Techniques for the Identification of Unknown Microorganisms

Techniques for the Identification of Unknow Micro beingnesssIdentification of unvalued MicroorganismsFor years and years, we provoke been trying to classify and understand microorganisms, and in doing so, we learned of certain techniques to draw them establish on what they look like, where they live, and what they eat. We employ this knowledge to derive straightforward tests to make a motion, and we apply a name to sign on down our choices, all in an effort to report a microorganism. Beginning in the early labs with the earliest microbiologists, the most putting green way to identify an organism is to look at it and look at what it does. In microbiology of bacteria, is it round (cocci) or rod- physiqued (bacilli)? Does it patsy purple ( constant of gravitation +) or rose-cheeked ( guanineme -)? Does it use milk sugar as food for thought, era creating an acidic product that fag be ascertained? These tests ground on the physiology/morphology and biochemistry fall u nder what is cognise as classic denomination1. Classic techniques focus on the ability of a bacterium to rear and re clear in certain conditions, such as the fact that insolence salts brush aside interact unfavorably with the peptidogly flush toilet cell walls of yard official bacterium, wherefore selecting against their initiateth. Most tests atomic number 18 used to specifically select for or oppose amidst the species, this in conjunction with a dichotomous key helps narrow down the choices until one is satisfactory. In contrast, modern methods of identification are based on the technological advances of recent years, namely, polymerase chain reaction (PCR) and genetics. The use of PCR to identify bacteria is rooted in the study of genetic coding, in sliceicular, ribonucleic acid. all(prenominal) bacterium has strands of RNA that is used to bind the mRNA for translation the 16s ribosomal RNA (rRNA)2. This sequence is conserved in all bacteria and can be used to ide ntify species after(prenominal) amplification through the PCR wreak. Since every species has a different genetic make-up, it is simply a matter of time for identification. exclusively for most purposes, getting samples to a PCR and running them takes quite some effort, and a lot of money, thus the simpler and more efficient method is the classical testing.At one point or another, we all have gotten the sore pharynx and cough. These symptoms are sometimes evidenced by microbes in the pharynx that shouldnt be there. bacteria such as Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhalis can infect the area and fount this sore feeling. A more serious offender, Streptococcus pyogenes, take a shits what is conveningly known as strep pharynx. These bacteria make the pharynx sore by harvesting erythrocytes and breaking them down as sources of food, in turn, destroying the lining and create the all familiar sore feeling1. A simple test on a blood supplemented agar-agar-agar helps reveal what source of pathogen is cavictimization the sickness. The agar is inoculated and incubated until growth is seen, and the plate pass oning show a pattern of hematolysis if compositors cased by one of the bacterial pathogens. Beta hemolysis is the ability to richly metabolize the cell and hemoglobin and will produce a move in sinker nearly the colonies. Alpha hemolysis is the partial breakdowns of hemoglobin into billiverdin, devising the halo appear scandalmongeringish/green. Gamma hemolysis is the absence of halo, as it means the organism cannot lyse erythrocytes1.ProceduresRespiratory dab assimilate a sterile cotton swab and swab the tonsilary section immunise blood agar by rolling the cotton tip in a ladder plate methodAfter the initial line, discard the swab in biohazard bin, and continue with a flamed inoculation loopSecond swabTake a sterile cotton swab and swab the tonsilary regionPlace the swab in a test tube weaken to 10-3 and 1 0-4Make a pour plate with nutrient agarUnknown identificationTo start to identify both unknown organism by classical methods, it is necessary to create an appropriate dichotomous key to be the tests. The first step to any key is the gram grease this fundamentally splits the frame of possible organisms in half, gram positive and negative. Further tests based on morphology and biochemistry will select and differentiate surrounded by common characteristics of species until one organism stands forth. At the start of every lab day, pert streak plates and broths are prepa bolshie to keep the organism isolated and fresh.Take a single village and prepare streak plates, one for 37C and one for 25C arrange broths aseptically in mistakable fashionDichotomous keyThe dichotomous key was prepa inflamed using only the organisms on the list provided by Dr. Maxwell.Gram shiteThe gram stain separates bacteria into two main groups, gram positive and gram negative. The peptidoglycan cell walls of gram positive organisms will absorb the quartz glass regal stain and appear purple under microscope inspection. The gram negative bacteria lose their outer membrane during the de alterization step and will absorb the safranin stain, appearing red.Prepare specimen by taking an isolated addiction and heat fixing on a swoop obligate crystal royal stain for 60 secWash with waterApply iodine fixer for 45 secWash with water clean with de colour inizer for exactly 8-10 secWash with waterApply safranin for 60 secIf gram positive, the next step in my dichotomous key is to differentiate based on morphology is it a cocci or bacilli? This step will split the organisms by shape, essentially cutting the number in half.Using the gram stain drop off, look at the shape of the specimen under 100x rock oil immersionBacilli are rod-shapedcocci are roundIf bacilli, run the catalase test. The catalase test will reveal if the organism has catalase, an enzyme used to breakdown ill oxidizing a gents such as total heat peroxide, which could be made as a result of metabolism. A drop of hydrogen peroxide is added to the specimen placed on a slide. A positive result will arrest bubbles to form, indicating presence of catalase.Put a colonization on a slideAdd hydrogen peroxide maintain bubbles/no bubblesIf catalase negative, as in no bubbles make, the organism is Lactobacillus acidophilus. The bacteria are part of healthy usual flora and aids in digestion3.If catalase positive, the endospore stain must be used for further classification. The endospore is a tough spore formed by some bacteria during harsh surrounds. The spore will be non-white green in contrast to a cell dyed red.Prepare a specimen by heat fixing on slideCover the slide with bibulous paperAdd methyl groupene downheartedPlace slide on steamer over a etna burnerSteam for 7-10 minutes, applying additional methylene blue devil to prevent the paper from ironicaling outRemove slide and paperCool for no mo re than 30 seconds and rinseCounterstain with safranin for 1 minuteRinse and blot dryIf endospore positive, the bacteria is Bacillus subtilis. The organism is lay out readily in the skank and can form a tough endospore when conditions become unfavorable. Bacillus is also a great model organism for gram positive studies4.If endospore negative, the organism is Corynebacterium diphtheriae. This bacterium is the cause of diphtheria, a respiratory infection.If the organism observed is cocci, run the catalase test. The catalase test will reveal if the organism has catalase, an enzyme used to breakdown harmful oxidizing agents such as hydrogen peroxide, which could be made as a result of metabolism. A drop of hydrogen peroxide is added to the specimen placed on a slide. A positive result will cause bubbles to form, indicating presence of catalase.Put a colony on a slideAdd hydrogen peroxideObserve bubbles/no bubblesIf catalase negative, the organism is Enterococcus faecalis. It is commo nly instal in the digestive tract of reality and is considered part of the normal flora. It does not produce catalase.If catalase positive, the use of mannitol salt agar is used to differentiate between the staphylococcus and micrococcus bacteria. Mannitol salt agar is used to select for staphylococcus. Staphylococcus aureus will produce a yellow halo. immunise the MSA plate with a isolated colonyIncubate at 37C for 48 hoursObserve growthIf a yellow chromaed halo around the colony is observed, it is either S. aureus white or S. aureus gold. The way to differentiate between the two is the color of the colony itself, which severally is, white or golden (yellow). S. aureus is part of the normal flora and what is the usually cause of infections and pimples.If the MSA plate shows no yellow halo, then it is either genus Micrococcus roseus or Staphylococcus epidermis. The way to differentiate between them is the color of the colony itself. Micrococcus will appear red in color collecti ble to a blusher it secretes5, while S. epidermis will be a white color. Both are normal skin flora.If the organism is gram negative, the first test to run is the lactose supplement phenol red broth. The purpose of this test is to indicate whether or not the organism can use lactose as a food source. Fermenting lactose will produce an acidic waste and cause the phenol red to revision color. A positive test will turn yellow.Inoculate a tube with an isolated colonyIncubate at 37C for 48 hoursObserve color changeIf the organism cannot enforce lactose, then perhaps it can use glucose as a food source. The glucose phenol red broth tests for a similar metabolic process as the lactose one.Inoculate a tube with an isolated colonyIncubate at 37o C for 48 hoursObserve color changeIf the bacteria are glucose negative, then it is either genus Pseudomonas (Burkeholderia) cepacia or Pseudomonas aeruginosa. The two can be differentiated by the pigment of the colonies. P. cepacia will appear a l ight purple color while P. aeruginosa will be white, and also produce a green pigment known as pyocyanin6.If glucose positive, a turn peddle must be prepared. The citrate pitch tests for the ability to use citrate as a nose candy source. A positive result will turn the green slant blue.Inoculate the slant with an isolated colonyIncubateObserve colorIf citrate negative, the bacteria is Proteus vulgaris. It is a bacterium that normally inhabits the intestines of humans and in certain cases can cause infections, albeit in most cases of immunocompromised individuals.If citrate positive, the organism is Serratia marcescens. It also produces a red pigment that can be used to identify it. The bacteria are found throughout environments and can be observed upon bathtubs that dont get washed often7.If the organism can ferment lactose, then the citrate test is prepared. The citrate slant tests for the ability to use citrate as a carbon paper source. A positive result will turn the green slant blue.Inoculate the slant with an isolated colonyIncubateObserve colorIf citrate negative, the bacteria is E. coli. This is one of the most used model organisms for gram negative studies in labs. The rod shaped bacteria can cause some food poisoning if ingested. But the best use of it is in the labs, as it is easy to care for, replicates fast, and genetically simple, allowing for genetic research8.If citrate positive, the bacteria could be either Enterococcus or Citrobacter. The test to differentiate between the two is a Methyl red Voges Proskauer media. The MR-VP is used to differentiate bacteria based on the ability to hydrolyze dextrose and create lasting organic acids during fermentation. The MR-VP test combines the two separate tests. The positive result for the methyl red test is a red color when the methyl red indicator is added to the media. The positive result for the Voges Proskauer test is a red color.Inoculate two MR-VPsIncubateAdd the MR reagent to one tube and o bserve any color change every 10 minsLook for any color change for the VP test before addition of reagentIf MR-VP positive, the organism is Enterobacter. E. aerogenes can cause infections and is considered pathogenic however it still does exist as part of the normal flora of the human intestines.If MR-VP negative, the organism is Citrobacter. They are ubiquitous in the environment and also inhabit the intestines.ResultsRespiratory swabsThe streak plate to identify organisms with hemolytic abilities showed growth of many different bacteria morphologies. Green and white colonies were observed to be growing on top of the plate. The media remained red throughout no indication of any white spots. The pour plates for were both too numerous to count.Unknown organismsThe organisms were isolated using a streak plate. Two isolations were obtained at two temperatures, 25o C and 37o C. the plate at manner temperature showed red pigmented colonies (B), while the body temperature plate showed th ick white colonies (A). A third unknown was given pre-inoculated (C).Three biochemical tests and two structural tests were used to identify the first unknown organism after isolation. A gram stain revealed that the organism was gram positive being violet under the 100x oil immersion lens. Also while under the microscope, it was revealed that organism A was cocci. A catalase test was done and organism A was compulsive to be catalase positive, producing bubbles after addition of hydrogen peroxide. The unknown bacteria were inoculated on a mannitol salt agar, which showed the halo, a positive result. To identify between the two staphylococci aureus, the pigment of the colonies was used white.A gram stain revealed that organism B was gram negative red under the 100x oil immersion lens. A lactose and glucose tube was inoculated in the same geological period to identify the ability to utilize those sugars. The organism was not able to metabolize lactose and was able to use glucose. A c itrate slant was inoculated, after 48 hours the butt and slant of the citrate slant was turned blue positive result.The third unknown was tested to be gram positive. A catalase test came out negative, producing no bubbles.ConclusionsRespiratory swabBecause no halo or clearing of any kind was observed in the media around the colonies, it can be safely said that none of the organisms in our throat swabs contained bacteria capable of hemolysis. This lack of hemolytic ability is known has da Gamma hemolysis. The pour plates were TNTC. This is probably because the dilutions performed were not enough too many organisms were taken per tonsil swab. At the time, both participants were mildly sick and had seasonal allergies, perhaps contributing to the amount of organisms per swab.Unknown organismsThree unknown organisms were presented for identification. Organism A was successfully identify as Staphylococcus aureus white. What lead to that identification is that S. aureus is a gram positiv e cocci that produces catalase and is able to use mannitol and grow on the mannitol salt agar and produce the halo it was determined that the color of the colonies was white as opposed to gold. S. aureus is a common bacteria found on the skin and part of human normal flora. The bacteria can cause minor infections of the skin and can sometimes cause serious diseases and infection if it reaches the systemic circuiti.Organism B was place as Serratia marcescens. It is a gram negative rod that can ferment glucose and citrate, but not lactose. It is a common environmental bacterium that can sometimes cause infections in skin and the urinary tract. S. marcescens is part of the family enterobacteriaceae and produces a red pigment that can be found on bathtubs and tiles, feeding on soap residuesii.Organism C was identified to be Lactobacillus acidophilus. It is a gram positive rod that does not produce any catalase for hydrogen peroxide breakdown. The bacteria are part of Lactobacillus whi ch ferments sugars into lactic acids. This bacterium is used often for the production of many dairy products such as cheese and yogurt. L. acidophilus is part of normal human flora and can be found on the skin or gut. Sometimes it is used as a probiotic supplement. It also prevents Candida from overgrowing in the female vaginaiii. All leash organisms were identified successfully with the use of classic techniques.