Germination and Heat Resistance of Bacterial Endospores B. Materials and Tools Ingredients:    Bacterial spores Each group chose one suspension of bacterial spores, namely Bacillus subtilis, B.cereus, B.licheniformis, B.polymyxa, B.macerans atau B.megaterium. Media / Chemicals  Order plates (4 plates each per group) I. Nutrient Agar (NA) b. NA + 5 % NaCl c. NA + 300 ppm NaNO2 d. Na + 5% NaCl + 300 ppm NaNO2  Liquid agar (5 tubes each per group): a. Nutrient broth (NB) b. NB + acid tartrate (pH 5.0) c. NB + 3% NaCl  Diluent solution 9 ml (3 tubes per group) Spore dye solution:   malachite green solution Safranin solution Tool :     Scales Pipettes, petri dishes, tubes, test tubes incubator water bath temperature 60o C and 80o C C. How it works Heat Resistance of Bacterial Spores A total of 0.1 mL of the initial spore suspension was pipetted and inserted into each in tubes containing 3 different types of media. For every medium 5 tubes were used, and the medium used was: a. NB b. NB + acid tartrate (pH 5.0) c. NB + 3% NaCl A total of 4 tubes of each medium were heated in a water bath with a temperature of 80o C, and removed after a heating time of 5, 10, 15 and 20 minutes (one unheated tubes of each medium were used as controls). All tubes were incubated at 28-30o C for 2-3 days, and observed Bacterial growth is indicated by the appearance of turbidity. Growth rate expressed relatively with signs (- = not growing, +, ++ or +++ ). Data from each group is collected and tabulated so that the influence of species can be seen bacteria and heating medium conditions against spore heat. Activation of bacterial spores The initial suspension of bacterial spores was given a heat shock at 60o C for 10 minutes, then pipetted 1 mL to make a 10-1 dilution.10-2 and 10-3 using, 9 mL diluent solution Bacterial Endospore Germination From the heated suspension and the resulting dilution, each is pipetted as much as 0.1 mL and made fertilization on four types of medium. Every tie The respective dilutions are fertilized into a medium consisting of: a. THEN b. NA + 5% NaCl c. NA + 300 ppm NaNO2 d. Na + 5% NaCl + 300 ppm NaNO2 By using a curved glass rod (hockey stick) that has ignited and then cooled, the suspension was dripped cup is evenly distributed over the entire surface. All upside down at 28-30o C for 2-3 days. The number been dipped in alcohol, on the top so that the plates were incubated of colonies formed was counted and expressed group is collected and for each spore species of NaCl and Na-nitrite in number of colonies/mL suspension. Data from each arranged in a table so you can see the data obtained tested. Observed the effect of bacterial species, addition on spore germination and bacterial cell growth. Endospore Staining Examples of spore staining will be: a. Initial spore suspension b. Sample of Nutrient Broth showing positive test (after incubation) From the bacterial suspension, a thin film is made on a glass object, then left dries in air and is fixed over fire slowly. Drip with Spore dye is malachite green and left for 20 minutes in the open air (without heating), or for 5 minutes on a water bath. Every time dye becomes dry, add new dye. Then the dye is washed off carefully liver using water for 20-30 seconds, and dripping with safranin for 30 seconds. After rinsing with water, then dry with absorbent paper. The preparation is then examined under a microscope using an oil lens immersion with a magnification of about 1000 times. Observed differences between vegetative cells with spores, the shape and size of the spores, and the location of the spores in the cell. Note: do the Heat Resistance section of Bacterial Spores first, pipette the suspension not yet heated into broth media, then the initial suspension used for Activation of Bacterial Spores and Germination of Bacterial Endospores.