Download 4a How to ID and Unk organism

HOW TO ID AN UNKNOWN ORGANISM: Note: we are not performing all of these tests;
I will describe the whole process to you but we will only do some of these tests. This whole
transcript will be on the Unit 2 exam.
METHOD OVERVIEW
Obtain your unknown stock, and check it for purity by performing a Gram stain. Then use it to
inoculate a working stock and a reserve stock. Each week, you check your working stock for
purity by performing another Gram stain before using it to inoculate the media to perform the
tests for that day. Use aseptic technique. You will not use the reserve stock unless your working
stock becomes contaminated.
Then determine the morphological characteristics of your unknown organism by performing a
Gram stain, motility stab, capsule stain. If you have a Gram positive rod, you will also need to do
a spore stain, 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.
GETTING STARTED
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, you need to go back to the original pure
culture and start again. If you only see one organism, the next step is to do a Negative stain with
India ink, which is the best way to see the arrangements of the organism. Record the shape
(rods or cocci?) and arrangement (singles, clusters, or chains?). Then use your unknown broth to
inoculate 3 broths, 2 slants, and 2 plates.
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”. The working stock tube is the one used to obtain
inoculums for other lab tests unless the stock becomes contaminated. Each WEEK, a new
working stock tube is made so the culture stays young. To make a new working stock, just
use the old working stock to inoculate a new tube. After growth is seen in the new tube the
following week, you need to check for purity by performing a Gram stain. Once you know your
new working stock is not contaminated, you can discard the old working stock tube. That means
you need to write dates on these tubes. The second TSA slant made on the first day will be the
reserve stock in case the working stock becomes contaminated.
1
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.
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
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.
2
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. 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. Controls are also made (positive and negative
results) for blood agar plates, motility stabs, and thio tubes. Now it is time to determine the
oxygen requirements by inoculating a sodium thioglycolate tube.
SODIUM THIOGLYCOLATE TUBES (OXYGEN REQUIREMENT)
This medium has an oxygen gradient, which means that most of the oxygen is at the top of the tube, and
the least amount of oxygen is at the bottom of the tube. To prepare this medium, a reducing agent called
Sodium thioglycolate was added, which removes the free oxygen by chemically binding with it.
Therefore, thioglycolate broth is called a REDUCING MEDIUM. It gets rid of the oxygen. There is also a
pink indicator dye called rasazarin that shows you where the oxygen is. Notice that the pink color is
only at the top of the tube. We have to be careful not to shake the tube, or we will aerate it (add more
oxygen). We need the oxygen gradient to be maintained for a successful test. The results of this test
determine what oxygen requirements your organism has.
Procedure:
1. Hold the thioglycolate tube carefully, taking care to move it gently without shaking,
jiggling, or stirring them (which introduces oxygen into the medium).
2. Label the tube with your name, the date, the organism, and “Thio” for Thioglycolate.
3. Put some of your unknown bacteria on a sterile loop and gently push the loop straight
down to almost the bottom of your tube. Do not touch the bottom as this may ruin the
loop, and do not introduce air by stirring or shaking the tube!
4. Gently pull the loop straight out of the tube and sterilize it.
1. STRICT AEROBES require oxygen to grow. There will only be growth on the
surface of the thio broth tube (pseudomonas and Bacillus megatarium)
2. STRICT ANAEROBES require the absence of all oxygen. There will only be
growth at the butt (bottom) of the tube (clostridium).
3. FACULTATIVE ANAEROBES grow best aerobically but do not require it.
Growth is throughout the tube, but is best at the top and decreases as one descends.
(E.coli, staph aureus)
3
PHYSIOLOGICAL TESTS
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. At the next lab period, you will also use this plate to perform the catalase and oxidase
tests.
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.
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.
UREA BROTH (Control = Proteus vulgaris)
This test checks for the enzyme called urease, which breaks urea down into ammonium and
carbon dioxide (water is not a product of this reaction). The ammonium will increase the pH. The
medium has a pH indicator called phenol red. When pH goes up, it will turn bright pink (positive).
CASEIN TEST (skim milk) (Control = Bacillus)
Some organisms produce an enzyme called caseinase (a protease), which breaks down the protein that
makes milk white. It breaks the protein down into small peptides that can be absorbed into the cell. Do
a heavy streak in the center. Positive is a clearing (halo) around the area of growth of the organism
because the milk is broken down and the white color disappears. Casein is what makes milk look
cheesy when it is left unrefrigerated.
4
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
LIPASE (control: positive = Staph aureus)
The Spirit Blue media has lipids. If the organism has the enzyme lipase, fatty acids will be released, and pH will
decrease (become acidic). This will precipitate the blue dye. A positive result is a dark blue streak in the center
of the plate where you inoculated it. If lipase if produced, the concentration of the blue will increase where it was
inoculated. Having clearing is NOT a positive test; it should be darker blue.
NOTE: If you have a Gram positive organism, and if it is not a spore former, you need to do an acid-fast stain. If
it is positive, you have a mycobacterium, which is not really a Gram negative organism. Negative stains are done
if there is a question about the arrangement of your organism. You can see their arrangements best with a negative
stain.
STARCH (Control = Bacillus)
This media has starch. Some molecules, such as starch, cannot be taken into a bacterial cell because the
molecules are so large. The organism can only use starch if it has an enzyme, called amylase, which can
hydrolyze (break down) the starch into simple sugars that can be absorbed into the cell. We will flood
the plate with iodine, which reacts with starch and turns it black. If the organism has the enzyme, there
will be no more starch left, so there is nothing for the iodine to react with. Therefore, the presence of
amylase will show up as a halo (area of clearing) around the organism (positive test). If the organism
could not use the starch, the starch forms a complex with iodine to give a black precipitate
around the organism. That means the organism is negative for amylase. NOTE: the black
color only lasts a few minutes, so you have to read the test right away before the color
disappears.
GELATIN (Control = Bacillus)
Some organisms produce an enzyme called gelatinase, which breaks down gelatin. If the gelatin is
broken down, it becomes liquefied, and can no longer solidify, even when cooled in the refrigerator.
Gelatin is a protein, so gelatinase is a protease. Gelatin is the only thing making the media solid. If it
remains liquid, even after refrigeration, it is positive. Solid is negative.
5
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 are used to identify an organism in the coliform group. A coliform is a gram
negative, aerobic or facultative anaerobic rod which produces gas from lactose within 48 hours.
The presence of some coliforms indicates fecal contamination. We will perform the indole test as
part of the SIM media. We performed the citrate test in the Simmon’s Citrate media. Now we
need to perform the MR-VP test to complete the IMViC series.
MR-VP TEST (Methyl Red/Voges-Proskauer)
We do two tests with this medium: The MR test and VP test. We will inoculate one MR-VP tube
today, let the culture grow until the next lab period, and then add 5 drops of Methyl Red to
perform the MR test. In the next lab period, we will inoculate a new MR-VP tube, let the culture
grow, and then add alpha-naphthol and potassium hydroxide reagents to perform the VP test.
We are looking for glucose fermentation. Bacteria convert glucose to pyruvate using different
metabolic pathways. One pathway produces unstable acidic products which quickly convert to
neutral compounds. Another pathway (the butylene glycol pathway) produces neutral end
products, including acetoin and 2,3-butanediol. A third pathway is the mixed acid pathway,
which produces stable acidic end products which remain acidic. If an organism produces a lot
of acid from the fermentation of sugars, it can override the buffer in the test media. If this
happens, the amber media will turn red. MR-VP broth differentiates organisms that are
single acid fermenters from organisms that are mixed acid fermenters because it contains
over-riding buffers that affect organisms that are single acid fermenters. An organism that
produces only one type of acid after sugar fermentation will not produce much acid, so the buffer
blocks the 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
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
6
VP TEST
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. The VP reagents are
called Barritts’s A (alpha-naphthol; 18 drops) and Barrett’s B (potassium hydroxide;
KOH, 8 drops). These are added to MR-VP broth that has been inoculated with an organism that
uses the butylene glycol fermentation pathway, the acetoin end product causes a rust or red
color (Gram negatives tend to do this). Therefore, red is a positive result, colorless is negative.
Shake gently after adding the reagents, wait 15 minutes, then shake again. (Control:
Enterobacter aerogenes = pos; E. coli = neg)
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.
7
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
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.
NOTE: PICK THESE TUBES UP FROM THE RACKS ONE AT A TIME AND LABEL THE
TUBE BEFORE YOU PICK UP THE NEXT TUBE. THEY ARE ALL THE SAME COLOR
AND YOU MIGHT GET THEM MIXED UP!
Each decarboxylase enzyme produced by an organism is specific to the amino acid on which it
acts. Therefore, we test the ability of organisms to produce arginine decarboxylase, lysine
decarboxylase, and ornithine decarboxylase using three different but very similar media.
If an organism is able to decarboxylate the amino acid present in the medium, alkaline
byproducts are then produced. Ornithine decarboxylation yields putrescine (named after its
putrid smell).
Lysine decarboxylation results in cadaverine (smells like a cadaver). These byproducts are
sufficient to raise the pH of the media so that the broth turns purple (in 48 hours). If you check it
in 24 hours, you might see that it is yellow because it fermented the glucose in the medium, but
that does not mean it is a negative test. You have to check it in 48 hours to allow the
decarboxylase activity to occur. If the pH becomes alkaline because the organism has the
decarboxylase enzyme, the media will turn purple in 48 hours (pos).
DNASE TEST (Control = Serratus marcescens or Staph aureus)
This tests for the presence of the enzyme, DNAse. It contains the indicator dye, Methyl green
complexed with DNA. Digestion of DNA releases the dye, so in otherwise green agar, a clear
halo formed around the growth indicates a positive test.
8
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
Get one SIM tube and use a needle to stab the media with your organism. Next lab period, we
will add 10 drops of Kovac’s reagent to the tube and check for three things on this one tube.
1) H2S (sulfur) PRODUCTION: Certain bacteria produce H2S from the enzyme cysteine
desulfurase. When the H2S reacts with ferrous sulfate in the medium, a dark
precipitate of iron sulfide is produced and the media will turn black (positive for H2S
production). (Control = Proteus vulgaris). Don’t get this mixed up with the phenylalanine
test, where the addition of ferric chloride turns the media green.
2) INDOLE PRODUCTION: Tryptophanase breaks tryptophan (an amino acid) down
into indole, pyruvic acid, and ammonia. If tryptophnase is present, the indole end
product reacts with the reagent we will add next time (10 drops of Kovac’s reagent). If a
red ring forms at the top of the tube, it is positive for indole, so the organism makes
tryptophanase. (Control = E. coli).
3) Motility: You have already done a motility test, but this media will show you again if
your organism is motile. The red dye is not in this media, so visualization is harder.
FERMENTATION BROTHS (Control = E. coli is AG)
We are looking for fermentation with acid (A) or acid + gas (AG). If there is fermentation, it will
be yellow. If there is gas, the inverted miniature tube inside the media will fill with a gas bubble.
If there is no fermentation, it is red, so record it as no change (NC) or Alk (protein digestion).
The medium has a Durham tube (a miniature tube that is inverted on the inside of the test tube).
If gas is produced, it will form a bubble inside the inverted tube. It also has phenol red as an
indicator. Phenol red turns yellow if acid is present, and red if bases are present.
Inoculate one each of the following tubes: glucose, lactose, mannitol, sucrose, and trehalose.
After 24 hours, if the inoculated medium is yellow, it fermented the sugar in that tube. It may or
may not have produced gas. Gas is produced during sugar fermentation, so when gas is present,
fermentation is present as well, but not all organisms ferment with gas. If it is yellow, record it as
(A). If it has gas in the Durham tube (a bubble that take up 10% of the tube, not a little bubble),
record it as (AG). If it did not turn yellow (stays red), you have to look at it again in another 24
hours. After 48 hours, if the media is still red, the organism is negative for fermentation of that
sugar. These tubes must be read in 24 hours, because in 48 hours, any change in color will
revert to the original color.
This is what happens:
Some organisms that ferment sugars can also digest proteins. When these organisms begin to
ferment a sugar, the media becomes acidic (yellow in 24 hours), which enables them to begin
digestion of the proteins which are in the media. When proteins are digested, the media becomes
9
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
AG
NC
NC
Without oil
AG
A
NC
Results
Ferments glucose
Oxidative
neither
Control
E. coli
Pseudomonas aeruginosa
Alcaligenes faecalis
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.
NOTE: Be able to match enzymes to their tests, substrates, reagents, and controls. You should
make a table to study from. In one column, put the name of the test. In the other columns, put the
substrates, products, enzymes, reagents, etc., and what a positive and negative result would look
like.
END OF MATERIAL FOR IDENTIFICATION OF UNKNOWN BACTERIUM
10