Micro Lab Unit 3 Notes

PRACTICE DICHOTOMOUS FLOW CHARTEach person in here is uniquely identified by their name (assuming there are not two people with the same name!). I can call Jim Smith, and only one person will respond. Each organism has a unique name, too. Their first name is their genus, and their second name is their species. But, unlike Jim Smith, I cannot just call out the name of a bacterium and expect it to respond.

So let’s say that I have a list of your names but I am not allowed to communicate with you. I need to be able to pick out Jim Smith and Suzie Slowpoke from this class. What tool would I need? It would be helpful if I had a list of characteristics that uniquely identified each person in this room. But if all I did was to look at each list of characteristics, and then look at each person to see if they match the list, it would be too hard if there were 40,000 people in this room.

Therefore, we need to make this list of characteristics into a dichotomous tree (called a dichotomous key). That means that for each branch of the tree there will be TWO AND ONLY TWO CHOICES. In this exercise, each deck will work as one group to make a dichotomous key that will uniquely identify each member in your group. The first branch of the key might be: Male or Female? That will divide out the sexes in the group. To further subdivide the males, you could ask about eye color. Can you ask: “Brown, blue, or green eyes?” NO! That is not dichotomous. You would have to say “Brown Eyes or Not Brown Eyes”. Then for the branch under “Not Brown Eyes”, you could ask “Blue Eyes or Green Eyes?”

You could also sort people out by age, height, hair color, hair length (don’t say long or short because that is subject to various interpretation; say “shoulder length or less vs. more than shoulder length). Note that you cannot sort by clothing because they might not be wearing that clothing next time. Decide what kinds of characteristics you want to use in your key.

After you go through each characteristic and sort people out, there should be only one person’s name at the end of each branch of the tree. There should not be any branches that are not necessary. When you key is done, I should be able to look at it, then look at any one person, and go through the tree to find that person’s name.

Work as a group to decide how to make your key, and each person should make their own copy of the same key. It might look something like the sample key below:

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SAMPLE DICHOTOMOUS KEY

BERGEY’S MANUAL

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Male

Eyes not brown

Eyes are green

Glasses Antonie van Leeuwenhoek

No glasses Edward Jenner

Eyes are blue

Glasses

6' tall or greater Louis Pasteur

Less than 6' tall

No glasses

Eyes are brown

Glasses Joseph Lister

No glasses Kirby Bower

Female

Glasses

Eyes are green

Hair shoulder legth or longer

Marsha Nirenberg

Hair shorter than shoulders

Margaret Pittman

Eyes are not green

Eyes are blue

25 years old or older

Rebecca Lancefield

Less than 25 years old

Eyes are brown

No glasses

5'6 or taller Alice Evans

Less than 5'6 Ruth Moore

In this unit, you will be given an unknown organism to identify to genus and species. The first thing you will be doing is a Gram stain to make sure only one organism is present. If you have a contamination, we need to know about it before we go any further. In that case, you might need to use selective media to get rid of the contaminating organism, while allowing yours to grow. Once you have performed the Gram stain, you will fill out a pink slip describing your organism’s Gram stain, shape, and arrangement, turn this slip in to your instructor to confirm that you are correct. After your pink slip is confirmed and returned, you can then go to your lab manual, Exercise 39, p. 268-269. If your organism is Gram positive, use the chart on p. 268. It will then ask you to decide if you have rods or cocci. Make that determination, then decide if you see spores or not. Continue to follow the tree until you figure out which GROUP NUMBER your organism fits into. If you have a Gram negative organism, the chart is on p. 269. Once you have identified your organism’s group, find the description of that group number in your lab manual, from pages 268-271. Each description will include a list of several organisms. From that description, you will be able to identify the GENUS of your organism. The description will also tell you what volume and section of Bergey’s Manual you need to look at to identify your SPECIES. Bergey’s Manual is a set of 4 books, but all of the organisms in this class will be in volumes 1 and 2 only. After you know the genus, go to the right volume and section in the Bergey’s books. You will see many pages that describe the many species for that genus…look for the table in that section so you can see at a glance all of the laboratory tests that differentiate each species. That will tell you which laboratory tests you need to perform to identify which species you have.

Bergey’s Manual is a collaboration of experts from all over the world. Phylogenetic classification of bacteria is being worked out by sequencing ribosomal subunits. Even with its discrepancies, the present edition of Bergey’s Manual is the official classification system for bacteria. In the edition we used of Bergey’s, organisms are grouped together in sections based on their phylogenetic relationship, according to their physiology (as demonstrated by the positive and negative lab tests we will perform). When you have an organism that is positive for a particular test, and you look it up in Bergey’s Manual, it may state that your organism sometimes is positive for that test, or that it generally appears in a certain way. Terms like “generally”, “usually”, and “sometimes” refer to the fact that results may vary from one isolate to another.

DESCRIPTIVE CHARTBefore you are given your unknown organism next lab period, we will go through a practice exercise. You will be given a descriptive chart with the laboratory test results already filled in. To see what this descriptive chart looks like, look in your lab manual at Exercise 34, p. 235. In class today, the instructor will hand out these charts, but they are already filled in for you. Start with the procedure listed above to identify the group by using your lab manual. Then identify the genus by using your lab manual. Then go to Bergey’s Manual, find the right table, look at the results of the lab tests on your descriptive chart, and identify your organism’s species. Write down your genus and species, and have the instructor check the key to see if you are correct.

Go to page 235 in your lab manual to see the blank descriptive chart. You will need to scan this and print THREE copies. The first descriptive chart (due date is on your schedule) will be when you can fill in the LEFT side of the page only. The second descriptive chart (due date is on the schedule) will be when you can fill in the RIGHT side of the page. It is okay to have incorrect

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answers on these charts; you do not lose any points for a wrong answer. However, do not leave any lines blank or you will lose points. After turning in each chart, your instructor will grade it and give you the correct answers for your organism so you can proceed with trying to identify your organism, using the correct data. The third descriptive chart should therefore contain all the correct answers. Make that chart nice and neat and turn it in along with your final report. It will serve as the results section of your paper. NOTE: on the back of your third descriptive chart, make a flow chart of how you identified your organism. You can use same format as the flow chart in your lab manual to get you started, but continue it with the details that enabled you to identify your organism to genus and species.

When you get your real organism in the next lab period, you will perform several tests on the first day, and in the following lab period, you will write the results on the left side of a blank descriptive chart. Then take your descriptive chart to the instructor, who will check to see if any of your results are in error before you go farther. Do not leave anything blank on your descriptive chart, or you will lose one point for each blank line. There are no points taken off for any wrong answers, just blank lines. Once your instructor has checked to make sure your results are what they should be for your organism, you can go on to perform the necessary lab tests listed on the right side of the page of your descriptive chart. When your results are in, you again turn in your descriptive chart to your instructor. Once you have correctly identified your organism, you will write a report on that organism. Make sure you are not absent during the next few weeks, because no one is allowed to read your tests or inoculate media for you. Remember, there is one day coming up that you will need to come in on the day after the inoculation to read the result.

JOURNALWhile you are performing tests to identify your organisms, you will need to keep a scientific journal to document each test and result. You will be given a handout to use as your journal. All of your writing on this document must be in pen, just like an actual scientific journal. You are not to use white-out for errors. You also cannot scribble out any errors. If you make a mistake, take your pen and make one single line through the word(s) you don’t want, then you can add the correct word(s). It is okay for your journal to be messy. NOTE: each student only gets ONE journal handout. You must bring it to class each lab period so you can see what observations you need to make and what to record. If you forget to bring it, you must use someone else’s to see what observations you need, write your answers on regular paper, then go home and transfer your answers to the journal, and do not forget to bring it again. Note: You must write the date you performed each test listed in this journal, even though there is no space that says to write the date. Download UNKNOWN REPORT GUIDELINE document from BB and bring it with you for the next lab period.

UNKNOWN REPORT GUIDELINE (Handout)This document shows you how to write your report. The PURPOSE statement is just 1-2 sentences. The purpose is to identify an unknown organism, using a series of tests. The Materials and Methods section will be the longest part of your report. You need to write paragraphs for each of these sections after each lab period. The MATERIALS section is just a list of the materials used during the entire project. That includes media, equipment, stains, reagents, etc. The METHODS section is in paragraph form. Each paragraph should have a heading. One heading should be “Maintaining Cultures”, with a paragraph below it describing what you did to maintain

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your cultures. Then write another heading regarding another method, with your paragraphs below it, etc. Note that the methods section should NOT contain any results.

Instead of writing a section for RESULTS, you will just be turning in a nice copy of your third descriptive chart, after you have recorded all of the data CORRECTLY, plus your journal, plus a flow chart that shows how you identified your organism. In the DISCUSSION section, talk about your organism, where it lives, any diseases it causes, and how you were able to come to the conclusion of what organism it is. Describe that it was positive for particular tests (describe the tests), and which tests were negative, and how those tests told you the name of the organism. When discussing your organism, you should also look at your descriptive chart and discuss the characteristics documented there. Your report will wind up being perhaps 11-13 pages (the method section is long), so start on the first day you get your organism.

Your unknown report is worth a total of 100 points. You will be graded on three things:1) Three points for your score on the pink slip for the Gram stain, cell shape, and

arrangements2) Two descriptive charts3) Your report, which also includes your third descriptive chart, flow chart, and journal

You will mainly be evaluated by the report. It is okay to not have the right organism because one of your tests did not come out right, as long as you followed Bergey’s Manual and came up with a logical conclusion of what your organism should be, based on your lab tests.

For the lab exam, you need to understand what media you used, why it was used, and what the results mean. You also need to know the reagents used, including the names of indicator dyes in the media, etc.

START UNKNOWN IDMETHOD OVERVIEWObtain your unknown stock, and check it for purity by performing a Gram stain. Then use it to inoculate a working stock and a reserve stock. Each week, you will check your working stock for purity by performing another Gram stain before using it to inoculate the media to perform the tests for that day. Use aseptic technique throughout this month. You will not use the reserve stock unless your working stock becomes contaminated.

Then determine the morphological characteristics of your unknown organism by performing a Gram stain, motility stab, capsule stain. If you have a Gram positive rod, you will also need to do a spore stain.

Next, determine the cultural characteristics of your organism by observing the growth patterns in broth and on agar slants and plates. Determine the optimal temperature and oxygen requirements, and determine what type of hemolysis your organism displays.

Next, determine the physiological characteristics of your organism. This will require about 18-20 individual tests to find out what enzymes your organism makes, its fermentation pathway, etc.

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GETTING STARTEDSelect a tube from the rack of unknown organisms, check what ID number it is, and write your name on the sign-up sheet next to the number of your unknown organism. Then write your name on the tube. Be careful not to contaminate this tube today. Once you have your unknown ID number recorded on your journal (lab handout), perform a Gram stain. Since the longest step is air drying, make 3-4 slides and allow them to air dry at the same time, but only use one to perform your Gram stain. That way, if your culture is not decolorized properly, you have several slides ready to go so you can perform another stain quickly. When you observe your organism under the microscope, check to make sure your culture is pure. Sometimes, the Gram stain becomes contaminated or your culture may be contaminated. If you see more than one organism, let your instructor know immediately. If you only see one organism, the next step is to do a Negative stain with India ink, which is the best way to see the arrangements of the organism. When you know the shape (rods or cocci?) and arrangement (singles, clusters, or chains?) go to the front of the lab are pink slips. Take one, and record your results. This exercise is worth 3 points towards your 100 pt. unknown report. On the pink slip, write whether your organism is Gram positive or negative (1 pt.), whether the organism is a rod or cocci (1 pt.), and whether the arrangements (1 pt.) are singles, clusters (staphylo), or chains (strepto). Then hand this pink slip to the instructor, who will tell you what the correct answers should be for your organism. Then record the correct results in your journal and draw the proper pictures there. You may NOT inoculate any tubes or plates until after your pink slip has been approved.

Once you have your pink slip approved, use your unknown broth to inoculate 3 broths, 2 slants, and 2 plates. When you are done with your original unknown broth, return it to the rack where you got it from. It will be stored in the refrigerator in case you lose your culture during the next few weeks. One of the slants you inoculate today will be the one you use to inoculate new media next week. Today is the only time that you will be using your original unknown broth tube.

CREATE A WORKING STOCK AND RESERVE STOCKInoculate 2 TSA slants by using a needle. Obtain the inoculum and place the needle in the TSA slant toward the bottom, and pull a straight line upwards on the surface of the slant. One of these slants will be labeled “working stock”. Your working stock tube is the one used to obtain inoculums for other lab tests after today. Your transfer schedule is this: Each WEEK, you will need to make a new working stock tube so your culture stays young. To make a new working stock, just use your old working stock to inoculate a new tube. After you see growth in the new tube the following week, you need to check for purity by performing a Gram stain. Once you know your new working stock is not contaminated, you can discard the old working stock tube. That means you need to write dates on these tubes. The second TSA slant you will make today will be your reserve stock in case your working stock becomes contaminated.

MORPHOLOGICAL CHARACTERISTICS:

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Gram stain, size determination, motility, capsule stain, spore stain

SIZE DETERMINATIONIf you have a Gram + organism, mix a loopful of it with a loopful of a Gram neg organism whose size is known. If you have a large organism, pick a large organism to compare it with. If you have a small organism, pick a small organism. Estimate the size of yours compared to the known.

MOTILITY TEST (positive is E. coli, negative is Klebsiella pneumoniae)Inoculate a motility stab. Use a needle to obtain the inoculum. Stab the needle into the motility medium, almost all the way to the bottom, then pull the needle back out in a straight line, backing the needle out of the same stab line you made going in. Remember, these need to be incubated at room temperature (25°C). If they are placed at room temperature, the flagella will detach, giving a false negative result for motile organisms. Also remember that motility media uses TTC as a terminal electr4on acceptor. If the organism can use it, the media will turn red, meaning the TTC has been reduced. If there is no red color at all, you will need to do a wet mount or hanging drop to observe the organism directly to determine if it is motile.

CULTURAL CHARACTERISTICS: Growth patterns, temperature, hemolysis, and oxygen requirements

GROWTH PATTERNSUse your working stock and reserve stock to observe the growth patterns of your organisms and record that information in your journal. The terminology to use is on p. 238 in your lab manual. When you have recorded the morphology on your reserve stock, hand it back in to the instructor next week, and it will be kept in the refrigerator. You will not use it except in emergency. Also use your TSB tubes from your optimum temperature experiment to determine the pattern of grown in broth (see page 238 of your lab manual).

DETERMINE OPTIMUM TEMPERATURE FOR YOUR ORGANISMInoculate one loop-full of your organism into 3 TSB tubes. Label one tube 25 °C, one tube 30 °C, and one tube 38°C. Make sure your name is on the tube. These tubes will be used to determine the optimal temperature for your organism. Use the spectrophotometer to calculate their optical density at the next lab period. The tube with the most growth (highest OD) is the temperature they prefer. Organisms that grow well in room temperature as well as body temperature might be opportunistic pathogens. These tubes can also be used to determine their pattern of growth in broth (see p. 238 of your manual).

HEMOLYSIS TEST (Controls: Beta = Staph aureus; gamma = E. coli, alpha = Strep bovis)Inoculate a blood agar plate. Streak for isolation again. You will use this plate to observe colony morphology and hemolysis patterns (see p. 358 in your lab manual). Beta hemolysis means the organism can completely lyse red blood cells and they digest the hemoglobin (pathogenic bacteria), so there will be clear areas around the colonies on your plate. Alpha hemolysis means the bacteria can oxidize the iron in the hemoglobin, which turns the colony green, with NO clear areas. Gamma hemolysis means the organism is non-hemolytic, so there will be NO clear areas, and the colony will not be green. At the front center deck, you can see the

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Controls (positive and negative results) for blood agar plates, motility stabs, and thio tubes. Now it is time to determine the oxygen requirements by inoculating a sodium thioglycolate tube.

SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)This medium has an oxygen gradient, which means that most of the oxygen is at the top of the tube, and the least amount of oxygen is at the bottom of the tube. To prepare this medium, a reducing agent called Sodium thioglycolate was added, which removes the free oxygen by chemically binding with it. Therefore, thioglycolate broth is called a REDUCING MEDIUM. It gets rid of the oxygen. There is also a pink indicator dye called rasazarin that shows you where the oxygen is. Notice that the pink color is only at the top of the tube. We have to be careful not to shake the tube, or we will aerate it (add more oxygen). We need the oxygen gradient to be maintained for a successful test. The results of this test determine what oxygen requirements your organism has.

Procedure:

1. Hold the thioglycolate tube carefully, taking care to move it gently without shaking, jiggling, or stirring them (which introduces oxygen into the medium).

2. Label the tube with your name, the date, the organism, and “Thio” for Thioglycolate. 3. Put some of your unknown bacteria on a sterile loop and gently push the loop straight down

to almost the bottom of your tube.  Do not touch the bottom as this may ruin the loop, and do not introduce air by stirring or shaking the tube! 

4. Gently pull the loop straight out of the tube and sterilize it. 

1. STRICT AEROBES require oxygen to grow. There will only be growth on the surface of the thio broth tube (pseudomonas and Bacillus megatarium)

2. STRICT ANAEROBES require the absence of all oxygen. There will only be growth at the butt (bottom) of the tube (clostridium).

3. FACULTATIVE ANAEROBES grow best aerobically but do not require it. Growth is throughout the tube, but is best at the top and decreases as one descends. (E.coli, staph aureus)

BEGIN PHYSIOLOGICAL TESTS

STREAK FOR ISOLATIONInoculate a TSA plate, using the streak for isolation method (draw 4 quadrants on the bottom of the plate, zig-zag the upper left quadrant, flame the loop, then draw the loop from quadrant 1 (Q1) into Q2 and zig-zag that second quadrant. Flame the loop, then draw the loop from quadrant Q2 into Q3 and zig-zag that third quadrant. Flame the loop, then draw the loop from quadrant Q3 into Q4 and zig-zag that final quadrant. You will use this plate to observe colony morphology on a plate (see p. 240 in your lab manual). At the next lab period, you will also use this plate to perform the catalase and oxidase tests.

CATALASE TEST (Control: positive = Staph aureus)

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Some facultative aerobes have the enzyme called catalase, which breaks down hydrogen peroxide (H2O2) into harmless water plus oxygen. Having this enzyme protects organisms from being destroyed by the H2O2 in the lysosomes of a white blood cell. Your instructor will lift the lid on your agar plate next lab period, and put one drop of H2O2 onto the colony. A positive test will show the oxygen bubbles rising up from the plate. That means the organism has the enzyme, so it is catalase +. See p. 249 in your lab manual. NOTE: do not get catalase mixed up with oxidase. Catalase breaks down into oxygen, but is it not the oxidase test!

OXIDASE TEST (Control: positive = Pseudomonas aeruginosa)Some aerobes have the enzyme called cytochrome oxidase, which is a molecule that is a terminal electron acceptor in the electron transport chain. Your instructor or lab technician will have a piece of paper inside a Petri dish at the side window. On a piece of paper, she will place one drop of the oxidase reagent Dimethyl-p-phenylene diaminic hydrochloride (this substance is carcinogenic, so you will not be using it). Then she will use a toothpick to obtain the organism from your TSA plate, and she will scrape the sample onto the drop of reagent on the paper. The test should be done in comparison to a positive control, because time is essential in the development of the test results. Count the number of seconds it takes to turn purple and record the time in your journal. If purple is observed at any time, it is positive for oxidase. If there is no color change, it is negative. See p. 249 in your lab manual.

NOTES: Even if you figured out what organism you have, you need to continue to perform the assigned experiments. Know which tests show what color on a positive test: Brown, Orange or Red, Blue, Yellow, Diffused black pigment, Pink ring on top, etc. Know what reagents are used, what substrates in the media are broken down, and what the products are.

CITRATE TEST (Control: positive = Enterobacter aerogenes)Citrate is the sole carbon source in this medium. If the organism can use citrate as its only carbon source, the medium will become basic. The medium starts out green and turns blue if it is a positive test. It may only be blue at the top, which is still positive. Acid = green (negative) and base = blue (positive). A negative tube will also show no growth. Ingredients in the medium include

1) Indicator dye is Bromthymol blue, which is green when acids are present2) Nitrogen source is Ammonium salts instead of peptones in order to test the ability

of an organism to use a single specific carbon sourceThis is the reaction: Citrate + NH4 increase in pH, turns the slant blue

LIPASE (control: positive = Staph aureus)The Spirit Blue media has lipids. If the organism has the enzyme lipase, fatty acids will be released, and pH will decrease (become acidic). This will precipitate the blue dye. A positive result is a dark blue streak in the center of the plate where you inoculated it. If lipase if produced, the concentration of the blue will increase where it was inoculated. Having clearing is NOT a positive test; it should be darker blue.

NOTE: If you have a Gram positive organism, and if it is not a spore former, you need to do an acid-fast stain. If it is positive, you have a mycobacterium, which is not really a Gram negative organism. Negative stains are done if there is a question about the arrangement of your organism. You can see their arrangements best with a negative stain.

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STARCH (Control = Bacillus)This media has starch. Some molecules, such as starch, cannot be taken into a bacterial cell because the molecules are so large. The organism can only use starch if it has an enzyme, called amylase, which can hydrolyze (break down) the starch into simple sugars that can be absorbed into the cell. We will flood the plate with iodine, which reacts with starch and turns it black. If the organism has the enzyme, there will be no more starch left, so there is nothing for the iodine to react with. Therefore, the presence of amylase will show up as a halo (area of clearing) around the organism (positive test). If the organism could not use the starch, the starch forms a complex with iodine to give a black precipitate around the organism. That means the organism is negative for amylase. NOTE: the black color only lasts a few minutes, so you have to read the test right away before the color disappears.

GELATIN (Control = Bacillus)Some organisms produce an enzyme called gelatinase, which breaks down gelatin. If the gelatin is broken down, it becomes liquefied, and can no longer solidify, even when cooled in the refrigerator. Gelatin is a protein, so gelatinase is a protease. Gelatin is the only thing making the media solid. If it remains liquid, even after refrigeration, it is positive. Solid is negative.

IMViCThis stands for a series of tests:

1) Indole2) Methyl Red3) Voges-Proskauer4) Citrate

The small “i” does not stand for anything; it just makes pronunciation easier.The IMViC tests are used to identify an organism in the coliform group. A coliform is a gram negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours. The presence of some coliforms indicates fecal contamination. We will perform the indole test as part of the SIM media. We performed the citrate test in the Simmon’s Citrate media. Now we need to perform the MR-VP test to complete the IMViC series.

MR-VP TEST (Methyl Red/Voges-Proskauer)We do two tests with this medium: The MR test and VP test. We will inoculate one MR-VP tube today, let the culture grow until the next lab period, and then add 5 drops of Methyl Red to perform the MR test. In the next lab period, we will inoculate a new MR-VP tube, let the culture grow, and then add alpha-naphthol and potassium hydroxide reagents to perform the VP test.

We are looking for glucose fermentation. Bacteria convert glucose to pyruvate using different metabolic pathways. One pathway produces unstable acidic products which quickly convert to neutral compounds. Another pathway (the butylene glycol pathway) produces neutral end products, including acetoin and 2,3-butanediol. A third pathway is the mixed acid pathway, which produces stable acidic end products which remain acidic. If an organism produces a lot of acid from the fermentation of sugars, it can override the buffer in the test media. If this happens, the amber media will turn red. MR-VP broth differentiates organisms that are single acid fermenters from organisms that are mixed acid fermenters because it contains over-riding buffers that affect organisms that are single acid fermenters. An organism that produces only one type of acid after sugar fermentation will not produce much acid, so the buffer blocks the

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media from changing color. But if the organism produces many different kinds of acids, it overrides the buffer and causes the color to change.

MR = METHYL RED TESTMethyl Red is a yellow colored pH indicator which turns red if the organism uses the mixed acid fermentation pathway, which is that pathway that produces stable acidic end-products. If positive, the enzyme present is formic hydrogenylase. The acids will overcome the buffers in the medium and produce an acidic pH. When methyl red is added, it will go from yellow to red, which is positive for an organism that uses the mixed acid fermentation pathway. (Control: E. coli = pos; Enterobacter aerogenes = neg)

NOTE: Methyl red differs from Phenol red Methyl Red: starts off yellow, turns red when acids are present (indicating glucose fermentation)

Used in MR-VP test (the first part of the test) for mixed acid fermentation

Phenol Red: starts off red, turns yellow when acids are present (indicating glucose fermentation)Used in Urea broth and in the Fermentation broths

VP TESTThe VP test is an indirect method of testing for non-acid end products of glucose fermentation. It detects organisms that utilize the butylene glycol pathway and produce acetoin. We cannot test for butylene glycol, but we can test for acetoin. The VP reagents are called Barritts’s A (alpha-naphthol; 18 drops) and Barrett’s B (potassium hydroxide; KOH, 8 drops). These are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol fermentation pathway, the acetoin end product causes a rust or red color (Gram negatives tend to do this). Therefore, red is a positive result, colorless is negative. Shake gently after adding the reagents, wait 15 minutes, then shake again. (Control: Enterobacter aerogenes = pos; E. coli = neg)

UREA BROTH (Control = Proteus vulgaris)This test checks for the enzyme called urease, which breaks urea down into ammonium and carbon dioxide (water is not a product of this reaction). The ammonium will increase the pH. The medium has a pH indicator called phenol red. When pH goes up, it will turn bright pink (positive).

CASEIN TEST (skim milk) (Control = Bacillus)Some organisms produce an enzyme called caseinase (a protease), which breaks down the protein that makes milk white. It breaks the protein down into small peptides that can be absorbed into the cell. Do a heavy streak in the center. Positive is a clearing (halo) around the area of growth of the organism because the milk is broken down and the white color disappears. Casein is what makes milk look cheesy when it is left unrefrigerated.

NITRATE REDUCTION TEST (control is E. coli)

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If nitrate (NO3) has an oxygen molecule removed, it has been reduced. The new molecule is nitrite (NO2). Nitrite can also be reduced to nitrogen gas (N2) if it loses oxygen. The reactions look like this:

NO3 (nitrate) NO2 (nitrite) N2 (nitrogen gas)

If an organism has the enzyme called nitrate reductase, it can reduce nitrate like this:NO3 (nitrate) NO2 (nitrite)

Is this enzyme clinically important? Not really. Some of you have brown eyes and some of you do not have brown eyes. It serves as a way of classifying organisms on a flow chart.

The nitrate broth we started with contains nutrients plus NO3, and it is clear. If is reduced, the tube is still clear, so how can we tell if NO3 was reduced to NO2? If the organism has nitrate reductase, it will reduce NO3 to NO2 so there will be no more NO3 present , just NO2. First, we add reagent A to the tube. Reagent A will bind to NO2 , forming a complex. However, this complex is clear also, so it does not tell us anything. Then we add Reagent B, which turns the complex a red color. If you add reagents A + B and the tube turns red, the organism has nitrate reductase.

However, some organisms with that enzyme reduce NO3 all the way to N2. They take all of the nitrate and reduce it all the way to nitrogen gas, as seen in this equation:

NO3 (nitrate) NO2 (nitrite) N2 (nitrogen gas)

In this case, there will be no NO3 or NO2 in the tube, so there is nothing for reagent A to react with, and reagent B will not turn the tube red, even though the organism has the nitrate reductase enzyme. Therefore, if you add Reagent A + B, the tube will be clear, yet the organism has the nitrate reductase enzyme. So, although a red color is a positive test, a colorless tube is NOT a negative test.

When the tube is colorless, there are two possibilities:1) The organism does not have nitrate reductase, and there is still NO3 in the tube2) The organism has nitrate reductase, and there is no NO3 or NO2 in the tube.

If a tube is colorless after adding Reagents A + B, we need to test the tube to see if there is NO3 in the tube. We do this by adding a little zinc powder by scooping some on the flat end of a toothpick and adding it to the tube. Zinc will react with NO3 if it is present (reduces any residual nitrate to nitrite) and it will turn red. Zinc is used to confirm a negative test.

Reagent A is sulfanilic acidReagent B is alpha naphthalamineReagent C is zinc powder

DECARBOXYLASE BROTHS

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A +B Red is positive A +B +C Red

is negative

(Controls for ornithine: Enterobacter aerogenes = pos; Klebsiella pneumoniae = neg)This tests for the presence of the enzyme decarboxylase. This test is useful for differentiating the Enterobacteriaceae. This enzyme removes and digests the acidic carboxyl group (COOH) from amino acids, plus cleaves off NH3, which will raise the pH. The pH indicator is bromcresal purple. The media is made to start out slightly acidic (pH 6). Bromcresal purple is yellow when acids are present and purple when bases are present.

Three tubes are inoculated. Each tube contains glucose plus one amino acid; either lysine, arginine, or ornithine. The carboxylase reaction requires an anaerobic environment, so each tube needs to be covered will a layer of sterile mineral oil to prevent air from reaching the culture.

NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR AND YOU MIGHT GET THEM MIXED UP!

Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine decarboxylase, and ornithine decarboxylase using three different but very similar media.If an organism is able to decarboxylate the amino acid present in the medium, alkaline byproducts are then produced. Ornithine decarboxylation yields putrescine (named after its putrid smell).Lysine decarboxylation results in cadaverine (smells like a cadaver). These byproducts are sufficient to raise the pH of the media so that the broth turns purple (in 48 hours). If you check it in 24 hours, you might see that it is yellow because it fermented the glucose in the medium, but that does not mean it is a negative test. You have to check it in 48 hours to allow the decarboxylase activity to occur. If the pH becomes alkaline because the organism has the decarboxylase enzyme, the media will turn purple in 48 hours (pos).

DNASE TEST (Control = Serratus marcescens or Staph aureus)This tests for the presence of the enzyme, DNAse. It contains the indicator dye, Methyl green complexed with DNA. Digestion of DNA releases the dye, so in otherwise green agar, a clear halo formed around the growth indicates a positive test.

PHENYLALANINE AGAR SLANT (Control = Proteus vulgaris)We are looking for the enzyme, phenylalanine deaminase, which removes an NH2 group from cysteine to produce pyruvic acid, ammonia, and hydrogen sulfide. When 5 drops of ferric chloride is added to this, it will turn green, indicating a positive test. A negative test stays yellow. Don’t get this mixed up with the SIM media, where ferrous sulfate turns the media black.

SIM MEDIA

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Get one SIM tube and use a needle to stab the media with your organism. Next lab period, we will add 10 drops of Kovac’s reagent to the tube and check for three things on this one tube.

1) H2S (sulfur) PRODUCTION: Certain bacteria produce H2S from the enzyme cysteine desulfurase. When the H2S reacts with ferrous sulfate in the medium, a dark precipitate of iron sulfide is produced and the media will turn black (positive for H2S production). (Control = Proteus vulgaris). Don’t get this mixed up with the phenylalanine test, where the addition of ferric chloride turns the media green.

2) INDOLE PRODUCTION: Tryptophanase breaks tryptophan (an amino acid) down into indole, pyruvic acid, and ammonia. If tryptophnase is present, the indole end product reacts with the reagent we will add next time (10 drops of Kovac’s reagent). If a red ring forms at the top of the tube, it is positive for indole, so the organism makes tryptophanase. (Control = E. coli).

3) Motility: You have already done a motility test, but this media will show you again if your organism is motile. The red dye is not in this media, so visualization is harder.

FERMENTATION BROTHS (Control = E. coli is AG)NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR AND YOU MIGHT GET THEM MIXED UP!

We are looking for fermentation with acid (A) or acid + gas (AG). If there is fermentation, it will be yellow. If there is gas, the inverted miniature tube inside the media will fill with a gas bubble. If there is no fermentation, it is red, so record it as no change (NC) or Alk (protein digestion).

The medium has a Durham tube (a miniature tube that is inverted on the inside of the test tube). If gas is produced, it will form a bubble inside the inverted tube. It also has phenol red as an indicator. Phenol red turns yellow if acid is present, and red if bases are present.

Inoculate one each of the following tubes: glucose, lactose, mannitol, sucrose, and trehalose. After 24 hours, if the inoculated medium is yellow, it fermented the sugar in that tube. It may or may not have produced gas. Gas is produced during sugar fermentation, so when gas is present, fermentation is present as well, but not all organisms ferment with gas. If it is yellow, record it as (A). If it has gas in the Durham tube (a bubble that take up 10% of the tube, not a little bubble), record it as (AG). If it did not turn yellow (stays red), you have to look at it again in another 24 hours. After 48 hours, if the media is still red, the organism is negative for fermentation of that sugar.

These tubes must be read in 24 hours, because in 48 hours, any change in color will revert to the original color.

This is what happens: Some organisms that ferment sugars can also digest proteins. When these organisms begin to ferment a sugar, the media becomes acidic (yellow in 24 hours), which enables them to begin digestion of the proteins which are in the media. When proteins are digested, the media becomes alkaline, and the media will turn back to red. If you want to know if it fermented the sugar, you need to read the tube in 24 hours.

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Suppose a student did not observe their tube right away, and then they see that it is red but it has gas. Since the gas is present, that indicates that it probably fermented the glucose (turned yellow at 24 hours, but he missed it), and then the organism proceeded to digest the protein, turning the media alkaline (back to red again). That would explain why it was red, but has gas (gas is produced during the fermentation process).

OXIDATION-FERMENTATION (O-F) TEST FOR GLUCOSEWe are looking for the ability to ferment or oxidize glucose. The pH indicator is Bromthymol blue, which is yellow when acid is present. You will STAB two O-F tubes of glucose. One tube will need a layer of sterile oil to create an anaerobic environment so we can check for fermentation. The other tube will not have oil, so we can have an aerobic environment to check for oxidation. Next time, you will see if it turns yellow. If it is yellow, record it as “A “ (acid present). If there is gas in the tube, also record “G”. If there was no change (stayed green), write “NC”.

With oil Without oil Results ControlAG AG Ferments glucose E. coliNC A Oxidative Pseudomonas aeruginosaNC NC neither Alcaligenes faecalis

NOTE: According to your lab manual flow chart, know what the first test is after a person has identified if their organism is Gram positive (do a spore stain) or Gram negative (use a thioglycollate broth to determine oxygen requirements).

Note: We are boiling down all you tubes today. DO NOT THROW AWAY THE DURHAM TUBES! To collect them, pour out your fermentation tubes through the strainer provided at the trash can. That will cause the Durham tubes to fall into the strainer. Do not clean them; just leave them in the strainer. Many students break test tubes while cleaning up these tests. This happens because they are trying to handle too many tubes at once. Be careful!

NOTE: Be able to match enzymes to their tests, substrates, reagents, and controls. You should make a table to study from. In one column, put the name of the test. In the other columns, put the substrates, products, enzymes, reagents, etc., and what a positive and negative result would look like.

END OF MATERIAL FOR IDENTIFICATION OF UNKNOWN BACTERIUM

EXAMINATION FOR COLIFORMS IN WATER

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Pubic drinking water supplies must be monitored for the presence of coliforms (bacteria that are found in our bowels). Coliforms are Gram negative facultative rods that ferment lactose, producing acid and gas. If ingested, they cause serious illness. If they are present in drinking water, the CDC will shut down the facility until the problem is cleared up. The test for coliforms in water has three phases:

1) Presumptive Test2) Confirmation Test3) Completed Test

Presumptive Test (work in pairs)Place 10 ml of water into each of three tubes of double-strength lactose.Place 1 ml of water into each of three tubes of single-strength lactose.Place 0.1 ml of water into each of three tubes of single-strength lactose.NOTE: this is not a serial dilution. The same amount is in each tube.These need to incubate for 18 hours, then we will take them out of incubation and place them in the refrigerator. At the next lab period, and count the number of tubes in each set that have gas. Then we will use a table (Ex 45, p. 314) to calculate the Most Probable Number (MPN). Then we will go on to the confirmation test, because right now, all we know is that we have bacteria that ferment lactose. They may not be Gram negative rods.Most Probable NumberSuppose you had 2 tubes with gas in the first and last set, and 3 with gas in the second set.Your MPN would be 43. That is the number of organisms per 100 ml.

NOTE: The table in the lab manual does not contain the combination 3-3-3, so if that is the combination you get, use the combination 3-3-2 in your lab manual.

Confirmation TestWe will use two media which are selective and differential. Each group of two people will work with one of each plate. Use the water sample to streak for isolation on the plates.

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To use the table, write down the number of positive tubes in the first set, the number of positive tubes in the second set, and the number of positive tubes in the third set. Then find that number combination on the table and record the MPN for that combination. This is just a small section of the table.

No. of Tubes Positive in MPN in theinoculum of the

middle set oftubes

firstset

middleset

lastset

2 3 1 362 3 2 432 3 3 533 0 0 233 0 1 393 0 2 643 0 3 953 1 0 43

EMB is selective because it has eosin and methylene blue, which only grow Gram negative organisms. It is differential because it shows lactose fermentation. Colonies with a black center is a positive confirmation test, since only coliforms will form colonies with black nuclei on EMB agar. When they show a green metallic sheen, that is positive for lactose fermentation.

MacConkey’s agar is selective because it has crystal violet and bile salts, which only grow Gram negative organisms. It is differential because it shows lactose fermentation by turning a pink color. Pink is positive for lactose fermentation.

Whether the confirmation tests were positive or not, we go on to the completed test, because right now, all we know is that we have Gram negative bacteria that ferment lactose. The may not be rods.

Completed TestInoculate a lactose broth and a TSA slant. Next time we will do a Gram stain. Take a needle; touch it to one of the colonies and do a Gram stain to check for morphology. If we have a Gram negative rod that we know is a lactose fermenter, which means we either have Enterobacter (not a coliform) or E. coli (which is a coliform). Now we have to do an IMViC test to see which one we have.

Indole Methyl Red V-P CitrateE. coli + + - -Enterobacter - - + +

P-GloYou should have downloaded the PGlo handout. Work in groups of four.

The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is only expressed in the presence of arabinose, which makes the transgenic organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.

pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)-GFP, the green fluorescent protein

P-Glo is a transformation exercise. We briefly talked about plasmids and transformation in lecture. Some organisms have the ability to take up DNA from their environment. We will use E. coli and

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force it to take up DNA in the form of a plasmid. In E. coli, there is a promoter region for ampicillin resistance. Another promoter region contains the gene for the sugar, arabinose (inducible operon). E. coli requires arabinose to turn the gene off.

We have a plasmid which contains the gene from a jellyfish that allows the organism to glow under florescent light. We are going to introduce the gene into the E. coli, and grow them in plates (LB instead of TSA). Two plates will have E. coli which are ampicillin resistant, two plates will have E. coli which are not ampicillin resistant. One of each of those plates will have E. coli which has had the plasmid inserted, the other will not. One of the other two plates (positive; contains the plasmid) will contain arabinose, the other will not. We will be incubating in the presence of ampicillin, and that will select for those organisms which can take up the plasmid. We will also be incubating for the presence of the sugar, arabinose, because it is required to turn the gene on. If there is no gene for ampicillin resistance or arabinose, it should not be able to glow.

Take one colony and place it in one of each of the pink and blue tubes.The technician will put plasmid in the pink tube. Put both of the tubes into ice for 10 minutes to force the transformation. Each tube also contains calcium chloride, which pokes holes in the outer membrane, allowing the DNA to gain access to the inside of the cell. Now we have to shock the cell to cause the DNA to be taken up. To do this, put them into the water bath at 57 degrees for 50 seconds, then back on ice for 2 minutes. Then collect your tubes and add 250 µl of LB broth to both tubes and incubate at room temperature for 10 mins. This allows for replication of the cell and incorporation of the plasmid. Then pipette 100 µl from the blue tube to the two negative plates (contain no plasmid; one has arabinose and one does not), and pipette 100 µl from the pink tube to the two positive plates (contains plasmid; one has arabinose and one does not). Next lab, we will expose them to UV light to excite the proteins and see which ones show green fluorescence. The only plate that should glow is the one with the plasmid plus arabinose.

PlatesLB/amp positive (has plasmid)LB/amp/arabinose; positiveLB/amp negative (before transformation, they have no amp resistance. Should not glow)LB negative (just a media like TSA; will grow, but no glow)

The promoter region (can turn off and on; is an operon. It is turned off and on by arabinose).The non-transformed cells do not have the ampicillin resistance gene.

Negative Positive

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ELISA TESTEnzyme-linked immunosorbent assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antigen in a sample. An unknown amount of antigen is affixed to a surface, and then a primary antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

ELISA tests could also use an antibody instead of the antigen. In this case, there will be two sets of antibodies, so we call them primary and secondary antibodies. The primary antibodies will be attached to the plastic plate, and then the secondary antibodies will attach to the primary antibodies. The secondary antibodies will then be conjugated to the enzyme, horseradish peroxidase, which will create a color change when a substrate is added. An ELISA test can tell us whether or not particular antigens or antibodies are present in the sample (qualitative). However, we cannot measure how many antigens or antibodies are present (quantitative) unless we perform a serial dilution.

For this exercise, you will receive a fluid sample that you pretend is from your body. It is labeled with a number; write that down. One of these samples is positive for an antigen (we are pretending it is positive for HIV). Then you will go around the class (between decks too!) and pretend to transfer body fluids. You put your fluid in someone else’s tube, mix it, and take half of it back. After the whole class has donated once, then go around and donate a second time. Keep track of who you donated to, and who you received donations from. You can donate only twice but you can be the recipient as many times as you want. After performing the ELISA test, if your sample is positive, track down where you might have gotten it from. Try to figure out who had the positive sample in the beginning.

ProcedureThe 96 well plates have letters down the left side and numbers across the top. ELISA uses positive and negative controls. The first three wells on the top row are used for positive controls, the next three for negative controls. The patient’s fluid samples are done in triplicate, so each student will take up three wells. Decide with the other people in your deck as to who will use which of the leftover wells in the plate; write your well numbers down on a table. ELISA’s are run in triplicate for several reasons: to circumvent the possible false negatives, to circumvent the possible false positives, and to increase statistical significance of the reactions.

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Either antigen or primary antibody

Secondary antibody

Enzyme: horseradish peroxidase

Once everyone in your deck has added their fluid to their assigned wells, let them incubate for 5 minutes and wash it with a buffer that will wash out any unbound antigen. Then put in a primary antibody (since we are pretending the antigen is HIV, the primary antibody would have to be an anti-HIV antibody). Let it incubate for 5 minutes to allow it to bind to the antigen if the antigen is present. Wash again with wash buffer, which will wash away any unbound antigen. Then add a secondary antibody. These are anti-human antibodies; antibodies against human antibodies. These secondary antibodies also have a horseradish peroxidase (HRP) enzyme attached to them. Allow the tray to incubate another 5 minutes, then wash with the buffer. Now add the substrate, which will bind to the HRP if HRP is present. When HRP comes in contact with the substrate, the color changes to blue. If the blue color appears, it means that the substrate found HRP to bind to. If HRP is present, the secondary antibodies must be present. If the secondary antibodies are present, that means the primary antibodies are present. If the primary antibodies are present, that means the antigen is present, so a color change is positive for the antigen (which we are pretending is HIV).

END OF MATERIAL FOR UNIT THREE

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