Yeast and mold count is a horizontal method to estimate the total viable (living) yeast and mold colonies present in a food sample. It is important and widely used to assess food quality, product safety, shelf life of food samples in laboratories. Yeasts are unicellular fungi and molds are multicellular fungi that widely grows on food samples.They can grow on foods as well and produce several toxins like mycotoxin, aflatoxin etc which have severe adverse effect on human health.
Chemicals,Glasswares & Instruments Used :
- 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.
- Vortex : Used to proper mixing or homogenising the diluted sample.
- Autoclave : For sterilization of equipments 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 150 °C±2 for about 2 hours.
- 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.
- Incubator(25°C±1): Incubators are equipment used in biological laboratories to grow, maintain or revive yeasts and molds. Incubators have specific temperature range optimum for growth .
- Sterile Petri Plates : Growth media is pour into this plates.
- 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.
- Micropipette(1 ml): Use to precisely measure the diluted sample.
- Measuring Cylinder : To measure distilled water during media preparation.We use 10 ml measuring cylinder for this test.
- Distilled Water : To prepare media. Distilled water should be autoclaved & contamination free.The conductivity of distilled water should be <5 μS/cm to prevent contamination
- 70% Isopropyl Alcohol or 70% IPA:Use as sanitizer to perform testing properly. Preparation described previously.
- 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 & It’s Working Principle :
| Dilution Water /Peptone Water | Dilution water is a non-selective liquid medium to dilute the concentration of sample. We use commercially purchased(HiMedia) dilution water to dilute the sample. |
| Yeast Extract Dextrose Chloramphenicol Agar (YEDCA) | YEDCA is a selective agar to isolate yeast and mold from a food sample. YEDCA usually has a lower pH (6.6±0.2) because yeasts and molds usually grows on a slightly acidic pH. This media contains an antibiotic (Chloramphenicol) that effectively kills any bacteria present in the sample and inhibit bacterial growth. |
Sample Storage:
| Storage | Any cooked food is considered as perishable item as high water content, available nutrients and optimum growth conditions can increase the bacterial number easily. So, for any cooked food it is recommended to transport the sample in a sealed and ice-box. On arrival to the lab, the sample shall be stored very carefully under refrigerated condition (4°C-8°C) |
| Preparation | Bring the sample in room temperature before performing the analysis for yeast and molds being viable |
Procedure:
| Dilution Water Preparation | Preparation described previously |
| Yeast Extract Dextrose Chloramphenicol Agar (YEDCA) Preparation | Dissolve 40.1 grams in 1000 ml distilled water. Boil the medium to dissolve the agar completely. Sterilize the agar by autoclaving at 15 lbs pressure & 121°C for 15-20 minutes. |
| Sample Preparation & 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. 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 Yeast Extract Dextrose Chloramphenicol Agar (temperature shall be between 45°C-55°C so that neither the bacteria or fungi dies due to high temperature nor the media get solidified) and swirl the plate clockwise and counterclockwise for at least 10 times for proper mixing and distribution all over 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 25°C±1 for 120 hours. |
| Observation | After 120 hours bring the plates out of the incubator and count the colony observed in each plates. Make a separate count of yeast colonies, which are characterized, as white smooth, moist, elevated surface colonies. Count moulds colonies, which are recognized by their profuse growth of hyphae.It is recommended that start the counting from 3rd day as it allows enough time to visible growth of yeast & mold as they may over countered by faster-spreading mold growth on 5th day. |
| Calculation | Calculate the total yeast & mold count using the following formula when Total number of colonies counted is more than 15. N = ∑C/(2.2 x d x v) N= Total Yeast & Mold Count ∑C = Total number of colonies counted 2.2 = Dilution constant d = Dilution Factor v = Volume of Inoculum For Total number of colonies counted within 4-15 use this formula N= ∑C/vd N= Total Yeast & Mold 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 |
Purpose Of Testing:
| Food Safety and Quality | High level of yeast and mold can spoil food, alter taste, texture and smell. Some molds produce mycotoxins that are extremely harmful for human health. |
| Shelf Life Assessment | Yeast and mold counts help predict how long a product will remain safe and acceptable for consumption. |
| Regulatory Compliance | Many food items and food industries must meet criteria limits for yeast and molds. |
| Detect Processing and Storage Failure | Any packaged food usually contains zero or very low yeast and mold count other than any fermented food or food with added probiotics. A high yeast and mold count usually indicates a failure in packaging, storage facility or unhygienic environment. Sometimes contamination may occur during food processing also. |
| Evaluation of Food Safety | Sometimes yeast and mould overgrowth indicates acidification and/or fermentation of the food. These kind of foods shall not be consume. |
Avoid the following mistakes to get proper result :
- The food shall weigh precisely 10 grams or as specified by another specification. A different weight could change the value. Assume that 100 yeasts and molds are present in 10 g of a sample. A sample weighing 9 grams will have a value of less than 100.
- Before testing, the viability, productivity, and specificity of the media must be verified by streaking with Certified Reference Material (CRM) of Candida albicans.
- The media must be between 45°C and 55°C. Cooler temperatures may solidify the media, while warmer temperatures may destroy bacteria.
- To avoid external contamination, the analyst must wear a mask and gloves. The number of mold and yeast is increased by external pollution.
- To reduce the danger of contamination, every work should be done in a sterile setting with sterile tools. To avoid contamination, keep the workplace (where testing is done) at 25°C±2.
- Before analysis, the sample should be properly mixed to ensure that the microorganisms are distributed evenly throughout the matrix.
- To inhibit the growth of bacteria and promote the growth of yeast and mold, the pH of the medium needs to be slightly acidic, between 6 and 6.6 ±0.2.
- Every microbiological test must be carried out in a clean, sterile setting. To minimize external contamination, all microbiological analyses should be conducted in a separate laboratory.
What we can conclude from this testing?
Yeast and mold count is an important microbiological parameter to check the total number of yeast and fungal count in food samples because it is a key parameter to determine the safety of a food sample. Higher yeast and mould count indicates that the food is contaminated and not safe to consume. Also, a higher yeast and mould count signifies the alteration of pH of the food and that the food is fermented. By using the method mentioned above, you can easily perform the Yeast and Mould 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– Frequently Asked Questions:
Q. What is yeast and mold count in food samples?
Yeast and mold count is a microbiological test that estimates the total number of viable yeast and mold colonies present in a food sample, usually expressed as colony forming units(CFU) per gram.
Q. Why it is important to test for yeast and mold in food?
- To ensure food safety and quality
- High count indicate spoilage or poor hygiene
- Some molds produce mycotoxins, which is harmful for human health
- Required for regulatory compliances in several food industries.
Q. Which foods are most susceptible to yeast and mold contamination?
Dairy products, Baked foods, Confectionary foods, Fruits vegetables and juices, Dried foods, Meat and meat products, fish items.
Q. What is the difference between yeast and mold?
Yeasts are unicellular, fast grower, often found in human skin which are mainly less pathogenic. They often cause fermentation in foods. Whereas molds are multicellular, fuzzy colonies, usually slow grower. Molds can cause several toxins that are highly pathogenic for human health.
Q. What environmental condition can cause yeast and mold growth on foods?
Yeasts and molds can grow over a wide range of temperature and pH. They can grow from 8°C to 35°C. Yeast and molds can grow in lower water content and even in low pH as 4.5. That’s why yeast and molds can grow in refrigerated conditions too.
Q.Which foods can be tested by this method?
This method can be applied to analyse total Yeasts and molds 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?
Yes,by following above mentioned method,you can easily test the Yeast and Mold Count at any laboratory or manufacturing industry with availability of the equipments & media.You can also 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)