4: Pipetting and Probiotics Experiment

Glass serological pipet

• We will use 5 ml, 10 ml, and 25 ml sizes
• Controlled using a Pipet-Aid = a pump that draws up or dispenses liquid
• The pump may be electronic, scrolling, or bulb

Micropipette (aka pipetman or pipettor)

• We will use 2 μl, 20 μl, 100 μl, and 1000 μl sizes
• P2: measures volumes less than 2.00 μL
• P20: measures volumes between 2.0 μL (minimum) and 20.0 μL (maximum)
• P100: measures volumes between 10.0 μL (minimum) and 100.0 μL (maximum)
• P1000: measures volumes between 100.0 μL (minimum) and 1000.0 μL (maximum)
• Draws up and dispenses liquid using a disposable plastic tip

Which pipet should you use?

• Generally, choose the smallest possible instrument to measure the volume you need all at once
  • example: if you need 3.5 ml of water, it is best to use a 5 ml serological pipet rather than multiple uses of a P1000
  • example: if you need 95 μl of water, it is best to use a 100 μl (P100) pipettor rather than a P1000
• The exception is if you need a precise amount that is slightly more than 1 ml, use a P1000 and pipet in 2 steps.
  • example: if you need precisely 1450 μl of water, pipet 750 μl then 700 μl, or some other combination that totals 1450 μl.
  • This situation is uncommon, however, and usually you should select the instrument that allows you to measure the entire volume at once.

For all Serological Pipette Usage:

• Carefully insert a clean serological pipet into the nose of the Pipet-Aid so that the numbers face you when you hold it.
  • Be sure to handle the pipet only near the top, so that your hands will not contaminate the portion that will enter the solution
• Insert the pipet into the solution and draw up the desired amount, measuring at the bottom of the meniscus
  • Note that usually the measurement numbers are in reverse order, so if you want to measure 2 ml using a 5 ml pipet, you may need to draw up your liquid to the mark that is labeled 3 ml. Some pipets are marked differently, so pay attention to each one you use.
  • Caution: Never allow any liquid to enter the nose of the Pipette-Aid
• Insert the filled pipet into the target container and dispense the liquid
• Carefully remove the used serological pipet from the Pipet-Aid and dispose as instructed by your professor.

To Set the Volume

Turn the knob on top of the plunger or use the volume adjustment wheel (Figure 2). You will probably need to release the scroll lock on the pipette before you can scroll. Caution: Never try to set pipettor outside its proper range, which is printed on the top of the pipettor!

To Put on the Tips

Gently but firmly press the end of the pipettor onto a pipet tip. P1000s use the large tips; P100s and P20s use the smaller tips; P2s use tiny tips. You should always change tips between samples.

To Suck Up (Aspirate) Liquid

Using your thumb, depress the plunger to the first stop or point of resistance. While holding the plunger down, insert the pipet tip into the solution. Slowly and smoothly release the plunger to draw up the desired amount. Caution: Never allow any liquid to enter the barrel of the pipettor!

To Squirt Out (Expel) Liquid

Insert the filled pipet tip into the target container, just below the surface of any existing liquid, and depress the plunger all the way to the second stop. To prevent bubbles, while holding the plunger down, remove the pipettor and tip from the target container, then release the plunger. Check the pipet tip to make sure you successfully dispensed all the liquid without leaving any drops stuck to the tip.

To Eject the Tip

Point the pipette tip over the waste container. Eject the tip into the waste container by pressing the tip ejector button.

Probiotics

In an average 1 gram of soil sample or 1 ml of sea water there could be 10,000 to 100,000,000 bacteria. We humans also have an incredibly diverse and essential microbiome in/on our bodies. Your skin is estimated to have 1 million bacteria per cm² (Chen and Tsao, 2013) and your digestive tract is even more jam packed with bacteria and approximately half of your poop are bacteria (micropia)! We need these bacteria for our health and digestion and they are involved in protecting us from infection, contributing to our digestion, impacting our mental health, and are responsive to our treatment of them. Especially after taking antibiotics, which kill a large proportion of our natural microbiome alongiside the dangerous pathogens, we might need to re-build our microbiome. One way of doing that is to consume probiotics.

In our lab today, we will measure the bacterial concentrations in two types of probiotics. Probiotics are live microorganisms (bacteria and fungi/yeasts) that are consumed intentionally to improve health. These organisms are often lactic acid producing bacteria including Bifidobacterium and Lactobacillus species which make the gut more acidic, increase the diversity of gut bacteria, and improve digestive health. Scientists are conducting clinical trials to learn more about the roles that probiotics can play in the treatment of human gut diseases and recent findings have suggested that probiotics and help shape the microbial community in the digestive tract leading to improved health and control of some digestive tract diseases (Kim et al., 2019). A variety of studies have also identified a link between consumption of probiotics and improved mental health including a reduction in depressive symptoms (Jarbrink-Sehgal and Andreasson, 2020).

Additionally, physicians often recommend that patients consume probiotics either as part of their diet or as supplements during or after antibiotic treatment. This recommendation comes because during a course of antibiotics, both the pathogenic (harmful) bacteria and the normal gut microbiome (helpful) bacteria are killed. Some patients have digestive-tract complications, which can be prevented by consumption of probiotics (McFarland, 2014). These probiotics can come in many forms either as over-the-counter supplements or as foods that naturally contain bacteria including yogurt, kefir, sauerkraut, kimchi, and kombucha. The advertisements on yogurt and probiotics list bacterial counts in the BILLIONS!!

However, not all probiotics are created equal. Because these are foods and supplements, they are not regulated by the FDA and vary widely in effectiveness. Different strains of probiotics may have different physiological effects on their human hosts and some might be better for some health concerns rather than others. Also, it is not always clear what concentration of bacteria are needed to be most effective. Finally, not all probiotics contain the number of bacteria stated on the label. Some manufactures may over-inflate their numbers, sometimes the units are hard to compare, and since these are living organisms, they can be impacted by changes in the environment like changes in temperature.

Our project today will be to look at two types of probiotics – one supplement and one yogurt with live and active cultures. In our experiment today, we will determine:

• The number of live bacteria: colony forming units/gram (CFU/g)
• Compare this to the advertised value
• Compare the supplement to the yogurt to determine which contains more living bacteria

To culture the bacteria in the probiotics, we will spread a diluted soil sample on a plate and look for the appearance of colonies. Just as you'll see spots of mold on an old piece of bread, you can see a bacterial colony on agar plates. A colony is a pile of genetically identical bacteria that arise from a single cell. Single cells that give rise to a colony are referred to as CFUs (colony forming units). As this single cell reproduces, the mass of dividing cells eventually becomes large enough such that a macroscopic colony becomes visible to the naked eye. By the time you can see a colony on a plate it contains approximately 1 million cells. Colonies are our way of seeing into the microscopic world, and each bacterial type has specific colony features that can be recognized by experienced microbiologists.

The probiotics that we will be starting with will have billions of bacteria per gram in their initial source and therefore be way too concentrated to form individual colonies on a plate. If we tried, the bacteria would be so crowded that it would completely fill the plate that we were using and form a "lawn" of bacterial growth. We would never be able to count something this concentrated. Instead, we will dilute them and spread out the CFUs so that they grow into non-overlapping colonies. To do this, we will dilute the bacterial samples repeatedly in a solvent to decrease the number of cells in any sample. We will then spread a small volume (100μL) on an agar plate to separate and spread out the bacteria all over the surface of the plate where they will grow into single colonies (Figure 4). Our work today will use a particular type of media (food source for bacteria) that is specific for Lactobacillus species as these are the bacterial organisms most common in our probiotics.

Protocol 1: Learning to Use the Pipettes

During the semester, we will perform experiments that will require us to transfer small volumes of liquid with precision and accuracy. Measuring small quantities (1 mL or less) of sterile broth, solutions, or bacterial cultures, can be accomplished using an instrument known as the Micropipette. This first activity will familiarize you with how to use a micropipette. Please refer to the pipetting guide in your lab manual.

Protocol 2: Aseptic Technique

Aseptic or sterile technique is a central concept in microbiology and may be the most important part of working in a microbiology lab. The goal of aseptic technique is to promote practices that:

• prevent the contamination of cultures, lab supplies, and equipment with bacteria or fungi from the environment, and
• prevent the contamination of individuals working in the lab with potentially pathogenic bacteria.

Aseptic technique requires constant attention until it becomes second nature. Practicing these skills and paying full attention to the steps is crucial to making proper aseptic technique a habit. The health and safety of everyone in the lab and the accuracy of your experiments depend on these principles. Here are some general guidelines for practicing aseptic technique:­

Protocol 3: Labeling

Labeling materials and supplies are a critically important aspect of science. With multiple plates, tubes, and samples, it is easy to become confused and lose track of things between and within experiments without the proper labeling practices. Here are a few guidelines for labeling:

Protocol 4: Serial Dilution

Adapted from: Serial Dilution Protocols: Jackie Reynolds – Microbe Library

The advertisements on our yogurt and probiotics list bacterial counts in the BILLIONS!! Because this density is so high, it would be almost impossible to get an exact count of the cells by examining them directly. Instead, microbiologists estimate the cell density through colony forming units (CFUs). To perform this calculation, the sample must first be diluted in water or a saline solution to keep the bacteria alive. An initial dilution of 1g of yogurt in 9mls of water (to make 10ml total) would create a 10-fold or 1:10 dilution. This will spread out the bacteria in 10x the volume, thereby making them further apart. This sample is still too concentrated with bacteria to be able to accurately count them. Therefore, we will continue separating the bacteria by making serial dilutions of the sample in 10-fold increments until they are easier to work with and count. Once we reach a reasonable dilution, we can spread the sample out on a solid medium that will feed the bacteria. Each CFU (viable bacteria that can grow in this environment) in the sample will then grow into a colony and we can count them and, using the dilution and plating information, calculate the CFUs present in each initial sample. For example, if we serially dilute 1 g of yogurt by a factor of 10³, spread and incubate the dilution on a solid medium, and then observe 130 colonies, we would obtain 130 × 10³ or 1.3 × 10⁵ CFUs/g of soil. This number represents the number of viable cells, i.e., cells in an environmental sample that can survive lab conditions and grow in culture.

Figure 5. Serial dilution and plating schematic. A series of 10-fold dilution is made from the soil sample. Each plate therefore has 1/10 of the number of bacteria from the previous plate. This makes is much easier to count the colonies. Image modified from Alves and Cruvinel, 2016.

Protocol 4: Spreading a Plate

One method of distributing bacteria evenly over the surface of an agar plate medium is commonly referred to as the spread plate method. Classically, a small volume of a bacterial suspension is spread evenly over the agar surface by using a sterile bent glass rod or glass beads as the spreading device. The goal is to distribute the bacteria evenly so that they are well separated and can be counted or sampled.

Some things to take note of in the plate spreading are:

• Be sure that your plates are dry before spreading. If they are too wet it will cause the bacteria to run into each other and smear.
• Choose the serial dilutions to plate thoughtfully. If you have too many bacteria on the plate you won't be able to count them next week. If you have too few, you won't have enough isolated colonies to get accurate counts.

NOTE: For yogurt I recommend plating the 1:10 through 1:100,000 dilutions. For the supplement capsule, I recommend plating the 1:10,000-1:100,000,000 dilutions.

• Plate the correct volume of sample. I recommend ~ 100 μL (0.1 mL). This is enough to fill the plate so that the bacteria spread out, but not so much that the plate is drippy.
• Work swiftly, some bacteria attach to the plate surface quickly, so be sure to spread as soon as you put on the liquid or you will have all your bacteria clumped wherever your dropped them.
• Use aseptic technique to prevent contamination from bacteria in the air. Work near your flame, keep the tubes and plates closed, and change tips whenever they touch something.
• Start plating from the most dilute suspension to the most concentrated. This way, you don't need to change tips as each sample has more bacteria than the prior one.

Video Demonstrating Serial Dilution

Counting Colonies

Look at your plates in order of concentration and you should see a predictable drop in CFUs/plate as a result of your 10-fold dilution series. If your plates differ from this substantially, let your instructor know.

The goal is to have between 30 and 300 colonies on a plate. Too many and it is too hard to count, too few and the count can be very inaccurate.

We want to work backward from this to determine the INITIAL number of bacteria in the initial sample of probiotics. To do this, we have to account for each set of dilutions we did, but since we know all the volumes, this becomes a multiplication calculation.