INTRODUCTION
Klebsiella pneumoniae belongs to Klebsiella species of Enterobacteriaceae and constitutes the normal flora of the human mouth and intestine. K. pneumonia is the most predominant and clinically critical organism. Infections with K. pneumoniae are mostly contracted from the hospitals and principally occur in patients with damaged host defenses. The objective of this study is to perform techniques to identify the bacterium in the given sample and confirm its presence by staining and microscopic analysis.
Streaking may be performed on the clinical sample using Mac Conkey agar or blood agar with the goal of isolating colonies of the organism from the sample .
Morphological Evaluation. The clinical isolate was tested for morphological properties using MacConkey Agar and incubated at 37 o for 24 hours with subsequent Gram staining. Bacterial suspension was made in normal saline for Gram staining . Smear was made using a glass slide that was further air dried and fixed using gentle heating method. The slide was swamped with crystal violet stain for 1 minute, after which the stain was washed off with extra tap water. Gram’s iodine was added on to the slide for 1 minute, followed by washing and destaining with ethyl alcohol. Finally, the slide was counter stained with safranin stain for 30 seconds. Last step was rewashed, air drying and observing under the microscope.
Motility Test
The motility and growth of organism was also evaluated. To confirm the lack of motility attributing structures such as flagella, a motility test was performed, that included inoculation and incubation a semi-solid agar deep test tube .
API 20E
A single isolated colony of the organism obtained from a pure culture was cultured in sterile distilled water and suspension was prepared . The commercially available API 20 E Biochemical strip containing a panel of 20 separate sections are then treated with the suspension. Sterile oil is then added in some sections, followed by water and API test strip was then added to the tray. The tray was then incubated at 370 for 18 to 24 hours.
RESULTS
Upon incubation for 24 hours, the inoculum cultured and streaked on the MacConkey agar culture plate revealed the presence of Gram-negative, random rod-shaped bacteria (Figure 1.). They exhibited a characteristic yeasty odor and mucoid appearance of colonies.
Figure 1. Mac Conkey Agar plate showing rod-shaped Klebsiella pneumoniae
The motility test demonstrated stationary bacteria that persisted at the site of inoculation.
The API test revealed positive reactions for decarboxylation of amino acids such as arginine, lysine and ornithine to form amines. After incubation, the results exhibited a color change from yellowish-orange to purple, suggesting an alkaline pH (>8.0) caused due to the decarboxylation of the amino acids . The carbohydrates such as glucose, sorbitol, melibiose, amygdalin rhamnose, mannitol, inositol, sucrose, and arabinose were examined by the fermentation reaction as reflected by the yellow color of the acid reaction.
DISCUSSION
Enterobacteriaceae is the genus of bacteria that include gram-negative, rod-shaped bacilli that are facultative anaerobic. K. pneumoniae, belonging to this family of bacteria is a pathogenic bacterium, which many cause infections of respiratory tract such as pneumonia, infections in the brain like meningitis or blood infections such as septicemia. These diseases may be treated by use of antibiotics that depend on the organ involved . As proposed, these experiments have confirmed the occurrence of K. pneumoniae in the given sample. One of the complications of the API system was that, in case of glucose reactions, the color of the reaction would occasionally return from positive to negative, giving rise to false negative reactions. These errors may be prevented in future by performing the test in replicates and keeping a track of the time of reactions.
Works Cited
Acharya, T. API 20E Test System: Introduction, Procedure Results and Interpretations. 6 May 2015. <http://microbeonline.com/api-20e-test-system-introduction-procedure-results-interpretations/>.
Pathogen Profile Dictionary. 2010. <http://www.ppdictionary.com/bacteria/gnbac/pneumoniae.htm>.
Qureshi, S. Klebsiella Infections Treatment & Management . 6 October 2015. <http://emedicine.medscape.com/article/219907-treatment>.
Sikarwar, A S. "Identification of Klebsiella Pneumoniae by Capsular Polysaccharide Polyclonal Antibodies ." Internationational Journal of Chemical Engineering and Applications (2011): 2(2): 130-134.
