Download Micro 2 transcripts to be made into flashcards

Chapter 45 notes: Skin bacteria
The dry keratin layers of the skin are not easily colonized by most microbes. Sebum from oil gland and
salts in perspiration inhibit the growth of most microbes. Only a few types of bacteria (normal resident
microbiota) can tolerate the dry keratin and the salty (hypERtonic) perspiration of the skin. The most
common type of bacteria found on the skin are Gram positive, salt-tolerant organism such as
Staphylococcus aureus. More bacteria are found in moist areas, such as the axilla and the sides of the
nose. Transient microbes may be present on the hands and arms because of contact with the environment,
but they do not live on our skin for long. Propionibacterium live in hair follicles on sebum produced by
our oil glands. The propionic acid they produce maintains the acidic pH of our skin (pH 3-5), which
suppresses the growth of other bacteria. Most bacteria on the skin are Gram positive and salt tolerant.
Mannitol salt agar (MSA) is selective for salt-tolerant organisms and it is also considered to be a
differential media because mannitol-fermenting organisms will produce acid, turning the indicator in the
media yellow. Staphylococcus aureus is part of the normal microbiota of the skin and is a pathogen. If the
skin becomes broken, it can invade and cause disease. Both pathogenic and non-pathogenic species of
Staphylococcus produce catalase and collagenase, and they are all oxidase negative. Staph aureus makes
an enzyme called coagulase, which clots the blood (clumping), whereas other species of Staphylococcus
do not produce this enzyme and are not pathogenic. If MSA grows an organism, we know it is
Staphylococcus, but then we do a coagulase test to see if it is Staph aureus or not.
Chapter 46 notes: Respiratory tract bacteria
The lower respiratory tract (larynx, trachea, bronchial tubes, and alveoli) is normally sterile, but the upper
respiratory tract (nose and throat) is in contact with the contaminated air we breathe. The throat is a
warm, moist environment, allowing many bacteria to thrive. It is normal to find Staphylococcus,
Streptococcus, Neisseria, and Haemophilus as normal microbiota in the throat. However, we do not often become ill
because of their antagonism toward each other. They suppress each other’s growth by competing for nutrients and
producing inhibitory substances. Streptococcal species are the predominant organisms in throat cultures, and some
species are the major cause of sore throats. Pathogenic bacteria may produce hemolysins, which breaks down red
blood cells.
HEMOLYSIS TEST (Controls: Beta = Staph aureus; gamma = E. coli, alpha = Streptococcus bovis)
Inoculate a blood agar plate using the streak for isolation method. You will use this plate to observe hemolysis
patterns. Beta hemolysis (Streptococcus pyogenes, Staphylococcus aureus) means the organism can completely
lyse red blood cells and they digest the hemoglobin (they are pathogenic bacteria), so there will be clear areas
around the colonies on your plate. Alpha hemolysis (Streptococcus pneumoniae) 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.
Streptococci that are alpha or gamma hemolytic are usually normal microbiota, whereas beta-hemolytic streptococci
are usually pathogens. More than 90% of streptococcal infections are caused by Beta-hemolytic Group A
Streptococcus pyogenes, which is sensitive to the antibiotic bacitracin, whereas other streptococci are resistant to it.
EVALUATION OF MSA PLATE
Growth on MSA indicates Staphylococcus (it’s the only genus that grows on high salt levels; that’s what
makes MSA selective media), and if the organism can ferment mannitol (a sugar), the waste product (an
acid) will turn the pH indicator yellow (that’s what makes the agar differential).
The yellow color in the media will differentiate the species of Staphylococcus. Staph aureus and Staph
saprophyticus (yellow colonies) are mannitol fermenters, while Staph epididymis and Micrococcus (white
colonies) do not ferment mannitol. You could then do a coagulase test (S. aureus would be the only one
positive).
24
If you then placed a Novobiocin antibiotic disc on the non-fermenting colonies and a clear zone develops,
the organism is Staph epidermidis. A Gram stain would show Micrococcus as being Gram positive cocci
that are very tiny.
Then do a catalase test to differentiate streptococci (negative) for staphylococci (positive).
Take a toothpick, scoop up a colony, and place it on a glass slide. Use a pipette to add a drop of hydrogen
peroxide to the colony. Bubbles are a positive test, which means that organism has the catalase enzyme,
so it can break down hydrogen peroxide in the lysosomes of our WBCs; that is a virulence factor.
H2O2  H2O + O2 (water and bubbles)
HOW TO ID AN UNKNOWN ORGANISM
Your goal with the enteric unknown is to determine the genus of the organism you have.
Individual tests can be performed in separate tubes (conventional method) or all the tests can be
performed in one specially designed Enterotube (rapid identification method). Comparisons
between rapid identification methods and conventional methods show that they are equally
accurate. 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 (see sample table below).
CHARACTERISTICS OF ALL ENTEROBACTERACEAE
- Gram negative rods
- Oxidase negative
- Catalase positive
- Fermentation of glucose (positive)
- Reduce nitrate (NO3; an inorganic substrate in the API tube. Reduction = positive)
- facultative anaerobes
- If they are motile, they have peritrichous flagella, so they can run and tumble.
25
METHOD OVERVIEW
Determine the morphological characteristics of your unknown organism by performing a Gram
stain, motility stab, capsule stain, and negative stain. If you have a Gram positive rod, you will
also need to do a spore stain, since only Gram positive rods make spores (only certain species).
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 1820 individual tests to find out what enzymes your organism makes, its fermentation pathway, etc.
MORPHOLOGICAL CHARACTERISTICS:
Gram stain, size determination, motility, capsule stain, spore stain
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.
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 electron 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. NOTE: an
Enterotube can test for MRVP, citrate, indole, carbohydrate and protein catabolism, but a
motility test cannot be performed in an Enterotube.
NOTE: After a person has identified if their organism is Gram positive, the next test is to
do a spore stain. If it has no spores, then you need to do an Acid Fast stain to see if it is
Mycobacteria. After a person has identified if their organism is Gram negative, the next
test to do is to use a thioglycollate broth to determine oxygen requirements.
CULTURAL CHARACTERISTICS:
Growth patterns, temperature, hemolysis, and oxygen requirements
GROWTH PATTERNS
Use 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 in your lab manual. When you
have recorded the morphology on your reserve stock, it will be kept in the refrigerator. You will
26
not use it except in emergency. Also use your TSB tubes from your optimum temperature
experiment to determine the pattern of grown in broth.
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.
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.
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)
27
PHYSIOLOGICAL TESTS
NOTE: Know which wells have what color of a positive test: Brown, Orange or Red, Blue,
Yellow, Diffused black pigment, Pink ring on top, etc.
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 +. 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. On a piece of paper, place one
drop of the oxidase reagent Dimethyl-p-phenylene diaminic hydrochloride (this substance is
carcinogenic). Then use a toothpick to obtain the organism from your TSA plate, and 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.
UREA BROTH (Control = Proteus vulgaris)
When protein is digested, the pH becomes a base. This test checks for the enzyme called urease,
which breaks the protein urea down into ammonium (a base) 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), negative is yellow. Urea is made when
proteins are broken down. The kidneys are supposed to filter the urea and put it into the urine for
elimination. If the kidneys malfunction, urea can build up in the blood and be very toxic. The smell
of OLD urine is from ammonia.
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.
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.
28
CITRATE TEST (Control: positive = Enterobacter aerogenes)
Citrate is a salt of citric acid. It is a part of the Kreb’s cycle. In this medium, citrate is the
sole carbon source. If the organism can use citrate as its only carbon source, the medium will
become basic (blue) because an acid (citric acid) is breaking down. The medium starts out green
and turns blue (basic pH) if it is a positive test. The pH indicator is Bromthymol blue, which is
green when acids are present. 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.
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.
GELATINASE (Gel Test) (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.
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
SIM tube with organism is used. Check for three things on this one tube.
1) H2S PRODUCTION (Hydrogen sulfide): Some organisms use sulfur as their terminal
electron acceptor in the electron transport chain. These bacteria have the enzyme
thiosulfate reductase, which reduces thiosulfate (the substrate in the medium) and
produces H2S gas (a rotten egg smell). When the H2S reacts with iron sulfate in the
medium, a dark precipitate of iron sulfide is produced and the media will turn black
29
(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: The enzyme tryptophanase breaks tryptophan (an amino
acid) down into indole (which contributes to the smell of feces), pyruvic acid, and
ammonia. If tryptophnase is present, the indole end product reacts with Kovac’s
reagent. If a red ring forms at the top of the tube, it is positive for indole, so the
organism makes tryptophanase. Kovac’s reagent has alcohol in it. Alcohol is lighter than
water, so when the test is positive and turns red, the red ring floats to the top of the tube.
(Control = E. coli).
3) Motility: This media will show you if your organism is motile.
TDA: Tryptophan deaminase; tests for the presence of pyruvic acid. A dark brownish red is
positive and yellow is negative. The substrate is tryptophan. tryptophan deaminase (breaks off an
amine group). The substrate is tryptophan (an amino acid; also found in turkey. This amino acid
might encourage sleep, which is why you might feel sleepy after a Thanksgiving turkey meal.
The TDA tests for the product: endopyruvic acid. Reagent (ferric chloride: FeCl3) is added to the
tube. A positive result turns dark brown with a reddish hue.
30
IMViC
This stands for a series of tests:
1) Indole
2) Methyl Red
3) Voges-Proskauer
4) Citrate
The small “i” does not stand for anything; it just makes pronunciation easier.
The IMViC tests were developed as a means of separating members of the Enterobacteriaceae, particularly the
coliforms, to determine if drinking water is contaminated with sewage. 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. Coliforms are only found in the GI tract of warm-blooded animals. 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.
Anaerobic respiration is called fermentation. We are looking for the ability of the organism to perform 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.
MR = METHYL RED TEST
A positive MRVP broth will be red.
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. 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 (Voges-Proskauer) TEST (Control: Enterobacter aerogenes = pos; E. coli = neg)
The VP test is an indirect method of testing for an organism that ferments glucose using the butylene glycol
pathway. Glycolysis forms pyruvic acid, which undergoes fermentation and produces acetoin, which can then be
taken into one of several different fermentation pathways, depending on the organism. In the butylene glycol
pathway, the end product is 2, 3 butanediol. We cannot test for that, but we can test for acetoin. If acetoin is present,
it turns red (positive) and colorless if negative. A positive test means the organism uses that particular pathway for
fermentation. .
31
The VP reagents are called Barritts’s A (alpha napthol, a carcinogen!) and Barrett’s B (potassium hydroxide;
KOH, a very caustic base, found in draino). If acetoin is present, it will turn a rust or red color (Gram negatives
tend to do this). Therefore, red is a positive result, colorless or brown is negative.
NITRATE REDUCTION TEST (control is E. coli)
Add one drop of Nitrate A and then one drop of Nitrate B. Red color is positive. If there is no color change after
adding the two reagents, add zinc powder. If it does NOT turn red it is positive. If the zinc powder makes it turn red,
it is negative.
Nitrate is reduced to nitrite, which can be further reduced to nitrogen. 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.
After you add the two reagents, if it turns red, the test is positive for nitrite, so that means the organism reduced NO3
to NO2. If it does not turn red, it might be because there is no nitrate present for the organism to reduce, so we
cannot say it is a negative test yet.
To find out if nitrate is present, add zinc powder. That will reduces nitrate to nitrite and produce a red color. This
time, if it turns red, it means there was nitrate present during the first part of the test and the organism did not reduce
it, which means the organism is unable to reduce nitrate. Therefore, the nitrate reduction test is negative. If there is
NO color change after adding zinc powder, the organism already reduced the nitrate, so the test is positive for nitrate
reduction.
The nitrate broth we started with contains NO3. If the organism has nitrate reductase, it will reduce NO 3 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 NO 2 , forming a complex.
However, this complex is clear, 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. 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. 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
32
DECARBOXYLASE BROTHS
(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.
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).
ADH: the substrate is arginine. The indicator is phenol red. If the organism has the enzyme arginine
dehydrogenase , it can break down arginine (an amino acid), test is positive (red). Negative is yellow.
(argentine dehydrolase) breaks down argentine, which is an amino acid. All amino acids have a carboxyl
group (with a free radical attached to the carbon) and an amino group. When ADH breaks down
argentine, it becomes ornithine, which is more basic. A pH indicator (phenol red) is present in the well.
The pH of phenol red is 6.8, which is close to neutral. If the pH goes down, it is acid (yellow), and if the
pH goes up, it is basic (red). Phenol red, in the presence of ornithine, gets redder, although in the first 24
hours it may still be just orange. The substrate of the ADH test is arginine, because that is what is broken
down. The product is ornithine. Since ornithine is more basic, the phenol red turns red.
LDC: Lysine decarboxylase. The substrate is lysine, another amino acid. If the organism has the enzyme
lysine decarboxylase, it will take lysine and break it down into cadaverine (rotten flesh smell). Red is
positive and yellow is negative.A decarboxylase breaks off the carboxyl group. The result in this case is
cadaverine. When an animal dies, there is a lot of protein breakdown, called putrefaction, and give that
stink of “death”. Cadaverine contributes to that smell. Lysine breaks down into cadaverine, which is more
basic, so phenol red becomes redder. 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).
ODC: Ornithine decarboxylase. This tests for decarboxylation of ornithine (an amino acid) into putricine
(putrid smell). Red is positive and yellow is negative. Ornithine was the PRODUCT of the ADH well, but
in the ODC well, it is the SUBSTRATE. The product here is putricine, which is also a product of
putrefaction and has that death smell. It is more alkaline (basic), so phenol red turns red. Therefore, in
tubes 2, 3, and 4, we are looking for red as a positive result.
33
FERMENTATION BROTHS (Control = E. coli is AG)
If an organism has the ability to ferment sugars, the end products of the fermentation process are
acids. 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.
These are different carbohydrates. 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.
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 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 Without oil
Results
Control
AG
AG
Ferments glucose
E. coli
NC
A
Oxidative
Pseudomonas aeruginosa
NC
NC
neither
Alcaligenes faecalis
34
LITMUS MILK
Know how to record the results of this test, and know what each of the following looks like: AR,
ACR, AGCR.
Litmus is a pH indicator that turns color only when oxidized. Reduced litmus is colorless. If litmus is
reduced, it will be colorless, so the white milk shows through. This is recorded as “R”.
If it turned pink, it fermented lactose and is acidic. Record this as “A”
If it is bluish, the pH is basic, record it as “K”
If it formed an acid curd (tilt the tube and the milk doesn’t spill), record it as “C”
If you see tracks of gas in a curd, also record “G”.
If the casein protein in the milk was broken down and it is clear on the top and brown in the tube,
that is proteolysis, recorded as “P”.
LACTASE: this tests for an enzyme that breaks down milk sugar (lactose) into glucose and
galactose. If the bacteria have this enzyme, the test is positive (yellow), but the organism is not
pathogenic. Only pathogens are missing this enzyme (negative is clear).
TSI (Triple Sugar Iron Agar)
This medium contains three sugars (below), iron (Fe ++), thiosulfate (oxidized sulfur), and
phenol red (indicator where acid is yellow and basic is red).
10x Sucrose (1%) (sucrose is a plant sugar made of glucose and fructose)
10x Lactose (1%)
1x Glucose (0.1%)
Note that there is 10x more Sucrose and Lactose than there is glucose.
NOTE: When protein is digested, the pH becomes basic because the byproducts are amino acids,
which break down into CO2 and ammonia. When sugars are fermented, the pH turns acidic
because the byproducts are acids.
Know how to record the results of this test, and know what each of the following looks like:
A/A,G, K/A,G K/A,G, H2S K/A, H2S A/A, H2S
A/A,G, H2S
Test results are recorded for the slant and the butt. “A” is for acid, and “K” is for base.
A TSI slant that is all yellow is recorded as A/A
A TSI slant that is red at the slant and yellow at the butt is recorded as K/A
If either sucrose or lactose is fermented, slant will be A/A because there is so much acid present,
it overwhelms the little bit of ammonia that was there.
If just glucose is fermented, more protein was degraded, so the ammonia will be stronger and
show up basic, K/A
If the tube has black in it, the iron was reduced and H2S formed.
If the tube has produced enough gas to separate the agar, it is positive for gas.
An A/A tube with black is recorded as A/A + H2S.
The same tube that also has gas is recorded as A/A + H2S + G
35
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 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.
36
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).
37