Download Micro Lab Unit 3 Notes

Micro Lab Unit 3 Notes
PRACTICE DICOTOMOUS FLOW CHART
Each 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
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BERGEY’S MANUAL
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 268271. 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 CHART
Before 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
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schedule) will be when you can fill in the RIGHT side of the page. It is okay to have incorrect
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.
JOURNAL
While 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 GUIDLINE document from BB and bring it with you for
the next lab period.
UNKNOWN REPORT GUIDLINE (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
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should be “Maintaining Cultures”, with a paragraph below it describing what you did to maintain
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
arrangements
2) Two descriptive charts
3) 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 ID
Select 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 if 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
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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.
DETERMINE OPTIMUM TEMPERATURE FOR YOUR ORGANISM
Inoculate 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).
CREATE A WORKING STOCK AND RESERVE STOCK
Inoculate 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. 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 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. You will use these two slants to observe the pattern of growth in a slant (see p. 238
of 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.
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.
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STREAK FOR ISOLATION
Inoculate 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.
SIZE DETERMINATION
If 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.
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
controls (positive results) for blood agar plates, motility stabs, and thio tubes. Now it is time to
inoculate a sodium thioglycolate tube.
OXYGEN REQUIREMENT TEST:
SODIUM THIOGLYCOLATE TUBES
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.
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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)
CATALASE TEST (Control: positive = Staph aureus)
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 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.
SIMMON’S 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 present
2) Nitrogen source is Ammonium salts instead of peptones in order to test the ability
of an organism to use a single specific carbon source
This is the reaction:
Citrate + NH4  increase in pH, turns the slant blue
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SPIRIT BLUE (control: positive = Staph aureus)
This 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.
STARCH AGAR (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 STAB (Control = Bacillus)
Some organisms produce an enzyme called gelatinase, which breaks down gelatin. If the gelatin is
broken down, it 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.
IMViC
This stands for a series of tests:
1) Indole
2) Methyl Red
3) Voges-Proskaur
4) 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.
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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
media from changing color. But if the organism produces many different kinds of acids, it
overrides the buffer and causes the color to change.
Methyl 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. 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
The 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. When the VP reagent is called
Barritts’s reagent, and includes the use of two substances: alpha-naphthol (18 drops) and
potassium hydroxide (KOH, 8 drops), which 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).
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NITRATE REDUCTION TEST (control is E. coli)
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 tube
2) 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 acid
Reagent B is alpha naphthalamine
Reagent C is zinc powder
A +B Red is
positive
A +B +C Red
is negative
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SKIM MILK AGAR (Control = Bacillus)
Some organisms produce an enzyme called casienase (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.
DECARBOXYLASE BROTHS
(Controls for ornithine: Enterobacter aerogenes = pos; Klebsiella pneumoniae = neg)
The decarboxylase 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).
DNA AGAR PLATE (Control = Serratus marcescens)
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
12
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
Get one SIM tube and use a needle to stab the media with your organism. Next lab period, we will
add 10 drops of Kovak’s reagent to the tube and check for three things on this one tube.
1) H2S (sulfur) PRODUCTION: Certain bacteria produce H2S from cysteine. 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 trypophase 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
13
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.
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).
OXIDATIION-FERMENTATION (O-F) TEST FOR GLUCOSE
We 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
AG
NC
NC
Without oil
AG
A
NC
Results
Ferments glucose
Oxidative
neither
Control
E. coli
Pseudomonas aeruginosa
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.
EXAMINATION FOR COLIFORMS IN WATER
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 Test
2) Confirmation Test
3) Completed Test
14
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 Number
Suppose 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.
No. of Tubes Positive in MPN in the
first
set
middle
set
last
set
inoculum of the
middle set of
tubes
2
3
1
36
2
3
2
43
2
3
3
53
3
0
0
23
3
0
1
39
3
0
2
64
3
0
3
95
3
1
0
43
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.
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 Test
We 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.
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.
15
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 Test
Inoculate 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.
E. coli
Enterobacter
Indole
+
-
Methyl Red
+
-
V-P
+
Citrate
+
P-Glo
You 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
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.
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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.
Plates
LB/amp positive (has plasmid)
LB/amp/arabinose; positive
LB/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 TEST
Enzyme-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.
Enzyme: horseradish peroxidase
Secondary antibody
Either antigen or primary antibody
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.
Procedure
The 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
18
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.
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).
19