Transcription
[Music] hello blood talk fans today's topic continues from last week's video where we talked about differentiating types of bacteria between gram-positive and gram-negative bacteria. once we know what types of bacteria cause infections, we can now treat the infections accordingly. but to treat an infection more effectively, we need to know which antibiotic works and the dosage. therefore, we'll be talking about antibiotic sensitivity tests by Kirby-Bauer methods or these diffusion methods today. I will include how to make McFarland turbidity standards to prepare for inoculum in this video as well. Without further ado, let us click that like button, share, subscribe, and don't forget to click that notification bell.
A quick overview of why we need to perform antibiotic susceptibility tests. Infections can be caused by either gram-positive or gram-negative bacteria. Knowing which type of bacteria causes infection will help determine the right antibiotic for each type of infection. We would not want to just give a broad-spectrum antibiotic randomly because that would contribute to creating bacterial resistant strains, sometimes we refer to it as superbugs, and we will need even stronger antibiotics to keep up with it. Eventually, we may not have anything to treat the infection. Antibiotic sensitivity tests will tell us which antibiotic works and the doses that need to treat each infection.
Why do we have to prepare an inoculum before we can perform an antibiotic sensitivity test? We will have to prepare an inoculum. McFarland turbidity standard is a method widely used to standardize an inoculum. The reasons we have to prepare an inoculum is to standardize the number of bacteria being tested. False susceptible results may occur if too few bacteria are tested. False resistant results may occur if too many bacteria are tested. Just in FYI for today's contents, the words susceptibility and sensitivity tests are being used interchangeably.
How to prepare McFarland turbidity standards. The most commonly used is the 0.5 McFarland standard, which contains 99.5 milliliters of 1% sulfuric acid and 0.5 milliliter of 1.175% barium chloride. Prepare this solution in the same type of tubes as the one you will use to prepare your bacteria suspension. The results will be slightly turbid. Store the mixture at room temperature in a dark area for later use. A 0.5 McFarland standard provides turbidity comparable with that of a bacteria suspension. Bacteria that has the same turbidity as a 0.5 McFarland standard would contain approximately 1.5 x 10^8 colony-forming units per milliliter or CFU/mL.
[Music] A modification of McFarland is to use latex particles because it is more stable and can provide comparable turbidity tests as the original McFarland standard mixture. To standardize the inoculum, the bacteria suspension is vortexed and compared to the turbidity directly with the 0.5 McFarland standard under adequate lighting. The two are positioned side by side with the 0.5 McFarland standard against a white card containing several horizontal black lines. The turbidities are compared by looking at the black lines through the suspension. If the suspension is too dense, it would be more difficult to see the lines through the bacterial suspension than through the 0.5 McFarland standard. In this case, you can either add sterile culture broth or saline to dilute the bacteria suspension until the turbidity is the same level. If the bacteria suspension is too light, more organisms may be added until the turbidity reaches the same as the 0.5 McFarland standard. Once the inoculum suspensions are standardized, the suspension should be used within 15 minutes of preparation.
Now that we have our bacteria suspensions, we can prepare for sensitivity tests by disk diffusion or Kirby-Bauer method. Let's talk about the disk diffusion methods a little bit before we go into the procedures. Kirby-Bauer test is a qualitative method. It is done by growing a lot of bacteria with antibiotic discs. Allows the bacteria to grow for about 16 to 18 hours, depending on how fast your bacteria grow. The clear media surrounding the disc indicates that the antibiotic inhibits bacterial growth. The purpose of the diffusion test is to determine the susceptibility to the antibiotic of the bacteria. This test tells you how sensitive or resistant the bacteria of interest is to various antibiotics. This helps the physician in selecting treatment options for the patients. Doctors can choose the most effective antibiotic to kill an infectious microorganism. If the organism is sensitive to an antibiotic, it will not grow in a zone of inhibition. Resistant bacteria will be able to grow close to the disc. Since the resistant bacteria is relative, some strains are more resistant than others, so we cannot just say which one is susceptible and which one is resistant by just looking at the zone of inhibitions or the zone around the disc. We will have to measure the inhibition zones and compare it to the table established by CLSI and FDA to determine that the bacteria is susceptible, intermediate, or resistant. A Mueller-Hinton agar plate is usually used for antibiotic sensitivity tests. Why Mueller-Hinton agar is used for antibiotic sensitivity tests? Let's take a look at these agar properties. First, Mueller-Hinton agar is a non-selective, non-differential media. Since it is non-selective, most of the organisms will grow on this plate. This way, we will not have bias when the zone of inhibition is measured. Second, Mueller-Hinton agar contains starch. The starch serves two purposes in this media. First, it absorbs toxins released from bacteria as the bacteria grows, so that the toxins will not interfere with bacterial growth and antibiotic diffusion. Second, starch serves as an energy source for the bacteria. Third, Mueller-Hinton agar is a loose agar. This allows for a better diffusion of antibiotic than other types of media agar, since the antibiotic can be diffused freely, a true zone of inhibition can be established.
Procedures for antibiotic sensitivity tests by Kirby-Bauer methods. Step 1: Label the plate. Step 2: Making the bacteria suspension by selecting a few isolated colonies of bacteria and suspending the bacteria in media broth or saline. We select a few colonies, not just one big colony, because we want to make sure that we get a well representation of the culture. If some of the bacteria develop resistance, then we will have a better chance of catching it, or if the patient is infected with more than one type of bacteria, we will have a higher chance of catching it if we get a few colonies instead of just one big colony. Step 3: Compare the bacterial suspension with 0.5 McFarland standard, making sure that the suspension is the same turbidity as the 0.5 McFarland standard. Step 4: Take a sterile swab and dip it once in the prepared bacteria suspension.
[Music] Step 5: Make a bacterial lawn by swiping the bacteria from left to right, from top to bottom. Step 6: Turn the plate 45 degrees and repeat swiping the bacteria from left to right, from top to bottom. Once finished, repeat this step two more times to ensure that the bacteria is equally distributed and there is no area that is left out. Do not forget to use the swab to go around the ring of the media.
[Music] Step 7: Sterilize forceps by dipping it in alcohol and pass it through flames. Step 8: Pick up an antibiotic disc and place it on the plate. Step 9: Use the forceps to press on the antibiotic disc lightly to make sure that the disc will not fall off the plate during incubation. Step 10: Repeat step 7 to 9 until you place all the antibiotic disc types. Step 11: Keep in mind that this step should be complete within 15 minutes of finishing making the bacterial lawn.
[Music] Step 12: Incubate the plate by storing it inverted at 35 degrees Celsius for 16 to 18 hours. It is a good practice to have a control plate incubated with each batch to ensure that the bacterial growth condition was not a factor and bacteria has sufficient time to grow before reading a zone of inhibition.
[Music] A control plate is made in the same manner as the testing plate, but instead of using patient sample to make a lawn of bacteria, a control plate containing control organisms, E. coli and Staphylococcus aureus, are usually used as control. After 16 to 18 hours, examine the plate to make sure that the organism has grown sufficiently. We do that by looking at the control plate. The organism must show growth that covers the whole plate or almost covers the whole plate. A control plate should be read prior to reading the patient plate. Measure the zone of inhibition from the control plate and then compare to the sensitivity table. If the inhibition zone is correct according to the table, then the patient plate may be read. If the QC is not passed, the patient testing results are not acceptable and cannot be reported. I will talk about how to troubleshoot if the QC fails at the end. If the organism only shows individual colonies, it is not acceptable. This may be an indication that you did not make a good lawn of bacteria.
[Music] If the growth is satisfactory, the diameter of each inhibition zone is measured using a ruler or caliper. An easy way to read the plate is to invert the plate over a black surface so you can see the inhibition zone easier. The inhibition zone is measured in millimeters for each antibiotic, then compared to interpretive tables from CLSI documents, and the results are interpreted as susceptible, intermediate, or resistant. Here is an example of the table from CLSI.
[Music] [Music] Interpretations of this diffusion test: Sensitive means infection treatable by the normal doses of the antibiotic. Intermediate means infections may respond to a higher dosage. Resistant means unlikely to respond to usual doses of the antibiotics.
[Music] If you see individual colonies in the inhibition zone, do not ignore that. The individual colonies could be from your culture. You may have other organisms mixed in with your sample, or you may have some that is resistant to that antibiotic. Let's work on some examples on how to interpret antibiotic sensitivity tests. What are the results of antibiotic sensitivity tests for antimicrobial agents A, B, and C? First, we have to measure the zone of inhibitions in millimeters. Second, we compare the diameter in millimeters to the susceptibility tables. Keep in mind that when you are using the table, double-check the organism names and the type of antibiotic. Each has their own specific diameter. Here is the table that we will be using for the example.
[Music] Let's look at the first sample. This A looks like 21 millimeters to me. Let's write it down and take a look at the table. This A, 21 millimeters, falls in the intermediate range. Next, this B. It looks like it will be susceptible because the zone is quite large. However, that is a mistake. You cannot just look at it. You have to measure the zone of inhibition to know if the organism is resistant, intermediate, or susceptible. Okay, it is 30 millimeters. When I compare to the table, this B is resistant. Antibiotic B will not work for the patient with this infection at this dosage. Next, this C looks like 15 millimeters. When I compare to the table, it is susceptible.
Troubleshooting the disk diffusion problem. A control plate is used to make sure that the results are valid. When control fails, there are steps that can be taken to troubleshoot the disk diffusion problem. If the zones are universally too large on control plates, this means you'll notice that all the antibiotic discs give a larger than normal zone of inhibition. This could mean a few things: inoculum is too light, the agar is too thin, or nutritionally poor media. Why is that? If the inoculum is too light, then it would take the bacteria longer to grow to cover the whole plate, but the antibiotic diffuses at the same rate. This leads to a larger zone of inhibition than expected. When the agar is too thin, the antibiotics spread more laterally, which leads to a larger zone of inhibition. If the media is nutritiously poor, the bacteria is not at its optimal growth condition. Growth rate decreases, so a large zone of inhibition is observed. If you double-check everything and they are correct, which means the suspension is not too light, the agar is not too thin, the agar is nutritiously adequate, and the organism is not a slow grower, get a new control vial. Stock your control organism fresh because your control organism may be contaminated, therefore the result is not as expected. If the zones are universally too small on control plates, it means inoculum is too heavy or agar depth is too thick. The same thinking as when you get zones universally too large, just reverse the reason.
That was all for antibiotic sensitivity test and McFarland standard. There are other methods for sensitivity tests such as E-test, dilutions, genetic testing. If you want to know more about any of these testing, please let me know. I can cover those topics next. Thank you for staying with me until the end. If you have any burning questions, please feel free to leave me a comment down below. Lastly, if you have not done so, please like, share, subscribe, and click that notification bell. I will see you in the next episode of Blood Talk. As always, remember, your blood tells you the story of your health. Thanks for watching. Bye.
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