Total Aerobic Counts & Bacillus cereus - 23F --Food Microbiology Laboratory (Com

Reflect in ePortfolio Download Print BI 218 Food Microbiology Total Aerobic Counts & Bacillus cereus Originated by: Allan Richardson Revised by: Yvonne Henry & Samiha Mohammad Sharif Uncontrolled when printe d 1 THEORY: Purpose and Background This method is applicable to the enumeration of viable aerobic microorganisms and specifically Bacillus cereus in foods. The aerobic plate count is commonly used as an indicator of the microbial level in a food sample. The method detects bacteria that form colonies on Trypticase Soy Agar (TSA), or Standard Methods Agar (SMA), also known as Plate Count Agar (PCA) at 35±1°C within 48 hours . Not e: not all microorganisms produce c olonies under these conditions; s ome b acteria for instance do not grow at 35°C, do no t grow under aerobic conditions, or require mor e than 48 hours to form colonies . The method generally allows the enumeration of mesophilic bacteria (optimal gr owth temperature between 20 - 45°C). However, under specific incubation temperatures, psychrophilic bacteria (optimal growth at or below 15°C) and thermophilic bacteria (optimal growth temperature above 45°C) can be analyzed. By using different media s, microbiologists can select for specific types of bacteria . The aerobic colony count (ACC), also known as the standard plate count, total plate count or aerobic plate count, estimates the number of viable microorganisms per gram or milliliter of product , or per swab. A portion of sample is mixe d with an agar medium and incubated under specified condi tions of time and temperature. The agar medium can be pre - made and inoculated as spread plates, or melted and maintained at 45°C - 55°C and inoculated in a pour plate method or with sample - ready 3M Pet rifilm. The assumption is that each viable microorganism cell will multiply , and give rise to a visible colony, which can be cou nted. The refore, the final count is expressed as colony forming units (CFU /mL or g ). The Petrifilm Plates are a little different them the pour plate or spread plate methods because they are considered “Sample Ready”. This means that they contain modified Standard Methods nutrients; a cold - water - soluble gelling agent; and 2, 3, 5 - triphenyl tetrazolium c hloride (TTC) indicator. One milliliter of a sample or its preparation is added directly to the plate. When pressure is applied to the overlay film with a plastic spreader, the sample will be spread over a surface area of 20 cm 2 . The gelling agent is all owed to solidify, and the plates are incubated at 35±1°C for 48±3 hours. All colonies will appear red due to the addition to TTC which is reduced. All red dots, regardless of their size and intensity, on the plate are counted and reported as ACC. The count ing limit on a Petrifilm AC plate is 250 colonies, any more colonies cannot be accurately counted. A common microbe found in a wide variety of foods is Bacillus. Bacillus species have the ability to reduce themselves to endospores which results in the bacteria staying in a dormant (inactive) state for prolonged periods of time. Though not all Bacillus species are considered pathogenic to humans, Bacillus cereus is a recogni zed foodborne pathogen that produces at least two types of well characterized toxins (i.e. an emetic type and the diarrheal type). Being ubiquitous it gains access to foods and multiplies under favourable conditions, producing toxins. At level >10 6 cells/g a significant enterotoxin may be produced resulting in food poisoning. This method also employs a plating technique onto a selective agar. The number of B. cereu s per gram or mL of the food is calculated using PEMBA or Brilliance Bacillus Agar and reported as CFU/mL or g. Bacillus cereus is not easily distinguished from closely related bacteria in the B. cereus group. B. mycoides, B. thuringiensis, and B. anthracis. Atypical strains of B. cereus are variable in expression of motility and hemoly sis and will need further testing to identify the isolates (Note: not performed in this experiment) . In this lab we will be using a general growth media which can support the growth of all types of bacteria called TSA and a media that specifies for Bacillus species called B rilliance Bacillus agar . EQUIPMENT AND SUPPLIES: • 99mL PO 4 in bottle • 9.0mL PO 4 buffer • 60mL TSA in small media bottles (tempered) • 3 Petri plates • 3 Bacillus agar plates: Brilliance Bacillus Agar • 1- APC 3M Petrifilm OBJECTIVE : • To practice aseptic sampling and homogenization techniques • To observe and enumerate B. cereus on TSA and B rilliance Bacillus agar to differentiate between TAC and selective/differential media METHODS: D ay 1 S terility control plates (as a class – done by lab monitor or 1 selected pair ): • CONTROL #1 Diluent control: Make a pour plate with TSA using one m L of the buffer diluent to check the sterility of the buffer • CONTROL #2 Agar Controls: Pour plates with TSA agar but no sample, to check the sterility of the agars PROCEDURE A: S AMPLING , HOMOGENIZING, & DILUTING 1 of 5 Automatic Zoom https://e.centennialcollege.ca/d2l/le/content/1047309/viewContent/12761768/View 2023-10-28, 3 : 41 PM Page 2 of 3