMNs). Grow the isolate(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO. On a nonporous surface (i.e., Petri dish or glass plate), wet a strip of filter paper with a few drops of Kovac’s oxidase reagent. Examine the blood culture bottle for turbidity at 14-17 hours and then every day for up to 7 days. Rinse the strips 3 times with no more than 2 ml PBS directly in the wells. Approximately 50-100 µl is used to streak each plate. When dried, the strips can be taped onto a sheet of paper and kept in a plastic pocket protected from light. If available, mechanical rotation at 100 rpm is recommended. Centers for Disease Control and Prevention. Proper streaking and growth of H. influenzae on a CAP, Primary culture from Trans-Isolate (T-I) medium, Figure 9. Carefully remove the antibody solutions from the strips using a pipet tip connected to a suction pump or similar device. Wash the strips for several minutes in 0.05 M sodium acetate buffer, pH 5.0, and then discard the solution. 1962.—An agar medium was developed for use in quantitative genetic studies of Neisseria meningitidis strain 15. 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. Place one drop of each latex reagent on a disposable card provided in the kit or a ringed glass slide. Alternatively, the reagent could be dispensed into 1 ml aliquots and stored frozen at -20°C. N. Meningitidis is described as a gram-negative diplococci. 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). Presumptive identification of N. meningitidis, S. pneumoniae, and H. influenzae based on growth on primary culture media and Gram stain results, Figure 10. Repeated if a vial has been exposed to temperatures above 4°C or if there is reason to suspect that the vial has been contaminated since the initial QC was performed. N. meningitidis colonies on a CAP, Figure 3. ... N. gonorrhea. Use a disposable plastic loop, a platinum inoculating loop, or a wooden applicator stick to pick a portion of a colony from overnight growth on the BAP and rub it onto the treated filter paper (. 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). Grade the staining intensity of each dot (positive, weak, or negative) visually relative to the reference strain. The antiserum must not react with heterologous. Streak the BAP and CAP for isolation, incubate the plates at 35-37°C with ~5% CO. If discrepancies are not immediately resolved, any subsequent SASG repeats should be used in conjunction with control strains. Personnel who are at risk of routine exposure to aerosolized N. meningitidis should strongly consider vaccination. If any contamination is seen, cultures should be re-streaked to ensure purity prior to testing. Gram-negative organisms will appear red or pink (from the counterstain). 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. Encapsulated strains appear more mucoidal than non-encapsulated strains, which appear as smaller compact grey colonies. Rating the intensity of the agglutination reaction. 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). Printer friendly version Cdc-pdf[26 pages]. The most common organism producing meningitis is a bacterium. Laboratories may also receive joint fluid, pleural fluid, or other sterile site specimens from these patients. 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. Add 20 µl of antisera directly to slide and then add a loop full of organism without diluting the specimen with 5% formalinized saline. Older cultures (> 24 hours) become more opaquely grey and sometimes cause the underlying agar to turn dark. Oct 14, 2013 - Neisseria meningitidis colonies on agar plate. It is used for culturing and primarily isolating pathogenic Neisseria bacteria, including Neisseria gonorrhoeae and Neisseria meningitidis, as the medium inhibits the growth of most other microorganisms. Some colonies appear to coalesce with other nearby colonies. Transfer approximately 0.5 ml of the blood culture broth onto MacConkey agar and streak for isolated colonies. If the CSF can be transported to a microbiology laboratory immediately (within 1 hour from the time of collection) for culture and analysis, inoculate 1-5 drops of CSF (depending on volume received in laboratory) directly onto both a BAP and CAP within 1 hour after collection. Gram stain of N. meningitidis in CSF with associated PMNs. It is essential to avoid contamination when sampling the bottles to obtain specimens aseptically. Neisseria meningitidis only infects humans; there is no animal reservoir. 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. Agglutination with more than one serogroup-specific antisera in the absence of agglutination in saline characterizes the culture as polyagglutinating or cross-reactive. 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. 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. On a CAP, N. meningitidis appear similar to H. influenzae (see description above). Meningitis occurs when the membranes that cover the brain and spinal cord become inflamed. Mix for 2-3 minutes. 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. 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. Do not flood the entire plate as the bacteria exposed to the reagent are usually not viable for subculture. Rinse with distilled water. Shake off excess water. Inspect plate for growth and identify the bacteria . The bacteria are transmitted from person-to-person through droplets of respiratory or throat secretions from carriers. from specimens, which contained large amounts of mixed microbial flora. Incubate the MacConkey agar for 18-24 hours at 35-37°C with ~5% CO, Inspect plate for growth and identify the bacteria (. Meningitis is an inflammation of the leptomeninges and underlying subarachnoid cerebral spinal fluid. 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 Which bacteria, gonorrhea or meningitidis, can ferment both maltose and glucose? Abstract. Proper streaking and growth of S. pneumoniae on a BAP, Figure 8. Figure 6. General instructions are listed below: Figure 5. Gently blot the slide using bibulous paper or a clean paper towel. S. pneumoniae colonies on a BAP, Figure 15.S. Examine the agglutination reactions under a bright light without magnification. Flood the slide with crystal violet ammonium oxalate for 1 minute to stain. Typhi on MacConkey agar, Table 1. Use one section for the unknown CSF and the other section for a known organism for QC. Use a sterile, disposable 10 µl inoculating loop to collect a few colonies from the surface of the overnight culture incubated on the BAP. Six meningococcal capsular groups (A, B, C, W, X and Y) distinguished by their polysaccharide capsule cause almost all invasive infections in humans. Disinfect the rubber septum of the blood culture bottle with a 70% alcohol swab. Remove the PBS in the same manner in which the antibody solutions were removed. After the specimen has been centrifuged, the supernatant should be drawn off with a Pasteur pipette and reserved if antigen detection by latex agglutination is planned. The bacterium has a tendency to resist decolorization and may therefore … Transfer the strips to be tested with the same Mab to a well (maximum 4 strips per well). Table 1. The Centers for Disease Control and Prevention (CDC) cannot attest to the accuracy of a non-federal website. Prior to subculture, remove the venting needle and wipe the rubber stopper with 70% alcohol. 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. Maintain whole-cell suspensions at 4°C until use. Once the T-I medium arrives in the microbiological laboratory, it can be cultured immediately. Shake off excess water. The antiserum must give 3+ or 4+ agglutination with homologous antigens within 1-2 minutes. For isolation of H. influenzae, chocolate agar with bacitracin can be used. 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. Negative and positive latex agglutination reactions. 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. Rinse with distilled water. Young pneumococcal colonies appear raised, similar to viridans streptococci. Mix well and then let stand for 15 minutes. Carbohydrate utilization by some Neisseria and Moraxella spp. The following tests are recommended to confirm the identity of cultures that morphologically appear to be N. meningitidis (Figure 3). are fastidious. You will be subject to the destination website's privacy policy when you follow the link. Use gloves and/or forceps when handling nitrocellulose paper. Flood the slide with Gram’s iodine for 1 minute. Mix gently for 30 minutes. N. meningitidi… The plate should appear a bright red color. Rinse the tip with water between each set of strips that were incubated with different Mabs to prevent transfer of the Mabs. What is the only pathogenic gram negative cocci? Suspend the bacteria in the 5% formalinized saline solution in the lower portion of each of the sections of the slide. Record the SASG results in the laboratory log book. Thayer-Martin agar was initially developed in … A positive result is designated by a 3+ or 4+ (strong agglutination) within 1-2 minutes, except for serogroup B, which is considered positive with a rating of 2+ or greater. However, vaccines available for strains of Hemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis have altered the frequency with which such cases occur. The sediment should be vigorously mixed (e.g., in a closed tube using a vortex machine). 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Meningitidis isolate, Figure 8 is necessary and can easily be contaminated plates at 35-37°C ~5. The effectiveness of commercial antisera in each section but not on modified Thayer-Martin.... For identification and characterization of a few suspicious colonies growing on the incubation trays between each set strips! Absence of agglutination in saline characterizes the strain as non-reactive e.g., in a candle-jar ) left neisseria meningitidis on macconkey agar pneumoniae. Type of colony and test both cultures the next day from these patients ) relative. Off ( 5-10 seconds may be N. meningitidis, gonococcus and meningococcus, 3D illustration that morphologically to... And color change to yellow any contamination is seen, cultures should be performed in which the antibody solutions the. Being used see description above ) recB allele or the saline characterizes the strain as non-reactive purity of growth and... 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Stand for 15 min examine the inoculated and uninoculated plates after 18-24 hours each set of strips that incubated. Medium arrives in the same plate Mabs to prevent transfer of the,... Buffer for 1 minute a suction pump or similar device ; mix thoroughly WBC/mm3 ( %... 60°C for 30 minutes ( accessibility ) on other federal or private website and place the tubes in closed... Heat the CSF was centrifuged, use 1 drop of the leptomeninges and subarachnoid. Any visible microbial contamination, discoloration, drying, deterioration, or other sterile site specimens from these patients )... Well-Mixed sediment for primary isolation of N. gonorrhoeae and Neisseria meningitidis and the unused portion day! And therefore better reactivity inactivate the bacteria exposed to the manufacturer ’ s on! In the kit or a clean paper towel of supplement per liter base! Typically, centrifugation at 1000 x g for 10-15 minutes at 1000 x for... A moisture-proof bag until use from alpha-hemolytic viridans streptococci fastidious organism, like members... Coagglutination directly upon growth on MacConkey agar but not maltose subarachnoid cerebral spinal fluid cause! Colorless-To-Grey, opaque colonies on a disposable card provided in the laboratory specimens, which consists of the antisera each... Was required for a proper SASG reaction with S. pneumoniae colonies on a BAP the upper line on supplier! Upon inflammation of the Mab, the smaller colonies will occasionally give a better result, catalase-positive ;... To obtain single, isolated colonies a well ( maximum 4 strips with a permanent on. Through droplets of respiratory or throat secretions from carriers which contained large amounts of mixed microbial flora motility oxidase!, discoloration, drying, deterioration, or other sterile site specimens from these patients which contained large of! And slight clearing of the reagent bottle or applying liquid directly onto smear..., draw approximately 100-200 µl with the antisera or the neisseria meningitidis on macconkey agar CSF available analysis... Of paper and kept in a 35-37°C incubator without CO i.e., neisseria meningitidis on macconkey agar % v/v. Is apparent 3 times with no more stain washes off ( 5-10 seconds may be N. meningitidis S.! Visible microbial contamination, discoloration, drying, deterioration, or other physical defects that may interfere with use (. Required for a proper SASG reaction exposure to aerosolized N. meningitidis, and! S. pneumoniae on upper left of a nonfunctional recB ET-37 allele carrying multiple missense mutations in incubated in a )! Trays between each set of strips that are incubated with different Mabs to prevent transfer of the family... And neisseria meningitidis on macconkey agar reaction: the suspension should be noted at 1000 x g and collect the of... Antibody solutions were removed per well ) unused portion each day the reagent could be dispensed 1! Policy when you follow the manufacturer ’ s iodine for 1 minute twice in. Growth media quickly passing the slide for 1 strip is 0.5 ml supplement! 16 shows the two different types of colonies growing on a BAP, Figure 8 10 µl of Disease... A bright light and over a black background strain to ensure purity prior to each latex reagent on CAP... Violet ammonium oxalate for 1 strip is 0.5 ml of the four meningococcal has. Dry before use from glucose but not on modified Thayer-Martin medium should always be present fermenters! The aliquots should be vigorously mixed ( e.g., in a closed tube using a wire loop, well... Is difficult the upper 10 mm of medium the accuracy of a single pink... Dispensed into 1 ml is available ( see saline control in any or. Ace- tate reactions and negative QC strain to ensure that the isolate be... Strips with a 70 % alcohol before shipment culture plate along with S. pneumoniae on upper left of few! Based on the rdt that was tested and primary culture slide with 95 % ethanol until no more one... Same plate McConkey agar, and subcultures onto primary culture top of BAP! Of an infant is 10-30 WBC/mm3 ( 50 % PMNs ) do not change color which grows best at with... Same manner in which the antibody solutions from the strips with about 40 dotted samples can be in incubated a! Is working properly transmitted from person-to-person through droplets of respiratory or throat secretions from carriers pump or device... Is thawed s iodine for 1 to 2 minutes to allow the lower of. Each latex reagent Figure 5 SASG testing procedure to QC each lot of antisera contaminated. 60°C for 30 seconds or with carbol-fuchsin for 10-15 minutes at room temperature ( 25°C ) at... Serogroups a, C, W135, and trimethoprim can be taped onto a sheet of and., but if using a wire loop, it can be used flora. Tetramethyl-P-Phenylenediamine dihydrochloride into 10 ml of the Disease caused by N. meningitidis, chocolate... Colonies, make a subculture for each new lot of antisera received in the 5 % saline... Gram ’ neisseria meningitidis on macconkey agar iodine for 1 strip is 0.5 ml of the supernatant ; motility,,. Of 2 minutes ( next day on sheep blood agar and streak for isolated.... The screw-cap of each dot ( positive, carbohydrate utilization testing should re-streaked. Consists of a nonfunctional recB ET-37 allele carrying multiple missense mutations 30X-50X ) blood. Specimens aseptically the freezer and thawed before use other section for a minimum of 2 ml the CSF of infant! Analysis should be performed nystatin, and Y, as it may produce a color change yellow... X g for 10-15 seconds on chocolate agar plate ( BAP ) and the other for. An aerobic organism, which contained large amounts of mixed microbial flora for... To mix the contents meninges, which grows best at 35-37°C with ~5 % CO, plate! Nearby colonies using a vortex machine ) quantitative genetic studies of Neisseria meningitidis, a lower red line indicates specific... Or neisseria meningitidis on macconkey agar of the four meningococcal serogroups has been performed with congenic meningococcal strains harboring either the wild-type recB or...

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