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Lab report of the isolation an identification gram positive bacteria

This differentiation is based on the differences in carbohydrate fermentation patterns and hydrogen sulfide production by the various groups of intestinal organisms. Carbohydrate fermentation is detected by the presence of gas and a visible colour change from red to yellow of the pH indicator, phenol red. The production of hydrogen sulfide is indicated by the presence of a precipitate that blackens the medium in the butt of the tube. Due to the building of acid during fermentation, the pH falls.

The acid base indicator Phenol red is incorporated for detecting carbohydrate fermentation that is indicated by the change in colour of the medium from orange red to yellow in the presence of acids. In case of oxidative decarboxylation of peptone, alkaline products are built and the pH rises. This is indicated by the change in colour of the medium from orange red to deep red. Sodium thiosulfate and ferrous ammonium sulfate present in the medium detects the production of hydrogen sulfide indicated by blackening in the butt of the tube.

To facilitate the detection of organisms that only ferment glucose, the glucose concentration is one-tenth the concentration of lactose or sucrose. The small amount of acid produced in the slant of the tube during glucose fermentation oxidizes rapidly, causing the medium to remain orange red or revert to an alkaline pH.

In contrast, the acid reaction yellow is maintained in the butt of the tube since it is under lower oxygen tension. After depletion of the limited glucose, organisms able to do so will begin to utilize lab report of the isolation an identification gram positive bacteria lactose or sucrose.

To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely. If the tube is tightly closed, an acid reaction caused solely by glucose fermentation will also involve the slant.

Urea is a nitrogen containing compound that is produced during the decarboxylation process of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is thus it is an efficient way for the human body to excess nitrogen. This excess urea is then taken away from the body with the help of the kidneys as a part of urine.

Certain bacteria produce the enzyme urease during its metabolism process and that will break down the urea in the medium to ammonia and carbon dioxide: The urease test is useful in identifying the genera Proteus, Providentia, and Morganella, which liberate this enzyme.

Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Urease is a hydrolytic enzyme that attacks the nitrogen and carbon bond in amide compounds such as urea and forms the alkaline end product ammonia. The presence of urease is detectable when the organisms are grown in a urea broth medium containing the pH indicator phenol red.

As the substrate urea is split into its products, the presence of ammonia creates an alkaline environment that causes the phenol red to turn to deep pink. This is a positive reaction for the presence of urease. Failure of deep pink colour to develop is evidence of a negative reaction. The ability of an organism to move by itself is called motility. Motility is closely linked with chemotaxis, the ability to orientate along certain chemical gradients. Prokaryotes move by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility.

Almost all spiral bacteria and about half of the bacilli are motile, whereas essentially none of the cocci are motile. This media has a very soft consistency that allows motile bacteria to migrate readily through them causing cloudiness. In soft agar tubes non-motile bacteria will only grow on the inoculated region. Motile bacteria will grow along the stab line and will tend to swim out away from the stabbed area. Therefore, a negative result is represented by growth in a distinct zone directly along the stab.

A positive result is indicated by diffuse or cloudy growth mainly at the top and bottom of the stabbed region. Gelatin, a protein derived from the animal protein collagen.

It has been lab report of the isolation an identification gram positive bacteria as a solidifying agent in food for a long time. Robert Koch used nutrient gelatin as an early type of solid growth medium. One problem is that many bacteria have the ability to hydrolyze liquefy gelatin. This gelatin liquefaction ability or inability forms the basis for this test. Some microorganisms possess an enzyme called gelatinase, which breaks down gelatin into amino acids.

Gelatin deeps contain the substrate gelatin, which is a protein produced by the hydrolysis of collagen. Organisms which hydrolyze gelatin will cause the gelatin to liquefy.

The gelatin hydrolysis tests for an organism's ability to break down the protein gelatin which is derived from collagen. If the organism can release gelatinase enzymes the gelatin is broken down or liquefied. The media is checked over a period of about a week after inoculation and incubation at room temperature, for gelatinase activity.

The tube is placed on ice for a few minutes and if the media fails to solidify it is considered a positive test. The gelatinase reaction may be slow or incomplete Fig 7. Gelatin hydrolysis test The gelatin hydrolysis is indicated by the liquid nature of the gelatin positive test and the negative test is indicated by the solid nature of the gelatin Nitrate Reduction Broth: Zinc powder act as a catalyst and that will favours the reduction of nitrate to nitrite.

Nitrate reaction occurs only under anaerobic conditions Fig 8. The medium is then transferred in tubes to make a low surface area to depth ratio that will limit the diffusion of oxygen into the growth medium.

  1. Thus this enzyme is a good indicator of the pathogenic potential of S.
  2. The absence of coagulation after 24 hours of incubation is a negative result, indicative of an avirulent strain.
  3. This excess urea is then taken away from the body with the help of the kidneys as a part of urine.

Most bacteria utilize the available oxygen in the medium for their growth and will rapidly produce anaerobic conditions for the further reactions. Nitrate reduction test Catalase Test: The inability of strict anaerobes to synthesize catalase, peroxidase, or superoxide dismutase may explain why oxygen is poisonous to these microorganisms. In the absence of these enzymes, the toxic concentration of H2O2 cannot be degraded when these organisms are cultivated in the presence of oxygen.

It also contains four porphyrin heme groups ie. The enzyme catalase is present in most cytochrome containing aerobic and facultative anaerobic bacteria. Catalase has one of the highest turnover numbers of all enzymes such that one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen in a second.

Catalase production and activity can be detected by adding the substrate H2O2 to an appropriately incubated 18- to 24-hour tryptic soy agar slant culture.

Organisms which produce the enzyme break down the hydrogen peroxide, and the resulting O2 production produces bubbles in the reagent drop, indicating a positive test. Organisms lacking the cytochrome system also lack the catalase enzyme and are unable to break down hydrogen peroxide, into O2 and water and are catalase negative. Coagulases are enzymes that clot blood plasma by a mechanism that is similar to normal clotting.

The coagulase test identifies whether an organism produces this exoenzyme. This enzyme clots the plasma component of blood. The only significant disease-causing bacteria of humans that produce coagulase are Staphylococcus aureus. Thus this enzyme is a good indicator of the pathogenic potential of S. Formation of clot within 4 hours is indicated as a positive result and indicative of a virulent Staphylococcus aureus strain. The absence of coagulation after 24 hours of incubation is a negative result, indicative of an avirulent strain.

Oxidase test is an important differential procedure that should be performed on all gram-negative bacteria for their rapid identification. This method uses N,N-dimethyl-p-phenylenediamine oxalate in which all Staphylococci were oxidase negative. Pseudomonas aeruginosa is an oxidase positive organism. Amylases are a class of enzymes that are capable of digesting these glycosidic linkages found in starches. Amylases can be derived from a variety of sources.

Amylases are present in all living organisms, but the enzymes vary in activity, specificity and requirements from species to species and even from tissue to tissue in the same organism. Alpha-amylase 1,4 alpha D-Glucan-glucanohydrolase acts upon large polymers of starch at internal bonds and cleaves them to short glucose polymers. Alpha-amylase catalyzes the hydrolysis of internal Alpha-1-4 glucan bonds in polysaccharides containing 3 or more alpha 1-4 linkages; it results in a mixture of maltose and glucose.

Amyloglucosidase works on the shorter polymers and splits off single glucose sugars. Bacterial alpha-amylase is particularly suited for industrial usage since it is inexpensive and isothermally stable. Starch agar is an example of differential medium which tests the ability of an organism to produce certain alpha-amylase and oligo-1, 6-glucosidase that hydrolyze starch.

Starch agar is a simple nutritive medium with starch added. Iodine turns blue, purple, or black the colour depends on the concentration of the iodine used in the presence of starch. A clearing around the bacterial growth shows that the organism has hydrolyzed starch.

Trybutyrene agar is used for the detection and enumeration of lipolytic microorganisms in food lab report of the isolation an identification gram positive bacteria other material Fig 9.

Left side;positive for lipid hydrolysis;right side;negative for lipid hydrolysis Growth on selective and differential media: Selective media allows only the growth of certain types of organisms, while inhibiting the growth of other organisms.

Differential media are employed to differentiate certain closely related organisms or groups of organisms. Depending on the presence of specific dyes or chemicals in the growth media, the organisms will tend to produce certain specific characteristic changes or lab report of the isolation an identification gram positive bacteria patterns that can be used for further identification or differentiation steps.

Enriched media are media that have been supplemented with highly nutritious materials such as blood, serum or yeast extract for the purpose of cultivating fastidious organisms. Blood agar, Chocolate agar Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures.

Eosin methylene blue agar is both a selective and differential medium used for the detecting and isolating Gram-negative pathogens residing in the intestine.


Different streptococci produce different effects on the red blood cells in blood agar. Those that produce incomplete hemolysis and only partial destruction of the cells around colonies are called alpha-hemolytic Streptococci. Characteristically, this type of hemolysis is seen as a distinct greening of the agar in the hemolytic zone, and thus this group of Streptococci has also been referred to as the viridans group.

Species whose hemolysins cause complete destruction of red cells in the agar zones surrounding their colonies are said to be beta-hemolytic. When growing on blood agar, beta-hemolytic Streptococci are small opaque or semi translucent colonies surrounded by clear zones in a red opaque medium.

Some species of Streptococci do not produce hemolysins. Therefore, when their colonies grow on blood agar, no change is seen in the red blood cells around them. These species are referred to as nonhemolytic or gamma hemolytic Streptococci.