RNAi regulates cancerous gene expression

One of the premises for the study of relatively rare inherited cancers is the assumption that understanding the molecular basis of these malignancies will result in better understanding and treatment strategies of the more common malignancies. While this has not yet resulted in new therapies, the management at diagnosis of many patients with an inherited predisposition has been radically altered. Thus, a working knowledge of cancer genetics will allow the clinician not only to appropriately manage patients, but also to comprehend the major advances in the field of oncology (1).

At the molecular level, cancer is caused by mutations in DNA, which result in aberrant cell proliferation. Most of these mutations are acquired in age-dependent manner and occur in somatic (uninherited) cells. However, some people inherit mutations in the germline (the gamets and their precursors). Inherited or genetic forms of cancer may be due to mutations in genes that directly control cell growth or apoptosis (1).  Cancer results from an accumulation of multiple genetic alterations that either inhibit or enhance normal cellular processes. Through a process of continual genetic evolution a cell requires new phenotypes able to proliferate, invade, disseminate throughout the body, escape immune surveillance and resist treatment. Many genetic alterations have been recognized in human tumors. Some of these confer a survival advantage and hence lead to clonally expansion of the founder cell. Most are frequent (e,g. p53 mutation) found in a wide variety of tumors. Other genetic aberrations are clearly related to individual tumors and either reflects the predisposition in such tissues to these genetic changes or the alterations required to overcome that tissue’s normal regulatory control (2).

Cancer genes

Many genes whose alteration is associated with carcinogenesis have been identified. Although initially known as oncogenes, the identification of inhibitory genes has led to the classification of cancer genes as either oncogenes or tumor suppressor genes.


Oncogenes are derived from normal cellular genes called proto-oncogenes. Proto-oncogenes were first elucidated in RNA tumor virus, and are now known to encode proteins that are crucial for normal cellular growth regulation including growth factors, components of the intercellular signaling pathways, DNA binding proteins, cell surface receptors and components of the cell cycle progression pathways. Oncogenes have been identified in human tumors which affect most components of signal transduction pathways such as growth factors (ERB-B2), GTP-binding proteins (ras) and nuclear transcription factors (myc). Proto-oncogenes become activated by:

  • Mutation.

Alterations of the nucleotide sequence lead to either an alteration in the amino acid sequence, or premature termination of translation may produce a protein with abnormal function.

  • Chromosomal rearrangement. 

Chromosomal translocation may lead to the formation of a novel fusion protein or loss of normal control of proto-oncogene expression.

  • Amplification .

Multiple copies of a proto-oncogene, may result from dysregulated chromosomal replication. This in turn leads to inappropriate high levels of [removed]2).

As well as initiating an oncogenic process, oncogenes are thought to be important in the maintenance of solid tumors. Inherited mutations of oncogenes are rare- it is thought that most of these would be lethal. However, mutations in the RET oncogene and the MET oncogene have been described and cause inherited susceptibility to malignancy (multiple endocrine neoplasia type 2 and familial papillary renal cell carcinoma ) (1). The ras pathway is central to the transmission of a growth factor signal from cell membrane to nucleus. Activation of three ras genes (Ha-ras, Ki-ras, N-ras) by point mutation is the most frequent dominant oncogene abnormality in human cancer. The ras mutations are frequently seen in bladder, lung and colorectal carcinoma (2).

The myc family genes (c-myc, L-myc, N-myc) are frequently activated by amplification to form oncogenes. The myc in combination with associated proteins (MAX, MAD) acts as a transcription factor controlling the expression of genes associated with many tumors such as small-cell lung carcinoma and neuroblastoma. A chromosomal translocation in Burkitt’s lymphoma leads to c-myc activation (2).

The mdm-2 oncogene does not produce a component of the transduction pathway of a growth signal. The mdm-2 protein acts by binding and inactivating genes such as p53 (an example of an oncogene affecting a tumor suppressor gene). The mdm-2 over expression has been identified in soft tissue sarcomas (2).

Tumor suppressor genes   

These are defined as genes in which mutation or other genetic modification leads to a loss of function which is then associated with tumor formation. The normal function of the protein product of these genes is generally central to the control of cell division and differentiation. Typically, both copies of a tumor suppressor gene must be affected to lead to loss of the protein’s function that is, the gene acts recessively.

Inactivation of a tumor suppressor gene may result from:

  • Deletion.

Deletion of both copies of a tumor suppressor gene (homozygous deletion) may occur. Deletion of a single copy with inactivation of the other allele by alternative means is the most frequent mechanism leading to inactivation of a tumor suppressor gene. The deletion of the single remaining allele by molecular techniques leads to a loss of heterozygosis, the characteristic marker of a region of the genome in which tumor suppressor gene is located.

  • Mutation.

Mutation of one or both alleles may result in tumor suppressor gene inactivation.

  • Epigenetic

Inactivations of a tumor suppressor gene by means which do not alter the genetic sequence of the genome are known as epigenetic changes. For example, inappropriate methylation may lead to a loss of expression and hence inactivation of the cell cycle regulator p16 (2).

A small number of genes has been identified which act as tumor suppressor genes. These include:

  • p53: the protein product of the p53 gene has a molecular weight of 53 kDa and is central to the control of the cell cycle and the response of the cell to stress such as DNA damage. p53 functions principally by controlling transcription of other genes which control pathways such as the cell cycle, DNA repair and apoptosis. Loss of normal p53 function is central to the pathogenesis of many malignancies; p53 mutations are the most common identified in human cancer, present in more than 50% of tumors. Germ-line deletion of one copy of the p53 gene leads to an increase in tumor incidence (Li-Fraumeni syndrome).
  • pRb: the retinoblastoma gene (Rb1) produces a protein with an essential role in the control of the cell cycle; loss of normal function leads to a loss of control of the G1/S checkpoint and hence inappropriate proliferation (2).

RNA Interference (RNAi)

Geneticists have been puzzled by long RNA molecules that are made by mammalian genomes but do not code for protein. RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. It is a precious question what do they do?

 Two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific RNAs (mRNA) and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general. The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of 20 nucleotides that are called siRNAs. Each siRNA is unwound into two single-stranded (ss) ssRNAs, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA. The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a  Lentiviral Delivery of designed shRNA’s and the mechanism of RNA interference in mammalian cells(3).

microRNAs – the ubiquitous, short noncoding RNAs that regulate gene expression-are known to affect the levels of both messenger RNA(mRNA) and protein. Because protein production is dependent on the presence of mRNA, it has been difficult to establish the relative contributions of miRNA –mediated mRNA cleavage versus translational repression.  Guo, et al (4) found that miRNA act mainly by destabilizing target mRNAs, rather than by inhibiting their translation. 

The first miRNAs were characterized in the early 1990s. However, miRNAs were not recognized as a distinct class of biologic regulators with conserved functions until the early 2000s. Since then, miRNA research has revealed multiple roles in negative regulation (transcript degradation and sequestering, translational suppression) and possible involvement in positive regulation (transcriptional and translational activation).  miRNAs are short ribonucleic acid  molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. The human genome may encode over 1000miRNAs, which may target about 60% of mammalian genes and are abundant in many human cell types. (5).

miRNA and cancer

By affecting gene regulation, miRNAs are likely to be involved in most biologic processes. Different sets of expressed miRNAs are found in different cell types and tissues. Aberrant expression of miRNAs has been implicated in numerous disease states, and miRNA-based therapies are under investigations (5). In many organisms, including humans, miRNAs have also been linked to the formation of tumors and dysregulation of the cell cycle. Here, miRNAs can function as both oncogenes and tumor suppressor gene (3).

Several miRNAs have been found to have links with some types of cancer. miRNA-21 is one of the first miRNAs that was identified as an oncomiR.  A study of mice altered to produce excess c-Myc — a protein with mutated forms implicated in several cancers — shows that miRNA has an effect on the development of cancer. Mice that were engineered to produce a surplus of types of miRNA found in lymphoma cells developed the disease within 50 days and died two weeks later. In contrast, mice without the surplus miRNA lived over 100 days. Leukemia can be caused by the insertion of a viral genome next to the 17-92 arrays of miRNAs leading to increased expression of this miRNA.  Another study found that two types of miRNA inhibit the E2F1 protein, which regulates cell proliferation. miRNA appears to bind to messenger RNA before it can be translated to proteins that switch genes on and off. By measuring activity among 217 genes encoding miRNA, patterns of gene activity that can distinguish types of cancers can be discerned. miRNA signatures may enable classification of cancer. This will allow doctors to determine the original tissue type which spawned a cancer and to be able to target a treatment course based on the original tissue type. miRNA profiling has already been able to determine whether patients with chronic lymphocytic leukemia had slow growing or aggressive forms of the cancer. Transgenic mice that over-express or lack specific miRNAs have provided insight into the role of small RNAs in various malignancies. A novel miRNA-profiling based screening assay for the detection of early-stage colorectal cancer has been developed and is currently in clinical trials. Early results showed that blood plasma samples collected from patients with early, resectable (Stage II) colorectal cancer could be distinguished from those of sex-and age-matched healthy volunteers. Sufficient selectivity and specificity could be achieved using small (less than 1 mL) samples of blood. The test has potential to be a cost-effective, non-invasive way to identify at-risk patients who should undergo colonoscopy (5). 

John Rinn and Maite Huarte at the Broad Institute in Cambridge, Massachusetts, and their colleagues reported that one long non-coding RNA is important in a cell’s response to the protein p53. Best known as a tumor suppressor, p53 controls the transcription of many genes. The team showed that it also triggers the production of several long non-coding RNAs and that one of these, lincRNA-p21, stifles the expression of many genes further downstream in the p53 response pathway, and promotes cell suicide. It seems to do this by associating with a second protein, hnRNP-K. The authors propose that other proteins like p53 activate long non-coding RNAs that help to silence genes. 

Once, it all seemed so beautifully simple. Our DNA, we thought, consisted of a set of recipes, or genes, for making proteins, and once we had identified them all and worked out what they do, we would be a long way towards understanding what makes us what we are. For starters, rather than each gene coding for one protein, they often code for many. The coding parts of genes come in pieces, like beads, or exons, after RNA copies are made, a single gene can potentially code for tens of thousands of different beads, or exons, after RNA copies are made, a single gene can potentially code for tens of thousands of different proteins, although the average is about five. Recent studies suggest up to 95% of our genes may be alternatively spliced in this way (6).

It is the way in which genes are switched on and off, though, that has turned out to be really mind-bloggling, with layer after layer of complexity emerging. Early studies suggested that gene activity was regulated mainly by transcription factors –protein that binds to DNA, blocking or boosting the production of RNA copies of a gene and thus the amount of protein that genes produces (6).

While transcription factors do play a big role, cells also produces a wide variety of RNAs that, rather than coding for a protein, control gene activity. Some dubbed small interfering RNAs (siRNAs), from complexes that seek out and destroy RNA copies of genes with a complementary sequence, preventing protein production. microRNAs work in a similar way but are not specific, controlling the activity of many genes simultaneously. Piwi-acting RNAS, meanwhile, shutdown the parasitic genes that litter our genome to stop them wreaking havoc, though it’s not clear how (6). John Avise of the University of California, Irvine argued that splicing mistakes and errant miroRNAs play a role in some cancers. On the Brightside, discoveries like siRNA could lead to potent new treatments for all kind of diseases (6).

A large scale genetic analysis of different malignant samples has identified thousands of cancer-related mutations. Of these, most are thought to be significantly mutated, implying possible pathogenic roles in   abnormal protein synthesis or gene silencing by the activation of RNAi. siRNA technology is now extensively recognized as a powerful tool for the specific suppression of gene expression and is presently being used by researchers in a wide range of disciplines for the assessment of gene function.


  1. Lalloo, F. In: Genetics for oncologists, The molecular genetic basis of oncologic disorders, ed.     Cold Spring Harbor Laboratory,  Remedica Publishing, London, (2002).
  2. Hall, G.D, Patel, P.M, Protheroe, A.S. Key topics in Oncology,  ed. Selby. P. J., Bios Scientific Publishers Limited, (1998).
  3. http://en.wikipedia.org/wiki/RNA_interference
  4. Guo, H et al. Nature, 466: 835-840 (2010).
  5. http://en.wikipedia.org/wiki/MicroRNA
  6. Le Page, M. June NewScientist. 34-35 (2010).


The beginning of many wars are prophesied in 2011 and the trigger is probably a large volcano in America which causes great loss of crops and reduced sunlight worldwide. This is also the time that our sun may begin its new solar maximum which has traditionally brought many new wars and revolutions to mankind from this increased social energy and activity.   But wars and the rumors of wars might be greatly reduced if this is foreseen and measures taken by major and minor governments to reduce this natural massive environmental damage from a volcano and the increased solar activity.

One extinct volcano may someday soon explode in 2011 in America according to remote viewers.  If this is so might this warning be actually an opportunity to prepare to mitigate this event and try new things to end this massive worldwide calamity.

A large tanker fleet that sprays large clouds of mists of fresh or salt water above a volcanoes massive dust and ash blooms worldwide might actually be very beneficial to farmlands if it can be extracted by the artificial rainfall by a massive air tanker fleet.  And might the oceans benefit by massive blooms of algae to sequester carbon dioxide from the volcanic dust seeded to all the oceans.  Then by man’s own doing massive starvation might be avoided from the reduced sunlight and massive disruption of commerce and farming and fishing.  And possibly great resource wars for food and water might be muted and ended quickly instead of lingering for many years in the world.

Mankind has the means to foresee these greater and lesser events in time now with (CRV) controlled remote viewing but few people use this information except the NWO elite, major militaries, and big business which have even more direct knowledge to future events.  It is the will to use this knowledge correctly and wisely that is lacking in our leaders.  To intentionally allow mass starvation of billions to cull the Earth of perceived excessive populations is immoral.  This withholding of high technology and assistance is harmful, shortsighted, and unnecessary.  When mankind can mute the excesses of nature we should at least try our best to do so instead of remaining silent.  The Club of Rome and other population studies have convince the elite that the world is overpopulate.  But perhaps it is so because the elite themselves have withheld the high technologies that are needed by all to end pollution and poverty. 

One thing I learned as an organic gardener is that I could grow large amounts of foods even in winter in any climate if I made a greenhouse 1/3 underground and insulated the side ground with 4 to 6 inches of foam from the surrounding frozen soils.  This simple cheap passive greenhouse construction can be the basis for massive northern yearround greenhouses in any climate.  The the blue and red LED grow light technologies can supply enough cheap grow lighting to make these greenhouses perform well in winter if extra light is needed.  This is the newer greenhouse technologies of most all future cities of man unless we are smarter and mute natures super volcanoes of the future.  It will be this or starvation for most people of Earth if nothing is done.

We need to try other things instead of only building massive seed banks, underground bunkers, and underground cities filled with bulk foodstuffs for the elite and militaries who know the coming future.  Might it be wiser to try new things to mute these massive environmental disasters rather than allowing it to run its course.

Studies are needed now to determine the best place to drop down the volcanic ash from the spray planes from above and how best to do this and when.  Many months and years might be needed to monitor an active volcano with many tanker planes to knock down the ash blooms. But would not a great human endeavor of great good be wiser than allowing great starvation and wars and the many uncertain consequences to the future?  

Men and worlds can become masters of their destiny in time but we must start now to do so.  If not we will become the slave who only reacts to current events. Do you want to see the masses of people starving to death in your undergrownd cities on your TV screens deep underground or do you want to see the many tanker planes saving most of the people from starvation and wars?  What will be your legacy to the world which you will become known.

Nature may have other less harsh plans of population reduction in December 2012.  If the radiations of solar flares and CMEs are too great many people may become sterile or less fertile if not shielded from this in underground havens for a short time.  So nature may have other plans for humanity which are less harmful and more humane than the elite’s do nothing policies for 2011 and 2012.

When the lights go out for a time in 2012 the great cosmic silence will be heard once more.  The worldwide electronic smog ending will bring a world of wonder when people can experience their true selves and begin anew.  People under 30 will experience many new psychic abilities as the electronic smog of cell phones, TVs, radios, mircrowaves, and a thousand others are gone.  And it will be a time of great wonders as well as loss but we will all see the great benefits of it.

Earthern and Pot Culture Method to Check the Stability of Marine Azotobacter in Soil


Among the three major habitats of the biosphere, the marine realm which covers 70% of the earth’s surface provides the largest inhabitable space for living organisms. The study of marine bacterial diversity is important in order to understand the community structure and pattern of distribution (Surajit Das et al 2006). For many years, the filamentous blue-green algae (cyanobacteria) were believed to be primarily responsible for N2 fixation in oceanic waters because low or negligible in situ rates were observed in their absence and there was a correlation of in situ N2 fixation with light intensity. However, evidence has been accumulating which documents the importance of bacterial N2 fixations in many and diverse marine habitats ( MARY LOU GUERINOT et al 1985) . It is commonly assumed that marine bacteria, since they live in the sea, must be Salt-tolerant organisms. ZoBell and Upham define marine bacteria as being bacteria from the sea which on initial isolation required seawater in the medium for growth. Therefore analysis of marine water will provide the effect of salts on the growth of marine Azotobacter. Biofertilizers are the source of microbial inoculants, which have brought hopes for many countries both economically and environmentally. Azotobacter sp is free living, known to fix atmospheric nitrogen. There are different strains of Azotobacter each has varied chemical, biological and other characters. Azotobacter and Azospirillum are two other efficient bacteria. The response of these organisms in increasing crop yield has been commonly experienced. These are the biofertilizers in the cultivation of most crops. Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well-manured soil with high organic matter content. Experiments with Azotobacter cultures and crop plants at the Indian Agricultural Research Institute, New Delhi, lead us to believe that significant increases in growth and yield of wheat, rice and vegetable crops could be obtained in pot trials. However, under field conditions, such uniform trends towards increases in yield are not always reproducible. We carried out pot culture experiment in order to assess the effects of Azotobacter isolated from marine source on the growth of Black gram. Their shoot length, root length and their chlorophyll content were measured.


Sample collection:

Samples of surface water were collected in the area of Thundi region (Palk Bay) .Sample collection was accomplished at the interval of approximately 20 days

Surface –water samples (at depths of 1-2 m) were collected in sterile tube containing Azotobacter selective medium. Sediment samples were collected separately in broth medium. (Table .1 and 2)

Chemical parameter of sea water:

Collected water samples were analyzed for total hardness i.e the presence of magnesium and calcium by EDTA (0.01 M Ethylene diamine tetra acetic acid) titration method. Total Chlorine content was analyzed by Mohr method.

In EDTA method 60 ml of water sample was pipetted to an Erlenmeyer flask. About 2ml of buffer solution (mixture of ammonium chloride and ammonium hydroxide), was added to the sample. A few drops of indicator(Eriochrome black) were added and the solution was gently stried. The EDTA solution was taken in the burette and titrated with water sample until the color of the solution turns red to purple to blue. As soon as the color of the solution turned blue, stopped the titration and record the final level of EDTA solution in the burette. Finally the experimental concentration of calcium and magnesium ions in the unknown water sample was calculated. The hardness of water sample can be classified using a sum of all the calcium and magnesium ions in solution.

In Mohr method 20 ml of sodium chloride (0.01 M) solution was pipette in 250 ml Erlenmeyer flask. Approx 2ml of dipotassium chromate indicator was added to the solution. Solution was turned bright yellow color. Silver nitrate (0.01 M) solution was taken in the burette. The known chloride was titrated with silver nitrate until the color changed from bright yellow to brick red color (swirl the flask constantly to see the uniform color). Finally the experimental concentration of chloride in the known solution was calculated.

To determine unknown chloride, 5 ml of water sample was taken in 250 ml Erlenmeyer flask. 2ml of indicator (dipotassium chromate) was added. Silver nitrate (0.01 M) solution was taken in the burette. The water sample was carefully titrated with silver nitrate solution. Near the end point drop by drop was added from the burette as soon as the color of the solution turned yellow to red, stopped the reaction and recorded the final level of silver nitrate solution in the burette. Finally the experimental concentration of chloride in the unknown solution was calculated.

Media preparation:

Different selective media were used for the isolation of Azotobacter sp from marine source. As the isolates are of marine origin, the media were prepared by adding 3 % sodium chloride (NaCl). Media used for the isolation of nitrogen fixing organism (Azotobacter) from marine sources were: (Table 3)

1). Jensen’s Agar Medium (with 3% NaCl)

2). Azotobacter Agar Medium (with 3% NaCl)

3). Burk’s Medium (with 3% NaCl)

4). Marine agar medium.

Processing of samples (Kannan, 2002):

10 ml of water sample were mixed with 90 ml of sterile distilled water it gave 10-1 dilution. From the 10-1 dilution, the sample was decimally diluted up to 10-9 dilutions. By using spread plate technique, 0.1ml of diluted sample was plated in a sterile Petri plates, containing selective media. The plates were incubated at room temperature (28º C) for 48-72 hours.

Identification of isolates:

Gram’s staining (Kannan, 2002)

Gram staining reactions were recorded from heat fixed smears of fresh cultures.

Catalase test:

Catalase test was performed by adding 3% hydrogen peroxide drop by drop to the slant of fresh Azotobacter culture. Presence or absence of bubbling was recorded.

Phase contrast microscopic observation:

Motility and cell shape were determined by direct observations of wet mounts of fresh broth cultures, using phase- contrast microscopy. (Table 4)

Acetylene Reduction Assay:

Individual colonies were picked, purified, and assayed as pure cultures for nitrogenase activity, using N-deficient medium. This technique is an indirect method of measuring nitrogen fixation at a point of time. This method provides a simple, inexpensive, highly sensitive and non-destructive procedure for measuring rates of nitrogen fixation. Cultures were randomly selected for this assay. Serum bottles with rubber stoppers were collected, cleaned and sterilized. 30 ml of the sterilized Azotobacter broth was transferred to each bottle .The organisms were inoculated in the medium and incubated at 28º C for 3-4 days .after incubation 10 ml of nitrogen gas , 3 ml of acetylene gas was injected in to the serum bottles using syringe ( N2 replaces the air inside the bottle). Incubated the bottles for over nite at 28º C. at the end of the incubation period, 0.5 ml of the gas sample was withdrawn from the bottle and injected in to a gas chromatograph with FID system with 80-100 mesh Poro PAK/ propack Q column. The column temperature was maintained at 80º C, detector temperature at 100º C and injector temperature at 120º C . The carrier gas used was nitrogen with a flow rate of 30ml/ sec, for flame ionization hydrogen and zero air at the rate of 30ml/sec .the area of ethylene peak was recorded for each culture. Randomly selected samples which showed maximum enzyme activity were selected for pot culture experiment.

Analysis of garden soil for Chemical and nutrient content for pot culture experiment:

Garden soil was collected from rhizosphere region. Collected soil was analysed for the presence of N, P, K, Copper, manganese, iron, and zinc.(Table 5)

Pot culture experiment :

The nitrogen fixing ability of the isolated Azotobacter sp was determined in garden soil by pot culture experiment by assessing the growth of black gram. After 7 days of sowing various characteristics of growth such as root and shoot length was measured and chlorophyll content was estimated. Experiment was carried out in GRD College. Coimbatore.

Healthy viable seeds were selected for the experiment .Each pot contains 50 viable seeds. 10-12 kg of finely processed soil was filled in each pot .sterilized the pots with soil at 15 lb pressure for 4 hrs. The broth containing active culture of Azotobacter (1 × 109 cells) was selected. Five efficient strains were selected based on acetylene reduction assay for the experiment. The broth cultures of the selected Azotobacter sp were observed under phase contrast microscope prior to inoculation. Pots were selected for the experiment was thoroughly cleaned with disinfectant. Pots were filled with right combination of soil.

The healthy seeds were selected. Those seeds were mixed with 3ml of Azotobacter inoculums and 3ml of cool rice porridge. Then the seeds were dried

Fifty seeds were sown in each pot. The pots were watered every day. The control pot was devoid of the bacterial inoculums. The effects of bacterial inoculums on the growth of plant root, shoot length were measured on the 7th, 14th, 21st day of plant growth.

Growth characters:

1. Percentage of germination

2. Shoot length

3. Root length

Percentage of germination:

The germination rate of all treated and control plant was calculated by using the following formula: (Table 6)

Number of seeds germinated

Percentage of germination = —————————————– × 100

Number of seeds sown

Shoot length:

The shoot length of the plant was measured in centimeter (cm) scale on 7th, 14th, 21st day of sowing from ground level to the shoot tip. (Table 7)

Root length:

The plants were uprooted without disturbing the root system, and then the roots were washed with tap water to remove the soil particles. The length of the root was measured in cm scale. (Table 7)

Biometric analysis; Estimation of chlorophyll :

Weighed 1g of leaf was finely cut in to pieces; tissues were ground to a pulp with the addition of 20ml of 80% acetone. Then centrifuged at 5000 for 5 min and transferred the supernatant to the 100ml volumetric flask. This procedure was repeated until the residue turned colorless. Finally the volume was made to 100ml with 80% acetone. The absorbance was read at 645,663nm against the solvent (80% acetone) blank.( Table 8a and 8b)


Totally 100 samples were collected in marine region of both water and sediments at the intervals of approximately 20 days (Table: 1).

Table :1 The total samples collected from marine region.

samples water sediment

1st time 10 10

2nd time 15 5

3rd time 15 5

4th time 15 5

5th time 15 5

Total 70 30

Out of 70 marine water samples collected, all the 70 samples were showed the presence of Azotobacter, but only 23 marine sediments out of 30 were showed the presence of Azotobacter (Table no: 2).

Table no 2: presence of Azotobacter sp (in percentage).

Source No of samples POSITIVE (Presence of Azotobacter) Percentage (Presence of Azotobacter)

Water 70 70 100

Sediment 30 23 76.6

Azotobacter sp is a gram-negative soil–dwelling organism with a wide variety of metabolic capabilities which includes the ability to fix atmospheric nitrogen by converting it to ammonia. These bacteria possess the highest cellular respiratory rate of any known organism. Their rapid consumption of oxygen allows them to grow well and to fix nitrogen under extreme aeration condition. (Page et al. 1988).

Initial isolation of marine bacteria prefers sea water or 3 % NaCl to fresh water in the medium for growth (ROBERT A. MACLEOD 1965).

The total hardness of water represents primarily the total concentration of calcium and magnesium ions expressed as calcium carbonate. Hardness may range from 0-100 of parts per million. Mg++ to maintain the respiratory activity of cell Azotobacter, an organism stable in water suspension.

Water analysis result showed that the total hardness of water was 20200 ppm and total chloride content was 18273.98 ppm. Zobell and upham define marine bacteria as being bacteria from the sea which on initial isolation required seawater in the medium for growth.

Table: 3 colony morphology of Azotobacter

Media Details

Jenson’s medium Large, circular, mucoid, watery due drop like colonies.

Azotobacter agar medium Small, circular, mucoid and watery colonies in a medium

Burk’s medium Surface Pellicle formation, turbidity indicating the Heavy growth of Azotobacter.

Marine agar medium Small, circular, smooth edged, raised elevated colonies were observed

Table4: characteristics of Azotobacter sp.

Test Result

Gram’s staining Gram negative rod shaped cells were seen

Catalase test Air bubbles were seen

Phase contrast microscopy Motile cells were seen/rarely non-motile cells were seen with different morphology.

The colony morphology of Azotobacter strain is found to be varying based on the selective media used for isolation.

Studies on the rates of nitrogen fixation were greatly enhanced by development of the acetylene reduction assay (Hardly et al., 1968). This assay is based on the fact that nitrogenase enzyme will reduce acetylene to ethylene. The rate of formation of ethylene is a measure of nitrogenase or nitrogen –fixing activity. Ethylene can be conveniently assayed with great sensitivity using a gas chromatography. In this study acetylene reduction was performed and their peak values were noted. Based on this assay the organism was selected for pot culture experiment.

Table: 5 Chemical and nutrient analysis of garden soil

The garden soil was tested for micro and macro elements.


pH 6.9

Electrical conductivity(dSm-1) 0.446

N(kg/ha) 98

P(Kg/ha) 14.5

K(Kg/ha) 275

Copper(ppm) 0.84

Manganese(ppm) 6.32

Iron(ppm) 8.04

Zinc(ppm) 1.04

pot culture experiment :

Five efficient strains were selected for pot culture experiment based on acetylene reduction assay.

Table 6: Percentage of germination

Result showed 85 percent of germination

pot culture seed germination in %

control 72

pot A 80

pot B 81

pot C 70

pot D 86

pot E 82

Table: 7 shoot and root length

Shoot and root length of the plant were measured, which ranged from 21.4 –

26.3 cm and 7.6-12.2cm respectively.

pot culture Shoot length(cm) Root length(cm)

7th day 14th day 21st day 7th day 14th day 21st day

CONTROL 8.3 18.0 20.0 7.2 9.2 11.2

POT A 8.9 21.0 23.0 7.6 9.6 11.6

POT B 9.5 22.1 24.2 8.5 9.8 11.1

POT C 7.3 19.2 21.4 8.0 9.7 11.5

POT D 9.1 24.4 26.3 8.2 9.8 12.0

POT E 9.2 22.6 25.0 8.5 9.7 12.2

Biometric analysis:

Estimation of chlorophyll by spectrophotometric method:

Table 8a : The optical density value at 645nm ranged from 0.102 – 0.202 OD and 0.266 – 0.562 OD at 663 nm

pot culture OD AT 645 nm OD AT 663 nm

control 0.103 0.302

pot A 0.156 0.423

pot B 0.202 0.562

pot C 0.182 0.522

pot D 0.154 0.455

pot E 0.102 0.266

Table 8a: Estimation of total chlorophyll content:

Pot culture Chlorophyll a Chlorophyll b Total chlorophyll mg/g

control 0.3558 0.0949 0.4502

pot A 0.4952 0.1592 0.6543

pot B 0.6594 0.1995 0.8587

pot C 0.6139 0.1724 0.7862

pot D 0.5364 0.1397 0.6759

pot E 0.3103 0.1091 0.4194

The total chlorophyll ranged from 0.4194-0.8587 mg total chlorophyll/g tissue.

The pot culture experiment results showed that, inoculation with Azotobacter influence the growth of black gram by increasing their shoot and root length and chlorophyll content.

Experiments with Azotobacter cultures and crop plants at the Indian Agricultural Research Institute, New Delhi, lead us to believe that significant increases in growth and yield of wheat, rice and vegetable crops could be obtained in pot trials. Experiment on soil Azotobacter on the growth of maize was carried out by N.A Hegazi(1979) result showed significant increase in the count of Azotobacter in 6 –week- old plant.

A pot culture experiment was conducted by C.V Kanchan to evaluvating the effects of Azotobacter inoculants on the yield of wheat. M.A kader(2002) was conducted a pot culture experiment on straw. He found significant increase in root growth by the treatment of Azotobacter .

S.K.Kavimandan (1986) was carried out a pot experiment with an Azotobacter chroococcum along with 50 Kg N /Ha. He found an adverse effect of bacterial inoculation on the yield of wheat. Choudhury .A(2005) carried out pot culture experiment on three rice cultivars with eight different N2 fixing bacteria strains with the objective to find out effective nitrogen fixer. He found that Azospirillum appeared to be the best followed by Pseudomonas and Azotobacter when inoculated to rice variety. Ravikumar et al (2004) found inoculation with Azotobacter in mangrove soil increase seedlings,. Root biomass, shoot biomass ,total chlorophyll of plant . thus azotobacterisation is beneficial in raising vigorous seedlings of mangrove in coastal wetlands.


A marine sample indicates that the concentration of nitrogen-fixing organisms is much lower in oceanic environments than in coastal environments. However, even at low densities, active population of nitrogen- fixing microorganism over vast areas of the open ocean could contribute substantially to the nitrogen inputs in the world’s ocean (Zehr et al 1998)

This study revealed that marine Azotobacter can be cultivated in laboratory condition, which provides more information on growth pattern on different media. Water analysis result showed high concentration of calcium, magnesium, chloride content.

Acetylene reduction assay was performed and checked the enzyme activity of randomly selected samples and were used for pot culture experiment. The pot culture experiment showed significant increase in shoot, root length of the plant. Hence marine Azotobacter can survive in soil and fix atmospheric nitrogen. Marine Azotobacter can be used as a suitable biofertilizer in order to reduce the usage of chemical fertilizer which is potent harmful substances mainly petrochemicals.


Bedford, R.H, 1933. Marine bacteria of the northern pacific ocean. The temperature range of growth. Contrib. can. Bio. Fisheries 8: 433-438.

Burk, D., and Horner, C.K. 1940. Molybdenum and calcium in Azotobacter nutrition. Proe. Third Intern. Congr. Microbiol. (New York), p 489-490.

CHOUDHURY . A 2005. Screening of rice cultivars and diazotrops combination for better N(2) fixing system. Indian journal of plant physiology Vol 10 p 82-85

Eisenstarh A., K.J. McMahon and Roma Eisenstarh, 1949. Department of Bacteriology, Oklahoma. A cytological study of pleomorphic strain of Azotobacter with the electron and phase Microscope and the Robinson Nuclear – Staining Technique.

Guerinot, M.L., and Patriquin D.G. 1981. The association of N2-fixing bacteria with sea urchins. Mar. Biol. Vol 62: 197-207.

Hans W.Parel. Microbially Mediated Nitrogen Cycling. Techniques in Microbial Ecology, P 4-27 – ND.

Hardy R.W.F. 1968. Acetylene ethylene assay for nitrogen fixation: Laboratory and field evaluation. Plant physiology vol.43, 1185-1207.

Hegazi, M. Monib and Vlassak K, 1978. Effect of inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under subtropical conditions, vol.38 No.4. P 621-625.

James. A. Coyer, Alejandrocabello – Pasini, Hewson Swift and Randall. S. Alberte, 1996. N2 fixation in marine hetrotrophic bacteria dynamic of environmental and molecular regulation. Vol 93: P 3575 – 3580.

Jensen. H.L. 1954, The Azotobacteriaceae. Bacteriological Rev. 18: 195-214.

S.K Kavimandan 1986 Influence of rhizobia,azotobacter and blue green algae on n content and yield of rice .Vol 96 133-135

M.A Kader 2002. Effects of Azotobacter inoculant on the yield and nitrogen uptake by wheat ,Journal of biological sciences , vol 2(4) p 259-261

Lewis I.M. 1937. Cell inclusions and life cycle of Azobacter J. Bacteriol 34: 191-205.

MacLeod. R.A. and Onofrey, 1957. Nutrition and metabolism of marine bacteria III. The relation of sodium and potassium to growth J. Cell. Comp. Physiol. Vol 50: 398-409.

Maria GT – Rubio, Sandra AVP., Jaime Bernal – Castilo, Patrica Martinel- Nieto, 2000. Association Latinoamericana de microbiologia. Vol 42: 171-176.

Mary LG and Rita R Colwell 1985, Enumeration, isolation and characterization of N2 fixing bacteria from sea water. Department of Microbiology, University of Maryland. Vol 50 No.2.

Murray C.M.J.P. Riley: and T.R.S. Wilson 1969. The solubility of gases in distilled water and sea water I Nitrogen. Deep sea. 16: 297-310.

Page W.J. and H.L. Sadoff, 1975. Relationship Between Calcium and Uronic Acids in the Encysment of Azobacter vinelandii. Journal of Bacteriology, Vol.122, No.1 p 145-151.

Postgate, J.R (1982). Fundamentals of nitrogen fixation (Cambridge Univ. Press. Cambridge, U.K).

Rai. M.K, Handbook of Microbial Biofertilizers, an imprint of the Haworth Press, Inc. New York, London, Oxford.

Ramos J.L and R.L. Robinson, 1985. Isolation and properties of mutant of Azobacter chroococum defective in aerobic nitrogen fixation J. Gen. Microbial. 131: 1449-1458.

Ravikumar et al 2004. Nitrogen fixing Azotobacter from mangrove habitate and their utility as marine biofertilizera, journal of experimental marine biology and ecology Vol312 p5-17

Richter. O. 1928. Natrium: Ein notwendiges N’ahrelement fur eine marine mikroarophile Leuchbakterie. Anz. Oesterr. Akad. Wiss. Math. Naturw. KI. 101: 261-292.

Robert A Macleod, 1965. The question of the existence of specific Marine bacteria, department of bacteriology, American society for microbiology, McGill University, Canada. Vol.29 No.1.

Robinson G.G.C.L.L. Hendzea and D.C. Giillespie 1973. A relation between heterotrophic utilization of organic acids and bacterial population in West Blue iake, Manitoba, Limnon, Oceanogr 18: 264-269.

Tyler. M.E., M.C.Bielling and D.B. Pratt, 1960. Mineral requirements and other characters of selected marine bacteria Jou. Gen. Microbiol. Vol.23: 153-161.

Vela G.R. and Rosenthal R.S., 1972. Effect of Peplon on Azotobacter Morphology, America Society for Microbiology. Vol.111 No.1.

Zobell. C.E and Upham H.C., 1994. A list of marine bacteria including description of sixty new species. Bull. Scripps Inst. Oceanography. Vol. 5: 239-292.

Standard method for the examination of water and waste water, 16th edition, APHA, AWWA, WPCF.

* Cristian G.D. Analytical Chemistry 4th Edition, J. Wiley and Sons.

* Harris D.C. Quantitative Chemical Analysis 5th Edition, W.H. Freeman.

* History of nitrogen fixation. From: Biology 446, Uni. Of Watterloo (Biology 446).

* http://www.indiaagronet.com/

* http://www.thekrib.com/plant/co2/hardness-larryfr.,html.

* The microbial world: The nitrogen cycle and nitrogen fixation produced by Jim Deacon, Institute of cell and molecular biology, the University of Edinburgh.

* Determination of hardness of water method WHO/M/26.RI, revised 10 Dec. 1999.

* htt://www.nalms.org/

Carlson, R.E. and J. Simson, 1996. A coordinators Guide to volunteer lake Monitoring Methods, North American lake Management Society, P 96.

* Determination of water hardness by EDTA Titration from Gannon University SIM.

* http:/www.bact.wisc.edu/The world of Microbes.htm


Pages from web.centre.edu.

* http://www/tau/ac/

The nitrogenous complex

* http://www.bookrags.com

Azotobacter compete article

*Shri Dorji Tenzing Bhutia, 2004. Joint Director Skims, Biofertilizers for Nutrient Management in Organic Production of Agri / Horticultural crops.

* http://cos.colstate.edu/stokes/chlorophyll.htm.


Chlorophyll : why?

** Atlas R. and Bartha R, 1998. Microbial Ecology Fundamentals and Applications 4th Edition Benjamin Cummings. Menlopark. Ca.694pp.

** Camphell. N. 1993. Biology 3rd Edition. Benjamin Cummings, Redwood City, Ca 1190.

** N.S.Subba Rao, Soil Microbiology and plant growth 4th edition.

** Robert L. Tate, 1995. Soil Microbiology. Jhon Willey and Sons, Inc.

** Jan Dirk Van Elsas, Jack T. Trevors, Elizabeth, M.H.Willington 1997, Modern Microbiology.

**S.Sadasivam and A.Manikam, 2004. Biochemical methods, 2nd Edi., Centre for plant molecular biology, TNAU/

(* Net reference)

(** Book Reference)

History of the ,clean Water Act, and What Caused Its Failure

By Peter Maier, PhD, PE

August 2008

Prior to 1972 states had their own ,water pollution, regulations, but since they were different, industries in ‘clean’ states moved to ‘dirty’ states. This led to employment loses in the ‘clean’ states and Congress was asked to set national ,water pollution, standards.

When reading the historical discussions prior to the actual ,CWA,, it becomes clear that the Act was not yet be able to set, sewage treatment, standards, but in stead established a principle in order to achieve a goal that when somebody uses water, it should be returned at least in the same or better conditions, hence the ultimate goal of the Act to eliminate all ,water pollution, by 1985. 

It was also realized that such a goal was not yet achievable, since the only technical term used in the legislation was demanding ‘secondary treatment’, without any further definition, but which was supposed to be 85% treatment. 

The legislation also selected a ‘technology-based’ program, in stead of a ‘water quality-based’ program, as it was felt that this would allow local politicians to manipulate local treatment requirements, thus avoiding the purpose and goal of the Act itself.

A technological-based program meant that everybody treating wastewater has to do so with the best treatment available, while a water-quality based program means that treatment standards could be determined by the water quality of the receiving water bodies.

The Act also acknowledged that ‘secondary treatment’ would not any longer be acceptable if better treatment would become available and incorporated special legislation to allow EPA to set stricter treatment standards to achieve the ultimate goal of 100% treatment. The Act also provided funding for R&D to achieve better treatment than the initial required ‘secondary treatment’.

When EPA implemented the ,CWA, it established the NPDES (National Pollution Discharge Elimination System) permit system and established 85% treatment of two commonly used pollution tests, the TSS (Total Suspended Solids) and the BOD5 (Biochemical Oxygen Demand test after 5 days) test.

The BOD5 test was widely used worldwide, but what was forgotten was the fact that the 5-day test was mainly used as a timesaver and only measured the pollution caused by fecal waste. When EPA assumed that the BOD5 of raw sewage is 200 mg/l to establish the ‘secondary treatment’ standards, it only addressed 40% of the ultimate BOD, which is 500 mg/l.

By setting 85% BOD5 treatment standards, EPA ignored all the ,water pollution, caused by nitrogenous (urine and protein) waste. For those interested in how the BOD test should be applied, visit www.petermaier,net and look in the Technical PDF file.

Using the BOD5 test without any nitrogen data, does not allow the real performance evaluation ,sewage treatment, plants nor to determine the real waste loadings on receiving water bodies.

Although EPA acknowledged the problems with the test in 1984, in stead of correcting the test, it allowed an alternative test and officially ignored the ,water pollution, caused by nitrogenous waste, while this waste, like fecal waste, not only exerts an oxygen demand, but also in all its forms is a nutrient for algae and other aquatic plant life. Utah States’ Science Council in 1984, recommended correcting this essential test, but their recommendation was rejected.

Nitrogenous waste, called a nutrient, according to EPA’s 1992 “National Water Quality Inventory Report to Congress” is now causing mayor problems in the nation’s rivers, lakes and estuaries.

The sad conclusion is, that solely due a lack of understanding of an essential pollution test, the ,Clean Water Act,, the second largest federally funded public works program, was a failure and that nobody seems to either care or can be held accountable.

3 Skin Nutrition Secrets For Beautiful Skin

No matter how vigilant you are, your skin takes a beating. It’s a tough world out there – and your skin is on the front lines.

However, all is not lost. You don’t have to resign yourself to your skin showing every battle it has waged with the elements.

Nor do you have to suffer further abuse through lasers, needles or surgery.

Put these 3 natural skin renewal steps to work. And revel in what you see when you look in the mirror.

The Most Important Skin Nutrient: Water

Your skin needs water more than anything else.

Consider this: Newborns’ skin can have as much as 83% water content. [1] Young children have about 65% water concentration in their skin. However, as we age, our bodies just can’t seem to hold onto it as well. An adult woman averages about 55% water.[2]

Now it’s hard to change how your body retains water as you get older. But no matter what, you can make sure you replenish your water supply more quickly by drinking more.

When you’re well hydrated, you’ll enjoy fuller, smoother skin. Wrinkles and sags start to disappear as your skin stretches tautly across your face.

But the benefits go even further…

  • Your body uses water to help remove toxins from your body.
  • Water increases blood flow, making it easier for your body to bring nutrients to your skin.
  • Good hydration increases your immune system’s mobility, making it easier for your body to repair damaged areas.

Simply by drinking more water you’ll make it easier for your body’s innate repair crews to do their work on your skin.

Essential Skin Nutrition

From collagen-production to immunity, your skin depends on good nutrition to keep doing its job… and looking good while doing it.

Key nutrients for your skin include:

  • Vitamin C – Vitamin C is a key nutrient in collagen production. Even more intriguing, recent laboratory studies indicate that vitamin C may actually turn on genes in your skin cells responsible for regeneration.[3]
  • Vitamin A -Recent research shows that vitamin A seems to increase the production of two key skin health components – procollagen, the precursor to collagen, and glycosaminoglycan. Glycosaminoglycan seems to help your skin hold onto water, help with collagen production and reduce wrinkles.[4]
  • Nucleic Acids – Anti-aging pioneer, Dr. Ben Frank, author of the classic book Eat and Grow Young, pointed out, healthy cell replication requires a good supply of nucleic acids. Dr. Frank noted that a diet high in nucleotides resulted in less wrinkles and better skin tone.[5]
  • Beta Glucans -Clinical studies have demonstrated these special immune-stimulating molecules can help burn patients heal,[6] protect your skin from UV light[7] and speed up overall skin renewal.[8]

By giving your skin these essential nutrients, you help your skin take care of itself.

Restore Your Skin

Beaten up by the elements, your skin is constantly regenerating itself. Cell by cell, your body keeps rebuilding this outer frontier. On average the entire outer layer of your skin is replaced every week.

Your body requires tremendous nutrition simply to fuel this constantly changing set of cells.

And it also requires very specific nutrition that stimulates and supports cell replication. Nutrition like chlorella growth factor (CGF).

Growth factors are found throughout nature, in plants and animals alike. They are the chemical signals that promote wound healing and tissue growth, through cell replication and differentiation.

Chlorella growth factor (CGF), found only in chlorella, stimulates renewal exceptionally well. And CGF’s renewing effects don’t stop with chlorella. It’s been shown to safely speed up tissue repair and healing in our skin. In lab studies, researchers have demonstrated that it even seems to slow down aging at the cellular level.[9]

This Special Food Nourishes And Renews Your Skin

Hydrate, nourish and renew. Your skin needs you to take on all 3 of these tasks.

When it comes to nourishment and renewal, the tiny green algae – chlorella – gives you extraordinary skin nutrition.

With vitamin C… rich in vitamin A… Full of beta glucans… with more concentrated nucleotides than any other food… and the only source of CGF…

Chlorella feeds your skin for regeneration.

  • You can apply chlorella topically in a skin care cream. Your skin will eat it up directly.
  • And you can also take it as a skin supplement. It’s a tasty addition to your diet.

Either way, your skin will respond beautifully. Add this special alga to your skin care routine and you’ll love what you see in the mirror.

[1] Friis-Hansen BJ et al. Total Body Water In Children. Pediatrics 1951 7(3).

[2] The Water In You. US Geological Survey.

[3] University of Leicester (2009, September 9). New Role Of Vitamin C In Skin Protection; Relevance To The Cosmetics Industry. ScienceDaily.

[4] Rafi R et al. Improvement of naturally aged skin with vitamin A (retinol). Arch Dermatol. 2007 May;143(5):606-12.

[5] Skin Care Pioneer. Life Extension Magazine. January 2003.

[6] Delatte SJ et al. Effectiveness of beta-glucan collagen for treatment of partial-thickness burns in children. J Pediatr Surg. 2001 Jan;36(1):113-8.

[7] Zulli F et al. Improving skin function with CM-glucan, a biological response modifier from yeast. Int J Cosmet Sci. 1998 Apr;20(2):79-86. doi: 10.1046/j.1467-2494.1998.171740.x.

[8] Pillai, R.; Redmond, M.; Röding, J. Anti-Wrinkle Therapy: Significant New Findings in the Non-Invasive Cosmetic Treatment of Skin Wrinkles with Beta-Glucan. Int. journal of cosmetic science, Volume 27, Number 5, October 2005, pp. 292-292(1).

[9]Makpol S et al. Chlorella Vulgaris Modulates Hydrogen Peroxide-Induced DNA Damage and Telomere Shortening of Human Fibroblasts Derived from Different Aged Individuals. Afr J Tradit Complement Altern Med. 2009; 6(4): 560-572.

Source by Michael E. Rosenbaum

Purify Water with Chlorine Tablets

As soon as summers start approaching, spending plenty of time in a swimming pool is the only thing one thinks of. Whether there is a swimming pool in the house, clubs or hotels, cleaning is always the most essential thing, which should be done on a periodical basis. Though, cleaning a swimming pool is a difficult task, it has to be done to prevent it being a breeding home for organisms like algae and bacteria.

The cleaning of a swimming pool is done by draining all the water from it and putting in swimming pool chemicals that help in removing dirt and organisms like algae and bacteria. Using chlorine tablets is of course an important step of the cleaning process and is done after fresh water is filled in the pool.

These days chlorine tablets are available in the market that is known by the name of Tri-chloro or Trichloro-s-triazinetrione. It is a stabilized form of chlorine that has become popular in the last few years.

How is Chlorine Made?

Chlorine is made by the process of electrolysis in which electricity is passed through salt (2Nacl) and water (H2O) to form gaseous chlorine. This chlorine is then used for a number of things such as bleaching and chlorination of swimming pools, etc.

What is Chlorination?

Chlorination is the process done to purify the water of a swimming pool. Chlorine tablets are used to keep the pool water safe for swimmers who at times complain of illnesses like ear, nose and throat infections. Swimming pool chlorine is also used with a view to serve as a preventive measure for the diseases like meningitis to occur.

How a chlorine tablet sanitizes the pool water is an important point to discuss. Let us highlight some vital facts on it.

When a chlorine tablet is inserted in a pool, it fuses into the water to give a chemical reaction that produces HOCl (Hypochlorous acid) and HCL (Hydrochloric acid).

The former form of chlorine is supposed to be the most essential part that helps in the cleaning process of a swimming pool.

Molecules of chlorine ions mix with water and kill the micro organisms like bacteria and algae while the sanitizing the swimming pools.

The percentage of chlorine that is found in swimming pools is definitely safe for people, but excess levels of it can cause serious health hazards. Although, chlorine tablets are absolutely safe chemicals for swimmers, it can cause mild skin irritation in some cases.

Parameters for Drinking Water in Ontario

In June 2003 the province of Ontario published, and in 2006 they revised a Technical Support Document for Ontario Drinking Water Standards, Objectives, and Guidelines to outline minimum requirements for public drinking water. The document helps deal with new chemicals coming into the environment and high levels of soaps that have the potential to pollute public drinking water. Parameters such as taste, alkalinity, and pH are listed alphabetically as a guide for engineers because there is an ideal range for water treatment plant operation or general aesthetic water quality.

Microbes are often present in water and filters are used to screen them out, for they are associated with a number of waterborne diseases such as Typhoid fever, cholera, amoebic dysentery, and bacillary that can be transmitted by water. Giardia cysts, Cryptosporidium oocysts, protozoa, and viruses must be cleared by filters and cut off from nutrients entering the system. The re-growth of algae, protozoa, and other microorganisms can clog filters cause an unpleasant taste in the water. Iron bacteria can cause discoloration, turbidity, and taste problems.

The Document for Water Standards attempts to limit the effect of agricultural run-off and makes mention of the importance of keeping pesticides from entering raw water sources. Municipal water supplies all have low concentrations of synthetic organic chemicals due to the natural decomposition of biological matter however.

The term pH is an acidity parameter where the objective is to produce a water that is neither corrosive nor produces incrustation. The guideline for drinking water is to maintain between 6.5 and 8.5 pH acidity. Levels below 6.5 have been associated with corrosion.

The province of Ontario has published standards, objectives, and guidelines to assess the quality of drinking water throughout the province and to outline the responsibilities of owners/operators of drinking water systems. The water must meet quality of drinking water parameters such as Zinc, PCBs (in case they are present), Total Dissolved Solids, Turbidity, Temperature, Sodium, Sulphate, Organic Nitrogen, Xylenes, Toluene, Sulphide, Sulphate, Pentachlorophenol, Odor, Methane, Manganese, Iron, Hardness, Copper, Chloride, Aluminum, Dissolved Organic Carbon, Ethylbenzene, and more. Chemical standards parameters are more detailed with over thirty chemicals listed with objectives and guidelines.

Water sample tests are taken at various locations and the various parameters analyzed. There are companies providing ongoing monitoring services and equipment maintenance for businesses and individuals in the province, so if you need and search for the term water quality test Mississauga or water quality test Newmarketyou will find a company to help you with regular water monitoring in those areas.

You may need “water sampling Peterborough” to btain a water test kit or sampling services. You may need a well inspection in Vaughn, Schomberg, Innisville, York, Dufferin-Peel, King City, Orangeville, Guelph, or Halton so a quick search will bring up companies like Tapped In Consulting who specialize in water system management.

A company like this has professional engineers and hydrogeologists who can prepare the forms and complete the inspections of the water system. Water potability tests include microbiological tests that are carried out at their laboratories to assess parameters like E.coli and Total Coliform as well as heavy metals, major ions, and trace metals. An Engineering Evaluation Report will be issued showing that the water system is in compliance with provincial guidelines.

Making the Case for Keeping Aquatic Aquarium Plants

Anybody who has seen a well-maintained planted aquarium knows how beautiful aquatic aquarium plants can be.  They add a depth and richness that turn a plain fish tank into a wonderful aquarium.  However, many people are intimidated by live aquarium plants and wonder if they are worth the trouble.  Do they provide any benefit besides looking good?

First of all, adding live aquatic plants doesn’t necessarily mean creating a densely planted, rich waterscape.  An aquarium can be enhanced just by adding one plant, or a few.  Live plants can be mixed in with artificial plants, or the aquarium can go to the extreme of a so called “Dutch aquarium” in which the plants take center stage and the fish are secondary in the tank.

Beyond the aesthetics, why should one consider plants in the aquarium?  Plants are living things that serve an important purpose in the ecosystem of the aquarium.  Through the process of photosynthesis, plants take in CO2 and release oxygen that is used by the fish. Nitrate can be bad for the fish when levels get too high, but the plants use the nitrate in the gravel as food.  The plants provide shelter for smaller or shy fish, and this extra sense of security is reflected in healthier fish.  The plants also help keep algae growth in check by shading the tank somewhat.  Certain fish species like to eat plants, too, so they can provide a dietary supplement to some fish

Keeping live plants in the aquarium can be relatively easy if a few basics are kept in mind.  This one may sound obvious, but it is important to remember that plants are living things.  They have needs just like the fish in the aquarium do and if one keeps those needs in mind, then the plants will be no trouble to keep in the tank.

When picking plants out to put in the aquarium, do some research and find out what the preferred water conditions are for the plants, just as you would when choosing fish.  Some plants thrive in higher pH conditions, some like soft water, others want high temps, etc.  Most aquatic plants will live within a common range of water conditions, but just be aware that there may be some plants that will not be compatible with your desired setup.

The substrate in the tank is important for keeping aquatic plants.  The gravel should be at least 3″ deep so that the roots can grow unrestrained and anchor the plant well.  A natural colored quartz gravel mix with rocks 2mm to 4mm in size is a very good choice.  For plants that require some additional soil, they can be put in pots that can then be planted into the gravel.

Lighting is a necessary component to a planted aquarium, but in most cases a basic fluorescent bulb or bulbs will do just fine.  Some plants need higher intensity light and heavily planted tanks have greater needs, but that is beyond the scope of this article.  Two important things need to be kept in mind regarding the lights and the hood.  The first is that fluorescent bulbs lose significant intensity after about a year, so even though the bulbs are not burned out, they should be replaced every year.  Another often overlooked task is cleaning the glass underside of the aquarium hood.  It often gets covered with algae and residue from evaporation, so clean it every week to keep the light intensity in the tank from fading.

Periodically the plants should be trimmed to prevent overgrowth.  Fast growing plants can reach the surface of the tank and start to grow horizontally, thus shading out everything below.  Some basic small sharp scissors will work for the trimming.  Any dead leaves or dead plants should be removed from the tank quickly as the decomposing plant material is not good for the water conditions.

The beauty of a well-planted tank and all of the potential benefits of aquatic aquarium plants make a convincing case for adding plants to any aquarium.  Given the fact that their maintenance is no more demanding than any fish that are already in the tank, it is hard to come up with a reason not to try live plants in your tank.

Reduce Eye Wrinkles With 5 Natural Proven Ingredients That Can Revitalize Your Delicate Eye Skin

Most of us who are past 40 years old start thinking about how we can reduce the fine lines and wrinkles appearing around our eyes, which are starting to make us look older and tired.

Eye wrinkles affect everybody — naturally some people have better skin than others so their facial tissues don’t dry out and wrinkle as much.

Age is a determining factor as well as damage caused by too much exposure to the suns ultraviolet rays. Then there is the natural reduction of collagen and elastin proteins in your dermis structural layer, as well as a reduction of hyaluronic acid.

Now you know the main causes — how do you go about improving these very sensitive and noticeable tissues around your eyes without resorting to laser, surgery or injections?

Often the best solutions are the simplest and easiest to use. These days some new anti-wrinkle cream ingredients are very powerful and tiny enough to penetrate right through your outer epidermal layer and get down to where they are needed in your dermis, once there they can have a positive effect your wrinkles.

The following 5 natural eye wrinkle ingredients are worth taking a serious look at:
Homeo Age™ extract from Canadian algae, has been shown to reduce wrinkles around the eyes and also has other anti-aging properties helpful for the delicate skin around the eyes. It stimulates cellular growth to rejuvenate tissue cells at the molecular level.

Natural Vitamin E (alpha-tocopherol) has been shown in many scientific studies to reverse the effects skin aging. It is one of the powerful antioxidant that can reduce the appearance of fine lines and wrinkles.

Functional Keratin™ is very similar to protein found in human skin and is powerfully effective in rejuvenating the skin. When in this unique bio-available form it can penetrate down to your dermis to stimulate growth of new protein cells.

Nano-Lipobelle H EQ10 is a special ‘nano-emulsion’ form of CoenzymeQ10. CoQ10 is beneficial to your body in many ways. However this particular version of it is especially helpful and has been shown in scientific studies to produce a dramatic anti-wrinkle effect because it can increase collagen and elastin production in the skin

Babassu is a light, natural wax, which is excellent for moisturizing, softening and soothing dry itchy and inflamed skin. It creates an invisible barrier to keep out dirt and grime yet retains moisture.

When you’re next looking for an eye contour gel to help reduce wrinkles around your eyes, make sure you keep these powerful proven ingredients in mind.

If your interested to find out more on how the above unique substances can positively affect your eye wrinkles, visit my website below.

Discover the best natural skin care creams available today.

User Guide to Omega-3 fatty acids (How to choose the best omega-3 supplement for you)

Let me start by saying that Omega-3 is probably the one nutrition supplement you should take. Over the years numerous clinical trials has been made to assess the health benefits of Omega-3  fatty acids. The bottom line of the majority of these trails is that taking Omega-3 will be beneficial to your health whether you are healthy or suffering from various health conditions. The origin of our dependency in Omega-3 fatty acids is still unknown but the fact is that Omega-3 fatty acids are one of the major building blocks of our body.

 Back to the basics – what is Omega-3?

Omega-3 fatty acids are essential fatty acids: They are necessary for human health but the body can’t make them which means you have to get them through food. Omega-3 fatty acids usually refer to 3 forms of fatty acids: ALA (alpha-Linolenic acid), EPA (Eicosapentaenoic acid), DHA (Docosahexaenoic acid). Omega-3 fatty acids can be found in fish, such as salmon, tuna, and halibut, other seafood including algae and krill, some plants, and nut oils. omega-3 fatty acids play a crucial role in brain function as well as normal growth and development. They have also become popular because they may reduce the risk of heart disease. The American Heart Association recommends eating fish (particularly fatty fish such as mackerel, lake trout, herring, sardines, albacore tuna, and salmon) at least 2 times a week. Research shows that omega-3 fatty acids reduce inflammation and may help lower risk of chronic diseases such as heart disease, cancer, and arthritis. Omega-3 fatty acids are highly concentrated in the brain and appear to be important for cognitive (brain memory and performance) and behavioral function. In fact, infants who do not get enough omega-3 fatty acids from their mothers during pregnancy are at risk for developing vision and nerve problems. Symptoms of omega-3 fatty acid deficiency include fatigue, poor memory, dry skin, heart problems, mood swings or depression, and poor circulation.

Health Benefits associated with omega-3 fatty acids

Omega-3 in Childhood Brain Development

During the last trimester of fetal life and the first two years of childhood, the brain undergoes a period of rapid growth – the “brain growth spurt.” Nutrient insufficiency during this period can compromise brain function. DHA is one nutrient absolutely required for the development of the sensory, perceptual, cognitive, and motor neural systems during the brain growth spurt. The fundamental importance of DHA for brain development is beyond dispute. The neurons are continually forming axons and dendritic extensions with accompanying cell membranes. Growing membrane must be relatively fluid, and DHA is the most fluidizing element in cell membranes. Even the synapses that are the primary functional units of brain circuits are made from membranes preferentially enriched in DHA. The retina, functionally an extension of the brain, contains rods and cones with the most fluid membranes of all the body’s cell types; they are also highly enriched in DHA. Laboratory animals (rodents, primates) with experimentally induced omega-3 deficiencies show deficits in retinal structure, visual acuity development, and cognitive performance.

Perinatal Importance of DHA and EPA

Demand for DHA rises exponentially as the brain rapidly expands in the third trimester, and continues after birth as the baby interfaces with environmental stimuli. Infants born prematurely are at special risk for omega-3 insufficiency because they may not have benefited from a full trimester of the mother’s lipid stores. Preterm infants have very limited ability to Synthesize DHA from the shorter chain alpha-linolenic acid (ALA). After birth, omega-3 status depends on the infant’s innate lipid metabolism and dietary intake of breast milk or formula. Although DHA and EPA are prominent ingredients of breast milk, many infant formulas do not contain these nutrients. Supplementing the mother’s diet with ALA is not a reliable means for obtaining DHA. In one study, lactating mothers received 10.7 g/day of ALA from flaxseed oil for four weeks. Breast milk levels of ALA, EPA, and DPA (docosapentaenoic acid) increased, but not that of DHA. All infants, whether preterm or full term, seem to require dietary DHA for retinal development and normal visual function.

Treating Developmental Coordination Disorder/Dyspraxia

The importance of DHA/EPA for overall brain and motor development after birth is illustrated by dyspraxia, also known as developmental coordination disorder (DCD). DCD/dyspraxia involves specific impairments of motor function and seriously affects about five percent of school-aged children. DCD’s core motor deficits are often accompanied by difficulties with learning, behavior, and psychosocial adjustment that overlap with dyslexia and attention deficit/hyperactivity disorder (AD/HD) and often persist into adulthood.

Managing Attention Deficit/Hyperactivity Disorder

AD/HD is the most common childhood developmental disorder, with prevalence estimates ranging from 4-15 percent for school-age children in the United States and elsewhere. Often AD/HD persists into adulthood. Considerable damage to the individual, family, and society can be exacerbated by co-morbidity with many other disorders of behavior, learning, or mood. AD/HD children consistently exhibit abnormal fatty acid status. Typically, reductions have been found in DHA and total omega-3 some of which may persist into adulthood. Low omega-3 levels are associated with a range of behavioral and learning problems. Omega-3 deficiencies correlate with behavioral problems (conduct disorder, hyperactivity-impulsivity, anxiety, temper tantrums, sleep difficulties) and learning difficulties in children. Omega-3 status is likely to be more relevant to AD/HD and related behavioral disorders.

Clinical Experience with Autism

The emergent rationale for employing DHA/EPA for autistic spectrum disorder (ASD) and other pervasive developmental disorders (PDD) dates to 2001, with case histories provided independently by two research groups. Vancassel reported low DHA (measured in plasma phospholipids), 20-percent lower-than-normal total omega-3 in ASD children. Bradstreet and Kartzinel reported finding omega-3 fatty acid deficiencies in nearly 100 percent of ASD cases. Then in 2002, Hardy and Hardy claimed that, of 50 children diagnosed with PDD, 90 percent were deficient in red blood cell membrane DHA/EPA. Various integrative physicians working with ASD and PDD patients have integrated DHA and EPA into their comprehensive regimens. A group in Austria conducted a six-week trial with 13 children, ages 5-17 years, diagnosed with ASD and displaying severe tantrums, aggression, and self-injurious behavior. Intervention was 1.5 g/day DHA/EPA (700 mg DHA and 840 mg EPA) or placebo. The DHA/EPA was well tolerated and there was a trend toward significant improvement over placebo for hyperactivity

High cholesterol

People who follow a Mediterranean-style diet tend to have higher HDL or “good” cholesterol levels, which help promote heart health. Inuit Eskimos, who get high amounts of omega-3 fatty acids from eating fatty fish, also tend to have increased HDL cholesterol and decreased triglycerides (fats in the blood). Several studies have shown that fish oil supplements rich in omega-3 fatty acids reduce triglyceride levels.

High blood pressure

Several clinical studies suggest that diets or fish oil supplements rich in omega-3 fatty acids lower blood pressure in people with hypertension. An analysis of 17 clinical studies using fish oil supplements found that taking 3 or more grams of fish oil daily may reduce blood pressure in people with untreated hypertension.

Heart disease

One of the best ways to help prevent heart disease is to eat a diet low in saturated fat and to eat foods that are rich in monounsaturated and polyunsaturated fats (including omega-3 fatty acids). Clinical evidence suggests that EPA and DHA (eicosapentaenoic acid and docosahexaenoic acid, the two omega-3 fatty acids found in fish oil) help reduce risk factors for heart disease, including high cholesterol and high blood pressure. Fish oil has been shown to lower levels of triglycerides (fats in the blood), and to lower risk of death, heart attack, stroke, and abnormal heart rhythms in people who have already had a heart attack. Fish oil also appears to help prevent and treat atherosclerosis (hardening of the arteries) by slowing the development of plaque and blood clots, which can clog arteries. Large population studies suggest that getting omega-3 fatty acids in the diet, primarily from fish, helps protect against stroke caused by plaque buildup and blood clots in the arteries that lead to the brain.


People with diabetes often have high triglyceride and low HDL levels. Omega-3 fatty acids from fish oil can help lower triglycerides and apoproteins (markers of diabetes), and raise HDL, so eating foods or taking fish oil supplements rich in omega-3 fatty acids may help people with diabetes. Another type of omega-3 fatty acid, ALA (from flaxseed, for example) may not have the same benefit as fish oil. Some people with diabetes can’ t efficiently convert ALA to a form of omega-3 fatty acids that the body can use.

Rheumatoid arthritis

Most clinical studies examining omega-3 fatty acid supplements for arthritis have focused on rheumatoid arthritis (RA), an autoimmune disease that causes inflammation in the joints. A number of studies have found that fish oil helps reduce symptoms of RA, including joint pain and morning stiffness. One study suggests that people with RA who take fish oil may be able to lower their dose of non-steroidal anti-inflammatory drugs (NSAIDs). Laboratory studies suggest that diets rich in omega-3 fatty acids (and low in the inflammatory omega-6 fatty acids) may help people with osteoarthritis. New Zealand green lipped mussel (Perna canaliculus), another potential source of omega-3 fatty acids, has been reported to reduce joint stiffness and pain, increase grip strength, and improve walking pace in a group of people with osteoarthritis. An analysis of 17 clinical trials looked at the pain relieving effects of omega-3 fatty acid supplements in people with RA or joint pain caused by inflammatory bowel disease (IBS) and painful menstruation (dysmenorrhea). The results suggest that omega-3 fatty acids, along with conventional therapies such as NSAIDs, may help relieve joint pain associated with these conditions.

Systemic lupus erythematosus (SLE)

Several studies suggest that EPA and fish oil may help reduce symptoms of lupus, an autoimmune condition characterized by fatigue and joint pain.


Some studies suggest that omega-3 fatty acids may help increase levels of calcium in the body and improve bone strength. Some studies also suggest that people who don’ t get enough of some essential fatty acids are more likely to have bone loss than those with normal levels of these fatty acids. In a study of women over 65 with osteoporosis, those who took omega 3 supplements had less bone loss over 3 years than those who took placebo. Many of these women also experienced an increase in bone density.


Several studies have found that people who took omega-3 fatty acids in addition to prescription antidepressants had a greater improvement in symptoms than those who took antidepressants alone. Studies are mixed on whether omega-3 fatty acids alone have any effect on depression.

Bipolar disorder

In a clinical study of 30 people with bipolar disorder, those who took fish oil in addition to standard prescription treatments for bipolar disorder for 4 months experienced fewer mood swings and relapse than those who received placebo.

Skin disorders

In one clinical study, 13 people with sun sensitivity known as photo dermatitis showed less sensitivity to UV rays after taking fish oil supplements. In another study of 40 people with psoriasis, those who took EPA with their prescription medications did better than those treated with the medications alone.

Inflammatory bowel disease (IBD)

omega-3 fatty acids can help reduce symptoms of Crohn’ s disease and ulcerative colitis, the two types of IBD. Some studies suggest that omega-3 fatty acids may help when added to medication, such as sulfasalazine (a standard medication for IBD).


Omega 3 may be beneficial to asthma patients. In a clinical study of 29 children with asthma, those who took fish oil supplements rich in EPA and DHA for 10 months reduced their symptoms compared to children who took placebo.

Macular Degeneration

A questionnaire given to more than 3,000 people over the age of 49 found that those who ate more fish were less likely to have macular degeneration (a serious age-related eye condition that can progress to blindness) than those who ate less fish. Similarly, a clinical study comparing 350 people with macular degeneration to 500 without the eye disease found that those with a healthy dietary balance of omega-3 and omega-6 fatty acids and more fish in their diets were less likely to have macular degeneration.

Menstrual pain

In one study of 42 women, they had less menstrual pain when they took fish oil supplements than when they took placebo.

Colon cancer

Eating foods rich in omega-3 fatty acids seems to reduce the risk of colorectal cancer. For example, Eskimos, who tend to have a high-fat diet but eat significant amounts of fish rich in omega-3 fatty acids, have a low rate of colorectal cancer. Animal studies and laboratory studies have found that omega-3 fatty acids prevent worsening of colon cancer. Preliminary studies suggest that taking fish oil daily may help slow the progression of colon cancer in people with early stages of the disease. If you have colorectal cancer.

Breast cancer

Although not all experts agree, women who eat foods rich in omega-3 fatty acids over many years may be less likely to develop breast cancer. More research is needed to understand the effect that omega-3 fatty acids may have on the prevention of breast cancer.

Prostate cancer

Population based studies of groups of men suggest that a low-fat diet including omega-3 fatty acids from fish or fish oil help prevent the development of prostate cancer.

Dietary Sources of omega-3

Fish, plant, and nut oils are the primary dietary source of omega-3 fatty acids. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are found in cold-water fish such as salmon, mackerel, halibut, sardines, tuna, and herring. ALA is found in flaxseeds, flaxseed oil, canola (rapeseed) oil, soybeans, soybean oil, pumpkin seeds, pumpkin seed oil, purslane, perilla seed oil, walnuts, and walnut oil. Other sources of omega-3 fatty acids include sea life such as krill and algae. Although ALA is a form of omega-3 it is less effective form since it has to be converted to EPA and DHA. Studies show that conversion rate of ALA to DHA (most essential form of omega-3) are between 0.2% (in men) -10% (in women) only.

Available Forms of omega-3

Both EPA and DHA can be taken in the form of fish oil capsules. Be sure to buy omega-3 fatty acid supplements made by established companies. Do not rely on companies that state that their products are free of heavy metals such as mercury, lead, and cadmium. Look only for companies who publish a COA (certificate of analysis) for their product. Most companies that do not publish their COA do so because their product does not comply with the industry standards such as IFOS (international fish oil standard) and CRN (council for responsible nutrition).

How to Take It?

Dosing for fish oil supplements should be based on the amount of EPA and DHA, not on the total amount of fish oil. Supplements vary in the amounts and ratios of EPA and DHA. A common amount of omega-3 fatty acids in fish oil capsules is 0.18 grams (180 mg) of EPA and 0.12 grams (120 mg) of DHA. Five grams of fish oil contains approximately 0.17 – 0.56 grams (170 -560 mg) of EPA and 0.072 – 0.31 grams (72 – 310 mg) of DHA. Different types of fish contain variable amounts of omega-3 fatty acids.


There is no established dose for children. Omega-3 fatty acids are used in some infant formulas.  Children should avoid eating fish that may be high in mercury, such as shark, swordfish, king mackerel, and tilefish.


Do not take more than 3 grams daily of omega-3 fatty acids from capsules without the supervision of a health care provider. 

  • For healthy adults with no history of heart disease: The American Heart Association recommends 0.5 gram daily of EPA and DHA.
  • For adults with coronary heart disease: The American Heart Association recommends an omega-3 fatty acid supplement (as fish oils), 1 gram daily of EPA and DHA. It may take 2 – 3 weeks for benefits of fish oil supplements to be seen.
  • For adults with high cholesterol levels: The American Heart Association recommends an omega-3 fatty acid supplement (as fish oils), 2 – 4 grams daily of EPA and DHA. It may take 2 – 3 weeks for benefits of fish oil supplements to be seen.

Author choice product

There are many high grade omega-3 supplements out there, the choice of supplement should be based on the following guide lines:

  • Product grade – look for high grade clean omega-3 supplement
  • COA – look for  a published certificate of analysis
  • Industry standards – product specifications shall comply with IFOS and CRN demands
  • Omega-3 content – look for high amount of Omega-3 fatty acids (50%) preferably rich in DHA
  • Price – good omega 3 product doesn’t necessarily mean expensive (20$ a month is adequate)

My choice of omega-3 supplement comes from a New Zealand based company who to this author opinion produce one of the best omega-3 supplements on the market today