Techniques for the Identification of Unknown Microorganisms

Techniques for the Identification of isolated Micro beingsIdentification of Unknown MicroorganismsFor years and years, we have been trying to classify and show microorganisms, and in doing so, we learned of certain techniques to identify them based on what they look like, where they live, and what they eat. We physical exercised this knowledge to derive simple tests to run, and we employ a key to set down our choices, all in an effort to identify a microorganism. Beginning in the earliest labs with the earliest microbiologists, the most common way to identify an organism is to look at it and look at what it does. In microbiology of bacterium, is it round ( coccus) or rod-shaped (bacilli)? Does it stain purple ( gravitational constant +) or expiration (gram -)? Does it use lactose as food, while creating an acidic product that send away be observed? These tests based on the physiology/morphology and biochemistry fall under what is known as classic appellative1. Classic techni ques focus on the ability of a bacterium to grow and reproduce in certain conditions, such as the fact that bile salts butt interact unfavorably with the peptidogly depose booth walls of gram positive bacteria, therefore selecting against their growth. Most tests are apply to specifically select for or strike out between the species, this in conjunction with a dichotomous key helps narrow down the choices until wholeness is satisfactory. In contrast, modern methods of identification are based on the technological advances of upstart years, namely, polymerase chain reaction (PCR) and genetics. The use of PCR to identify bacteria is rooted in the study of genetic coding, in lineamenticular, RNA. Every bacterium has strands of RNA that is used to reserve the mRNA for translation the 16s ribosomal RNA (rRNA)2. This sequence is conserved in all bacteria and jackpot be used to identify species after amplification done the PCR process. Since every(prenominal) species has a diff erent genetic make-up, it is simply a matter of time for identification. But for most purposes, getting samples to a PCR and running them takes quite just ab out(p) 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 terrible pharynx and cough. These symptoms are sometimes caused by microbes in the pharynx that shouldnt be there. Bacteria such as Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhalis can infect the area and cause this sore feeling. A more serious offender, Streptococcus pyogenes, causes what is commonly known as strep throat. These bacteria make the pharynx sore by harvesting erythrocytes and respite them down as sources of food, in turn, destroying the lining and ca employ the all familiar sore feeling1. A simple test on a blood supplemented agar-agar-agar helps come upon what source of pathogen is causing the sickness. The agar is inoculated and in cubated until growth is seen, and the plate entrust show a pattern of hemolysis if caused by one of the bacterial pathogens. of import hemolysis is the ability to fully metabolize the cellular phone and hemoglobin and will produce a clear halo around the colonies. Alpha hemolysis is the partial(p) breakdowns of hemoglobin into billiverdin, making the halo appear yellow/ reverse lightning. Gamma hemolysis is the absence of halo, as it means the organism cannot lyse erythrocytes1.ProceduresRespiratory swab acquit a sterile cotton swab and swab the tonsilary regionInoculate blood agar by rolling the cotton tip in a band 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 tubeDilute to 10-3 and 10-4Make a pour plate with nutrient agarUnknown identificationTo start to identify any unknown organism by classical methods, it is necess ary to produce an appropriate dichotomous key to accompany the tests. The first step to any key is the gram stain this essentially splits the number of possible organisms in half, gram positive and negative. Further tests based on morphology and biochemistry will select and differentiate between common characteristics of species until one organism stands out. At the start of every lab day, fresh streak plates and broths are prepa fierce to keep the organism isolated and fresh.Take a single settlement and prepare streak plates, one for 37C and one for 25CPrepare broths aseptically in similar fashionDichotomous keyThe dichotomous key was prepa ruby- ruby using only the organisms on the list provided by Dr. Maxwell.Gram stainThe gram stain separates bacteria into two main groups, gram positive and gram negative. The peptidoglycan cell walls of gram positive organisms will absorb the crystal violet stain and appear purple under microscope inspection. The gram negative bacteria lose th eir outmost membrane during the de likenessization step and will absorb the safranin stain, appearing red.Prepare specimen by taking an isolated colony and heat fixing on a slideApply crystal violet stain for 60 second baseWash with waterApply iodine fixer for 45 secWash with waterRinse with decolorizer 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 undercut the number in half.Using the gram stain slide, look at the shape of the specimen under 100x crude 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 harmful oxidizing agents such as atomic number 1 peroxide, which could be do as a result of metabolism. A drop of hydrogen peroxide is added to the specimen placed on a slid e. 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, as in no bubbles formed, the organism is Lactobacillus acidophilus. The bacteria are part of healthy normal flora and aids in digestion3.If catalase positive, the endospore stain must be used for get along classification. The endospore is a tough spore formed by some bacteria during harsh environments. The spore will be dyed green in contrast to a cell dyed red.Prepare a specimen by heat fixing on slideCover the slide with bibulous paperAdd methylene bluePlace slide on steamer over a Bunsen burnerSteam for 7-10 minutes, applying incremental methylene blue to prevent the paper from drying outRemove slide and paperCool for no more than 30 seconds and rinseCounterstain with safranin for 1 minuteRinse and blot dryIf endospore positive, the bacteria is Bacillus subtilis. The organism is found readily in the soil an d can form a tough endospore when conditions become unfavorcapable. Bacillus is also a enceinte 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 commonly found in the digestive tract of humans 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 differe ntiate between the staphylococcus and micrococcus bacteria. Mannitol salt agar is used to select for staphylococcus. Staphylococcus aureus will produce a yellow halo.Inoculate the MSA plate with a isolated colony comprehend at 37C for 48 hoursObserve growthIf a yellow colored 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 respectively 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 Micrococcus roseus or Staphylococcus epidermis. The way to differentiate between them is the color of the colony itself. Micrococcus will appear red in color due to a pigment it secretes5, while S. epidermis will be a white color. Both are normal trim flora.If the organism is gram negative, the first test to run is the lactose supplement phenol red broth. T he 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 change color. A positive test will turn yellow.Inoculate a tube with an isolated colonyIncubate at 37C for 48 hoursObserve color changeIf the organism cannot utilize 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 Pseudomonas (Burkeholderia) cepacia or Pseudomonas aeruginosa. The two can be tell by the pigment of the colonies. P. cepacia will appear a light purple color while P. aeruginosa will be white, and also produce a green pigment known as pyocyanin6.If glucose positive, a change state burden must be prepared. The citrate slant tests for the ability to use citra te as a carbon 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 a lot7.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 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 c an 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 search8.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 stable 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 observe any color change every 10 minsLook for any color change for the VP test in the lead addition of reagentIf MR-VP positive, the organism is Enterobacter. E. aerogenes can cause infections and is considered pathogenic howeve r 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. greenish 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 in like manner 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 thick white colonies (A). A third unknown was given pre-inoculated (C).Three biochemical tests and two morphological tests were used to identify the first unknown organism after isolation. A gram stain reveale d 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 determined 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 period to identify the ability to utilize those sugars. The organism was not able to metabolize lactose and was able to use glucose. A citrate 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 bubb les.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 gamma hemolysis. The pour plates were TNTC. This is believably 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, possibly contributing to the amount of organisms per swab.Unknown organismsThree unknown organisms were presented for identification. Organism A was successfully identified as Staphylococcus aureus white. What lead to that identification is that S. aureus is a gram positive 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 b acteria found on the bark 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 identified 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 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 which 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 s kin or gut. Sometimes it is used as a probiotic supplement. It also prevents Candida from overgrowing in the female vaginaiii. All three organisms were identified successfully with the use of classic techniques.