(1) Preparation and homogenization of the sample for Total Plate Count test and Escherichia coli
Principle:
Liberation of bacterial cells that may be protected by food particles and to reactivate the cells viability of bacteria that may be reduced due to unfavorable conditions in the food. Preparation and homogenization of the sample intended that bacteria well distributed in the food sample set.
Equipment:
(a) appropriate homogenization equipment (blender) with a rotation speed of 10000 rpm to 12000 rpm;
(b) Autoclaves
(c) electric heater;
(d) the balance sheet capacity of 2000 g calibrated to the nearest 0.1 g;
(e) measuring flask 1000 ml, 500 ml, and 50 ml calibrated;
(f) The glass trophy sterile;
(g) sterile Erlenmeyer;
(h) Bottle sterile diluent;
(i) Pipette volumetric sterile; 10.0 mL and 1.0 mL calibrated, fitted with a bulb and pipettos
(j) the reaction tube; and
(k) spoon, scissors, and a sterile spatula.
Diluting solution:
Butterfield’s Phosphate-Buffered Water dilution (BPB);
(a) KH2PO4 34 g
(b) Distilled water 500 ml
Adjust the pH with NaOH to pH 7.2, align the volume to 1000 ml with distilled water. Sterilization at 121 ° C for 15 minutes. Store in refrigerator to make a diluting solution of 1.25 mL of stock solution was diluted with distilled water to a volume of 1000 ml, then put in a bottle of 450 ml diluent and into as many as 9 ml test tube and sterilized at 121 ° C for 15 minutes.
Homogenization example:
(a) Weigh 50 g sample aseptically into the diluent bottle that had contained 450 ml of sterile diluent in order to obtain a dilution of 1:10, and
(b) Shake the mixture several times so homogeneous.
(2) The number of total plate (35oC, 48 hours)
Principle
Mesophyll aerobic bacterial growth after the sample was incubated in the appropriate seeding for 48 hours at a temperature (35 ± 1) ° C.
Equipment:
(a) Incubator (35+ 1) ° C calibrated;
(b) Oven / dry sterilizer calibrated.
(c) autoclave;
(d) circulating water bath (45+) oC
(e) Equipment colony counter;
(f) Tally register;
(g) 160 mL diluent bottle, made of borosilicate glass, with a rubber stopper or a screw cap plastic;
(h) measuring 1 mL sterile pipettes with 0.1 mL scale equipped bulb or pipettor; and
(i) Petri dish glass / plastic sterile (minimum size 15 mm x 90 mm).
Hatcheries and thinners:
Plate count agar (PCA)
(1) Tryptone 5 g
(2) Yeast extract 2.5 g
(3) Glucose 1 g
(4) To 15 g
(5) 1000 ml of distilled water
Dissolve the above materials into a 1000 ml with distilled water and adjust the pH to 7.0. Put it in a bottle. Sterilized using an autoclave at 121 ° C for 15 minutes.
Ways of working:
(a) Make a dilution rate as needed such as in Figure 1 using a diluent solution Butterfield’s Phosphate-Buffered Water dilution (BPB);
Figure 1. The level of dilution using a diluting solution dilution Butterfield’sPhosphate-Buffered Water (BPB)
(b) Pipette each 1 ml of dilution rate (F) 10-1 to 10-4 in a sterile petri dish in duplicate,
(c) Pour 12 ml to 15 ml of PCA medium that is still liquid with a temperature (45 ± 1) ° C into each Petri dish,
(d) Shake the Petri dish with caution (rotary and rocking forwards, backwards, to the right and to the left) so that the sample and seeding evenly mixed and solidified;
(e) Work on blank examination by mixing dilution water for each sample examined,
(f) Allow the mixture solidifies in a petri dish,
(g) Put all the petri dish upside down into the cabinet incubator at 35 ° C for (48 ± 2) hours, and
(h) Record growth colony (n) in each petri dish containing 25 to 250 colonies colonies after 48 hours.
How to calculate:
(a) Select a petri dish from a dilution that shows the number of colonies between 25 colonies of up to 250 colonies per petri dish. Calculate all the colonies in Petri dishes using a colony counter. Calculate the average number of colonies and multiply by the dilution factor. Express the result as the number of bacteria per gram;
(b) if one of two Petri dishes are a smaller number of colonies from 25 colonies to or greater than 250 colonies, count the number of colonies are located between 25 colonies of up to 250 colonies and multiply by the dilution factor. Express the result as the number of bacteria per gram;
(3) Escherichia coli
Principle:
Growth of Escherichia coli is characterized by the formation of gas in the Durham tube, followed by biochemical tests and further referenced in Table APM (Figures Most Likely).
Equipment:
(a) Incubators (35 ± 1) ° C;
(b) a water bath with a closed circulation system, (45.5 ± 0.2) ° C;
(c) Rack for test tubes;
(d) Pipette measuring 10 mL and 1 mL of 0.1 mL scale;
(e) the diluent bottle made of borosilicate glass, with screw cap / plastic;
(f) Test tube and tube Durham;
(g) glass petri dish the size of 15 mm x 100 mm or plastic size 15 mm x 90 mm, sterile; and
(h) Needle ose (inoculation), with an inner diameter of approximately 3 mm.
Seeding diluent and reagents:
(a) Lauryl sulfate tryptose (LST) broth / Lauryl tryptose (LT) broth;
(b) Brilliant green lactose bile (BGLB) broth 2%;
(c) Escherichia coli (EC) broth;
(d) Levine’s eosin methylene blue (L-EMB) in order;
(e) Plate count agar (PCA);
(f) Gram stain;
(g) Tryptone (tryptophane) broth;
(h) Reagent Kovacs’;
(i) Methyl red – voges Proskauer (MR – VP) broth;
(j) voges Proskauer reagents;
(k) A solution of methyl red;
(l) Koser’s citrate broth;
(m) Peptone diluents 0.1%;
(n) Reagent indole;
(o) 40% potassium hydroxide solution;
(p) Buffer fields phosphate buffered dilution water;
(q) alpha naphtol solution of 5%; and
(r) Crystal creatine.
Ways of working:
APM – Test predictions at Escherichia coli:
(a) Make the preparation and homogenization of the sample as in 10.1,
(b) Inoculate each 1 ml of each dilution rate (a solution of 10-1, 10-2 and 10-3) into three tubes Laurryl sulfate tryptose (LST) broth in which there are inverted Durham tube. Hold the dropper so that the lower end of the pipette attached to the tube. Let the pipette contents flowing 2 seconds to 3 seconds. Pipette not blown to remove its contents,
(c) Insert the tubes into the incubator at 35 ° C for
(48 ± 2) hours,
(d) Observe these tubes on the ke- hour (24 ± 2). If there is a tube that had been containing gas, turbid then the tube is declared “positive”,
(e) tubes containing gas that has not been declared “negative”, continue incubation for 24 hours,
(f) Note the gas formation after incubation (48 ± 2) hours, and the tube declared “positive”, and
(g) Conduct a test tube affirmation of all that is positive for the test prediction.
APM – Test affirmation for Escherichia coli:
(a) Transfer of one eye of each tube Ose LST positive EC broth tube into discrete,
(b) EC Inkubasikan tubes into the circulating water bath, for (24 ± 2) hours at temperature (45.5 ± 0.2) ° C, the tube that has formed gas expressed “positive”,
(c) If negative, Inkubasikan and check back in an hour ke- (48 ± 2). If the gas has been formed, the tube is declared “positive”, and
(d) Perform complete testing of all test tubes positive for confirmation.
Full test for Escherichia coli:
(a) Shake tubes positive EC broth carefully,
(b) Take Ose colony of one eye, then streaking / embedded in the cup so that the L-EMB, such that the resulting colonies were separated by a minimum distance of 0.5 cm,
(c) L-EMB Inkubasikan cup for 18 hours up to 24 hours at a temperature (35 ± 1) ° C,
(d) check the vials for the presence of green colonies with or without metal lightning,
(e) From each cup L-EMB, move up to 5 colonies suspicious on the tube for oblique PCA,
(f) Inkubasikan tubes so that the slant for 18 hours up to 24 hours at a temperature of 35 ° C and used for the next test,
(g) Prepare a Gram stain of every culture. E coli is a rod-shaped gram-negative and no berspora to be tested using IMViC reactions like this and should be inoculated back into LST broth tubes to confirm the existence of gas production.