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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

and MacConkey agar. possible Gram negative organisms are Enterobacter aerogenes. During this procedure a smeared is prepared by placing a loopful of the unknown organisms to a slide. the gram stain procedure is performed.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. these organisms are some of the leading causes of infections in humans and animals. and Neisseria flavescens. The following are some possible Gram positive organisms that may be identified as the unknown organism: Staphylococcus aureus. slightly pass the . Also. Also. Materials and Methods DAY 1 On day 1. aseptic techniques and procedures will be used to help identify the Gram positive and Gram negative organisms in the mixed culture. it is important to understand the biochemistry and metabolic properties of these organisms. First. Furthermore. One organism will be a Gram positive and the other will be a Gram negative. All of the organisms that are listed above are important because they play a role in the world around us. Proteus vulgaris. Therefore. This report addresses various procedures that will enable one to determine two unknown organisms. S. Columbia CNA agar with 5% sheep blood. the following experiments were performed: Gram stain. epidermidis. it is also critical to understand how they develop and function by using a variety of tests and procedures. Selective and differential media streak plate technique with Tryptic soy agar (aerobic and anaerobic). Before obtaining the organisms use aseptic techniques by flaming the loop before obtaining the culture and letting it cool for about 30 seconds. The unknown that is used in this experiment is the blue test tube. and Enterococcus faecalis.

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

For further determining the Gram negative organism. The following lab will help determine if enzymes or enzymatic pathways is present. for this experiment an oxidase test strip and a slide is needed. After this experiment disinfect the lab bench with Quat 50.Identification of Gram positive and Gram negative Organisms in a Mixed Culture are incubated for 24-48 hours at 37 degree Celsius. Last. the IMVIC series experiment is conducted. The SIM agar deep will be incubated at 37 degree Celsius for 24 hours. This test is conducted by placing a drop of water onto the slide. a loopful of the organisms from the MacConkey agar is streaked onto the slant of Simmon’s citrate medium (remember aseptic techniques by flaming the loop before obtaining the organisms). . obtain a methyl red-Voges Proskauer broth tube. Next. This will determine a positive or negative reaction. A small amount of bacteria is aseptically transferred to the pink to gray area of the strip (McPherson 180). Next. The MRVP tube will be incubated for 48 hours at 37 degree Celsius (199). Next. organisms from the MacConkey agar are transferred (aseptically) and placed onto the slide and mixed with water. Aseptically transfer organisms from MacConkey agar to MRVP tube. Then. 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. the oxidase test is determined for Gram negative. The citrate medium is labeled and incubated for 24 hours at 37 degree Celsius. 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 ¾ of the way to the bottom” (McPherson 199).

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

Using parafilm cover the tube. Also. add 5 to 10 drops of nitrate reagent and nitrate reagent B to each tube (McPherson 212). The oxidase test concluded that there was no change because there was no change in color on the oxidase strip. Apply 0. In next two days further procedures will need to be conducted for completing this experiment. The tubes should be placed in the disposal baskets. on Day 3 the nitrate reductase experiment. There was no growth on the MacConkey agar. There was growth on TSA aerobic and anaerobic plates. Last.2 ml of 40% KOH to the test tube (199). The catalase . 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 50.6 ml of alpha-naphthol solution and 0. The growth on the Columbia CNA agar was gamma hemolysis. Add five drops of Kovac’s reagent to the SIM tube and observe for a red color (199). discard in the appropriate places and disinfect the lab bench with Quat 50. The tubes will be incubated for 48 hours at 37 degree Celsius (McPherson 211). two experiments need further processing. Day 3 Day 3. Transfer 2.5 ml of MRVP broth to a glass tube (199).Identification of Gram positive and Gram negative Organisms in a Mixed Culture negative (MacConkey agar) is inoculated into one nitrate broth with the loop. add zinc dust to tube (212). Results Tests for Gram positive: Gram stain was purple cocci under microscopy. Look for a gas to appear. After each experiment on this day remove labels. Last. If the tube is no color. First. obtain the SIM tube from the IMVIC series experiment. obtain the MRVP broth tube to perform a Voges Proskauer test. Observe and look for a red color. add five drops of methyl red reagent and observe for a red color (199).

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

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

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

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

Elizabeth. Microscopy and a Survey of Microorganisms. 2nd ed. LLC. Introduction.Identification of Gram positive and Gram negative Organisms in a Mixed Culture Works cited Fish McPherson. 37-242. 2011. . Eden Prairie: Bluedoor. Print.

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