Sampling

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 • 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 + 225 mL wt:vol; 25 mL + 225 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 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: • No dilution • 25 CFU/ml; 250 CFU/g • -1 dilution • 250 CFU/ml; 2500 CFU/g

11. procedures • Homogenize sample (25 g + 225 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 (25-250 colony/plate) • Calculate bacterial number in food sample • Solid food: average CFU number x dilution x 10 • E.g. 56 x 103x 10 = 560000 = 5.6 x 105 CFU/g = 5.75 log10 CFU/g • Milk:average CFU number x dilution • E.g. 56 x 103 = 56000 = 5.6 x 104 CFU/g = 4.75 log10 CFU/mL

14. Materials and methods • Food sample: • 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 (25-250 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 ................. 5.0 g Yeast Extract ………................................ 2.5 g Dextrose............................................... 1.0 g • Agar .................................................... 15.0 g