EM effects of preventing Staphylococcus aureus from propagation
Minako Matsumori
October 1995

I. Objective
Mastitis is the increase in the number of debilitating and disease bearing cells within udder milk, affecting the qualities of the milk. The financial impact of each case of mastitis has been calculated at some 50,000 yen. The primary pathogenic bacteria in mastitis is Staphylococcus aureus, which is involved in 60% of all cases, while the streptococcus accounts for about 30% of the total. The remaining 10% appears to be caused by corynebacterium, coliform bacteria and fungus.

A primary characteristic of the main culprit, Staphylococcus aureus, is the appearance of growths in the mammary gland, which, once infected, is difficult to treat and quickly becomes a chronic problem. Therefore, this study reviewed preventing or suppressing the growth of Staphylococcus aureus through the use of effective microorganisms (EM).

II. Materials and Methods Materials
Milk: Taken on 16 October 1995 from cows infected with mastitis
Kyusei EM1: Manufactured by the Products Department of the International Nature Farming Research Center. Main microorganisms include : lactic acid bacteria, photosynthetic bacteria, yeast fungi, filamentous fungi, and actinomycetes. Manufactured on September 6, 1995.
Molasses: Beet molasses
Mannitol Salt Agar with Egg Yolk: (Meat extract, pepton, sodium chloride, mannitol, phenol red, agar, and egg yolk).
Mannitol Salt Liquid Medium: (Meat extract, pepton, and mannitol.)
PS Latex "Eiken": Test for coagulase production, and will be used in identifying Staphylococcus aureus. Coaglase is a special enzyme produced by Staphylococcus aureus. PS Latex is floating in buffer and sensitized with human plasma.
Rabbit Plasma "Eiken": Test for coagulase production, and will be used in identifying Staphylococcus aureus. It is consisted from frozen 1ml of rabbit plasma.

Methods
1. The isolation and identification of Staphylococcus aureus and its cultivation The milk is diluted in sterilized saline solution in seven steps from 10-1 to 10-7. The diluted liquid is inoculated into the mannitol salt agar with egg yolk, then cultivated for approximately 43 hours at 37oC. Colonies which grow by incubation are analyzed and identified Staphylococcus aureus using PS latex. The identified Staphylococcus aureus is cultivated for approximately 24 hours at 37oC in a mannitol salt liquid medium.

Directions for the mannitol salt agar with egg yolk
Dissolve the mannitol salt agar in distilled water, place into autoclave to undergo high pressure sterilization. Add 10 ml of a septically removed egg yolk. (Egg yolk is added because the culture will become cloudy as a result of the Staphylococcus aureus reacting with the yolk.) The completed solution is poured into sterilized Petri-dishes.

Directions for making the mannitol salt liquid medium
Dissolve the mannitol, pepton, and meat extract into distilled water, place into autoclave to undergo high pressure sterilization.

Usage of rabbit plasma "Eiken"
Add 7 ml of sterile physiological saline solution to dried plasma, and dissolve. Divide 0.5 ml of this plasma solution into a test tube. Concurrently, the amount of one platinum loop of cultured analyte in the agar medium for one night are portioned into the test tubes mentioned above, then mixed with the plasma solution. It is cultured for three hours at 37oC, and examined. It is deemed positive if the plasma solution is coagulated.

Usage of +PS Latex
The analyte is smeared on a slide and mixed with physiological saline solution. Then the PS latex solution is dropped on it, and it is agitated gently for one minute. If cohesion takes place within one minute, it is considered positive, while if cohesion does not take place, it is negative.

2. Cultivation of EM and the growth inhibition test of Staphylococcus aureus by EM
This culture is diluted with a sterilized physiological saline solution in eight steps from 10-1 to 10-8. These diluted solutions are inoculated onto the mannitol salt egg yolk agar and are then cultured at 37oC for approximately 20 hours. The colonies formed on the culture are counted, and then the number of bacteria of the culture that is diluted by 10-4 is calculated. This culture, diluted by 10-4, is called A. 1.8 ml of EM is stirred in with 9 ml of sterilized molasses diluted 50 times (1% pepton, 0.1% meat extract are added) and cultured for one day at 37oC. This liquid culture is called B. The mixture of one ml of A and one ml of B, and eight ml of the 50 times dilution of sterilized molasses (1% pepton, 0.1% meat extract are added) is called No. 1. In addition, a mixture of one ml of A and nine ml of the 50 times dilution of sterilized molasses (1% pepton, 0.1% meat extract are added), is called No. 2. No. 1 and No. 2 are both cultivated for a day at 37oC. The cultured solution is diluted with a sterilized saline solution in eight steps from 10-1 to 10-8. These diluted solutions are cultured at 37oC for two days, on a mannitol salt agar with egg yolk. Afterwards, the number of colonies are counted, and the number of bacteria found in No. 1 and No. 2 are calculated.

III. Results
1. The separation and identification of Staphylococcus aureus and its cultivation
The milk is diluted with a sterilized saline solution in seven steps from 10-1 to 10-7. These diluted solutions are inoculated to mannitol salt agar with egg yolk and are cultured for approximately 43 hours at 37oC. As a result, seven colonies appeared on the inoculated plate containing the milk that was diluted at 10-1. Using the PS latex and rabbit plasma, these colonies were identified as both being positive. Also, these colonies were yellowish, and the area around them showed the egg yolk reaction. Due to the decomposition of the mannitol and the production of acid, thus the agar culture changed in color from red to yellow. Therefore, it was conclusive that the bacteria present was Staphylococcus aureus.

2. Cultivation of EM and the growth control of Staphylococcus aureus using EM.
The Staphylococcus aureus is cultured in a mannitol salt liquid medium, after which the presence of Staphylococcus aureus is carefully counted. Within this, the number of living bacteria counted were 5.8x107 per ml. When this mannitol salt liquid medium is diluted into 10-4 saline solution, the number of bacteria as 5.8x103 per ml. Sample No 1 and No 2 are cultured for one day. In counting the density of the presence of Staphylococcus aureus, Staphylococcus aureus were not found in No. 1 (with the presence of EM), but were found in No. 2 at 8.2x107 (Figure 1).

IV. Conclusion
This experiment allowed us to confirm that EM successfully prevent growth of Staphylococcus aureus, and can sterilize them. However, the conditions in the average barn are different from those of the experiment (temperature, concentration of molasses, and the amount of EM, etc.). Therefore eradication of Staphylococcus aureus from within a barn is not a given.

It is necessary for more data to be collected, and for further experimentation. Staphylococcus aureus also grows outside the mammary gland under such conditions as a milking machine in poor condition, and excessive milking, which results in cuts and sores developing on the nipple, as well as milk being spilled on the floor. Infections occur usually during milking, and is spread through hands, cloths, and milking units that come in contact with the infected cow. Steps effective in preventing the spread of infective mastitis are disinfecting the nipple, treatment, and destroying the animal.

Figure 1 The growth inhibition of Staphylococcus aureus when EM are used.