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

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.

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)

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

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

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; 2500 CFU/g

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)

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

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

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

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.

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.

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

PCA Pancreatic Digest of Casein ................. 5.0 g Yeast Extract ………................................ 2.5 g Dextrose............................................... 1.0 g Agar .................................................... 15.0 g