Introduction: Testing for Microbes in Soil (Sterilized) Friendly Types.

Picture of Testing for Microbes in Soil (Sterilized) Friendly Types.

DANGER:

Because what I am doing may have harmful impacts (rare) but I did research on the soil before proceeding, I strongly RECOMMEND that you don’t repeat this experiment in any way. Some types of soil may have bacteria that could cause illness if you have an overactive or underactive immune system. Play it safe and don’t repeat. If you repeat it at your own RISK. I checked the company and reports and found info that the company sterilizes the soil and then bacteria are most likely added. NOT all companies would do this and this is the RISK and sometimes pathogenic (if you have an immune disorder) or worse bacteria could be found in the soil. This project is for Educational Purposes Only. I have done 3 years in Biotechnology and 2 years in Waste and waste water management. I gained precautions and skills this way too.

Phenol and many strong acids are used. Both are extremely dangerous if misused. 93% sulfuric acid can cause severe burns on contact and phenol is a chronic poison cable of being absorbed through the skin and it may cause poisoning if enough of the liquid is absorbed through the skin and it can cause burns too. If the solution of phenol catches fire very dangerous phenol fumes can be released. I keep flammable dyes with alcohol including phenol away from any flames during use.

Safety: Although what I am doing is extremely rarely harmful
there are small risks associated with it. NEVER take water or soil near duck ponds or compost soil since this increases the risk of being exposed to pathogenic bacteria. Also never take samples from old canned food in case of exposure to bacteria that can cause botulism. You can inhale potentially lethal spores this way!!!!Never take samples for fun from dead animals, etc due to diseases and pathogenic diseases (fungi, protozoa, bacteria, etc). Check for epidemics for that before you test if you are going to repeat.

Also for all chemicals dispose of them properly. For example with 78% sulfuric acid if you have left over 100 ml add the 100 ml (add acid to water) to 1000 ml of H20. This will give you 1.3 molar concentration. Then add baking soda carefully to neutralize the acid. Heat may be released so be careful.

If you use dyes that are potentially environmentally dangerous like Malachite green then dilute them and use yeast or activated carbon to neutralize the chemical. Yeast is a living organism that can absorb 50% or more of the dye even if the yeast is dead or alive still the yeast afterward are sent to a waste center.

Protocol.

Take about 10 g of soil for 150 ml of water (this can
include tap water) and shake very well every 30 minutes. Filter with coffee paper and collect the clear liquid. Do this outside to prevent any contamination from occurring.

Gram staining Procedure.

Warning:

These chemicals are toxic and Iodine is both corrosive and toxic. Wear gloves, apron, and safety goggles.

Take a slide and add a smear of bacteria that you are trying to culture. Heat fix the slide with heat from a hot plate or an alcohol lamp. Carefully add crystal violet chemical to the heat fixed smear for 30 seconds. Wash off the excess crystal violet by running water down the slide and not directly on the smear since this will cause most of the smear to come off.

Add
gram Iodine to the smear for 30 seconds and wash off the same method as before. Add ethanol a few drops on the smear to decolorize the smear. Finally, add the Safranin O to the smear. Wash off with water and put a cover slip on and let it stand for 10 minutes. Go from 40 x until you see something and then 100 X and finally 1000 x. Place a drop of cedar oil (IMMERSION OIL) over the smear.

Methods
of endospore stain. I made a 2% 500 ml Malachite green solution and bought a 100 ml of Safranin O 1% solutions. Protocol. Make a heat fixed smear. Using a hot plate or heat source place the slide under boiling water and add 1% Malachite green to the smear. Make sure that the dye doesn’t run dry. After 2-5 min under the steam wash with water

Like the gram stain procedures. Don’t add the water directly to the smear! Then add drops of Safranin O 1% to the smear

Warning:
Malachite green is considered a mutagenic agent and is severely irritating to skin and may cause eye damage if exposed to the eyes. I dispose of small amounts of 0.002% with baker yeast and sugar to absorb the dye and send it to the Eco center.

Just be prudent since that water would contain lots of
bacteria inside it. Wear all the PPE (like nitrile gloves, goggles, rubber apron, etc) before doing this.

Next, I will explain the Phenol red broth. Usually, I add 0.08 g of Phenol red to 6.5 g of sugars like (Fructose, Glucose, Maltose, Lactose, Xylitol, and Sucrose). I add 8 grams of phenol red powder to the flasks and measure it with a graduated flask 100 ml with tap water.

Phenol red is an indicator normally at ph 7 (it’s orange to
red) and when the organism produces an acid the ph drops below 6 the indicator turns yellow. This will tell you if the bacteria will ferment the sugar. If no sugar can be fermented the solution should be orange to orange red. Always record the initial color with the label.

Methyl red test for Bacteria:

Here methyl red is used when I grew nutrient broth with 100 ml of water with a sample of the bacteria. This means that I added liquid with bacteria from the soil into the nutrient agar. Then days later I added 1.5 ml of 0.02% methyl red which equaled 0.003 g of methyl red. Another way would to add 3 grams of methyl red to 100 ml in a graduated flask 100 ml and do a serial dilution 1/10 two times.

Pyruvate ions that the organism converts glucose into pyruvate through the glycolysis cycle turn the ph near 4. This changes the color from orange-yellow to red.

The control was nutrient agar with methyl red on the left and the right was the bacteria that produced pyruvate acid and turned the medium red.

Urease Test with Urea 2% and Nutrient agar 5% with phenol red.

Here I will show how to make a 2% urea nutrient agar broth. I did not and could not afford nutrient broth so I used 5% nutrient agar with 2% Urea with a trace of phenol red and filled up to 100 ml. When a 2% urea 5% nutrient agar broth changes due to bacteria in oxygen if it is positive it will convert Urea into Ammonia and CO2 thus raising the ph turning the solution pink

. This must be done within 5 days to prevent a false positive.

DANGER: Using Urea as a nitrogen source means THAT YOU
CANNOT mix it with bleach. Doing so increases the risk of creating Nitrogen Trichloride a potentially explosive material. Diammonium phosphate, Ammonia, Urea should NEVER be mixed with bleach. Alcohols, Copper sulfate, Acetone can be used to kill the bacteria instead. Always review MSDS before use.

Triple sugar iron agar.

Hydrogen Peroxide Catalase Procedure:

Here we are testing to see if the bacteria produces catalase enzyme. Catalase breaks down Hydrogen peroxide into water and oxygen. I am using 35% Hydrogen peroxide. It is just fizzed more when the reaction is positive. Warning: Hydrogen Peroxide is Corrosive (Quite) at these concentrations. Be aware of false positives in this case too.

O/F test Protocols and procedure:

O/F test. The O/F test determines if a bacteria can convert glucose into acids in an anaerobic environment or aerobic environment. 2 ml of 0.04% Bromothymol blue with nutrient agar or broth is added. 13 to 24 hours a color change from green-blue to yellow for both O/F conditions means this bacteria can ferment in both environments. This test is to prove that you can grow bacteria with nutrient broth in 24 hours.

The bacteria were free of other pathogens and I did them in an open area with lots of air to allow no contamination to occur. The control was water but if it was nutrient agar then this would have wasted nutrient agar if the unknown bacteria was airborne due to contamination.

Acetate/Citrate/Tartrate Bromothymol
blue broth procedure:

Here I will test levels of salts of acetate, citrate, and tartrate in simmon citrate like a medium. Below is the recipe.

Recipe per L of Citrate Bromothymol blue agar....

Magnesium sulfate (Heptahydrate) ...... 0.2 grams.

Ammonia dihydrogen phosphate........ 1.0 grams.

Dipotassium phosphate........ 1.0 grams

Sodium chloride........ 5.0 grams.

Bromothymol blue....... 0.08 grams.

Agar......... 15 grams.

However, since I don’t have Ammonia dihydrogen phosphate I used Diammonium phosphate and tri-potassium phosphate or trisodium phosphate. You can buy TSP as a cleaner or mix Potassium carbonate or sodium carbonate with weak phosphoric acid to form these salts. I only used 100 ml so I made stock solutions of stronger salt and did serial dilutions. For example, for 0.1 g of Diammonium phosphate, I added 10 grams of salt to 100 ml and added 1 ml to a 100 ml solution of water.

Acetate and Potassium Tartrate salts are substituted for different tests. Potassium tartrate can be made by mixing potassium bitartrate 37 g in 250 ml of near boiling water and adding 15 g of hot calcium carbonate. This will make approx. 22 g of potassium tartrate. Not many bacteria can take 2-5 g of tartrate salt and convert it into lactic acid, acetic acid, and CO2.

For each acid salt Bromothymol blue broth, I added at least 2
g of each acid salt and then added 3 grams of sea salt. (I did have sodium chloride as pure salt.)

Here the label with 11% glucose is the test and the right
flask with the corrosive and poison symbol is the control with a few drops of 1 M ammonium hydroxide. This picture is the final result and at the beginning, the solution was green at a ph of 6 and turned blue (weak base) after 13 hours. I wrote the labels in an old lab book so I could remember which one was the control and which one was the test result.

Not all lactobacillus bacteria can grow in acetate or citrate Bromothymol blue broth. Yogurt bacteria were mixed with sodium citrate Bromothymol blue broth remained yellow-green. No color change so these bacteria cannot convert citrate into acetoin and CO2 as a final product.

Below is the result
for potassium tartrate Bromothymol blue broth. The color was light blue initially and a deeper blue color did not occur. Indicating that this unknown bacteria cannot use tartrate salts a carbon source.

Here I am preparing 5 g of tartaric acid by heating 13 g of potassium bitartrate at 70 degrees C with 10 g Calcium carbonate. This forms CO2 (gas) and Potassium tartrate and Calcium tartrate and water. Then the deposit of calcium tartrate is mixed with 0.65 moles per L of Sulfuric acid equiv. Here is a picture of prep of 0.65 m/L of sulfuric acid from 13 moles per L sulfuric acid. Calcium chloride is added to the solution of boiling water with calcium carbonate and potassium bitartrate.

The picture above proves that the tartrate Bromothymol blue
the broth is positive and the unknown bacteria can produce CO2 and lactic acid plus acetic acid and turn blue.

Acid fast stain protocol and explanation:

Here you can test a negative for several bacteria. It is very unlikely that organisms that cause TB would be found in packaged soil and secondly, TB has been nearly wiped out these days. Finally, TB requires a high concentration of fat to grow if TB was present the gram stain bacteria would appear faint hollow color and that was clearly not the case.

Danger: Phenol found in the acid fast procedure is a chronic poison and has no antidote if absorbed through the skin. It is highly corrosive and can cause burns and possibly poisoning if exposed to skin. Even though the bottles allow drop by drop to be released always wear gloves and the proper PPE while and after handling. Also be careful about an alcohol flame while using it if you do. In drop form, it would be difficult to get massively exposed to the required phenol for injury or death. Don’t be reckless with this chemical.

Protocol:

Make a smear by using an inoculating loop to take a large sample from the agar and place it on a slide with a magic marker circle and place the inoculating loop with a bit of water. Heat fix the slide and take a hot plate and heat up 200 ml of water. Wait until the water is steaming then add the slide directly under the slide. Add a few drops of carbonyl fushol to the slide. Let the dye soak for 3-5 minutes. Next, wash off excess carbonyl fushol with distilled water. Then add a few drops of acid alcohol to the slide to discolor the smear. Finally, add a few drops of 1% methylene blue in ethanol. Wash off with excess water and let the put on a cover slip and place the slide to dry for 10 minutes. As with the endospore stain and the gram stain view at 1000x with oil immersion. Below I am showing a ring was the slide would go.

The final thought as Discussions:

Rod was gram positive for many of the bacteria the bacillus and the Clostridium require protein (amino acids). Methyl red test was positive indicating that this bacteria can ferment glucose have water as a control, this because this bacteria may be airborne and with Nutrient agar, it may contaminate the control too. The negative loafer methylene blue stain proves that the bacteria in question is not related to corynebacteria. Many of the bacillus bacteria also produce biofilms in aerobic conditions or when nutrients have been depleted.

Clostridium bacteria can produce biofilms and
others. It is not Clostridium since the odor would smell like sulfur (complex sulfur molecules convert H202 into H20 without catalase and this bacteria cannot grow with glucose or other sugars. Some species of Bacillus can tolerate 7% salt and it is possible with genetic variance can tolerate up to 10% salt. Bacillus Lentimorbus cannot produce catalase. B.L cannot ferment lactose but many other sugars it can ferment like glucose. However, sucrose and other complex sugar B.L cannot ferment. Also, the soil had no known outbreaks of any types of diseases and the soil is screened before they are allowed to sell it to people. [citation needed] this means that the soil doesn’t have any pathogenic bacteria. Also, Triple Iron agar did not turn black indicating the presence of Hydrogen sulfide in 1 day. The bacteria in present cannot use sulfur to produce from other chemicals made from sulfur.

Test with Simon citrate agar and acetate Bromothymol blue agar is needed as well. Evidence that similar bacteria to BL can use both citrate and possibly acetic acid as a fuel source. However, Bacillus sphaericus is different than Bacillus L this means it may or may not be able to use acetic acid as a fuel! No endospore was found after 26 hrs so it may be L. reuteri PTA5289. [34] This is because it fits no xylose use, sucrose positive only after 3 days, glucose (positive) test, and fructose and so on. It is also catalase negative. It can ferment many sugars and produce acids from glucose with oxygen. [36]The L.r can ferment glycerol too. Methyl red test requires oxygen with glucose to produce acid from glucose. It could be positive for methyl red. Urease test for different strains of Lactobacillus reuteri may be able to use it or maybe it cannot. Some species of this L.r can have urease while other do not. Urease test was negative.

Nitrate and Nitrite test both were negative and evidence that lactobacillus r may or may not be able to convert ammonia into nitrate or nitrite.

For the use of
For the use of citrate or acetate in the cytoplasm of the bacteria, it can use both. Some species can ferment acetic acid but not citric acid. It doesn’t match some non-pathogenic Listeria or Arthrobacter because listeria doesn’t match characters for sugars and is catalase positive while archeobacteria form odd shaped that was not seen under the microscope. While archaebacteria are funny shaped. Other data said it cannot be any other bacteria like Actinomyces, etc. Certain species of L.r can tolerate up to 20% salt. It may be Bacilius.L if unknown bacteria can ferment maltose but L.r strain after 72 may ferment maltose. The control plates with Nutrient agar and phenol red (orange control) turned pink and this may say that the bacteria can be airborne and jump from one place to another!! This points to Bacillus L found naturally or inoculated in the soil. However, it may be just contaminated the air with that bacteria that landed on the plate (control). L.r reacts weird with sugars AND tolerates a higher concentration of salt 10-20%. B.L does not fit the salt tolerance or catalase negative result.

Ammonia is produced when ammonium phosphate and ammonium citrate/acetate is consumed. Citrate or acetate is consumed producing pyruvate and CO2. The pyruvate is converted into acetoin and CO2. The Na ions from the sodium citrate or sodium acetate produce sodium carbonate plus ammonia so this raises the ph. Sodium carbonate with ammonium citrate/ acetate forms ammonium carbonate which converts slowly into ammonia and ammonia bicarbonate.

Both citrate and
acetate Bromothymol blue was positive in 24 + hours. However, L.r depending on the strain may use citrate or acetate ions as a carbon source. Some bacteria can convert tartaric acid and salts into lactic acid but most lactobacillus bacteria cannot convert tartaric acid into CO2 and lactic acid. Some LAC can convert tartaric acid into other weaker acids and CO2. It is not known if Potassium bitartrate can be used and converted into acids and CO2. It may or may not. Final evidence that Potassium bitartrate cannot be used as a fuel source. Since no color change had occurred converting potassium bitartrate another into lactic acid and produce CO2. Some other evidence occurred that said that lactobacillus bacteria can use Diammonium phosphate as a Nitrogen and phosphorous source I am growing yogurt bacteria in a citrate Bromothymol blue broth. Evidence still exists that lactobacillus bacteria can use Diammonium phosphate as a fuel. It cannot be lactobacillus r due to these reasons. I don’t know what type of bacteria it is. However other evidence proves that is L.r due to the fact that only a few species of Lactobacillus can use Diammonium phosphate and L.r can. Potassium tartrate made from potassium bitartrate and calcium carbonate in 350 ml hot water solution. The tartaric acid made from potassium and calcium tartrate reacting with 0.65 moles per L Sulfuric acid and draining excess fluid to get the crystals reacted with sodium bicarbonate to neutralize excessive H2SO4. A solution of 2 g of sodium tartrate with 100 ml of content from Simmon citrate agar recipe with excess salt was made. In 13 hours the solution turned dark blue!! Other data proves that most likely L.r can use Diammonium phosphate and similar chemicals as a Nitrogen and Phosphorous from trisodium phosphate. The reason why these test occurred less than 24 hours was due to the carbon source being quite high. I was afraid of contamination from the air even though a Penicillium (Harmless version) was growing. If I would use 0.2 g Carbon source instead of 2-5 g Carbon source it may take 3-4 days and contamination may be the result.

Final Conclusion:

Tests for Bacillus L are more accurate than Lactobacillus R. Because I don’t know how L.r responds to urease, Nitrate, nitride, Methyl red or Acetate Bromothymol blue and Simmon citrate broth. The L.r could use these. It can tolerate high salt and has no endospores. The sugars do ferment or don’t depend on the sugars used. Finally, catalase test is negative. Thus it could be either a Lactobacillus like L.r. At first for the soil experiment, 1 took several tries.


L.r fits more due to several factors. See discussions. They respond to glucose but not fructose (slow response), no response to xylose, etc and is catalase negative. This with the other possibility of testing positive for MR, testing negative for Urease and other types of test may indicate that it is L.r. For L.r the O/F test was positive. Since acids are produced when glucose is burned then the methyl red test would be positive. Lactobacillus r test positive for methyl red, no response from xylose, slow response from fructose and sucrose as well as lactose without air. The only one which grew in 3 days was glucose and it did not grow well since it lacked protein. For Lactobacillus r it was also catalase negative. Inconclusive or not sure results were the Nitrate-Nitrite test, Citrate Bromothymol blue test, Acetate Bromothymol blue test, TSI results and others. Since some species can use acetate and citrate may be the bacteria that is L.r can use both acids as a carbon source. Tartaric salts (Potassium tartrate) Bromothymol blue broth turned light blue and acts as a negative. Bitartrate was negative while sodium tartrate Bromothymol blue broth turned light blue to dark blue in 13 hours. Also, I used extra salt and tartrate to enhance the broth and to make it take less time.

Another important factor is the salt tolerance it either could be Bacillus L or Lactobacillus r but Bacillus L hates high salt concentration while Lactobacillus bacteria in general loves salt. So perhaps Lactobacillus r loves salt concentrations as high as 20%.

Safety Note:

If you are going to repeat what I have done then adhere to the warnings that I gave you. Also note that results may vary depending on what bacteria is found to the packaged soil. Again, Never take from samples that include feces like duck pond water, or any area of your body that can expose you to potentially pathogenic bacteria. This includes samples from lake water and compost soil. This comes from general precautions that I learned in Biotechnology advanced diploma and waste and waste management programs.

Here copper sulfate 10 grams per 400 ml is used to kill the bacteria. Articles on Listeria said that copper ions can significantly kill Listeria. If it can kill listeria than non-pathogenic bacteria like Lactobacillus r can be killed this way. Non- Sporing bacteria are killed by boiling the copper sulfate solution at 100 degrees C for 20 minutes. Heat and moisture deactivate the spores plus it is more likely to inhibit the bacteria through copper sulfate. Copper sulfate in hot boiling solution will kill and convert bacteria (friendly) into spores that could be killed by steam. None of the bacteria produce spores to heat and copper sulfate will produce death zone for the bacteria.

Placing a tight lid on a beaker with a hot solution will prevent the steam from leaving thus exposing the dry steam to reheat that may kill the bacteria spores. Normally it is not recommended to add bleach to ammonia to kill bacteria but Nitrogen trichloride can be avoided at a high pH (Like adding Lye) to the solution.

References:

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