Identification of unknown Bacteria - Research Paper Example

Identification of unknown Bacteria The purpose of the experiment was to identify unknown bacteria from mixed culture #18 using biochemical and morphological test: The tests done include: Culturing and Gram staining for Morphology. The biochemical tests were: Lactose fermentation, Tryptophan digestion (Indole), Orinithine, methyl red and mannitol test. Basing on the results the most likely bacteria were: for the unknown A, was Klebsiella pneumoniae which is a typical rod shaped bacterium, a Gram-negative, lactose fermenting, facultative anaerobic, encapsulated and non-motile. For the unknown B: It was likely to be Salmonella Typhimurium bacteria which is a typical nonspore-forming rods, facultative anaerobic, motile and negative, They ferment glucose and utilize citrate as carbon source and are lactose and sucrose non fermenters. These tests have been fully explored in the next section. INTRODUCTION With regards to the aim of the experiment, which is to identify unknown bacteria from mixed culture #18 using biochemical and morphological test, the two organisms identified had distinct characteristics which were looked for following laboratory tests. Normally, Gram staining is employed to differentiate 2 bacteria groups with different cell wall constituent. The method has the ability to differentiate Gram negative and Gram positive bacteria group; thus the bacterial cells are either colored red or violet. Gram positive bacteria is shown by Violet staining due to a thick layer of peptidoglycan existing in the cell wall of the bacteria, thus the crystal violet is retained by these cells on a slide. Consequently, Gram negative bacteria is shown by red staining due to a thin layer of peptidoglycan existing in the bacteria cell wall, hence crystal violet is not retained by bacterial cells. (Winn et al,2006) MacConkey Agar is used to distinguish Gram- negative, lactose-fermenting organisms from organisms that are non-fermentative .It is an inhibitory and differential medium that contains Crystal violets, bile salts, inhibitory agent and neutral red. Neutral red acts as a pH indicator. Klebsiella pneumoniae bacteria is known to be lactose fermenters hence unknown A while Salmonella Typhimurium bacteria is not lactose fermentor thus unknown B. The orinithine decarboxylase test is important for Enterobacteriaceae differentiation. (MacFaddin, 2000).  MR-VP broth for Methyl Red test consists of peptone, phosphate buffer and glucose. The unknown bacterium that is able to perform mixed-acid fermentation generates enough acid that overcame the broth buffering ability; hence this results to a decreased pH (Harley, 2005) Mannitol medium is used to differentiate mannitol fermenters from non fermenters. It contains mannitol and phenol red indicator. For Coagulase-positive bacteria, yellow colonies can be observed with yellow zones, similarly small red or pink colonies can be produced for Coagulase-negative bacteria with no change of color to the medium. (Winn et al, 2006) MATERIALS AND METHODS GRAM’S STAINING Gram’s staining was done on both unknown A and B as follows: Reagents used include: Crystal violet, Gram's Iodine, decolorizer (acetone), Safranin (secondary stain) and water. Bacteria Cell slides to be stained were prepared. Heating the sample on the slide was carried out by passing the slide with a sample drop on it carefully through a Bunsen burner thrice. Crystal violet was added to the fixed sample slide then incubated for a minute. Slide rinsing was done for at most five seconds to remove unbound crystal violet. Addition of Gram's iodine for one minute was carried out to fix crystal violet to the bacteria cell wall. The slides were rinsed with acetone for 3 seconds. Safranin was added and incubation done for one minute. Later the slide was washed with gentle water stream for 5 seconds. The primary stain will be retained if it is Gram positive bacteria and the secondary stain will not be taken (safranin), hence it will result to violet appearance under a microscope. In the test the bacteria was Gram negative, the primary stain was lost and the secondary stain was taken resulting to red appearance after viewing under a microscope. BIOCHEMICAL TEST DETERMINATION OF THE UNKNOWN A Three biochemical tests were done in the determination of this unknown A, they include: lactose, Indole and Orinthine decarboxylase tests LACTOSE FERMENTATION TEST MacConkey Agar was used in the determination of this unknown. The unknown isolate A was streaked on the MacConkey agar and incubated for 48 hours at 37oc.Later the colour change was observed and recorded INDOLE TEST Unknown bacterium A was inoculated on an agar medium with tryptophan. Trypticase soy agar was used.  Incubation was done for 48 hours at 37oc to allow bacterial growth. ORINTHINE DECARBOXYLASE TEST Reagents used were: tube medium of MIO, Inoculating needle, Primary isolation medium, Incinerator and Kovac’s reagent. The medium was inoculated with isolated colonies. Four Kovac’s reagent drops were added to the tube to test for Indole production and development of pink to red color was looked for and the results were recorded. The reagent was added after the results of Orinthine and motility had been recorded. DETERMINATION OF UNKNOWN B BIOCHEMICAL TESTS Three biochemical tests were done in the determination of this unknown A, they include: lactose, Methyl Red and Mannitol tests LACTOSE FERMENTATION TEST The test procedure was similar to that of the unknown A METHYL RED TEST Two MR-VP broths were used. One broth was inoculated aseptically. The other broth was left uninoculated (a control). Incubation was done at 37oC for 48 hours. Later Methyl Red drop was added to each broth. The color change was observed and results recorded. MANNITOL TEST Mannitol salt agar was used. The plate was streaked with the unknown B and incubated for 48 hours at 37Oc.Colour change was observed and the results recorded accordingly. RESULTS For both the unknown A and B, the result on Gram’s staining was negative, reddish-pink coloration was observed. Under biochemical tests; For the unknown A, Lactose test was Positive with yellow coloration observed as shown in figure 1, Indole test was negative with no red coloration observed after addition of Kovacs’ reagent as shown in figure 2, Ornithine test was as well negative with yellow coloration observed. For the unknown B, Lactose test was negative with red coloration, Methyl red test was Positive with red coloration, Mannitol test was Positive as well with yellow coloration observed as shown in figure 3 Figure 1: Lactose fermenting bacteria was indicated by yellow colonies Figure 2: Shows Indole test results after addition of Kovacs’ reagent Figure 3: Shows mannitol test results observed. DISCUSSION Three processes were employed in Gram staining: Crystal violet staining, decolorization with acetone, and counterstaining with safranin. The three processes included: On the slide, bacterial cells were stained with Crystal violet dye. Gram’s iodine solution was added (potassium iodide and iodine). This lead to complex formation (iodine -crystal violet). The complex molecule is larger than the original stain crystal violet and water insoluble iodine. Acetone as a decolorizer was added to the slide, acetone is dehydrated by peptidoglycan layer resulting to it being tightened and shrunk. The crystal complex can’t pass the peptidoglycan layer that is tightened duet to its big size, thus it is trapped in the Gram positive bacteria cell wall. Consequently, the outer membrane of gram negative bacteria degenerates and Gram negative peptidoglycan thinner layer can’t retain the big crystal violet-iodine complex leading to loss of color. Safranin as a counter stain (weakly water soluble) was placed on slide; hence the cell wall was stained red. Due to its lighter color than crystal violet, the Gram positive bacteria cells purple color cant disrupted it. Hence, the decolorized Gram negative bacterial cells stained red. In the experiment the bacteria was Gram negative, Crystal violate stain was lost and safranin stain was taken up leading to red coloration under a microscope. Under lactose fermentation test, the MacConkey Agar was used to distinguish Gram- negative, lactose-fermenting organisms from organisms that are non-fermentative .It is an inhibitory and differential medium that contains Crystal violets, bile salts, inhibitory agent and neutral red. Neutral red acts as a pH indicator. Klebsiella pneumoniae bacteria is known to be lactose fermenters hence unknown A while Salmonella Typhimurium bacteria is not lactose fermentor thus unknown B. The orinithine decarboxylase test is important for Enterobacteriaceae differentiation. With regards to methyl Red test used to determine unknown B, MR-VP broth consists of peptone, phosphate buffer and glucose. The unknown bacterium that was able to perform mixed-acid fermentation generated enough acid that overcame the broth buffering ability; hence this resulted to a decreased pH. Bacteria which perform other types of fermentation can’t overcome the buffering broth capacity. CONCLUSION The selective and differential media were able to select the unknown bacteria A and B growth as well as they had the ability to differentiate between species biochemically. Basing on the results the most likely bacteria were: for the unknown A, was Klebsiella pneumoniae which is a typical rod shaped bacterium, a Gram-negative, lactose fermenting, facultative anaerobic, encapsulated and non-motile. For the unknown B: It was likely to be Salmonella Typhimurium bacteria which is a typical nonspore-forming rods, facultative anaerobic, motile and negative, They ferment glucose and utilize citrate as carbon source and are lactose and sucrose non fermenters. Works Cited Harley, J. P. (2005).  Laboratory exercises in microbiology, 6th ed. McGraw Hill, New York, NY. MacFaddin, J. F.  (2000).  Biochemical tests for identification of medical bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA.  Winn, W., S. Allen, W. Janda, E. Koneman, G. Procop, P. Schreckenberger, and G. Woods.  (2006). Color atlas and textbook of diagnostic microbiology, 6th ed. Lippincott Williams & Wilkins, Philadelphia, PA.     Read More