Lab 5: Metabolic Activities of Bacteria PDF

Lab 5 Metabolic activities of bacteria  Laboratory Exercise 5: Metabolic Activities of Bacteria Objectives 1. Learn how to use differential and selective media to identify bacterial species. 2. Be able to perform the following biochemical tests and understand how they work: carbohydrate...

Lab 5 Metabolic activities of bacteria  Laboratory Exercise 5: Metabolic Activities of Bacteria Objectives 1. Learn how to use differential and selective media to identify bacterial species. 2. Be able to perform the following biochemical tests and understand how they work: carbohydrate fermentation, gelatin hydrolysis, starch hydrolysis, casein hydrolysis, urea hydrolysis, citrate utilization, catalase activity, tryptophan hydrolysis (indole test). 3. Be able to relate the results of the tests performed to the different types of metabolic activities carried out by individual bacterial species. Key Terms: fermentation, amylase, gelatinase, caseinase, urease, citrate permease, catalase, indole, tryptophanase, Kovac’s reagent, BBL Dry Slide, agar slant (locate butt and slant), Durham tube, Simmon’s citrate  Many bacterial species appear identical under the microscope Additional tests are needed to identify them to species Metabolic tests can distinguish bacteria with similar appearance Bacteria vary in their ability to produce enzymes Determines what chemicals they can break down/use as energy sources Knowing which enzymes a bacterial species can produce tells us something about its genetic make-up WHY? What are two metabolic tests that we have looked at already? (Hint: lab 2)  Carbohydrate Fermentation Bacteria Glucose Lactose Sucrose Some bacteria have the ability to ferment simple Proteus vulgaris AG NEG A sugars or carbohydrates, and produce acids AG AG NEG and/or alcohol. Escherichia coli A NEG A Gas may be produced as well Bacillus subtilis Media: 3 types of Fermentation tubes Streptococcus A A A Glucose broth faecalis Sucrose broth Lactose broth All contain phenol red: a pH indicator that is red at neutral pH Inoculation: use loop to transfer bacteria into broth Results: Yellow color- positive for acid production (A) May or may not have air bubble in Durham tube (A or AG) Red: negative (N) Note: we will use lactose only for the identification of unknowns https://www.youtube.com/watch?v=vJcL9f52Zuk Hydrolysis of Gelatin The Gelatin (protein) is broken down into peptides and amino acids by the enzyme Gelatinase. Media: gelatin deep Inoculation: use inoculation needle to stab into the media Results: ( + ) for Gelatinase activity – Liquid ( - ) for Gelatinase activity – Solid Note: tubes should be cooled before reading to ensure Any liquid is not the result of melting https://www.youtube.com/watch?v=xFUdG8NHmhs  Hydrolysis of starch Starch is broken down into dextrin, glucose, and maltose (monosaccharide and disaccharide). Iodine is used to detect the presence of starch (changes to a blue/black color when starch is present). The presence of starch means the bacteria do not produce the enzyme needed to break it down. Media: starch agar plate Procedure: spot inoculation of bacteria onto agar plates After incubation, add iodine to surface of agar plate If the bacteria is (+) for amylase activity, it will break down the starch – no black color, no change in the color of the iodine. If the bacteria is (-) for amylase activity, it doesn’t break down the starch – black color forms (starch still present in media). Bacillus subtilis is positive for amylase activity; no starch is found near the growth, so no color change in iodine has occurred. This bacteria did not break down starch (amylase negative); so blue/black color change is seen after iodine is added. https://www.youtube.com/watch?v=zFhMbXSgve8&t=40s Hydrolysis of Casein Casein is a milk protein. Caseinase (a proteinase) breaks down the casein into amino acids. Media: skim milk plate Inoculation: spot inoculation onto surface of plate Results: ( + ) for casein activity – clear zone around bacterial growth neg ( - ) for casein activity – no clear zone pos Note: casein test is not used for identification of unknowns https://www.youtube.com/watch?v=c3tx4oY1ncQ Hydrolysis of Urea Urease and breaks down urea into NH3 and CO2. Alkaline pH is detected. Media: urea slant Inoculation: surface of slant. Leave cap loosened Results: ( + ) for urease activity – pink color. ( - ) for urease activity – no color change (peach or yellow color). Note: yellow color is a sign of an acidic pH and is NOT positive for urease https://www.youtube.com/watch?v=KAFXB8TBARw&t=86s Citrate Utilization https://www.youtube.com/watch?v=LZ_mVPRLayQ&list=RDCM UCIzOHtZa0iOH49ez1O5mReQ&start_radio=1&t=7 Citrate permease allows citrate to enter bacterial cell In Simmons Citrate media, Citrate is the only source of carbon Only bacteria that can take it in can grow on this media The products: alkaline (detect by pH change) Media: Simmon’s Citrate has bromothymol blue: green at neutral pH, turns blue in alkaline pH Inoculation: stab and streak- with inoculation needle Results: ( + ) for citrate permease– blue color (alkaline pH) ( - ) for citrate permease– no change (green color).  Catalase activity Catalase is an enzyme that cells produce when they live in an oxygen environment. H2O2 (hydrogen peroxide) is broken down into H2O and O2 by catalase. Media: TSA slant (not pictured here) Inoculation: surface of slant Results: After incubation, add hydrogen peroxide ( + ) for catalase activity –– bubbles as free O2 forms ( - ) for catalase activity – no change. https://www.youtube.com/watch?v=C_p8iA2TlfM Hydrolysis of Tryptophan (Indole test) Tryptophan is an amino acid. Tryptophanase breaks down tryptophan into indole, NH3 (ammonia) and pyruvate. In the media we detect the indole. To detect Indole we use Kovac’s reagent (BBL Dryslide test) Kovac’s embedded in the dryslide- safer than using liquid Media: Tryptone broth Inoculation: add bacteria to broth with loop Results: After incubation, use sterile cotton swab to add bacteria to dryslide After adding bacteria to dryslide Positive result Negative result ( + ) for Indole – pink-red color ( - ) for Indole – No pink-red color https://www.youtube.com/watch?v=m27wzevB9bA  Lactose Bacteria Gram stain Morphology Arrangement Fermentation Gelatinase Amylase Urease Citrate permease Catalase Tryptophanase (Simmon’s citrate) (Indole test) Bacillus subtilis Corynebacterium xerosis Enterobacter aerogenes Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Proteus vulgaris Staphylococcus aureus Streptococcus faecalis Serratia marcescens