Biological Tests (ARIBA PDF)

Tests detecting Hydrolytic enzymes Starch Hydrolysis test Urea Hydrolysis test Casein Hydrolysis test Gelatin Hydrolysis test DNA Hydrolysis test Lipid Hydrolysis test  HYDROLYSIS or HYDROLYTIC REACTIONS Reactions that use water to split complex molecules are called...

Tests detecting Hydrolytic enzymes Starch Hydrolysis test Urea Hydrolysis test Casein Hydrolysis test Gelatin Hydrolysis test DNA Hydrolysis test Lipid Hydrolysis test  HYDROLYSIS or HYDROLYTIC REACTIONS Reactions that use water to split complex molecules are called hydrolysis or hydrolytic reactions. The enzymes required for these reactions are called hydrolytic enzymes.  Starch Hydrolysis Materials  one starch agar plate  Gram iodine ( from gram stain kit)  Recommended organisms bacillus cereus E.coli  Medium Recipe  Beef extract 3.0 g  Soluble starch 10.0 g  Agar 12.0 g  Distilled or Deionized water 1.0 L  Procedure  Inoculate a starch agar plate with two organisms.  After incubation you will add a few drops of iodine to reveal clear areas surrounding the growth.  Iodine will not color the agar where the growth is , only the area surrounding the growth. LAB ONE 1 : using a marking pen divide the starch agar plate into three equal sectors. Be sure to mark on the bottom of the plate. 2 : Label the plate with the names of the organisms , your name , and the date. 3: spot inoculate two sectors with the test organisms. 4: invert the plate and incubate it aerobically at 35 C for 48 hours. LAB TWO 1: Remove the plate from the incubator and before adding the iodine note the location and appearance of the growth. 2: cover the growth and surrounding areas with gram iodine. Immediately examine the areas surrounding the growth for clearing. Usually the growth on the agar prevent contact between the starch and the iodine so no color reaction takes place at that point. Beginning students some times look at this lack of color change and judge it incorrectly as a positive results therefore examining the agar for clearing look for a halo around the growth not at the growth it self. 3: Record your results in the chart provided on the data sheet.  Applications Starch agar used for cultivating Neisseria and also isolate and identify Gardnerella vaginalis. It aids in differentiating members of genera corynebacterium , clostridium , bacillus.  Urea Hydrolysis  Urea is a product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and carbon dioxide by bacteria containing the enzyme Urease.  urea agar was formulated to differentiate urease positive bacteria from urease negative bacteria.  Urea hydrolysis to ammonia by urease positive organisms will overcome the buffer in the medium and change it from orange to pink.  The agar must be examine daily during incubation.  Rapid urease positive organisms will turn the entire slant pink within 24 hours.  Weak positive may take several days.  Urease negative organisms either produces no color change in the medium or turn it yellow.  Materials  Four urea broths  Four urea agar slants  Fresh slant cultures of Ecoli proteus vulgaris Klebsiella pneumonia Medium Recipes Urea agar  Peptone 1.0 g  Dextrose ( glucose ) 1.0 g  Sodium chloride 5.0 g  Potassium phosphate 2.0 g  Urea 20.0 g  Agar 15.0 g  Phenol red 0.012 g  Distilled water 1.0 L  Urea broth  Yeast extract 0.1 g  Potassium phosphate 9.1 g  Urea 20.0 g  Phenol red 0.01 g  Distilled water 1.0 L Procedure LAB ONE 1: obtain 4 tubes of each medium. Label three with the names of the organisms , your name and the date. Label the fourth tube of each medium “control “. 2: inoculate three broths with heavy inocula from the test organisms. Do not inoculate the control. 3: streak inoculate three slants with the test organisms , covering the entire agar surface with heavy inoculum. Do not inoculate the control. 4: incubate all the tubes aerobically at 35 c for 24 hours. LAB TWO 1 : remove all tubes from the incubator and examine them for color changes. Record all broth results and any rapid positive and slow positive agar test results and in the charts provided on the data sheet. Discard all broth tubes and positive agar tubes in an appropriate autoclave container. 2: return any negative agar tubes to the incubator. Inspect them daily for pink color formation for up to 6 days. 3 : enter your agar results daily in the chart provided.  Application This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease. Urinary tract pathogens from the genus proteus may be distinguished from other enteric bacteria by their rapid urease activity.  Casein hydrolysis test Casease is an enzyme that some bacteria produce to hydrolyze the milk protein Casein the molecule that gives milk its white color. when broken down into smaller fragments the ordinarily white casein loses its opacity and become clear. The presence of casease can be detected easily with the test medium milk agar. When plated milk agar is inoculated with a casease positive organism , secreted casease will diffuse into the medium around the colonies and create a zone of clearing where the casein has been hydrolyzed. Casease negative organisms do not secrete casease and thus do not produce clear zones around the growth.  Materials One milk agar plate Fresh cultures of : Bacillus cereus Escherichia coli Medium Recipe Skim Milk Agar Pancreatic digest of casein 5.0 g Yeast extract 2.5 g Powdered non fat milk 100.0 g Glucose 1.0 g Agar 15.0 g Distilled water 1.1 L  Procedure Inoculate a single milk agar plate with a casease positive and a casease negative organism. LAB ONE 1: using a marking pen divide the plate into three equal sectors. Be sure to mark on the bottom of the plate. 2: label the plate with the names of the organisms , your name and the date. 3: spot inoculate two sectors with the test organisms and leave the third sector uninoculated as a control. 4: invert the plate and incubate it aerobically at 35 c for 24 hours. LAB TWO 1: examine the plates for clearing around the bacterial growth. 2: record your results in the chart provided on the data sheet.  Application The casein hydrolysis test is used for the cultivation and differentiation of bacteria that produce the enzyme casease.  Gelatin Hydrolysis Test Gelatin is a protein derived from collagen a component of vertebrate connective tissue. Gelatinases comprise a family of extra cellular enzymes produced and secreted by some microorganisms to hydrolyze gelatin. The Presence of gelatinase can be detected using nutrient gelatin. When a tube of nutrient gelatin is stab inoculated with a gelatinase positive organism secreted gelatinase will liquefy the medium. Gelatinase negative organisms do not secrete the enzyme and do not liquefy the medium. A 7 day incubation period is usually sufficient to see liquefaction of the medium. However gelatinase activity is very slow in some organisms. All tubes still negative after 7 days should be incubated an additional 7 days.  Materials  Three nutrient gelatin stabs  Ice bath  Fresh cultures of Bacillus subtilis Escherichia coli Medium Recipe Nutrient Gelatin  Beef extract 3.0 g  Peptone 5.0 g  Gelatin 120.0 g  Distilled water 1.0 L  Inoculate two nutrient gelatin tubes and incubate them for up to a week. Gelatin liquefaction is a positive result but is difficult to differentiate from gelatin that has melted as a result of temperature. Therefore be sure to incubate a control tube along with the others. If a control melts as a result of temperature refrigerate all tubes until it re solidifies.  Procedure LAB ONE 1: obtain three nutrient gelatin stabs. Label each of two tubes with the names of the organism , your name and the date. Label the third tube “ control”. 2: stab inoculate two tubes with heavy inocula of the test organisms. Do not inoculate the control. 3: incubate all tubes at 25 c for up to 1 week. LAB TWO 1 : examine the control tube. If the gelatin is solid the test can be read. If it is liquefied place all tubes in the ice bath until the control has re solidified. 2: when the control has solidified examine the inoculated media for gelatin liquefaction. 3: record your results in the chart provided on the data sheet.  Application This test is used to determine the ability of a microbe to produce gelatinases. Staphylococcus aureus which is gelatinase positive can be differentiated from s.epidermidis.  DNA Hydrolysis Test An enzyme that catalyzes the depolymerization of DNA into small fragments is called deoxyribonuclease or DNase. ability to produce this enzyme can be determined by culturing and observing an organism on a DNase test agar plate. DNase test agar contains an emulsion of DNA , peptides as a nutrient source and methyl green dye. The dye and polymerized DNA form a complex that gives the agar blue green color. Bacterial colonies that secrete DNase will hydrolyze DNA in the medium into smaller fragments unbound from methyl green dye. This results in clearing around the growth.  Materials  One DNase test agar plate  Fresh cultures of : staphylococcus aureus staphylococcus epidermis Medium recipe  tryptose 20.0 g  Deoxyribonucleic acid 2.0 g  Sodium chloride 5.0 g  Agar 15.0 g  Methyl green 0.05 g  Distilled water 1.0 L  Procedure LAB ONE 1: using a marking pen , divide the DNase test agar plate into three equal sectors. Be sure to mark the bottom of the plate. 2 : label the plate with the names of the organisms , your name and the date. 3: spot inoculate two sectors with the test organisms and leave the third sector as a control. 4: invert the plate and incubate it aerobically at 35 c for 24 hours. LAB TWO 1: examine the plates for clearing around the bacterial growth. 2 : record your results in the table provided on the data sheet.  Application DNase test is used to distinguish serratia species from enterobacter species. Staphylococcus aureus from other staphylococcus aureus species.  Lipid Hydrolysis test Lipid is the word generally used to describe all types of fats. The enzymes that hydrolyze fats are called lipases. Bacteria can be differentiated based on their ability to produce and secrete lipases Variety of simple fats can be used for this determination , tributyrin oil is the most common constituent of lipase testing media because it is the simplest triglyceride found in natural fats and oil. Tributyrin agar is prepared as an emulsion that makes the agar appear opaque. When a plate is inoculated with a lipase positive organism , clear zones will appear around the growth as evidence of lipolytic activity. If no clear zone appear the organism is lipase negative.  Materials  One tributyrin agar plate  Fresh cultures of : Bacillus subtilis Escherichia coli Media recipe  beef extract 3.0 g  Peptone 5.0 g  Agar 15.0 g  Tributyrin oil 10.0 ml  Distilled water 1.0 L  inoculate a tributyrin agar plate with two organisms one of which will produce a clearing around the growth.  Procedure LAB ONE 1 : using a marking pen divide the bottom of the plate into three equal sectors. 2 : label the plate with the names of the organisms , your name and the date. 3: spot inoculate two sectors with the test organisms , leaving the third sector un inoculated as a control. 4: invert the plate and incubate it aerobically at 35 c for 24 to 28 hours. LAB TWO 1 : examine the plates for plates for clearing around the bacterial growth , and record your results in the chart provided on the data sheet. If no clearing has developed re incubate for 24 to 28 hours.  Application The lipase test is used to detect and enumerate lipolytic bacteria especially in high fat dairy products. A variety of other lipid substrates including corn oil , olive oil and soybean oil are used to detect differential characteristics among members of Enterobacteriaceae , clostridium and staphylococcus.