Lab Test 1: Lab 1

General information about the ___ (a.k.a. the "Pipetman") The ___ are the single most commonly used instrument in the BSCI lab and every student must know how to use them accurately. They are also very expensive and easily broken by those who do not know how to use them. It will be assumed that you will know how to use a micropipetter in courses and labs after you finish BSCI 1510L. ___ button ___ adjustment knob volume ___ plastic ___ disposable ___

micropipetter micropipetters plunger volume indicator shaft tip

Practical Considerations Related to Counting Colonies There is no ___ amount of diluted suspension that must be spread over the surface of the agar plate. However, a volume of less than about 25 µl is difficult to spread uniformly over a typical plate. Volumes of greater than about 500 µl are not hard to spread, but tend to pool toward one side of the plate if the plate isn't level and that amount of liquid takes a long time to be absorbed by the agar. ___ µl is a very convenient amount to spread over a plate with a diameter of about 8 cm. Not only is it easy to spread uniformly and is easily absorbed, but it also ___ the calculation of the titer. It is standard to specify the titer as cells per cm3 (i.e. per ml = 1000 µl). If 100 µl is plated rather than one cm3, then the titer inferred using the counts and total dilution factor is on the basis of per 100 µl rather than per 1000 µl. To convert the inferred titer to the standard units, one simply multiplies the count by ___. Although in theory any number of colonies on a plate can be counted, in practice it is best to try to count colonies on a plate having between about 20 and 200 colonies. If a plate has fewer than 20 colonies, the task of counting becomes very easy and there is little likelihood of miscounting. However, the fewer the number of colonies measured, the more likely ___ non-representative sampling is to affect the accuracy of the measurement. As the number of colonies approaches 10, the resulting calculated titer effectively has only one significant digit of precision, which translates into a very poor estimate. On the other hand, if a plate having a diameter of about 8 cm has more than 200 colonies, accurate counting becomes more difficult because it becomes difficult to keep track of which colonies have already been counted. Additionally, the higher the density of colonies on a plate, the more likely it is that two bacteria will fall so close to each other that their two colonies merge and appear to be a single colony, resulting in an ___. Fortunately, a tool called a ___ ___ makes it easier to count dense colonies on a plate. A colony counter is a relatively simple electrical device having a wire placed in the agar near the edge of the plate, and a metal probe (connected to the counter by another wire) used to complete the circuit each time it is poked into the agar at the location of a colony. Each time the circuit is completed, the counter increments the displayed count by one. The lighted stage of the counter is divided into a 1 cm grid that can be used to help keep track of which sections of the plate have been counted (Fig. 6). Using a colony counter, it is practical to count colonies numbering in the ___. Although it is preferable to count plates having ___ than about 200 colonies, it is possible to estimate colony numbers ranging up into the thousands under certain conditions. This can be done by counting a known ___ of the plate (e.g. half or quarter) or known area of the plate (one or more centimeter squares on the grid) and then scaling the count up to the area of the whole plate. For example, if a quarter of the plate was counted, the count would be multiplied by four. If 3 cm2 of a plate having a total area of 51 cm2 was counted, then the count would be multiplied by 51/3. In order to make this kind of estimation accurately, the colonies must be spread ___ over the plate so that it is safe to assume that the part of the plate that was counted was representative of the plate as a whole. If any part of the plate has a streak of solid bacteria, then the entire plate becomes impossible to use in a titer determination since it is not possible to know how many colonies were ___ in the streak. Here are some rules of thumb for counting colonies: If there are a few colonies (about 100 or less) on the entire plate, count ___ colony. If there are an intermediate number of colonies (100 to 400), use the ___ on the counter to divide the plate into quarters. Count the number of colonies in ¼ of the plate, then multiply by 4 to estimate the total number of colonies on the plate. If there are a large number of colonies (greater than about 400), count the number of colonies in a certain number of representative 1 __ squares (use the grid on the counter as a guide; Fig. 6), then use the area of the plate (π r2) to estimate the total number of colonies on the plate. Rulers are available in the lab drawers. If the colonies form a continuous sheet (known as a "lawn"; Fig. 7), it is ___ to obtain a count. At this point you may be wondering how it is possible to know how much to dilute the stock suspension in order to arrive at a plate having between 20 and 200 colonies. The answer is that it is not possible to know! For that reason, microbiologists usually inoculate a ___ of plates that differ in dilution factor by as much as an order of magnitude each. It is assumed that some or many of the plates will be useless because they either contain few or no colonies, or contain too many overlapping colonies to count. That is fine as long as at least one plate falls into the countable range. Since the colony densities on all of the plates are related by known dilution factors, ___ of the countable plates can be used for a titer estimate. If the microbiologist has an expectation of the approximate titer of the stock solution based on prior experience, the number of plates in the dilution series could be as few as two or three. But it is better to make too many plate dilutions than too few, because producing no countable plates means repeating the experiment and waiting at least a day for the colonies on the new plates to develop.

particular 100 simplifies 10 random undercount colony counter hundreds fewer fraction uniformly overlapping every grid cm impossible series any

General information about the micropipetter (a.k.a. the "Pipetman") A micropipetter is an adjustable microliter (µl) pipette (Fig. 2). There are several different models and sizes available, but you will be using only 3 of these, the P-__ micropipetter, the P-___, and the P-___. Each pair of students should always have a set of the three micropipetters in a drawer at their lab station. The pipetters should be numbered to correspond with your drawer number. If a particular size of pipetter is missing from your drawer, check nearby drawers for one with your ___ ___ written on it. (DO NOT take pipetters out of drawers that do not have your drawer number on them! If you still cannot find the pipette that is missing from your drawer, ask your TA to assist you.) The number on each indicates the ___ ___ (in µl) that can be measured with the micropipette. NEVER adjust the micropipette for a setting above this number! Exceeding the maximum volume will break the micropipetter, which costs over $___ to replace. The volume indicator consists of a three-number dial and is read from top to bottom. The three digits indicate the volume selected and are colored black and/or red. The ___ numbers on the P-20 pipette show microliters; the ___ numbers show tenths of microliters. The P-200 pipette has only ___ numbers and thus shows only the number of microliters. The P-1000 has a ___ digit to represent one thousand microliters, followed by ___ numbers to indicate hundreds and tens of microliters. Note: The first (red) digit on the P-1000 will always read zero, except in the case when it is set for its maximum volume of 1000 microliters. In that case, it will read 1, 0, 0. It is possible to measure some volumes using more than one type of micropipetter. For example, 20 µl could be measured by setting the P20 to 2,0,0 or the P200 to 0,2,0 or the P1000 to 0,0,2. However, the most precise measurements will be made using the ___ possible pipetter. In this example, that would be the P20.

20 200 1000 drawer number maximum volume 250 black red black red black smallest

What is Titer? A common task in the field of microbiology is to determine the titer of a suspension of ___. Functionally, cell titer is similar to ___ and generally the same kinds of calculations that can be done with concentrations can be done with titers. However, concentration is appropriate for ___, whereas cell cultures are ___. Concentration is expressed in grams or moles per unit volume, while titer is expressed in ___ per unit ___ (usually per cm3). A fundamental difficulty in measuring bacterial titer is that it requires "___" something that cannot be seen with the naked eye and is visible only under high ___ through a microscope. For this reason, indirect methods are typically used. Two of the most common methods are ___ (week 3 and 5) and ___ (this week).

bacteria concentration solutions suspensions cells volume counting magnification spectrophotometry plating

1.9 explain how standard error of the mean is affected by increasing sample size standard error ___ as n increases

decreases

1.10 calculate the mean, standard deviation and standard error of the mean for a sample using Excel use ___ statistics

descriptive

Serological Pipette Serological pipettes come in a variety of sizes, generally from 0.5 ml - 25 ml, and are made of ___ (nondisposable) or ___ (disposable). In general, we will use ___ pipettes and we will always use them with a ___ pipette pump. NEVER pipette by ___! Note: Sometimes the white rubber ___ (into which the pipettes are inserted) detaches from the pipette pump when the pipette is removed and remains stuck on the end of the pipette. Before you discard disposable serological pipettes, check to be sure that you are not throwing away the seal (Fig. 1) - it can easily be re-attached to the pump.

glass plastic disposable mechanical mouth seal

Sterile Techniques LO1.3 use sterile techniques to manipulate a bacterial culture without contamination and to inoculate sterile medium on a Petri plate using a spreader. 1. The non-___ portion of the instruments that you use to transfer inoculants should ___ touch any non-___ object. This is to prevent bacteria and fungi in the environment from getting into the sterile media and also to prevent the bacteria that you are culturing from getting onto lab equipment and other students. Do not remove disposable sterile ___ or loops from their packaging until immediately before you use them. Open their package at the ___ end, touch only the handle and as you remove the implement, do not let it touch the place where you grasped the packaging to open it. Never lay a spreader or loop on the bench top outside of its packaging. After using it, place the spreader or loop back inside of its package and place that in an ___ bag. If you wish to attach a serological pipette to its pump ahead of use, open only a small portion of the non-tip end of the plastic sleeve and leave the rest of the pipette undisturbed in the sleeve while you attach the pipette to the pump. You can then place the assembly on the benchtop and maintain sterility until you pull the pipette out of the sleeve. Immediately after use, place contaminated implements in the appropriate waste container (i.e. the autoclave bag). 2. ___ containers (screw capped tubes containing broth and plates containing agar-based media) should be left ___ except during the brief interval that you are inoculating them or removing a sample. 3. Agar plates should be stored lid side ___ to prevent condensation from dripping onto the media. The exception to this is immediately after adding liquid inoculants or media additives (such as X-gal and IPTG). You should allow the liquid to be ___ completely before inverting the plates. 4. A bacterial colony on a plate results from a ___ bacterium that was present when the plate was ___. Thus all of the many bacteria in the colony should be identical to the one from which they originated. Starting a pure broth culture from a plate requires touching only a ___ colony with the inoculating loop. The colony should be widely separated from others to prevent accidentally touching two colonies (and therefore having the possibility of producing a mixture of two types of bacteria in the broth culture). The colony should also be perfectly ___. An oblong colony may have formed from the merger of two colonies that were near each other when they started to grow. 5. When using the colony ___, be sure that you remove the lid and place the ground wire in contact with the ___ of the agar. Since you will destroy the sterility of the plate when you touch the probe tip to the colonies, do ___ use the counter on any plates from which you plan to later culture colonies. When you have finished counting the plates, wipe the tip of the probe with a ___. 6. When you no longer need cultured plates, place them in the special ___ bags. Do not place them in the regular trash. Likewise, do ___ put regular trash in the autoclave bags. Liquid bacterial waste should be poured into the designated ___ and the empty tubes should be placed in an autoclave bag. Container with small amounts of liquid bacterial waste (e.g. microfuge tubes) can be placed directly in the autoclave bags without emptying the liquid.

handle never sterile spreaders handle autoclave Media closed down absorbed single inoculated single round counters edge not tissue autoclave not containers

Goals of BSCI 1510L It is a common (but ___) belief that the primary purpose of BSCI 1510L lab is to ___ the topics being studied in the lecture course with which it is associated (BSCI 1510). In reality, there are several goals that we try to achieve in BSCI 1510L: 1. To increase your proficiency in the use of ___ and ___ common in modern cell and molecular biology laboratories. When you are finished with this course, we hope that you will be able to begin working in a research lab in the BSCI department or Medical Center (as many students do) without making a fool of yourself. 2. To introduce you to principles of the major components of the scientific process: experimental ___, statistical ___, and scientific ___. Not only will this course help to prepare you to do well in subsequent courses and undergraduate research at Vanderbilt, but it will also make you a more savvy, ___ and informed consumer and citizen. 3. To improve your background and understanding of ___ concepts in cell and molecular biology. We have this goal in common with the lecture course. However, rather than concentrating on the details of these concepts, we are more likely to be focused on how the concepts apply in a laboratory setting and how laboratory techniques are used to discover the details of these concepts. This means that we will study some concepts at a different time than when they are covered in lecture or that the concepts may be organized in a different way. We will also typically apply higher order thinking skills such as application, evaluation and synthesis during our analyses of the results of experiments.

incorrect review equipment techniques design analysis writing skeptical major

Experimental Goals to learn to operate commonly used ___ equipment to gain experience with Microsoft ___ to understand how scientific ___ are located and cross-referenced to prepare a range of concentrations by ___ to learn ___ technique and common microbiological techniques to understand the nature of sample ___ and the quantities used to describe it

laboratory Excel papers dilution sterile variation

Practical Considerations Related to Bacterial Suspension Dilutions What volumes should be used to make the serial dilutions? There is no exact answer to this question but you should use volumes that are: ___ enough to be accurately measured obtained with the most ___ measuring instrument available measured in a way that doesn't waste a lot of materials. Using a P20 micropipetter, it is possible to accurately measure volumes as small as 5.0 µl if you are careful. Although the P20 has marks that allow it to be set to the nearest 0.02 µl, due to the physical limitations of you and the instrument, you are probably really measuring to an accuracy of about 0.2 to 0.5 µl. Thus a 5.0 µl measurement is probably accurate to about 10% and any smaller measurements would be even worse. (We will commonly measure out volumes as small as 1 µl later in the semester, but in those cases we aren't doing quantitative work and for those materials it is OK for the volumes to be approximate.) What if you want to measure 19 µl? Any of the micropipetters could be set for that value, but a P20 would be precise to a few tenths of a microliter, a P200 would be precise to a few microliters, and a P1000 would be precise to around ten microliters. Thus the P___ would be the best choice. What about 25 µl? That is higher than a P20 can measure, so you will either have to use a P200 or add together two volumes (e.g. 5 and 20 µl) using a P20. For convenience, use the P___. What about 1 ml? You could measure that with a 10 ml serological pipette, but you would be lucky to achieve a precision of 0.1 ml (i.e. 100 µl) with that instrument. On the other hand, a P1000 set on 1000 µl (i.e. 1 ml) would be precise to about 10 µl. So use the P___. The bottom line is that you should use the smallest instrument that has the capacity to go up to the volume that you need to measure. Now let's say that you need to make a 10 000-fold dilution and intend to plate the resulting suspension. You could measure out 10.0 ml of buffer using a serological pipette (technically 9.999 ml, but you can't measure that precisely with a serological pipette!), then add 1 µl (i.e. 0.001 ml) of the stock suspension using a P20 micropipetter. That would be a really bad idea for two reasons. There would be a relatively high percent error in measuring out the 1 µl (probably 20-50% error). You would also be making about a hundred times more solution than the 100 µl that you need to spread on the plate. On the other hand, you could achieve the same thing if you diluted 10.0 µl of stock suspension in 990 µl of buffer in a 1.5 ml microfuge tube, then repeated the dilution using 10.0 µl of the resulting diluted solution into another 990 µl of buffer in a second tube. In this case, your percent error would probably be only a few percent if you measured carefully and vortexed each dilution to mix it thoroughly. In addition, you would only be using about 2 ml of buffer (still way more than you need for one plate, but better than the 10 ml in the first example).

large precise 20 200 1000

General Note About the Location of Materials The ___ are always kept in the drawers (A) at your lab station, and also microfuge racks (B). Please return them there when you are finished using them. ___ are in a box on the top of the bench. Some frequently used materials may be placed on your bench, e.g. boxes of micropipette ___. Sometimes materials that must be kept cold are placed in ___ buckets at your bench. Other materials are usually placed on the supply bench at the ___ of the lab. Test tube racks are also available in the wood cabinet in the front of the lab. In situations where you need gloves, they are available in a rack on the wall by the supply bench in the back of the lab. Listen to instructions given in the introduction for materials placed in special locations. The materials are located in such a way as to make it efficient to get them to you. Do ___ line up at the back of the lab to get things at the beginning of the period. That's a waste of time! Get things as you ___ them. For this lab, the following materials will be at the ___ of your bench to be shared with the other students: boxes of pipette ___ (small for P20/P200 and large for P1000) microcentrifuge ___ (at bench to be shared with others) Please keep them in the center of the bench where others can find them. They are sterile, so follow the ___ procedures described in the "Microbiology 101" page. In particular, do not stick your hand in the microfuge tube beaker and keep the lid closed on the pipette tip boxes.

micropipetters Tissues tips ice back NOT need center tips tubes sterile

Important S.I. Units It will be assumed throughout this course that you are familiar with the common S.I. (i.e. metric) units: grams, liters, meters, and molarity as well as their prefixes from nano- through kilo-. You should be able to convert between units using a systematic method (such as ratios or the factor-label method) and perform calculations with appropriate unit manipulations. Since it is assumed that you already have these skills, they will not be taught so if you are having trouble in this area, please seek help from your TA or the course director. The vast majority of the measurements in this course will be made in either ___ (ml) or ___ (µl) and you should be able to instantly and effortlessly convert between these two units in your head. You also must know how big a centimeter and millimeter are without referring to a ruler.

milliliters microliters

Microcentrifuge Tubes Microcentrifuge tubes (also called Eppendorf tubes or "microfuge" tubes for short) are commonly used to hold and ___ small amounts of liquid. (They may or may not actually be used in a microcentrifuge.) In this laboratory, one may assume that microfuge tubes are ___ and free of contaminants (such as protein, DNA or RNA). In order to keep the tubes uncontaminated by materials from your skin, you should ___ remove a tube by reaching your hand directly into the storage beaker. Rather, remove the ___ lid and pour one (or a few) microfuge tubes into the lid, pick up the one tube that you will use (being careful to only touch the ___ of that tube), and return the rest of the tubes by pouring them in the beaker from the foil lid. Replace the foil lid to avoid possible ___ contamination.

mix sterile NOT foil outside airborne

Vortexer A vortexer (or "Vortex Genie") is a device used to facilitate ___. To use a vortexer in ___ mode (as we nearly always do), make sure that the switch is set to pulse and press the tube containing the solution to be mixed down on the rubber cup on the top of the machine. The cup will vibrate as long as it is ___. The speed of the vortexer can be ___ by rotating the knob on the front. Usually the best speed is the fastest speed that will not slop the liquid out of the top of a tube (if it is ___). When do you use a vortexer? A vortexer can be used to mix ___ amounts of liquids in a ___ tube or ___ tube. A vortexer is particularly useful in ___ centrifuged bacterial pellets which may be difficult to dislodge. Most bacteria (such as the Escherichia coli that we commonly use) are quite ___ and are not harmed by the agitation. In contrast, larger cells that are more fragile may be ruptured by the agitation. A vortexer should never be used to mix solutions containing ___, because the forceful agitation can ___ the enzyme's activity. In the case of enzyme solutions, the "___ flick" method of mixing is more appropriate. In either case of mixing, if the liquid is dispersed on the sides of the tube, the microfuge tube may need to be spun in a microcentrifuge for a few seconds to collect the liquid in the bottom of the tube again. Also, for very tiny amounts of liquid (fewer than about ___ µl) it is usually more effective to mix the solution by simply sucking the liquid in and out of the micropipette tip several times.

mixing pulse depressed adjusted open small test microcentrifuge resuspending robust enzymes decrease finger 20

Microcentrifuge Although we will not be using a microcentrifuge ("microfuge") for this week's experiment, we will use it commonly later in the semester. The purpose of a microfuge is the ___ of a vortexer. We use a microfuge when we want to force a ___ material into a ___ in the bottom of a microfuge tube. Two materials that we commonly centrifuge are ___ and precipitated ___. Because of the high speed at which the microfuge operates, it is critical that the centrifuge be "___" and that the lid be closed properly before turning it on. In order to balance the microfuge, each tube in the microfuge must have another tube directly opposite of it in the rotor. If other students are also centrifuging a similar amount of material as you, you can just balance your tube with somebody else's. However, if there are an odd number of tubes to be centrifuged, then you must put a "dummy" tube opposite the odd sample tube. If the tube contains a negligible amount of material (i.e. less than __ microliters), you can simply put a closed, empty tube opposite the unbalanced tube. However, if the volume is significant (more than about __ microliters), then you should place a volume of water in the dummy tube that is approximately equal to the volume of the sample it is meant to balance. Knowing which type of microfuge you are using is equally as important as balancing the tubes inside of it. Our lab has a ___ of microfuges which have a variety of lids. In some cases, the rotor must be covered with a plastic cap before closing the lid and you must do so before turning the machine on. It is easy to tell when someone has not followed the guidelines listed here because a loud buzzing or whirring sound can be heard when the microfuge is turned on. An unbalanced centrifuge can "walk" (vibrate) its way off of the tabletop and crash to the floor. Given the cost of a microfuge (over a thousand dollars), we do not want this to happen Although the microfuges can be set for a variety of speeds, we usually just turn them on at ___ speed. The timers on some of the microfuges are not very good, so for short spins of less than a minute, it is often easiest to just turn the microfuge on with the timer set for a long time, then turn the knob to "Off" ___ after timing the spin with a watch or digital timer. (The ___ length of the spin is usually not very critical.) Depending on the ___ of suspended material, you may or may not be able to see the pellet in the bottom of the tube. Because the tube is held in the rotor at an angle, the pellet is not located in the bottom (tip) of the tube. It is oriented off to one ___ of the tube (note: Figure 6.) By noticing the orientation of the hinge of the microfuge tube lid (i.e. toward the center or toward the side), you can ___ the location of the pellet even if you can't see it. Usually, after pelleting a suspension, one wishes to remove the ___ by either pouring or sucking it out with a micropipetter. By knowing the position of the pellet, one can take care not to dislodge the pellet with the tip of a micropipetter. Fortunately, bacteria are relatively "sticky" and so bacterial pellets rarely fall out of their tube. However, large DNA pellets can sometimes slide out and be lost with the supernatant, so ___ care is needed with them.

opposite suspended pellet bacteria DNA balanced 20 20 variety full manually exact amount side predict supernatant more

The Semester Project In addition to the regular weekly lab exercises, in-class assignments and homework, a significant component of this class (about 10% of your grade) will be carrying out a group ___ project under the mentorship of your TA. The general focus of the experiment will be the ___ of antibacterial soap. Over the course of the semester there will be several phases to the experiment: obtaining ___ skills and knowledge, ___ the experiment, carrying out the experiment and collecting ___, data analysis and ___ a scientific paper describing the results. A more extensive timeline and description of the project can be found in the folder titled "Experimental Design Project" (also found here) This week you will begin learning techniques and background that you will need for the project as well as for other weekly experiments. Specifically, you will learn basic ___ and bacteriological lab skills, ___ background, ___ skills and have your first exposure to the scientific literature. You will continue to add to these skills through the 6th week of lab, after which you will carry out the actual experiment. Because you will use Excel, statistics and scientific literature throughout this semester and on into the second semester of the course, the background information on these topics has been placed in the Excel Reference and Statistics Manual and Scientific Literature Guide. At places in the text describing the experiments, you will find links to appropriate sections of these appendices for background and instructions. You are responsible for doing the corresponding ___ from the appendices that are associated with the background for particular experiments. There will be ___ questions over the material in the appendices as well as the regular background. You will eventually read ___ of the appendices as part of reading assignments, so it's fine to read ahead in them at any time. However, make sure that you have read at least through the reading assignment for a particular week.

research efficacy background designing data writing pipetting statistical Excel readings prelab all

Determining Titer by Dilution, Plating and Counting Colonies LO1.5: describe the origin of a bacterial colony and explain why a plate count provides a measure of the titer of the culture used to inoculate the plate. Although ___ readings are a quick and easy way to measure bacterial suspensions, they provide only a ___ measure of titer. While there are some general rules of thumb relating absorbance units to actual numbers of bacteria per cm3, the exact relationship could vary depending on the type of bacteria, type of culture medium, etc. To determine a more ___ relationship requires an actual ___ of the number of bacteria present in a certain volume of medium. This presents some logistical challenges given that the bacteria are practically invisible and that there may be over a million of them present in each cm3 of suspension! Fortunately, a ___ method has been developed for counting bacteria. This method is known as ___ dilution and plating. Before making serial dilutions, the bacteria suspension must be thoroughly ___. This is important because unlike solutions, which remain uniformly mixed indefinitely, suspended materials eventually settle down to the ___ of their containers. If a dense bacterial suspension is left to stand undisturbed overnight, many bacteria can be seen as a white pellet or cloudy mass in the bottom of the tube. Even over shorter time periods the titer of the suspension at the bottom of the tube will be higher than the titer at the top. The best way to re-suspend bacteria for a more accurate count is to use a ___. In our lab the vortexers (a.k.a. Vortex Genies) are sitting on the top of each bench. To ___ the number of bacteria present in a suspension to a reasonable number, the suspension must be ___ a great deal. As a rule of thumb, you can expect that an overnight culture of Escherichia coli will have around 1 x 108 cells per cm3. To reduce this number to a few hundred cells per cm3 would require measuring a very tiny amount of stock suspension into a very large volume of culture medium. It is ___ to do the dilution in several ___ with each dilution using a small fraction of the previously diluted suspension. (Keep in mind that each dilution will introduce systematic ___ into the final measurement. This error should be kept to a minimum by careful measurement and thorough mixing after each dilution.) Each dilution can be described by comparing the volume of suspension before the dilution with the volume present after the dilution. The dilution ___ is the ratio of the ___ volume over the final ___ volume; it is always a fraction less than one. For example, if 10 µl of suspension is added to 990 µl of buffer (for a total final volume of 1000 µl or 1 ml), the dilution factor would be 10 µl/1000 µl or 0.01 or _/100 . A dilution factor can be used to calculate the titer of the final suspension by multiplying the titer of the initial suspension by the dilution factor. (Dilution factors can also be used to calculate final concentrations of solutions from initial concentrations.) The reciprocal of the dilution factor (i.e. the final volume over the initial volume) is also used to describe a dilution. For example, the previous example can also be described as a ___-fold dilution. The total effect of serial dilutions can be calculated from the product of the dilution factors of each of the individual dilutions. For example, two 100-fold dilutions followed by a dilution of 5 µl into 20 µl (final volume of 25 µl; dilution factor 0.2) would produce a combined dilution factor of 0.01 x 0.01 x 0.2 = 0.00002 which is a 50 000-fold dilution. Because of the very large and very small numbers involved, it is usually advisable to use scientific notation (i.e. dilution factor=2x10-5 and 5x104-fold dilution). LO1.4: calculate the dilution factor for a particular dilution and use it to calculate the titer of the resulting suspension. ___ over ___ total volume To get final titer: ___ factor multiplied by the ___ titer After the number of the bacteria present in the diluted suspension has been reduced to a reasonable level, they must be ___. Because of the small size of the bacteria, this would be impossible to do directly- like searching for tiny needles in a very large haystack! Fortunately, there is a simple way to determine how many bacteria were present in a volume of suspension. A known volume of suspension is spread over the surface of a Petrie plate containing nutrient agar. At the location on the agar plate where each bacterium comes to rest, a colony of bacteria will begin to grow radially from the location of the inoculating bacterium. After incubating the plate overnight at 37˚C, the colonies will be of sufficient size that they can easily be seen by the naked eye (Fig. 6). By counting the colonies on the plate, the number of bacteria present in the suspension on the previous day can be ___. These bacteria represent only a tiny fraction of those present in a cm3 of the original undiluted solution, but by considering the amount of solution plated and the combined dilution factor of all of the dilutions used to create the plated solution, the titer of the original solution can be inferred. One common misconception is that the conditions under which the plate is maintained will affect the titer calculation. The amount of time that the plate is incubated at 37˚C will affect the ___ of the colonies (i.e. longer incubation will make the colonies larger and vice versa), but won't affect the ___ of colonies. Likewise, after the overnight incubation the plates can be stored in a refrigerator or cold room (i.e. about 5˚C) indefinitely until they are counted. Their growth rate at that temperature is so slow that the size of the colonies changes little over any reasonable amount of time - again there is no effect of time on the ___ of colonies.

spectrophotometer relative exact count standardized serial mixed bottom vortexer reduce diluted better steps error factor initial total 1 100 initial final dilution original counted inferred size number number

Learning Objective 1.8: explain the difference between what is described by the standard deviation of a population and what is described by the standard error of the mean. STANDARD DEVIATION: ___ of data in population STANDARD ERROR OF MEAN: The standard deviation of ___ means, each resulting from a certain number of ___, is given the special name: the standard error of the mean (or S.E.). measures how far the sample ___ of the data is likely to be from the true population mean.

spread many samples mean

Experimental Procedure: Pipetting Test LO1.2 accurately measure and dispense a specified volume using a micropipetter First ___, immerse, then release, then ___ stop and ___ stop

stop first second

LO1.1 relate the digits displayed on a micropipetter with the volume they represent P20 P200 P1000 Refer to the prelab assignment where you determined the micropipetter settings for the following volumes: P20: 6.2 µl P20: 12.5 µl P200: 33 µl P200: 150 µl P1000: 240 µl P1000: 1 ml red: ___ on 20 ___ on 200 ___ on 1000

tens, ones, decimal hundreds, tens, ones thousand, hundreds, tens 0 6 2 1 2 5 0 3 3 1 5 0 0 2 4 1 0 0 last none first

Instructions for using a micropipetter 1. To set the volume: Hold the Pipetman in one hand and turn the ___ adjustment knob with the other hand until the correct volume shows on the digital indicator. DO NOT ADJUST THE DIAL PAST THE ___ SETTING OR FORCE THE WHEEL WHEN IT DOES NOT TURN EASILY. 2. ___ a disposable tip to the pipette shaft. YOU MUST ___ HAVE A TIP IN PLACE BEFORE USING A MICROPIPETTER!!! The tips come in two sizes (Fig. 3). The ___ size fits either the P20 or the P200. The ___ size fits only the P1000. Press ___ to ensure an airtight seal. (You will know that you do not have the tip on the pipette firmly enough if you are unable to draw any liquid up.) Remove the tip from its storage box slowly to prevent accidentally jerking out the entire rack of tips and scattering them on the floor. 3. Depress the plunger to the ___ stop (Fig. 4, left). This part of the stroke will allow you to draw up the calibrated volume displayed on the digital volume indicator. DO ___ press all the way to the second stop (Fig. 4, right)! Doing so is one of the most common pipetting mistakes and will result not only in inaccurate measurements, but may waste an entire bench's worth of a critical reagent. 4. Holding the Pipetman vertically, ___ the disposable tip into the liquid to be measured. 5. Allow the plunger to return ___ to the up position (Fig. 4, center). NEVER LET IT ___ UP! 6. Withdraw the ___ from the liquid and pull the filled tip along the ___ of the stock container to remove excess liquid from the outside of the tip. You should be able to see liquid in the pipette tip (Fig. 5, left). 7. Dispense the Sample: Touch the tip against the ___ wall of the receiving vessel and depress the plunger slowly all the way to the ___ stop (bottom of stroke, Fig. 4, right), expelling any residual liquid in the tip (Fig. 5, right). Frequently, very small volumes must be mixed with larger volumes of water or a solution. In this case, the small volume should be expelled with the tip below the surface of the solution. The residual liquid coating the inside of the tip can then be ___ out by drawing solution into the tip and expelling it several times. Drawing solutions in and out of the tip is also useful for mixing microliter quantities. 8. With the plunger fully depressed, withdraw the Pipetman from the vessel carefully, THEN allow the plunger to ___ to the up position. Unless the same solution is to be measured into a clean container, ___ the tip. A ___ tip should be used for each sample to prevent carryover or contamination.

volume MAXIMUM Attach ALWAYS small large firmly first NOT immerse slowly snap tip side side second rinsed return discard fresh