29 colonies were counted using standard counting rules and the results were
expressed as colonyhrm
2
.
3.3.4.2 Microbial Contamination of Jamu Gendong Bottle
Fifty mL of buffer phosphate was rinsed into the investigated bottle. Serial dilutions were prepared to achieve readable numbers of colonies
according to BAM 2010 in the range of 25-250 colonies. These dilutions generally were 10
-1
up to 10
-6
. Ten mL of the investigated sample was aseptically transfered to 90
mL of diluent 10
-1
, then 1 mL from previous dilutions was aseptically dispensed into the next dilutions. One mL of the last three dilutions was
aseptically dispensed onto duplicate Petri dishes using a pipette and sterile tips. Fifteen to twenty mL of plate count agar which was tempered
– heated and kept at 45 ± 1ºC in the liquid state was aseptically poured over
pour-plate method and immediately mixed thoroughly and uniformly by a circular and side-to-side motion of plates on flat level surface. After
solidified, the agar plates were inverted and incubate promptly for 48 ± 2 h at 35°C.
∑ ∑
3.3.4.3 Product Total Plate Count BAM, 2010
Serial dilutions were prepared according to to achieve readable numbers of colonies according to BAM 2010 in the range of 25-250
colonies. These dilutions generally were 10
-1
up to 10
-7
. Total plate count evaluation procedures are as stated in 4.2. The
colonies were counted using standard counting rules and the results were expressed as CFUmL. Counts computed should be in the range of 25-250
colonies.
∑ [
where: N = Number of colonies per ml or g of product
∑ C = Sum of all colonies on all plates counted n1 = Number of plates in first dilution counted
n2 = Number of plates in second dilution counted d = Dilution from which the first counts were obtained
30
3.3.4.4 Staphylococcus analysis BAM, 2010
One mL portions of determined dilutions of samples was transferred using a sterile pipette to 3 tubes of 9 mL Trypticase Soy broth
TSB containing 10 NaCl. The tubes were then incubated at 35
o
C for 48 ± 2 h. Tube showing growth turbidity was vortexed and a loopful was
streaked on Baird-Parker medium. The plates were incubated for 48 h at 35
o
C to obtain isolated colonies. Colony suspected of being Staphylococcus is circular, smooth,
convex, moist, and gray to jet-black colored, frequently associated with an outer clear zone. The confirmed tube samples were reported as MPN of
Staphylococcusml, according to the three series MPN table shown on Appendix 18.
3.3.4.5 Salmonella analysis BAM, 2010
Investigated samples 25 mL samples were placed into 225 mL of buffered peptone water BPW for the pre enrichment and was incubated
at 35ºC for 24± 2 h. An aliquot of 1 mL was taken from the pre enrichment showing growth turbidity to the TT broth. While the RV
broth was inoculated with a 0.1 mL aliquot of the pre enrichment and were then incubated at 42ºC for 24± 2 h, and the TT broths at 35ºC for 24± 2 h.
After which, the broths were streaked 3 mm loopful 10 µl onto Bismuth Sulfite BS, Hektoen Enteric Agar HEA, and Xylose Lysine
Deoxicholate XLD agar plates and are incubated at 35ºC for 24 h.
Typical Salmonella sp. colonies release H2S and appear completely black or have black centers when plated on BS, HEA, and
XLD agar plates. Atypical colonies may appear yellow on XLD and HEA and green, clear or mucoid on BS plates. Indicative colony of Salmonella
sp. were selected and stabbed into a Triple Sugar Iron agar TSI and Lysine Iron Agar LIA slant, and incubated at 35ºC for 24 ± 2 h.
Salmonella in culture typically produces alkaline red slant and acid yellow butt, with or without production of H
2
S blackening of agar in TSI. In LIA, Salmonella typically produces alkaline purple reaction in
butt of tube with or without production of H
2
S.
3.3.4.6 pH
Sample was prepared and immersed by electrode to take the pH reading, allowing time for the meter to stabilize. Afterwards, the electrode
was rinsed with distilled H
2
0 and dried to repeat duplo on a fresh portion of the sample.
3.3.4.7 Statistical Analysis
Data analysis was performed with Microsoft Excel 2010 software application. A statistical significance was determined by two paired t-Test
Analysis of variance.