12/15/2017

Mixing Human Pheromone Products - the Basics 

Many pheromone users try to mix two pheromone products, then fail, then go back to using standalone products. Here you can learn some basics facts about pheromone mixing. Naturally, it is much easier to create a pheromone mix if you know what you are mixing. The only way to know this is to use every pheromone product on its own first and see what reactions that pheromone product - cologne, perfume or oil - gives in specific situations.
 




You may be inquisitive as to where we got the matter for writing this article on pheromones in pen ink. Of course through our general knowledge, and the Internet!


Burgess, J. G., H. Miyashita, H. Sudo, and T. Matsunaga.1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: Localization of activity to the chromatophores. FEMS Microbiol. Lett. 84: 301 - 306.

When it comes to pheromone mixing, you must know that some pheromone products are oil-based and some are alcohol-based. For example Primal Instinct and Scent of Eros use oil, while The Edge and New Pheromone Additive use alcohol base. If you use an atomizer and even if you shake your atomizer there is a chance the products won't mix well. To be able to mix different pheromone products, you'll need a calibrated glass dropper. It's not expensive, and it is available at the nearest pharmacy. Some pheromone products are stored in a roll-on bottle, others sometimes even in an open mouth bottle, however, some products have an integrated dropper you can use.

We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.

Mckenney, D., K. E. Brown, and D. G. Allison. 1995. Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J. Bacterial 177: 6989 - 6992.

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities - sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

Starch hydrolysis test, Protein hydrolysis test, Lipid hydrolysis test, Oxidative / Fermentative test, Salt concentration (0%, 3%, 5%, 7%, 10%), TCBS, Growth in Temperature, 42??C and 47??C

Mearns - Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 - 157.

Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

In this experiment among 17 Producer and Inducer strains only 3 of them have responded to the quorum sensing principle. (BrB+/BrB-), (GcC+/GcC) and (SA+/SA-)

Bainton, N. J., P. Stead, and S.R. Chhabra. 1992. N-(3-oxohexanoyl)-L-homoserine Lactone regulated carbapenem antiobiotic production in Erwinia carotovora. Biochem. J. 288: 997-1004.

When it comes to pheromone mixing, you must know that some pheromone products are oil-based and some are alcohol-based. For example Primal Instinct and Scent of Eros use oil, while The Edge and New Pheromone Additive use alcohol base. If you use an atomizer and even if you shake your atomizer there is a chance the products won't mix well. To be able to mix different pheromone products, you'll need a calibrated glass dropper. It's not expensive, and it is available at the nearest pharmacy. Some pheromone products are stored in a roll-on bottle, others sometimes even in an open mouth bottle, however, some products have an integrated dropper you can use.

A. Live cells of producer and inducer strains B. Live cells of producer strain alone C. Live cells of inducer strain alone In culture system A 200ul of 16 hours old broth culture of both producer and inducer strains were added to the 15 ml of nutrient broth.

Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55: 165-199. Msadek, T. 1999. When the going gets tough: survival strategies and environmental signaling network in Bacillus subtilis. Trends Microbiol. 7: 201-207.

ANTIBIOTIC ASSAYS Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37??C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

The results of Vanderivere and Kirchman 1993 suggest that the addition of increased surface by adding sand will induce the exopolymer synthesis through the high cell density dependent system. In the same way the bacterial organisms attached to the surface of seaweed shows alteration of genes expression may be due to the response to the high competitive environment. When cell density increases the competition for space and nutrients are also increased. So the existing bacteria were forced to protect themselves in this competitive environment. Normally in this condition the bacteria will be activated to induce the expression of certain hidden genes in genetic material through quorum sensing. The quorum sensing is principles were active compounds (autoinducer) from bacterial cell will promote the expression of a particular hidden gene of other bacterial organism in a stressed condition.

The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing - controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I - generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

Whiteley, M., K.M.Lee, and E.P.Greenberg.1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc.Natl.Acad.Sci.USA.96:13904-13909.

Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

Bassler, B.L.1999. Curr.Opin.Microbiol.2: 582-587.* Bernan, V. S., M. Greenstein, and W. M. Maiese. 1997. Marine microorganisms as a source of new natural products. Adv. Appl. Microbiol. 43: 57 - 89.

GcC- GcD- GcE- GcF- Gc A+/GcA- Gc B+/GcB- Gc C+/GcC- Gc D+/GcD- Gc E+ /GcE- Gc F+/GcF- GcA- GcB- GcC-

The basic idea about pheromone mixes is that by adding products together, you can maximize their effectiveness. And, if you have a mix that simply does not work, don't put more stuff into it - it is unlikely that this will make it work. Most mixes are very simple - and made of two products only. Some popular mixes include Scent of Eros (contains androstenol) plus NPA (contains androstenone), or Androstenone Pheromone Concentrate (APC) plus New Pheromone Additive.

Liming, Y., G. K. Boyd, and J. G. Burgess. 2002. Surface induced attachment induced production of Antimicrobial compounds by marine epibiotic Bacteria using modified Roller Bottle. Mar. Biotechnology. 4: 356 - 366.

Even though the Tongat Ali plant is protected in Malaysia herbal suppliers there have been aggressively marketing the product in the United States using scientific studies that boast of the testosterone enhancing properties of the product. Due to the increased demand and decreasing availability of the plant prices have skyrocketed and counterfeits have begun to appear and like yohimbe most of the Tongat Ali found on the market now are basically worthless with nowhere near the amount of active ingredient needed to be effective therefore if you want to be sure you are indeed getting a pure source have it shipped directly to you from Indonesia.

Burgess, J. G., E. M. Jordan, M. Bregu, and A. Mearns-spragg. 1999. Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70: 27-32.

Patterson, G. L., and C. Ml. Bolis. 1997. Fungal cell - wall poly sacchraides elicit an antifungal secondary metabolite in the Cyanobacterium Scytonema ocellatum. J. Phycology. 33: 54 - 60.

REFERENCES Alim, I., and K. Yuto. 2003. Mc21-A, a Bacteriocidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenotica sp. nov. O-Bc 30T, against Methicillin -Resistant Staphylococcus aureus. Antimicro. Agents Chemoth. 47: 480 - 488.

You may be inquisitive as to where we got the matter for writing this article on pheromones in pen ink. Of course through our general knowledge, and the Internet!



Icebreaker Pheromone Cologne



The collected seaweed samples were thoroughly washed with sterile seawater to removes the loosely attached bacteria/particles. Seaweed fronds were scrubbed with sterile cotton swabs to obtain epiphytic bacteria. Epiphytic bacterial organism in the swab were inoculated in sterile peptone broth (50% sea water) and incubated at 28??C in an incubated shaker (220 rpm / min) for overnight. After the incubation period the enriched cultures were serially diluted up to 10-8 concentration and 200 microlitre of each diluted samples were transferred into the nutrient agar plate (50% sea water). The plates were incubated at 28??C for 5 days and the plates with crowded colonies were selected. In the crowded plates those colonies, which showed the sign of inhibition zone around its margin to the neighboring colony, were selected and considered as producer strain. The neighboring sensitive colonies were treated as inducer strain. Both producer and inducer strains were streaked repeatedly until to get pure culture. The pure culture were properly labeled and subjected to the quorum sensing analysis.

ABSTRACT Bacterial biofilms are sessile communities with high cell density that are ubiquitous in natural, medical, and engineering environments; they are fascinating since they are primitive tissues with an advanced chemical communications network. Currently, there is an explosive amount of biofilm research, most of it with the ultimate aims of biofilm prevention, control, or eradication.The present investigation was aimed to study the cell signaling principle among seaweed epibiotic bacterial organisms , totally 54 bacterial isolates were made out of 20 seaweed species. Among 54 bacterial isolates 17 of them producer, another 17 of them inducer strains rest of 20 isolates were normal that have not showing any signs of activity. The 17 producers and 17 inducer strains were subjected to the cross species induction by quorum sensing principle and found that only 3 of them have responded to the cross species induction in production of antibacterial compound against the respective inducer strains. All the 3 producers and inducer were identified by biochemical analysis and surprisingly that all the 3 producer strain belongs to Genus Pseudomonas and inducer strains were belongs to Genus Vibrio. The supernatant obtain from mixed culture of Pseudomonas & Vibrio shows the antibiotic activity against the various Vibrio species which were isolated from other sources. Such as Vibrio from primary film, Vibrio from sediment Vibrio from seaweeds epibiotics. The obtain results clearly shows the particular Pseudomonas from seaweed epibionts has the capability of producing potential antibiotic compound against wide range of Vibrio species through quorum sensing.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

QUORUM SENSING EXPERIMENT NUMBER 1 In this present study, the producer and inducer strains were cross reacted to find out the production of antibiotic compound through quorum sensing. Totally three set of cultures were maintained as follows (along with one as control).

In this present work the totally 54 isolates were screened from 20 different seaweed species out of which 34 species were showed the signs of quorum sensing i.e. 17 producer and 17 inducer strains, but when these organisms where subjected to quorum sensing principle in mixed culture only 3 them have responded. So the present study reveals around 17% of bacterial species isolated from seaweeds and sediment were responded to quorum sensing. According to the results of bacteria isolated from marine algae surfaces indicated that the incidence of antibiotic producing strains from this habitat was 20% where as that from sea water was only a few percent. In the present study also reveals more or less the same ratio in Pseudomonas spp. was observed. Our results also reveals a results of Kell et al., 1995; Stead et al., 1996; they have said the culture supernatant of Pseudomonas sps known to contain AHLS which induces the production of phenazine antibiotics. In this investigation due to time constraint, It was not attempted to identify the active compound secreted by Pseudomonas through quorum sensing, which may leave the space for the further intensive research in future.

Dekievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 66: 4839-4849. Dimango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aerugniosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin-lnvestig. 96: 2204-2210.

Table 1. List of Seaweeds species collected for the present study SPECIES NAME FAMILY Halimeda gracilis Chlorophyceae Ulva lactuca Chlorophyceae

In gram positive bacteria peptide and derivative peptide based signaling molecules seem to be the predominant mode of communication. During high cell density the marine bacteria can produce enzymes, surfactants, toxins, and antibiotics by the chemical signal communication. Marine epibiotic bacteria are also known to produce compounds active against drug resistant hospital pathogen by the cross species induction method. Building on assays described by Austin (Billaud and Austin 1990) a screening procedure has been developed in which marine bacteria are challenged by exposing them to terrestrial bacteria prior to assay of antimicrobial compounds. Hence in this present investigation it is proposed to find out the ability of sea weed epibiotic bacterial organism to produce antibacterial compounds through quorum sensing.

Kell, D. B., A. S. Kaprelyants, and A. Grafen. 1995. Pheromones, Social behaviour and the functions of secondary metabolism in bacteria. Trends in Ecol. and Evol. 10: 126-129.

Br B+ /Br B- Br C+/Br C- Br D+/Br D- Br E+/Br E- Br F+/Br F- BrA- Br B- BrC- BrD- BrE- BrF- NIL 39

Allison, D. G., B. Ruiz, C. Sanjose, A. Jaspe, and P.Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbial Lett. 167: 179-184.

Rheinheimer, G.1992.Aquatic microbiology (Wiley, New York), 3rd Ed. Stead, P., B. A. M. Rudd, H. Bradshaw, D. Nobel, and M. J. Dawson. 1996. Induction of phenazine biosynthesis in cultures Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 140: 15-22.

Quorum sensing usually focused on the bacteria growing in homogeneous environment. However few studies have attempted to a study this principle in heterogeneous environment also. In this present investigation we have attempted to study both homogeneous as well as heterogeneous environment. In former one we have isolated producer strain in seaweed eipbionts and it shows inhibitory activity against the inducer organism at the same seaweed epibionts. Later producer strains from seaweed epibionts, were treated with various Vibrio organisms from different environment. The obtain result of this study shows that the producer strain are capable of secreting antibiotic compounds not only to their natural competitors in its own habitate but also to the pathogen inhabiting in a distant related environment.

Burgess, J. G., M. Elizabeth, M. Bregu, A. Meams-Sprugg, and K. G. Boyd. 1999 Microbial antagonism: a neglected avenue of natural products research. J. Biotechnology. 70. 27-32.

Hypnea valentiae Rhodophyceae Hypnea pannose Rhodophyceae Hypnea esperi Rhodophyceae Acanthophora spicifera Rhodophyceae ISOLATION OF EPIPHYTIC BACTERIA

GcA-, GcB-, GcC-, GcD- GcE-, GcF- Gracillaria edulis U1-, U2-, U3-, U4- Ulva lactuca SA- Sediment

Bryers, J. D., and W. G. Characklis. 1982. Processes governing primary biofilm formation. Bioeng. 24: 2451-2476 Burgess, J. G., K. Boyd, E. Armstrong, T. Pisacane, and D. R. Adams. 2003. The development of a marine natural product-based antifouling paint. Biofouling. 19: 197 -205.

BACTERIAL IDENTIFICATION The organisms responded to the quorum sensing process alone were identified by the following biochemical analysis.

RESULTS AND DISCUSSION: QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

All the cultures were incubated at 28??C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

The basic idea about pheromone mixes is that by adding products together, you can maximize their effectiveness. And, if you have a mix that simply does not work, don't put more stuff into it - it is unlikely that this will make it work. Most mixes are very simple - and made of two products only. Some popular mixes include Scent of Eros (contains androstenol) plus NPA (contains androstenone), or Androstenone Pheromone Concentrate (APC) plus New Pheromone Additive.

But on the northern tip of Sumatra (Aceh province of Indonesia) where the Tsunami occurred there still exist forests with a large areas of Tongkat Ali coverage. Tongkat ali from the Indonesia Aceh province can be purchased in wholesale and retail quantities from Sumatra Pasak Bumi.



The world of pheromones in pen ink is an interesting one. It is with this objective that this article on pheromones in pen ink was written so that people got to know more about it.
Author Dave Thomson
 
 
 


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