Presentation on theme: "Lab 4: Determination of Aerobic colony count in Foods"— Presentation transcript:
1 Lab 4: Determination of Aerobic colony count in Foods
2 ENUMERATION OF MICROORGANISMS IN FOOD ApplicationThis method is applicable to the enumeration of viable aerobic bacteria (psychrophilic, mesophilic and/or thermophilic bacteria) in foods.
3 The Aerobic Colony Count (ACC) estimates the number of viable aerobic bacteria per g or mL of product.A portion of the product is mixed with a specified agar medium and incubated under specific conditions of time and temperature.It is assumed that each viable aerobic bacterium will multiply under these conditions and give rise to a visible colony which can be counted.Psychrophilic bacteria: an organism which grows optimally at or below 15oC, which has an upper limit for growth at ca. 20oC, and which has a lower limit of growth of 0oC or lower.Mesophilic bacteria: an organism whose optimum growthtemperature lies within a range generally acceptedas ca oC.Thermophilic bacteria: an organism whose optimumgrowth temperature is > 45oC.
4 Materials and special equipment HClNaOHStomacherpH meterColony counting device
5 ProcedureDetermine which type of aerobic bacteria are being enumerated.Analyze each sample unit individually.The test shall be carried out in accordance with the following instructions:Handling of Sample UnitsDuring storage and transport, the following shall apply: with the exception of shelf- stable products, keep the sample units refrigerated (0-5oC).Sample units of frozen products shall be kept frozen.Thaw frozen samples in a refrigerator or under time and temperature conditions which prevent microbial growth or death.3. Analyze sample units as soon as possible afterreceipt in the laboratory.
6 Procedure Preparation of Media 1. Prepare plate count agar and dispense in appropriate quantities. Sterilize.3. Clean surface of working area with a suitable disinfectant.4. Clearly mark the duplicate Petri plates.
7 Procedure Preparation of Dilutions 1. Prepare sterile 0.1% peptone water diluent.2. To ensure a truly representative analytical unit, agitate liquid or free flowing materials until the contents are homogeneous.If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.3. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL (the analytical unit) into 225 mL of the required diluent, as indicated in Table I.If a sample size other than 25 g or mL is used, maintainthe 1:10 sample to dilution ratio, such as 11 (10) g ormL into 99 (90) mL.
8 Procedure4. In some instances it may be advantageous to prepare the initial dilution on a percent basis to obtain a more accurate test material weight than is attained by the dilution ratio method; i.e., a 10% solution (suspension) is represented by 10 g (mL) per 100 g (mL) of solution (suspension), whereas a 1:10 dilution is based on 10 g (mL) of product (solute) plus 90 g (mL) of diluent (solvent).
9 Procedure6. Check the pH of the food suspension. If the pH is outside the range of , adjust the pH to 7.0 with sterile NaOH or HCl. 7. Prepare succeeding decimal dilutions as required, using a separate sterile pipette for making each transfer. 8. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.
11 ProcedurePlating1. Agitate each dilution bottle to re-suspend material that may have settled out during preparation.
12 ProcedureIncubation Incubate plates in the inverted position for 48 h ± 4 h. Incubation temperature is dependent on the growth temperature requirements of the target organisms (for psychrophilic bacteria: oC, for mesophilic bacteria: oC, and for thermophilic bacteria: 55oC). Avoid crowding or excessive stacking of plates to permit rapid equilibration of plates with incubator temperature.
13 Counting Colonies1. Count colonies promptly after the incubation period.If possible, select plates with colonies (including pinpoint colonies).If plates contain colonies which spread, select a representative portion of the plates free from spreaders, if possible, and count the colonies in this area.The total count of the entire plate is estimated bymultiplying the count for the representative area countedby the reciprocal of the fraction of the plate counted;e.g., 30 colonies counted on 1/4 of area of the plate; countfor the whole plate: 30 x 4 = 120 colonies.
14 Differentiation of Colonies from Interfering Particles 1. Food particles such as meat, milk powder, etc., often interfere with the enumeration of the plates.This can be eliminated by making one extra plate of each dilution containing interfering particles and holding it under refrigeration as a control for comparison during counting.2. Alternatively, after incubation flood plates with 2 mL of 0.1% 2,3,5, tri-phenyl- tetrazolium chloride.Gently rock plates from side to side to cover the entire area with solution.Pour off excessive solution and allow the plates to remain at room temperature for 3 hrs. in an inverted position.The bacteria reduce the indicator to a formazan which colorsthe colonies red and aids in distinguishing the food particles.Colonies cannot be picked for isolation after this methodhas been used.
15 Recording ResultsCalculate the average count of the duplicate plates.