1. Sampling
Sampling (採樣)
必須使用無菌工具
保持低溫
樣品保存
樣品選擇

2. Sampling tools Sampling bags Sampling tube Sponge or cotton swab
Solid and liquid food or environmental samples
Sampling tube
Liquid samples
Sponge or cotton swab
For surface
Each group choose your own samples and collection tools. Food samples is preferred.

7. Food samples ready-to-eat food
It is better to store at room temperature for at least half day
Solid (e.g. sandwich, egg roll)
Liquid (e. g. soy milk, milk tea)

8. total plate count (TPC) aerobic plate count (APC) total mesophilic plate count
Total bacterial number is an important indication for food quality
Basic principle for TPC
Sampling size, 25 or 50 g
Solid sample
Add 9 times volume of buffer (25 g mL wt:vol; 25 mL mL vol:vol)
Homogenize
Homogenizer or stomacher

9. Materials and methods Food samples
store at room temperature
Depend on the food samples to decide the dilution range
Dilution range is from -1 to -4

10. Detection limit No dilution -1 dilution
1 CFU/ml; 10 CFU/g
-1 dilution
10 CFU/ml; 100 CFU/g
However, we need 25 to 250 CFU in one plate
Thus, reliable detection limit is:
25 CFU/ml; 250 CFU/g
250 CFU/ml; CFU/g

11. procedures
Homogenize sample (25 g mL phosphate buffer, 1:9 dilution)
Homogenize for 2 min
For milk, no homogenization
Decimally serial dilution
Place one mL of the undiluted homogenate into a Petri-dish (0 dilution)
Add agar to mix the sample: 混稀 pour plate
Place 0.1 mL of the undiluted homogenate onto a Petri-dish (-1 dilution) 塗抹: spread plate
Place 0.1 mL of -1 dilution the homogenate onto a Petri-dish (-2 dilution)

13. Incubate the plates at 37C for 24 (spread) to 48 (pour) hours
Count the colony ( colony/plate)
Calculate bacterial number in food sample
Solid food: average CFU number x dilution x 10
E.g. 56 x 103x 10 = = 5.6 x 105 CFU/g = 5.75 log10 CFU/g
Milk: average CFU number x dilution
E.g. 56 x 103 = = 5.6 x 104 CFU/g = 4.75 log10 CFU/mL

14. Materials and methods Food sample: One sample bag
Each group decide their own sample. Usually ready-to-eat food has not high amount of bacteria. Thus, buy them one day before and place at room temperature.
Don’t buy cookies, bread, any food item with package.
One sample bag

15. Dilution Buffer (PBS) 225 mL
Dilution tubes
Plate (TSA or PCA)-6 plates (spread)
The dilution will be decided on the class depending on the food item.
Stomacher
Test tube racks

16. procedures Purchase the food item and place into the sampling bag.
Record the time, place, food name, who sample it.
One the class, take 25 g from the food item. Do NOT just take one site, take several sites of the food item.
Add 225 mL (1:9), stomach the sample for at least 1 min.

17. Dilution the samples according to the dilutions.
Spread plates.
incubate at 37C for 24 hours.
Count the colony number at the suitable dilution ( CFU).
Calculate the bacterial counts in the sample.

18. Preparation of media(um)
Two types of agar
Tryptic Soy Agar (TSA)
Plate Count Agar (PCA)
Add agar powder into a bottle or flask according to the instruction on the bottle
TSA -40 g/L
PCA 23.5g/L

19. PCA
Pancreatic Digest of Casein g Yeast Extract ……… g Dextrose g
Agar g