The purpose of this experiment as to learn how to properly conduct certain methods for identification on a sample of an unknown bacterium. Each of tests’ results was essential in the process of elimination by giving clues and facts about the organisms make up and metabolism. The unknown in this experiment happened to be Salmonella typographic, a bacterium responsible for the most common type of food poisoning. Samples of unknown bacterium were handed out during lab. In this case, the unknown was identified as “number eight”.
The first and most important test in determining the identity is the most widely used test in microbiology, the Gram stain. This is done by staining a prepared slide of the bacterium with crystal violet, followed by Gram’s iodine to act as a mordant, a decontrolling solution, and a final counter stain of Gram’s Seafaring. Each of the biochemical tests that followed were performed within two class periods, the first to inoculate the particular media used in each test with the unknown bacteria, and the second to observe, analyze, and interpret the results after forty eight hours of incubation at thirty seven degrees Celsius.
Of all of the tests conducted during this experiment, some required only observation of color change in the reticular types of media or characteristics of the growth on the medium after incubation. However, most of the tests required one or more additional steps. These include the SIMI tubes, in which five drops of Kava’s was added to the top of the test tube to observe for presence of red color which indicates a positive indolent reaction.
The catalane test called for one drop of hydrogen peroxide to be dropped on a plate of nutrient agar inoculated with the unknown bacterium. If bubbles appeared immediately, this indicated a positive result for the enzyme catalane. As for the oxides test after incubation, a sterile toothpick was used to scrape some of the bacteria from the inoculated nutrient agar in the Petri dish and scraped on to the oxides dry slide. A positive result for oxides would result in the appearance of a blue-black color within ten seconds and no color change would be a negative result.
Another procedure that required further action was the Methyl Red- Vogues Proteases test. A millimeter of the inoculated broth was split in half between two empty sterile test tubes; five drops of Methyl Red reagent were added to one. A positive result in this test would be a tube that turns red due to very acidic end products from glucose fermentation. As for the other tube, five drops of reagent Barite’s A was added and the tube was shook, five drops of reagent Barite’s B was added and left alone for ten minutes.
The formation of a pink ring at the top of the tube that eventually worked its way down was indicative of a the breakdown of glucose to stratospherically. The protein hydrolysis test was one of the most simple. All that was to be done was refrigerating the inoculated gelatin medium at four degrees Celsius for thirty minutes. If the medium turned solid, this was a negative result for the enzyme gelatins and if the medium remained a liquid, this meant that the bacteria hydrolysis the gelatin protein and that is why the medium could not solidify, even at a cold temperature.
For the detection of the enzyme amylase was the starch hydrolysis test. After incubation, the streaked starch plate was flooded with iodine. The purpose of the iodine was to stain the starch a bluish-purple color. In the presence of amylase, a colorless zone surrounding the growth of the bacterium would be present where the starch was digested. The remaining tests are ones that were simply observed for positive or negative results based on certain characteristics after inoculation and incubation.
The reagents, enzymes involved, and possible outcome of each test are listed in the Table 1. Table 1: Materials, Purposes and Possible Results Test Media and/ or Reagents Used Enzyme Involved Appearance of positive Result Appearance of Negative Result Casein Hydrolysis Casein agar plate casemate Clear zone around bacterial growth No clear zone Catalane Test Nutrient agar plate catalane Immediate release of bubbles No bubbles Citrate Utilization
Simmons citrate agar slant/ brotherly blue Slant will turn royal blue color and growth will be visible Slant will remain green and no growth will appear Nutrient broth/ Phenol red color change from red to yellow shows acid production from sugar fermentation/ gas bubble trapped in Durham tube No color change/ no gas production; Darker red or hot pink indicate an alternative metabolism PR Deg red or hot pink indicate an alternative metabolism PR Such red or hot pink indicate an alternative metabolism Hydrogen Sulfide Production SIMI agar Black pigment along stab line or throughout tube indicates sulfur containing mini acids were metabolize No black pigment Indolent Production SIMI agar/ Kava’s reagent Cherry red color ay the top of the agar No development of color Motility Cloudiness throughout tube No cloudiness, agar is clear throughout tube Starch Starch agar plate amylase Clear zone around organism after iodine stain No clear zone after stain Nitrate reduction Mixed Acid fermentation (MR..) MRS.. Broth Tube turns bright red Tube turns yellow or bright orange Vogues Proteases (UP) Pink ring at top of the after 10 minutes No color change Oxides test TTS plate Oxides Appearance of blue-black color within 10 seconds No color change. Urea Hydrolysis Urea broth Urea broth will turn a hot pink color Broth will remain straw color ITS Triple sugar iron agar WA Slant and butt are observed separately for color change. If one or the other turns yellow- only glucose was fermented.
If both are yellow (acidic) then glucose as well as sucrose, lactose or all were fermented. Gas production will cause splits in agar. Black indicates metabolism of sulfur containing amino acids No color change/ no splits, bubbles, etc. Gelatin Gelatins Tube remains liquid after refrigeration Tube that remains solid after refrigeration Lipase Tributary agar Clear zone around growth The Gram stain result of the unknown bacterium “number eight” was Gram negative with the morphology of rod shape. The results from the series of remaining tests are clearly expressed in Table 2 and the strategy of determining the identity of the unknown is demonstrated by flow chart.
Observations Interpretation Gram Stain Pink rods Gram negative rods Negative for caseins Bubble immediately released Positive for catalane Bright royal blue Positive for citrate utilization PR Lace Dark red Negative for fermentation/ alternative metabolism used PR Deg Yellow/ bubble in Durham tube Positive for fermentation of dextrose/ gas produced PR such Yellow/ no bubble Positive for fermentation of sucrose/ no gas produced Hydrogen Sulfide Production Very black color Positive for HAS production Negative for indolent Positive for motility Negative for amylase Red developed immediately Positive for utilization of nitrate Turned red positive for highly acidic end products No color changes Negative for breakdown of glucose to stratospherically Table 2: Results Negative for oxides Negative for areas Breaks and bubbles in agar, black color
Positive for gas production/ positive for HAS Turned to solid during refrigeration Negative for gelatins Negative for lipase Unknown #8- Gram Stain Result: Gram negative rods Lactose Fermentation- Result: Negative Positive Negative Escherichia coli Shillelagh dysentery’s Interconnect arrogates Salmonella typographic Kielbasa pneumonia Serrated mercenaries Protests vulgarism Pseudonymous organisms Allegiances fiscals Citrate Utilization- Result: Positive Positive Negative Salmonella Typographic Shillelagh dysentery’s Indolent Test- Result: Negative Serrated mercenaries Positive Negative Salmonella hypothermia Allegiances fiscals Hydrogen Sulfide Production- Result: Positive Salmonella typographic pseudonymous organisms Areas Test- Result: Positive Salmonella typographic After a series of differential tests, the conclusion was made that unknown “number eight” was Salmonella typographic.
The initial test, the gram stain revealed that the bacterium was a Gram negative rod. The organism was inoculated in to several different types of media for the rest of the biochemical tests. Fortunately, all of the tests worked properly and were consistent with the typical nature of the organism. An alternative possible identification of the unknown bacteria could be Interconnect arrogates due to the close similarities in previous data, especially the results in the Dextrose and sucrose fermentation tests along with the citrate and nitrate utilization tests. Salmonella typographic belongs to the genus Salmonella. These bacteria are named after scientist who discovered them, Daniel Salmon.