There are many methods of measuring bacterial overgrowth and some are more complex than others. While some accuracy needs to be sacrificed when taking measurements, the simplest way is quite accurate and is commonly used. The best known techniques are the observation and counting of bacteria, the measurement of the wet and dry mass or the level of turbidity. The school laboratory should have all the necessary equipment and materials to conduct at least one of these experiments.
Steps
Method 1 of 3: Observe Bacteria Directly
Step 1. Gather the materials
There are some special tools you should have in addition to those commonly found in most biology labs. Preparing the containers and tools in advance allows you to complete the experiment without having to constantly search for what you need. It is important to know the intended use of each piece and to have a knowledge of elementary laboratory terms.
- Get a counting chamber. It is a device with a chamber, a slide and a built-in microscope, which is easy to assemble and use. You can purchase it at a lab supply or school supplies store. A manual should be included in the box to guide you through the process.
- Prepare a plate for inoculation on solidifiable substrate or for spatulation; these are containers where you can observe bacteria.
- The term culture refers to the artificial development of an organism for an experiment.
- Broth is the liquid medium in which culture grows.
Step 2. Use the spatula plate or for the curing substrate
You can also put the bacteria directly into the container to observe them under a microscope, just apply them on the plate; take note of the number of cells present.
Step 3. Make sure the sample has the right concentration
If there are too many bacteria, they overlap and you may not be able to count them accurately; if so, you should dilute the culture with more broth. If the concentration is too low, you do not have enough microorganisms for an accurate estimate, you must therefore filter the broth respecting the proper technique.
Step 4. Count the bacteria
The last step is the physical count. Look at the sample through the microscope lens of the counting chamber and write the number of cells you see; compare the result with those of the other tests.
Method 2 of 3: Measure Dry and Wet Mass
Step 1. Make sure you have the right equipment
This method involves the use of expensive machinery and a lot of time. Unless the lab has everything it needs, consider using another method; however, if possible, the measurement of dry and wet mass allows constant results. Here's what you need:
- Gravity convection stove;
- Aluminum weighing plate;
- Flask series;
- Laboratory centrifuge or filtration apparatus.
Step 2. Make sure the culture is in a flask
If not, pour it into this container; at this stage it should still be a broth, although it separates later.
Step 3. Dry an aluminum weighing pan in the laboratory oven
Alternatively, you can use an acetate cellulose filter membrane with a diameter of 47 mm and with pores of 0.45 µm. Whichever medium you decide to use, weigh it so you know the mass you need to subtract next, once the bacterial cells are arranged.
Step 4. Mix the contents of the flask into which you poured the culture to amalgamate it
The cells tend to settle to the bottom due to gravity; then mix it thoroughly to distribute them in suspension in the liquid and make the sample more uniform.
Step 5. Use a centrifuge to separate the bacteria from the broth
This tool quickly rotates the flask and a counterweight eliminating the liquid and leaving the culture. Read this article for more details.
Step 6. Scrape the bacterial residue and transfer it to the weighing pan
Throw away the broth as you no longer need it, but save the flask as you will still need it.
Step 7. Rinse the juicer and pour the water you use into the dish
Add a flask of rinse water to the bacterial cells to weigh the wet mass.
Step 8. Find the dry mass
Place the weighing pan in the laboratory oven and let the bacteria dry at 100 ° C for 6-24 hours, respecting the instructions of the specific instrument you are using and the weighing pan; make sure the temperature is not too high to avoid burning the cells. After the appropriate time, weigh the material remembering to subtract the mass of the plate.
Method 3 of 3: Measure the Turbidity Level
Step 1. Get the necessary equipment
You need a light source and a spectrophotometer that you can buy at a laboratory supply store; the machine should be equipped with a manual for its correct use. The equipment is inexpensive and easy to use; consequently, this method is one of the most common for measuring bacterial growth.
Step 2. Illuminate the sample
In simple terms, turbidity is the level of opacity of a liquid; you should get a value that is measured in NTU (Nephelometric Turbidity Units). Equipment may need to be calibrated before accurate nephelometry can be performed.
Step 3. Take notes
Turbidity corresponds to the amount of bacteria present in the sample. The spectrophotometer indicates the percentage of light transmission (% T); the higher the number, the clearer the sample (less bacteria). Compare various bacterial growth measurements obtained with different methods.
Warnings
- Since you are working with colonies of bacteria, take safety precautions, such as wearing safety glasses and gloves. You should also use a mask, especially if you don't know the type of microorganism you are breeding.
- Take precautions with any kind of bacteria, even if you believe it is harmless, by protecting all wounds, scrapes and cuts before starting.
