IF YOU CAN SEE THIS
MESSAGE YOU ARE NOT
IN “SLIDE SHOW” MODE.
PERFOMING THE LAB IN
THIS MODE WILL NOT
ALLOW FOR THE
ANIMATIONS AND
INTERACTIVITY OF THE
EXERCISE TO WORK
PROPERLY. TO CHANGE
TO “SLIDE SHOW” MODE
YOU CAN CLICK ON
“VIEW” AT THE TOP OF
THE PAGE AND SELECT
“SLIDE SHOW” FROM THE
PULL DOWN MENU. YOU
CAN ALSO JUST HIT THE
“F5” KEY.
Agar Plates
Antiseptic Dispenser
Swabs
Pencil
Microbe Samples
Bunsen burner
Incubator
37 0C
Loops
Loops
Click on the blackboard
to view a larger board for
discussion. Click on the
INDEX link below to
advance directly to a
staining lab.
Agar Plates
pH = 7
pH = 9
BACTERIAL STAINING
Most bacteria are difficult to see under the bright field of
a microscope. Bacteria are almost colorless (composed
primarily of water) and therefore show little contrast with
the suspension. To visualize bacteria, either dyes or
stains are used. Since staining of bacterial cells is
relatively fast, inexpensive, and simple, it is the most
commonly used technique to visualize bacterial cells.
Staining not only makes bacteria more easily seen, but it
allows their morphology
(e.g. size and shape) to be
visualized more easily. In some
cases, specific stains can be
used to visualize certain
structures (flagella, capsules,
endospores, etc) of
bacterial cells.
pH = 11
Incubator
pH = 5
pH = 3
There are several staining methods that are used
routinely with bacteria. These methods may be classified
as 1) simple and 2) differential. Simple stains will react
with all microbes in an identical fashion. They are useful
solely for increasing contrast so that morphology, size,
and arrangement of organisms can be determined.
Differential stains give varying results depending on the
organism being treated. These results are often helpful
in identifying the microbe. Commonly used
microbiological stains generally fall into one of two
categories
there
-10
C
0 -Cbasic stains or acidic
35 C stains (although
50 C
100 C
are a few stains such as India Ink which are neutral).
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
A basic dye is a stain that is positively charged and will
therefore react with material that is negatively charged.
Bacterial cells have a slight negative charge will
therefore attract and bind with basic dyes. Some
examples of basic dyes are crystal violet, safranin, basic
fuchsin and methylene blue. Acid dyes are negatively
charged and are repelled by the bacterial surface
forming a deposit around the organism. They stain the
background and leave the microbe transparent.
Nigrosine and congo red are examples of acid dyes.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
At first glance, the easiest way to stain bacterial cells
would appear to be simply mixing the bacterial
suspension with the dye and making a wet mount of this
mixture. Unfortunately, if you were to try staining
bacterial cells in this manner you would find that there
was too much background (unbound dye) to allow for
visualization of the cells. Therefore, you need to remove
the unbound dye. Simply washing off the dye would
result in removal of the cells along with the excess dye.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
Therefore, you need a mechanism to fix (adhere) the
cells to the slide before staining to allow for removal of
excess dye while keeping the cells on the slide. A simple
method is that of air drying and heat fixing. The
organisms are heat fixed by passing an air-dried smear
of the organisms through the flame of a gas burner. The
heat coagulates the organisms' proteins causing the
bacteria to stick to the slide. Be very careful not to over
heat the organisms when fixing them to a slide. This
distorts the sample of the organisms.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
Keep in mind the analogy of a fried egg. When you drop
a raw egg onto a cold frying pan, it has a certain shape.
Start heating it and the proteins (albumin) on the lower
surface of the egg precipitate and fix the egg to the pan.
At this point there has been a minimal distortion in the
shape of the egg as a whole; only a small percent of the
proteins have been precipitated. If you keep applying
heat, the shape of the entire egg will change and
eventually it will be reduced to charred remains. When
you heat fix a slide, you want to apply enough heat to
precipitate
proteins to allow
the cells 50
toCstick to 100
theC
-10
C
0 the
C
35 C
slide but not to drastically change the shape of the cells
(or reduce them to charred remains).
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Observe the links given below to bring you to
the VIRTUAL LAB that you wish to perform. If
you have performed all of the exercises, you
can click on END LAB.
Agar Plates
pH = 7
pH = 9
pH = 11
pH = 5
pH = 3
Simple Stain
Freezer
Refrigerator
Endospore Stain
-10 0C
0 0C
Negative Stain
Incubator
Incubator
Gram Stain
35 0C
50 0C
Capsule Stain
End Lab
Acid Fast Stain
Incubator
100 0C
Agar Plates
pH = 7
pH = 9
SIMPLE STAIN
Staining is a technique that is used to enhance contrast
in a microscopic image. Staining can also be used to
highlight structures for viewing. The simple stain can be
used to determine cell shape, size, and arrangement. The
simple stain is a very simple staining procedure
involving only one stain. The most common stains
(dyes) used are methylene blue, Gram safranin, and
Gram crystal violet. Basic stains, such as methylene
blue,
Gram0 Csafranin, or Gram crystal
violet
are useful
-10
C
35 C
50 C
100for
C
staining most bacteria. These stains will readily give up
a OH- ion or accept a H+ ion, which leaves the stain
positively charged. These positively charged stains
adhere readily to the cell surface, since the surface of
most bacteria are negatively charged.
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
SIMPLE STAIN PROCEDURE
1) Perform a bacterial smear of the given
bacterial culture
2) Allow the smear to dry thoroughly.
3) Heat-fix the smear cautiously by passing the
underside of the slide through the burner flame
two or three times. Overheating can distort the
cells.
-10 C
0 C
35 C
50 C
100 C
4) Saturate the smear with basic dye and let sit
for approximately 1 minute. We will use
methylene blue
5) Rinse the slide gently with water
6) Observe the slide under the microscope
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the SLIDES to bring one of
them to the rinse station. Now click on
the EYE DROPPERS to transfer 1 drop
of the bacterial broth to the slide. Click
on NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the BUNSEN BURNER to bring
the burner to the table. Next click on the
slide to fix the bacterial sample to the
slide by passing the slide through the
flame two or three times. Click on NEXT
when the sample has been fixed.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the bottle of methylene blue
dye in the shelves and flood the sample.
Let the dye and sample sit for 1 minute.
Next click on the water rinse bottle to
rinse the dye from the sample. Let air
dry. Click on NEXT when the sample has
dried.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from a Simple
Stain with methylene blue. Click on the
EYEPIECE of the microscope to view
the slide under High Power (400 X). You
will need to sketch a few of the bacteria
cells that you are viewing. Be sure to
indicate any colors you observe by
using colored markers (pens or pencils)
or by indicating
any color with a label.
37 0C
Click Here go to the
next staining
procedure
Agar Plates
pH = 7
pH = 9
ENDOSPORE STAIN
The endospore stain is a differential stain used to
visualize bacterial endospores. Endospores are formed
by some bacteria, such as Bacillus. By forming spores,
bacteria can survive in hostile conditions. Spores are
resistant to heat, dessication, chemicals, and radiation.
Bacteria can form endospores in approximately 6 to 8
hours after being exposed to adverse conditions. The
normally growing cell that forms the endospore is called
a vegetative cell. Spores are metabolically inactive and
-10
C
0 C They can remain viable
35 C
50 C
100 C
dehydrated.
for thousands
of
years. When spores are exposed to favorable conditions,
they can germinate into a vegetative cell within 90
minutes.
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Endospores can form within different areas of the
vegetative cell. They can be central, subterminal, or
terminal. Central endospores are located within the
middle of the vegetative cell. Terminal endospores are
located at the end of the vegetative cell. Subterminal
endospores are located between the middle and the end
of the cell. Endospores can also be larger or smaller in
diameter than the vegetative cell. Those that are larger in
diameter will produce an area of "swelling" in the
vegetative cell. These endospore characteristics are
consistent
the spore-forming
species
-10
C
0 within
C
35 C
50 C and can
100be
C
used to identify the organism.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Because of their tough protein coats made of keratin,
spores are highly resistant to normal staining
procedures. The primary stain in the endospore stain
procedure, malachite green, is driven into the cells with
heat and readily adheres to the endospore. Since
malachite green is water-soluble and does not adhere
well to the cell, and since the vegetative cells have been
disrupted by heat, the malachite green rinses easily from
the vegetative cells, allowing them to readily take up the
counterstain. This allows the endospores to be visible
with
green stain35and
cells
-10
C the malachite
0 C
C the vegetative
50 C
100 Cto
be visible with the counterstain.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
ENDOSPORE STAIN PROCEDURE
1) Perform a bacterial smear of the organism
you are given to stain
2) Saturate the smear with malachite green
3) Heat the slide gently over a Bunsen burner
for 5 minutes
4) Rinse the slide gently with water
-10 C
0 C
35 C
C
100 C
5) Counterstain
with safranin
for 250minutes
6) Rinse the slide gently with water
7) Observe the slide under the microscope.
Endospores will stain green. Vegetative cells
will stain red
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the SLIDES to bring one of
them to the rinse station. Now click on
the EYE DROPPERS to transfer 1 drop
of the bacterial broth to the slide. Click
on NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the bottle of malachite green
dye in the shelves and flood the sample.
The green dye will be visible in the
endospores. Let the dye and sample sit
for 1 minute. Click on NEXT when the
sample has dried.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Click on the BUNSEN BURNER to bring
the burner to the table. Next click on the
slide to fix the bacterial sample to the
slide by gently warming the sample
above the flame for 5 minutes. Click on
NEXT when the sample has been fixed.
Carbolfuchsin
Timer
5
2
3
4
1 min
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the water rinse bottle to rinse
the dye from the sample. Let air dry.
Click on the counterstain Safranin to
stain the sample for two minutes. The
red counterstain will be visible in the
vegetative bacteria. After the stain has
set click on NEXT.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the water rinse bottle to rinse
the counterstain safranin dye from the
sample. Let air dry. After the stain has
dried click on NEXT.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
The green
stained bacteria
are endospores
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from an
Endospore stain. Click on the
Themicroscope
red stained to view
EYEPIECE of the
bacteria
are (400 X). You
the slide under High
Power
vegetative
will need to sketch
a few of the bacteria
cells that you are viewing. Be sure to
indicate any colors you observe by
using colored markers (pens or pencils)
or by indicating
any color with a label.
37 0C
Click Here go to the
next staining
procedure
Agar Plates
pH = 7
pH = 9
CAPSULE STAIN
The capsule stain employs an acidic stain and a basic
stain to detect capsule production. Capsules are formed
by organisms such as Klebsiella pneumoniae. Most
capsules are composed of polysaccharides, but some
are composed of polypeptides. The capsule differs from
the slime layer that most bacterial cells produce in that it
is a thick, detectable, discrete layer outside the cell wall.
Some capsules have well-defined boundaries, and some
have fuzzy, trailing edges. Capsules protect bacteria
-10
C
0 C
35 C
50
C allow 100 C
from
the phagocytic
action of leukocytes
and
pathogens to invade the body. If a pathogen loses its
ability to form capsules, it can become avirulent.
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Bacterial capsules are non-ionic, so neither acidic nor
basic stains will adhere to their surfaces. Therefore, the
best way to visualize them is to stain the background
using an acidic stain and to stain the cell itself using a
basic stain. We will use India ink and Gram crystal violet.
This leaves the capsule as a clear halo surrounding a
purple cell in a field of black. The medium in which the
culture is grown as well as the temperature at which it is
grown and the age of the culture will affect capsule
formation. Older cultures are more likely to exhibit
capsule
production.
When performing
a capsule
stain
-10
C
0 C
35 C
50 C
100 on
C
your unknown, be sure the culture you take your sample
from is at least five days old.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
The ability to produce a capsule is an inherited property
of the organism, but the capsule is not an absolutely
essential cellular component. Capsules help many
pathogenic and normal flora bacteria to initially resist
phagocytosis by the host's phagocytic cells. In soil and
water, capsules help prevent bacteria from being
engulfed by protozoans. Capsules also help many
bacteria to adhere to surfaces and thus resist flushing. It
also enables many bacteria to form biofilms. A biofilm
consists layers of bacterial populations adhering to host
cells
and embedded
in a common
-10
C
0 C
35 C capsular
50 Cmass.
100 C
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
CAPSULE STAIN PROCEDURE
Agar Plates
pH = 7
pH = 9
1) Place a single drop of India ink on a microscope
slide
2) Add the organism to be stained and mix into the
drop of India ink
3) Spread the mixture on the slide to a thin layer
4) Allow the film to air dry. DO NOT heat or blot dry.
Heat will melt the capsule
5) CSaturate
violet
1 minute
-10
0 C the slide with crystal
35 C
50 for
C
100 C
6) Rinse the slide gently with water
7) Allow the slide to air dry
8) Observe the slide under the microscope. The
background will be dark. The bacterial cells will be
stained purple. The capsule (if present) will appear
clear against the dark background
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the slides to bring one of the
slides to the rinse table. Click on the
bottle of India ink dye in the shelves and
add 1 drop to the slide. Click on NEXT
when the dye has been added to the
slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the EYE DROPPERS to transfer
1 drop of the bacterial broth to the slide.
Allow the thin film to air dry. Click on
NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the Crystal Violet to saturate
the sample for 1 minute. Click on the
water rinse bottle to rinse the dye from
the sample. Let air dry. After the stain
has dried click on NEXT.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
The purple
stain is the
bacteria
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from a Capsule
stain. Click on the EYEPIECE of the
microscope to view
The the
cleared
slide under High
areaswill
in the
Power (1000 X). You
need to sketch
India ink
are the
a few of the bacteria
cells
that you are
capsules
viewing. Be sure to
indicate any colors
you observe by using colored markers
(pens or pencils) or by indicating any
color with a label.
Incubator
37 0C
Click Here go to the
next staining
procedure
Agar Plates
pH = 7
pH = 9
ACID FAST STAIN
The acid-fast stain is a differential stain used to identify
acid-fast organisms such as members of the genus
Mycobacterium. Acid-fast organisms are characterized
by wax-like, nearly impermeable cell walls; they contain
mycolic acid and large amounts of fatty acids, waxes,
and complex lipids. Acid-fast organisms are highly
resistant to disinfectants and dry conditions.
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
Because the cell wall is so resistant to most compounds,
acid-fast organisms require a special staining
technique. The primary stain used in acid-fast staining,
carbolfuchsin, is lipid-soluble and contains phenol,
which helps the stain penetrate the cell wall. This is
further assisted by the addition of heat. The smear is
then rinsed with a very strong decolorizer, which strips
the stain from all non-acid-fast cells but does not
permeate the cell wall of acid-fast organisms. The
decolorized non-acid-fast cells then take up the
counterstain.
-10
C
0 C
35 C
50 C
100 C
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
The acid-fast stain is an especially important test for the
genus Mycobacterium. There are two distinct pathogens
in this group: M. tuberculosis, the causative organism of
tuberculosis, and M. leprae, the causative agent of
leprosy.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
ACID FAST STAIN PROCEDURE
Agar Plates
pH = 7
pH = 9
1) Perform a bacterial smear
2) Flood the smear with carbolfuchsin
3) Heat the slide gently over the Bunsen burner for
5 minutes
4) Rinse the slide gently with water
5) Decolorize the slide with acid-alcohol until the
rinse runs clear
6) CRinse the
water 50 C
-10
0 C slide gently with
35 C
100 C
7) Counterstain with methylene blue for 2 minutes
8) Rinse the slide gently with water
9) Observe the slide under the microscope. Acidfast cells will stain fuchsia (pink or red). Non-acidfast cells will stain blue
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the SLIDES to bring one of
them to the rinse station. Now click on
the EYE DROPPERS to transfer 1 drop
of the bacterial broth to the slide. Click
on NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the bottle of Carbolfuchsin dye
in the shelves and flood the smear. Click
on NEXT when the smear has been
stained.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Click on the BUNSEN BURNER to bring
the burner to the table. Next click on the
slide to fix the bacterial sample to the
slide by gently warming the sample
above the flame for 5 minutes. Click on
NEXT when the sample has been fixed.
Carbolfuchsin
Timer
5
2
3
4
1 min
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the water rinse bottle to rinse
the dye from the sample. Let air dry.
Next click on the acid-alcohol
decolorizer to rinse the stain from the
slide. Next click on the rinse bottle
again to clear the acid-alcohol. Click on
NEXT when finished rinsing.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the Methylene blue dye in the
and saturate the sample for 2 minutes.
Next click on the water rinse bottle to
rinse the dye from the sample. Let air
dry. Click on NEXT when the sample has
dried.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
Acid Fast
bacteria will
stain red
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from an Acid
Fast stain. Click on the EYEPIECE of the
microscope toNon-acid
view thefast
slide under High
Power (400 X). bacteria
You willwill
need to sketch a
staincells
blue that you are
few of the bacteria
viewing. Be sure to indicate any colors
you observe by using colored markers
(pens or pencils) or by indicating any
color
with a label.
37 0C
Click Here go to the
next staining
procedure
Agar Plates
pH = 7
pH = 9
NEGATIVE STAIN
The negative stain is particularly useful for determining
cell size and arrangement. It can also be used to stain
cells that are too delicate to be heat-fixed. We will use
nigrosin as our negative stain. Nigrosin is an acidic
stain. This means that the stain readily gives up a H+ ion
and becomes negatively charged. Since the surface of
most bacterial cells is negatively charged, the cell
surface repels the stain. The glass of the slide will stain,
but the bacterial cells will not. The bacteria will show up
-10
0 C
35 C
50 C
100 C
asCclear spots
against a dark background.
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Negative staining is an excellent way to determine an
organism’s cellular morphology. Since the cells
themselves are not stained, their morphology is not
distorted in any way. The nigrosin provides a dark
background against which the shapes of the unstained
cells are clearly visible. This method provides a high
degree of contrast not available in most other staining
procedures.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
NEGATIVE STAIN PROCEDURE
Agar Plates
1) Place a single drop of nigrosin on a
pH = 7
pH = 9
pH = 11
pH = 5
pH = 3
microscope slide
2) Mix a sample of your organism to stain into
the drop of nigrosin
3) Spread the sample on the slide into a thin
Freezer
layer
-10 0C
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
4) Allow the film to air dry
5) Observe the slide under the microscope. The
bacteria will be clear and the background will
stain dark
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the slides to bring one of the
slides to the rinse table. Click on the
bottle of Nigrosin dye in the and add 1
drop to the slide. Click on NEXT when
the dye has been added to the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the EYE DROPPERS to transfer
1 drop of the bacterial broth to the slide.
Allow the thin film to air dry. Click on
NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from a
Negative stain. Click on the EYEPIECE
of the microscope to view the slide
under High Power (1000 X). You will
need to sketch a few of the bacteria
cells that you are viewing. Be sure to
indicate any colors you observe by
using colored markers (pens or pencils)
or by indicating any
color with a label.
37 0C
Click Here go to the
next staining
procedure
Agar Plates
pH = 7
pH = 9
GRAM STAIN
The Gram stain is the most important staining procedure
in microbiology. It is used to differentiate between gram
positive organisms and gram negative organisms.
Hence, it is a differential stain. Gram negative and gram
positive organisms are distinguished from each other by
differences in their cell walls. These differences affect
many aspects of the cell, including the way the cell takes
up and retains stains.
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
Gram positive cells take up crystal violet, which is then
fixed in the cell with iodine mordant. This forms a
crystal-violet iodine complex which remains in the cell
even after decolorizing. It is thought that this happens
because the cell walls of gram positive organisms
include a thick layer of protein-sugar complexes called
peptidoglycans. This layer makes up 60-90% of the gram
positive cell wall. Decolorizing the cell causes this thick
cell wall to dehydrate and shrink, which closes the pores
in the cell wall and prevents the stain from exiting the
cell.
of the gram staining
procedure,
gram
-10
C At the0 end
C
35 C
50 C
100 C
positive cells will be stained a purplish-blue color.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Gram negative cells also take up crystal violet, and the
iodine forms a crystal violet-iodine complex in the cells
as it did in the gram positive cells. However, the cell
walls of gram negative organisms do not retain this
complex when decolorized. Peptidoglycans are present
in the cell walls of gram negative organisms, but they
only comprise 10-20% of the cell wall. Gram negative
cells also have an outer layer which gram positive
organisms do not have; this layer is made up of lipids,
polysaccharides, and proteins. Exposing gram negative
cells
to the0 Cdecolorizer dissolves
-10
C
35 C the lipids
50 Cin the cell
100 C
walls, which allows the crystal violet-iodine complex to
leach out of the cells. This allows the cells to
subsequently be stained with safranin. At the end of the
gram staining procedure, gram negative cells will be
stained a reddish-pink color.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
Often, detecting the presence of microorganisms and
determining whether an infection is caused by an
organism that is Gram-positive or Gram-negative will be
sufficient to allow a doctor to prescribe treatment with an
appropriate antibiotic while waiting for more specific
tests, such as a culture, to be completed.
Agar Plates
pH = 7
pH = 9
pH = 11
Freezer
-10 0C
pH = 5
pH = 3
Refrigerator
0 0C
Incubator
Incubator
Incubator
35 0C
50 0C
100 0C
GRAM STAIN PROCEDURE
Agar Plates
pH = 7
pH = 9
1) Perform a bacterial smear of your given culture
2) Saturate the smear with crystal violet for 1
minute
3) Rinse the slide gently with water
4) Saturate the smear with iodine for 1 minute
5) Rinse the slide gently with water
6) Decolorize with Gram decolorizer for 3-5
seconds
7) CRinse the
-10
0 C slide gently with
35 Cwater
50 C
100 C
8) Counterstain with safranin for 1 minute
9) Rinse the slide gently with water
10) Observe the slide under the microscope. Gram
positive bacteria will stain purple. Gram negative
bacteria will stain red/pink
pH = 11
Freezer
0
pH = 5
pH = 3
Refrigerator
0
Incubator
0
Incubator
0
Incubator
0
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the SLIDES to bring one of
them to the rinse station. Now click on
the EYE DROPPERS to transfer 1 drop
of the bacterial broth to the slide. Click
on NEXT when you have allowed the
sample to dry on the slide.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the Crystal Violet to saturate
the sample for 1 minute. Click on the
water rinse bottle to rinse the dye from
the sample. Click on NEXT when you
are finished with the rinse.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the Iodine bottle to saturate the
sample for 1 minute. Click on the water
rinse bottle to rinse the dye from the
sample. Click on NEXT when you are
finished with the rinse.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the Gram Decolorizer bottle to
rinse the sample for 2-3 seconds. Next
Click on the water rinse bottle to rinse
the decolorizer from the sample. Click
on NEXT when you are finished with the
rinse.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the counterstain Safranin to
stain the sample for 1 minute. Next click
on the water rinse bottle to rinse the dye
from the sample. Let air dry. After the
slide has dried click on NEXT.
India Ink
Iodine
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Incubator
37 0C
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
Click on the microscope to bring the
microscope to the table. Next click on
the slide on the rinse table to bring the
slide to the microscope stage. Click on
NEXT when the slide is on the stage.
India Ink
Iodine
Gram Positive
Bacteria will
stain Purple
Crystal Violet
Methylene Blue
Malachite Green
Eye Droppers
Agar Plates
Microscope
Safranin
Nigrosin
Slides
Acid Alcohol
Gram Decolorizer
Loops
Water Rinse
Bunsen burner
Carbolfuchsin
You are viewing bacteria from a Gram
stain. Click on the EYEPIECE of the
Gram the
Negative
microscope to view
slide under High
Bacteria
will to sketch a
Power (400 X). You
will need
Pink
few of the bacteriastain
cells
that you are
viewing. Be sure to indicate any colors
you observe by using colored markers
(pens or pencils) or by indicating any
color with a label.
Incubator
37 0C
Click Here go to the
next staining
procedure
END