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CHAPTER 3 – BIOCHEMICAL TEST (✔ identification Gram negative bacteria,…
CHAPTER 3 – BIOCHEMICAL TEST
✔ identification Gram negative bacteria
● Indole production
● Methyl Red/Voges Proskauer
● Citrate
● H2S production in SIM
● Urea hydrolysis
● Motility
● Lactose fermentation
● Sucrose fermentation
● Glucose fermentation & gas production
● Oxidase test
✔ Staphylococcus identification tests (S.A from other species)
● BAP
● MSA
● Mstaph broth
● Coagulase
INDOLE PRODUCTION TEST
✔ Results: A red color in the alcohol layer indicates a positive reaction whereas negative test will show yellow colored ring of Kovac’s reagent.
✔ Method: inoculating the test organism into peptone and incubating it at 37ºC for 48-96 hours. Then add 0.5ml of Kovac’s reagent and shake gently
✔ Material: : Tryptone broth, Kovac’s reagent
✔ Principle (MOA): how red color is form? enzyme tryptophanase, degrade amino acid tryptophan to indole, pyruvic acid and ammonia (bact only use this)
✔ Purpose: to identify whether bact can produce enzyme (It tests for the bacteria species’ ability to produce indole)
Methyl Red (MR) Test
✔ Purpose: to identify the bact whether it able can produce acid due to fermentation glucose(tests for acid end products from glucose fermentation)
✔ Principle (MOA)
✔ Material: Glucose Broth, Methyl Red indicator
✔ Method: inoculate the test organism in glucose phosphate broth and incubate at 37ºC for 2 days. Then add 5 drops of 0.04% solution of methyl red, mix well and read the result immediately
✔ Results: Positive tests are bright red indicating a low pH and negative test are yellow. If the test is negative after 2 days repeat it after 5 days(a + result is red (indicating pH below 6) and a – result is yellow (indicating no acid production)
Voges Proskauer Test
✔ Purpose: tests for acetoin production from glucose fermentation.
✔ Principle(MOA): In the presence of potassium hydroxide and atmospheric oxygen, acetoin is converted to diacetyl, and alpha napthol serves as a catalyst to form a red complex
✔ Material: Glucose Broth, Voges Proskauer reagents (A: 5% Alpha-Naphthol, & ethanol, B: Potassium Hydroxide, & Deionized Water)
✔ Method: Inoculate glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of VP reagents
✔ Results: This test is usually done in conjunction with the methyl red test. An organism of the family Enterpbacteriaceae is usually MR positive or VP negative or vice versa(+ result is red after VP reagents are added (indicating the presence of acetoin) and a – result is no color change)
Citrate Utilization Test
✔ Purpose: to study the ability of an organism to utilize citrate as a sole source of carbon for the growth
✔ Principle(MOA): The blue color is due alkaline pH that results form the utilization of citrate. It turns the indicator in the medium from green to blue. (if bacteria use citrate it becomes blue, if not remain green.presence of bromothymol blue make the color change to blue)
✔ Material: Simmon’s Citrate Agar & pH indicator—bromthymol blue.
✔ Method: Inoculate slant with inoculating loop. After 24hours put in incubator
✔ Results: + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a – result remains green
Hydrogen Sulphide Production
✔ Purpose: to determine the ability to reduce sulfur into H2S.
✔ Principle(MOA): black color produced bc of iron , reagent sulpfate react change sulphide. H2S react iron change ferrous sulphide.
✔ Material: amino acid, ferrous sulfate/peptonized iron
✔ Method: Stab SIM media with inoculating needle
✔ Results: H2S will react with the iron or ferrous sulfate and produce a black precipitate. A positive result has a black precipitate present and a negative result has no black precipitate
Urease Test
✔ Purpose: detects the ability o an organism to produce urease enzymes
✔ Principle(MOA): . Urease in the presence of water converts urea into ammonia and carbon dioxide. Ammonium makes the medium alkaline and phenol red indicator changes to purple-pink color.
✔ Material: Urea broth and phenol red indicator
✔ Method: Inoculate Urea broth with inoculating loop. It is incubated at 37ºC and examined after 4 hours and after overnight incubation.
✔ Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a bright pink color, and is positive. If test is negative, broth has no color change and no ammonia is made.
Sugar Medium [TSI agar]
Oxidase Test
✔ Purpose: This tests for the bacterial oxidase to catalyze the oxidation of oxidase reagent
✔ Principle(MOA): bacterial oxidase to catalyze the oxidation of oxidase reagent thus producing purple color.
✔ Material: oxidase reagent, filter paper
✔ Method: Put a drop of freshly prepared 1% solution of oxidase reagent on a filter paper, rub a few colonies of the test organism on the filter paper
✔ Results: A positive result is formation of purple color. All the members of the family Enterobacteriaceae are oxidase negative
Mannitol Salt Agar (MSA)
✔ Purpose: the bacteria’s ability to tolerate 7% salt concentration and ferment mannitol
✔ Principle(MOA)
✔ Material: MSA media and phenol red indicator
✔ Method: Inoculate an MSA plate using streak plate method and incubate 24-48 hours.
✔ Results:
● If the organism is tolerant to salt it will grow.
● If the organism is not tolerant to salt it will not grow.
● If the salt tolerant organism can ferment mannitol then there will be yellow zones around the colonies.
● If the salt tolerant organism cannot ferment mannitol then the media will remain pink
Coagulase
✔ Purpose: the bacteria’s ability to clot blood plasma using the enzyme coagulase
✔ Principle(MOA): clotting indicates bacteria produce coagulase.
✔ Material: This media contains rabbit plasma dissolved in buffer.
✔ Method: Inoculate rabbit plasma with one single colony. Break up colony and stir until blended in plasma. Incubate at 37 degrees C for 24 hours
✔ Results:
● If the organism is has coagulase it will clump the plasma.
● If the organism does not have coagulase it will not clump the plasma
Endotoxins
• the components of outer membrane of Gram negative bacteria. Endotoxins are lipopolysaccharides (LPS) in nature and are released from the bacterial surface by natural lysis of the bacteria or by disintegration of the organisms in vitro
• a mild amount of endotoxin may cause fever. Larger amounts of endotoxins can cause irreversible shock as seen in association with fulminating Gram negative bacteraemia
• most popular endotoxin detection systems are based on LAL,
Limulus amoebocyte lysate (LAL)
• Gram negative bacteria are characterized by their production of endotoxins, which consist of lipopolysaccharide (LPS) layer (outer membrane) of the cell envelope. The LPS is pyrogenic and responsible for some of the symptoms that accompany infections caused by gram- negative bacteria
• The (LAL) test employs a lysate protein from the blood cells of the horseshoe crab (Limulas polyphemous) the lysate protein is the most sensitive substrate known for endotoxins.
• performed by adding aliquots of food suspensions or other test materials to small quantities of a lysate preparation, followed by incubation at 370C for 1 hour. The presence of endotoxins causes gel formation of the lysate material.
Application:
• The LAL test is used to test for pyrogens
• Even very tiny levels of pyrogens cause a dangerous temperature rise, so any liquid going to be injected or fed into a patient's blood stream has to be tested for pyrogen contamination. Previously this was done by injecting the liquid into a rabbit and monitoring the animal's body temperature.
• The new LAL test uses white blood cells taken from the horseshoe crab which can detect the pyrogens in a test-tube
