MICROBIOLOGY LAB
TESTS
The following microbiology tests will be
performed in lab this semester
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Gram stain
Motility
EMB ( Eosin Methylene blue agar)
MacConkey agar
Carbohydrate fermentation ( glucose, lactose,
sucrose)
Gelatine liquefaction test
Mannitol salt
Indol
SIM ( indol, motility,sulfur reduction)
 IMViC
tests (Indole ,Methyl Red,VogesProskauer (VP) test,Citrate Test)
 Triple sugar Iron Agar
 Skim Milk agar
 Catalase
Gram stain
 Purpose:
The Gram stain is the most
commonly used differential stain for
determining cell morphology.
 The
gram stain divides most bacteria into
two main groups based on the amount of
the sugar: peptidoglycan present in the
bacteria cell wall.
Gram stain
Procedure

Procedure:
1. 1. clean your slide
2. Put ½ drop of water on the slide
3. Using a bacteria loop add bacteria to the
slide
4. Let slide air dry
5. Heat fix the slide
6. Add crystal violet to slide and let sit for 2
minutes.
7. Wash off under water
8. Add iodine to slide and let sit for 2 minutes
Procedure continue:
13.
Wash off under water
THIS IS THE MOST IMPORTANT STEP!!
Holding slide at an angle, carefully add
ethyl alcohol for 8 seconds
After 8 seconds are up, wash off alcohol
under water.
Add safranin red for at least 2 minutes
Wash off and blot dry
14.
Examine under microscope
9.
10.
11.
12.
Gram Positive bacteria
 Gram
positive bacteria stain purple and
have a thick layer of peptidoglycan in the
cell wall
Gram Negative bacteria
 Gram
negative bacteria stain pink and
have a thin layer of peptidoglycan in the
cell wall
Motility
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Purpose: “The
movement of bacteria is
detected by use of a
medium with low agar
concentration.
The medium is
inoculated with an
inoculating needle
Reading the test: An
organism that is motile
will grow out into the
surrounding medium
away from the stab line.
Motility
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Test Tube Results
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Test tube 1: Motile
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Test Tube 2:Nonmotile
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Test Tube 3：Motile
Motility Test
EMB ( Eosin Methylene
blue agar)
Purpose: This agar a is selective and
differential medium used for isolation
and differentiation among
Enterobacteriaceae.
EMB ( Eosin Methylene
blue agar)
EMB is selective for gram negative
bacteria meaning only gram
negative bacteria will grow on this
medium.
EMB ( Eosin Methylene blue agar)
 EMB
also contains lactose.
 Organisms which do not ferment lactose
will have colorless colonies
 Organisms which do ferment lactose will
have pink colonies.
 E. coli develops a metallic green sheet
of colonies
EMB with E. coli
Organisms which do ferment
lactose will have pink colonies.

.
Organisms which do ferment
lactose will have pink colonies
MacConkey agar
Purpose: MacConkey is both
selective and differential used to
differentiate between gram negative
and gram positive bacteria.
MacConkey agar
MacConkey is selective for gram
negative bacteria meaning only
gram negative bacteria will grow on
this medium.
MacConkey agar
 MacConkey
also contains lactose.
 Organisms which do not ferment lactose
will have colorless colonies
 Organisms which do ferment lactose will
have pink colonies
Organisms which do not ferment
lactose will have colorless colonies

Organisms which do ferment
lactose will have pink colonies
MacConkey Agar
Left: Shigella
- result
Right: Escherichia
+ result
MacConkey agar
1. Which plate could have E. coli on it?
2. Which plate shows the presence of a
lactose-fermenter?
3. Which plate could have Salmonella
on it?
A
4. Which plate shows the presence of a
lactose non-fermenter?
B
4. B
2. A
3. B
1. A
Answers:
Carbohydrate fermentation
( glucose, lactose, sucrose)

Purpose: This test is used to determine the
ability of an organism to ferment various simple
carbohydrates ( sugars)
 A small tube ( Durham tube) is inverted and
place in each larger tube of liquid medium
 The inverted tube is able to trap any gas
produced.
 The indicator, phenol red will turn yellow below a
pH of 6.8 9 showing fermentation) and darker
pinkish-red above pH 7.4 ( showing no
fermentation)
Carbohydrate fermentation
( glucose, lactose, sucrose)
 Reading
 Acid:
the Test:
(yellow): Positive for metabolizing
sugar + ( a color change has happened)
 Acid, Gas: ( yellow plus gas bubble):
Positive for metabolizing sugar and gas
production (+) ( color change plus bubbles)
 Negative: NO color change
Carbohydrate fermentation
( glucose, lactose, sucrose)
Carbohydrate Fermentation Tests
Glucose w/ Durham Fermentation Tube
Carbohydrate Fermentation Tests
Sucrose Fermentation
Gelatine liquefaction test
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
Purpose: This test is used to determine the
ability of the bacteria to produce gelatinases
that digest and liquefy gelatin.
Reading Test:
 Once the tubes are inoculated with the bacteria
and incubated, they will cause the gelatin to
melt.
 In order to determine whether or not the reaction
has taken place, the incubated tubes must be
placed on ice until the control tube is once again
solid.
Gelatine liquefaction test

Positive Test

Negative Test
Mannitol salt
 Purpose:
Mannitol salt agar is both
selective and differential.
 Principle: Mannitol salt helps determine
two characteristics of bacteria, whether
they are salt tolerant or not, and whether
they are able to ferment mannitol
Mannitol salt used to id
Staphlococcus bacteria

Mannitol salt agar is a selective and differential
primary culture medium useful in recovery and
identification of staphylococci from specimens
containing mixed flora.
 The high salt concentration (7.5%) inhibits most
gram-negative and gram-positive bacteria
except Staphylococcus species
The Staphylococcus aureus ferments mannitol and
turns the medium yellow. The Serratia marcescens
does not grow because of the high salt content
Streptococcus agalactiae does not grow on MSA
because of the high salt content. Staphylococcus
epidermidis grows but does not ferment mannitol.
Indol
 Purpose:
The indole test is used to identify
bacteria capable of producing indole.
 Reading
the Test:
 When Kovac’s solution is added to the
inoculated tube, a red layer will form when
indol is present = positive test.
 No red layer = negative test
Indol
With the Kovac’s reagent added

Positive Test

:
Negative Test
SIM Test
(Indol, motility, sulfur reduction)
 Purpose:
The SIM test performs three
different tests: motility, indole and sulfur
reduction.
 Motility and indole have been mentioned
earlier
 Sulfur reduction: This test is used to
identify those bacteria capable of reducing
sulfur.
SIM Test
(, sulfur reduction portion)

Black precipitate indicates a
positive test (for H2S
production)

No precipitate means
a negative test
IMViC tests
 The
IMViC tests are a group of individual
tests used in microbiology lab testing to
identify an organism in the coliform group.
 A coliform is a gram negative, aerobic or
facultative aerobic rod which produces gas
from lactose within 48 hours.
 The presence of some coliforms indicate
fecal contamination.
These four tests include
 Indole
test ( discussed earlier)
 Methyl Red test
 Voges-Proskauer (VP) test *
 Citrate Test
IMViC Series
Indole Test
broth with
tryptophan
Methyl Red test and VogesProskauer (VP) test *
 These
tests both use the same broth for
bacterial growth.
 The broth is called MRVP broth.
 After growth, the broth is separated into
two different tubes, one for the Methyl Red
(MR) test and one for the VogesProskauer (VP) test.
Methyl Red

The pH indicator
Methyl Red is added
to one tube and a red
color appears at pH's
lower than 4.2, and
indicated positive test.
IMViC Series
Methyl Red Test
VP test

The reagents used for the VP test are Barritt's A
(alpha-napthol) and Barritt's B (potassium
hydroxide).
 When these reagents are added to a broth in
which acetyl methyl carbinol is present, they turn
a pink-burgundy color (a positive VP test).
 This color may take 20 to 30 minutes to develop.
E. coli does not produce acetyl methyl carbinol,
IMViC Series
Voges-Proskauer Test
Leave uncapped for 15-20 minutes……..

MRVP broth
VP test

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Citrate Test
This test uses Simmon's citrate agar to
determine the ability of a
microorganism to use citrate as its sole
carbon source.
Simmons Citrate Test
 The
citrate agar is green before
inoculation, and turns blue as a positive
test indicator
IMViC Series
Citrate Utilization Test
-
+
IMViC tests

These IMViC tests are useful for differentiating
the family Enterobacteriaceae, especially when
used alongside the Urease test.
 Except for the lowercase “i”, which is added for
ease of pronunciation, each of the letters in
“IMViC” stands for one of these tests. “I” is for
indole; “M” is for methyl red; “V” is for VogesProskauer, and “C” is for citrate.
Triple sugar Iron Agar
 The
tube is inoculated by stabbing into the
agar butt (bottom of the tube) with an
inoculating wire and then streaking the
slant in a wavy pattern.
 Results are read at 18 to 24 hours of
incubation.
Triple sugar Iron Agar
 When
there is a yellow butt and a red slant
that indicates that the organism can
ferment glucose.
 The change in color from red to yellow is
due to the change in pH as lactic acid is
produced.
 The pH indicator used is Phenol Red
Triple sugar Iron Agar
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Nonfermentative
Gram-negative Bacilli

Positive test
Triple Sugar Iron
-/+/+
Starch Agar
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Starch Agar is basically an all-purpose medium
A plate of this medium is single streaked or spot
inoculated with the organism to be tested. After
incubation (preferably for several days) the
medium is flooded with Gram's iodine
 The iodine reacts with starch to form a dark
blue-colored complex.
 Any clear area around the growth of the
culture after the addition of the iodine indicates
the breakdown of starch by the organism due
to its production of amylase, an extracellular
enzyme.
Starch Agar and the Amylase Test
Starch Agar and the Amylase Test
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The clear (non-blue) area
around the culture on the
left indicates the absence
of intact starch – and
therefore a positive
reaction for the amylase
test.
The blue color extends up
to the culture on the right
which is thus seen to be
unable to break down
starch – a negative
reaction for this test.
Skim Milk agar
 Skim
Milk agar is used to detect the
presence of caseinase.
 Caseinase is an enzyme that catalyzes
the breakdown of casein into its individual
amino acids.
Skim Milk agar
 Negative
test
Skim Milk agar

This bacteria
produces
caseinase which
has resulted in
the breakdown
of the casein on
either side of the
bacterial growth.
This is seen as a
clear zone
Catalase

Catalase is an enzyme that converts
hydrogen peroxide into water and oxygen.
 The presence of catalase can be easily
detected by the slide method.
 A drop of 3 percent hydrogen peroxide is put
on a slide and the bacteria is emulsified in
the drop.
 The presence of bubbles is evidence of the
production of oxygen
Catalase
What is the name of the lab procedure demonstrated here?
Answer: Stabbing the butt!
Use the chart and/or the flow chart
your lab manual to figure out who I
am!
U
M
V
C
Answer: Escherichia coli
I
Use the and/or the flow chart on
of your lab manual to figure out
who I am!
U
M
V
C
Answer: Serratia marcesens
I
Use the chart and/or the flow
chart of your lab manual to figure
out who I am!
U
M
V
C
Answer: Enterobacter aerogenes
I