Microbiology Unknown Lab
0.1%
dextrose
TSI agar
1.0%
sucrose
Triple Sugar Iron Agar
A
(a) Red/red (no sugar
fermentation)
(b) Control
(c) Red/yellow (Glucose
fermented but lactose and
sucrose not fermented)
(d) Yellow/yellow (Glucose
fermented. Lactose and/or
sucrose fermented)
(e) Red/yellow with H2S
B
C
D
E
1.0%
lactose
Penol Red broths
A
B
C
D
E
A (acid with gas)…..B (acid)……C (uninoculated control)……D (no recation)… …E
(alkaline reaction)
O-F tests
Control
E. coli
Fermenter
Pseudomonas
Oxidizer
Alcaligenes
Nonsaccharolytic
Motility Test
Decarboxylation
uninoculated
Positive
Negative
Gelatin Hydrolysis
Simmons Citrate
A
B
C
A: Positive…Enterobacter
B: Negative…E. coli
C: Control-uninoculated
Phenylalanine Deaminase Test
neg
pos
Urea Hydrolysis
Positive
uninoculated
Negative
Methyl Red Test
A
A: Positive (E. coli)
B
B: Negative (Enterobacter aerogenes)
Voges Proskauer Test
A
A: Negative (E. coli)
B
B: Positive (Enterobacter aerogenes)
Indole Test
Negative
-
Positive
+
Catalase Test
+
Positive
Negative
GRAM
STAIN
General Microbiology (BIOL 187) Laboratory Report (Unknown Identification)
Page 1: Cover page
Include your name, the unknown number and you final Genus and species identification
Page 2: Test listing
I want a (+) / (-) listing of each test in a vertical, easily scanned format
Page 3: Discussion section
Detail, in complete sentences, why your results justify your identification and rule out other possibilities.
Page 4: About My Microbe section
Research your microbe and give me a several paragraphs about your microbe.
(Don't copy word-for-word from your textbook---This will be frowned upon!)
Page 5: Dichotomous key
Create a neat dichotomous key, which results in the correct separation of all 8 possible unknowns. This key should be
legible and sized to occupy a complete sheet of paper (nothing microscopic is acceptable here)
Page 6:Lab handout where you recorded your initial results
Include the handout from lab where your recorded your data as you read the test reactions.
Grading:
20 pts.
Correct Identification
10 pts.
Discussion section
7 pts.
"About my microbe section"
8 pts.
Dichotomous key
5 pts.
Neatness, accuracy and completeness of report
50 points total
Differential Identification of Enterobacteriaceae by Biochemical Tests.
Test
Indol
Salmonella
enteritidis
Citrobacter
freundi
Enterobacter
cloacae
Enterobacter
aerogenes
Proteus
vulgaris
Proteus
mirabilis
+
- or +
-
-
-
-
+
-
+
+
+
+
-
-
+
+
VogesProskauer
-
-
-
-
+
+
-
- or +
Simmons
Citrate
-
-
+ or -
+
+
+
- or +
+ or -
Hydrogen
Sulfide
-
-
+
+ or -
-
-
+
+
-
-
-
+ or -
+ or -
-
+
+
Motility
+
+ or - or +
(+)
d
Shigella flexneri
Methyl
Red
Urease
How do I interpret the table above??
Escherichia coli
+ or -
-
+
+
+
+
+
+
Gelatin
Hydrolysis
-
-
-
-
- or (+)
- or (+)
+
+
Lysine
decarboxylase
+ or -
-
+
-
-
+
-
-
Arginine
dihydrolase
d
d
+ or (+)
d
+
-
-
-
Ornithine
Decarboxylase
+ or -
-
+
- or +
+
+
-
+
Phenylalanine
deaminase
-
-
-
-
-
-
+
+
Gas from
Glucose
+
-
+
+
+
+
+ or -
+
Glucose
fermentation
+
+
+
+
+
+
+
+
Lactose
fermentation
+
-
-
+ or -
+
+
-
-
Sucrose
fermentation
d
-
-
d
+
+
+
- or +
Mannitol
fermentation
+
+ or -
+
+
+
+
-
-
Sorbitol
fermentation
+
- or +
+
+
+
+
-
-
Acid/Acid
Alk/Acid
Or
Acid/Acid
TSI
Acid/Acid
Alk/Acid
Alk/Acid
Acid/Acid
Acid/Acid
means 90% or more of the strains are positive
means 90% or more of the strains are negative
means most strains are positive but a significant number are negative
means most strains are negative but a significant number are positive
means a positive result may be weak or delayed in its occurrence
means different results can be obtained. Tests marked with a d would be unreliable for identification purposes.
Acid/Acid
Lets make a dichotomous key !
Where do I start?
Escherichia coli
Shigella flexneri
Salmonella
enteritidis
Citrobacter freundi
Enterobacter
cloacae
Enterobacter
aerogenes
Proteus vulgaris
Proteus mirabilis
Indol
+
- or +
-
-
-
-
+
-
Methyl
Red
+
+
+
+
-
-
+
+
Voges-Proskauer
-
-
-
-
+
+
-
- or +
Simmons
Citrate
Hydrogen Sulfide
-
-
+ or -
+
+
+
- or +
+ or -
+ or -
-
+
+
+ or + or +
+ or +
+
+
+
+
+
+
+
-
-
-
-
- or (+)
- or (+)
+
+
+ or -
-
+
-
-
+
-
-
d
d
+ or (+)
d
+
-
-
-
+ or -
-
+
- or +
+
+
-
+
-
-
-
-
-
-
+
+
+
-
+
+
+
+
+ or -
+
+
+
+
+
+
+
+
+
+
-
-
+ or -
+
+
-
-
d
-
-
d
+
+
+
- or +
+
+ or -
+
+
+
+
-
-
+
- or +
+
+
+
+
-
-
Acid/Acid
Alk/Acid
Or
Acid/Acid
Test
Urease
Motility
Gelatin
Hydrolysis
Lysine
decarboxylase
Arginine
dihydrolase
Ornithine
Decarboxylase
Phenylalanine
deaminase
Gas from
Glucose
Glucose
fermentation
Lactose
fermentation
Sucrose
fermentation
Mannitol
fermentation
Sorbitol
fermentation
TSI
Acid/Acid
Alk/Acid
Alk/Acid
Acid/Acid
Acid/Acid
Acid/Acid
Enterobacter cloacae
Enterobacter aerogenes
Methyl Red
-
Next…look for a test that
distinguishes between the 2
methyl red negtive microbes
+
E.Coli
Shigella flexneri
Salmonella
Citrobacter
Proteus vulgaris
Proteus mirabilis
Test
Indol
Methyl
Red
Voges-Proskauer
Simmons
Citrate
Hydrogen Sulfide
Urease
Motility
Gelatin
Hydrolysis
Lysine
decarboxylase
Arginine
dihydrolase
Ornithine
Decarboxylase
Escherichia coli
Shigella flexneri
Salmonella enteritidis
Citrobacter freundi
Enterobacter
cloacae
Enterobacter
aerogenes
Proteus vulgaris
Proteus mirabilis
+
- or +
-
-
-
-
+
-
+
+
+
+
-
-
+
+
-
-
-
-
+
+
-
- or +
-
-
+ or -
+
+
+
- or +
+ or -
-
-
+
+ or -
-
-
+
+
-
-
-
+ or -
+ or -
-
+
+
+ or -
-
+
+
+
+
+
+
-
-
-
-
- or (+)
- or (+)
+
+
+ or -
-
+
-
-
+
-
-
d
d
+ or (+)
d
+
-
-
-
+ or -
-
+
- or +
+
+
-
+
Phenylalanine
deaminase
-
-
-
-
-
-
+
+
Gas from
Glucose
+
-
+
+
+
+
+ or -
+
Glucose
fermentation
+
+
+
+
+
+
+
+
Lactose
fermentation
+
-
-
+ or -
+
+
-
-
Sucrose
fermentation
d
-
-
d
+
+
+
- or +
Mannitol
fermentation
+
+ or -
+
+
+
+
-
-
Sorbitol
fermentation
+
- or +
+
+
+
+
-
-
TSI
Acid/Acid
Alk/Acid
Alk/Acid
Acid/Acid
Acid/Acid
Acid/Acid
Acid/Acid
Alk/Acid
Or
Acid/Acid
The lysine
decarboxylase
test
distinguishes
between these
2 microbes
Methyl Red
Enterobacter cloacae
Enterobacter aerogenes
-
+
E.Coli
Shigella flexneri
Salmonella
Citrobacter
Proteus vulgaris
Proteus mirabilis
+
Lysine decarboxylase
Enterobacter cloacae
Enterobacter aerogenes
NOW…look for a test that
distinguishes between the 6
methyl red positive microbes.
Escherichia coli
Shigella flexneri
Salmonella enteritidis
Citrobacter freundi
Enterobacter
cloacae
Enterobacter
aerogenes
Proteus vulgaris
Proteus mirabilis
+
- or +
-
-
-
-
+
-
+
+
+
+
-
-
+
+
-
-
-
-
+
+
-
- or +
-
-
+ or -
+
+
+
- or +
+ or -
-
-
+
+ or -
-
-
+
+
-
-
-
+ or -
+ or -
-
+
+
+ or -
-
+
+
+
+
+
+
-
-
-
-
- or (+)
- or (+)
+
+
+ or -
-
+
-
-
+
-
-
d
d
+ or (+)
d
+
-
-
-
+ or -
-
+
- or +
+
+
-
+
Phenyl
alanine
deamin
ase
-
-
-
-
-
-
+
+
Gas from
Glucose
+
-
+
+
+
+
+ or -
+
Glucose
fermentation
+
+
+
+
+
+
+
+
Lactose
fermentation
+
-
-
+ or -
+
+
-
-
Sucrose
fermentation
d
-
-
d
+
+
+
- or +
Mannitol
fermentation
+
+ or -
+
+
+
+
-
-
Sorbitol
fermentation
+
- or +
+
+
+
+
-
-
TSI
Acid/Acid
Alk/Acid
Alk/Acid
Acid/Acid
Acid/Acid
Acid/Acid
Acid/Acid
Alk/Acid
Or
Acid/Acid
Test
Indol
Methyl
Red
Voges-Proskauer
Simmons
Citrate
Hydrogen Sulfide
Urease
Motility
Gelatin
Hydrolysis
Lysine
decarboxylase
Arginine
dihydrolase
Ornithine
Decarboxylase
The phenylalanine
deaminase test
has 4 negatives and 2
positives for the 6
microbes that are not
yet “keyed-out”
It would be a possibility
for the next choice.
Methyl Red
Enterobacter cloacae
Enterobacter aerogenes
-
+
-
+
Lysine decarboxylase
Enterobacter cloacae
E.Coli
Shigella flexneri
Salmonella
Citrobacter
Proteus vulgaris
Proteus mirabilis
-
+
Phenyalanine deaminase
Enterobacter aerogenes
E.Coli
Shigella flexneri
Salmonella
Citrobacter
(these 4 still need to be
“keyed-out” from each
other)
Proteus vulgaris
Proteus mirabilis
(these 2 still need to be
“keyed-out” from each
other)