Isolation and identification of unknown bacteria - Lab Report Example

?ISOLATION AND IDENTIFICATION OF UNKNOWN BACTERIA INTRODUCTION Identification of bacteria as a causative agents of diseases, epidemics, contaminated food and other sources of infection and illness is a process that has growing significance in the modern world. Bacteria are a causative agents for many diseases in humans and domestic animals that and can be a cause of significant health impact on the human population (Scallan et al. 2011). Bacteria also can contaminate and impair the process of food production and packagind that can lead to significant economic impact but more importantly can be a source of more widespread morbidity in human population and a mechanism of generation of widespread epidemics. With the appearance of multiresistent bacteria that are not responcive to a range of antibiotics it becomes necessary to identify the exact causative agent in order to identify the most effective antibiotic treatment (Bennett 2008). Identification of bacteria is also important in cases of food poisoning, infective diarhoea and other gastrointestinal diseases because by identifying the bacteria we can find the reservoir and the mechanism of contamination and therefore prevent any further morbidity and contamination. In our present project we will receive a pure culture of unknown bacteria and using the techniques and procedures that we learned in the lab we will try to identify the underlying microorganism. This procedure will help us practically learn the procedure and technique of identifying unknown bacteria. By this process we will understand the importance of accurate identification of bacteria and the necessary materials and methods that are necessary for this process. There are newer procedures of rapid identification of bacteria like mass spectrometry and computional analysis, microarray-based identification of bacteria and other that allow faster and affective method of identifying bacteria and other microorganisms however thay are much more expensive and require expensive machines for this process (Honisch et al. 2007) (Small et al. 2001). MATERIALS AND METHODS Aseptic technique We used standardized procedures in the process of cultivation of bacetia. We received pure culture of unknown organism. Using Bunsen burner the loop was sterilized by heating the entire wire into the flame of the burner until it glows with bright red or orange color and afterwards the loop was left to cool. The culture of uknown bacteria was held in inoculation tube in a form of liquid culture and the inoulation tube was sealed with sterile cap.. We maintained aseptic conditions during the handling of the culture. During the whole process the caps and the tubes were held in hand were not allowed to be contaminated by contacting the table or other source of environmental bacteria. The openings of the inoculation tube was sterilized by burning with the Bunsen burner twice in order to avoid contamination with environmental bacteria. With the cooled loop, carfully not to touch the sides of the tube we collected one loop of material and after that the edges of the tube were again burned with flame from the Bunsen burner and the caps were put . The loop was then inoculated on two separate TSA plates. The lid of the plates was opened with free hand and the material was seeded with gentle strokes of the loop using T streak method in order to allow optimal contidions for growth. The lid was then put back on the plates and the loop was again sterilized by burning until glowing bright red on the Bunsen burner. Gram stain For completing the Gram stain we needed Gram stain reagents (crystal violet, Gram's iodine, 95% ethyl alcohol, and safranin), microscope slides and bacterial cultures. In preparation of the Gram stain asepttci principles were used. Inocultion loop was sterilized by burning on a Bunsen burner until bright red color appeared. Five loop of the unknown colony were placed on a microscope slide and the material was sread even on the surface of the slide. The material was left to air-dry and room temperature. After this the slide was passed through flame of the Bunsen burner to fixate and kill the bacteria. By this process the bacteria are fixated on the glaas of the slide and the remaining procedures will not wash away the material. After cooling of the slide the process of Gram stain continued on a staining apparatus over a sink. First the slide was covered with 3-4 drops of crystal violet and was left for 30 seconds in order to allow the color to diffuse into the bacteria. Using clothespin the slide was gently rinsed in tap water and than shacked in order to remove the excess stain. After this the slide was returned on the stain apparatus and was covered with Mordant Gram’s iodine solution and left for 60 seconds. The slide was again gently rinsed in tap water and the excess water and dye was shaken of the slide. After this step the slide 95% alcohol was poured down the slide until the solvent was running clear in order to decolorize the slide and remove excess dye. We placed white paper towel under the slide in order to visualize stain running off of slide and the slide was rinsed in tap water and then was counterstained with 3 drops of safranin. The stain was left to fixate for 30 seconds. At the end the slide was again rinsed in tap water and excess water was shaken off (Penn, 2008). Isolation streak technique With isolation streak technique was used in order to isolate the unknown bacteria on a TSA medium that can further be analyzed for any unpurities, contamination and as a source for the consequent analysis that we will condust. Using aseptic techniques, a loopful of bacteria was removed from a pure culture. Before this step the loop was sterilized on a burning flame untl bright red hot. The lid of the Petri dishes was lifted out of the way, and keep angled over the dish. On the back of the dish 3 quadrants were drawn using a grease pencil. The loop was gently pressed on the agar surface in the first and larges section with enough pressure not to dig into the agar surface and the loop was gently swept back and forth across the agar surface on the top, outside edge of the dish. In a zig-zag manner the material was spreading the sample out in section. The loop was again heat-sterilized and allowed to cool. Then from the sterile part of the agar in the second quadrant the loop was swept in a zig-zag maner across the agar surface from section second quadrant into the first quadrant and back in one step without repetition the process. After this process the loop again was heat-sterilized and allowed to cool. Then again the loop was placed on the sterile part of the agal in the third quadrant and then was gently stroked on the agar surface, returning several times into the second quadrant and back until there was no space in the third quadrant. The dish was again covered, the loop was heat-sterilized and the dish was inverted and incubated. This same process was conducted on two different Petri dishes and the dishes were incubated in two different temperatures. One at room temperature and the other at 36 degrees Celsius for a period of 48 hours. Isolation In order to isolate the unknown bacteria from the incubation tube two Petri dishes were inoculated using T strike technique using aseptic techniques and they were incubated one at room and one at temperature of 35 degrees in a period of 48 hours. After the process of incubation the plates were examinated for favored bacterial growth. Bacterial growths on the second and third quadrant with favored bacterial growth, were then again obtained with a sterile loop and using t-streak technique were again inoculated on 2 TSA plates. The TSA plates were then incubated in separate conditions one at room temperature and one at 35 degreees Celsius for 96 hours. (Penn, 2008). Oxidaze test: In the oxidaze test the unknown bacterial colonies were tested for presence of cytochrome Oxidase activity over a prefabricated dye (tetramethyl-p-phenylenediamine). A sterile, cotton-tipped wooden spatula was immersed into a fresh dye solution and the excess dye was remowd by pressing the tip of the spatula against the side of the dye container. After this the cotton was pressed on on of the colonies located on the Petri dish and the results of the color change was then recorded on the surface of the cotton on the wooden spatula. Change in color within 10-20 seconds after the contact withthe colony was recognized as a positive oxidaze test. Lactose fermentatnion test: In the lactose fermentation test, one MakConkey agar slant was used. In the process of inoculation inoculums from the TSA slant was used using aseptic techniques. Inoculation loop was sterilized by heating on flame until bright red color. After the cooling of the loop a small colony of the TSA plate with the unknown bacteria was taken wis the sterile loop. Immideatelly the loop was striken of the MakConkey agar in one area. After this process the inoculation loop was sterilized by burning and after it cooled from the edge of the first area the material was striken on a second area of the plate. This process was repeated 3 time creaing 3 areas with different concentrations of material. The inoculated plate was then inverted and incubated at 37 degrees Celsius for 48 hours (Penn, 2008). Urea broth test: In the Urea Broth test, one Urea agar slant was used. In using inoculums from TSA slant solid culture, an inoculating needle was sterilized, allowed to cool and then stabbed into the Urea agar slant, not touching the butt. As the needle was taken out, the slant was also streaked. The inoculated slant was then incubated at 37 degrees Celsius for about 24 hours (Penn, 2008). RESULTS In performing the isolation process, it was certain that isolation was achieved, as any gram stains performed on the unknown bacteria were very apparent to have a majority of Gram-negative encapsulated rods. The colonies formed on the TSA plates also indicated isolation as all colonies had same characteristics throughout the TSA agar. The characteristics shown by the bacterium through colony formation, was that of an opaque muccoid appearance without sign of swarming. Colony size ranged from small to larger and muccoid, with viscis consistency, drop like and smooth forms. DISCUSSION Gram stain The Gram stain helps to distinguish between Gram positive thick-walled bacteria with up to 40 layers of peptidoglycan) and Gram-negative (thin-walled bacteria containing one to two layers of peptidoglycan) bacteria in the unknown pure culture. The results from the Gram stain indicated that the unknown bacteria is a Gram-negative bacterium, as it didn’t retained the crystal violet primary stain after the ethyl alcohol application. The unknown bacteria, in being Gram-negative, contains a thin wall of peptidoglycan that may be comprised one or two layers of this particular sugar and amino acid polymer. This is why this particular bacterium cannot hold the primary stain so well. Crystal violet stain is likely to wash away due to small permeation into the peptidoglycan layer (Penn 2008). Oxidaze test: The purpose of the citrate test is to differentiate between enteric bacteria that can utilize oxygen for energy production with an electron transfer chain. Our sample unknown bacteria was oxidaze negative therefore means that the bacterium does not contain cytochrome c oxidase and therefore cannot utilize oxygen for energy production with an electron transfer chain. Oxidaze test help us to conclude that the bacteria is propably from the family of enterobacteriaceae, because usually oxidaze negative bacteria are members of this family. We also can conclude that we need to complete other tests in order to further differentiate these unknown bacteria. Lactose fermentatnion test: The purpose of the lactose test test is to differentiate between enteric bacteria that can metabolize the lactose and produce change in the Ph of the medium of can produce some other end products of this metabolism like H2S that can then be identified. On MakConkey agar by utilizing the lactose available in the medium, lactose positive bacteria (such as Escherichia coli, Enterobacter and Klebsiella for example) will produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies. The bile salts precipitate in the immediate neighborhood of the colony, causing the medium surrounding the colony to become hazy (MacConkey 1905). The unknown bacteria obtained from the TSA agar was lactose positive which means that it can utilize lactose. This can help us in identification of the unknown bacteria and based on the appearance of the colonies, Gram stain, oxidaze test we can suggest that the unknown bacteria is from the Klebsiella spp. but there are other bacteria that are gram negative, oxidaze negative, lactose positive like Escherichia colli or Enterobacter aerogenes. That is why we need to do additional tests in order to confirm our findings. Urea broth test: Our Urea Agar Slants can be used to aid in the Recommended Procedure differentiation between members of the Enterobacteriaceae family based on their ability to hydrolyze the substrate urea . Urease is an important microbial enzyme concerned with the decomposition of organic compounds. Urease can act on the test substrate, urea, contained in the medium resulting in the release of ammonia. The release of this alkaline end product results in an up shift in the pH that is detected by the color indicator. The unknown bacteria that we tested were urease positive. Based on the previous tests that we conducted and the positive urease test we can also conclude that the unknown bacteria are Klebsiela Pneumoniae. Final Gram Stain In the final Gram stain there were no signs of contamination and it was very apparent the presence of Gram negative rods without signs of any other bacteria. There were many spores appearing as crystal particles on the Gram stain slide. The bacterium appeared the same as seen on the first Gram stain: Gram negative and had morphology of bacilli. Errors In doing the urea broth test we incubated the slants for a period of 24 hours. However there are bacteria that give rapid urea test what is strongly positive even after couple of hours like Proteus spp. and bacteria like Klebsiela or Enterobacter which manifest urease positive phenotype after incubation of 6 or more hours. This was another tool that could be used to specify the unknown bacteria and maybe help us identify it with smaller number of tests. However this did not affect the results because we positively identified K Pneumoniae based on the previous tests. CONCLUSIONS In conclusion to the project, it is believed that unknown bacteria contained the specimen Is Klebsiela Pneumoniae, which was concluded through the tests and indications like colony morphology and growth. Klebsiella pneumoniae is a Gram-negative bacteria without capability of motion, produces a capsule. On biochemical tests, it is lactose positive, facultative anaerobic oxidaze negative bacteria, ureaze positive rod shaped bacterium. In humans most commonly is found in the normal flora of the mouth, skin, and intestines and usually doesn't cause morbidity. It is the very member of the Klebsiella genus of Enterobacteriaceae and has great importance in clinical practice because it can be causative agent of multiple conditions and diseases (Ryan and Ray 2004). It can cause pneumonia in humans that can cause severe inflammation and destruction of the lungs. Pneumonia caused by Klebsiela pneumoniae is characterized by necrosis of the lung tissue, hemorrhagic, thick sputum with severe general condition of the patient. It can also cause infections f the urinary tract as cystitis, pyelonephritis, prostatitis and other diseases. This bacteria can cause really a wide range of conditions in the veins (thrombophlebitis), gastrointestinal tract (noncalculous cholecystitis, diarrhea, gastroenteritis etc.), in the bones (osteomielitis), in the nervous system (meningitis) and other tissues. This is why is very important to be able to accurately identify bacteria in order to start appropriate treatment. This is also important because as member of the enterobacteriacae family it is prone to developing antibiotic resistance and there is need to find the most effective treatment using antibiogram (Lledo et al. 2009).Klebsiela pneumonia is the most important member of the Klebsiella spp. and finding new and effective means of fast identification can help finding prompt and effective treatment for some of the conditions that this bacteria causes. References: Bennett, PM (March 2008). "Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria". British Journal of Pharmacology 153 (Suppl. 1): S347–S357. doi:10.1038/sj.bjp.0707607. PMC 2268074. PMID 18193080. Scallan E, Griffin PM, Angulo FJ, Tauxe RV, Hoekstra RM (2011). "Foodborne illness acquired in the United States—unspecified agents". Emerging Infectious Diseases 17 (1): 16-22. doi:10.3201/eid1701.P21101. Honisch C, et al. (2007) Automated comparative sequence analysis by base-specific cleavage and mass spectrometry for nucleic acid-based microbial typing. Proc Natl Acad Sci U S A 104: 10649–10654. Small, J., D. R. Call, F. J. Brockman, T. M. Straub, and D. P. Chandler. 2001. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays. Appl. Environ. Microbiol. 67:4708-4716. MacConkey AT (1905). "Lactose-Fermenting Bacteria in Faeces.". J Hyg (Lond) 5 (3): 333–79. PMID 20474229. Penn, J, 2005, Laboratory Manual for Topics in Biology. Shoreline Community College, pp. 37-119. Lledo W, Hernandez M, Lopez E, et al.(2009), Guidance for Control of Infections with Carbapenem-Resistant or Carbapenemase-Producing ‘’Enterobacteriaceae’’ in Acute Care Facilities. March 20, 2009 / 58(10);256-260 Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0838585299. Read More