TITLE: Granulate formulation protocol
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Bioformulation</span><span> is used in agriculture for various reasons like soil fertility, plant growth promotion, and suppression of phytopathogens. The bacterial inoculants are applied as a formulated product like powder, spray, or pellet. This is a green strategy that is being developed as a less harmful method to protect crops other than pesticides. In the paper published by </span><span style = "font-style:italic;">Alvarez et al, 2016</span><span>, they developed a talc-based powder formulation based on </span><span style = "font-style:italic;">Bacillus</span><span> B25 spores and evaluated some of its characteristics, like shelf life and efficacy against the pathogenic fungus</span><span style = "font-style:italic;"> Fusarium verticillioides</span><span style = "font-style:italic;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Colony Forming Units (CFU) </span><span>is a unit that is used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. It also depends on their ability to multiply under controlled conditions. In the paper published by </span><span style = "font-style:italic;">El-Hassan and Gowen, 2006</span><span>, they analyzed various formulations of </span><span style = "font-style:italic;">Bacillus subtilis </span><span>by counting the CFU of B. subtilis present in every formulated product.   </span></div><div class = "text-block"><span style = "font-weight:bold;">Fatty acid analysis</span><span> is an important means to characterize fats and oils and to determine the total fat content. The resulst can be used to identify the</span><span style = "font-style:italic;"> Bacillus mycoides</span><span> strain in the soil and determine its survival with the granulate formulation.  </span></div></div>

[STEPS]
?. [Bacterium Inoculum]
Grow a single colony of bacteria in an assay tube with   of Luria Broth (LB) medium
5 mL
?. [Bacterium Inoculum]
Incubate it in an orbital shaker at 200 rev.min-1 at   for  .
30 °C
?. [Bacterium Inoculum]
After bacterial growth, take a   Erlenmeyer flask and add   of LB medium in it.
500 mL
100 mL
?. [Bacterium Inoculum]
Add  of the culture () in the flask and incubate at  and 200 rev.min-1 for  , until an optical density of close to 1 is obtained.
1 mL
30 °C
?. [Spore Production]
Add  of Difco Sporulation Medium (DSM; 5gl-1 peptone, 3gl-1 yeast extract, 1gl-1 KCl and 0.12 gl-1 MgSO47H2O) in a  Erlenmeyer flask.
100 mL
500 mL
?. [Spore Production]
Sterilize this DSM medium at  and 1.5 psi for .
121 °C
?. [Powder Formulation]
Pack it in plastic bags and store it at room temperature.
?. [Spore Production]
Add  each of:  Ca(NO3)2,  MnCl2­.4H2O and  FeSO4
1 mL
?. [Powder Formulation]
Mix talc (which will be the carrier) with, carboxy-methyl-cellulose (CMC; 1% w/w), CaCO3 (15% w/w) and glucose (0.25% w/w) in powder form.
?. [Spore Production]
Inoculate with 1X106 c.f.u ml-1 of the bacterial strain. Keep the culture conditions at  and 200 rev.min-1 for .
30 °C
?. [Powder Formulation]
Mix the same material with the bacterial spore suspension and dry at  for .
55 °C
?. [Powder Formulation]
Autoclave the mixture at  and 15 psi for .
121 °C
?. [Powder Formulation]
Using sterile porcelain mortar and pestle pulverize the formulation.
?. [CFU determination]
Colony Forming Units (CFU) can be determined by estimating the OD of spore suspension using a tube-reading spectrophotometer adjusted at 1.978 [corresponding to 8.5 · 1010 CFU/ml] at 600nm absorbance wavelength
?. [CFU determination]
The formulation will be placed on sterile aluminum foil in pans and air-dried for  with occasional stirring in a laminar airflow cabinet.
?. [CFU determination]
Dried formulations (35% moisture content) of B. mycoides will be passed through a 250μm mesh sieve to attain the desired particle size.
?. [CFU determination]
Pack in sterilized polypropylene bags, seal and store at prior to use.
550 Room temperature
?. [CFU determination]
Count CFUs to estimate the number of viable propagules of B. mycoides using the standard dilution platin method.
?. [STD dilution method]
Take three   aliquots of the dried powder and place in   sterile PBST solution (this will include PBS +  Tween 20). Stir magnetically at high speed for . Now dilute this suspension with approximately and take   of this suspension and plate on Nutrient Agar (NA) media.
1 g
99 mL
0.2 mL
?. [Fatty acid analysis]
By performing saponification, methylation, and then extracting we can obtain fatty acid methylesters from wet biomass.
?. [Fatty acid analysis]
Next, separate the fatty acid methylester mixtures by using a microbial identification system. Peaks can be automatically integrated, and the Microbial ID will calculate the fatty acid names and percentages.