How to Test ACF at Home

"I am applying ACF on thousands of acres. I bought a microscope and tried to see the bugs, but I'm not sure what I'm looking at!"

We get this a lot. Although your microscope from Canadian Tire might not cut it, there are certain things you can do to evaluate a sample ACF-SR at home.

First, let's start with some information on the different types of microscopes:

With the range of microscopes noted above, only the compound microscope has some use in viewing bacteria, along with a practical price.

It is important to note that bacteria range in size from 1 to 10 microns (a millionth of a metre). Since the upper limit of magnification is 1000 times, there are many problems in attempting to identify bacteria with a compound microscope:

1. Bacteria are small (1 to 10 microns): In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.

2. Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.

3. Bacteria are transparent: Bacteria will show their colour only if they are present in a colony. Individual cells present on the slide are clear. Regular, bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.

4. Bacteria are difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt on the slide. Some bacteria also form clumps and can be difficult to see the individual cells.

Shapes of bacteria can be readily seen under a microscope at 400 times magnification.

Typical bacterial shapes are shown in the drawing below:

Of course, there are countless species of bacteria for each shape, making it impossible to identify species using a compound microscope. Even advanced techniques such as oil immersion, staining, heat fixing, etc., fail to identify species. Some actual examples are:

Above: Bacillus subtilis, mostly spore form, at 1000 times magnification.

Above: Bacillus thuringiensis, also 1000 times magnification. Not distinguishable from Bacillus subtilis.

Above: Bacillus licheniformis, unstained

The three photos show different species of Bacillus. Note that Bacillus are the largest bacteria contained in ACF products. As it is impossible to differentiate between even the largest Bacillus using a compound microscope, it is obviously impossible to differentiate between the smaller bacteria in ACF (Rhodopseuomonas and Nitrifying bacteria).

What Can Be Done to Evaluate ACF Bacteria?

Fortunately, there are some relatively inexpensive and well understood ways to evaluate ready-to-use or brewed AdvancedAg bacteria.

Total Plate Count

Particularly, with brewed ACF, farmers often ask how many bacteria are in the brewed product.

At the very start of the process, 10 pounds ACF Base are mixed into 1000 litres of water or so, and aeration begins. During the course of the next 48 to 72 hours, the various bacillus, photosynthetic and nitrifying bacteria in ACF are growing rapidly, reaching peak numbers in about 72 hours. At this time, the CFU in 1000 litres of brew will be a minimum of 500 million to a maximum of 10 billion per ml.

Fortunately, there are readily available tests kits that can verify this. A variety of commercially available test kits for total bacterial count are available. One such kit, the HPC Total Count

Sampler, is available from Amazon.com:

https://www.amazon.com/Millipore-Microbial-Count-Samplers-Red/dp/B00BB1FEDK

With kits such as this, a sample of brew is placed into the kit chamber, the kit is stored for 72 hours at 25 C, then it is read by looking for coloured colonies as shown below:

As ACF is highly concentrated, the sample has to be diluted before placing it into these test kits. For specifics of the dilution, provide your test kit information to your authorized distributor, and you will receive the specific dilution needed to read the ACF without falling into the TNTC (too numerous to count) range!

Nitrification

Nitrifying bacteria are very difficult to identify and they do not show up on the test kits such as the Millipore kit described above. The reason is that nitrifying bacteria, which convert NH3 into NO2 and then into NO3, do not consume organic carbon. Thus, they do not grow on standard media.

The best way to verify nitrifying bacteria is through verifying the function of nitrification. We provide interested customers with bottles of nitrification nutrient solution. The customers can take that solution and set up a test and control with ease. The steps are simple:

Set up a 10 litre aquarium style tank (one test, one control), both with aquarium style aerators and air stones. To both test and control tanks, add 100 ml of Nitrification Nutrient Solution. To each tank, add 9 litres of tap water. Then to the test tank, add one litre of final ACF brew while adding just water to the control tank.

You will need a standard aquarium style test kit to check ammonia, nitrite and nitrate. Check NH3, NO2, and NO3 at time zero (once you have just filled the test and control tanks). Repeat check at 3 days of aeration.

With the above steps, you will see that the nitrification proceeds rapidly in the test tank (with ACF product), while zero nitrification proceeds in the control tank.

Rhodopseudomonas Activity

Rhodopseudomonas is one of the most important bacteria in ACF. This bacterium works aerobically and aerobically. It fixes both nitrogen and carbon, meaning that it can convert atmospheric nitrogen into usable N for your crops (as well as converting CO2 into organic

carbon).

There is a very simple way to check for Rhodopseudomonas. Take a small sample of ACF-SR and place it into a clear bottle. Fill the bottle to the top, cap the bottle, and place it under direct light (a desk lamp will work well too). After about a week, the sample will turn a darker red, due to pigmentation formation by Rhodopseudomonas. This simple check tells you that Rhodopseudomonas is present, alive, and growing.

Improving Bioavailability of Organic Compounds

This is an extremely important function of ACF, as only simple and low molecular weight organics can be taken up by roots. Further, digestion of complex organics usually liberates NH3 that is otherwise bound up and not available to plants when in the form of protein. Use of special nutrient plates can demonstrate the ability of ACF to digest proteins, starches, and fats. The trick is that proteins, starches and fats are all large compounds, and that uptake of these compounds, or release of N or P bound up by these compounds, depends on digestion of the proteins, starches, and fats.

Fortunately, there is an easy test to show this! We can provide pre-made, sterile plates, plus some sterile tools to get the job done.

Supplies

• Sterile Loop

• Plate with Special Media

Sterile loops are individually wrapped, long plastic rods with a tiny loop at the end. They are dipped into brewed ACF, then using the loop end, the ACF is spread onto the special plates.

For best results, the loop is dragged across the surface of the plate, making the pattern shown here:

The plates we can supply include a protein that forms a white, opaque colour that you cannot see through. When the ACF bacteria break down the protein, a clear zone in shown around each colony. After the plate sits at room temperature for 3 to 5 days, a clear zone shows around each colony, proving that the protein was digested.

While best results will require some lab training, these tests are simple enough so that any interested person could see the basic properties themselves without going through an expensive lab.

For more on this, shoot us an email at support@advancedag.ca, or call our office at 403.752.0278.