LabReport on Bacteria Identification
HansChristian Gram, a Danish scientist, developed a method ofdifferentiating two types of bacteria based on the structuraldifferences in their cell wall. In his test, the Gram test, bacteriathat retain the crystal violet dye are called Gram-positive bacteria(NIAID). They do so because of a thick layer of peptidoglycan. Incontrast, those that do not retain the violet dye and are colored redor pink are called Gram-negative bacteria. Gram- negative bacteriaare more resistant to antibodies relative to Gram-positive because oftheir impenetrable cell wall. (Jim and Justin)
Gram-negativebacteria can decolorize to accept counterstain stain red or pink,they do not retain the Gram stain when washed with acetone andabsolute acetone. Gram-positive bacteria retain crystal violet dyeand stain dark violet or purple, they retain a blue or purple colorwith gram stain when washed with water and absolute alcohol. In aGram stain test, the bacteria are washed with a decolorizingcounterstain after being dyed with crystal violet. Unlike theGram-positive bacteria, the Gram-negative bacteria do not have anouter cell membrane. The Gram-positive bacteria cell wall is high inpeptidoglycan that makes it retain the crystal violet dye (TheMicrobial World).
Thefollowing is a diagram that differentiates Gram-positive andGram-negative bacteria by their cell wall structure.
Thisreport presents the process for identification of bacteria by, firstdifferentiating them into two main colonies based on Gram stain testand subsequently differentiating further each group using other testsas illustrated below.
Formy organism identification project, I selected the unknown tubelabeled eight which contained two different organisms. On the firstday, I performed a streak plate test to isolate the two bacteria intocolonies. In the streak plate, I labeled an agar plate with myinitials. Sterilized the inoculating loop, collected the bacteriafrom stock broth mixed culture and streak a series of zigzag linesalong one edge of the plate. I then re-sterilized the loop and pulledthe bacteria from one end of the first streak lines across a seconddirection of the plate. I repeated re-sterilization two more timesand pulled the bacteria from one end of the second streak linesacross the third quadrant of the plate and subsequently across thefourth quadrant of the plate.
Onthe second day my streak plate had great results, I was able todifferentiate my organisms. One looked like white dots, and the otherlooked like smoother lines. I subsequently performed two more streakplates to grow my bacteria separately. I labeled one unknown A andthe other unknown B.
Onday three, after successfully separating my bacteria into two purecolonies, I performed a Grain stain for each of the colonies. TheGrain stain verified that my unknown colony A was Grain positive(purple) – rod-shaped, and my unknown colony B was Grain negative(pink) rod. Based on this, I could eliminate all the coccus shapedorganisms from the list of the biochemical tests sheet. These wereEnterococcusfaecalis, Staphylococcus aureus, Staphylococcus epidermisand Micrococcusluteus.
Fromthe Grain stain test, the Grain positive bacillus shapedmicroorganism were possibly, Bacilluscereus, Bacillus megaterium,or Bacillussubtilis.I decided to perform a Simmons Citrate test. I simply sterilized theloop and streaked the surface of the Simmons citrate slant with theunknown organism A.I incubated the medium for 24 hours at 37C and observed the color change. The reaction for which it tested wasan anaerobic reaction. This was not sufficient to enable me isolatethe organism, I then performed a Methyl Red test. In the Methyl redtest, I incubated the broth for 24 hours at 37C and added five drops of the pH indicator methyl red.
Onthe fourth day, the result of the Simmons Citrate test was negative.The slant remained green. If the slant had changed from green toblue, the organism uses citrate as its singular source of carbon andthe result would be positive. If, following the incubation, the slantremained green, the organism lacks citrase, and the result isnegative. The Simmons citrate test eliminated Bacillus subtilis. Simmons citrate tests for the use of citrate as the sole source ofcarbon. The citrate agar slants contain sodium citrate, bromothymolblue, water, and sodium. If the organism utilizes citrate, an enzymecalled citrase catabolizes the citrate into oxaloacetic acid andacetic acid. The oxaloacetic acid is subsequently broke down intopyruvate, and the acetic acid is converted to carbon (IV) oxide. Thecarbon (IV) oxide reacts with the water and sodium in the media toproduce an alkaline sodium carbonate, which reacts with the pHindicator to produce a Caribbean blue color. From this test, theunknown organism A is possibly Bacilluscereusor Bacillusmegaterium.
Fromthe methyl red test, the result was negative. No bright red color wasobserved, indicating a negative result, thus eliminating Bacilluscereusand Bacillussubtilis.
Consideringthe overall experiment, I was only left with the Gram-positive,rod-shaped organism
Bacillusmegaterium.The unknown organism A was undoubtedlyBacillusmegaterium.
Sometests were also performed for the unknown organism B – bacteria aswell. My choice for this Gram was: Enterobacteraerogenes,Escheriacoli, Klebsiella pneumonia,Proteusvulgaris, Pseudomonas aeruginosa, orSalmonella typhimurium.For the unknown bacteria B, Gram – bacillus shaped bacteria I ran alactose test and a Mannitol test. The lactose test, tests for thepresence of the enzyme caseinase, which hydrolyzes the macromoleculecasein, present in milk into peptides that can be absorbed by thecell for use. I incubated the medium containing skim milk, peptone,and agar for 24 hours at 37 Cand observed the result. In the mannitol test, the mannitol salt agarselects and differentiates halophiles based on the ability to fermentmannitol. Mannitol salt agar contains a high salt percentage (7.5percent) to inhibit non-halophiles and a pH indicator that turns theagar yellow if the acidic by-products of the mannitol fermentationare present. Gram –negative and non-halophilic Gram- positivebacteria do not grow on mannitol salt agar. In incubated the mediumat 37C and observed the results.
Onthe fifth day, the lactose test was negative eliminating Enterobacteraerogenes, Escherichia coli,and Klebsiellapneumonia.The area surrounding the growth remained cloudy indicating that theorganism could not break down the casein, and thus the result wasnegative. Mannitol test was positive. The medium turned yellow,meaning organisms are positive for mannitol fermentation. Thiseliminated ProteusvulgarisandPseudomonasaeruginosa.Thus, unknown organism B was Salmonellatyphimurium.
Bacillusmegateriumis a rod-like, Gram-positive, a bacterium found in diverse habitats.(Bacteriol). Salmonellatyphimuriumis a rod-shaped, flagellated, Gram-negative bacterium (CDC). Theknowledge gathered from the experiment clearly agrees with the aboveinformation. Thus, the identification process was successfullyconducted, and I identified the unknown organism A as Bacillusmegateriumand the unknown organism B as Salmonellatyphimurium.
JimH. and Justin O. (2015). Gram-positiveBacteria.
NationalInstitute of Allergy and Infectious Diseases, NIAID (2012).Gram-negativeBacteria.
Gram-positivevs. Gram-negative Bacteria.
TheMicrobial World (2013). 2-25Thecell wall surrounds and holds in the microbe.
Centrefor Disease Control and Prevention, CDC (2014).Human Salmonella typhimuriumInfections Linked to Exposure to Clinical and Teaching MicrobiologyLaboratories.
J.Bacteriol. (2011). Journal of Bacteriology. Volume 193: 1641994213