Penicillium I Spores Effects With Metals.

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Introduction: Penicillium I Spores Effects With Metals.

Hello.

I am trying to observe what would happen if I combined specific metals with distilled water, some salt and sugar with cocoa (low chlorine) present for a specific fungus (Penicillium I). It is known that brass, copper, zinc, and silver disrupt enzymes of fungi, cell wall and DNA and RNA but how different is PI (a mold) compared to specific bacteria and fungi (very different).

How will specific metals like Lead, Arsenic, and Copper effect the fungi that I am testing? I am testing a 1 million spore per ml sample (pretty high) but necessary for this project.

Silver and some other metals (Well especially silver) react to form insoluble inert silver chloride with chloride ions so low chloride ions for these test as well as distilled water is necessary.

WARNING: Some molds are pathogenic and dangerous to your health. Also, arsenic salts and lead salts if you try to make them are extremely toxic and even cancer-causing level 1. They can be fatal if absorbed through the skin, etc even at concentrations as low as 1%. Please do not ADD acids to lead, arsenic, etc if you are going to repeat this!! The metals are a LOT easier to handle than the salts.

Step 1: Prep Work for Plates and Metals and Mold on Plates Part 1

Prep work for agar.

Every 5 plates have 20 ml of cocoa agar. It consists of 100 ml approx distilled water, 5.0 grams sucrose, 5.0 grams Cocoa with sodium bicarbonate, and 5.0 grams Agar Agar. Heat in a hot plate for 15-20 minutes with occasional stirring until it begins to broth and foam up, then put on the flask on a cool surface. For a 100 ml sample of agar, mix make sure the flask is at least double the volume. Pour the hot agar 80 deg C into the plates and close them to avoid other contamination. Allow them to solidify.

Wearing gloves place each metal (Lead, arsenic) into the gloves. A bit of transfer of lead ions from metal will be transferred to the arsenic, etc but this is a minor and unavoidable problem.

Make a concentrated sample of water with mold samples and allow to stir without air for a few days and filter giving the active mold sample. Prepare this sample outside at 5 degrees C in open space (Not indoors) to avoid spore potential contamination!

Place 2 ml of concentrate mold sample onto agar cocoa plates. Close the plates.

Step 2: Prep of Silver Metal Over Copper With Other Types of Metals.

Here I will show how to get nanosilver (Near nanosilver particles) with copper and silver nitrate in distilled water.

In 20 ml of distilled water with 5 g silver nitrate add 6 g of copper metal.

Here the equation (Chemical to it).

2AgNO3 (aq) + Cu (s) >>>>> 2Ag(S) + Cu(NO3)2(aq).

5 grams silver nitrate/169.87g times 107.8682 g (Silver) = 3.17 g silver produced with 6 g copper. 2.85 g of copper left over in this reaction.

Warning: Pure Silver Nitrate solutions are toxic (well over 10 g per L) perm stain hands black with exposure and are corrosive to skin and eyes. Wear gloves and goggles to protect yourself if you work with silver nitrate.

Step 3: Results of Metals Part 1 (Brass, Lead, Arsenic, and Copper).

Here is the picture for the third entry here are some results for 6 days.

Brass---- Growth complete with Aspergillus Niger.

Lead--- Very little growth of A. niger.

Arsenic -----Very low growth of Aspergillus niger.

Copper pipe- the complete growth of black mold.

These indicate that arsenic and lead even though they are less active than copper and brass can strongly inhibit the growth of Aspergillus niger!! It may be due to Arsenic metal slowly releasing As+3 ions and Pb+2 ions for lead and their toxicity on human cells (disrupting enzymes including DNA, ATP, etc) even in small amounts may be why these metals have inhibitory effects on fungi.

However, growth did occur slower than brass and copper than arsenic and lead indicating it just slowed down the growth a bit (the mold was not completely killed by lead or arsenic metal). There some evidence that these types of fungi like some bacteria can pump out metal ions from the cell. In bacteria, the antibiotics can be neutralized or pumped out. This is a possible explanation for the slow mold growth on these plates.

Step 4: Silver (II) Oxide-silver Copper Powder, Zinc Powder, Copper Powder, Cobalt Powder and to Metal Fungi Test on Cocoa Agar.

First to make Ultra 99% pure silver(II)-oxide and silver powder with copper.

Add to 4 degrees C distilled water (Room temperature also will work--- but the particle size will be different effecting your results) and add 5 g 98% silver (I) nitrate to the water. Clean all beakers with distilled water since any tap water, etc, if you use it, would convert silver nitrate (due to chloride ions from chlorine) into silver chloride that would ruin the result totally.

Then add 10 g of 99.99% copper powder and stir well being careful to avoid splashes or too rapid stirring. (Lab coat, gloves, and goggles (Prefer Faceshield) is better since the silver nitrate stains badly skin and is fairly corrosive too.

Decant the cold water into another beaker and treat the leftover sol as trace silver nitrate (special disposal). Put in 50 ml of cold distilled water again and heat gently 50-60 degrees C on a Hotplate until very little water is Left. Let the rest air dry and place in a bottle or sealable bag for storage. Overheating may change silver metal into oxides by air exposure but Low heat can significantly reduce this problem.

Buy copper powder, silver powder and cobalt powder from e-bay.

Add 5 g of sugar to 100 ml tap water with 5 g cocoa 99%, and 5 g Agar-agar. If you are going to make 200 ml you obviously have to double the grams of sugar, etc to fit the same ratio. Boil the agar solution in a beaker double the volume of your sample or flask until some foaming and boiling happen ( A bit) then let it cool for 5-10 minutes. Be careful Agar turns into a gel at 50 degrees so do not wait too long since you cannot simply heat the agar back up into a liquid again (or very easily).

Let the plates that you poured 10 ml of each (20 ml if you have them) and let them solidify at room temp for 20 minutes approx.

Add 5 g by weighing and taring of a digital scale (approx 5 g) give or take 0.1 grams of the silver(II) oxide -silver metal with copper, copper powder, zinc powder, cobalt powder and To metal to each plate. Note that To metal is NOT a powder but 5 g is the amount I will use.

Step 5: Results for Part 2 (SIlver Oxide-Copper, Copper, Zinc and Cobalt, Etc

Here are the results for the following metal powders. Here the concentration

of each metal is 5 grams.

Silver (II) Oxide-Copper Powder----- Little growth for 2 weeks.

Copper Powder ------- Little growth for 2 weeks.

Zinc Powder ------ Little growth for 2 weeks.

Cobalt Powder -----Little growth for 2 weeks.

Step 6: Discussions and Conclusions.

Well, I am very surprised about the Metal powder. I did not think especially Silver (II) Oxide-Copper, Zinc or Cobalt was very effective at preventing the mold from spreading across the plate in 2 weeks!!! I think the size of the particles (Nano) allowed the metal to enter the mold thus destroying vital enzymes of the cell wall, DNA, etc.

I tried researching how metals like this kill Mold and I got nothing out of it. Bacteria it interferes with DNA synthesis, Cell wall reformation, RNA synthesis, etc. So I assume the metal is toxic in a similar manner but I could be wrong on that.

The metals (Lead, Arsenic, etc) surface area was too big to allow the metal to pass into the cell or enough of the metal inside to kill the Mold. However, powder including copper did.

However later On 1 month or so the Mold slowly grew right over the metals (Powder) indicating it only slows it down and just like the other metal it does not kill them.

Conclusion.

2 Weeks of Metals like Arsenic, Lead, etc resulted in a complete growth of Black Mold (Aspergillus Niger) but the powder for 2 weeks results in some but it does not result in LAWN (Overgrowth of mold on the plate). The powder is far more effective at killing mold than regular metal is.

Eventually in a month lawn even over the metal powder grew over the agar.

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    4 Comments

    In 4 days of water plates with copper, no seeds grew. This could be due to the facts that the copper powder is also toxic to the seed or takes out moisture from the seed. Interesting concept but at least copper powder is not capable of allowing clover to grow.

    It been a few days under the grow light and it has failed.

    Here is some Common WIki knowledge for how copper kills microbes (Including fungi and bacteria).

    Examples of some of the molecular mechanisms noted by various researchers include the following:


    • The 3-dimensional structure of proteins can be altered by copper, so that the proteins can no longer perform their normal functions. The result is inactivation of bacteria or viruses[11]
    • Copper complexes form radicals that inactivate viruses.[12][13]
    • Copper may disrupt enzyme structures, and functions by binding to sulfur- or carboxylate-containing groups and amino groups of proteins.[14]
    • Copper may interfere with other essential elements, such as zinc and iron.
    • Copper facilitates deleterious activity in superoxide radicals. Repeated redox reactions on site-specific macromolecules generate OH- radicals, thereby causing "multiple hit damage" at target sites.[15][16]
    • Copper can interact with lipids, causing their peroxidation and opening holes in the cell membranes, thereby compromising the integrity of cells.[17] This can cause leakage of essential solutes, which in turn, can have a desiccating effect.
    • Copper damages the respiratory chain in Escherichia coli cells.[18] and is associated with impaired cellular metabolism.[19]
    • Faster corrosion correlates with faster inactivation of microorganisms. This may be due to increased availability of cupric ion, Cu2+, which is believed to be responsible for the antimicrobial action.[20]
    • In inactivation experiments on the flu strain, H1N1, which is nearly identical to the H5N1 avian strain and the 2009 H1N1 (swine flu) strain, researchers hypothesized that copper's antimicrobial action probably attacks the overall structure of the virus and therefore has a broad-spectrum effect.[21]
    • Microbes require copper-containing enzymes to drive certain vital chemical reactions. Excess copper, however, can affect proteins and enzymes in microbes, thereby inhibiting their activities. Researchers believe that excess copper has the potential to disrupt cell function both inside cells and in the interstitial spaces between cells, probably acting on the cells' outer envelope.[22]

    Currently, researchers believe that the most important antimicrobial mechanisms for copper are as follows:


    • Elevated copper levels inside a cell causes oxidative stress and the generation of hydrogen peroxide. Under these conditions, copper participates in the so-called Fenton-type reaction — a chemical reaction causing oxidative damage to cells.
    • Excess copper causes a decline in the membrane integrity of microbes, leading to leakage of specific essential cell nutrients, such as potassium and glutamate. This leads to desiccation and subsequent cell death.
    • While copper is needed for many protein functions, in an excess situation (as on a copper alloy surface), copper binds to proteins that do not require copper for their function. This "inappropriate" binding leads to loss-of-function of the protein, and/or breakdown of the protein into nonfunctional portions.

    https://en.wikipedia.org/wiki/Antimicrobial_properties_of_copper

    Here some results of trying to grow Clover on Cocoa agar plates with 2-3 grams copper powder on the plates. Not sure if this type of tissue culture will work. It prone to excessive drying, etc probably due to the copper.

    002.jpg

    Correction in 1 month the zinc, copper, and silver copper plates that had the powder it grew around the plate.

    003.jpg