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.
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.
Oxidase test used to differentiate
"Differentiation among meembers of the genera Neisseria and Pseudomonas
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.
TSI slant results only glucose fermentation results
"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
Results TSI (A/Ag)
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.
Results TSI (A/AH2S)
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
Results TSI (K/A)
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.
Resluts TSI (K/AH2S)
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
Results TSI (K/N)
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
Hydrogen Sulfide Test Mechanism
"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 in the SIM tube (hydrosulfide tube).
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 Production test
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.
Indole test procedure
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.
Methyl red Test
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.
Vogues-Proskauer Test results
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.
Citrate Utilization Test
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.
Citrate Utilization Test Results
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.
Urease Test results
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.
Starch agar plate
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.
Nutrient gelatin deep tubes
A organism that produces gelatinase which will hydrolyze the gelatin in the agar and turn into liquid.
Facultative anaerobes are usually fermenters of carbohydrates. Fermentation is best described by considering the degradation of glucose by way of teh Embden-Meyerhof pathway or GLYCOLYITC PATHWAY.
A typical carbohydrate fermentation medium contains
"1. Nutrient broth ingredients for the support of the growth of all organisms. 2. A specific carbohydrate that serves as the substrate for determing the organisms fermentative capabilities (glucose
Results carbohydrate fermentation test
If the tube changes to a yellow color indicates that the carbohydrates have been fermented and produced acidic waste products. In some cases the organism can produce CO2 gas in addition to fermentation which can be seen in the inverted tube.
Nitrate Reduction Test
Reduction of nitrates by sonme aerobic and facultative anaerobic microorganisms occurs in teh absence of molecular oxygen. The cell will use organic substances such as nitrates (NO3) or sulfates (SO4) to supply oxgen that is subsequently utilized as a final hydrogen acceptor during energy formation . Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium.
Reagents added to Nitrate Broth
Following incubation two reagents are added: sulfanilic acid (solution A) followed by Solution B alpha-naphtylamine. DO NOT CONFUSE WITH BARRITTS REAGENTS.
Results Nitrate Reduction Test
Color change means that it is nitrate positive without having to add reagents. If it is colorless then you add the 2 reagents: sulfanic acid and alpha naphtylamine. Now if the solution turns red with the addition of these it is positive for nitrate. If no color change is seen with the addtion of the reagents then you add zinc. If the addition of zinc does not lead to a further color change you have a TRUE POSITIVE. If it does change color means that you reduced it with zinc leading to a TRUE NEGATIVE.
"composed of both pathogenic and non pathogenic organisms. The major species are S. aureus
Infections associated with S. epidermidis
Skin lesions and endocarditis
Infections associated with S saprophyticus
been implicated wiht some urinary tract infections
Infections associated with S. aureus
"Often responsible for the formation of abscesses
Produced by S. aureus. Which causes clot formation leukocidin. which lyses white blood cells. hemolysin which are active against red blood cells. and enterotoxins which are responsible for a type of nontoxic nature
Mannitol salt agar
Medium is selective for salt-tolerant organisms such as staphylococci. Differentiation among the staphylococci is predicated on their ability to ferment mannitol. Uses the indicator phenyl red to show manitol fermintation with a yellow color
Mannitol salt agar results
"S. aureus strains. exhibit a yellow halo surrounding their growth. and nonfermentating strains do not. Is should be noted that other salt-tolerant microorganisms
Production of coagulase is indicative of an S. aureus strain. The enzyme acts within host tissue to convert fibrinogen to fibrin. In the coagulase tube test for bound and free coagulase. Clot formation within 4 hours is interpreted as a positive result and indicative of a virulent S. aureus. DONE IN RABBIT PLASMA
Staphlococcus aureus manitol results
Staphlococcus epidermidis test results
(+) growth in manitol (-) fermentation. Colonial pigmentation is white. Coagulase (-). DNase (-). Hemolysis (-). Novobiocin sensitity: sensititve
Their homolytic activity. The serologic classificiation of Lancefield
"An incomplete form of hemolysis
A complete destruction of red blood cells exhibits a clear zone of approximately 2 to 4 times the diameter of the colony. Streptococcus pyrogens and Streptococcus agalactiae
"Is indicative of the absence of any hemolysis around the colony. Most commonly
Group A streptococcus
"S. pyogenes referred to as Streptococcus pyogenes
Group B streptococcus
"S agalactiae Beta hemolytic. A resistant. CAMP test (+) S. pneumonia alpaha hemolytic. OP sensitive ""optochin. "
Group B streptococci produce a peptide CAMP substrance that acits in concert with the Beta hemolysis produced by some strains of Staphylococcus aureus causing an increased hemolytic effect. Streaking a blood plate with a Staphlococcus aureus and then perpendicularly streaking the Streptococcus (B) should produce an an arrow-shaped zone of hemolysis adjacent to th4e central streak of S. aureus growth.
Standard Qualitative Analysis of Water
1. Determines the presence of coliform bacteria in a water sample. 2. Obtains an index indicating the posible number of organisms present in the sample under analysis. 3. Confirms presence of coliform bacteria in a water sample for which the presumptive test was postive. 4. Confirms the presence of coliform bacteria in a water sample
"G-. Facultative anaerobe
Is specific for detection of coliform bacteria. Is a series of tubes (3 tubes used in lab) containing lactose fermentation broth. One set of 3 tubes had double strenght lactose broth and was inculated with 10ml of water sample. The next set of 3 tubes was single strenght lactose tubes innoculated with 1ml of water sample. The last set of 3 tubes of single strenght lactose was inculated with 0.1ml of the water sample. Then the tubes were incubated and the results were used to give a probility of amount of fecal contamination
Most Probably number Test (MPN)
Is conducted by determining the number of tubes in each group that show gas following the incubation period. Gives a probability of the amount of contamination
After the presumptive test is postive a eosin-methylene blue (EMB) agar or Endo agar is streaked from a positive lactose broth tube. EMB forms a complex that precipitates out onto the coliform colonies producing a green metalic sheen on the plate. Endo agar will show dark pink complex tht turns the E.coli colonies and the surrounding medium pink
"After a positive EMB eosin-methylene blue (EMB) test an isolated colony is picked up and grown in a lactose broth once again to look for the pressence of gas production. Along with a slant incoculated. If the lactose and tube produces gas and the gram stain from the slant reveals gram negative rods
4 methylum bellifferyl Beta D Glucuroni DE. Is a medium in which E. coli will glow under uv light. Substrate mug will be released by glucuronidase to make mug positive in UV
The enzyme used to break down ECMUG into the ultraviolet compound. Is used at the final step of water testing. The completed test
Citrate Test indicator
Bromethyl blue. Will turn blue in the pressence of alkaline environment. Citrate utilization produces pyruvate and CO2. CO2 causes the pH to increase
Nitrate Reduction Pathway
Nitrate (NO3) ---> NO2 (nitrite) where you add reagents for color change ---> complete (add zinc) ---> NH3 color change in tube ----> gas in tube
Eosin methylene blue. Will be a metallic green will be positive for coliforms. Selective Gram (-). Lactose positive (+)
Used to test for the bacteria Samonella typi. Test for pressence of a specific antibody for Salmonella typhi H flagella antigen (type d). Will take advantage of an agglutination phenomenon resultign from the aggregation of particulate antigens by a specific antibody.
Widal Test Procedure
Prepare a serial dilution (1:10 to 1:1280). This will be done by adding 0.9ml of .85 NaCl and .1 of serum making a 1:10 dilution. Then add 500 of this to another tube and mix with another 500 Normal saline. Continue this dilution. After double dilutions are made adding 500ml of the H FLAGELLA ANTIGEN. then incubate for 1 hour at 50%.
Results for Widal (postive)
Look for agglutination in the tubes. Highest one with particles (clumps on the bottom of it). When it turns homygenus.
First titer conducted
Second followup titer. If you get a x4 increase is positive result.
Strep vs Staph catalase test
Staph catalase (+) Strep catalase (-)
Alpha hemolysis. Optochin resistant
also known as a p disk works on Streptococcus pneumonia
Normal bacterial flora found in mouth
1. Corynebacterium (G+) CLUB SHAPPED. 2. Neisseria (G -) DIPLOCOCCI
Cell eat eating . When a netrophil or macrophage. Will be inside cell Staphlycoccus epidermidus
Unable to be eaten by cell can be due to capsule or pilus. Ex. Streptococcus pnuemonia.