2 MetabolismMetabolism is the sum total of all chemical reactions that occur in a cell.2 types of metabolic pathways; catabolism and anabolism.
3 catabolismThe breakdown of food material either through respiration or fermentation
4 AnabolismThe biosynthesis of materials needed by the cell such protein production or photosynthesis
5 Laboratory Aim today!To determine if our unknown sample carries out respiration (oxidative) or fermentation (fermentative).If our organism is fermentative, which sugars are specifically fermented?
6 O/F Glucose testThis test will be done to determine if our organism is oxidative or fermentative!O/F Glucose is a differential medium that contains three important ingredients1- glucose2- peptone3- brom-thymol blue pH indicator that turns yellow at acidic pH
7 Procedure1- you will inoculate 2 tubes with your unknown by stabbing the agar.2- to one tube, you will aseptically layer with a couple of drops of mineral oil. This prevents the diffusion of oxygen and helps establish anaerobic condition.
8 You will repeat the procedure with your two control organisms; E coli (facultative) and Pseudomonas (oxidative)A color change from green to yellow means a positive test. Due to pH indicator changing as a result of slight acid production due to respiration. Look for yellow near the top of tube if organism is aerobic!
10 Fermentative tests1- specific sugar tests such as glucose, mannitol and lactose.2- Mixed acid fermentation using MR-VP medium3- 2,3- butanediol fermentation4- citrate fermentation
11 Specific sugar fermentation reactions 1- we will use specific sugar broth with a Durham tube inside.2-The pH indicator phenol red will be used to detect the formation of acid that indicates fermentation.3- you will inoculate your unknown into a tube of glucose, lactose and mannitol!4- the control tube will be E.coli into a glucose tube.
13 Mixed acid fermentation Some species of bacteria such as Escherichia, Proteus, Salmonella, and Aeromonas can ferment glucose and produce acidic products such a lactic acid, succinic, acetic, and formic acid.In addition they can produce CO2 and H2 gas due to the presence of an enzyme called formic hydrogenlyase.
14 We will use the medium called MR-VP to inoculate our unknown and your control which is E. coli. MR-VP medium contains glucose, peptone, and dipotassium phosphate.Next week we will use methyl red to detect the presence of mixed acids!
15 2,3- Butanediol fermentation Some species of bacteria such as Enterobacter and Serratia can ferment glucose and produce a neutral end product such as 2,3-butanediol.This is important because usually if a type of bacteria is + for mixed acid it is – for neutral fermentation. It allows clinical microbiologists to distinguish between important gram – bacteria.
16 You will inoculate a tube of MR-VP medium with your unknown and the control organism which is Enterobacter aerogenes.Next week, we will discuss how we detect the neutral end product using Barritt’s reagent.
17 Citrate fermentationSome bacteria can break down citrate to oxaloacetate and pyruvate. These intermediates are then fermented to produce formate, lactate, acetate, acetoin, and carbon dioxide.
18 We will use Simmons citrate agar We will use Simmons citrate agar. It contains citrate and ammonium salts to serve as a source of nitrogen. As the organisms break down the citrate they must also break down the ammonium salts into ammonia. Ammonia raises the pH and the agar changes color from green to Prussian blue.
20 You will inoculate the Simmons citrate agar with your unknown and the control which is Enterobacter aerogenes. First streak with a needle and then stab it!
21 Oxidative tests 1- cytochrome oxidase test 2- catalase test 3- nitrate reduction
22 Cytochrome C oxidase test Aerobic organisms such as Pseudomonas carry out respiration and possess an enzyme called cytochrome oxidase that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen.You will streak half a TSA plate with your unknown and the other half with your control Pseudomonas aeruginosa.
23 Next week, we will use an artificial electron acceptor to detect the enzyme! The artificial acceptor is called N,N,N,N-tetramethyl-p-phenylenediamine!
24 Catalase testA dangerous by product of aerobic respiration is hydrogen peroxide. Aerobes produce an enzyme called Catalase that breaks down the hydrogen peroxide into water and oxygen.Next week, you will use hydrogen peroxide to detect the presence of the enzyme by your unknown. The TSA plate will also be used for this experiment. The control is a nutrient agar slant inoculated with S. aureus.
25 Nitrate reductionSome bacteria can use nitrates as the final electron acceptor instead of oxygen.Some bacteria such as Bacillus can reduce the nitrates to gas end products such as N2O or N2.Others such as E.coli can reduce the nitrates to nitrites.
26 We will assay for the final end product, gas or nitrite! We will use beef extract medium with potassium nitrate. A Durham tube will be in place to detect gas formation.You will inoculate a tube of beef extract with your unknown and the control E. coli.
27 Next week, we will detect gas formation by looking at Durham tube and detect nitrite formation using Nitrate reagent A and B. These 2 reagents react with nitrite to give you a deep red color!