Identification of Two Unknown Bacteria in Mixed Culture
A standard part of nearly all lab courses in introductory microbiology is an activity wherein
the student must use everything that has been learned in the course to identify an unknown bacterial
culture. Your ability to make aseptic transfers, perform the gram stain, identify cell morphology and
shape, and conduct metabolic testing is under examination in this process. Your ability to follow
the stepwise logic of a dichotomous key will also be tested. Overall, your strategy is to be
parsimonious; you should only do needful tests according to your key and should only need to do
them once. This Exercise and Report are worth 50 points towards your final grade.
These instructions replace those in Exercise 25, but we will be using the worksheets
attached to Exercise 25 in the lab manual for writing up the report. An example of the completed
lab report using the worksheet pages is attached to this guide to help guide you in recording
your data completely and writing up your final report correctly.
Making a Dichotomous Key
The first thing to prepare for an Unknown Identification exercise is to make a dichotomous
key. Dichotomous keys are written from known characteristics of the possible organisms you may
need to identify. The following six organisms will be used in this activity; you will be given a
mixture of two of them together. Characteristics that you can use in making a dichotomous key are
listed in the table. Abbreviations: A = Acid, G = gas, P = pink, W = white, +/- = Variable, - = No
Growth, - = No Growth.
-Bacillus
Enterobacter aerogenes
-Bacillus
Citrate utilz.
Lactose ferm.
Sucrose ferm.
Glucose ferm.
+/
A
-
A
-
-
-
+
-/+
-
+
-
+
-/
-
-
-
-
+/-
-
+
-
+/
AG
A
A
G
-
+
A
A
G
+
+
-
-
+/-
-
+
+
-
+/
A
G
AG
A
G
-
-
-
-
+/-
-
+
-
-
+/
-
-
-
-
-
-
-
-/-
+
+
-
-
+/
A
G
A
A
+
-
+
-
-/+
-
+
Urea
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 1
MR/VP
+
Motility
+
Indole
production
Catalase
Pseudomonas aeruginosa
Oxidase
Bacillus
Hemolysis
Klebsiella pneumoniae
Staphylococcus epidermidis
Abundant, opaque,
golden growth
Off-white, smooth,
small, round
White, moist,
glistening growth
Translucentcreamy, mucoid,
round
Abundant, thin,
white growth with
medium turning
green
Abundant, thick,
white, glistening
growth
Salt tolerant
Escherichia coli
+
Coccus
+
Coccus
Bacillus
Staphylococcus aureus
Culture
characteristics
on agar slant
Mannitol
fermentation
Organism
Gram
Stain, Shape
Table I. Morphological, Staining, and Metabolic Characteristics of Six Bacteria Used in This
Exercise
Dichotomous keys are charts that require decisions at branch points, much like a flow chart
in computer logic: If the answer to a question is yes, then do X; if the answer is no, then do Y.
Your job is to make the shortest key, with the shortest pathways, to a final identification of an
organism. Ideally, any pathway in your key should have no more than four steps (including gram
staining and cell morphology as separate steps) leading to an identification.
To make a dichotomous key, you first need to sort all the different species in your list into
two big groups based on common characteristics between them. Most microbiologists begin with
gram stain results, and I encourage you to do the same. Using the Example Dichotomous Key in
this Guide, you will see that you should begin your own key by writing out the names of all the
possible bacteria in the test near the top of the page. I used 8 bacterial names in my Example Key;
you should use the 6 organism names from Table I. I recommend using landscape (sideways) mode
in laying out a key since the wider the page, the better.
After listing all 6 potential species on your key sheet, you should next draw two arrows
downwards that show this group being separated by the gram stain. Write gram positive at the end
of one arrow and gram negative after the other. Follow each result with a list of bacteria still
remaining in each group. In my Example Key, I was able to divide my list of 8 bacteria into two
groups of four.
The next dichotomy to further separate the gram positives from each other, and the gram
negatives from each other, should be one of salt tolerance, lactose fermentation, or cell morphology
(bacilli or cocci) since you will get results of these tests from the selective media (MacConkey and
mannitol salt agar) you will use early in the exercise. The important thing is that you are subdividing
each group at every dichotomy into two subgroups of nearly equal size. If you separate off only one
species at a time at each dichotomy, your key will be far too long and will not be parsimonious.
In my example key, I decided to separate the gram negatives using galactose fermentation. I
decided on this test after discovering that the four gram negative bacteria differed in their
fermentation reactions to galactose. Half fermented the sugar and half did not.
That's the strategy you should employ at each step in the key: find a characteristic that differs
in the group of organisms you need to separate. Ideally this characteristic or test divides them into
roughly equal subgroups. You continue finding tests that subdivide those groups until you only
have one organism left per group.
You will need a completed dichotomous key by the first period of the Unknown ID activity.
The instructor will not let you progress further unless you can demonstrate that you have a
completed and correct dichotomous key to work from. Ultimately you will handwrite or type a
copy of your dichotomous key and (if typed) paste it onto the blank page of your lab manual (pg.
210). Try, if you can, to get it all on one page. Use the Example Key on the next pages an example.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 2
Here's the list of 8 bacteria and their characteristics that I used in making my Example
Dichotomous Key of Eight Bacteria on the following page. You are not using these bacteria in Ex
25; these are just for demonstration purposes.
Table II. Characteristics of 8 Example Bacteria Used to Make a Dichotomous Key
Organism
Characteristics
Alcaligenes faecalis
Gram negative bacillus, acid-fast negative, ferments galactose, catalase
positive, motile, alkaline with litmus milk, not hemolytic, H2S negative
Citrobacter freundii
Gram negative bacillus; acid-fast negative, does not ferment galactose,
catalase positive, non-motile, acid produced in litmus milk, not
hemolytic, H2S negative
Pseudomonas fluorescens
Gram negative bacillus, acid-fast negative, does not ferment galactose,
catalase positive, non-motile, alkaline in litmus milk, hemolytic, H2S
negative
Streptococcus pneumoniae Gram positive oval coccus found as streptococci, acid-fast negative,
galactose fermentation negative, catalase negative, non-motile, acid in
litmus milk, hemolytic, H2S negative
Mycobacterium smegmatis Gram positive club-shaped bacilli, acid-fast positive, galactose
fermentation negative, catalase-negative,, non-motile, alkaline in
litmus milk, catalase positive, not hemolytic, H2S negative
Mycobacterium leprae
Gram positive club-shaped bacilli, acid-fast positive, galactose
fermentation negative, catalase-positive, non-motile, alkaline in litmus
milk not hemolytic, H2S negative
Corynebacterium xerosis
Gram positive club-shaped bacilli, acid-fast negative, galactose
fermentation positive, non-motile, acid from litmus milk, catalase
positive, not hemolytic, H2S negative
Aeromonas hydrophila
Gram negative bacilli, acid-fast negative, ferments galactose, catalase
positive, motile, acid from litmus milk, not hemolytic, H2S positive
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 3
Be sure to list of all
the bacteria that
remain at each step of
the process. At the
start, you list all the
bacteria being
considered.
Use the name of the
actual test or
molecule being
detected (lipid
hydrolysis), rather than
using the name of the
medium (e.g. mannitol
fat medium)
Example Dichotomous Key to Identifying Eight Bacteria
Alcaligenes faecalis
Citrobacter freundii
Pseudomonas fluorescens
Streptococcus pneumoniae
Gram stain
negative
positive
Alcaligenes faecalis
Citrobacter freundii
Pseudomonas fluorescens
Aeromonas hydrophila
Mycobacterium smegmatis
Mycobacterium leprae
Corynebacterium xerosis
Streptococcus pneumoniae
Acid-fast staining
Galactose fermentation
positive
acid fast
negative
Alcaligenes faecalis Citrobacter freundii
Aeromonas hydrophila
Pseudomonas fluorescens
H2S production
positive
Aeromonas
hydrophila
You will be working
with nine bacteria, as
listed in Exercise 25 of
your lab book. This
example key was made
up using 8 bacteria
Mycobacterium smegmatis
Mycobacterium leprae
Corynebacterium xerosis
Aeromonas hydrophila
negative
Litmus milk
acid
Citrobacter
Alcaligenes
freundii
faecalis
alkaline
Pseudomonas
fluorescens
not acid fast
Mycobacterium smegmatis
Mycobacterium leprae
Catalase produced
positive
Mycobacterium
leprae
negative
Hemolysis
positive
Mycobacterium Streptococcus
smegmatis
pneumoniae
Figure 1. An Example Dichotomous Key Using Characteristics From Table II.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 4
Corynebacterium xerosis
Streptococcus pneumoniae
negative
Corynebacterium
xerosis
Getting Started on the First Day (Instructions replace those in Ex. 25 in the Lab Manual)
On the first day:
1. Pick up an assigned bacterial culture from the instructor. The culture may be provided as a
broth or an agar slant. You should ask whether the culture is < 24 hours old as this will
determine whether or not you can perform a reliable gram stain (re-read the Gram Staining test
in your lab book if you don’t understand why). You should see a mixed culture with one
coccus species and one bacillus species. Record your observations on pg. 211, including the
shape, arrangement, and gram stain reaction. Include the color of your gram stained cells too.
2. Write down in your lab book the number of your assigned culture. It is very important that you
do not lose this number, for it is exclusively yours.
3. Next, inoculate TS agar, MacConkey agar, and mannitol salt agar plates from your mixed
culture, with proper streaking technique to get isolated colonies. Incubate these at a
temperature and a time period consistent with mesophiles that we've grown before during this
lab. Part of your final score will be based on your ability to correctly streak a plate and get
isolated colonies.
4. Next, perform a gram stain on your culture at your earliest opportunity. You may need to
transfer and grow the culture on a TSA plate for the appropriate amount of time before it is
ready for gram staining. You may label your original culture (usual labeling rules) and place it
in the 10oC incubator (refrigerator) so you have a backup of your culture. Write the results of
your gram stain at the beginning of the worksheets for Ex 25 in the appropriate space for "gram
stain of original culture.
5. All plates, tubes, or other media that are to be placed in an incubator or the refrigerator must
minimally have your full name, your culture number, and your lab section (morning, afternoon,
evening). You should also write the date of inoculation on all media so you know when you
started the test. I’ll leave it to you to keep track of what medium is in the tube!
6. As part of your report, you will be keeping a handwritten log or journal of everything you do in
regards to your culture, including inoculation and incubation times and dates, reagents added,
colors seen, interpretations made, etc. Start writing down what you do on the first day in a
notebook that can be kept in a safe place; you cannot lose your journal.
7. Be sure you show appropriate actions in cleaning up your lab space and in working safely while
in the lab. I will be looking out for correct and safe placement of the Bunsen burners and
incinerators, appropriate placement of notes versus cultures, whether you keep your chair
pushed in, hair tied back (if appropriate), books and packs underneath, etc.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 5
The Second Lab Period
1. Interpret the results of your TSA, MacConkey, and mannitol salt agar plates and record the data
(colony color, morphology) on pg. 211. Did you get separation on the TSA? What type of
organisms should grow on MacConkey? What else can you learn from colony color on
MacConkey? Write down your results.
2. Gram stain a colony (note it's morphology!) on MacConkey and write down your results on pg.
211. Designate this as Organism #1. Is the gram stain consistent with what you expected on
MacConkey? What morphological information or metabolic ability has been tested on
MacConkey? Use your answers to these questions by recording your data in the table on pg. 211.
Include both color changes (if any) and the interpretation of this result.
3. Use your answers to these questions by recording your data in the table on pg. 211. Gram stain a
colony from the mannitol salt agar, and call this Organism #2 . Is the gram stain consistent with
what you expected on MSA? What morphological information or metabolic ability has been tested
on MSA? Use your answers to these questions by recording your data in the table on pg. 211.
Include both color changes (if any) and the interpretation of this result.
4. Subculture a colony of Organism # 1 and #2 a TS slant to grow pure cultures for next time. What
would be a good temperature and time for your organisms?
5. Look at your dichotomous key and determine what remaining tests you need to perform to
complete the identification. If you haven't already shown your instructor your completed and
corrected dichotomous key, do so early in this period. You will be asked to defend your choices of
tests based on what's in your key. You must do this before you are allowed to proceed with futher
testing.
6. Are you keeping careful records of the date and everything you see and do? I hope so, for you'll
need this detailed information to write up your final rationale for how you identified your bacteria.
7. Inoculate all the media that you need in order to complete the identification for next time. Find the
procedures for how to initiate and interpret each test from your lab manual. Remember, you can't
ask your lab partner (that is considered cheating, which comes with grade penalties).
8. Turn in your TSA streak plate for grading. Be sure it is properly labeled and includes your
culture number.
Third Lab Period
1. Interpret the tests you have set up and do any remaining "on-the-spot" tests like oxidase, catalase,
gram staining, etc. on your fresh colonies on TS agar slants. Write down all your data in the
appropriate spaces on pg. 211 and 212. Don't forget to include color changes as well as interpretation.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 6
2. Complete the identification of your organism. Begin writing up your report on pg. 213 using the
following example report as your guide. Remember to paste in or write in your dichotomous key on
pg. 210 AND show the pathway you took to reach an identification (See example lab report following).
How I will grade your Unknown ID Exercise:
UNKNOWN REPORT GRADING RUBRIC
Student:
Possible
Your
GRADED ITEM
Pts
Points
Dichotomous Key Flow Chart:
- Is the chart parsimonious with four or less steps needed to reach the IDs?
- Are all steps true dichotomies and not trichotomies?
- Did you use only the characteristics for the 9 bacteria that are listed in your lab manual
for this exercise?
- Are all nine bacteria separated out?
- Is path you took for Organisms #1 and #2 clearly marked on your key?
- Did you have the key ready by the start of the third lab period?
- Was the key draw onto or pasted onto blank pg. 210 in your lab manual?
10
Gram Stain and Cell Morphology, Organism assignments
- Did you fully describe the shape, arrangement, and gram color and interpretation for
both organisms?
- Was this information consistent with the assigned organisms (teacher grades this)
Streak Plate (TSA) - Graded by instructor
- Did student achieve isolation of colonies, especially of both types?
- Was the technique faulty or beyond the control of the student?
Experimental Results
~ For every test you did, is there BOTH visual AND interpretive
Information for each test result?
- Did you include the media used in each test, along with the temperature and time of
incubation?
- Did you include any problems you encountered along the way?
~ Did you follow your key and only do the tests your key said to do?
- Were results correctly interpreted? (Teacher grades this)
Correct ID
~ Was the unknown correctly identified? Verified by instructor?
~ If unknown ID incorrect, was it the result of student error (technique or
judgment) or a factor out of the student's control?
Final Report on pg. 213 & 214
- Did you neatly write or type and paste in your final report on these pages?
~ Did you include dates, media used, and results (positive or negative) for each test you
inoculated or interpreted?
~ Did you make appropriate conclusions based on your key and results?
~ Has you identified any errors in technique or judgment and suggested alternatives for
future work?
~ Is report written in a professional and scientific manner?
Neatness, spelling, grammar, nomenclature
~ Is report written in professional, objective manner--no personal pronouns
~ Have all scientific names been written using proper nomenclature?
~ Is grammar and spelling proper and accurate?
~ Is report prepared in a tidy fashion according to requirements?
5
5
10
5
10
5
If lab book submitted late (assign -20% per day to this report)
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 7
TOTAL
50
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 8
Dichotomous Key to Identifying Eight Bacteria
NOTES:
Alcaligenes faecalis Mycobacterium smegmatis
Citrobacter freundii Mycobacterium leprae
Pseudomonas fluorescens
Corynebacterium xerosis
Streptococcus pneumoniae Aeromonas hydrophila
Gram stain
negative
Alcaligenes faecalis
Citrobacter freundii
Pseudomonas fluorescens
Aeromonas hydrophila
Mycobacterium smegmatis
Mycobacterium leprae
Corynebacterium xerosis
Streptococcus pneumoniae
Acid-fast staining
Galactose fermentation
positive
Alcaligenes faecalis
Aeromonas hydrophila
H2S production
positive
Aeromonas
hydrophila
negative
Citrobacter freundii
Pseudomonas fluorescens
Litmus milk
Citrobacter
Alcaligenes
freundii
faecalis
Organism #2
acid fast
negative
acid
Don't forget to highlight he
exact pathway you followed
in your key to reach an
identification of BOTH
your organisms.
positive
alkaline
Pseudomonas
fluorescens
not acid fast
Mycobacterium smegmatis
Mycobacterium leprae
Catalase produced
positive
Mycobacterium
leprae
negative
Corynebacterium xerosis
Streptococcus pneumoniae
Hemolysis
positive
Mycobacterium Streptococcus
smegmatis
pneumoniae
The name of only one organism
should be found at the end of each
pathway. Be sure this scientific
name is spelled correctly and
written in proper scientific format.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 9
negative
Corynebacterium
xerosis
Organism #1
Name Philo Microbius
Partner
leave blank
EXERCISE
Date 4/25/14
Unknown #
25
16
Section MW 7:30-9:20 pm
Source
leave blank
Be sure to describe the color,
Laboratory Report: Worksheet
Final Report:
shape and
and arrangement
that you
actually see along with your
Clinical Unknown Identification
interpretation
Gram stain of original specimen: Two organisms were seen, a purple (gram positive) bacillus in
chains (concobacillus) arrangement and a pink (gram negative) coccus in groups of 6
(hexacoccal).
(describe cell shape, arrangement, and Gram reaction)
Gram stains of TSA plate subcultures: Organism #1 was a purple (gram positive) bacillus in
streptobacillus arrangement and Organism #2 was a purple (gram positive) coccus in
staphylococcal arrangement. Note: I made a mistake in gram staining and describing the cocci
on the original specimen.
(describe cell shape, arrangement, and Gram reaction)
Organism #1
Organism #2
Colony description
Test
White, round, entire margin,
convex
(TS agar or blood?)
Gram stain
Viewed on TSA
Purple (gram positive) of 24
hr old colonies grown at
37oC
Yellow, irregular, lobate margin,
flat
Viewed on TSA
Purple (gram positive of 86 hr old
colonies grown at 37oC
Colony appearance on MacConkey
No growth after 24 hrs at
37oC
Dark purple with metallic sheen
after 24 hrs at 37oC
Colony appearance on mannitol salt
agar (MSA)
Pink tinged whitish colonies,
round, entire, and elevated
after 24 hrs at 37oC
Yellow, irregular, lobate margin,
convex after 24 hrs at 37oC
Did not perform
Did not perform
Did not perform
Did not perform
Positive (blue, acid from on
MacConkey agar, 37oC, 24
hrs)
Negative XX: Not turbid, no
gas, yellow in glucose PR
ferm. tube.
Positive AG: turbid and gas,
yellow color in sucrose PR
ferm. tube.
Did not perform
Negative
turbid,
gas in tube
Be(no
sure
to describe
the
Special stains
capsule
endospore
These
LactoseWarning:
fermentation
usingresults,
media X
including the color change and
reactions are only examples
Glucose(not
fermentation
media
correct!)using
-- copy
theX
format of these examples.
colorusing
or physical
SucroseInclude
fermentation
media X
change relative to control,
what this means (positive or
Mannitolnegative),
fermentation
using
media X
what
medium
was
used, and for how long and at
what temp. it was incubated.
25-6 Exercise 25 Clinical Unknown Identification
color, or
physical characteristics that you
actually
see along with your
Did not perform
interpretation.
Negative XX: Not turbid, no gas,
yellow in sucrose PR ferm. tube.
Positive: AG: Turbid with lots of
gas using mannitol PR ferm. tube
211
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 10
Test
Organism #1
Organism #2
Did not perform
Did not perform
Indole production
Methyl red
Voges-Proskauer
Citrate utilization
Urea hydrolysis
Motility
Catalase test
Coagulase
Oxidase
Remained deep purple (non change
(negative for ) after adding methyl
red to culture in BBSF broth, 24 hrs
at 37oC
Changed from yellow to bright blue
(positive for esculitin) after adding
VP reagents to culture grown in
BBSF broth, 24 hrs at 37oC
Remained pink, no change (negative
for calbutrin) using supbacter media
grown in BBSF broth, 24 hrs at 37oC
Did not perform
No flagella visible (negative) after
staining with colortail dye; taken
from culture in thioglycollate, 72 hrs,
at 25oC
Bubbles appeared after adding
H2SO4 (positive for cats) to a sample
25 hrs old cultured in TS broth
Did not perform
Negative XX: Not turbid, no gas,
yellow in glucose PR ferm tube.
Warning: These results,
Changed from deep purple to hot
including
the color
change
pink (positive)
after adding
methyl
and reactions
are only
red to culture
in BBSF broth,
24
o
hrs
at
37
C
examples (not correct!) -Remained
yellow
copy
the (negative
format offorthese
esculitin) after adding VP reagents
examples. Include color or
to culture grown in BBSF broth,
o
physical
change
relative to
24 hrs at 37
C
control, what this means
Change from
pink toorgreen,
with what
(positive
negative),
gas (positive for calbutrin) using
medium was used, and for
supbacter media grown in BBSF
how
at what temp.
broth,
24long
hrs atand
37oC
was
incubated.
Did not perform
it
Warning: These results,
4-6 flagella
visiblethe
percolor
cell change
including
(positive) after staining with
and reactions
only
colortail dye;
taken fromare
culture
examples
(notatcorrect!)
-in thioglycollate,
72 hrs,
25oC
copy
the format
of these
No bubbles
appeared
after adding
H2SO4 (negative
forInclude
cats) to acolor or
examples.
sample 25 hrs old cultured in TS
physical change relative to
broth
control, what this means
(positive
or negative), what
Did
not perform
medium was used, and for
how long and at what temp. it
Positive AX: turbid but no gas,
was incubated.
red color in glucose PR ferm tube.
Consult your dichotomous key Consult the table compiled from class results at the end
of the second session and figure 23.1 to help you identify your organisms. (made from
the table of organism characteristics) to identify your organism.
Final Identification
Organism #1
Organism #2
212
Please change this. You should
be using your dichotomous key
Clostridium difficule
to direct you at each step as to
what test to perform (look up
what media you should use and
how to interpret it in your lab
Yersinia pestis
book) This exercise is open
book, open notes. You just can't
25-6 Exercise 25consult
Clinicalother
Unknown
Identification
students
in the lab.
You can always ask me if you
need help on something.
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 11
Final Report
1. What is the identification of your organism? Write out step-by-step the tests you did and why you did
them (support this with what your key indicates should be done next). Include the outcome (positive or
negative) of each test. Finish with the name of your organism. Discuss the process of identification
(reasons for choosing specific tests, any problems, and other comments).
Organism #1:
Please change this. You can number each of your steps. Be
sure to include lots of detail. It is better to go overboard and be
too detailed rather than too general. I won't fault you for being
too detailed.
1. On the first day, 4/20/12, I subcultured double unknown # 16 onto a TS agar plate and grew it for 48 hrs at 37. I
streaked this plate with the idea of isolating the two species from each other. Next, I gram stained the broth culture we were
given and discovered two species, a gram-positive coccus (staphylococcal arrangement) and a gram-negative bacillus
(single bacilli arrangement). Finally, I inoculated two additional selective /differential plates and streaked for isolation:
MacConkey agar and Mannitol salt agar.
2. On 4/22/12 I found that I had good isolation of two different colony morphologies. The yellow, convex colony with an
entire margin and round shape on TSA I designated as Organism #1. I gram stained #1 and found gram-positive cocci in
staphyococcal. arrangement. A gram-positive organism also grew on EMB agar (producing green colonies, meaning it was
galactose positive), but it did not grow on MacConkey. This result is consistent with the notion that gram-positive colonies
should grow on EMB, and that galactose fermenters should produce green colonies. I subcultured a colony of #1 from EMB
onto a TS agar slant to grow for next time at 37oC for 48 hrs.
My dichotomous key indicates that for a gram-positive coccus, I need to do the oxidase test next.
3. On 4/24/12 I performed the oxidase test on Organism #1, using colonies on my younger, TS agar slant culture. The result
was positive. Since I was able to do this during class time, I was able to move to the next test in my key, which is for
fermentation products from glucose (MRVP). I asked the instructor for an MRVP broth and inoculated it from the TS slant
for next time.
4. On 4/28/12 I performed the MR and VP tests according to the lab manual procedure and discovered it to be MR positive
but VP negative. I was surprised at the negative VP test since my key says all bacteria at this point should be VP positive.
I asked permission to do the VP test again, and was given a "virtual" result of VP negative from the instructor. So, given a
You should keep a log
positive MR and negative VP, my organism should be Proteus vulgaris.
or journal of all the
things you do each day
Organism #2
(in addition to test
1. On the first day, 4/20/12, I subcultured double unknown # 16 onto a TS agar plate and grew it for
48 hrsin
at the
37. tables
I streaked
results
on
this plate with the idea of isolating the two species from each other. Next, I gram stained the broth culture we were given
previous
pages.
You
and discovered two species, a gram-positive coccus (staphylococcal arrangement) and a gram-negative bacillus (single
write up your
bacilli arrangement). Finally, I inoculated two additional selective /differential plates and streakedcan
for then
isolation:
step-by-step process on
MacConkey agar and Mannitol salt agar.
pg. 213 and 214, or type
2. On 4/22/12 I saw a different type of colony (blue with entire margins) on my TSA, which presumably
wasand
the glue
gram-them
them up
negative bacterium I saw on the first day. I designate this as Organism #2. I gram stained this newer, younger culture but
into your lab book on
surprisingly found it to be a gram-positive bacillus in single arrangement. The difference in the cell morphology of
Be sure
Organism #2 compared to the first day may be due to the older age of the cultures given to us. Oldthese
cells pages.
produce unreliable
they are formatted to fit
gram stain results.
A gram-positive organism also grew on EMB agar (producing yellow colonies, meaning onto
it was these
galactose
negative),
pages!
but it did not grow on MacConkey. This result is consistent with the notion that gram-positive colonies should grow on
EMB, and that galactose non-fermenters should produce yellow colonies.
My dichotomous key indicates that for a gram-positive bacillus, I need to do the urease test next.
25-6 Exercise 25 Clinical Unknown Identification
213
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 12
Organism #2, continued
3. 4/24/12. For Organism #2, I need to do the sorbitol test next. I went to the instructor and asked for a sorbitol
fermentation tube with phenol red. I inoculated this for next time.
4. On 4/28/12 I learned that Organism #2 could produce both acid and gas from sorbitol; it was positive for sorbitol
fermentation. My next step in the key is to an esculiten production test. I went to the instructor and asked for mebatunin
media and inoculated this broth for next time.
5. On 4/30/12 I learned that Organism #2 was positive for esculiten production. According to my key, this means this is
Yersenia pestis.
214
25-6 Exercise 25 Clinical Unknown Identification
Guide to the Identification of an Unknown Bacterium – Methods and Report Format pg. 13