Download Unit 8: Oxygen Utilization - Central New Mexico Community College

Unit 8
Unit 8: Oxygen Utilization, Streak Isolation and Introduction to
Bacterial Identification
By Patricia G. Wilber, Karen Bentz and Heather Fitzgerald
Copyright Central New Mexico Community College, 2015
Introduction:
Microbes have varying oxygen requirements and usage abilities and these differences can be
used to help identify bacterial species. Two quick assays that you will be using in this lab to
check for oxygen usage in bacteria are the catalase and oxidase tests. They are often called
“spot tests” because they produce results immediately, or “on the spot”. You will not have to
wait for your bacteria to grow for 24 hours on a media to see the results. We can also use the
information from the catalase and oxidase tests in a more theory-based way to understand
more about metabolism in microbes and sharpen our critical thinking skills!
General Descriptive terms:
Obligate- requiring
Facultative- as needed
Terms describing oxygen utilization.
Obligate aerobes: Organisms that REQUIRE oxygen to survive. These organisms only get energy
from aerobic respiration, using oxygen as the final electron acceptor for the electron transport
chain. Mycobacterium tuberculosis and Nocardia asteriodes are obligate aerobes.
Many sources call Pseudomonas aeruginosa an obligate aerobe, but that is not correct. That
organism is a facultative anaerobe (see next category). None of the organisms we use in our lab
are obligate aerobes.
Facultative anaerobes: Organisms that can produce energy via aerobic respiration using oxygen
as the final electron acceptor OR, in the absence of oxygen, can perform fermentation or
anaerobic respiration, which uses something besides oxygen as a final electron acceptor such as
sulfate, SO42-.
These organisms tend to grow best in the presence of oxygen and go anaerobic facultatively
(i.e. as needed) when oxygen concentration is lower than they prefer. (The oxygen
concentration optima will vary by organism.) Many (but not all) of the organisms we use in lab
are facultative anaerobes.
Microaerophiles: Organisms that do best when environmental oxygen levels are lower (210%) compared to normal atmospheric levels (21%) and MAY also prefer elevated CO2 levels
(capnophiles). Microaerophiles REQUIRE oxygen to live and have an electron transport
Unit 8 Page 1
Unit 8
chain. Under oxygen tensions that are low for them, some will activate anaerobic pathways.
Campylobacter and Helicobacter species are microaerophiles. Microaerophiles can be
capnophilic also and both Campylobacter and Helicobacter are capnophilic.
Streptococcus species are NOT microaerophiles despite the fact that disease articles often
refer to Streptococci as microaerophiles. This is probably because some Streptococcus
species have enzymes that are more effective at breaking down blood under low oxygen
conditions, so are more pathogenic (which is unrelated to oxygen utilization for the
production of energy) under low oxygen conditions. Streptococcus species are facultative
anaerobes.
Capnophiles: Organisms that grow well in higher than atmospheric levels of CO2 (0.03%). Some
need the extra CO2 and other species just tolerate it well. Campylobacter species are
capnophiles (prefer 10% CO2) as well as microaerophiles. Haemophilus is also a capnophile
(prefers 5% CO2) and a microaerophile.
Obligate anaerobes: Organisms that do not use oxygen and furthermore, they are poisoned by
normal (21%) levels of atmospheric oxygen. Oxygen kills them. We do not have any of these in
lab because they are hard to grow. Clostridium species are obligate anaerobes. One reason
Clostridium difficile forms endospores when exposed to air is that it would otherwise be killed
by the high oxygen levels.
Aerotolerant anaerobes: Organisms that live by fermentation and do NOT use oxygen for
metabolism. However, unlike obligate anaerobes, aerotolerant anaerobes are not killed by
oxygen. They just “ignore” it.
Unit 8 Page 2
Unit 8
Figure 8-1. Growth patterns for various oxygen utilization strategies.
 Apply the terms above to the tubes below and defend your answers.
 Also determine which tubes contain organisms that perform aerobic respiration.
 Answers at the end of the Unit.
Retrieved 6/8/15 from http://en.wikipedia.org/wiki/Microaerophile. This picture is in the public domain.
Tube
#
Oxygen
utilization
category
Aerobic
Defense (write why you made your choices for columns 2 &
respiration? 3 here)
Yes or No
1
2
3
4
5
Answers are at the end of the Unit.
Streak Isolation from a mixed broth.
In medicine, samples from patients are rarely
delivered in a pure culture, so perfecting a streak isolation and being able to recognize
different colonies is really important!
Unit 8 Page 3
Unit 8
Day I
I.
Streak Isolation using a mixed Broth.
Materials


1 Chocolate agar plate per student.
Bacterial Culture: 0.5 ml Streptococcus pyogenes (Spy) mixed with 5 ml Bacillus
megaterium (Bm) in one T-Soy broth tube.
NOTE: Bm grows on top of the broth, some stuck on the sides and some clinging to the
bottom. You MUST STIR this broth with your sterilized loop in order to be sure to get
enough of the Bm mixed with the Spy on your loop to get both on your streak isolation!
Procedure
1.
2.
3.
4.
5.
6.
Refer to Unit 3 to review the streak isolation procedure from a broth.
A broth tends to be easier to streak from than a plate.
STIR your mixed broth thoroughly with your sterilized loop before streaking!
Tap your loop to reduce bacteria before performing the streak isolation.
Perform your streak isolation on your chocolate agar plate.
Be sure to label your plate correctly.
General procedure for the Streak Isolation Technique.
B, 10 streaks through A
A, 1 cm
smear
C
E
D
Figure by Patricia G. Wilber
Unit 8 Page 4
Unit 8
II.
Catalase Test
Introduction
Hydrogen peroxide (H2O2) is found in all living aerobic cells and is produced during aerobic
metabolism. Hydrogen peroxide is toxic in high concentrations. Most cellular compounds
are damaged by the oxidizing effects of H202. For example, when DNA or phospholipids (in
the cell membrane) are damaged, cells are stressed and may die. To help prevent these
unwanted consequences, many organisms that perform aerobic metabolism produce the
catalase enzyme to detoxify most of the intracellular H2O2 by breaking it into water and
oxygen. (Some produce other enzymes like peroxidase that carry out the same general
reaction to detoxify H2O2.)
In the catalase test, we expose bacteria to a solution of H2O2 and if bubbling is observed, the
organism has catalase. The catalase enzyme is hydrolyzing H2O2 into water and oxygen (the
bubbles) and giving off heat. If there is no bubbling, the organism is negative for the catalase
test which means the organism does not produce the enzyme catalase.
In cells that metabolize aerobically, H2O2 is produced as a byproduct of energy production.
Therefore, organisms that produce the enzyme catalase must belong to an oxygen
utilization category that includes aerobic metabolism. Organisms that are catalase positive
might be obligate aerobes (these are always catalase positive) or they might be facultative
anaerobes (these are often catalase positive although some facultative anaerobes are
catalase negative). While microaerophiles do perform aerobic metabolism, they are always
negative for the catalase test.
Organisms that are negative for the catalase test (no bubbling) lack the enzyme catalase.
HOWEVER, since there are other enzymes (like peroxidase) that break down H2O2 that was
made via aerobic respiration, a negative catalase test result does not allow us to rule out any
oxygen utilization category except obligate aerobe (because all obligate aerobes produce
catalase and are positive for the catalase test ). We do know that if an organism tests
negative for the catalase test that it lacks catalase.
Some organisms use hydrogen peroxide as a poison. For instance, human white blood cells,
called neutrophils, can release H2O2 along with other chemicals against some types of
bacterial infections. Other organisms produce catalase to protect themselves from hydrogen
peroxide attacks. In the cases where the organism is anaerobic (does not perform aerobic
respiration) but does produce catalase as a defense mechanism, the amount of catalase
produced is too low to register in the catalase test, and the organism shows a negative
catalase test result.
The catalase test is also used clinically to differentiate Staphylococcus species (catalase +,
facultative anaerobes) from Streptococcus species (catalase -, facultative anaerobes) and
Clostridium species (catalase - some are aerotolerant anaerobes and some are obligate
anaerobes) from Bacillus species (catalase +, facultative anaerobes).
Unit 8 Page 5
Unit 8
The catalase enzyme has four active sites for binding H2O2, so one catalase molecule can turn
four hydrogen peroxide molecules into four water and two oxygen molecules (and release
some heat in the process)
CATALASE
(Enzyme)
4H2O2 --------------------> 4H2O + 2O2
The equation above reflects what really happens, but usually equations are reduced, so this is
the reduced form:
CATALASE
(Enzyme)
2H2O2 --------------------> 2H2O + O2
Oxygen Utilization
Category
Obligate aerobe
Facultative anaerobe
Microaerophile and
Capnophile
Aerotolerant anaerobe
Obligate anaerobe
Respiration
Catalase reaction
Yes, does aerobic
respiration
Yes, does aerobic
respiration, and well as
anaerobic
Yes, does aerobic
respiration, some have
anaerobic pathways
Only anaerobic
Only anaerobic
Always catalase positive
Video Links:

Catalase Test https://youtu.be/mtK91MOd650
Video created by Corrie Andries and Karen Bentz
Unit 8 Page 6
Some are catalase positive,
some are catalase negative
Always catalase negative
Always catalase negative
Always catalase negative
Unit 8
Materials: (per pair)




1 Wooden dowel/stick for every TWO cultures tested (use both ends)
3% solution of Hydrogen peroxide
Glass Slides
Cultures
o Enterococcus faecalis (Ef)
o Streptococcus mitis (Sm)
o Pseudomonas aeruginosa (Pa)
o Bacillus megaterium (Bm)
o Staphylococcus saprophyticus (Ss)
Procedure:
1. Put your slide on the black table top.
2. Pour a dime-sized puddle of hydrogen peroxide on the slide.
3. Using your wooden dowel, put the flat end on top of one single colony and push gently
so some bacteria sticks to the dowel. Do not gouge the agar.
4. Place the end of the dowel with the bacteria on it into the hydrogen peroxide on the
slide and let it sit there. DO NOT STIR. Do not remove it. (Bubbling will occur
immediately if the test is positive.)
5. Observe and record your results in the table provided.
6. Dispose of the used dowel and slide in the Sharps container.
Results and Interpretation:
Figure 8-2. Positive catalase result.
Figure 8-2. Negative catalase result.
Photographs by Heather Fitzgerald
Unit 8 Page 7
Unit 8
Positive test result: Vigorous and immediate bubbling as the dowel with the bacteria is put
into the hydrogen peroxide.
 A positive result indicates the organism tested produces the enzyme catalase and can
break hydrogen peroxide into water and oxygen.
 A positive result also means the organism has the ability to perform aerobic respiration
and therefore might be an obligate aerobe, or a facultative anaerobe.
(Microaerophiles, which do perform aerobic respiration, are always catalase negative.)
Negative test result: No or very little bubbling when the dowel with the bacteria is put in the
hydrogen peroxide.
 A negative result means the organism tested lacks the enzyme catalase and cannot
break hydrogen peroxide into water and oxygen using that enzyme (because it does
not have that enzyme).
 Organisms that test negative for the enzyme catalase are not obligate aerobes
(obligate aerobes are all catalase positive), but an organism that is negative for the
catalase test might be a facultative anaerobe, a microaerophile, a capnophile, an
aerotolerant anaerobes or an obligate anaerobe.
 Note: Obligate anaerobes are poisoned by oxygen, so if a specimen was growing on a
petri dish or a tube in our lab and was not in special anaerobic media, the organism
CANNOT be an obligate anaerobe.
 Also, a negative result DOES NOT guarantee the organism is anaerobic. Organisms can
produce other enzymes that break down hydrogen peroxide. For example, organisms
in the genus Streptococcus test negative for catalase, but these organisms are NOT
anaerobes.
 Note: Streptococcus species are, according to most authorities, facultative anaerobes,
but articles, especially disease articles, often refer to Streptococci as microaerophiles.
This is probably because some Streptococcus species have enzymes that are more
effective at breaking down blood under low oxygen conditions. Streptococcus species
also have an incomplete electron transport system and only use oxygen as a final
electron acceptor under certain conditions (such as on a blood rich medium). Thus,
they DO perform aerobic respiration, sometimes. However, they DO NOT produce
catalase. Instead they produce a peroxidase to break down hydrogen peroxide.
Unit 8 Page 8
Unit 8
Table 8-1. The results and interpretation of the catalase test.
Organism Name
Is the
organism
tested
Gram + or
Gram -?
(simply
look this
up on pg
22)
Catalase test
Results
(bubbles =
positive; no
bubbles =
negative)
Does the
organism
produce the
enzyme
catalase?
(Y or N)
Does the organism
have the ability to
perform aerobic
respiration?
(Y or N or can’t tell)
Post Activity Questions
1. Based on the results you collected, are any of the organisms you tested aerotolerant
anaerobes? (“Maybe” is an acceptable answer.) Defend your answer.
2. Based on your results and the material in the introductions, could any of the organisms
you tested be obligate aerobes? (“Maybe” is an acceptable answer.) Defend your
answer.
Unit 8 Page 9
Unit 8
3. Based on your, results can you use the catalase test to distinguish all Gram(+)
organisms from Gram(-) organisms? Defend your answer.
4. Clinically, when is the catalase test often used?
Unit 8 Page 10
Unit 8
Oxidase Test
III.
Introduction
Indophenol oxidase, IF PRESENT, participates in the cytochrome oxidase complex which is the
last step of the electron transport chain (ETC). The ETC is the final process of aerobic and
anerobic respiration. The cytochrome oxidase complex oxidizes cytochrome C (causes Cyt C to
lose electrons) and reduces oxygen (causes O to gain electrons) which can then combine with
H+ to generate water as shown. Since this enzyme works with oxygen it is only useful for
aerobic respiration processes.
This is the equation:
Indophenol oxidase
(Enzyme)
O2 + 4 H+ + 4 e- -------------> 2 H2O
but it is usually reduced to
Indophenol oxidase
(Enzyme)
½ O2 + 2H++ 2e- -------------> H2O
Only SOME bacteria with an ETC have this version of indophenol oxidase. Other bacteria have
other oxidases. Also, remember that some bacteria lack an ETC altogether, so those would not
have indophenol oxidase in any form.
Figure 8-4. The electron transport chain. Note the cytochrome oxidase complex. This is where
indophenol oxidase will be IF the organism has that enzyme. When the Oxidase Test is positive,
indophenol oxidase is present.
NADH
Dehydrogenase
Complex
+
Cytochrome
b-c Complex
+
H
+
H
+
+
H
+
Periplasmic
+
H
Hspace
H
+
H
Cytochrome
Oxidase
Complex (cyt a,a3)
ATP
Synthase
+
+
+
H
+
H
H
H
+
H
H
-
e
e
Cytochrome C
-
Q
eNADH
e
+H+
+
H
NAD+ +
H+
+
+
-
H
H
+
H
+
H
2e- + 2H+ + ½ O2  H2O
Cytoplasm of bacterial cell
Image created by Patricia G. Wilber, 2015
Unit 8 Page 11
ATP
ADP + Pi
Unit 8
If indophenol oxidase is present in the ETC of an organism, our oxidase test works by providing
a phenylenediamene oxidase reagent which is oxidized by the enzyme indophenol oxidase. The
oxidation process turns the reagent purple within 10-30 seconds. Organisms that show a
positive reaction (purple) in the oxidase test therefore have an ETC with indophenol oxidase
and we have confirmed that they can perform aerobic respiration. They may be obligate
aerobes, facultative anaerobes, capnophiles or microaerophiles.
In the diagnostic lab, oxidase and catalase tests are often performed and reported together.
The oxidase test is also useful to help distinguish the oxidase positive Pseudomonas, Nesseria,
Helicobacter and Campylobacter species from other Gram-negative, rod, shaped bacteria.
Pseudomonas aeruginosa is an extremely hardy species that can colonize a wide variety of
habitats, including hypoxic (without oxygen) environments. Many sources state that organisms
such as P. aeruginosa, that are catalase positive and oxidase positive, are obligate aerobes, but
this is incorrect. Pseudomonas aeruginosa is actually a facultative anaerobe and in the
immunocompromised, can be a dangerous, hard to control pathogen in the blood, lungs and
wounds.
Nesseria gonorrhea, the organism that causes gonorrhea, is oxidase positive, although the test
is not important for diagnosis of that infection.
Helicobacter pylori, a stomach bacterium found in about 4% of the U.S. population and a
causative agent of ulcers and stomach cancer, is also oxidase positive.
Most Enterobacteriaceae (bacteria found in the intestines) are oxidase negative.
Oxygen Utilization
Category
Obligate aerobe
Facultative anaerobe
Microaerophiles and
capnophiles
Aerotolerant anaerobe
Obligate anaerobe
Respiration
Oxidase reaction
Yes, only performs aerobic
respiration
Yes, performs aerobic
respiration, and well as
respiration without oxygen
Yes, does aerobic
respiration, and well as
respiration without oxygen
Does not perform aerobic
respiration
Does not perform aerobic
respiration; poisoned by
oxygen
A few species are positive
Unit 8 Page 12
A few species are positive
A few species are positive
Always oxidase negative
Always oxidase negative
Unit 8
Video Links:

Oxidase Test https://youtu.be/F_ehe8l9m-Y
Video created by Corrie Andries and Karen Bentz
Materials: (per pair)





Wooden dowels/sticks
Oxidase reagent (in an ampule)
Paper towels
Bibulous paper
Cultures
o Enterococcus faecalis (Ef)
o Streptococcus mitis (Sm)
o Pseudomonas aeruginosa (Pa)
o Bacillus megaterium (Bm)
o Staphylococcus saprophyticus (Ss)
Procedure:
1. Put some bibulous paper on a paper towel.
2. Using your wooden dowel, put the flat end on top of one single colony of the bacteria
being tested (to get a single species in case of contamination) and push gently so some
bacteria sticks to the dowel.
3. Place the end of the dowel on the bibulous and gently grind the end of the bacteria onto
the paper.
4. Put a few drops of oxidase reagent on the bibulous paper next to the scrubbed in
bacteria and let the reagent bleed over into the bacteria.
5. Observe your results. A positive reaction will turn the paper purple within 5-30 seconds
and a negative reaction will be no color, yellowish or sometimes pinkish within the 30
second time frame. Do not record results obtained after 30 seconds.
6. Record your results in Table 8-2.
7. Dispose of the bibulous paper and paper towels in the biohazard bucket and the wooden
dowel in the sharps container.
Precautions:




Use a platinum or wooden stick.
Use fresh (less than 24 hours old) colonies (those provided in this class are fresh).
Colonies should be at room temperature prior to testing.
Use ONLY colonies grown on non-selective, non-differential media. If you try the
oxidase test using bacteria grown on a MacConkey’s plate, for example, the result
virtually always appears positive because of the purple crystal violet in that medium.
Unit 8 Page 13
Unit 8
Results and Interpretation:
Figure 8-5. Oxidase positive result
Photograph by Andrea Peterson, 2015
Figure 8-6. Oxidase negative result.
Photograph by Andrea Peterson, 2015
Positive test result: The bibulous paper turns purple within 5-30 seconds. DO NOT consider the
result positive if the color change occurs later than 30 seconds.
If the organism is positive for the oxidase test, the organism has the enzyme indophenol
oxidase. Since the enzyme indophenol oxidase is part of the utilization of oxygen in the ETC,
the organism CAN perform aerobic respiration. Therefore an oxidase positive organism might
be an obligate aerobe, a facultative anaerobe (like Pseudomonas), a capnophile or a
microaerophile (like Campylobacter, which is also a capnophile).
Unit 8 Page 14
Unit 8
Negative result: If there is no color change to purple within 30 seconds (disregard color
changes after 30 seconds), the oxidase test is considered negative. Therefore the organism
tested lacks the enzyme indophenol oxidase.
If the result is negative, we have ONLY learned that the organism tested lacks the enzyme
indophenol oxidase.


A negative result DOES NOT guarantee the organism is anaerobic. There are other
enzymes that can be used in place of indophenol oxidase in the cytochrome oxidase
complex! For example, organisms in the genus Proteus test negative for the oxidase
test. These organisms are NOT anaerobes. They are facultative anaerobes and they do
have an ETC that is used in the final step in aerobic metabolism.
A negative result DOES NOT mean that the organism lacks an ETC. Even anaerobic
organisms have ETCs. They just do not use oxygen as the final electron acceptor! For
example, Clostridium perfringens, which is anaerobic and can cause gas gangrene, uses
nitrate (NO3- ) as its final electron acceptor.
Organisms that test negative for the enzyme oxidase might be obligate aerobes, facultative
anaerobes, microaerophiles, capnophiles, aerotolerant anaerobes or obligate anaerobes.
However, recall the obligate anaerobes are poisoned by oxygen so if a specimen was growing
on a petri dish or a tube in our lab and was not in special anaerobic media, the organism
CANNOT be an obligate anaerobe.
Table 8-2. The results of the oxidase test.
Organism Name
Is the
organism
Gram(+) or
Gram(-)?
Use pg. 22
to look this
up.
Oxidase test
Results within 30
seconds (purple =
positive; no color
change =
negative)
Unit 8 Page 15
Does the
organism
produce the
enzyme
indophenol
oxidase? (Y or N)
Does the
organism have
the ability to
perform aerobic
respiration?
(Y or N or maybe)
Unit 8
Post Activity Questions:
1. Based on the results you collected from the oxidase tests are any of the organisms you
tested aerotolerant anaerobes? (“Maybe” is an acceptable answer.) Defend your
answer.
2. Based on the results of your oxidase test, list any of the organisms that MIGHT be
obligate aerobes. Defend your answer(s).
3. True or False: A negative oxidase means the organisms is anaerobic. DEFEND YOUR
ANSWER.
4. Use the results key below to answer the questions and identify George’s organism. (You
can meet George below.)
A. George is a 2192 student. (There. You met him!) He did a catalase test on an
unknown organism that is one of those in the Results Key at the end of this unit, and
found bubbling. Using the Results Key, list all the organisms that show bubbling and
all that do not show bubbling. You may abbreviate the organism names.
Unit 8 Page 16
Unit 8
Circle the list that contains the one organism that George tested.
Dichotomous Key Question: Did the organism bubble as a result of the Catalase
test?
Yes: (List those that should bubble)
No: (Those that should not bubble)
B. George next did a Gram stain and found he had a purple rod. This test results in
TWO questions for the identification process.
Use your circled list above to make a new list (because that is the list that contains
the one organism George has).
Dichotomous Key Question: Is the organism Gram(+)?
Yes: (thus George might have)
(hint: bubbled, purple)
No: (George doesn’t have)
(hint: bubbled but is not purple)
Dichotomous Key Question: Is the organism bacillus in shape?
Yes: (thus George might have)
(hint: bubbled, purple, rod)
No: (George doesn’t have)
(hint: bubbled, purple, not a rod)
Unit 8 Page 17
Unit 8
C. Finally, George did an oxidase test and he got purple on his bibulous paper. He
should now be able to identify his organism.
What is the Dichotomous Key Question?
Write the full name of his organism in proper scientific format.
How do you know this is George’s organism?
Results Key for Post Lab Q4.
Organism
Cell
Shape
Gram(-) Bacteria
Escherichia coli
rod
Haemophilus
haemolyticus
rod
Proteus vulgaris
rod
Shigella flexneri
rod
Psuedomonas
aeruginosa
Citrobacter
freundei
Klebsiella
pneumoniae
Serratia
marcescens
rod
rod
rod
rod
Gram(+) Bacteria
Bacillus subtilis
rod
Staphylococcus
aureus
Bacillus
megaterium
Staphylococcus
saprophyticus
Streptococcus
mitis
Streptococcus
pneumonia
Streptococcus
pyogenes
Enterococcus
faecalis
cocci
rod
cocci
cocci
cocci
cocci
cocci
Catalase
Oxidase
(+)
(-)
(+)
(+)
(+)
(-)
(+)
(-)
(+)
(+)
(+)
(-)
(+)
(-)
(+)
(-)
(+)
(-)
(+)
(-)
(+)
(+) or (-)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
Unit 8 Page 18
Unit 8
DAY 2
I.
Streak Isolation
Results and Interpretation.
Hopefully, your results show clear isolation like this: (You have different species though and
this is a blood plate, not a chocolate agar!)
Plate by Leanna Gutierrez, Bio 2192, Spring 2016.
On your plate, hopefully you see some big grayish, flat and not shiny colonies. These are Bm.
Hopefully you also see some tiny green colonies. These are Spy.
We are getting closer and closer to the midterm and the final project. BOTH require a streak
isolation. For the midterm, you will streak isolate a single species from a plate or a broth
(depends on the term). For the final project you will streak isolate from a broth containing two
species mixed together.
Insert a photo of your streak isolation here. Compare it to the picture above.
Unit 8 Page 19
Unit 8
1. Do you have two isolated colonies? (your instructor can help you determine this.)
2. If not, why not? Ideas:
a. You did not stir the broth adequately before streaking. The Bm grows on the top
of the broth, so it just might have been missed.
b. You used too much bacteria so one over-ran the other.
3. If you did not get isolated colonies find a class mate that has them and really look at the
colonies. They are very different from each other. The Bm is flat and big and grayish is
not shiny. The Spy is tiny and greenish.
If, at this point, you still cannot perform a correct streak isolation pattern and achieve isolated
colonies, YOU MUST figure out why this is so. In any case, answer the following questions.
The main things to consider are:
1. Have you mastered the basic pattern? YES or NO.
a. If no, look at the pattern in U3, look up streak isolations online and practice the
pattern on a piece of paper to develop muscle memory.
2. Are you using too much bacteria? YES or NO.
a. If yes, USE LESS! Touch your loop to a solid colony if using a plate; do not scoop
it. Tap the excess liquid off your loop if using a broth.
3. Did you sterilize your loop between sections? YES or NO.
a. If no, just remember to use that incinerator!
4. Are you overlapping too much? YES or NO.
a. If yes practice the pattern with (see 1) with less overlap between sections. You
must overlap some, though. No overlap = no bacteria to spread to the next
section.
Unit 8 Page 20
Unit 8
Post Lab Questions
1. Based on the results of your catalase test and the oxidase test, list one organism you
tested that could be a facultative anaerobe. Defend your answer using the data you
have collected.
2. Using the Results Key below, fill in the following table
Catalase Test
Oxidase Test
bubbles
purple
bubbles
not purple
no bubbles
purple
No bubbles
Not purple
List up to two possible
organisms
Possible oxygen utilization
categories for these oxidase and
catalase results
3.
Answers for Figure 8-1.
Tube # Oxygen utilization
category
1
obligate aerobe
Aerobic
Respiration?
Yes or No
YES, aerobic
Defense
growing only at top where there is oxygen
Unit 8 Page 21
Unit 8
2
obligate anaerobe
3
facultative anaerobe
NO, not
aerobic
YES, aerobic
4
microaerophile
YES, aerobic
5
aerotolerant
anaerobe
NO, not
aerobic
growing only at bottom where there is no oxygen
growing throughout the tube but the population size is bigger
at the top where there is oxygen
grows best with small amounts of oxygen; many
microaerophiles are also capnophilic
grows equally well throughout the tube without regard to
oxygen concentration
The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms,
our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our dean, John
Cornish.
Unit 8 Page 22