over view on culture media

An appropriate culture media is needed for the proper growth and maintenance of a microorganism that is done in the laboratory. These specialized culture media are important in the identification and isolation of microorganism. Each microorganism varies in terms of their needs, such as energy, carbon, nitrogen, phosphorus, sulfur, and various minerals that they acquired in their specific culture media. It is necessary to have knowledge in identifying the normal habitat of microorganism so that it would be easier in selecting specific culture media that is suitable for them.  

Complex media is composed of peptones, meat extract and yeast extract. A partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources produces protein hydrolysates called peptones. Meat extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals. Both meat and yeast extract are aqueous extracts of élan beef and brewer’s yeast. They serve as sources of carbon, energy, and nitrogen.

 Selective media favor the growth of particular microorganisms. Bile salts or dyes like basic fuschin and crystal violet favor the growth of gram negative bacteria by inhibiting the growth of gram positive bacteria without affecting the gram negative organisms. Endo agar, eosin methylene blue agar, and Mac Conkey agar are wisely used for the detection of E. coli.  

Morpholigical Description of E.coli

Escherichia coli is quickly identified in urine by the process of hemolysis that occurs in blood agar.  It is determined in a differential media such as EMB having a morphological feature of an iridescent “sheen”. It gives positive test for indole, lysine decarboxylase, and manitol fermentation and produces gas from glucose.

Culture Media Contains Nutrients for Culture Growth

Since Pasteur and Koch, microbiologists have tried to mimic the natural environment of bacteria in order to grow them in a laboratory. Beef broth is one medium used for cultivation of bacteria, or nutrient broth which contains water, beef extract and peptine, a nitrogen preparation from plant or animal sources. Agar is used to solidify the medium, upon the addition of this, the product is called nutrient agar.

"Agar is a polysaccharide derived from marine red algae. Introduced by the school of bacteriology of Robert Koch, agar is a unique colloid that remains liquid until cooled top below approximately 36˚ C. This allows for mixing blood with culture media for determination of haemolytic reactions. The solidified medium can be used to cultivate bacteria, isolate pure cultures, or accomplish other tasks, such as a medium for measuring bacterial growth. Once solidified, agar will remain solid at room temperature. It will not melt until it reaches a temperature of 85˚C, making the material excellent for growing thermophilic bacteria on nutrient agar. Agar adds no macronutrients to the nutrient medium. Sometimes it is valuable to use semi-solid medium, such as when testing bacterial motility. In this case, a lower concentration of agar is added to the medium to make it stiff but not as solid as nutrient agar"

  Nutrient broth and nutrient agar are examples of chemically undefined medium or complex medium.  We cannot be certain of the exact components or the quantity thus it is called complex. We do not precisely know what carbon and energy sources are present nor what other factors that facilitates growth are present. Complex media are commonly used in the teaching laboratory. Because you simply want to grow bacteria and are not concerned what specific nutrients are needed to accomplish this.

Another type of medium is a chemically defined medium or synthetic medium. In this type of medium, the components are known. This is commonly used when understanding the growth conditions necessary for bacterial growth.

Biuret Protein Assay

The bond between the amino group and the carboxyl acid group on adjacent amino acids in a protein is a peptide bond. When the Biuret reagent (1 percent solution of copper sulfate) is added to a solution containing peptide bonds, the solution turns a violet color. The violet color is a positive test for the presence of protein. The more intense the color, the greater the number of peptide bonds that react.

Principle:

Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent.

Equipment:

In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 450 nm. Glass or polystyrene (cheap) cuvettes may be used.

methodology

Reagent

A formula for biuret reagent is (per liter final volume) 9 gm Sodium potassium tartrate (f.w. 282.22), 3 gm Copper sulfate x 5 H2O (f.w. 249.68), 5 gm Potassium iodide (166.0), all dissolved in order in 400 ml 0.2 M NaOH (f.w. 40.0) before bringing to final volume. The volume can be scaled up or scaled down of course. Discard if a black precipitate forms.

Assay

1. Volumes sample, reagent can be scaled up/down and/or volume ratios varied, as with any assay.
2. Warm up the spectrophotometer 15 min. before use.
3. Prepare standards from bovine serum albumin, preferably calibrated using absorbance at 280 nm and the extinction coefficient. Using 5 ml color reagent to 1 ml sample a recommended range is 0.5 to 20 mg protein.
4. Prepare a reference tube with 1 ml buffer.
5. If possible, dilute unknowns to an estimated 1 to 10 mg/ml with buffer; a range of dilutions should be used if the actual concentration cannot be estimated.
6. Use 1 ml sample per assay tube
7. Add 9 ml Biuret reagent to each tube, vortex immediately, and let stand 20 min.
8. Read at 550 nm.

Analysis

Prepare a standard curve of absorbance versus micrograms protein (or vice versa), and determine amounts from the curve. Determine concentrations of original samples from the amount protein, volume/sample, and dilution factor, if any.

Comments

The color is stable, but all readings should be taken within 10 min. of each other. As with most assays, the Biuret can be scaled down for smaller cuvette sizes, consuming less protein. Proteins with an abnormally high or low percentage of amino acids with aromatic side groups will give high or low readings, respectively.
For Bovine serum albumin we typically obtain a linear relationship between absorbance and amount protein over a range of 0.5 to 20 mg protein. The assay has not been reliable for amounts below 0.5 mg, however the actual sensitive range may extend beyond the upper limit.

References

• Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949.
• Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957.
• Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940.
• Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269.
• Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.

All About E.coli

Organism: E-coli

Habitat (reservoir) –normal bowel flora of humans and others animals may also inhabit female genital tract.

Mode of transmission

-          Varies with the type of infection. For non-gastrointestinal infection, organism may be endogenous or ma spread person to person, esp. in the hospital setting ; for gastrointestinal infection, transmission ode varies with the type of E-coli and may involve fecal-oral spread bet. Humans via contaminated food or water or consumption of undercooked beef or milk colonized cattle

Virulence Factor:

Several,including endotoxin , capsule production and pili that mediate attachment to host cells.

Colunial appearance ofen beta hemolytic on blood agar, but most other genera are non- hemolytic.

Colonial appearance and characteristic :

  Mac – LF; flat, dry, pink colonies with a surrounding darker pink area of precipitated bite salts.

HE- yellow

XLD- yellow  

Bacterial and Archaeal Cells Reproduce Asexually

The interval time between successive binary fission of a cells of population of cells I kwon the generation time (double time) . Under optimal condition , some species have a very fast generation time; for others, it is to much slower.

The generation time is useful in determining the amount of time that passes before disease symptom appear in an infected individual; faster division times often mean shooter incubation period for a disease. For example, suppose you eat an undercooked hamburger contaminated with pathogens E.coli 057:H7, which has one of the shortest generation times – just 20 minutes under optimal conditions.

*The number of E. coli cells progress from 1 cell to 2 million cells in a mere 7 hours.  The J-shaped growth curve gets steeper as the hours pass.  Only a depletion of food, build up of waste, or some other limitation will halt the progress of the curve.

Gas producing bacteria. The Genus  aerobacter . The organism belonging to this group are of intestinal and soil origin: Escherichia coli, motile ad without capsule: and Aer. Aerogenes, nin-motle and frequenty  capsulated when grown in milk. And they do not produce spores an and are gram-negative, differing in this latter respect from the other  lactic bacteria. These organism grow readily on ordinary cultured media. The colonies on agar or gelatin plates are easily differentiated from those of the preceding group because they are much larger and inclined to be slimy. The optimum growth temperature is above blood heat, but the organism grow well at lower temperature. In the presence of suitable carbohydrates these organism can developed under anaerobic conditions, but when deprived of sugars they are aerobic.

Presumptive Method. Advantage is taken of certain of the physiological characters of E. coli to differentiate it from the other organism. The media commonly used lactose broth and lactose bile in fermentation tubes. When various amount of water to be examined are inoculated into fermentation tube containing lactose broth kept at blood heat for twenty four hours, gas will be produce on those tubes in which E. coli is present.  It seems safe to infer that E. coli I not present if the gas is not produced in any tubes inoculated. The reverse, however, that E, coli must necessarily be present whenever gas is not produced, does not hold gold for there are other organisms which can ferment lactose with the production of acid gas. The test is more definite when a 2 percent of solution of lactose bile substituted for the lactose broth in tubes. The bile inhibits the growth of the most organism not of intestinal origin. Gas production, under these conditions, therefore in the large proportion cases means that this organism is not present. These tests are termed presumptive tests, because while they enable one to recognized waters which are suspicious, they do not certainly identify the organism responsible for the gas in the suspicious cases.  This permits one, in the words, to divide waters tested into two groups and those which are certainly good and those which are suspect.  The latter require further examination.

The presumptive test is made by inoculation of varying dilutions of the water being assayed into a series of fermentation tubes of lactose broth.  In cases in which detection of coliform organisms in a considerable amount of water, such as fifty or hundred milliliter quantities, is attempted large tubes or bottles must be used in which have been placed amount of double or quadruple strength lactose broth so that upon dilution by the addition of within approximately twenty-four hours at 35 degree -37 degree C is presumptive evidence that coliform organisms were present in forty eight hours but not in twenty hours, the results is classified as doubtful, and a confirmed or completed test is carried through.  If no gas is produced, it is assumed that no coliform bacteria were present in the inoculums.  All cases of doubtful reaction, and preferably positive presumptive reactions as well, should be tested further by use of confirmation test.  A confirmation may be made by transfer of a loop of the broth to any one or more of several media.  It may be streaked on the surface of Endo agar or on eosin methylene blue agar in plates, or inoculated into a tube of brilliant green lactose bile.  If colonies typical of Escherichia coli are produced on the agar or if gas is produced in the brilliant green lactose bile, the presence of coliform organism is confirmed.  If typical colonies only are produced, transfers of such colonies should be made and pure cultures grown on agar slant and in lactose broth.  If the organism belongs to the coliform group stained preparation will show it to be rod-shaped, gram-negative and without spores and gas should be produced in lactose broth.

Identification of Escherichia coli.  Many methods have been devised for the differentiation of the colon bacillus either directly from water or from lactose broth or lactose bile tubes discussed above.  When water containing E coli is plated on agar containing 1 percent lactose and sufficient litmus solution to color it blue, and is then incubated for twenty four hours at blood heat, the lactose fermenting colonies will be found to be surrounded by a red zone.