3.

Take 1 ml of liquid soil suspension from 3rd, 4th, 5th and 6th test-tubes and pour aseptically onto the plates/Petri dishes containing the medium. Soil suspensions with different dilutions will ensure the growth of micro organisms depending on their population and, thus, could be taken for further purification and multiplication from the appropriate tube. 4. Spread the aliquots (soil suspension) of different dilutions on the plates. 5. Invert the plates, and incubate them at 28-30 C for 3-4 days. 6. Microbial colonies with a transparent zone will develop. 7. Take a single colony and streak it on phosphate-containing medium plates, and incubate at 28 C for 3-4 days. 8. Take the single colony from the above plates having clear zones of solubilization, and maintain them on slants, as this provides a larger surface area for growth. After proper identification, PSMs from the colony so developed can be taken and multiplied in order to obtain a pure culture through the inoculation and fermentation process. Identification Phosphate-solubilizing micro-organisms can be either bacteria or fungi. The following species are more effective: bacteria: Bacillus m egaterium , Bacillus polym yxa , Bacillus pulvijaciens , Pseudom onas striata , Pseudom onas rathonis ; fungi: Aspergillus niger , Aspergillus aw ori am , Penicillium digitatum . The bacterial species are aerobic and heterotrophic. Cell size is 1.1-2.2 m, and the cells are rod-shaped. The transparent zone around microbial colonies indicates the extent of phosphate solubilization and the effectiveness of the microbes. INOCULATION OF CULTURE MEDIUM This is a method by which micro-organisms are transferred from any source (purified culture) to the medium for their multiplication in a laboratory. To avoid any contamination, inoculation is done in a laminar air-flow chamber aseptically as follows: 1. Select a pure culture of the micro-organism to be multiplied. 2. Take a specific culture medium in a sterilized flask/tube

(already prepared and kept ready). 3. Hold the pure culture tube between thumb and forefinger in such a way that the cotton plug of the tube is towards the body of the worker.

Chapter 7 - Biofertilizer assay and production

4. Remove the cotton plug from the tube/flask containing the culture, take the pure culture from the tube using a sterilized platinum needle and transfer it to the medium in the flask/tube. Plug the inoculated tube/flask, keep it in the incubator at 28 2 C for 3-5 days to allow the microbes to grow. When a large volume of culture medium is required for mass production of biofertilizer, multiplication is done in large flasks (2-5 litres) in a rotary shaker or in fermenters. FERMENTATION The sterilized medium is inoculated with a pure culture of the desired microorganism as described above. This is called a broth, and it will be used for the further multiplication of micro-organisms and their commercial production. The broth is put in fermenters of the requisite size depending on the amount of broth required to be used in the production of biofertilizer. The broth is aerated continuously by forcing sterile air through a porous stainless steel tube at 1012 litres of air per hour. The aeration requirement of microbes varies from species to species. When the number of microbes reaches 10 8-10 9 cells/ml, it is considered ready for mixing with the carrier. A large amount of inoculum reduces the time required to reach maximum viable numbers and, therefore, reduces the risk and effects of contamination. Inoculum levels generally vary from 0.1 to 1 percent 6 (sometimes 5 percent). Generally, the process should provide 10 7 10 bacteria per millilitre of culture medium at the beginning of fermentation. The fermentation time also varies from 6 to 18 hours depending on species, growth conditions and the initial amount of microbes taken from the mother culture. Apart from large-capacity steel fermenters, broth can also be fermented/ incubated in large conical flasks (2-5 litres), which are mounted and shaken continuously on a rotary shaker for 12-36 hours in order to achieve the same population

of microbes as in steel fermenters. MEASUREMENT OF MICROBIAL GROWTH Microbial cells are usually counted using a Petroff-Hauser bacterial counter (after placing it under a phase-contrast or dark-field microscope). This counter consists of a thick slide containing one block, which is divided into ten sub-blocks with grooves. The depth of each groove is 0.2 mm. Each sub-block has the capacity to retain a definite number of micro-organisms. Thus, the number of microbes in a block can be determined. It is observed that 1 ml of liquid pure culture of bacterium medium may contain about 5 000 million bacteria. The growth of any micro-organism (e.g. bacteria) can also be measured by counting the number of colonies developed on Petri dishes. Colony counting is a commonly practised method in microbiological laboratories. There are many techniques for counting viable cells that are able to divide and form offspring. The usual method is to determine the number of cells in a given sample capable of forming colonies on a suitable agar media. The method involves: serial dilution;

 spreading of diluted suspension on plates, and counting of colony-forming units on plates. Serial dilution A laminar air-flow chamber is used in order to achieve serial dilution of the broth culture of the strain or biofertilizer sample suspension. For plate counts, the countable range is generally 30-300 cells/ml. To achieve this concentration, the procedure is: 1. Set out 8 tubes, each containing 9 ml of sterile water. 2. Dilute 1 ml of broth culture or biofertilizer sample suspension -1 (1 g of sample in 9 ml of water) in steps (10 to 10 -8 ) with a sterilized 1-ml serological pipette equipped with a rubber bulb of 1 ml capacity. 3. Suck up broth culture or sample suspension from tube 1 to the 1-ml mark. 4. Immediately expel the broth culture or sample suspension back into the tube with sufficient vigour to effect a thorough mixing. 5. Repeat sucking up and expelling 5 times, and then transfer 1 ml to tube 2. 6. Take a new sterile pipette, attach the rubber bulb, and remove 1 ml to tube 3. 7. Repeat this procedure using a fresh sterile pipette each time until the dilution series is completed. 8. After completion of serial dilution, put an aliquot of the diluted sample on a pour plate, spread plate or drop plate with specified nutrient agar media (as described below). After incubation at 28 2 C for 3-5 days, each viable cell will give rise to one distinct colony, which is counted and calculated for the number of viable cells per millilitre of suspension. The microbes so grown are taken for further multiplication and testing for their efficiency/quality. Spreading of diluted suspension on plates and colony counting Pour plate method In this method, sterilized molten medium is kept ready in conical flasks placed in a water-bath at a constant temperature of 48 C. The procedure is: 1. Remove dry sterilized Petri dishes from paper packs and stack them in a laminar air-flow chamber.

2. For each dilution, three Petri dishes will be required. Stack the plates in sets of three each and label them with the help of a glass marker pen. 3. Using a fresh sterile pipette, pour 1 ml of aliquot from the last dilution (say, 10 -8) into each of the three Petri dishes. 4. Using the same pipette, pour similar aliquots from the next two dilutions (say, from 10 -7 and 10 -6) into three Petri dishes for each dilution. 5. Pour about same volume of molten medium into each of the plates. 6. Immediately after pouring, move the plates gently in a whirling motion to mix the contents. 7. Allow the medium to solidify, and incubate at 28 2 C for 3-5 days. 8. Count the colonies after 3-5 days.