5-nitrate.handout

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Feb 20, 2024

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Aaliyah Stevenson, Esha Patel, Nolan Odell, Eesha Parekh Nitrate reduction Case Study for group work In Los Angeles in 1997, a stocked cash drop vault for the Dunbar armored car service was robbed of $18.9 million dollars. The guard was ambushed by 5 masked people who then broke into the vault, emptied the cash into a U-Haul, and drove away. Police tailed the U-Haul until shots were fired from the passenger side window and the police officer driving was hit in the shoulder. Minutes later, a second police car spotted the abandoned, empty U-Haul in a nearby neighborhood. Two individuals were taken into custody for questioning and their clothing was tested for gunshot residue, the small particles that are emitted from the back and the muzzle of a fired weapon. Nitrates are present in fresh gunpowder and nitrites are present in the residue from burned gunpowder. The modified Greiss test, a variation of the nitrate reduction test, is used in crime scene investigations because it does not require a broth solution and will detect the nitrite compounds formed when gunpowder is fired. 1. The clothing from both suspects are subjected to a modified Griess test in the state crime lab. For suspect #1 – there is no color change on the fabric with the addition of solutions A and B (Figure A below). The fabric does turn red when zinc is added. Identify what nitrogen containing molecules are present on the shirt. Can this shirt fabric be used as evidence that the suspect fired or handled a gun? Explain. If the fabric color changes to red when zinc is added, that means that nitrate compound is present in the fabric because addition of zinc reduces the nitrate to nitrite and nitrite reacts with sulfanilic acid. This clearly tells that the shirt fabric can be used as evidence to prove that the suspect fired or handled the gun 2. For suspect #2 – there is a color change on the fabric with the addition of solutions A and B (Figure B below). Identify what nitrogen containing molecules are present on the shirt. Can this shirt fabric be used as evidence that the suspect fired or handled a gun? Explain. If red color comes up after the use of solutions on the fabric, it means it contains some compound of nitrite and as explained nitrite reacts with sulfanilic acid to produce diazotized sulfanilic acid which reacts with a naphthylamine to form red azo dye. The shirt fabric can be used as evidence to prove that the suspect fired or handled a gun. 1
3. When presented with the results of the Modified Greiss test, both of the suspects claim to have been handling ammonium nitrate fertilizers at their jobs with a landscape company. Could this be an explanation for the results in either suspect’s modified Greiss test? Which suspect? Explain. This could be an explanation for the results of the modified Greiss test for suspect B. The red color after the addition of zinc was present on suspect B so there was nitrate present that could have been from the nitrate fertilizer or the gunpowder. 4. You call the suspects employer (a landscape company) and learn that the suspects were fired over a month ago. In light of that and other incriminating evidence, the suspects are formally charged with the robbery and kept in custody. When their hired lawyers begin to build their case, it is discovered that the reagents used in the modified Greiss test were expired 3 days prior to their use. The manufacturer guarantees results on reagents so recently expired but the lawyers ask the state crime lab to demonstrate that the reagents are working properly so the test results can be upheld in court. Because the crime lab is next door to the state infectious disease lab, it’s decided that microbes will be used as positive and negative controls to ensure that the modified Greiss test reagents are working properly. The four microbes listed below were subjected to a nitrate reduction test with fresh reagents. Results of that test are below. Fill in the missing information on each microbe in the table. MICROBE DESCRIPTION (include gram stain, shape, and arrangement) COLOR CHANGE W ADDITION OF A AND B COLOR CHANGE W ADDITION OF ZINC POSITIVE/NEGATIVE FOR NITRATE REDUCTASE? Staphylococcus aureus Cluster Gram positive Cocci yes n/a Positive Pseudomonas aeruginosa Gram negative, rod, single monotrichous arrangement no no Positive Neisseria gonorrhea gram negative, round, diplococci arrangement No yes Negative 2
Select a microbe that is positive for nitrate reduction and a microbe that is negative for nitrate reduction for use in testing the expired reagents in your modified Greiss test. List your selected positive control microbe and describe its ability to reduce nitrates into nitrites or further reduction to molecule nitrogen. List your selected negative control microbe. Positive control microbe: Staphylococcus aureus This microbe is able to reduce nitrates to: nitrite Negative control microbe: gonorrhea 5. The state infectious disease lab agrees to test the positive and negative control microbe in a modified Greiss test with the expired crime lab reagents. The lab technician performing the test sprays the lab bench with ethanol, an extra precaution to protect from Covid which is also studied in the lab, and the labels fall off of the microbe culture tubes. In order to find the which culture is which, the lab tech inoculates both the positive and negative control cultures in thioglycolate broth with casein and yeast extract added to provide a source of nitrogen. List the types of metabolism your positive and negative control are capable of performing and their expected growth pattern in the thioglycolate + nitrogen tube. Type of metabolism (aerobic respiration, anaerobic respiration, and/or fermentation) Expected growth pattern in a thioglycolate + nitrogen tube Positive control microbe: Staphylococcus aureus Anaerobic respiration expected heavy growth on top of the tube, and growth throughout the top but not as heavy as on top. Negative control microbe: Neisseria gonorrhea Relies on aerobic respiration There will be growth only on top of the tube. 3
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The following day, the lab tech looks at the tubes and the positive and negative control growth patterns in the thioglycoate + nitrogen tubes are clearly distinguishable. These control microbes are subjected to the modified Greiss test using the expired crime lab reagents. The expected results are observed for positive and negative control microbe (color change, no color change respectively), proving that the reagents work, the evidence can be used in court, the criminals go to jail, the stolen money is recovered, the lab tech gets a big raise, and the day is saved. 4