MMBB255 Week 2 Experiments
1
Staining and Enrichment
I. Objective
The goal of this experiment is to learn about four types of staining techniques (simple, differential, negative,
and structural) that are used to aid in identification of bacteria plus an introduction to the use of controls. You
also will begin to enrich for and examine different types of microbes that may be present in materials that you
might encounter every day.
II. Background
Staining techniques provide valuable tools for visualizing microbes (bacteria and fungi) in the light
microscope that are otherwise difficult to see due to their small size and transparent nature as you saw last week.
Stains are dyes that have three characteristic functional groups: (1) an aromatic group (i.e. benzene ring), (2) a
chemical group called a chromophore, which lends a color to the aromatic group (the aromatic group with a
chromophore is called a chromogen), and (3) an auxochrome group, which allows the compound to combine
either electostatically or covalently with the target thereby staining it. This process, which is typically
irreversible, is similar to what happens when berry juice, which is intensely colored, stains fabric. An example
of this is shown below.
Chromophores
O2N
Aromatic Chemical
(Benzene)
NO2
NO2
Chromogen
(Trinitrobenzene, yellow color)
Auxochrome
(Ionic Interaction)
OH
O- + H+
OH
O2N
NO2
NO2
Yellow Stain
(Trinitrohydroxybenzene, Picric Acid)
Types of Dyes
A. Acidic dyes have an affinity for positively-charged components in the cell because their auxochrome
group is negatively charged (anionic). Example: Picric Acid (shown above).
B. Basic dyes have an affinity for negatively-charged components in the cell because their auxochrome
group is positively-charged (cationic). Example: Methylene Blue
MMBB255 Week 2 Experiments
2
4 Types of Staining Techniques
1. Simple stain
A simple stain employs a single stain that is used primarily to examine shape and arrangement of cells.
Basic stains such as methylene blue, crystal violet, or carbolfuchsin, are often used for this type of staining
because they will react with negatively-charged molecules in the cell, i.e. nucleic acids and cell wall
components.
2. Differential stain
A differential stain uses two contrasting dyes to distinguish between different types of organisms.
Examples include the Gram stain and the acid-fast stain. There are four terms generally used for multi-dye
staining: primary stain, mordant, decolorizer, and counterstain. For example the Gram stain separates
Gram positive (Gm+) from Gram negative (Gm-) bacteria. Nearly all bacteria can be classified as either Gm+
or Gm-. This is a fundamental type of stain used routinely in clinical situations to classify and differentiate
microorganisms. Four reagents are used for Gram staining and are applied in the following order: CV-I-E-S
1. Crystal Violet, the primary (purple) stain.
2. Iodine, the mordant which forms a complex with crystal violet.
3. 95% Ethanol, the decolorizing agent.
4. Safranin, a red counterstain.
Bacteria such as Staphylococcus aureus and Streptococcus pyogenes are Gm+ because they have thick
cell walls and retain the primary stain, crystal violet - once it complexes with iodine, after decolorization. So
when the ethanol is added, the crystal violet-iodine complex is not washed away. In contrast, bacteria such as
Escherichia coli and Salmonella Typhimurium are Gm-. Gm- organisms have thin cell walls and an outer
membrane, and so they do not retain the crystal violet-iodine complex after rinsing with ethanol. Instead,
they can take up the red safranin stain. Hence, if you stain a sample containing a mixture of Gm+ and Gmcell types with the four reagents described above, you will find a mixture of dark purple/blue cells (Gm+)
and reddish/pink cells (Gm-).
3. Structural stain
This type of stain is used to identify particular cellular structures. Some examples: a flagellar stain
enables one to see the flagellar filaments which some cells use for movement; a capsular stain used to
visualize capsule material (generally a polysaccharide coat that some cells produce); a spore stain to detect
spores (specialized resting cells); and a nuclear stain to examine the nucleus.
4. Negative stain
As the name implies these types of stains will color everything except the object of interest. In this lab we
use it to visualize capsular material surrounding a cell; therefore a Negative/Structural stain combination.
Capsules will be seen as clear areas after applying the negative stain India ink; the cell and background are
colored while the capsule remains colorless.
Enrichments
An enrichment is a culturing technique that is designed to enhance the growth of one (or more) type of
organism from a sample in which a number of different organisms may be present. For example, a typical soil
sample is likely to contain various types of Aspergillus fungal spores (a eukaryote), myxobacteria spores (Gram
negative prokaryotes of the genera Myxococcus, Stigmatella, and relatives), bacillus spores (Gram positive
prokaryotes of the genus Bacillus), pseudomonads (prokaryotes of the genus Pseudomonas), rhizobia
(prokaryotes of the genera Rhizobium and Bradyrhizobium), and Nitrobacter, just to name a few. Notice that
many of the groups mentioned are spore-forming microbes. Spores are designed to withstand long periods of
hardship, when nutrients are not available or when the temperature is not compatible with growth. Because
spores are heat-resistant forms of these microbes, heat treatment can be used to enrich for the spore-formers
such as Bacillus, Myxococcus and Aspergillus. Additionally, if you wanted only prokaryotic spore formers in the
soil sample, you could use a heat-treatment to destroy the non-spore cell types and add an antibiotic to
selectively destroy the eukaryotic Aspergillus cells that would survive heat-treatment. In this type of enrichment,
the antibiotic of choice would be an antibiotic that specifically targets a eukaryotic cell type, but not a
prokaryotic cell type. Fundamental differences between prokaryotic and eukaryotic cells make this type of
antibiotic selection possible.
MMBB255 Week 2 Experiments
3
Cellular Morphology Revisited
Important information can be gleaned by examining cells in the microscope. Samples containing living
microbes can be used to determine cell size, cell morphology, and if cells possess certain types of motility.
Interactions between cells can also be examined with living specimens. Stained, heat-fixed preparations of cells
also are useful for determining cell size and cell morphology.
Recording microscopic observations:
Always record: 1. What total magnification 2. What color(s) (Note: if you have any degree of color
impairment, please inform your teaching assistant so that we can assist you in color
determination.) 3. Size (W x L) 4. Arrangement (see below) 5. Morphology (see below).
Cell Size Examples
Name
Haemophilus influenza
Streptococcus pneumoniae
Escherichia coli
Bacillus megaterium
Oscillatoria
Epulopiscium fishelsoni
Saccharomyces cerevisiae
Paramecium
Type of Organism
bacterium, a prokaryote
bacterium, a prokaryote
bacterium, a prokaryote
bacterium, a prokaryote
bacterium, a prokaryote
bacterium, a prokaryote (a symbiont
of the sturgeonfish)
yeast, a eukaryote
protozoan, a eukaryote
Cell Morphologies
Size
0.25 µm x 1.2 µm
0.8 µm
1 µm x 3 µm
1.5 µm x 4 µm
8 µm x 50 µm
50 µm x 600 µm
8 µm
30µm x 150 µm
MMBB255 Week 2 Experiments
4
Tuesday’s Procedures
A. Follow-up for Your Streaks – Work Individually
1. Examine both plates (TSA and ME); you should see different colony types growing on each. Describe 2
of the predominant colonies for each plate using the terminology found in last week’s lab (page 9).
2. Practice your streaking technique by streaking a TSA plate with the E.coli/S.aureus mixture.
3. Gram stain the 2 colonies you described in step 1, remember to use the 5 needed observations and your
conclusion (i.e. Gram positive or negative). See part B. Use the E.coli/S.aureus mixture on the same slide
as your colonies for a control (i.e. to make sure your staining technique worked).
Colony Observations
Streaking Results
Cell Observations
TSA
TSA
ME
ME
B. Gram staining (Bartholomew procedure) – Work Individually
1. Prepare smear. See the procedure in a and b and the slide diagram below. E. coli is a Gm- rod and S.
aureus is a Gm+ coccus; these are your positive and negative controls and are mixed together. Air dry.
Heat fix.
(a) Making a slide from a broth culture:
Using a sterile inoculating loop (your lab instructor will demonstrate), aseptically place a drop of
the broth cell culture on a clean, dry glass slide, spreading it in the size of a dime. You can place 2-3
samples on each slide. If problems are encountered in finding cells after staining it may be useful to
apply the culture without spreading (takes longer to dry)- highly recommended.
(b) Making a slide from a colony on solid medium:
For microscopic examination of cells from a colony, the cells must be suspended in broth, buffer, or
water. Using a loop, place a small drop of liquid on a glass slide (water is ok to use, but note that
some cells might swell in water). Next, aseptically touch a sterile (but cooled) inoculating loop,
needle, or a sterile toothpick to the surface of the colony to collect some cells. You need only a tiny
amount of cells — barely enough to see on the end of your loop, needle, or toothpick. Transfer these
cells to the drop of liquid on the glass slide and mix the cells with the liquid uniformly in a quarter
size area.
E. coli/ S. aureus
ME #1 Colony
ME #2 Colony
E. coli/ S. aureus
TSA #1 Colony
TSA #2 Colony
MMBB255 Week 2 Experiments
5
2. Add 1° stain - crystal violet. Place the heat-fixed smear on the staining tray. Cover with Hucker’s
crystal violet (HCV or CV) for one minute. Wash gently with water (tap water or deionized) to remove
some dye. (leave some of the dye behind)
3. Add mordant - iodine. Flood with Gram’s Iodine (Burke’s formula is best) for one minute. Wash with
water
4. Decolorize with 95% ethanol, use one of the two ways (1 or 2) listed below:
a. Timed: add ethanol to the surface of the slide while it is flat on the staining tray for 4-7 seconds for
thin smears or 10-30 seconds for thick smears.
b. Visual: drop ethanol over smear, while holding the slide at an angle, until you can no longer see dye
coming off the smear.
5. Stop the decolorization by washing gently with water
6. Add the counterstain - safranin. Cover the smear with safranin, a red dye, for at least one minute
(more is better). Very lightly wash with water and blot with a Kimwipe and allow to dry.
7. Visualize. Examine the stained preparations under your microscope at 1000X: place the microscope
slide on the microscope stage and focus with the 10X objective. Rotate to 40X, then partially rotate the
objective to the 100x objective and add a small drop of oil to the center of the smear. Then fully rotate
to the 100X objective. Draw the cellular morphology and arrangement, make sure to include the five
observations. Cells that retain the crystal violet will be purple/blue and are considered Gram positive.
Cells that lose the crystal violet will take up the safranin dye and will be red/pink. Red/pink cells are
considered Gram negative.
POTENTIAL PROBLEMS: (this is why controls are needed)
• If the smear is too thick, the reagents will not penetrate evenly (seen as a mix of red and purple). This
is a very common problem when staining from a colony.
• Washing too vigorously can remove cells and/or dye — safranin is particularly easy to remove,
making Gram negative cells difficult or impossible to see.
• Decolorizing too much or little.
• Washing too much of the crystal violet off with water.
• If the sample pH is acidic it must be neutralized with 1% NaHCO3 and rinsed prior to staining.
Observations at 1,000 X
E. coli
S. aureus
(negative control)
Gram Reaction
(purple is +, red is -)
(positive control)
Colonies
See table in Part A
See table in Part A
Cell observations
(draw and describe
using the 5 criteria)
MMBB255 Week 2 Experiments
6
C. Negative Stain (Capsule Stain) using India ink – Work Individually
Background: There are a variety of different stains to allow the visualization of bacterial capsules and
glycocalyx (slime layer). Capsules and glycocalyx are found external to the cell wall (or outer membrane of
Gram negative (Gm-) organisms) and are usually composed of polysaccharides. Capsules have a very defined
shape and boundary, while slime is irregular in shape and has an indistinct boundary. This staining technique
works well if the capsule is well developed - one third the diameter of the cell or larger. However, when the
capsules are not well developed they are very difficult or impossible to detect with the above staining techniques
and would require specific growth media that stimulate capsule production in order to prove or disprove
capsules.
1. Place a drop of India ink on a slide. Sterilize
the loop. Aseptically add a loopful of the
Klebsiella pneumoniae cells from a plate culture
to the ink drop and mix evenly. Prepare
Citrobacter freundii cells similarly. C. freundii
does not produce a capsule (negative control)
whereas K. pneumoniae does produce a capsule
(positive control shown in the figure to the right
using a slightly different procedure).
2. Place a cover slip on the drop. Press down
firmly on the cover slip with a Kimwipe until the
suspension is about one cell thick (a thin film
under the cover slip with a grayish appearance).
Dispose the Kimwipes into the biohazard bag and
wash your hands; this is the only time you would
dispose the Kimwipes into the biohazard bags.
3. Examine in the microscope at 1000X before the preparations dry out; only clear areas one third larger
than the cell diameter should be considered as positive for capsules (anything smaller is doubtful or
negative).
Observations at 1000X
Klebsiella pneumoniae
(positive control)
Citrobacter freundii
(negative control)
MMBB255 Week 2 Experiments
7
Thursday’s Procedures
A. Complete any staining procedures not finished on Tues.
B. Do an acid-fast stain (the Ziehl-Nielsen Procedure) – Work Individually
Background: Acid-fast organisms, such the clinically important Mycobacterium tuberculosis, are Gram+ bacilli
that stain poorly (generally seen as a gradient of purple) because they contain mycolic acids. They may appear
(if you can see them at all) as “Gram-neutral” or “Gram-ghosts” because they did not pick up either crystal
violet or safranin. To identify these hard to Gram stain organisms, alternative stains have been devised that take
advantage of the large amounts of mycolic acid or waxes
found in the cell wall of these bacteria. The principle
involved in the stain is that the heating (consider it as
a mordant) of the slide allows carbolfuchsin greater
penetration into the cell wall. The mycolic acids and
waxes form a complex with the basic dye which then
Mycobacterium
Escherichia coli/Sa
fails to wash out with mild acid decolorization.
Acid-fast staining procedure:
1. Prepare smear. Using your loop, aseptically
make a smear of Mycobacterium smegmatis,
from a plate, and the E.c./S.a. mix (non acidfast organisms) from a broth, on a clean glass
slide as shown in the diagram. Heat-fix the
cells to the slide and place on a staining tray.
Place a piece of towel just smaller than the
size of the slide on the prepared slide and
saturate the towel with carbolfuchsin, the
primary stain (see the figure on the right).
2. Steam ('mordant') the slide over a Bunsen
burner , place a towel under the burner to
catch any drips, for 4-5 minutes (longer is
better) by holding the slide with the
clothespin and dragging the slide above the
flame back and forth until you see it steaming
— only seen when examined away from the
flame. Do not allow the towel to dry by keeping it moist by adding more carbolfuchsin. Do not boil the
stain. Allow the stain to remain on the slide for 2-5 more minutes without heat.
3. Rinse with water (here the tap works best), pulling the towel off gently and the clothespin. Decolorize by
running acid alcohol over the smear (2-5 seconds for light smears, 10-30 seconds for thick smears, or
using the visual method of dripping it over the smear until no more color comes off). Rinse with water.
4. Cover smears with crystal violet, the counterstain, and allow them to stand for one minute. Rinse with
water, blot with Kimwipes, and air dry.
Cell Observations 1000X
Mycobacterium smegmatis (+ Control)
Escherichia coli/S. aureus (- Control)
MMBB255 Week 2 Experiments
8
C. Microbial diversity: Enrichment/isolation of aerobic and thermophilic spore-forming
bacteria. – Work in Pairs
Background: The aerobic spore-forming bacilli (rod-shaped organisms) are very easy to isolate due to the spore
that is resistant to heat and desiccation. We use the spores’ resistance to heat to enrich for spore formers. Spores
are everywhere. You should see spores at various stages of development in your cultures in the lab. We will
grow our enrichments at two temperatures, 30°C or mesophilic and 60°C or thermophilic.
1. Place a pinch of the soil sample and a spatula point of soluble starch into a tube containing 2 ml of 1%
yeast extract solution, mix well. Cap the tube and place in a heating block set at 100°C for 5 min. Cool.
You just enriched for spore formers.
2. Aseptically streak for isolation. Mix the tube then remove two loops full of liquid from the tube with
inoculating loop to inoculate the first sector of a plate of TSA (trypticase soy agar). Finish your streak for
isolation with your loop.
3. Invert the TSA plate and incubate at 30°C until next Tuesday. You just isolated your aerobic, sporeforming mesophiles.
4. Wrap parafilm** around the cap of the tube to prevent evaporation and incubate at 60°C until next
Tuesday. You are enriching for thermophilic aerobic spore-formers.
D. Microbial diversity: Enrichment/isolation of myxobacterial fruiting bodies. – Work in Pairs
Background: Rabbit droppings (dung) provide a rich source of spore-forming bacteria, including the
myxobacteria because rabbits are coprophagic (which means that they ingest their own dung). In soil, the
myxobacteria produce fruiting bodies, which are dense packing’s of myxobacterial cells that have differentiated
into spores. These fruiting bodies and associated spores stick to dung and subsequently are eaten by a rabbit.
Spores survive passage through the stomach so the dung that is released contains many spores. When the
conditions are favorable, the spores germinate (this is analogous to seed germination) and become vegetative, or
growing, cells.
1. Inoculate. Obtain one agar plate containing 30µg/ml cycloheximide and amphotericin B; the prokaryotic
myxobacteria are resistant to the action of cycloheximide and amphotericin, while this antibiotic will
inhibit other eukaryotic organisms. Using a flame-sterilized forceps (tweezers), remove 3 pieces of rabbit
dung and press them gently into the agar. Dampen with a few drops of water (use a Pasteur pipette). Pour
one tube containing 5 ml of soft agar (0.7% w/v agar) maintained at 55°C in a waterbath (this is called
tempering*) so that each piece of dung is partially covered; keep the soft agar at 55°C until ready for
use. Wrap a piece or parafilm** around the rim of the petri dish to keep the inside moist
2. Incubate the plate upright at 30°C for several weeks until you see yellow or orange mounds on the dung.
3. Week 4 has the instructions for further analysis.
*Tempering is used to cool/hold agar solutions just above their solidifying temperature (47°C in this class);
you must at least boil agar in order to get it into solution. Tempering makes the agar cool enough
(without solidifying) not to kill organisms, degrade heat sensitive compounds like antibiotics, cut down
water vapor condensation, and warp heat sensitive plastics.
**Parafilm is a laboratory stretchable wrap. It is used to seal openings. It is a barrier to water molecules but
not to gas molecules (i.e. does not keep O2 out).
MMBB255 Week 2 Experiments
9
Week 2 Study Questions
1. Write out the steps for doing a Gram stain from memory. What is positive and negative?
2. Name and spell two organisms; one that is Gram positive and one that is Gram negative such as the controls
we used.
3. Why do some bacterial cells stain Gram positive while other stain Gram negative? In other words, what is the
cellular difference between the two cells types that allows this staining difference?
4. What types of staining procedures are there (4) and give an example of all.
5. Why was the acid fast stain developed? What is the mordant? Give an example of an organism that is positive
for acid-fast. What Gram reaction does the organism have?
6. Why do we temper agar? Try to list three reasons.
7. Parafilm is used to prevent what?
8. How did we enrich for spore formers? How did we isolate mesophilic and thermophilic spore forming
organisms?
9. How does the enrichment for Myxobacteria work?
10. What is the purpose of controls?