Unknown identification

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  INTRODUCTION Identification of an unknown bacterium is one of the crucial steps in helping a clinician to identify a disease. It is vital information from which the clinician will base all the treatments and consequent procedures. It allows a medical technology to apply all the cultural and biochemical procedures that he acquired inside the laboratory. It may be done in a traditional method or by using an automated identification system like BBL Crystal ID ™. In the traditional metho d, manual procedures should be done, starting from the gram staining, growth in the appropriate medium and several biochemical tests in order to identify a single bacterium. Nowadays, the most commonly used method in the identification is using the BBL Cry stal ID™ where we are only required to prepare a broth culture of the bacteria and pour them in a test kit where several tests are already included. After which codes will be provided and will be encoded in a database where the specific organism will be given. TEST PERFORMED FOR THE IDENTIFICATION OF UNKNOWN Growth on MacConkey Agar and Mannitol Salt Agar Materials:    Alcohol lamp    Inoculating loop    Broth culture of the unknown bacteria    Mannitol salt agar    MacConkey’s agar   Procedure: 1.   Flame the inoculating loop bringing it all to red, to disinfect and let it cool. When it’s cold enough, fish out a loopful and inoculate using simple streak method on the agar. 2.   After streaking, cover the culture media, label it properly. 3.   Incubate at 37 degrees celsius for 18-24 hours. Results: MSA: there is growth and color change on the medium (red to yellow) MAC: no growth Interpretation: On MSA, pathogenic organism ferments mannitol, thereby changing the color of the medium from red to yellow. On MAC, there is no growth indicating that the given organism to us is gram positive. Principle:  Mannitol Salt Agar (MSA) is a selective medium that favors the growth of staphylococci and for differentiation between pathogenic and non-pathogenic types. MSA contains 7.5% NaCl and staphylococci are characteristically able to tolerate this high salt concentration. MSA also contains the sugar-alcohol mannitol and the pH indicator phenol red. Generally pathogenic staphylococci are able to ferment the mannitol, lowering the pH, and thereby turning the indicator yellow. The non-pathogenic staphylococci do not ferment mannitol and the medium remains pink in their vicinity. MacConkey Agar (MAC) is a differential plating medium, selective for Gram negative organisms, used primarily for detection and isolation of enteric bacteria. Gram positives are generally inhibited by crystal violet in the medium. Isolated colonies of lactose-fermenting bacteria are brick red in color and may be surrounded by a zone of precipitated bile. This reaction is due to the action of the acids, produced by fermentation of lactose, upon bile salts present in the medium and subsequent absorption of neutral red. Non-lactose fermenters are colorless and transparent. Gram Staining Materials:    Broth cultures of the unknown bacteria    Staining rack    Microscope    Cedar wood oil    Gram’s stains  a.   Crystal Violet b.   Grams Iodine c.   95% ethyl alcohol d.   Safranin Procedure: Bacterial smear and Gram’s staining:  1.   Prepare bacterial smear of the unknown bacteria. 2.   Flood the bacterial smear with crystal violet 10 seconds. (wash with running tap water) 3.   Flood with Gram’s Iodine 10 seconds. (wash with water)  4.   Decolorize with 95% ethyl alcohol until thinnest parts of the smear is colorless. (wash with water) *the third step is the most critical and most affected with technical variations in timing and reagents. So you should be careful* 5.   Flood with Safranin. 10 seconds (wash with water) 6.   Air dry. Microscopic Examination:  1.   Examine the bacterial smear using the brightfield microscope under Oil Immersion Objective. 2.   Draw and write down your observation. Result: Gram positive cocci inclusters  Interpretation: The results in microscopic examination, if the bacteria is a gram positive bacteria it will not decolorize with ethanol and will take up the color of the primary stain which is the crystal violet resulting to a purple color while a gram negative bacteria do decolorize with ethanol and take up the color of the counter stain which is the safranin resulting to a pink to red color. Principle: Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal violet-iodine complex that occurs during staining, while Gram-negative cells have only a thin layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol, and Gram-negative cells do decolorize. This allows the Gram-negative cells to accept the counter stain safranin. Gram-positive cells will appear blue to purple, while Gram-negative cells will appear pink to red. Catalase test Materials:    Glass slides    3% sodium peroxide    Inoculating loops    Alcohol lamp Procedure: 1.   From the NAS (nutrient agar slant) emulsify 1-2 colonies with saline solution on a clean glass slides 2.   Add 1-2 drops of 3% sodium peroxide 3.   Observe for presence of bubbles 4.   Report result. Results: Catalase positive   (rapid formation of bubbles)  Interpretation: Positive reactions are evident by immediate effervescence or bubble formation. No bubble formation means no catalase enzyme to hydrolyze the hydrogen peroxide represents a catalase-negative reaction.  Principle: Catalase is an enzyme, which is produced by microorganisms that live in oxygenated environments to neutralize toxic forms of oxygen metabolites; H 2 O 2 . The catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide and protects them. Anaerobes generally lack the catalase enzyme. Catalase mediates the breakdown of hydrogen peroxide H 2 O 2  into oxygen and water. Coagulase Materials:    Glass slides    Test Tube    Plasma    Inoculating loop    Alcohol lamp Procedure: A. Slide Method 1.   Place 1-2 drops of plasma on the glass slide then add 1-2 loopfuls of the the tested organism. 2.   Mix the cultured organism into the plasma. 3.   Report results. (+) presence of agglutinin (- ) absence of agglutinin B. Tube Method 1.   Place 0.5 mL of plasma into a tube and add 1-2 loopfulsof the tested organism. 2.   Mix and incubate at 37˚ C. observe for coagulation  ever 30 mins for 2 hours. 3.   Report results. (+) coagulation observed within 2 hours. If no coagulation is observed after 2 hours, reincubate for 24 hours. (-) no coagulation after 24 hours RESULTS:  (+) slide test: the tested organisms show agglutinins RESULTS: (+) tube test: he tested organism showed coagulation INTERPRETATION:  Our organism showed positive results for both the slide and tube coagulase test. In the slide coagulase test, agglutinin is observed while on the tube coagulase test, gel like formation is observed.

DM2000 verzija 2

Jul 23, 2017
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