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Tests to Identify Unknown Pathogenic Bacteria

Paper Type: Free Essay Subject: Biology
Wordcount: 2110 words Published: 18th May 2020

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The purpose of the unknown report was to identify a pathogenic bacteria by applying all the method we have learned in the microbiology lab. It is crucial to identify a pathogenic bacteria in order for the physician to start the appropriate treatment for the patient.

Rob had recently undergone lobectomy two weeks ago. While at the hospital, he has been complaining of new symptoms, including frequent and painful urination. Based on Rob’s symptom, I suspected that Rob might have urinary tract infection by nosocomial from surgery. For instance, he might have infected by a catheter or other medical equipment used during surgery.

According to Metri B.C., et al., urinary tract infection remains to be the most common nosocomial infection which approximately 40% of all hospital-acquired infection. The symptoms of urinary tract infection include a burning sensation when urinating, frequent urge to urinate, cloudy, dark, or bloody urine (WebMD). Urinary tract infection occurs mostly to women because women’s urethra is shorter than men, which bacteria can quickly reach the bladder. However, urinary tract infection can occur to anyone during in hospital by using an infected catheter or other medical equipment.

Based on Rob’s symptom, I am going to conduct a test to find out which bacteria caused Rob’s symptom. 

Results Table



Gram Staining

Simple Stain

Bacillus, diplobacilli

Streak Plate

Contaminated, White Colony formed

EMB Agar

Black colony formed, Lactose fermented

Fluid Thioglycolate

Facultative Anaerobe

SIM Medium:

Indole Production

Hydrogen Sulfide




Phenol Red Broth (Maltose)


Citrate Test


Phenylalanine Deaminase Test

Motility Medium


2nd Motility Medium


Urease Test

2nd Urease Test


Decarboxylation Test (Arginine)

Lysin Decarboxylation Test





The first procedure was to get the pure culture by streak the unknown bacteria into a nutrient agar. First, I used my hands to swirl the tube around because most of the bacteria were at the bottom of the tube, so by swirling the tube will let the bacteria mixed well in the tube. Then, I used a Bunsen burner to sterilize all of my loop and needle for all the test. After I sterilized the loop, I began to collect a bacterium from the unknown broth. I streaked four times on the nutrient agar. Then, I incubated my streak plate for 37-degree Celsius for 24 hours. I examined my streak plate had a creamy white color, round with raised margin, and smell like almond. Then. I performed gram and simple staining to start the identification process. The gram stain and simple stain were used to check the gram reaction, shape, and arrangement of the bacteria (Chess 75). I used my streak plate for gram staining because gram staining was always best to use from a fresh sample. I began the gram staining on the microscopic glass slide. As a result, my unknown bacteria showed mostly pink color; however, I noticed there was a different color in my slide. So, I showed my gram stain picture to Professor Igoe to hear her opinion on my gram stain. She immediately noticed that my streak plate was contaminated and she recommended me to use Eosin Methylene Blue Agar (EMB). EMB agar is a selective medium for the isolation of gram-negative enteric bacteria (Chess 395). Also, the medium could differentiate bacteria with lactose fermented, which showed blue-black colonies (Chess 395). I used my unknown broth to streak into EMB agar, and I incubated for 37 degrees Celsius for about 24 to 48 hours. When I checked my EMB agar, the black colony was formed around the agar. I confirmed that my unknown was gram negative and also lactose fermented. Then, I performed a simple stain of my unknown bacteria. I used the streak EMB agar plate onto the slide. At first, I saw cocci with the arrangement of a streptococcal, short chain. I asked my colleagues to verify my result. Each colleague had a different opinion on my simple stain result. So, I reperformed the simple stain. However, I couldn’t distinguish the shape and the arrangement of my unknown culture. I did a third simple stain, and I finally found the shape and the arrangement of my unknown bacteria. It was a bacillus and diplobacilli arrangement. Then, I tested for Fluid Thioglycollate Medium, which was to distinguish the unknown to be either aerobe or facultative anaerobe (Chess 375). I used my needle to inoculate the tube with the appropriate organism. If my unknown grew on top of the tube will result as aerobe, grew on the bottom will result in anaerobe, and if my unknown bacteria grew in the middle of the tube will result in facultative anaerobe. My results came out as a facultative anaerobe since it was most of the tube was filled with turbid. I performed SIM Medium, which was to differentiate bacteria based on the hydrogen sulfide, indole production, and motility (Chess 415). I used the needle to inoculate the tube by stabbing the tube. After incubated a SIM tube, I found out that my tube had a growth radiated from stab line. Also, the tube formed black precipitation. I couldn’t distinguish the motility of the medium due to black precipitation was heavily formed inside the tube. After I reviewed my tube, I applied Kovacs reagent to see indole production. My SIM tube remained yellow after I added Kovac reagent. So, I had a negative result for indole. I filled out biochemical test paper to perform motility test since I couldn’t distinguish the motility of the unknown. The result showed a test tube that had growth throughout the medium. Afterward, I tested for citrate test. I used a needle to make a single streak up the slant of the test tube. Once the test tube incubated for 24 hours, the result showed that my test tube came out positive, which showed a blue color. From this point, I narrowed my selection of genus was either Citrobacter, Enterobacter, Salmonella, or Providencia. I continued my test with phenylalanine deaminase test. Phenylalanine deaminase test used to differentiate the medium whether my unknown has the enzyme phenylalanine deaminase (Chess 477). I inoculated my unknown media to nutrient agar. After the incubation period was over, I added 4 to 5 drops of ferric chloride. The result came out as negative, which showed a yellow color on the plate. Since I streak my plate with EMB agar came out as positive, my unknown had lactose fermented, which was positive for lactose. Also, I have already done my citrate test, which resulted positive, so I continued my test for urease test. Urease test was performed to determine if an organism to produce a urease which hydrolyzed carbon dioxide and ammonia (Chess 491). I used a needle to inoculate the test tube heavily. The result of the urease test showed that the media remained yellow. From this point, I concluded that my unknown genus would be Enterobacter. I continued my test with arginine. Arginine test was to determine the enzyme that catalyzes the removal of an amino acids’ carboxyl group (Chess 473). I used a loop to inoculate the test tube. Also, I added 3 to 4 millimeters of sterile mineral oil as a reagent. The resulted of the arginine test showed yellow, which was negative for arginine. Afterward, I used a loop to inoculate medium for lysin decarboxylation test. The result of the lysin showed that the tube remained yellow, which was negative for lysin. So, I tested my unknown with Gelatin test. Gelatin test used to differentiate organisms can enzymatically liquify gelatin from those that cannot (Chess 499).  I used a needle to inoculate the test tube with a single stab to the bottom. After incubated the test tube, I sat my test tube into the ice for 15 minutes. The result showed that gelatin became solid. Based on all my results, it pointed out that the unknown organism that Rob had was an Enterobacter intermedius. However, E. intermedius was not a pathogenic microbe, which cannot harm a human. So, I had to do an additional test for my unknown. I retested urease and motility test. I incubated both tests for 24 hours, and I had the urease test result showed positive in which the test tube showed pink. Based on my retest result, I determined that my unknown genus was Citrobacter. Since indole production showed negative from the test, I tested for raffinose test. The purpose of the raffinose test was to see if the organism can ferment raffinose as a carbon base source. I used a loop to inoculate the test tube for three times to see a reaction. Moreover, I added oil as a reagent after the test tube was incubated for 48 hours. The result of the raffinose test came out as a positive, which showed yellow in the test tube.


Based on all the test I have done, now I can conclude that my unknown organism that Rob had a Citrobacter freundii. Citrobacter freundii is a gram-negative, facultative anaerobe, motile bacteria. Citrobacter freundii causes urinary tract infection mostly by nosocomial infection from the hospital. Therefore, Rob got infected by Citrobacter freundii due to infected catheter or other medical equipment that used during surgery. Citrobacter freundii infection can be treated by antibiotic medication such as aminoglycosides, fluoroquinolones, carbapenems, and cefepime (Wang et al.)

Works Cited

  • Chess, Berry. Laboratory Application in Microbiology: A Case Study Approach Thrid Edition. McGraw Hill Education, 2015.
  • Cowan, Majorie Kelly. Microbiology: A Systems Approach Fifth Edition. McGraw Hill Education, 2018
  • Igoe, Jessica. “Urinary Tract Infections.” Microbiology 002, https://canvas.pasadena.edu/courses/1094178/pages/urinary-tract-infections?module_item_id=11325358
  • Metri BC, Jyothi P, Peerapur BV. Antibiotic resistance in Citrobacter spp. isolated from urinary tract infection. Urol Ann. 2013 Oct-Dec;5(4):312-3. doi: 10.4103/0974-7796.120295. PubMed PMID: 24311921; PubMed Central PMCID: PMC3835999.
  • Ranjan, K P, and Neelima Ranjan. “Citrobacter: An emerging health care associated urinary pathogen.” Urology annals vol. 5,4 (2013): 313-4.
  • Wang, Jann-Tay, and Chang, Shan-Chwen. “Citjrobacter species.” Antimicrobe, http://www.antimicrobe.org/b93.asp
  • “Urinary Tract Infection (UTI): Symptoms, Causes, Diagnosis, Treatment.” WebMD, WebMD, www.webmd.com/women/guide/your-guide-urinary-tract-infections#1-3.


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