Micro Lab II - Sheet1.csv

During aerobic respiration microorganism produce hydrogen peroxide and in some cases an extremely toxic superoxide. Organisms capable of producing catalase rapidly degrade hydrogen peroxide. 2H2O2 ----catalase---> 2H2O + O2. This tests by adding H2O2 and to look for the pressence of O2 ( bubbles forming).

Enzyme used to breakdown Hydrogen peroxide into water and free O2. 

Alcaligues facealis G- (+). Psuedomonas aerugose G- (+). Staphylococcus aureus G+ (+). Enterococcus faecalis G+ (-).

Tests to see if the final electron acceptor is O2 and cytrochomes. Uses the reagent p-Aminodimethylaniline oxalate and will turn a purple color for a positive result. The reagent will be used as an artificial substrate donating electrons and thereby becoming oxidized in the presence of the oxidase adn free oxygen. Shows the pressence of oxidase activity.

Catalyzes the oxidation of reduced cytochrome by molecular oxygen resulting in the formation of H2O or H2O2.

"Differentiation among meembers of the genera Neisseria and Pseudomonas

Pseudomonas aeuogonsa G- (+). Stapylococcus G+ (-). Enterococcus facillis G+ (-). Escherisha coli G- (-). Alcaligues facealis G- (-).

Used as the final electron acceptor for the oxidase test. Will turn a purple color in the presence of cytochrome oxidase production.

All gram negative bacilli capable of fermenting glucose with the production of acid.Will be catalase positive but oxidase negative. Are normally highly motile because of peritrichous flagella.

Triple sugar iron agar test. Slant contains lactose and sucrose in 1% adn glucose (dextrose) in a centration of 0.1% . Has an acid base indicator of phenol red that will change the medium color red to yellow indicating the presence of acid.THE SLAB IS INOCULATED BY THE STAB METHOD.

"Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in agar butt). The organisms preferentially degrade glucose first. Since this substrate is present in minimal concentration

Acid over acid gas. Meaning that the slant will be streaked with a yellow discoloration (top layer). Then the layer underneath will be yellow due to acid production from fermentation and will have air pockets (bubbles) signifying gas was made. ORGANISMS: E.COLI. ENTEROBACTER AEROGENES. KLEBSIELLA PNEUMONIAE.

Will have acid on the slant will yellow discoloration. The lower part will have a yellow color with a dark black bottom signifying that it can use H2S.ORGANIMS: CITROBACTER FREUNDII. PROTEUS VOLAGARIS

Alkaline or acid. The top slant will be a red/pink color. Inside the tube will be yellow. This yellow in the inside means that the organism can only use glucose and not sucrose or lactose. . SHIGELLA FLEXNERI.

Alkaline or acid and H2S. The top will be a red pink color where the bottom will be a slight yellow and have a black discoloration . meaning that it can only use glucose and can use H2S. SLMONELLA TYPHIMURIUM

Alkaline over no change. Will be light pink on top and the same red color on the bottom. Happens when the organism is both oxidase positive and catalase positive meaning it can not do fermentation. PSEUDOMONAS AERUGINOSA. Not part of he enterobacteriaceae family

"There are two major fermentation pathways by which some microorganisms are able to produce hydrogen sulfide. (H2S). 1. The reduction (hydrogentation) of organic sulfur present in the amino acid cysteine. This is a component of the peptones in the medium. These peptones are degraded by a enzyme called DESULFURASE and an sulfur atom is lost and reduced by hydrogen. 2. The reduction of inorganic sulftates such as the thiosulfates

Is the tube in which a Hydrogen sulfide test is conducted. Contains peptones and sodium thiosulftate as the substrate ferrous sulfate (FeSO4) which will behave as teh H2S indicator. This will combine with the gas produced by forming an insoluble black ferrous sulfide preciptate that is seen along the line of the stab production.

Motility is recognized when the culture growth is not restricted to the line of inoculation. A motile organism will move away from the stab line spreading out in the tube.

"Indole

Tests to see if the microbe can oxidize tryptophan (an essential amino acid) with the enzyme trptophanase. When trryptophan is broken down it turns into indole. Indole is colorless and is detectable by adding Kovac's reagent. This will produce a cherry red reagent layer which is composed of P-DIMEHTYLAMINOBENZALDEHYDE. BUTANOL. AND HYDROCHLORIC ACID.

Obtain a SIM tube and then inoculate with needle via a stab. After incubating this test will tell you if the organism can oxidase H2S (hydrogen sulfide) by a black line where the stabe was. If the organism is motile by the spreading away from teh stab line. And if the organism can utilize tryptophan by adding Kovacs reagent and looking for the presence of indole at the top of the tube. This will be seen as a red color.

Used to check presence of a concentration of acid as the end products of the fermentation of glucose. Test is usually used to separate E.coli and E. aerogenes. The low ph of 4 is maintained by E. coli but E. aerogenes can enzymatically convert these acids to nonacid end products. When the methyl red is added in a pH of 4 it will turn red signifying a positive result. If it is added at a pH of 6 still shows the presence of alcohol but remains yellow indicating a negative result.

Determines the capability of some organisms to produce nonacidic or neutral end products such as acetylmethylcarbinaol from the organic acids that result from glucose fermentation. This test uses Barritts reagent which consists of a mixture of alcoholic alpha-naphthol and 40% potassium hydroxide solution. To detect the presence of acetylmethylcaribanol must be oxidized to diacetyl compound. If the acetylmethylcarbanol is present a red or rose color will appear.

A positive result is a red or rose color in the MR-VP medium. This is done with the addition of Barritt's reagent and will develop in about 15 minutes after the activation of acetylmethylcarbanol and represents a positive result.

In the absence of fermetable glucose or lactose some microorganisms are capable of using citrate. This depends on the presence of citrate permease that allows the transport of citrate in the cell. Citrate is the first major intermediate in teh Krebs cycle and is produced by teh condenstation of active acetyl with oxaloacetic acid. . Citrate is broken down by the enzyme citrase.

In a simmons citrate slant agar a streak is made on top of the slant and then incubated. The slant has a dark green color and a positive result will show growth on the surface and the slant will turn blue.

Tests for the presence of urease in a organism. Urease is a hydrolyic enzyme that attackes the nitrogen and carbon bonds in amide compounds such as urea and forms the alkaline end product ammonia. The urea broth medium contains a pH indicator phenol red.

The phenol red in the urea broth medium will change to a dark pink color in an alkaline environment created by the breakdown of urea into ammonia. No color change indicates a negative result

"Is selective for gram negative bacteria and differential for lactose fermenting gram negative bacteria. Inhibits gram + growth by the use of crystal violet in the medium. The incorporation of bile salts

is a high-molecular weight branching polymer composed of glucose molecules linked together by glycosdic bonds. The degradation of this macromolecule requires the presence amylase. This turns the long chain into shorter ones which can be broken down utilmately into maltose. This is down by the enzyme maltase.

The medium is composed of nutrient agar supplemented with starch. The detection of the hydrolytic activity within the plate to determine is the starch is still present in the plate. If starch is NOT used when iodine is placed on the colonies that use starch a blue-black color will appear around the colonies in the medium. If the starch is used a clearing will be seen around the colonies when the iodine is placed on them. Showing a positive result.

Are high-molecular weight compounds possessing large amount of energy. The breakdown of lipids are done by lipases which cleave the ester bonds in these molecules.

"Used to determine the hydrolytic activities of the exoenzyme lipase. Tributyrin forms an emulsion when dispered in teh agar producing an opaque medium. Organisms that produce lipase will show a zone of LIPOLYSIS

Casein is the major milk protein and is a macromolecule composed of amino acid subunits linked together by peptide bonds. They are broken into amino acids by the process called peptonization or proteolysis. This is done by the enzymes called proteases by cleaving the peptide bonds.

Is used to see if the organism produces the exoenzymes proteases which will break down the lactose in the plate. A positive result will in a ZONE OF PROTEOLYSIS around the bacterial growth.

Gelatin is a protein produced by hydrolysis of collagen is nutritional value is questionable since it lacks the essential AA tryptophan (indole test).Liquefaction is accomplished by some micro organisms.

A organism that produces gelatinase which will hydrolyze the gelatin in the agar and turn into liquid.