In the last year of my specialization (science-mathematics) I have to do a thesis. I opted for a biological thesis, more specifically about the different biological food processes such as spoilage, mold, fermentation, bacteria, food poisoning and how these can all be avoided as much as possible. I would like to do a little research myself. I wanted to test certain foods for their bacteria count. For example, I wanted to test a piece of meat, fish, a piece of fruit, … for their numbers of bacteria when stored in the refrigerator, at room temperature and in the freezer. With this I hope to support my entire thesis.
Now I actually have some problems counting the bacteria. Some say I have to use a breeding ground for this. I don’t know exactly where to get it and how to use it. Others advise me to make a breeding ground myself, but I am missing a number of ingredients for that. Still others say I should just put the foods to be examined under a microscope and the colonies should be visible to the naked eye. I have a (3D) microscope with which I can perform some experiments, but it does not magnify strongly enough for bacterial colonies.
Answer
Dear Nouchka,
Indeed, the most common method of counting bacteria is by using culture media. To count bacteria with a microscope, you cannot simply put your food under the microscope: you need a special type of microscope slide (= counting chamber) and a microscope that magnifies 1000x.
Plating bacteria on nutrient media is simpler, but requires specialized equipment. Keep in mind that potentially pathogenic bacteria will grow on your petri plates. Therefore, first consult with your teacher before you start this; you will also need laboratory equipment from your school, such as pipettes and test tubes.
For your experiment it is best to buy ready-made dishes (petri plates) with nutrient medium, which are available from specialized companies such as bd, duchefa, vwr and many others. It is advisable to use a general culture medium, such as Luria Bertani (LB) agar, on which most bacteria grow. You will have to work as sterile as possible: disinfect the surface you are working on with alcohol and work as close as possible to a burning flame. Disinfect your hands regularly and keep the petri plates and test tubes you use closed as much as possible. If you use glass test tubes, you can sterilize them in an old bottle sterilizer.
Dissolve 10 grams of your food in 10 ml of sterile water (or sterile physiological solution) by shaking or mixing. Label 8 sterile test tubes of 10-1 to 10-8 and fill each test tube with 9ml of sterile water. Shake the test tube with your food thoroughly (this is 100) and add 1ml of this solution to the 10-1 test tube. Now shuffle the 10-1 test tube thoroughly and transfer 1ml of this into the 10-2 test tube, and so on. Make two petri plates from each of these dilutions by placing 0.1 ml of liquid on one plate (shake first). Spread this liquid with a sterile spatula until the plate is dry; smooth the liquid away from the edges. You can sterilize a spatula by dipping it in alcohol and then holding it in a flame; let your spatula cool down before you put it on your plate. Clearly write the food, the dilution and any other information on each plate with an alcohol marker. You can pack the plates in plastic foil to avoid contamination. Place the plates upside down in a dark place at a constant temperature. Many bacteria grow best at 28°C, pathogens usually at 37°C. Count the number of colonies per plate daily: some bacteria will be visible after a day, others only after a week or more. The plates on which 20 to 200 colonies grow are the most reliable: take these counts to calculate back how many bacteria were in your food (For example: if 100 colonies grow on the plates of the 10-3 dilution, then there were 100*10*1000= 1000000 bacteria in your food, or 100000 per gram.
Good luck!
liesbet
Answered by
ir. Liesbet D’hondt
flow cytometry of microorganisms in plants
Burg. van Gansberghelaan 96 box 1 9820 Merelbeke
http://www.ilvo.vlaanderen.be
.