Total plate count (TPC), also known as Total Aerobic Plate Count or sometimes as Standard Plate Count is a horizontal method to estimate the total viable (living) bacterial count present in a food sample in a specific time. This is done to know the the total bacterial load of any food sample. Usually most of the food samples have some bacterial load. Though fermented foods like idli, yoghurt, dahi etc have a higher bacterial count. This is done by pour plate technique. Pour plate technique allows to grow aerobic and facultative anaerobic bacteria to grow. To enumerate the exact number of total bacterial count, foods need to dilute serially.
Scope of testing of Total Microbial Plate Count of Food Samples :
This method can be applied to analyse total microbial plate count of wide range of food samples, solid food items like cooked food,ready to eat foods,packaged foods ,Cereals & grains, bakery & confectionery,herbs & spices etc. Semi solid food items like jam,jellies,yogurt & liquid food items like milk & milk based products ,oils etc.
INSTRUMENT USED in TOTAL MICROBIAL PLATE COUNT (TPC) OF FOOD SAMPLES
- Weight Balance : To weigh the media that supports the growth of bacteria.
- pH meter: Used during media preparation.Proper pH ensure the expected microbial growth.
- Autoclave : For sterilization of equipments & media at 121°C & 15 lbs pressure( it is the minimum & standard temperature which kill microorganism & its spore) and for disinfection of all equipments at same temperature & pressure after completion of testing.
- Hot Air Oven: For drying of equipments.It is generally used as dry sterilization at 160 °C for about 1 hour.
- BioSafety Cabinet: Use to perform the whole testing in a sterilized environment.The testing should performed inside the biosafety cabinet only otherwise contamination can be occured which ultimately leading to the inaccurate result.
- Bunsen Burner or Spirit Lamp: Generate heat which is use to sterilize forcep for avoid contamination.
- Bacteriological Incubator(30°C ±1): Incubators are equipment used in biological laboratories to grow, maintain or revive bacteria or microorganisms.Incubators have specific temperature range optimum for growth and aerobic or anaerobic conditions. Incubators are also available with shelves & fan with specific distance to ensure uniformity of temperature throughout the chamber.
- Vortex machine : Samples should be homogenised using vortex machine.
CHEMICAL, EQUIPMENT & GLASSWARE USED in TOTAL MICROBIAL PLATE COUNT (TPC) OF FOOD SAMPLES:
1. Sterile Petri Plates : To give microorganisms a clean, nutrient-rich environment in which to develop and expand so that specialists can examine and observe them. Growth media is pour into this plates.
2. Sterile Conical flask : To prepare media.Capacity of the flask will depend on the amount of media prepared.Here we use 250ml capacity for 100ml media.
3. Measuring Cylinder : To measure distilled water during media preparation.We use 100 ml measuring cylinder for this test.
4. Distilled Water : To prepare media,distilled water should be autoclaved & contamination free.The conductivity of distilled water should be <5 μS/cm.
5. 70% Isopropyl Alcohol or 70% IPA:Use as sanitizer to perform testing properly. Add 70 ml isopropyl alcohol in 30ml of distilled water.
6. Gloves,Cap & Mask : To maintain proper sterility inside the biosafety cabinet.Bare hands can contaminate the equipments which affect the result accuracy. If you don’t wear gloves & masks can affects on analyzer’s body also.
MEDIA USED & ITS WORKING PRINCIPLE DURING TOTAL MICROBIAL PLATE COUNT (TPC) OF FOOD SAMPLES:
| DILUTION WATER | Dilution water is a non-selective liquid medium to dilute the concentration of sample. We use commercially purchased dilution water to dilute the sample. 10 g of samples to be mixed with 90 ml of dilution water to make the concentration to 10-1. Any alteration in sample quantity will hamper the bacterial count. |
| PLATE COUNT AGAR | A non-selective solid growth media should be used to perform this test. Culture media can be broadly distinguish in 2 category as selective media (supports growth of any specific bacteria, like Macconkey agar for Escherichia coli) and non-selective media (supports growth of wide variety of bacteria). In this method we use commercially purchased Plate Count Agar (PCA), alternatively another non-selective media like nutrient agar or Mueller Hinton Agar (MHA) can be used.PCA contains all basic nutrients for bacterial growth like protein and carbohydrate, thus it non-specifically supports growth of a wide range of aerobic and facultative anaerobic bacterial growth. |
SAMPLE PREPARATION & STORAGE DURING TOTAL MICROBIAL PLATE COUNT (TPC) OF FOOD SAMPLES:
| Storage | Samples should be carried in temperature controlled cooled box with ice pack (to inhibit microorganism growth) and shall be analysed immediately after receiving in lab.If not, samples should be stored and refrigerated immediately at 4°C-8°C.This temperature inhibit microbiological activities which states is as close to reality. |
| Preparation | Bring the sample in room temperature(25°C±2) before processing to make the micro-organisms viable. |
PROCEDURE FOR TOTAL MICROBIAL PLATE COUNT (TPC) OF FOOD SAMPLES:
| Media Preparation: | Dissolve 3 g of peptone water medium (Dilution Water) in 200 ml of distilled water and dispense 90 ml in a big 250 ml glass conical flask and 9 ml in 9 test tubes. Take 23.5 g of Plate Count Agar in 1000 ml distilled water. Put them in autoclave and sterile the media in autoclave at 121°C for 15-20 minutes. |
| Sample Preparation and Serial Dilution: | Add 10 g of sample with the 90 ml dilution water, mark it as 10-1 and mix it with a vortex machine. Hand vortex may be done as an alternative. Solid & hard food samples shall be homogenised using vortex machine whereas semi solid or soft foods like cake ,milk & milk products ,liquid food products can be hand vortexed. Keep it for 5-10 minutes to suspend the food particles at the bottom of the flask to avoid contamination. Transfer 1 ml from this to a 9 ml tube and mark this dilution as 10-2. Make this serial dilution up to 10-6 or as required. |
| Plating: | Remove the lids of the plates and dispense 1 ml from each dilution tube to sterile petri dish. Now pour the Plate Count Agar (temperature shall be between 45°C-55°C so that neither the bacteria dies due to high temperature nor the media get solidified) and swirl the plate clockwise and counter clockwise for at least 10 times for proper mixing and distribution all overt the plate. Close the lids of the plates and keep it for 30 mins to solidify the media. Perform the test in duplicate set (dispense 1 ml from each dilutions to 2 plates and mark them as set 1 and set 2 for better evaluation). |
| Incubation: | Incubate the plates in incubator at 30°C±1 for 72 hours. |
| Observation: | After 72 hours bring the plates out of the incubator and count the colony observed in each plates. Any bacterial colony will appear as white coloured colony. Plates will more than 300 colonies shall be rejected for counting. |
| Calculation: | Calculate the total plate count number using the formula N = ∑C/(2.2 x d x v) N= Total Plate Count ∑C = Total number of colonies counted 2.2 = Dilution constant d = Dilution Factor v = Volume of Inoculum Use this formula when Total number of colonies counted is more than 15. For Total number of colonies counted within 4-15 use formula N= ∑C/vd N= Total Plate Count ∑C = Total number of colonies counted d = Dilution Factor v = Volume of Inoculum For Total number of colonies counted within 1-4 express the result as <40 cfu/gm or Total number of colonies counted less than 1 express the result as <10 cfu/gm The total number of colonies counted on agar plate is 58. Dilution Factor 10-1 Volume of inoculum 1 ml |
PURPOSE OF TESTING FOR FOOD SAMPLE’S TPC:
| Enumerate Bacterial Load | Total plate count is the key analysis to enumerate the viable bacterial load in a food samples. Food samples may be sterile or non-sterile depending on the quality of food. Also, some food may contain certain amount of bacterial load like fermented foods. In fact some fermented foods like dahi, yoghurt or any other food, fortified with probiotics must have a bacterial load. Thus this method is essential to enumerate the bacterial count. |
| Evaluate Food Quality | Usually a low bacterial count (TPC) indicates that the food is safe for consumption whereas a high bacterial count (TPC) indicates that the food has been spoiled and not safe to consume. |
| Detect Processing and Storage Failure | Any packaged food usually contains zero or very low bacterial count other than any fermented food or food with added probiotics. A high bacterial count usually indicates a failure in packaging, storage facility or unhygienic environment. Sometimes contamination may occur during food processing also. |
| Shelf Life Evaluation | Lower the total plate count higher the shelf life. A high total plate count indicates nearer shelf life of a sample. |
HANDLING MISTAKES SHOULD KEPT IN MIND DURING THE TEST OF TPC:
- The weight of the food should be exactly 10 gm or as mentioned in any other specification. A higher or lower weight may alter the value. Suppose 10 g of a chocolate sample contains 1000 bacteria. If anyone take 9 gm of sample then it will give value less than 1000.
- All the works should be done in a sterile environment and using sterile equipment to minimise contamination risk. Maintain the temperature of workplace(where testing is performed) at 25°C±2 to prevent contamination.
- Sample should be mix thoroughly before analysis to allow even distribution of bacteria along the matrix.
- Media control must be done to check the viability, productivity and specificity of the media prior testing.
- The temperature of the media shall be between 45°C-55°C. Warmer media may kill bacteria and cooler temperature may solidify the media.
- The analyst shall wear gloves and mask to prevent outside contamination. Outside contamination enhance increase the total plate count.
- All microbiology test shall be performed in a sterile and uncontaminated environment. Any microbiology analysis should be done in a separate lab dedicated only for microbiological analysis to reduce external contamination.
- Glasswares should be cleaned properly as mentioned earlier.
CONCLUSION OF TOTAL PLATE COUNT TEST IN FOOD SAMPLE:
Total plate count is an important microbiological parameter to check the total number of viable bacterial count in food samples because it is a key parameter to determine the safety of a food sample. Higher total plate count means higher bacterial load in a food which indicates that the food is contaminated and not safe to consume. A lower TPC indicates that the food is okay to consume. By using the method mentioned above, you can easily perform the Total Plate Count test in any lab or factory or in other places that has the right equipment and media. This testing process gives you accurate results, which are really important for quality control in many areas. Plus, if you find the procedure confusing, you can look at the real-time photo included with this text, which shows you each step and makes it easier to understand. Also, if you want professional help or need more advanced testing, you can contact Pro Research & Testing Laboratory for your testing needs.
FAQ ABOUT THE TOTAL PLATE COUNT TEST OF FOOD SAMPLE:
Q. What is Total Plate Count?
A. Total Plate Count or TPC is the total number of viable bacteria present in a food sample. TPC does not distinguish between pathogenic and non pathogenic bacteria but it is a process that only enumerate the total number of bacteria present in a sample.
Q. What is called viable bacteria?
A. Viable are the living bacteria that are capable of growing and reproducing under suitable condition.
Q. Who shall do TPC?
A. Any individual or organisation intended to check the quality of food, Quality of manufacturing and processing or to check the shelf life can perform the TPC.
Q.Why foods get contaminated?
A. Foods are often get contaminated due to mishandling or introduction of harmful bacteria from outside Sometimes dirty equipment under-cooked foods contaminated water use for cooking may cause foods get contaminated.
Q.Which foods can be tested by this method?
A.This method can be applied to analyse total microbial plate count of wide range of food samples, solid food items like cooked food,ready to eat foods, packaged foods ,cereals & grains, bakery & confectionery,herbs & spices etc. Semi solid food items like jam ,jellys,yogurt & liquid food items like milk & milk based products ,oils etc.
Q. Can I do this test in any laboratory or manufacturing units?
Ans:Yes,by following above mentioned method,you can easily test the Total Plate Count at any laboratory or manufacturing industry with availability of the equipments & media.If you can’t understand the procedure you can check our real time photo attached with this writing or also you can reach to Pro Research & Testing Laboratory for the testing purposes.
How We Verified This Testing/Research Procedure :
This testing procedure is done under qualified analyst .Continually monitored by expertise.Repeatedly testing is always done to get accurate result.
Written by
Anwesha Das (M.Sc Microbiology,BU)
Designation – Microbiologist
Reviewed by
Sramana Ghosh (M.Sc. Microbiology, MAKAUT)
Designation – Quality Manager
Verified By
Tathagata Talukdar (M.Sc,Microbiology) University of Calcutta
Designation – Senior/Chief Microbiologist
Experience – 12 Years + of experience including medical microbiology (NABL 15189) and general microbiology (NABL 17025)