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12_BIOCHEMICAL CHARACTERIZATIONS
USING GRAM NEGATIVE BACILLI
Identification and characterization of micro-organisms usually begins with an
examination of their growth characteristics and requirements for growth such as colony
characteristics, gram reaction, oxygen requirements, temperature requirements, etc.
However, these observations can only take us so far in identifying most organisms and so
the microbiologist must rely upon an examination of the metabolic capabilities of a microbe
to obtain definitive characterization.
Since microbes have such diverse capabilities for metabolizing unique substrates,
biochemical analysis is one of the methods of choice for elucidation of their identity.
Biochemical characterization examines the materials that microbes utilize for their
survival. It includes an examination of what the microbe uses as a source of carbon;
whether it is an autotroph or heterotroph, and how it obtains energy – phototroph or
chemotroph, and more. Experiments examine whether the organism ferments sugars and
if so, which ones and what kind of byproducts they produce – acids, gas, alcohol or a
mixture of neutral products. In addition, biochemical characterizations use a variety of
tests to determine which kinds of enzymes might be present in a particular microbe. For
example, do they make the enzyme urease, do they digest starch with the enzyme amylase,
do they hydrolyze fats or certain amino acids like tryptophan? By examining the
organism’s biochemical activities one can gain an understanding of the particular organism
and how its unique metabolic “options” allow it to thrive in its own particular environment.
In this lab you will begin a biochemical characterization of several well-known
organisms. You will use this information collaboratively with your classmates to begin
constructing a table of characteristics for these microbes that will ultimately help you in
the identification of your two unknown organisms for your double unknown project. These
are the types of tests that will be available for characterization during your double
unknown project later in the quarter.
NOTE: To prepare for this lab, prepare a pre-lab flow chart in your lab notebook as
usual – before period 1, make sure to prepare for Period 1 AND before period 2 including
notes about how to interpret test results. ALSO, note that the homework assignment (a
second document posted online) is due at the START of lab on the 1st day of this lab.
PERIOD 1
Materials per table of 4:
Broth culture of one of the “known” organisms – make a note of which one your
instructor provides to your table.
1 Phenol Red Glucose Broth
1 Phenol Red Lactose Broth
1 Phenol Red Mannitol Broth
1 SIM Tube
1 Gelatin Deep
1 Tryptone Broth
1 TSA plate
1 Urease Slant
1 Starch Plate
2 MRVP Tubes
1 Citrate Tube
1 Nitrate Tube
1 Kligler’s Iron Agar Slant
1 Motility Deep
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Procedure:
1. Each lab team will characterize at least one of the known Gram-negative organisms to
be assigned by the instructor. Record the name of this organism in your lab notebook.
2. In general one loopful of the organism from the broth should be used to inoculate each
of the following tests, except a needle should be used for stabs.
Glucose, Lactose, and Mannitol sugar fermentation tests--inoculate the glucose,
lactose, and mannitol fermentation broths with separate loopfuls of the organism.
Each fermentation broth contains the appropriate sugar and the pH indicator Phenol
Red. If the sugar is fermented then acid is produced which in turn causes the pH
indicator to change color from red to yellow. In addition, each tube contains a small
inverted tube called a Durham tube that traps gas produced during fermentation of
the carbohydrate (i.e., sugar). (Part of Ex. 36 starting on p. 251 p. in Benson lab
manual – use Figure 36.7 in Benson lab manual for interpretation of results)
Gelatin— inoculate the gelatin deep by stabbing it using a needle with a sample of
your assigned organism by going nearly to the bottom of the deep. Try to go in and
out along the same line for the inoculation. If the organism makes the enzymes to
hydrolyze gelatin, the solidifying properties of gelatin will be lost causing it to liquefy.
(Part of Ex. 35 starting on p. 247 – use Figure 35.3 in Benson lab manual for
interpretation of results)
Urease slant-inoculate the urease slant media with one loopful of the organism
streaked in a line across the surface of the slant, and then use a needle to stab the
media with a sample of your assigned organism. The urease tube contains the pH
indicator phenol red. If the organism has the enzyme urease it will break the urea in
the tube down into ammonia, which is alkaline. Phenol red in an alkaline environment
turns a bright pink (cerise) indicating a positive result. (Part of Ex. 37 starting on p.
263 in Benson lab manual – use Figure 37.4 in Benson lab manual for interpretation
of results)
Citrate tube-inoculate the citrate slant media with one loopful of the organism
streaked in a line across the surface of the slant, and then use a needle to stab the
media with a sample of your assigned organism. Citrate tubes contain citrate as a
sole carbon source and the pH indicator Bromthymol Blue. If the organism can utilize
citrate as a sole carbon source, the byproducts are alkaline and the tube will change
from green to a deep royal blue. Since this only occurs aerobically, be sure to leave
the cap loose slightly loose during incubation. (Part of Ex. 36 starting on p. 251 p. in
Benson lab manual – use Figure 36.9 in Benson lab manual for interpretation of
results)
Starch plate-streak a loopful of organism in a straight line across ½ of the starch
plate. Streak a loopful of Bacillus subtilis provided by your instructor in a straight
line across the other ½ of the starch plate as a control. Label the plate with which
organism you’ve put on which side. This test determines the presence of the enzyme
amylase. (Part of Ex. 37 starting on p. 263 in Benson lab manual – use Figure 37.2a
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in Benson lab manual for interpretation of results).
MRVP broth (this broth contains the media that is used for 2 different tests: the MR
(Methyl Red) test and the VP (Voges-Proskauer) test) – inoculate TWO MRVP broths
with one loopful of organism (next time you’ll complete the MR test on one tube and
the VP test on the other). These tests are used to determine the organism’s ability to
ferment sugars into neutral byproducts such as acetoin (VP test) or into a variety of
acids (MR test). (Part of Ex. 36 starting on p. 251 p. in Benson lab manual – use
Figure 36.8 in Benson lab manual for interpretation of results for these 2 tests)
Indole test/Tryptone Broth (Tryptophan degradation) – inoculate the typtone broth
media with one loopful of organism. Some microbes have the enzyme tryptophanase
which confers the ability to degrade the amino acid tryptophan to the product indole.
(Part of Ex. 37 starting on p. 263 in Benson lab manual – use Figure 37.5 in Benson
lab manual for interpretation of results)
Motility test – inoculate this semisolid medium by stabbing using a needle with a
sample of your assigned organism. Try to go in and out along the same line for the
inoculation.
Nitrate test – inoculate the nitrate broth with one loopful of organism. Some
microbes have the enzyme nitrate reductase, which allows them to reduce nitrate
(NO3-) to nitrite (NO2-) – they use nitrate as their final electron acceptor during
anaerobic cellular respiration. These microbes are part of the group of bacteria called
nitrifying bacteria, due to their role in the nitrogen cycle. (Part of Ex. 36 starting on p.
251 in Benson lab manual – use Figure 36.12 in Benson lab manual for
interpretation of results)
Kligler’s Iron Agar – inoculate the slant with one loopful of the organism streaked in
a line across the surface of the slant, and then use a needle to stab the media to the
bottom with a sample of your assigned organism. This is a multiple test medium that
will detect the fermentation of glucose and lactose and the production of hydrogen
sulfide (H2S). It has phenol red as a pH indicator. (Part of Ex. 38 starting on p. 269 in
Benson lab manual – use Figure 38.1 in Benson lab manual for interpretation of
results)
TSA plate – perform a streak isolation of your assigned organism on this TSA plate.
Bacteria taken from colonies on this plate will be used to perform the catalase and
oxidase tests in the next lab period. (Part of Ex. 36 starting on p. 251 p. in Benson
lab manual – use Figure 36.10 (oxidase test) and Figure 36.11 in Benson lab manual
for interpretation of results for these 2 tests)
3. Incubate all of the biochemical tests at 37C – almost all tests can be incubated for 2448 hrs at 37oC, but check your Benson lab manual for information citrate and urea
hydrolysis tests that may need to go longer than that. All of your table’s plates can be
placed in the 37oC white tub, and all of your table’s tubes should be placed together on
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the racks that will be incubated at 37oC – share 1 rack with another table.
4. Return your table’s bacterial stock to the refrigerator, just as you got it at the start of
the lab period.
Note: Biochemical tests may be added/removed based on availability as determined by
your instructor.
Materials:
Kovac's reagent
Methyl Red
Voges Proskauer I reagent
Voges Proskauer II reagent
Iodine
PERIOD 2
3% hydrogen peroxide (H202)
Oxidase reagent
Nitrate A reagent
Nitrate A reagent
zinc dust
Procedure:
1. Observe biochemical tests for growth. If there is no growth then the test is not valid
and must be repeated; growth is a prerequisite for all of these biochemical tests. Except
as where indicated for particular tests, a positive result should be written as + and a
negative result should be written as –. NOTE that in addition to recording observations
of growth in your notebook, you should also record what you see such as color change,
and write the test result (recording this information will allow you to refer back to your
observations of color change, etc., in the future to see if you have interpreted the test
correctly and for troubleshooting purposes).
2. Read and record observations for the biochemical tests as follows (and for each test
also refer to the page #s and figures in your Benson lab manual listed above for
Period 1 Procedures to help interpret each test). When recording your results, you
should refer to uninoculated media to help determine if colors have changed, and ALSO
to look at test results of other tables – you are expected to be able to recognize and
interpret positive and negative results for each test. In general, try to minimize your
exposure to open tubes and the reagents you are adding.
Glucose, Lactose, and Mannitol sugar fermentation tests- Since these nutritionally
complete media support the growth of most common bacteria, growth (turbidity)
should be obvious. If the bacterium metabolizes the sugar, the acidic end products of
fermentation lower the pH, which in turn changes the color of the pH indicator from the
original red to yellow. In addition, some organisms produce gas as an end product of
fermentation; gases produced will be seen trapped in the Durham tube. Record the
results as follows:
NG (no growth) - no turbidity visible; test must be repeated
NR (no reaction) - growth present but no change in the color indicator (Phenol Red)
A (acid) - acid produced as indicated by the yellow color
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AG (acid and gas) - gas produced in addition to acid
Gelatin liquefaction - Some bacteria have enzymes that can break down the protein,
gelatin. Since gelatin melts at 37C your gelatin is liquid regardless of whether the
gelatin was broken down. Place your tube on a green rack in the refrigerator for 10
minutes, then make observations. If the gelatin is still liquid after 10 minutes of
immersion then record the result as positive.
Urease-Examine the urease tube for any change in color. A bright pink color indicates
that the organism has the enzyme urease. Urease breaks the urea into an alkaline
biproduct, ammonia, which raises the pH and turns the phenol red indicator pink.
Citrate – If the organism can use Citrate as a sole carbon source the media will become
alkaline and the Bromthymol Blue pH indicator will change from green to a deep blue.
Starch Hydrolysis-To test for the presence of the enzyme amylase, pour enough iodine
on the plate the plate – you can gently swirl to get iodine to cover the whole surface.
Iodine binds to starch to produce a black precipitate. If the organism has the enzyme
amylase, the starch surrounding the area of growth will have been hydrolyzed, leaving a
clear area that does not form the black precipitate.
MR and VP tests-Methyl Red/Vogues Proskauer tests look for the ability to ferment
glucose into either mixed acids (MR test) or neutral products such as butanediol (VP
test). Note that these are 2 different tests that use the same media.
1: To perform the MR test add several (10-15) drops of Methyl Red indicator to one
of the MRVP tubes. Mix the tube well and look for the color to remain red. This
indicates that the organism can ferment the sugar into mixed acids.
2: To the other MRVP tube add 15-20 drops of Voges Proskauer (VP) reagent I
(alpha-naphthol) and 15-20 drops of VP reagent II (40% KOH). Mix well and then
let stand undisturbed on a rack on your lab benchtop. The appearance of a pink
color within a few minutes to 30 minutes indicates a positive test. The color should
deepen with time.
Tryptone Broth (Indole test) – This test assesses the ability of microbes to break down
the amino acid tryptophan using the enzyme tryptophanase – indole is one of the
breakdown products of this reaction. Add 10-15 drops of Kovac’s reagent and look for
the formation of a red ring at the top of the tube, which indicates that the organism is
positive for the enzyme tryptophanase.
Motility – Observe for growth, which you will see as a red coloration. Determine if
motility occurred by looking if the red is visible in the media away from the stab line. If
only the stab line itself is red, this is a negative result for motility. If there is red also
away from the stab line, this is a positive result for motility. Note that some motilitypositive organisms move faster than others, resulting in variation in the extent of
growth away from the stab line.
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Nitrate test – This test determines whether microbes have the enzyme nitrate
reductase, which is used to convert nitrate to nitrite during anaerobic cellular
respiration.
Nitrite reacts with Nitrate A and Nitrate B reagents to form a red color. Thus, if a
red color is visible after adding Nitrate A and Nitrate B reagents, the microbe has the
enzyme nitrate reductase.
If there is no red color at this point, there are 2 possibilities: either the microbe lacks
nitrate reductase and the nitrate broth still contains nitrate, or the microbe has nitrate
reductase and has further converted the nitrite to gases such as ammonia or nitrogen
gas – these gases are not detected by this test.
To distinguish between these 2 possibilities, you will add zinc dust to the broth –
zinc dust converts any remaining nitrate to nitrate. Thus, if the broth turns red after
adding zinc dust, this indicates that there was nitrate in the tube, meaning the organism
does not have nitrate reductase. On the other hand, if the broth does not change color
after adding the zinc dust, this indicates that there was no nitrate in the broth, meaning
the organism does have nitrate reductase.
1. Inspect tubes for growth. If no growth, record NG as the result.
2. To each tube containing growth, add the following and mix well after each
addition: 20 drops of sulfanilic acid (“Nitrate A”) + 20 drops of alphanaphthylamine (“Nitrate B”)
3. If the broth turns red, this is a positive nitrate reductase result. NOTE: It is
suggested that you allow the broth to stand for a few minutes, mixing frequently, so
that the reagents penetrate adequately. Only then should you make a decision on
color change.
4. If the broth does not change color after standing for a few minutes, add a few
particles of zinc dust with a toothpick. Mix well.
a. If broth turns red, this is a negative nitrate reductase result.
b. If broth does not turn red, this is a positive nitrate reductase result.
Kligler’s Iron – Use information in the Benson lab manual to determine the test result.
Note that this test determines whether bacteria can ferment glucose, can ferment
lactose, and can use peptones as energy sources. This test also determines whether
bacteria can produce hydrogen sulfide – a positive test for hydrogen sulfide production
is a black precipitate in the tube. Note that a positive reaction for Hydrogen Sulfide may
obscure motility determination.
TSA plate – use colonies on the plate to perform catalase and oxidase tests as described
below:
Catalase - Place a drop or two of 3% hydrogen peroxide on a clean glass slide. Pick up a
sample of Staphylococcus aureus culture provided by instructor and mix well in the
hydrogen peroxide solution. For this test, you should pick up more bacteria than you
usually do – try to pick up the equivalent of 1-2 medium sized colonies. This will serve
as a positive control for this test – Staphylococcus aureus should give a positive result
for catalase. Discard the glass slide when done. Now repeat this procedure using a new
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slide with a sample from the edge a colony of your table’s assigned organism. Observe
for bubbling. Discard the glass slide when done. Catalase is an enzyme that breaks
down hydrogen peroxide, a toxic byproduct of aerobic respiration via the reaction 2 H2O2
 2 H2O + O2 . Bubble formation (production of oxygen gas) constitutes a positive test. All
organisms that aerobically respire must make catalase. The lactic acid bacteria, which
include the Gram positive Streptococcus, are obligate fermenters and even though they
grow in the presence of oxygen, do not produce catalase. The catalase test, therefore, is an
important step in distinguishing the lactic acid bacteria from other Gram positive cocci.
Do not perform the catalase test directly on a colony on a plate because media ingredients
including yeast extract and blood are catalase positive.
Oxidase – Use a sterile swab to apply a sample of the bacterium to an oxidase strip (one
end of the strip has the reagent – apply to that end!!!). Look for a change to a dark color
(purple or black) – which indicates a positive reaction for oxidase (only record as
positive if it occurs within the first 30 or so seconds – if it only turns color after that it is
considered a negative result).
3. Your instructor will either ask your table of 4 for results, or will instruct you to use your
information and the information of your classmates construct a table of the biochemical
activities of these “known” organisms. Use this table as a starting point for the
identification of your unknown organisms. You will need to record ALL results for ALL
organisms. The results posted are valid for these particular organisms only. Different
strains of the same genus and species may vary slightly in their biochemical reactions.
Note: your instructor will specify how to do this step 3.
4. Discard all plates and tubes properly.
NO POST-LAB QUESTIONS TO FOLLOW. INSTEAD BIOCHEMICALS HOMEWORK
SHOULD HAVE BEEN COMPLETED BEFOREHAND, AND NOTE THAT YOU WILL ALSO BE
TESTED ON THESE TESTS ON THE NEXT EXAM, INCLUDING WHAT EACH TEST IS
TESTING FOR AND HOW TO INTERPRET EACH TEST.
FOR YOUR NOTEBOOK: IT IS REQUIRED TO HAVE A PRE-LAB (include all required
information but be concise!) AND OBSERVATIONS FROM ALL TESTS COMPLETED.
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