Staining of bacteria forms the foremost and the most important step in the identification of bacteria. Gram staining: differentiates bacteria into two types Gram positive and Gram negative bacteria Gram positive bacteria can be either cocci or bacilli or vibrios. Gram negative bacteria can be either cocci or bacilli.
Motility testing
Motility testing is performed by preparing a wet mount and is then observed under the microscope.
Biochemical tests
The staining will be followed by use of various biochemical reagents and tests to get closer to the identification of bacteria. There are many biochemical tests available for bacterial identification. The biochemical tests that will be used for identification of Bacillus spp. are as mentioned below
…show more content…
Keep the petri dish cover available. Using a sterile inoculating loop or wooden applicator stick, collect a small amount of bacteria from a well-isolated 18- to 24-hour colony and place it onto the microscope slide. Be careful not to pick up any agar. Using a dropper or Pasteur pipette, place 1 drop of 3% H2O2 onto the bacteria on the microscope slide. Do not mix. Immediately cover the petri dish with a lid to limit aerosols and observe for immediate bubble formation. Observing for the formation of bubbles against a dark background enhances readability.
(b) Coagulase test
Purpose
The coagulase test differentiates strains of Staphylococcus aureus from other coagulase-negative species. The coagulase test can be performed using two different procedures - Slide test and tube test. For both tests, clumping or clots of any size indicate a positive response.
Procedure:
The slide test is performed by preparing a suspension of bacterial cells mixed into a drop of rabbit plasma on a microscope slide. If bound coagulase is present on the bacterial cells, then the presence of plasma will cause the bacterial cells to clump. The tube coagulase test is performed by mixing bacterial cells into a larger volume of plasma in a small test tube. Formation of a clot will be noted within 24 hours for a positive
…show more content…
A positive diagnostic test rests on the generation of alkaline by-products of citrate metabolism.
Procedure
Use a fresh (16- to 18-hour) pure culture as an inoculation source. Pick a single isolated colony and lightly streak the surface of the slant. A needle is the preferred sampling tool in order to limit the amount of cell material transferred to the agar slant. Avoid using liquid cultures as the inoculum source. Citrate utilization requires oxygen and thus screw caps, if used, should be placed loosely on the tube. Incubate at 35oC for 18 to 48 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green colour to
The following tests according to the lab manual were performed: gram stain, fermentation tubes, methyl red, vogues proskauer, sulfur, indole, motility and growing it up on MacConkey agar. The gram stain was performed incorrectly the first time. This is because the decolorizer was not on the bacterium slide for long enough, giving a false outcome.
In this experiment, an unknown bacterium was given to each individual student. The main purpose of this lab was to identify the given unknown bacteria going through a series of biochemical tests as one of the gram negative bacteria among six different Gram negative bacteria Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Salmonella typhimurium. At the very beginning, streaking method; T-streak technique was used to isolate the pure colonies. For the morphological identification of unknown bacteria, Gram Stain Method was done. Biochemical tests that were conducted for the experiment
To perform this test, a small drop of water is placed on a clean microscope slide. A metal loop that has been properly sterilized in the blue flame and allowed time to cool is used to
There are many reasons for knowing the identity of microorganisms. The reasons range from knowing the causative agent of a disease in a patient, so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all of the methods that have been learned so far in the microbiology laboratory class for the identification of unknown bacteria. The identification process can be completed with a series of deferential stains and biochemical tests. Creating a dichotomous key helps to limit the amount of biochemical tests done on an unknown organism and by observation
The purpose of this experiment is to distinguish and indentify an unknown bacterium. There are several tests that can help one eliminate and narrow down the options. The most useful test, and the very first one done, is a gram stain. This test will tell whether the bacterium is gram-positive or gram-negative. After the type of gram stain is identified, the tester has a wide array of differentiating tests at their disposal. Based on the results from these tests, and the numerous others that are available, one can accurately establish the identity of an unknown bacterium.
The oxidation fermentation test was used to differentiate if the organism utilizes lactose, mannitol, glucose and citrate aerobically (oxidation) or anaerobically (fermentation). A methyl red test was performed to determine if the organism carried out mixed-acid fermentation when supplied glucose. A Voges-Proskauer test was performed to evaluate if the unknown was able to ferment glucose into butanediol. A citrate test was performed to determine if the unknown organism was able to break down citrate into ammonia. An oxidase test was then performed to determine if the unknown culture was oxidase positive or negate.
The test showed a clear zone around the colonies thus determining that the bacteria can break down starch meaning it was a positive result. With a positive starch hydrolysis result the next task was to perform a VP. The results from this test came back negative because there was no color change in the broth. My next step was to check for a swollen cell. I found that the cell was swollen looking from the spore stain slide.
The chart below shows the biochemical tests of the gram stain below. Test performed Purpose Materials used Observation Results Gram stain test Gram reaction to specimen Crystal violet, iodine, alcohol safranin Pink/purple colored Gram positive cocci/ gram negative rods After the gram stain showed the specimen as Gram positive cocci, and Gram negative rods, more tests are necessary. Test performed Results Enterotube II Gram negative rod: Citrobacter
Picture 3. This picture shows the stock cultures for both colony 1 and colony 2. The left test tube shows the growth from colony 1 on the TSA slant. The right test tube shows the growth from colony 2 on the TSA slant.
The identification of unknown bacteria are used in labs by microbiologist for the purpose of studying bacteria that cause diseases, diagnosis of diseases and for treatment of the diseases. Therefore, it is important for them to effectively identify the right bacteria that is causing harm to people, animal or environment and treat it successfully. Hence, discussing the different type of test used and the processes of how one could identify the unknown bacteria.
For the Gram stain, I used a microscope slide, wash bottle of water, clothespin, and the reagents crystal violet, Gram’s iodine, ethyl alcohol, and safranin to observe whether the organism was Gram negative or positve. The method was placing and heat fixing a loopful of the unknown organism on the slide. The organisms were then stained with crystal violet for a minute, rinsed off, flooded with iodine for a minute, rinsed off, decolorized with alcohol for 30 seconds, and then finally stained with the counter-stain, safranin, for a minute. I streaked the unknown onto a TSA plate and incubated at 35 C. With a pure colony, we performed a second gram stain procedure and inoculated it into a TSA slant. I used BCP Lactose broth and a loop to perform a BCP Lactose test. The broth was inoculated with a loopful of the unknown and incubated
(4 pts) 3. You are handed two slides that have been prepared with your organism. One has been properly Gram stained and the other has been properly Acid-Fast stained.
Whenever there is an unknown disease caused by microorganisms, tests are usually made in order to identify the organism causing the disease. There are several tests that need to be made and they include tests such as performing a gram stain, streaking a plate to isolate colonies, inoculating a broth culture, inoculating API strip, and performing oxidase and catalase tests. Having knowledge on how to identify these tests are of high importance in the medical field so it would be to the advantage of those individuals who know how to examine microorganisms and be able to identify it by correctly performing tests on organisms.
Each test performed in the lab on theses unknown bacteria have a very specific significance. With each test performed correctly the lab officer is able to move closer towards a proper identification of the unknown bacteria. After performing Gram staining and deciphering if the unknown was gram positive or negative the lab officer was then able to proceed to the next step of identification. The gram positive unknown’s reaction to the catalase test informs the tester of the Genus theyre working with. This indicates which tests to perform next. The MacConkey agar is a selective medium that only allows the growth of gram positive bacteria confirming the results received from the gram staining procedure. The NaCl growth test indicates whether or not the organism is able to
Half of each tube’s contents are poured into a new test tube each respectively after the tubes are incubated for 1 hour. One set of tubes is tested for: