Identification of Two Unknown Bacteria in a Sample
Margaret Manuel
BIO 253
November 6-18, 2013
Instructor: Professor Sara Houser
Jefferson College of Health Sciences
INTRODUCTION
The objective of this exercise was to isolate and determine the identity of two unknown bacteria, one a gram negative rod, and the other a gram positive cocci, in a simulated clinical urine specimen. In order to achieve this objective, it was necessary to perform all steps in an orderly manner, record observations accurately, and arrive at conclusive identification based on the data recorded. The purpose of this process was to utilize laboratory skills learned during the course, successfully apply them in practice, and arrive at a conclusion through critical thinking. The rationale behind the exercise is that in practice, the same standards of accuracy apply. Accurate identification of organisms in a urinalysis is crucial before a diagnosis can be made. Subsequently, a treatment strategy cannot even begin until the diagnosis is sound. Not only is accurate identification important, but it must be done in a timely manner.
BACKGROUND
A simulated clinical urine specimen was supplied along with various materials for performing diagnostic tests. Detailed instructions of the exercise were provided. In previous weeks, "Identification of Enteric Gram Negative Rods" was an experiment assigned in preparation for the current exercise. A table of results obtained from that exercise was a key diagnostic reference in the current project. The previous exercise had been a group effort. The current project, an individual assignment, is being utilized to evaluate comprehensive lab skills.
HYPOTHESIS
If bacteria species in a clinical specimen are isolated through gram staining, then identity of each can be determined through the results of diagnostic tests.
MATERIALS AND METHODS
November 6th, First Session: A simulated clinical specimen, identified as U11, which was a mixed culture, was provided. As instructed in the exercise, a gram stain was performed in order to observe both organisms.
Next, the following agar plates were streaked with the culture and then incubated at 37 degrees Celsius for 24-48 hours.
Trypticase Soy Agar
Mannitol Salt Agar
Eosin Methylene Blue.
November 7th, Second Session: Examined agar plates for growth. Observations described in RESULTS section. A second gram stain of original specimen was prepared and examined.
November 8th, Third Session: Colonies growing on TSA and EMB plates were observed. Using a swab, re-inoculated MSA plate with U11 and placed plate back in incubator.
November 11th, Fourth Session: Gram stains were prepared again from TSA and EMB plates. A gram stain slide was prepared from small growth on the MSA plate. Photographs and observations documented. A TSA slant was inoculated with gram positive growth from the MSA plate which was then returned to the incubator.
November 12th Fifth Session: Grams stains were again made from EMB and TSA plates. The second TSA slant was then inoculated with the isolated gram negative organism.
November 13th, Sixth Session: Growth in slants and on plates were again observed and recorded. Gram stains were made and data recorded.
November 14th, Seventh Session: Gram stains from growth in slants were recorded.
November 15th, Eighth Session: The following test tubes were inoculated with gram negative growth from the TSA slant:
Glucose, Dextrose, Motility, SIMS, citrate, and Trypt. All tests were returned to the incubator.
November 18th, Ninth Session. Observed results of tests and consulted several textbook and online sources in order to identify organisms.
November 20th, Tenth session, After erroneous identification, prepared to repeat tests as necessary. Re-gram staining samples from TSA slants confirmed isolation. The gram positive slant revealed possible contamination as gram negative rods were observed as well as gram positive cocci. A fresh TSA slant was requested so that a pure stock culture could be established . The MSA plate by this time appeared to be drying out, but a few new specks of white growth were seen. A sample colony was looped from the MSA plate and a gram stain was performed. After isolation was confirmed, the new TSA slant was inoculated from the same colony.
The slant was placed in the incubator. Meanwhile, a loop of sample was obtained from the gram negative stock culture and inoculated into the urea test tube, which had not been previously used.
November 21th, Eleventh session. Previous gram negative tests were reevaluated. Urea test result recorded. Catalane test performed on sample from new Gram positive slant. Based on data and observations of new tests, a second identification of both organisms was made and was confirmed to be correct.
RESULTS
November 6th. Gram stains of the sample U11 revealed only gram negative rods.
November 7th. Growth on agar plates was observed.
Manitol Salt Agar, no growth Eosin Methylene Blue Trypticase Soy Agar
A second gram stain of original sample shows mostly gram negative rods and possibly some
Gram positive cocci can be seen in this slide as well as gram negative rods.
.
November 8th Plates were observed for growth again. TSA shows abundant growth with golden looking, waxy, slightly raised appearance. The growth on EMB is very dark purple, almost black. There is still no growth or evidence of fermentation on MSA plate. As a result, I consulted lab assistant who suggested re-inoculation.
November 11th. Gram staining of TSA and EMB plates continued. There were still enough dark, rounded organisms observed to postpone further testing until isolation of gram negative rods confirmed. The MSA plate now had a pinpoint sized, white colony. A gram stain was done from a portion of the colony which when magnified, revealed gram positive cocci:
A TSA slant was then inoculated with a portion of the colony growing in the MSA plate and placed in the incubator.
November 12th. A gram stain from a colony on the EMB plate finally yielded isolated gram negative rods. As a result, a portion of the colony was inoculated into a TSA slant.
November 13th Gram stains from both slants continue to confirm isolated organisms.
November 14th Gram stains from both slants still confirm isolated organisms.
November 15th No further observations, but results observed from previous days facilitated beginning of gram negative tests.
November 18th Tests for dextrose and lactose were yellow proving fermentation. Motility was negative, showing growth only at the center of inoculation site.
Far left: Dextrose, Lactose and SIMS are negative. Citrate (blue tube) appears negative, but was re- assessed as positive later. Center: Motility is negative. Far right: Indole is negative. I Are negative. Center: Motility is negative.The indole test did not change in color. The SIMS test was yellow. The citrate slant was slightly bluer. The urea test was not performed. A sample of the Gram positive slant did not bubble when catalase was added.
Far left: Dextrose, Lactose and SIMS are negative. Citrate (blue tube) appears negative, but was re- assessed as positive later. Center: Motility is negative. Far right: Indole is negative.
I
Are negative. Center: Motility is negative.
November 20th A faulty conclusion resulted in repeated testing. New growth was observed on the nearly dried MSA plate. A gram stain confirmed the presence of gram positive cocci
A new TSA slant was inoculated from the MSA colony and incubated.
November 21 A sample from the new gram positive slant tested positive for catalase. Gram negative test for urea was positive. Citrate was also re-evaluated as positive. As a result, the organisms were re-classified and confirmed to be correct this time.
DISCUSSION AND CONCLUSION:
The organisms present in sample U11 were the gram negative rod Klebsiella pneumonia and gram positive cocci, Staphylococcus epidermis. K. pneumonia is a common pathogen in urinary tract infection, S. epidermis would be the contaminant, as it is often seen in hospital acquired infections, especially in cases where patients have indwelling urinary catheters.
S. Epidermis seemed difficult to locate in the initial gram stain of U11
It was also slow growing, requiring much more incubation time than the gram negative organism. This factor delayed inoculation of the TSA slants and further testing. Time could have been saved by inoculating the TSA slants as soon as EMB and MSA plates showed growth. EMB is a differential media designed to inhibit growth of gram positive bacteria. The salt in MSA kills gram negative bacteria, thus making it selective for gram positive organisms. In hindsight, isolating the organisms did not have to be such a lengthy process and days of gram staining could have been avoided. Morphology of growth on agar plates offered early clues. The dark colonies on EMB only superficially resembled Escherichia coli, which was the incorrect identification given on November 18. E.coli on EMB has a metallic sheen. The color of the colonies is greenish black. The colonies in this test were dark purple to black and were opaque, not metallic.
Klebsiella on EMB does not have a metallic sheen.
Likewise, the gram positive organism was also incorrectly guessed on November 18. Enterococcus faecalis would have fermented the mannitol on MSA, causing yellow, halo –like colonies to grow. In contrast, S. epidermis grows discreetly and has a white grainy appearance like salt or sugar. Cell morphology of E. faecalis is characterized by small, chained groups. , not the grapelike clusters observed in this exercise. Slow growth during the isolation should have been an indication to allow more time for the stock culture in the TSA slant to incubate. A false negative resulted during the first catalase test, probably caused by inadequate time for the stock culture to grow.
Had this been an actual clinical setting, it would have been a crucial mistake to misdiagnose both organisms. The need for adherence and accuracy is important.
References
Kaiser, G. (2008) Doc Kaiser's microbiology home page. K. pneumonia on EMB. Retrieved … from:http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab3/embklebsiella.html
LeBoffe, M andPierce, B.(2012) Microbiology: laboratory theory and application. . .. .. ……...Morton Publishing. Englewood, CO
Microbiology laboratories: microbes in our world and what they do. Typical results biochemical ……...tests.Retrievedhttp://inst.bact.wisc.edu/inst/index.php?module=book&func=disp ……..layarticle&art_id=123
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