What are the requirements of a scientific experiment? How do scientists design their investigations? In this lesson, we'll use the work of Avery and Griffith to explore the process of experimental design.
Checklist for Experimental Design
Have you ever wanted to design your own house? How would you go about doing that? You'd probably think of exciting things to do with the architecture, right - like spiral staircases or rooftop patios? But, no matter what kind of house you wanted, you'd have to follow certain rules. Your house would need a roof, walls, doors and windows. You'd have to account for the air ducts that comprise your heating and air conditioning systems. You'd also need to build in the plumbing and electrical supply lines.
Just like builders have rules for designing their homes, scientists have criteria for designing their experiments. When a scientist is planning to run an experiment, s/he has to make sure s/he fulfills certain requirements. If s/he doesn't, then the work won't be valued by the scientific community. In this lesson, we're going to discuss the rules that scientists have to follow and see how these rules played out in the famous experiments by Oswald Avery and Griffith.
Let's begin by talking about the rules that scientists have to follow when designing their experiments. First of all, an experiment must be designed to answer the question that the scientist is trying to solve. There should be a definite purpose for running the experiment - something concrete that the scientist wants to determine.
A scientific experiment must use objective data.
Next, the experiment must provide objective results. There has to be data collected from the experiment that is measured in some objective way. A scientist can't say things like, 'this chemical looked reddish-purple' or 'that mouse grew a lot faster than the other one.' A scientist has to use numbers and units to describe his findings, and his experiment has to facilitate the collection of that data.
Finally, experiments have to control for multiple variables. That is, if more than one variable is affecting the experiment, then only one variable should be affecting it at a time. Scientists make sure this happens by incorporating a control into the experiment. A control is a means of ensuring that only one factor is being tested at a time. Those are the main requirements that a scientist should consider. Now, let's find out how the Avery and Griffith experiments were designed to follow the rules.
Introduction to the Griffith ExperimentFrederick Griffith
Griffith worked with two strains of pneumococcus: one that had a protective sugar coat and one that didn't. The one with the sugar coat was called the S bacteria because it looked smooth under the microscope. The one without the coat was called the R bacteria because the lack of coating made it look rough. Mice always died when injected with the S bacteria because the mouse immune system couldn't penetrate the sugar coating. But, mice injected with the R bacteria didn't die. They survived because their immune systems could kill off the unprotected R bacteria.
Griffith knew that an infection of S bacteria would kill a mouse. But, if he first killed the S bacteria by heating it and then injecting the dead bacterial cell parts, the mouse survived. So, there were two ways a mouse could survive being injected: if it got dead S bacteria or if it got living R bacteria.
However, Griffith also discovered that if he mixed living R cells with dead S cells and then injected the mouse with the mixture, the mouse would die! When Griffith examined the blood of the dead mouse, he found living S bacteria inside. He knew this was significant because he had not injected any living S bacteria.
Somehow, the R bacteria had changed into S bacteria and caused a lethal infection in the mouse. Griffith didn't know what had happened, but he concluded that some kind of transforming principle was responsible for the change in the R bacteria.
Mice survived when injected with dead S or live R bacteria, but died when injected with both.
Designing Avery's Contribution
Now it was time for Oswald Avery to take the stage. He made it his mission to continue Griffith's work and identify what that transforming principle was. Avery and his colleagues set out to design an experiment that would answer the question about the transforming principle. Avery knew they would need to first identify all of the possible factors that could be responsible for transforming the bacteria. Then, they would have to show that one - and only one - of those factors was doing the transforming.
Avery's list of candidates included the sugar coat on the S cells, the bacterial proteins and the bacterial nucleic acids (including RNA and DNA). Remember, scientists didn't know at this point that DNA was the genetic molecule. For Avery, it was just one of many possibilities. He also had to consider another possibility: the immune system and blood of the mouse itself. Armed with this list of candidates, Avery could now continue designing his experiment to identify the transforming principle.
Since he was going to have to test so many different factors, Avery had to make sure he could separate each factor from the rest. In other words, he had to control for multiple variables. Avery had to be able to test each factor to see if it was the transforming principle without including any of the other factors. So, this is what he did.
First, he separated the mouse from all of the other factors. He wanted to see if he could get transformation if the live R cells and the dead S cells were mixed together in a test tube, not inside of the mouse. Avery wanted to mix live R cells with all of the substances inside the dead S cells. He heat-killed the S cells just like in Griffith's experiment. But, to expose the insides of the dead S cells, he had to lyse, or break open, the cell membranes with detergent.
The resulting lysate, which was a solution of all the cell parts mixed together, was then mixed in with the live R cells. The mixture yielded live S cells, demonstrating that the transforming principle was something in the lysate, not something in the mouse.
When live R cells and lysate from dead S cells were mixed, live S cells resulted.
Next, Avery tested whether the sugar coat was the transforming principle. He used an enzyme to destroy the sugar coat inside the lysate and found that the resulting mixture could still transform bacteria. This told him that the live R bacteria were not just making their own sugar coat out of the pieces from the dead S cells - they were getting transformed by something else.
Then Avery took the same lysate and used protein-digesting enzymes to eat up all the protein molecules floating around in there. You see, back then many scientists thought that proteins were the source of genetic information. But, even when all the proteins had been destroyed, the lysate was still capable of bacterial transformation. So, the transforming principle wasn't the sugar coat or the proteins. It had to be one of the other things left in that lysate.
Avery and his colleagues were able to isolate the nucleic acids in the lysate by precipitating them with alcohol. They mixed the precipitate with water and tested it for the transforming ability by mixing in the live R cells. Sure enough, the nucleic acids contained the transforming principle. But, Avery knew that this mixture contained both types of nucleic acids: DNA and RNA.
To rule out RNA, he destroyed it with a ribonuclease enzyme. The solution still had the transforming principle. At this point, Avery was pretty sure that DNA was the culprit because DNA was just about the only thing left. But, to make sure, Avery destroyed the DNA with the enzyme deoxyribonuclease. The resulting mixture was not able to transform live R bacteria into S bacteria! So finally, Avery was certain that DNA was the transforming principle.
Results of Avery's Work
We can see that Oswald Avery successfully answered the question and controlled for multiple variables in his experiment. But, what about providing objective results? How did Avery know for certain whether transformation was happening at each little step? All I've told you so far is whether the mixtures were capable of transformation. But, how did Avery know? What were his objective results?
By looking at the bacteria inside the test tubes, Avery could see whether the R bacteria had changed into S bacteria. He didn't have to measure how many of each bacteria strain there were. He simply had to state whether or not he saw the live S strain. Avery's objective results were a series of 'yes' or 'no' answers to the question, 'Did transformation occur in the absence of this factor?'
Avery had objective results for the experiment in the form of yes or no answers.
Avery and his colleagues really put their brains to the test in designing this famous experiment. Not only did they have to follow the criteria for good experimental design, they also had to use what they knew about culturing bacteria, separating substances and destroying molecules with specific enzymes. In the end, the work of Frederick Griffith and Oswald Avery helped move science forward by proving that DNA was the source of genetic information.
All scientists have to follow certain criteria when designing their experiments. A good scientific experiment answers a question, provides objective results and controls for multiple variables. We can look at Griffith and Avery's experiments to see how scientists meet these criteria. Frederick Griffith's work led to the discovery of a transforming principle that could change bacteria from one strain to another.
Oswald Avery set out to identify what that transforming principle was. He designed an experiment that would test all of the possible factors. Avery successfully controlled for multiple variables in his experiment by eliminating all of the extra possibilities and only testing one at a time. His work provided objective results in the form of 'yes or no' data on whether or not each factor could transform bacteria. Finally, Avery's work answered the question by demonstrating that DNA was the transforming principle. Together, Avery and Griffith paved the way for more scientists to study the genetic properties of DNA.