Lab 3 Isolation Methods PDF

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Lab 3 Isolation Methods...

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Lab 3: Isolation Methods In this lab you will be learning how to isolate individual bacteria.

Background Isolation Streak Plate Samples from patients or the environment are usually mixtures of a number of different bacteria. Most laboratory work done with bacteria requires pure cultures of a single organism, so the members of mixed populations must be separated from each other in some way. This can involve spread plates, pour plates, or isolation streak plates. The isolation streak plate is a widely used method to separate individual bacteria from mixtures. There are a number of somewhat different isolation streaking patterns, but they all have the same purpose. The goal of the streaking pattern is to isolate individual bacteria from the mixture as a result of having fewer and fewer bacteria as you continue around the plate. Eventually the concentration of bacteria gets so low that a single bacterium or a small group of cells comes off the loop to form single isolated colonies on the surface of the plate (figure 3.1). Usually, each colony arises from a single bacterium that divides repeatedly over time to form a mass of millions of cells. Therefore, all the bacteria in the colony should be the same. These single colonies can then be picked off the plate with your inoculating loop for further testing.

Figure 3.1 The technique we will use is known as the quadrant technique, which uses the four-streak pattern (figure 3.2). If doing this in a lab, you would then incubate the agar plate for 24 hours after you streak it and evaluate whether you managed to isolate single bacterial colonies.

Figure 3.2

Be sure to flame your loop every time you turn the plate before you make your next streak. And always label your plates before you begin.

Figure 3.3 Here are some additional types of streak isolation methods in figure 3.4 below.

Figure 3.4 Labeling. You must carefully label your plates and tubes. Always label the BOTTOM of the plate so that, even if lids get accidentally switched or broken, you know what was in each plate. Likewise, do not label tubes on the cap.

Background Standard Plate Count The standard plate count method is a commonly used method for estimating the number of microbes in a sample. In this method, the original sample is diluted through a series of bottles or tubes. A set volume (aliquot) of the dilutions is then put onto plates, and the number of colonies on the plates is counted after incubation for 24 hours or more. The number of bacteria that were in the original sample can be calculated by multiplying the number of colonies on the plate by the dilution factor. Greater accuracy can be obtained by counting two or three plates and averaging the counts. In the standard method, only plates containing between 30 and 300 colonies are considered to be valid and countable. Plates on which fewer than 30 colonies are growing are labeled TFTC, too few to count. Plates on which more than 300 colonies are growing are labeled TNTC, too numerous to count. The assumption is that each colony arises from a single bacterial cell. Because this may not always be true, counts in the surface plate count method are reported as colony-forming units, or CFU. An advantage of the surface plate count method over directly counting microbes under the microscope is that both living and dead cells look the same under the microscope, but only living cells will grow on a plate. Plate counts, therefore, provide a better indicator of disease risk.

The standard plate count method is very widely used to estimate the number of bacteria in environmental samples, drinking water, and various juices and foods. This is used to determine the safety of where you swim and what you eat or drink. Safety standards from various federal or state agencies are usually expressed as a maximum allowable cells or CFU per milliliter (mL) or CFU per 100 mL.

Background- Dilutions Dilution of the sample is necessary to reduce the number in a sample to a level that can be counted. Dilutions are all about ratios and how to express them. The formula for calculating a dilution can be stated as the volume of sample/(volume of sample + volume of diluent). If you add 1 mL of sample to a 9 mL tube of diluent, your dilution is 1/(1 + 9) or 1/10 (figure 3.5). The way to express 1/10 in scientific notation is 10−1. This is commonly written as a 1:10 dilution. If you add 0.1 mL of a sample to a 9.9 mL tube of diluent, your dilution is 0.1/(0.1 + 9.9) or 0.1/10 or 1/100 or 1:100 (figure 3.6). The way to express 1/100 in scientific notation is 10−2.

Figure 3.5 Single dilutions (a) Transferring 1 mL into a 9.0 mL tube −1 gives a 1:10 (10 ) dilution. (b) Transferring 1 mL into a 99 mL bottle −2 gives a 1:100 (10 ) dilution.

Figure 3.6 Serial dilutions: Transferring 1 mL into the first 9 mL tube gives a 1:10 −1 (10 ) dilution. Transferring 1 mL from the first 9 mL tube into the second one gives an additional 1:10 dilution to give a total dilution of −2 1:100 (10 ). A third transfer would give a −3 total dilution of 1:1,000 (10 ).

In serial dilutions (one dilution followed by another), you multiply all the dilutions used to get the final dilution of that sample (figure 3.7, next page). If you make a 1/100 dilution in your first sample and then use that to make a 1:10 dilution into your second sample, the dilution in your second sample is (1/100) × (1/10) or 1/(10 × 100) or 1/1,000 (expressed as 10−3).

Calculating Cell Concentration The reason for doing a standard plate count is to find out the concentration of bacteria in the sample being tested. The formula for calculating cells per milliliter (CFU) in the original sample is the number of colonies on the plate × 1/(final dilution). For example: If a plate with a 10−6 final dilution has 86 colonies on it, your cells per milliliter are 86 × (1/10−6). 1/10−6 = 1,000,000 or (106). So 86 × (1/10−6) = 86 x 106 (1,000,000). Expressing this correctly in scientific notation gives us 8.6 × 107 cells per milliliter.

10-1

10-2

10-3

10-4

10-5

Figure 3.7 Colony numbers decrease the sample is serially diluted.

Measuring Bacterial Growth (Quantification using Optical Density) Bacterial growth can be measured either qualitatively or quantitatively. There are many instances when it is critically important to know the number of bacteria present in a sample (quantitative). There are a variety of methods that can be used individually or in combination with other techniques to provide an accurate measurement of viable cell numbers. One method involves using the colony counter to physically count each colony; another utilizes the spectrophotometer. A spectrophotometer is an instrument that can be used to determine bacterial cell numbers. A spectrophotometer measures the cell density or turbidity of a sample as a function of absorbance. The theory behind spectrophotometry is simple. The machine directs a beam of light through a sample. Light either scatters, is absorbed, or passes through the sample (figure 3.8). A sensor detects how much light passes through the specimen. Sensor data are presented numerically or graphically (needle scale) as either absorbance or transmittance. The more cells there are in the sample, the more light is absorbed by the cells and the less light passes through the sample.

Figure 3.8 Measuring absorbance with a spectrophotometer. In the upper image, there are no cells in the sample (blank). The absorbance value is zero. In the lower image, a large number of cells are present. They absorb a large amount of light, which translates to a large absorbance value. The amount of light that passes through the sample is measured using a detector that displays this value in units of absorbance.

Spectrophotometric measurements reflect either the light absorbed (absorbance, turbidity) by the sample or the light transmitted (transmittance) through a culture. Both of these measurements are proportional to the number of bacteria in a solution. The more turbid the culture, the more bacteria are present. Absorbance is a measurement of the amount of light absorbed or dispersed by the specimen. Absorbance is directly proportional to cell number. The more cells that are present in the sample, the higher the absorbance. Transmittance measures how much light passes through the sample. High transmittance values indicate that more light is passing through the sample, which also means there are fewer cells in the sample. Transmittance values are inversely proportional to cell number.

Lab 3 Activities For this lab you will conduct a case study on a real world problem, Listeria food poisoning. Read through the case study below. You will be asked questions throughout that you should determine the answer to assist you with your lab assignment; you won't turn these answers in for credit. After reading the case study, complete the lab assignment 3 in Blackboard. *Note: This is a fictitious case study, I did not base it on a real person. There is also a quick online lab simulation for plate streaking at the very end.

Case Study: Blue Hell Ice Cream Sunny is five months pregnant and has been craving ice cream her entire pregnancy. While visiting family in Texas, she is introduced to Blue Bell ice cream and eats a big bowl of chocolate chip cookie dough flavored ice cream. Forty-eight hours later, Sunny experiences vomiting, diarrhea, cramping and nausea. She is not overly concerned as pregnancy does weird things to the body. After suffering through two days of gastrointestinal symptoms, Sunny arrives in the Parkland Hospital ER presenting with a stiff neck, loss of balance, muscle aches, fever and confusion. After questioning Sunny, the nurse quickly tells the doctor on shift that Sunny has been eating a lot of Blue Bell ice cream; they are all aware of the recent Listeria outbreak. The doctor immediately orders the nurse to perform a blood draw from two different venipuncture sites. *Critical thinking moment: why would the doctor have the nurse take two blood draws from two different sites? Listeria monocytogenes is the gram-positive bacteria responsible for causing Listeriosis. It is a food-borne pathogen that can cause serious infection in pregnant women and immunocompromised individuals. Most healthy individuals never know they are infected with L. monocytogenes as they are asymptomatic. One feature that makes L. monocytogenes a successful food-borne pathogen is that it grows over a broad temperature range, including refrigeration and survives freezing temperatures. L. monocytogenes is not transmissible between people, except mother to fetus, which makes Sunny's exposure particularly dangerous. After the blood draw, Sunny is given an IV of high dose ampicillan (2 g every 4 hrs). Pregnant woman infected with listeria often become bacteremic (bacteria in the blood). Meningeal symptoms, neurological findings or mental status changes may suggest this diagnosis. Sunny's blood samples and test samples from the ice cream manufacturing factory are sent to the lab where you work. You and your colleagues get to work identifying the bacteria in the samples. One scientist uses the patient's blood sample to complete an isolation streak plate, then grows up one colony on blood agar plates to check for beta-hemolysis, a signature of L. monocytogenes. You will learn more about blood agar and hemolysis in following labs. Your job is to estimate the number of microbes in a manufacturing plant sample using the standard plate count method.

You are to test a sample of condensation that was dripping from the ceiling into the ice cream vats in the factory. Before beginning your standard plate count, you must first dilute your sample using a serial dilution. You add 1 ml of the condensation water sample into 9 mls of broth (Dilution A). What dilution factor is this? Next, you take 1 ml of Dilution A and add it into 9 mls of broth (Dilution B). What dilution factor is this? You complete six total dilutions (A - F) each time adding 1 ml into 9 mls of broth. What is your final dilution? You plate 1 ml of Dilution F onto three agar plates (1 ml per plate). Your results show 53 colonies, 57 colonies and 49 colonies. What is your average colonies? What is the concentration of bacteria in your sample? *Note: I don't know the actual technical process of testing condensation for pathogenic exposure in a food manufacturing plant. I designed this fictitious case study to meet our course needs. That is the end of your case study. Yes, Sunny is doing fine and the baby is healthy thanks to the quick thinking nurse at Parkland! _______________________________________________________________________________________ What really happened with Blue Bell ice cream? A team from the South Carolina Department of Health randomly sampled ten products from a local Blue Bell distribution center in January of 2015. Two of the ten samples tested positive for listeria. To be sure, the team went back and collected 30 more samples. All 30 of the samples tested positive for listeria. The team uploaded their findings into Pulsenet, a database of DNA fingerprints the Center for Disease Control monitors to identity outbreaks nationwide. Dr. Robert Tauxe, deputy director of CDC's Foodborne Disease Division verified that the listeria germs found in the South Carolina ice cream matched illnesses in a hospital in Kansas. That hospital was Via Christi St. Francis in Wichita. Listeria had sickened five of their patients over the past year, but the hospital couldn't figure out where it was coming from. Four of the five of the patients had been served milkshakes made with Blue Bell ice cream, the fifth patient had a different Blue Bell product from the hospital; three of those patients died. No details were released regarding the patients' deaths. Each patient was admitted to the hospital for different ailments, it was never released whether listeria was the actual cause of their deaths, probably because it is very difficult to prove what the exact cause of death is if you are already suffering multiple ailments. The Kansas Department of Health tested 45 samples from the remaining Blue Bell products in the Via Christi Hospital and found another hit. The Pulsenet database also matched five other cases that came from three different states going back to 2010. A low level outbreak was going on for five years completely undetected. Pulsenet enabled the CDC to trace the tainted ice cream to Blue Bell's main plant in Brenham, Texas and another Blue Bell factory in Broken Arrow, Oklahoma. When the FDA inspected Blue Bell's main plant in Brenham, Texas, in March, they found a number of violations detailed in a report, including condensation dripping into the ice cream, dirty equipment, and paint chipping from the ceiling directly above an ice cream mixer. Blue Bell didn't issue the voluntarily recall of their products until April 2015. Several workers in the plant came forward stating the conditions in the Texas plant were not clean or up to code, yet they passed every health code check they had which occurred several times a year. Answer to critical thinking moment: Increasing the number of blood cultures obtained increases the likelihood of isolating the offending organism and can be lifesaving. When two sets of blood culture are obtained and a pathogen is identified from both cultures, it is highly unlikely that the organism cultured is a contaminant.

Additional Lab Activity: You will simulate streaking a plate using the website below.

1. Copy and paste the following address into your browser. http://learn.chm.msu.edu/vibl/content/streakplate.html 2. Click on Module (underlined in blue) to begin your simulation. 3. Click Description and read the information. Click Menu when you are done. 4. Click Steps. These are the steps you will need to take to complete the simulation so make sure you know them! 5. Click Menu and then Start to begin the simulation. Remember flame your loop first to sterilize it, then pick a colony, then streak quadrant 1, flame, streak quadrant 2, flame, streak quadrant 3, flame, streak quadrant 4. Did you get any colonies? Which quadrant(s) were they in? 6. You are done with the simulation. Try is as many times as you like....