Favour Peters Em Osias

EXERCISE 3D Biochemical Tests (Group 4) Steffi Muriel Justine Millare Oslyn Opilas Jade Mendoza

Jamie Pooten Tanya Philipp

Introduction: For this lab experiment, we are using different culture media to find out the biochemical properties of our given microorganism. We are using Triple Sugar Iron (TSI), Methyl Red (MR), Sulfide-Indole Motility (SIM), Voges-Proskauer (VP), Citrate Test (SC), and Lysine Iron Agar (LIA). Obj: Identify the biochemical properties of each microorganism in each test. Get familiar with each culture media. Questions: 1. A. FW A19 FW A10 FW A18 FW A11 FW IV3 FW SHB4 FW IV4 FW BH5 TSIA Yellow but red slant Yellow but red slant Yellow but red slant Yellow Yellow Yellow but red slant Yellow but red slant Orange but yellow slant MR positive positive Positive Positive Positive Positive Positive positive VP Positive Positive Positive Positive Positive Positive Positive positive SC Negative Negative Negative Positive Negative Positive Negative Positive LIA Positive Positive Positive Positive Positive Positive Positive positive SIM Motile Motile Motile Not motile Motile Motile Motile Motile

B. Procedure Sulfur Indole Motility Test 1. Inoculate a SIM tube with the organism to be tested. 2. Incubate for 48 hours. 3. Add kovacs reagent to the top of the tube, pink/red color formation indicates indole formation. 4. Black percipitate in the medium indicates hydrogen sulfide formation. 5. Growth away from the stab line indicates motility. Triple Sugar Iron Agar (TSI) Test

1. With a sterilized straight inoculation needle touch the top of a well-isolated colony 2. Inoculate TSI Agar by first stabbing through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant. 3. Leave the cap on loosely and incubate the tube at 35 in ambient air for 18 to 24 hours VP Test Using sterile technique, inoculate each experimental organism to the appropriately labeled tube of medium by means of loop inoculation. Incubate the cultures for 24-48 hours at 37C. The experiment should be conducted in the LAF. Arrange the materials required for the experiment in the LAF. 1. Sterilize the loop vertically in the blue flame of the Bunsen burner till red hot. Heat from the base of the wire first and slowly move towards the loop (tip). Heat the wire until it is red-hot. 2. From the rack, take the test tube containing the Tryptic Soy Broth(TSB) cultures that has been kept for 24 - 48 hours. 3. Remove the cap from the TSB tube and flame the neck of the tube.

4. Using aseptic technique take a loop full of the organism from the TSB (tryptic soy broth). 5. Again flame the neck of the tube and replace the cap and place the tube in the test tube rack. 6. Take two sterile MR-VP broth tubes, one named Test and the other Control. 7. Remove the cap of the MR-VP broth tube named 'Test' and flame the neck of the tube. 8. Inoculate the MR-VP broth with the inoculation loop containing the inoculum from the TSB. 9. Again flame the neck of the MR-VP tube and place it in the test tube rack. Inoculate only the broth in the tube named 'Test' using aseptic technique. Leave the broth in the tube named 'Control' uninoculated. 10. Incubate both the tubes (Test and Control) for 24 to 48 hours at 37C. 11. Remove the broths from the incubator. 12. Remove the cap and add 10 drops of Barritt's A reagent and 10 drops of Barritt's B reagent to each broth. 13. Shake gently for several minutes. 14. Red color formation within 15 to 20 minutes is a positive result. No red color formation after 15 to 20 minutes is a negative result.

MR Test

1. Obtain two MR-VP broths from the back shelf.

2. 3. 4. 5. 6.

Inoculate one broth using aseptic technique. Leave the other broth uninoculated (this will be a control). Incubate at appropriate temperature (whatever temperature your organism grows well at). Incubate for two to five days. Obtain your broths from the incubator. Add a dropper full of Methyl Red to each broth. Methyl Red is found in small dropper bottles on the back shelf. Observe the color (which should develop within a few minutes).

SC Test 1. 2. 3. 4. Prepare medium as described above or as directed by the manufacturer. Allow tubes to warm up to room temperature prior to inoculation. Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single isolated colony and lightly streak the surface of the slant. A needle is the preferred sampling tool in order to limit the amount of cell material transferred to the agar slant. Avoid using liquid cultures as the inoculum source. 5. Citrate utilization requires oxygen and thus screw caps, if used, should be placed loosely on the tube.

LIA Test The LIA slant is inoculated by stabbing the agar to within 1/4 inch of the butt of the tube and streaking the slant. The slant is then incubated at 35C for 1824 hours.

C. Positive results TSIA- It is positive if the color change to red or as long as there has a red pigment. MR- It is positive if there is a solid ring on top. VP- It is positive if there is a white solid, meaning there is a presence of bacteria. SC- It is positive if the color changes to blue. SIM- It is positive if there is a white precipitation.

D. Negative results TSIA- It is negative if the color change to yellow. MR- It is negative if there is no solid ring on top. VP- It is negative if there is no white solid, meaning there is a presence of bacteria. SC- It is negative if the color changes to green. SIM- It is negative if there is no white precipitation. 2. A. E.coli- positive acids are produced throughout the tube and the tube will turn yellow, gas is also produced. The Triple Sugar Iron or (TSI) test is a microbiological test roughly named for its ability to test microorganism's ability to ferment sugars and to produce hydrogen sulphide. The positive result given by Escherichia coli is indicated by the red color after the addition of methyl red reagent (MR). (VP) The negative result given by Escherichia coli is indicated by the lack of color change after the addition of the reagents. (SC) A negative result is indicated by a lack of growth and no color change. (SIM) A positive indole result is indicated by the red layer at the top of the tube after the addition of Kovcs reagent. B. S.aureus- It exhibits acidic fermentation and Glucose and lactose and/or sucrose fermentation (TSI). (VP) It color change to pink, meaning that it has butanediol fermentation and can split glucose to acetoin. Color change to red meaning that it can ferment glucose to a mixture of the following organic acids: formic acid, acetic acid and lactic acid (MR). (SIM) It is positive because the growth in citrate medium or growth with color change to blue, meaning it can utilize citrate as the only carbon source. C. B.subtilis- It is negative because the color changed to yellow meaning ti has a pH 6 (MR). (VP) It is positive because it changed to red color indicating the presence of acetoin. (TSI) It has a yellow butt and red slant meaning it is acid positive result. (SIM) It has no red coloration in the surface meaning it is motile. D. N.gonorrhoaea- It is has negative results in all test which means it has a pH6 because the MR results to yellow color change. For VP, the results involve in the absence of red tinge and acetoin. It also has a red hue, representing the indole negative end result for SIM. And as for TSI, it is acid negative result. 3. Based on the results of the biochemical tests performed on th eight unknown bacteria, these are the probable identity: Salmonella typhimurium , Pseudomonas aeruginosa , Streptococcus pyogenes , E.coli, S.aureus, B.subtilis, and N.gonorrhoaea. I chose these bacteria because they are commonly used bacteria in the lab and they match with the given results/data. Discussion: In this lab, we are involved in different culture media, Triple Sugar Iron (TSI), Methyl Red (MR), Sulfide-Indole Motility (SIM), Voges-Proskauer (VP), Citrate Test (SC), and Lysine Iron Agar (LIA). Results were observed after 24 hours of incubation and are interpreted in the following paragraphs along with explanations of the workings of each media type used in this lab. Every bacterial species in every type of media test had growth present. Conclusion:

We identified the biochemical properties of the unknown microorganism that was given to us. Through the culture media, we are able to find these things out. Each test represents as our guide and helped us throughout this experiment. There was a lot of color changing so we have to look closely to each test tube. Always remember to always heat the loop before and after use in order to become sterile. There are a lot of culture media test out there and what we did was just a few of them and these tests can help us in the future experiments.

Reference http://dpuadweb.depauw.edu/~cfornari/F97GR4/discuss.htm http://www.odinity.com/biochemical-activities/ http://www.microbelibrary.org/component/resource/laboratory-test/3203-citrate-test-protocol http://web.clark.edu/tkibota/240/Unknowns/MethylRed.htm http://amrita.vlab.co.in/?sub=3&brch=76&sim=215&cnt=2 http://microbeonline.com/triple-sugar-iron-agar-tsi-principle-procedure-and-interpretation/