5: STAINING TECHNIQUES Gram staining can also be a diagnostic tool which can

Unstained bacterial cells are nearly transparent guide medical practitioners as to what type of
when observed by light microscopy and hence, are antibiotic can be used to treat a particular infection.
difficult to see. However, most bacteria can be easily
stained. Staining involves preparing a smear by 2. Why are the steps in Gram staining so
spreading a drop of an aqueous suspension of cells carefully standardized?
on a glass slide, allowing it to dry in air, fixing it with Gram staining is standardized so as to reduce the
gentle heat, and then applying staining reagents. errors that may arise from an inconsistent gram
Dye as stains are used to make microorganisms staining technique. This is to also ensure that
more easily seen, to show certain cell structures, or to scientists around the world follow the same protocol
reveal their chemical nature. Basic dyes have a so that they would arrive to a similar conclusion.
colored ion that is positively charged which, this,
complexes with the negatively charged components 3. What is the influence of pH and age of the
of the bacterial cell. Examples are crystal violet, cultures on Gram reaction?
methylene blue and safranin. The opposite is true It is important to use fresh, young and actively
with acidic dyes which have a negatively charged growing cultures when doing Gram staining. Older
colored ion which binds more to the background. cultures may have breaks in the cell wall and often
There are several types of stains. A simple stain is give gram variable results where a mixture of pink/red
sufficient to visualize gross morphology like size and cells are seen among blue/purple cells.
shape. Differential stains distinguish different group Moreover, bacterial cultures should be cultivated
of bacteria based on certain stained cell components. within their optimum pH range because deviations in
An example of this is the Gram stain. In Gram this physicochemical factor induces changes in the
staining, the Gram-positive bacteria are differentiated structure of the bacteria, particularly the cell wall.
from the Gram-negative ones by virtue of their
thicker cell wall which, thus, absorb the primary stain 4. Why is staining an important procedure
and resist decolorization. The other type of stain is the in viewing microorganisms and other
structural stain which targets specific parts of the cell, types of cells?
allowing the visualization of that particular component. One of the difficulties in viewing microorganisms is
that there is only a slight contrast in appearance
The Gram-positive organisms appear blue or deep between the cells and their environment. To solve this
violet; the Gram-negative, red or pink. problem, staining techniques are being done to add
contrast and give color to the cells for easy viewing.

5. Cite some examples of the application of

differential staining in the field of
Microbiology.
One of the best example of a differential staining
method is the Gram staining. This staining
differentiates Gram positive from Gram negative
bacteria. Some other examples include endospore
staning, flagella staining, acid-fast staining and
capsule staining.

6. What color would the bacterial cells have

when viewed microscopically after the
Guide Questions:
following treatments:
1. What is the value and importance of
Gram staining?
Gram staining is a very important preliminary step in
the initial characterization and classification of
bacteria. Through this, many organisms in the
Domain Bacteria are differentiated according to their
cell wall composition (Gram-positive and
Gram-negative).
 6: ENUMERATION OF CULTURABLE • The assumption made in the viable counting
MICROORGANISMS procedure is that each viable cell can grow and divide
Microorganisms are ubiquitous. They grow by to yield one colony. Thus, colony numbers are a
increasing their number in a population and each reflection of cell numbers.
reproduction of the cell reproduces the entire
organism. Population growth is measured by tracking
changes in the number of cells or changes in the level
of some cellular components (proteins, nucleic acids,
or the dry weight of the cells themselves. In this
exercise we will consider the two common
measures of cell growth: cell counts and turbidity,
the latter of which is a measure of cell mass.
The accurate enumeration of microorganisms in a
population is important for successful isolation,
cultivation and characterization. The most common
method of enumerating bacteria is by doing a
standard or viable plate count. In this method, a
known volume of a sample is serially diluted, and
each dilution is plated onto a suitable agar medium.
The colonies that form on the plates are then counted,
permitting the estimation of the microbial population.
This method has two known variations: the spread
and pour plating method.

A. Turbidimetric Methods
• A suspension of cells looks cloudy (turbid) to the eye
because cells scatter light passing through the
suspension.
• The more cells that are present, the more light is
scattered, and hence the moreturbid the suspension
• Cell mass is assessed
• Since cell mass is proportional to cell
number, turbidity can be used as a measure of cell
Guide Questions:
numbers and can also be used to follow an increase
1. What are the basic differences between
in cell numbers of a growing culture.
the streak plate and the pour plate
• Turbidity is measured with a spectrophotometer
methods of isolation?
Streak plating is the spreading over of bacteria on the
B. Microscopic Counts
surface of a semi- solid, agar-based nutrient medium
• A total count of microbial numbers can be achieved
in a Petri dish such that fewer and fewer bacterial
using a microscope to observe and enumerate the
cells are deposited at widely separated points on the
cells present in a culture or natural sample.
surface of the medium and, following incubation,
Microscopic counts can be done on either samples
develop into colonies.
dried on slides (using Petroff-Hauser counting
Pour plating is used to count the number of
chamber) or on samples in liquid (using flow
microorganisms in a mixed sample, which is added to
cytometer).
a molten agar medium prior to its solidification.
Streak plate will produce surface colonies, while pour
plate will produce surface and sub-surface colonies.
C. Viable Counts
• A viable cell is one that is able to divide
2. How do surface colonies differ from deep
and form offspring, and in most cell- counting
or buried colonies?
situations, these are cells we are most interested in.
Surface colonies are aerobic, while buried colonies
Viable count or plate count is used to determine the
may be anaerobe, microaerophilic, facultative
number of cells in a sample capable of forming
anaerobes or aerotolerant microbes.
colonies on a suitable agar medium.
 3. In the spread plate method, why is the medium
volume plated usually limited to not more • viability lasts for approx. 1 month
than 0.1 mL? • active culture stored at 4 deg C
If the sample is more than 0.1 mL, the excess liquid • prone to contamination
does not soak or absorb in and may cause the • risk of mutation increases with frequency of
colonies to coalesce as they form, making them sub-culture
difficult to count.
MINERAL OIL PRESERVATION
4. Why are Petri dishes incubated in an • Availability of air is restricted, reducing the metabolic
inverted position? rate
If incubated in a normal position, evaporation will take • For bacteria , yeasts, molds, mushrooms
place in the media. This will result to water droplets • NOT for obligate aerobes
accumulation on the lid of the dish. These droplets • Longevity: 1 -3 years
can fall to the culture media, causing contamination or • Genetic stability: poor
spreading of colonies. • Cost: low

5. Why is it necessary that the diluents with PRESERVATION IN STERILE WATER

culture be shaken before diluting? • Simple and inexpensive
Diluents with culture must be shaken in order to make • For bacteria , yeasts and molds
sure that the cells are evenly distributed in the entire • Longevity: 5-10 years
diluent. This will provide a homogenous culture with a • Genetic stability: moderate
constant concentration of bacterial culture. • Cost: low

7: STANDARD AND GROWTH CURVE PRESERVATION IN STERILE SOIL

• For sporulating bacteria and fungi
BACTERIAL GROWTH • e.g., Bacillus spp, Streptomyces spp
In prokaryotic cells, growth is defined as an increase • Longevity: 5-20 years
in the number of cells. Microbial cells have a limited • Genetic stability: moderate
life span, and a certain species is maintained as a • Cost: low
result of the continuous growth of its population. The
growth curve describes the entire growth cycle of a ULTRA-LOW FREEZING AT -80°C USING
bacterium and is made up of four phases: lag, log GLYCEROL
(exponential), stationary, and death. The phases of • Cryoprotectant is used (glycerol)
bacterial growth reflect events in a population of cells, • For bacteria, yeasts, multicellular fungi
not in individual cells. Since cell numbers in a • Longevity: 4-5 years
bacterial culture can quickly become massive, there is • Genetic stability: moderate
a necessity to use quantitative methods. • Cost: medium

8: CULTURE PRESERVATION TECHNIQUES LIQUID DRYING (L-DRYING)

• Liquid is dried without freezing
IMPORTANCE OF PRESERVATION • A protective agent is used (MSG, skim, milk,
• To maintain VIABILITY medyo-inositol, raffinose, honey)
• To maintain CELL INTEGRITY • For bacteria, yeasts, molds, algae
• To maintain GENETIC STABILITY • Longevity: 4-40 years
• Genetic stability: good
QUALITY CHECKS • Cost: high
VIABILITY: Check growth on appropriate medium
PURITY: Check for presence of contaminants CRYOPRESERVATION USING LIQUID NITROGEN
IDENTIFY: Check distinguishing characteristics • Deep freezing at -196°C
• A cryoprotectant is used
CULTURE PRESERVATION TECHNIQUES (DMSO)
PERIODIC TRANSFER • Safest storage for bacteria, fungi, bacteriophages,
• simplest method protozoa, viruses, plant, and animal cells
• culture is re-streaked onto fresh • Longevity: infinite
• Genetic stability: good
• Cost: high

MAIN FACTORS TO CONSIDER IN SELECTING

THE RIGHT PRESERVATION METHOD
• Availability of materials and equipment • Cost of
materials and labor
• Type of microorganism
• Required longevity
• Desired stability
• Value of microorganism

NOTES
• There is no universal method for successful
preservation of all microorganisms
• Different microorganisms, exhibit different responses
to stresses imposed by the preservation method and
resuscitation
✔ A culture should therefore be preserved using at
least two methods to be assured of back-up culture.