Growth Requirements of E. coli

In growing a bacteria there must be consideration of factors that helps the cell grow, or synthesize. Growth factors that include all the nutrients required by the organism to be cultivated, and such factors as pH, temperature and aeration. Agar,is a good medium where microbes are capable to grow in them, this is an extract form marine alga.

First factor to consider is its nutrients,A very good medium includes the exact and accurate qualities and amount of concentrations in every component f the medium, to make the bacteria adapt to its new environment. Otherwise, it is much cheaper and simpler to use natural materials such as yeast extract, protein digest, or similar substances. A factor to consider is the Hydrogen Ion Concentration (pH)A, where it is very important to determine the optimum range they can grow in. Most organisms grow best at a pH of 6.0-8.0, although some forms have optima as low as pH 3.0 and others have optima as high as pH 10.5. Another factor is temperature, microorganisms vary on the range of the temperature they are capable to grow in with such as psychrophilic forms grows best at low temperature( 15-20 C) mesophilic forms grow best at 30-37 C; and most thermophilic forms grow best at 50-60C. Proper aeration & ionic Strength & Osmotic Pressure are also factors affecting the growth of bacteria.

Escherichia coli occurs in enormous numbers in normal feces and is widely distributed in the intestinal canal of animals and humans. Ordinarily it does no harm.

Extensive studies of bacteria in the feces of infants with diarrhea indicate that many cases of infant diarrhea are due to certain particular kinds of Escherichia coli. These pathogenic E. coli can be distinguished from less harmful varieties by immunologic studies of their antigenic nature. 14 enteropathogenic E. coli serotypes are generally recognized. In a study by Rantz it is pointed out that in patients with urinary infections, about 45% of these infections were acquired in the hospital as the result of catheterization.

Since Escherichia coli is always present in feces, and since other species of Escherichiae tribe frequently accompany it and closely resemble it, this tribe is frequently referred to as "the coliform group." They are easily cultivated and recognized and usually remain alive in foods and water for considerable periods of time. The coliform group is therefore commonly sought after in bacteriologic examinations of water, milk, and food as evidence of fecal pollution, rather than Salmonella or Shigella.

Enterotoxic E. coli

Some strains of E. coli produce two kinds of soluble endotoxin, one heat-labile and antigenic, that acts somewhat like cholera toxin, causing severe diarrhea in humans. These enterotoxic strains should be considered as a distinct group of enteropathogenic E. coli; they differ serologically from the common H and O varieties that do not produce any soluble exotoxin.

Since the coliforms rapidly ferment lactose with the production of acid and gas, measured portions of the suspected water are put into broth containing lactose. If gas is present after 24 to 48 hours, it is not necessarily due to coliform species. Several other organisms also produce gas from lactose. Plates or tubes of selective medium are therefore inoculated from the lactose-broth tubes showing gas. After incubation, colonies resembling those of the cloriform group are selected for pure-culture study and are subjected to simple biochemical tests that easily identify them.

source:  Revised by Robert Fuerst, PhD. (1978). Frobisher and Fuerst's Microbiology in health and disease. (14th ed.). West Washington Square, Philadelphia: W.D. Saunders Company.

source :Brock,Thomas D. Biology of microorganisms.6th ed. Englewood Cliffs, N.J. 

Kjeldahl Method

Introduction

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

Determination of Protein Concentration


1.Kjeldahl method
-a food digested with strong acid so that it releases nitrogen which can be determined by suitable titration techniques.
-does not measure the protein content easily
-Conversion factor is needed to convert the measured nitrogen concentration to a protein concentration


Principles:
A. Digestion
- sample is heated in the presence of sulfuric acid (oxidizing agent), anhydrous sodium sulfate, and catalyst.
- digestion converts any nitrogen in the food into ammonia and other organic matter to carbon dioxide and water.
- ammonia gas remains in acid solution because it is in the form of ammonium ion which binds to the sulfate ion to form ammonium sulfate.  


B. Neutralization
- the solution in the digestion flask is then made alkaline by addition of sodium hydroxide which converts  the ammonia sulfate into ammonia gas
- the ammonia gas moves out into receiving flask which contains an excess of boric acid
- in receiving flask. ammonium ion and boric acid to borate ion


C. Titration
- titrate with standard sulfuric or hydrochloric acid using a suitable indicator
- the concentration of hydrogen ions required to reach the end-point is equivalent to the concentration of nitrogen
- once nitrogen content has been determined, it is converted to a protein content using appropriate conversion factor

Advantages:
-universal
-high precision
-good reproducibility 


Disadvantages:
-does not give a measure of true protein
-different proteins need different concentration factors
-the use of concentrated sulfuric acid at high temperature
-time consuming


2. Enhanced Dumas method
-Principles: A sample of known mass is combusted in an high temperature to release carbon dioxide, water and nitrogen
- Nitrogen content is measured by separating nitrogen from carbon dioxide and water by using a column
-also used conversion factor to determine protein content


Advantages
-faster
-does not need toxic
-easy to use
-samples can be measured automatically


Disadvantages:
-high initial cost
-does not give a measure of true protein
-small sample size make its difficult to obtain a representative sample


3. Methods using UV-Visible spectroscopy
-use either the natural ability of proteins to absorb (or scatter) light or chemically or physically modify proteins to make them absorb (or scatter) light in the region
- calibration curve of absorbance versus protein concentration must be built first
-main difference: the chemical groups which are responsible for the absorption or scattering of radiation


A. Direct Measurement at 280nm
-Principle: tryptophan and tyrosine absorb ultraviolet  light strongly at 280 nm
-use the same wavelength to measure protein concentration
-Advantages: simple to carry out, non-destructive and no special reagents are required
-Disadvantages: nucleic acid absorb strongly at 280 nm
-To overcome: measure the absorbance at two different wavelengths.

Kjeldahl Method for Determining Nitrogen

There are three main steps in K jeldahl method namely digestion, distillation and titration.

Digestion- is usually done by boiling a homogenous sample in concentrated sulfuric acid and the end result is an ammonium sulfate solution. The general equation for this step that is shown below:  

Organic N + H2SO4    →

(NH4)SO4 + H2O + CO4 + other sample matrix byproducts

In distillation additional excess base to the digestion product in converting NH4 to NH3 as indicated in the follow up equation. NH3 is recovered by distilling the reaction product.

ammonium sulfate	heat	ammonia gas
(NH4)2SO4 + 2NaOH	→	2NH3 + Na2SO4 + 2H2O

Titration

Titration quantifies the amount of ammonia in the receiving solution. The amount of nitrogen in a sample can be calculated from the quantified amount of ammonia ion in the receiving solution.

There are two types of titration—back titration and direct titration. Both methods indicate the ammonia present in the distillate with a color change.In back titration (commonly used in macro Kjeldahl), the ammonia is captured by a carefully measured excess of a standardized acid solution in the receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change until the solution is "back titrated" with base.

ammonia	standard sulfuric acid acid		excess ammonium sulfate	sulfuric acid
2NH3 +	2H2SO4	→	(NH4)2SO4 +	H2SO4
(no color change)

ammonia sulfate	measured excess acid	measured sodium hydroxide		ammonium sulfate
(NH4)2SO4 +	H2SO4 +	2NaOH	→	Na2SO4 + (NH4)2SO4 + 2H2O
(color change occurs)

In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical reaction is:

ammonia gas	boric acid		ammonium- borate complex	excess boric acid
NH3 +	H3BO3	→	NH4 + H2BO-3 +	H3BO3
(color change occurs)

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solutions changes.

ammonium- borate complex		sulfuric acid		ammonium sulfate		boric acid
2NH4	+	H2BO-3	+	H2SO4 (NH4)2SO4	+	2H3BO3
(color change occurs in reverse)

The boric acid method has the advantages that only one standard solution is necessary for the determination and that the solution has a long shelf life.

Sources:   

Cole –Parmer Technical Library Cole (September 2, 2011). http://www.coleparmer.com/techinfo/techinfo.asp?htmlfile=KjeldahlBasics.htm&id=38

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.