Isolation and Identification of Unknown Microorganism

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Bacteria can be found almost everywhere on earth and affects humans/animals in a good and often dangerous forms. In fact we couldn’t survive without some bacteria present in our body helping to digest our food and fighting off foreign microbes. Bacteria were also among the first life forms to appear on Earth. discovered by Dutch Scientist Antonie van Leeuwenhoek during the 1670’s. Proper patient diagnostic testing are very important in the medical field. It makes it possible to identify microbes causing an infectious/ non infectious diseases and to perform relevant and reliable testing to improve patient care and treatments. In order to classify my unknown microbe I will begin by performing a gram stain test which will allow me to narrow down the genus and species based on the metabolism, structure, and other elements.

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I will be examining the possible microb affecting 8 year Jeannie, who visits the hospital with her mother after reporting symptoms of what appears to be food poisoning. Jeannie has been suffering from high fever, diarrhea, and abdominal cramps for the last five days without any improvement. These symptoms appear to be related with a gastrointestinal infection. Bacterial gastroenteritis causes inflammation in your stomach and intestines resulting from poor hygiene, consuming contaminated food or water , or close contact with someone who is already infected. Possible infections related to the

GI include (Salmonella, Shigella, Giardia, E.Coli, Cryptosporidium, Campylobacter, Adenovirus, Yersinia enterocolitica, Clostridium difficile… ect). Symptoms include: diarrhea, vomiting, abdominal pain , dehydration , blood in your stools, fever and loss of appetite. The goal of this unknown report is to identify the unknown bacteria/virus using a variety of tests, applications, procedures and skills.

Identification (Materials and Methods)

The first steps taken in the identification process were to perform a streak isolation on a nutrient plate from the unknown organisms in the culture using the aseptic techniques I learned previously in the lab manual. Streak isolation is used to separate a mixture of bacteria to pure culture. After incubation , isolates were observed and recorded as showing no yellow/ cloudy color. A Gram stain procedure was then performed by transferring a drop of the suspended culture to be examined on a slide with an inoculation loop. After this slide was prepared I added Crystal Violet, Iodine, decolorizer, and Safranin. The colony was identified as gram negative single Bacillus exhibiting rod shapes. I was then able to continue the process by following the appropriate page containing gram negative organisms. Next I wanted to check the aerotolerance of my bacteria by conducting a Fluid Thioglycollate test. Results showed growth throughout the tube, primarily in the middle which determined that is was a facultative anaerobe. These organisms are capable of producing ATP through aerobic respiration and then converting back to anaerobic respiration depending on the environment. Moving along , a SIM test was performed. SIM is a unique process which tests for three different parameters ; (sulfur, indole,motility). The sulfur reduction test is especially useful in differentiating enteric organisms. A large group of gram negative bacteria characterised by a facultatively aerobic are commonly found in the intestines. this information lead me in the right direction for my patient who seems to be infected with a gastrointestinal infection. My result were; Motility positive , Indole negative, and H2S positive. At this point these characteristics are leaning towards Salmonella. additionally I performed a series of differential tests to determine if a certain microbe produces that enzyme needed for carbon and energy source for growth. These tests included; Citrate Utilization, Lactose fermentation, Arginine Dihydrolase, Lysine Decarboxylase and Phenylalanine Deaminase.

My results were observed and recorded; Citrate (+), Lactose (+), Arginine (+), Lysine Decarboxylase (-) and Phenylalanine Deaminase (-). The Citrate Utilization test was performed to limit and seperate my potential unknown into a category of species. I was limited down to; Citrobacter, Enterobacter, Salmonella, and Providencia. Organisms which can utilize Citrate as their exclusive carbon source use the enzyme Citrate-Permease to transport the citrate into the cell. These organisms also creates an Alkaline environment in the medium by converting the Ammonium Dihydrogen Phosphate to Ammonia and Ammonium Hydroxide (unknown). My observed results indicated positive when the medium turned a light blue color. Not being able to fully distinguish between the light blue and green , I purposely re-incubated my medium in hopes of seeing the color more effectively. After the following day I was positively able to determine this color to be very dark blue for citrate. My next series of tests were conducted in order to separate members of the species Proteus, Morganella, and Providencia from other species of Enterobacter, Salmonella, and Escherichia. This was done with the Phenylalanine Deaminase Test. After incubation 5 drops of Ferric Chloride was added to the media and if phenylpyruvic acid was produced, it would react with the ferric chloride and turn dark green. My results indicated negative as the medium remained a straw yellow color. This tests lead me to the Enterobacteriaceae species which ultimately started my search for the causative agent affecting Jeannie. Moving along I continued by conducting the Arginine Dihydrolase test which is used to see if the microbe can use the amino acid Arginine as a source of carbon and energy for growth (Unknown). My results were observed and recorded as positive for Arginine Dihydrolase as the medium changed to a purple color.

As my experiment came a to a close I was faced with a difficult decision during my identification process. because class time prohibited and limited the process, I was unable to conduct the last steps which would have been the Gelatin Hydrolysis Test. This is used as a differential medium that tests the ability of an organism to produce Gelatinase an exoenzyme , that hydrolyzes gelatin. This test required longer than usual time of incubation for more sufficient results. It is also helpful to note that gelatin is a commonly known component of several foods like marshmallows, jello, gummy worms, frosting, M&M’s …. Ect. but mostly it’s a protein derived from connective tissue. Ultimately I was able to narrow down between Enterobacter Cloacae and Enterobacter Amnigenus. After careful research amongst the two I was able to identify my causative agent as Enterobacter Cloacae which is the most common of all Enterobacter Genus causing gastrointestinal infections. It also showed similar results to my own observations.

Enterobacter Cloacae

The Genus Enterobacter are a group of gram negative bacteria arranged in rod shaped Bacillus belonging to the Enterobacteriaceae family. This bacteria is classified as a facultative anaerobe which means they are able to survive in both Aerobic and Anaerobic environments. Also presenting significant motility. Their ubiquitous in nature allows them to be present in most animals often resulting in worldwide distribution in soil, sewage, water, vegetables and in the feces of healthy persons. In humans, Enterobacter species acts as opportunistic (disease-causing) pathogen. In many cases , Enterobacter Cloacae and Enterobacter Aerogenes is associated with nosocomial acquired, causing opportunistic infections in hospitalized patients or those with a debilitated immune system (Rogers). it is possible that Jeannie’s symptoms worsened after visiting the hospital and her symptoms continued to last for five days.

E. Cloacae is among the most common of the enterobacter species. Enterobacteriaceae have been associated as a foodborne pathogen frequently causing illness in neonates and children between three to fours years old. This is due to spoilage of foods and drinks (milk, dairy products, meats, poultry, fish, seafoods, fruit, vegetables…ects) which makes it a possibility as to why Jeannie’s mother first believed that she was suffering from food poisoning. The growth and metabolic activity of Enterobacteriaceae in food will result in off flavors, odors, and other deviations due to the enzymatic breakdown of proteins or lipids, production of nutrients and gas production (Baylis 29). Infections of the gastroenteritis such as eating contaminated foods can lead to abdominal cramps, diarrhea and even fever. All the signs that Jeannie experienced. Treatment options for Jeannie include Antibiotics, Intravenous (IV) Therapy, Dietary supplement and even Surgery. According to the John Hopkins Point of Care Information Technology Center, Enterobacter cloacae has a tendency to be resistance during the course of the treatment, therefore at least two antibiotics should be prescribed simultaneously. Commonly used antibiotics include, Aminoglycosides and Ciprofloxacin. For more serious infections, antibiotics should be administered every six to eight hours until fever retreats, after which the drugs may be given orally. For mild infections, oral administration every six to eight hours for at least 14 days can help cure the infection (Hopkins). Patients whose bloodstreams are affected by E. Cloacae can lead to low blood pressure can be given IV therapy and surgery depends on the severity of the infection. Some obvious preventions of this infection would be to practice proper hand washing techniques and monitor the temperature , PH and atmosphere of refrigerators or anywhere food is stored.


The identification of an unknown is such an important process when diagnosing patients and setting up treatment form them. After comparing my data and results from all tests along with the morphology, shape and gram staining I was able to come to a conclusion that the solution in the Unknown #15 test tube is Enterobacter Cloacae. Though the Gelatin test set me back a bit , I was able narrow down possible solutions that lead to Gastroenteritis. 8 year old Jeannie presented signs of ingesting contaminated food and treatment of prescribed medications will help often blocking the acid in the stomach.

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