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Identification of Gram positive and Gram negative Organisms in a Mixed Culture

Identification of Gram positive and Gram negative Organisms in a Mixed Culture


By Ashley Hathaway
July 6, 2011 Microbiology 210 lab report

Identification of Gram positive and Gram negative Organisms in a Mixed Culture

Introduction
The purpose of this experiment is to identify possible organisms in an unknown mixed culture. This report addresses various procedures that will enable one to determine two unknown organisms. One organism will be a Gram positive and the other will be a Gram negative. The unknown that is used in this experiment is the blue test tube. The following are some possible Gram positive organisms that may be identified as the unknown organism: Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. Also, possible Gram negative organisms are Enterobacter aerogenes, Proteus vulgaris, and Neisseria flavescens. All of the organisms that are listed above are important because they play a role in the world around us; these organisms are some of the leading causes of infections in humans and animals. Therefore, it is important to understand the biochemistry and metabolic properties of these organisms; it is also critical to understand how they develop and function by using a variety of tests and procedures. Furthermore, aseptic techniques and procedures will be used to help identify the Gram positive and Gram negative organisms in the mixed culture.

Materials and Methods DAY 1


On day 1, the following experiments were performed: Gram stain, Selective and differential media streak plate technique with Tryptic soy agar (aerobic and anaerobic), Columbia CNA agar with 5% sheep blood, and MacConkey agar. First, the gram stain procedure is performed. During this procedure a smeared is prepared by placing a loopful of the unknown organisms to a slide. Before obtaining the organisms use aseptic techniques by flaming the loop before obtaining the culture and letting it cool for about 30 seconds. Also, slightly pass the

Identification of Gram positive and Gram negative Organisms in a Mixed Culture culture across the flame to prevent contamination (McPherson 38). Spread the organisms over the slide using the loop. Then, let the smear air dry at room temperature(approximately 15 to 30 minutes); after it is air dried, heat fix smear by passing the slide over the flame three times(McPherson 49). A Gram stain consists of 4 important steps, primary stain, and mordant, decolorize, and counterstain. First, apply crystal violet to culture on slide and wait 30 seconds. With water, wash the slide off for 5 seconds. Secondly, apply Iodine to slide and let the mordant stay on the slide for 60 seconds. Again, after 60 seconds, wash the slide with water for 5 seconds. Thirdly, use ethanol to decolorize the slide by letting it slide over the smear instead of directly on top of the smear. During this step, do not decolorize too much. Last, apply safranin to the slide and let it sit for 60 seconds then wash with water for 5 seconds (McPherson 57). After the gram stain, use bibulous paper to blot the slide dry to use under microscopy with oil immersion lens at 100X magnification. This will help determine the morphology and Gram stain of the organism. Also, on day 1 the streak plate technique is performed, Two TSA plates (1 labeled aerobic and anaerobic), a Columbia CNA agar, and MacConkey agar plate are used. Use a wax pencil to mark 4 quadrants on each plate. Aseptically transfer a loopful of culture to quadrant 1 of the TSA aerobic plate and spread the bacteria over the surface of the agar within quadrant 1. Flame the loop and let it cool for 10 seconds. Drag the loop from quadrant 1 to quadrant 2 and streak. Flame loop again and let it cool for 10 seconds again. Then, drag the loop through quadrant 2 into quadrant 3 and streak. Flame loop and let it cool for 10 seconds. Last, drag loop through quadrant 3 to quadrant 4 and streak quadrant 4 by zig zagging crosses that dont overlap (McPherson 67). Repeat the procedure above using the remaining agar plates. Be certain that the labels are written properly with the condtions and on the correct side of the agar plate. All plates

Identification of Gram positive and Gram negative Organisms in a Mixed Culture are incubated for 24-48 hours at 37 degree Celsius. After this experiment disinfect the lab bench with Quat 50. DAY 2 The Gram stain results and selective and differential media results help determine the next tests towards indentifying a Gram negative and Gram positive. Next, the IMVIC series experiment is conducted. For further determining the Gram negative organism, a loopful of the organisms from the MacConkey agar is streaked onto the slant of Simmons citrate medium (remember aseptic techniques by flaming the loop before obtaining the organisms). The citrate medium is labeled and incubated for 24 hours at 37 degree Celsius. Next, obtain a sulfide-indolemotility agar deep and aseptically transfer a loopful of the organisms from the MacConkey agar and inoculate into the SIM agar deep by stabbing the medium approximately of the way to the bottom (McPherson 199). The SIM agar deep will be incubated at 37 degree Celsius for 24 hours. Last, obtain a methyl red-Voges Proskauer broth tube. Aseptically transfer organisms from MacConkey agar to MRVP tube. The MRVP tube will be incubated for 48 hours at 37 degree Celsius (199). The following lab will help determine if enzymes or enzymatic pathways is present. Next, the oxidase test is determined for Gram negative, for this experiment an oxidase test strip and a slide is needed. This test is conducted by placing a drop of water onto the slide. Then, organisms from the MacConkey agar are transferred (aseptically) and placed onto the slide and mixed with water. A small amount of bacteria is aseptically transferred to the pink to gray area of the strip (McPherson 180). This will determine a positive or negative reaction.

Identification of Gram positive and Gram negative Organisms in a Mixed Culture Furthermore, the catalase test experiment will need a bottle of 3% Hydrogen Peroxide and two glass slides. This procedure is performed for Gram negative and Gram positive. Aseptically transfer a loopful of bacterial growth from the MacConkey agar (Gram -) onto the glass slide. Apply a drop of 3% Hydrogen Peroxide and watch for bubbling to appear. Again do the same procedure except aseptically transfer a loopful of bacterial growth from the Columbia CNA agar (Gram +) onto the glass slide. As stated previously, apply hydrogen peroxide to see bubbling appearance (McPherson 218). Following the catalase test, the urease test is performed. For this test two urea slant agars are needed. Label one urea slant for Gram negative and the label the other slant for Gram positive. Aseptically transfer bacterial growth from the CNA agar and streak the organism on the surface of the urea slant, labeled Gram positive. Again, aseptically transfer bacterial growth form the MacConkey agar and streak the organism on the surface of the urea slant. Both urea slant tubes of Gram negative and Gram positive are incubated for 24 hours at 37 degree Celsius (McPherson 204). For the DNase test, two methyl green plates are needed. Label one for Gram negative and label the other agar Gram positive. Use aseptic techniques and flame the loop until orange. After the loop cools for 30 seconds, spot inoculate the Gram negative organism onto the center of the plate. Again, perform the same experiment using the Gram positive organism onto the center of the plate. Check for labeling and incubate the plates for 24 hours at 37 degree Celsius (McPherson 234). Last, the nitrate reductase procedure will need two tubes of nitrate broth. Label one Gram positive and the other Gram negative. Each organism from Gram positive (CNA agar) and Gram

Identification of Gram positive and Gram negative Organisms in a Mixed Culture negative (MacConkey agar) is inoculated into one nitrate broth with the loop. The tubes will be incubated for 48 hours at 37 degree Celsius (McPherson 211). In next two days further procedures will need to be conducted for completing this experiment. After each experiment on this day remove labels from day 1 procedures of those that are not needed and discard in the appropriate places and disinfect the lab bench with Quat 50. Day 3 Day 3, two experiments need further processing. First, obtain the SIM tube from the IMVIC series experiment. Add five drops of Kovacs reagent to the SIM tube and observe for a red color (199). Last, obtain the MRVP broth tube to perform a Voges Proskauer test. Transfer 2.5 ml of MRVP broth to a glass tube (199). Apply 0.6 ml of alpha-naphthol solution and 0.2 ml of 40% KOH to the test tube (199). Using parafilm cover the tube. Observe and look for a red color. Last, add five drops of methyl red reagent and observe for a red color (199). Also, on Day 3 the nitrate reductase experiment, add 5 to 10 drops of nitrate reagent and nitrate reagent B to each tube (McPherson 212). If the tube is no color, add zinc dust to tube (212). Look for a gas to appear. After each experiment on this day remove labels, discard in the appropriate places and disinfect the lab bench with Quat 50. The tubes should be placed in the disposal baskets.

Results
Tests for Gram positive: Gram stain was purple cocci under microscopy. There was growth on TSA aerobic and anaerobic plates. The growth on the Columbia CNA agar was gamma hemolysis. There was no growth on the MacConkey agar. The oxidase test concluded that there was no change because there was no change in color on the oxidase strip. The catalase

Identification of Gram positive and Gram negative Organisms in a Mixed Culture test resulted in catalase presence (there was bubbles). Results for Urease test showed fuchsia color change so urea is utilized. The nitrate reductase test showed a red color so nitrate was reduced to nitrite. The DNase test confirmed that DNase is produced. Tests for Gram negative: Gram stain was pink rods under microscopy. There was growth on TSA aerobic and anaerobic plates. There was bacterial growth on the MacConkey agar. The oxidase test concluded that there was no change in color on the oxidase strip. The catalase test resulted in catalase presence (bubbles). Results for the urease test showed yellow color so urea is not degraded by the organism, which is a negative reaction. The nitrate reductase test showed no color even after the addition of zinc dust. The DNase test confirmed that DNase is not produced. The citrate permease results showed that the medium changed from green to blue. The SIM agar deep resulted in no red color after adding Kovacs reagent. A Voges Proskaer test resulted in the development of a red color. However, the methyl red test did not have a red color, so the acids did not lower the pH of the medium. A table is listed below that summarizes the results from day 1, 2, and 3 experiments. Gram positive cocci + + +; gamma Gram negative rods + + + + + + -

Morphology TSA aerobic TSA anaerobic Growth of Columbia CNA aerobic Growth on MacConkey Oxidase Indole Methyl red Voges Proskaer Citrate Permease Nitrate Reductase Urease Catalase DNase

+ + + +

Identification of Gram positive and Gram negative Organisms in a Mixed Culture

Discussion
The goal of this experiment was to identify two organisms in a mixed culture. The purpose of using the inoculations techniques was to minimize contamination of the media and organisms. This is known as aseptic techniques in which a Bunsen burner flame was used to incinerate any organisms (McPherson 38).All inoculations were performed to acquire data that suggests enzymes or metabolic properties where present. In order for the bacterial growth to develop, all procedures were incubated at 37 degree Celsius over a specific period of time. The purpose for the incubation conditions was to allow the organisms to grow and react with the different mediums that were used to identify specific biochemical properties. Temperature has a great effect on the organisms growth. After viewing under microscopy of 100X the organisms that I found was purple cocci. With this in mind, I chose this because there were very small purple circles on the slide so therefore my decision was Gram positive cocci. From this procedure, it helped me eliminate the possible unknown organisms, Clostridium perfringens and Corynebacteruim xerosis. The organisms grew on the TSA agars under aerobic and anaerobic conditions. The Gram positive organisms grew on the Columbia CNA agar with a gamma hemolysis. This eliminated the S.aureus possible unknown organism. When I performed the catalase test bubbles begin to appear so this informed me that catalase is present. Next, the urease test showed a fuchsia color change that indicated that urea is utilized. Furthermore, after the urease and catalase test, I was able to conclude that E.faecalis is not the possible unknown organism. The nitrate reductase test did show a red color. A red color indicates that the nitrate was reduced to nitrite. After this test I concluded that my Gram positive unknown is S.epidermidis. However, when I performed the

Identification of Gram positive and Gram negative Organisms in a Mixed Culture DNase test the results were positive. This is suppose to be negative for my chosen unknown. Therefore, an error had occurred when performing this test. The only organism that tests positive for DNase is S.aureus, which was eliminated because of its beta hemolysis pattern. Possible errors that may have occurred when I conducted this experiment are wrongful observation of the methyl green plate and growth on the Columbia agar. In detail, I did not recognize a positive or negative reaction correctly. Above all, I concluded that my Gram positive unknown organism is S. epidermidis. I determined Gram negative by observing pink rods under 100X microscopy. Therefore, possible unknown organisms were E. aerogenes, P.aeruginosa, or P. vulgaris. The organism grew on the TSA aerobic, TSA anaerobic plates and the MacConkey agar. The oxidase test concluded that there was a negative reaction so this eliminated possible unknown organism P. aeruginosa. Bubbles appeared in the catalase test. I did the catalase test to see if catalase was present. The urease test confirmed the unknown because it showed a yellow color which means that the urea is not degraded by the organism. Therefore, my unknown for Gram negative was E. aerogenes. However, I continued to perform more procedures to clarify my Gram negative biochemical properties. After being incubated for 24 hours at 37 degrees Celsius, the citrate permease procedure showed a color change from green to blue, which is seen in the E.aerogenes organism. Therefore, the organism used citrate a carbon source and it contains citrate permease and citrase. I did this test to clarify my unknown to make certain that P.vulgaris is not a possible organism. The following procedures were performed to test for the biochemical properties of E.aerogenes. Next, the nitrate reductase test showed no color even after the addition of zinc dust, so the organism reduced nitrate to ammonia or nitrogen gas. The DNase test confirmed that DNase is not produced and there is no change in the medium. The SIM agar deep resulted in no

Identification of Gram positive and Gram negative Organisms in a Mixed Culture red color after adding Kovacs reagent. Last, a Voges Proskaer test resulted in the development of a red color, therefore, acetoin is present. However, the methyl red test did not have a red color, so the acids did not lower the pH of the medium. Overall, my Gram negative unknown is E. aerogenes. In conclusion, the organisms that were identified are Gram positive S. epidermidis and Gram negative E. aerogenes.

Identification of Gram positive and Gram negative Organisms in a Mixed Culture

Works cited Fish McPherson, Elizabeth. Introduction. Microscopy and a Survey of Microorganisms. 2nd ed. Eden Prairie: Bluedoor, LLC, 2011. 37-242. Print.