CLASS No.

ORGANIZATION AND GUIDANCE ON WORK IN

BACTERIOLOGICAL LABORATORY.
METHOD OF MICROSCOPY.
Experiment 1. Preparation of a Bacterial Smear. Using Simple Staining Methods
for Direct Observation of Bacteria in a Sample
1. Bacteriological laboratory. Structure and rules.
Bacteriological laboratory is an important structure in the system of
diagnostic microbiology services of practical and diagnostic public health
institutions. Students study the course of microbiology, virology and
immunology in the bacteriological laboratory, which has the same equipment
as practical bacteriological laboratories. Therefore it is very important to study
structure, equipment and organization of working place of microbiologist on
the first class. Besides, students have ability to master widely used
microbiological method – smear preparation, staining with aniline dyes, and
microscopy with immersion objective.
The signs and symptoms of some infectious diseases may be specific for
the particular microorganisms, e.g. the specific spasm of mastication muscles
of the tetanus and the characteristic rash of chickenpox. But many infections
are unspecific, and any of several different pathogenic organisms may be the
cause of an illness such as sore throat, bronchitis, pneumonia, meningitides,
diarrhea, wound sepsis and fever. In those cases, help of microbiologic
laboratory is required to elucidate the cause. The reliability of that help
depends on the correct techniques being used in collecting the appropriate
specimen from the patient, and doctors must be properly instructed in those
procedures. The precise identification of the patient’s pathogenic organisms is
generally necessary for the effective use of selective chemotherapeutic drugs.
In other words, doctor has to identify and treat specific infections rather than
clinical syndromes. Since, moreover, different strains of many bacterial species
differ in their susceptibility to particular drug. It is usually desirable for the
bacterium isolated from the patient to be tested for its drug sensitivity in the
laboratory
Types of laboratories. Depending from their designation, microbiological
laboratories may be bacteriological, parasitological, mycological, virological,
immunological and special (for diagnosis of particularly virulent infections).
Table 1.1 Equipment and materials used in laboratory

Equipment and materials Use

Loop Routine inoculation of agar
slopes/deeps and small volumes of
liquid media (up to ca 10 cm3); making
streak plates

Spreader Making spread/lawn plates

Forceps Transfer of sterile paper/antibiotic

discs; also plant material, e.g. short
lengths of root with nodules

Pipette Transfer of measured volumes/drops of

culture/sterile solutions
A glass pipette is supplied with a
dispensing device.
A pipetting device (sampler) is supplied
with tips of respective volume

Test tube Small volumes (ca 5–10 cm3) of liquid

media/agar slopes/sterile solutions for
inoculation

Petri dish (plastic/glass) Plastic: pre-sterilised for

streak/spread/lawn/pour plates;
Glass: can be re-used after sterilisation
by hot air oven

Test-tube rack A piece of equipment that is used to

hold upright multiple test tubes at the
same time
Bunsen burner Sterilisation of wire loops and (with
alcohol) metal forceps and glass
spreaders

Autoclave Sterilisation of media, solutions and

equipment before use and contaminated
items afterwards; melting solidified
agar media for use

Hot air oven Sterilisation of glass Petri dishes and

pipettes and paper discs (but not
essential as autoclaves serve virtually
all needs)

Discard pots containing Disposal of used pipettes and slides of

disinfectant microscopical preparations

Microwave oven Melting solidified agar media for use

(but not in vessels with metal caps and
not for sterilisation)
Incubator Incubation of cultures (with adjustable
temperature range)

Water bath Suitable temperature for keeping melted

agar media molten for use (ca 50 °C);
accurate temperature control
Thermometer Checking incubator/water bath
temperatures
pH meter Checking and adjusting pH values of
media
Cupboard Storage of culture media and stock
cultures
Refrigerator Storage of heat-labile materials
Laminar flow hood The laminar flow hood provides an
aseptic work area while allowing the
containment of infectious splashes or
aerosols generated by many
microbiological procedures.

Microscope slides, Microscopical observations

cover slips,

immersion oil,

staining tray and bridge,

Culture media ingredients Stock of a range of culture media in

Disinfectants
Alcohol
Autoclave indicator tape
Non-absorbent cotton wool
dehydrated form (tablets/powder);
available as complete media and as
separate ingredients
Treatment of work surface before and
after use and spillages; disposal of
used pipettes and microscope slides; in
soap form for hand washing
Sterilisation of metal forceps and glass
spreaders by ignition
Changes colour in response to heat to
distinguish those items that have
received heat treatment (but is not an
indicator of effective sterilisation)
Plugs for test tubes, flasks and pipettes
 1.2 General laboratory rules
The microorganisms used for instruction in this course are pathogenic for
humans or animals. The safety of every student depends upon the
conscientious observation of rules that must be followed by all who work in
the laboratory. Certain precautions must be followed to avoid endangering
health of neighbors and those who clean the laboratory. Any student who is in
doubt about how to handle infectious material should consult an instructor.
The following rules must be observed at all times.
1. Always wear a laboratory coat when working in the laboratory
classroom.
2. Don’t put anything in mouth which may have come in contact
with infectious material.
3. Smoking, eating and drinking in the laboratory are not permitted
at any time.
4. Mouth pipetting is not permitted under any circumstances. Use the
safety pipetting devices which are provided. Dispose of used pipettes in the
appropriate receptacle. Any infectious material which may accidentally fall
from pipettes to the laboratory bench or floor should be covered with a
disinfectant and reported to instructor immediately.
5. Any spilled or broken containers of culture material should be
thoroughly wet down with a disinfectant and then brought to the attention of
an instructor
6. Report at once an accident which may lead to a laboratory
infection.
7. The microscope is both an expensive and delicate instrument, so
treat it accordingly. Always, at the end of each laboratory period, carefully
clean oil from the objective and condenser lenses, align the low power dry
objective with the condenser and rack condenser up and body tube down. You
will be held personally responsible for any defect found on microscope when
it is recalled at the semester's end.
8. When laboratory work is finished, dispose all used glassware and
cultures in the appropriate vessel. Wash hands thoroughly with soap and water
before leaving the laboratory.
9. Do not throw refuse of any kind into the sink. Use the provided
containers.
10. Be sure all burners are turned off at the end of the laboratory
period. Double check to be sure that handles on all gas outlets are in the off
position.
11. The inoculating loop should be heated until red hot before and
after use. Always flame loop before you lay it down.
 12. Always place culture tubes of broth or slants in an upright
position in a tray. Do not lay them down on the table or lean them on other
objects. They may roll onto the floor and break. All culture containers which
are to be incubated should bear the following notations: 1) initials (or last
name of the student) and group number, 2) specimen (name of organism or
number of unknown) and 3) date. When using Petri plates, these notations
should be entered on the bottom half, not the lid. Unless otherwise directed, all
plates are to be inverted, all plugged tubes should have the plugs firmly set
into the tubes.
13. Laboratory attendance is mandatory.

1.3 Methods of decontamination: sterilization vs disinfection

Table 1.2 Main terms
Sterilization means the complete destruction of all the micro-organisms
including spores, from an object or environment. The methods
of sterilization are used in a microbiology laboratory, surgical practice
and hospital service, pharmacies, etc. Nutrient media, glassware,
containers and instruments used for the collection of specimens and
isolation of organisms have to be sterile before using.
Disinfection is the process of reducing or eliminating pathogenic
microorganisms or viruses in or on material so that it no longer
presents a hazard. Disinfection refers to the use of a physical process or
a chemical agent (a disinfectant) to destroy vegetative pathogens but
not bacterial endospores. It is important to note that disinfectants are
normally used only on inanimate objects because, in the
concentrations required to be effective, they can be toxic to human and
other animal tissue. Disinfection processes also remove the harmful
product of microorganisms (toxins) from materials.
Disinfectants Products or biocides used to reduce the number of viable
microorganisms, or bioburden, on or in a product or surface
to a level previously specified as appropriate for its intended
further handling or use.
Antiseptic A biocide or product that destroys or inhibits the growth of
microorganisms in or on living tissue (eg, skin) or biologic fluids
(eg, mucosal secretions).

Aseptic free of, or using methods to keep free of, microorganisms.

Septic characterized by the presence of pathogenic microbes in living

tissues or associated fluids.
Preservation The prevention of multiplication of microorganisms in formulated
products, including pharmaceuticals and foods.

Pasteurization The process of heating food or other substances under

controlled conditions of time and temperature to kill pathogens and
reduce the total number of microorganisms without damaging the
substance.

1.3.1.Methods of Sterilization:
Physical Agents

 Steam – Used in machines called autoclaves. Autoclaves use steam heated

to 121–134 °C (250–273 °F). To achieve sterility, a holding time of at least 15
minutes at 121 °C (250 °F) or 3 minutes at 134 °C (273 °F) is required. Autoclave
treatment inactivates all fungi, bacteria, viruses and also bacterial spores. Pressure
cooking food is also steam sterilization though it is not that thorough.
 Heating – Under heating flaming, incineration, boiling in water,
tindalization, dry heat. These methods inactivate and kill microorganisms in
objects like glass, metals. Boiling in water for 15min inactivates viruses and kills
most vegetative bacteria. However it has no effect on the spores. Tindilization
means boiling for 20 minutes and then cooling, again re-boiling and cooling for
three times. This method is more effective on sporulating bacteria than just boiling.
Dry heat method can be used on powders and items that bear very high them of
heat.
Sterilisation of equipment and materials
 Wire loop
Heat to redness in Bunsen burner flame.
 Empty glassware and glass (not plastic!) pipettes and Petri dishes
Either: hot air oven, wrapped in either greaseproof paper or aluminium and held at
160 °C for 2 hours, allowing
additional time for items to come to temperature (and cool down!).
Or: autoclave/pressure cooker.
Note: plastic Petri dishes are supplied in already sterilised packs; packs of sterile
plastic pipettes are also available but cost may be a consideration.
 Culture media and solutions
Autoclave/pressure cooker.
 Glass spreaders and metal forceps
Flaming in alcohol (70 % IDA).
  Radiation – Electron beams, X-rays, gamma rays, or subatomic particles are
used for sterilizing disposable medical equipment, such as syringes, needles,
cannulas, IV sets and biological safety cabinets between uses.
 Sterile filtration - Clear liquids that would be damaged by heat,
irradiation or chemical sterilization can be sterilized by mechanical filtration.
Fileration is done through pores that are smaller in size than the organism in
question and this has to be done very slowly.
Chemical Agents

 Chemical sterilization – Chemicals like Ethylene oxide, Ozone, Bleach,

Glutaraldehyde and Formaldehyde, Phthalaldehyde, Hydrogen Peroxide, Dry
sterilization process, Peracetic acid and Silver are used in varying degrees.
Products that can get damaged due to heat are subjected to chemical sterilization
for e.g. biological materials, fiber optics, electronics, and plastics. Ethylene oxide
gas and Ozone gas oxidize most organic matter. Though bleach and
Glutaraldehyde and formaldehyde solutions is used as a disinfectant, it’s a much
more concentrated in sterilization also infected item is left immersed for long
duration for effective sterilization. Dry sterilization process with chemicals is
useful for sterilizing plastic bottles medical and pharmaceutical applications.
A. Alcohols.
Ethyl alcohol, isopropyl alcohol, and n-propanol exhibit rapid, broad-spectrum
antimicrobial activity against vegetative bacteria, viruses, and fungi but are not
sporicidal. Activity is optimal when they are diluted to a concentration of 60% to
90% with water.
B. Aldehydes
Glutaraldehyde is used for low-temperature disinfection and sterilization of
endoscopes and surgical equipment. It is normally used as a 2% solution to achieve
sporicidal activity. Formaldehyde is bactericidal, sporicidal, and virucidal.
C. Biguanides
Chlorhexidine is widely used in handwashing and oral products and as a
disinfectant and preservative. The Mycobacteria, because of their unique waxy cell
envelope, are generally highly resistant to these compounds.
D. Bisphenols
The bisphenols are widely used in antiseptic soaps and hand rinses. In general, they
are broad spectrum but have little activity against Pseudomonas aeruginosa and
molds. Triclosan and hexachlorophene are bactericidal and sporostatic.
E. Halogen-Releasing Agents
The most important types of chlorine-releasing agents are sodium hypochlorite,
chlorine dioxide, and sodium dichloroisocyanurate, which are oxidizing agents that
destroy the cellular activity of proteins. Hypochlorous acid is the active compound
responsible for the bactericidal and virucidal effect of these compounds. At higher
concentrations, this group is sporicidal. Iodine is rapidly bactericidal, fungicidal,
tuberculocidal, virucidal, and sporicidal. Iodophors (eg, povidone-iodine) are
complexes of iodine and a solubilizing agent or carrier, which acts as a reservoir of
the active I2.
F. Heavy Metal Derivatives
Silver (Ag+) sulfadiazine, a combination of two antibacterial agents, Ag+ and
sulfadiazine, has a broad spectrum of activity. Binding to cell components such as
DNA may be responsible for its inhibitory properties.
G. Organic Acids
Organic acids are used as preservatives in the pharmaceutical and food industries.
Benzoic acid is fungistatic; propionic acid is both bacteriostatic and fungistatic.
H. Peroxygens
Hydrogen peroxide has broad-spectrum activity against viruses, bacteria, yeasts,
and bacterial spores. Sporicidal activity requires higher concentrations (10–30%)
of H2O2 and longer contact times.
I. Phenols
Phenol and many phenolic compounds have antiseptic, disinfectant, or preservative
properties.
J. Quaternary Ammonium Compounds
These compounds have two regions in their molecular structures, one a water-
repelling (hydrophobic) group and the other a water-attracting (hydrophilic) group.
Cationic detergents, as exemplified by quaternary ammonium compounds(QACs),
are useful antiseptics and disinfectants. QACs have been used for a variety of
clinical purposes (eg, preoperative disinfection of unbroken skin) as well as for
cleaning hard surfaces. They are sporostatic; they inhibit the outgrowth of spores
but not the actual germination process. QACs are also mycobacteriostatic and have
an effect on lipid-enveloped butnot lipid-nonenveloped viruses.
K. Vapor-Phase Sterilants
Heat-sensitive medical devices and surgical supplies can be effectively sterilized
by vapor-phase systems using ethylene oxide, formaldehyde, hydrogen peroxide,
or peracetic acid.

1.4 Aseptic procedure (sterile technique) in microbiology.

1.4.1. Work with a Bunsen burner: main rules
Bunsen burner, device for combining a flammable gas with controlled
amounts of air before ignition; it produces a hotter flame than would be
possible using the ambient air and gas alone.
 Bunsen burners produce an open flame with two regions: The primary
flame, a small inner cone, is a pale blue flame; and the secondary flame,
almost colourless flame, seen as a larger, outer cone. The hottest part of the
Bunsen flame, which is found just above the tip of the primary, inner, flame,
reaches about 1500 °C. Therefore, extreme safety measures are needed when
handling a Bunsen burner.

burner tube/barrel

stopcock/ neddle valve

Fig 1.3. Main parts of a Bunsen

burner

Risks:
 Starting a fire at the department.
 Gas explosion.
 Burns by open flame.
 Burns by touching hot materials.
 Cuts by shattered glassware.

!!! Before working with fire in the lab, be sure that:

 any long hair is tied back and that
 shirt sleeves do not hang down.
!!! Prior to lighting the burner, set up supplies near a gas supply valve and
have all required equipment within reach:
 Bunsen burner
 rubber tube connecting burner to gas supply
 matches

!!!Handle the burner by its base (After the burner is lit, the barrel itself will be
too hot to handle)

!!!Never leave a lit Bunsen burner unattended

 Starting a Bunsen burner
1. Double-check that the stopcock needle valve) is in the “off” position
and gas is not supplied into a burner.
2. Open the main gas supplying valve. Turn the
handle of the gas supply main so that it is in line, or
parallel, with the outlet and supply hose.
3. Open the gas supplying valve located near your
working place.
4. Strike a match, open the stopcock and light a Bunsen burner. Light the
match and slowly run it up the side of the barrel until it ignites the gas. (This is
done for safety. If the match was held directly over the barrel, a high flame could
burn the hand.) Shake match out and place it in a trash can.
5. Adjust the flame. Once the burner is lit, the stopcock in the base can be
manipulated to control the height of the flame. The flame should be adjusted so
that there is a clear blue flame surrounding an inner blue cone.
 Twist the collar to adjust the flame’s temperature. The collar controls the
amount of air entering the barrel, which determines the temperature of the flame.
Close the collar so that no air enters the barrel for the coolest flame, or a safety
flame. When you want to heat something, open the air ports until the flame is the
right color. The flame gets hotter as it turns blue, and is hottest when it’s almost
invisible.
!!!!Once the flame is lit, it must be tended at all times. If you need to
leave the burner, for any reason, extinguish the flame by turning the gas valve
handle to the OFF position.
6. Close the needle valve by turning it clockwise or transversal to
barrel axis. Close the valve completely to cut off the gas supply.
7. Turn gas supplying valve located near your
workplace into off position. Turn the valve handle so it’s
perpendicular to the gas line and hose.
8. Turn off the gas main. Turn the valve handle so
it’s perpendicular to the gas line and hose.
9. Before you leave the lab, double check the gas
main to ensure you’ve turned off the gas.

1.4.2. Using a wire loop

Wire loops are sterilised using red heat in a Bunsen
flame before and after use. They must be heated to red hot
to make sure that any contaminating bacterial spores are
destroyed. The handle of the wire loop is held close to the
top, as you would a pen, at an angle that is almost vertical.
This leaves the little finger free to take hold of the cotton
wool plug/screw cap of a test tube/bottle.
1.4.3. Flaming procedure
The flaming procedure is designed to heat the end of the loop gradually because
after use it will contain culture, which may ‘splutter’ on rapid heating with the
possibility of releasing small particles of culture and aerosol formation.
1. Position the handle end of the wire in the light blue cone of the flame. This is the
cool area of the flame.
2. Draw the rest of the wire upwards slowly up into the hottest region of the flame,
(immediately above the light blue cone).
3. Hold there until it is red hot.
4. Ensure the full length of the wire receives adequate heating.
5. Allow to cool then use immediately.
6. Do not put the loop down or wave it around.
7. Re-sterilise the loop immediately after use.

Hint
If a loop does not hold any liquid the loop has not made a complete circle. To
correct the problem, first ensure that the loop has been sterilised and then reshape
the loop with forceps. Do not use your fingers because of the possibility of
puncturing the skin.

1.4.4. Flaming the neck of bottles and test tubes

1. Loosen the cap of the bottle so that it can be removed
easily.
2. Lift the bottle/test tube with the left hand.
3. Remove the cap of the bottle/cotton wool plug with the
little finger of the right hand. (Turn the bottle, not the
cap.)
4. Do not put down the cap/cotton wool plug.
5. Flame the neck of the bottle/test tube by passing the
neck forwards and back through a hot Bunsen flame.
6. After carrying out the procedure required, e.g.
withdrawing culture,replace the cap on the bottle/cotton
wool plug using the littlefinger. (Turn the bottle, not cap)
1.4.5. Aseptic transfer of material in a loop

Fig. 1.2. Aseptic transfer.

After the tube is recapped at the

end, the loop is resterilized
before being taken out of service

NOTES ON ASEPTIC TECHNIQUES

You will be working with many pathogenic species of bacteria in the
laboratory. Therefore, you must learn to use careful aseptic technique at all
times, both to protect self, and classmates, and to avoid contaminating
cultures.
Remember that bacteria are in the air as well as on skin, the counter,
and all objects and equipment that have not been sterilized.
The most important tool for transferring cultures is the wire inoculating
needle or loop. It can be quickly sterilized by heating it to red hot in a bunsen
burner flame. Adjust the air inlets of the burner so that there is a hotter inner
cone and the outer, cooler flame. A dry needle may be sterilized by holding it
at a 30o angle in the outer part of the flame. A wet loop with bacteria on it
should first be held in the inner part of the flame to avoid spattering, and then
heated until red hot in the outer part of the flame. Always flame the loop
immediately before and after use! Allow it to cool before picking up an
inoculum of bacteria. If the loop spatters in the agar or broth, it is too hot.
Hold the loop or wire handle like a pencil.
2. Method of Miscroscopy. Types of microscopes

A complex of bacterioscopic, bacteriological, serological, allergic and biologic

techniques is used in the microbiological diagnosis of bacterial infections.
Depending on the nature of the infectious disease, one of them is used as the
main one, while the other are supplementary. Microscopy is an important part
of the examination of many specimens.
The Light Microscope
The resolving power of the light microscope under ideal conditions is about
half the wavelength of the light being used. (Resolving power is the distance
that must separate two point sources of light if they are to be seen as two
distinct images.) With yellow light of a wavelength of 0.4 μm, the smallest
separable diameters are thus about 0.2 μm, i.e., one-third the width of a typical
prokaryotic cell. The useful magnification of a microscope is the magnification
that makes visible the smallest resolvable particles. Several types of light
microscopes are commonly used in microbiology:
Bright-Field Microscope
Bright field microscopy is the simplest of all the light microscopy techniques.
Sample illumination is via transmitted white light, i.e. illuminated from below
and observed from above. Limitations include low contrast of most biological
samples and low apparent resolution due to the blur of out-of-focus material.
Phase Contrast Microscope
This microscope imposes the contrast and makes evident the structure within
the cells that differ in thickness or refractive index. The difference in the
refractive index between bacteria cells and the surrounding medium makes
them clearly visible. Retardation, by a fraction of a wavelength, of the rays of
light that pass through the object, compared to the rays passing through the
surrounding medium, produces phase difference between the two types of
rays.
Dark-Field Microscope
Dark field microscopy is a technique for improving the contrast of unstained,
transparent specimens. Dark field illumination uses a carefully aligned light
source to minimize the quantity of directly transmitted (unscattered) light
entering the image plane, collecting only the light scattered by the sample.
Fluorescence Microscope
A fluorescence microscope is an optical microscope that uses fluorescence and
phosphorescence instead of, or in addition to, scattering, reflection, and
attenuation or absorption, to study properties of organic or inorganic
substances.
Differential Interference Contrast (DIC) Microscope
Employ a polarizer to produce polarized light. The polarized light beam passes
through a prism that generates two distinct beams; these beams pass through
the specimen and enter the objective lens, where they are recombined into a
single beam. Because of slight differences in refractive index of the substances
each beam passed through, the combined beams are not totally in phase but
instead create an interference effect, which intensifies subtle differences in cell
structure. Structures such as spores, vacuoles, and granules appear three-
dimensional.
Confocal Scanning Laser Microscope (CSLM)
CSLM couples a laser light source to a light microscope. A laser beam is
bounced off a mirror that directs the beam through a scanning device. Then the
laser beam is directed through a pinhole that precisely adjusts the plane of
focus of the beam to a given vertical layer within the specimen. By precisely
illuminating only a single plane of the specimen, illumination intensity drops
off rapidly above and below the plane of focus, and stray light from other
planes of focus are minimized. Thus, in a relatively thick specimen, various
layers can be observed by adjusting the plane of focus of the laser beam. Cells
are often stained with fluorescent dyes to make them more visible.
The CSLM is equipped with computer software to assemble digital images for
subsequent image processing. Thus, images obtained from different layers can
be stored and then digitally overlaid to reconstruct a three-dimensional image
of the entire specimen.
The Electron Microscope
Beams of electron are used instead of beam of light, used in light microscope.
The object which is held in the path of beam scatters the electrons and
produces an image which is focused on a fluorescent viewing screen. Gas
molecules scatter electron, therefore it is necessary to examine the object in a
vacuum. As the wavelength of an electron can be up to 100,000 times shorter
than that of visible light photons, electron microscopes have a higher resolving
power than light microscopes and can reveal the structure of smaller objects.
Scanning Probe Microscopes
Measure surface features by moving a sharp probe over the object’s surface.
The scanning tunneling microscope and the atomic force microscope are
examples, which enable scientists to view atoms or molecules on the surface of
a specimen. For example, interactions between proteins of the bacterium
Escherichia coli can be studied with the atomic force microscope.

Table 1.3. Comparison of Various Types of Microscopes

Type of Maximum Resolution Description
microscope magnification (nm)
Extensively used for the
Bright-field 1,500x 100-200 visualization of micro
organisms; usually
necessary to stain
specimens for viewing
Dark-field 1.500X 100-200 Used for viewing live
microorganisms,
particularly those with
characteristic morphology;
staining not required;
specimen appears bright on
a dark background
Fluorescence 1,500X 100-200 Uses fluorescent staining;
useful in many diagnostic
procedures for identifying
microorganisms
Phase contrast 1.500X 100-200 Used to examine structures
of living microorganisms;
does not require staining
DIC 1.500X 100-200 Is particularly useful for
observing unstained cells
because of its ability to
generate images that reveal
internal cell structures that
are less apparent by bright-
field techniques.
TEM (trans- 500,000- 0.1 Used to view ultrastructure
mission 1,000,000 of microorganisms,
electron – X including viruses; much
microscope) greater resolving power and
useful magnification than
can be achieved with light
microscopy
SEM 10,000- 1-10 Used for showing detailed
(scanning 100,000X surface structures of
electron microorganisms, produces a
microscope) three-dimensional image

2.1. Structure and main parts of a bright-field microscope.

Eyepiece: The lens the viewer looks through to see the specimen. The eyepiece
usually contains a 10X or 15X power lens.
Diopter adjustment: Useful as a means to change focus on one eyepiece so as to
correct for any difference in vision between your two eyes.
Body tube (Head): The body tube connects the eyepiece to the objective lenses.
Arm: The arm connects the body tube to the base of the microscope.
Coarse adjustment: Brings the specimen into general focus.
Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses.
Objective lenses: One of the most important parts of a compound microscope, as
they are the lenses closest to the specimen.
A standard microscope has three, four, or five objective lenses that range in power
from 4X to 100X. When focusing the microscope, be careful that the objective lens
doesn’t touch the slide, as it could break the slide and destroy the specimen.
Specimen or slide: The specimen is the object being examined. Most specimens
are mounted on slides, flat rectangles of thin glass. The specimen is placed on the
glass and a cover slip is placed over the specimen. This allows the slide to be easily
inserted or removed from the microscope. It also allows the specimen to be
labeled, transported, and stored without damage.
Stage: The flat platform where the slide is placed.
Stage clips: Metal clips that hold the slide in place.
Stage height adjustment (Stage Control): These knobs move the stage left and
right or up and down.
Aperture: The hole in the middle of the stage that allows light from the
illuminator to reach the specimen.
On/off switch: This switch on the base of the microscope turns the illuminator off
and on.
Fig. 1.3 Main parts of a bright-field microscope.
https://www.microscopemaster.com/parts-of-a-compound-microscope.html

Illumination: The light source for a microscope. Older microscopes used mirrors
to reflect light from an external source up through the bottom of the stage;
however, most microscopes now use a low-voltage bulb.
Iris diaphragm: Adjusts the amount of light that reaches the specimen.
Condenser: Gathers and focuses light from the illuminator onto the specimen
being viewed.
Base: The base supports the microscope and it’s where illuminator is located.

2.2 The setting up of a microscope

It is a basic skill of microbiology yet it is rarely mastered. Only when it is done
properly can the smaller end of the diversity of life be fully appreciated and its
many uses in practical microbiology, from aiding identification to checking for
contamination, be successfully accomplished. The amount of magnification of
which a microscope is capable is an important feature but it is the resolving
power that determines the amount of detail that can be seen.
Microorganisms include bacteria, actinomycetes, fungi, protozoa and
microscopic algae. Due to their difference in size they are observed using
different magnification and sample preparation techniques.
Bacteria and yeast
Yeast can be seen in unstained wet mounts at magnifications ×100. Bacteria
are much smaller and can be seen unstained at ×400 but only if the microscope
is properly set up and all that is of interest is whether or not they are motile. A
magnification of ×1,000 and the use of an oil immersion objective lens for
observing stained preparations are necessary for seeing their characteristic
shapes and arrangements. The information gained, along with descriptions of
colonies, is the starting point for identification of genera and species, but
further work involving physiology, biochemistry and molecular biology is then
needed.
Moulds
Routine identification of moulds is based entirely on the appearance of
colonies to the naked eye and of the mycelium and spores in microscopical
preparations. Mould mycelium and spores can be observed in unstained wet
mounts at magnifications of ×100 although direct observations of ‘mouldy’
material through the lid of a Petri dish or specimen jar at lower magnifications
with the plate microscope are also informative (but keep the lid on!). Routine
identification of moulds is based entirely on the appearance of colonies to the
naked eye and of the mycelium and spores in microscopical preparations.
Protozoa and algae
Protozoa and algae are large organisms and therefore are readily visible at a
magnification of ×10 to ×100 in unstained wet mounts. A magnification of
×100 is advantageous for observing natural samples that contain a variety of
organisms, particularly as many are very motile. Identification of algae and
protozoa is based entirely on their microscopical appearance. The common
algae are green and non-motile; diatoms have a brown, sculptured outer layer
of silica and move slowly. Protozoa are colourless and most are motile. Hay
infusions and cloudy vase water are rich in algae and protozoa, but clear
samples of water are rarely rewarding.

Hints
Adjust the iris diaphragm to achieve optimum balance between definition
and glare. Do not control light intensity by moving the sub-stage condenser,
the position of which should be to focus the light on the specimen. Re-adjust
the iris diaphragm for each objective lens.
For looking at wet mounts of living specimens of protozoa, algae, moulds
and even yeasts, the low power objective lens (×10) is often adequate but also
necessary for locating and centering on an area of interest before turning to the
high power objective lens (×40). Without altering the focus, turn to the high
power lens and then finely re-focus.
Use the oil immersion objective lens for examining stained preparations
of bacteria. Put one drop of immersion oil onto the preparation; a coverslip is
not required. Remove the slide and wipe the oil immersion lens clean at the
end of the practical session.

2.3 Principle of immersion microscopy

Oil immersion is a technique used to increase the resolving power of a

microscope. This is achieved by immersing both the objective lens and the
specimen in a transparent oil of high refractive index, thereby increasing the
numerical aperture of the objective lens.
Immersion oils are transparent oils that have specific optical and viscosity
characteristics necessary for use in microscopy. Typical oils used have an
index of refraction around 1.515. An oil immersion objective is an objective
lens specially designed to be used in this way. Total power of magnification
formed by the combined lenses (objective and ocular) is product of the
separate powers of each lens:
Power of objective x Power of ocular = Total magnification.

Table 1.4. Calculation of microscope total magnification

Power of objective Power of ocular Total magnification
40x high dry objective 10 x 400 x
100x oil immersion 10 x 1000 x
objective
10x low power 20x 200 x
objective

Fig. 1.4. Principle of immersion microscopy.

 3. Stained preparations
A ‘smear’ of bacteria or yeast is made on a microscope slide, fixed, stained, dried
and, without using a coverslip, examined with the aid of a microsope. Aseptic
technique must be observed when taking samples of a culture for making a smear.
A culture on agar medium is much preferable to a liquid culture for making a
smear. A smear that is thin and even enables the shape and arrangement of cells to
be clearly seen and ensures that the staining procedure is applied uniformly. There
are two broad types of staining method:
(1) a simple stain involves the application of one stain to show cell shape
and arrangement and, sometimes, inclusions that do not stain, e.g. bacterial
endospores;
(2) a differential stain involves a sequence of several stains, sometimes with
heating, and includes a stage which differentiates between either different parts of
a cell, e.g. areas of fat storage, or different groups, e.g. between Grampositive
and Gram-negative bacteria.

3.1. Procedure for making a bacterial smear.

1. Clean a plain microscope slide thoroughly: remove fat applying a soap and
then cleaning it off with a fiber-free cloth.
2. Label a microscope slide with a marker pen to record the culture being used,
date and initials; this is also a useful reminder of which side of the slide is
being used.
3. Flame a wire loop to ensure that no culture accidentally remains from a
previous operation.
4. Transfer one or two loopfuls of sterile water on to the centre of the slide.
5. Flame loop and allow to cool.
6. Using aseptic technique, transfer a very small part of a single colony from a
plate or slope of agar medium into the sterile water on the slide.
If the amount of culture on the loop is easily visible you have taken too much!
Fig. 1.5. Sampling of the bacterial culture.
7. Make a suspension of the culture in the sterile water on the slide and
thoroughly but gently spread it evenly over an oval area of up to 2 cm
length.
8. Flame the loop.
If it is necessary to use a liquid culture or sample, the use of sterile water to
prepare the smear will probably be unnecessary and may result in a smear
with too few cells.
9. Dry the suspension by warming gently over a Bunsen burner flame and then
‘fix’ it by quickly passing it through the flame a few times.

Fig. 1.6. Heat fixation of

a slide.
 This is called a heat-fixed smear; it should be visible to the naked eye as a
whitish area. Fixing is necessary to ensure that cells adhere to the slide and to
minimise any post-mortem changes before staining.
The smear is now ready to be stained.
3.4.2. Dyes (stains)
Stain or dye is the synthetic chemical which is derived from nitrobenzene or
aniline. Stains are used commonly in microbiology to increase the contrast
between microorganisms or parts of its and the background,so that it can be easily
visible. The process of giving colour to particular organism or components of its is
known as staining.
Each stain or dye is composed of three components.
i) Benzene ring – the colourless part of a dye and it is basic structural component
of a dye.
ii) Chromophore – the functional group of a dye that give colour to the stain.
iii) Auxochrome – the group that gives ionic property to the stain.
Benzene ring and chromophore is collectively known as chromogen.
Types of stains:
Based on the nature of chromogen, there are three types of stain.
1. Acidic stain (Anionic stain)
2. Basic stain (Cationic stain)
3. Neutral stain
Acidic stain (Anionic stain)
Chromogen of acidic stain is negatively charged. so, it is also known as Anionic
stain. Acidic stain are used to stain the positively charged components such as
background staining. Acidic stain can not stain bacterial cell due to repulsion of
same charge.
Examples: Eosin, Nigrosin, India ink
Basic stain (Cationic stain)
Chromogen or coloured part of basic stain is positively charged. so, it is also
known as cationic stain. Basic stain are used to stain negatively charged
components such as bacterial cell.
Examples: methylene blue, safranin, malachite green,basic fuschin, crystal violet
Neutral stain:
In neutral stain, both cation and anion are coloured, such that net charge is neutral.
Neutral stain are actually is a salt of acidic and basic stain.
Examples: giemsa stain.
3.4.3. Simple method for smear staining.
1. Put the slide with the fixed smear uppermost on a staining rack over a sink
or staining tray.
2. Thoroughly cover the smear with stain and leave for, usually, 3-5 minutes.
3. Hold the slide with forceps (optional but avoids stained fingers), at a 45°
angle over the sink.
4. Rinse off the stain with tap water.
 5. Blot-dry the smear with filter/fibre-free blotting paper using firm pressure,
but not sideways movements that might remove the smear.
6. Examine under oil immersion.
7. When finished, dispose of slides into discard jar.
8. Suitable stains include basic dyes (i.e. salts with the colour-bearing ion, the
chromophore, being the cation), such as methylene blue, crystal violet and
safranin.

Fig. 1.7. Procedure for simple staining

Pre-lab questions: (questions to be answered before doing the experiment.

The answers are due at the beginning of the experiment):

1. What safety measures must be followed when handling a Bunsen burner?

2. What device is used to fix a wire loop if necessary?
3. What device is used to sterilize wire loops, metal forceps and glass
spreaders?
4. Why do we sterilize a wire inoculating needle or loop immediately before
and after use?
5. What is the difference between sampling slant and broth cultures?
6. What type of microscope is used in this lab work?
7. How do we set a microscope?
8. What type of objective lens is used to observe shapes and arrangements of
bacterial cells?
9. How do we fix bacterial cells in this lab work?
10.Which dye do we use for simple staining of a bacterial smear?
 THE PRACTICAL PART INCLUDED IN YOUR VIDEO LAB.

Materials (per lab group)

 Bunsen burners
 Staining trays and bridges
 Test tubes with sterile water
 Loops
 Slides
 Soap
 Fiber-free cloth
 Fuchsine
 Wash bottles
 Blotting paper
 Immersion oil
 Alcohol
 Microscopes
 Discard jar
 Marker pen
 Forceps
 Empty tubes with plugs/caps
 Empty Petri dish
 Micrococcus luteus cell culture
 Serratia marcescens cell culture
 Bacillus megaterium cell culture

Experimental procedure:
Main task 1:
1. Start the Bunsen burner properly according to para.1.4.1.
2. Flame a loop according to para. 1.4.2. and 1.4.3.
3. Position a tube and then two tubes in one hand properly. Flame their necks
according to para. 1.4.4.
4. Obtain a loopful using a sterile technique according to para.1.4.5.
5. Hold and open a Petri dish using one hand.
6. Turn off the Bunsen burner properly according to para. 1.4.2.
7. Familiarize with a structure of the autoclave, dry-air sterilizer, incubator.

Main task 2: Prepare the smears from different agar cultures and examine
them using an immersion oil objective of light microscope.

Step 1. Start a Bunsen burner at your workplace.

Step 2. Adhering to aseptic procedure requirements prepare a bacterial smear
for three provided cultures of microorganisms: Micrococcus luteus, Serratia
marcescens, Bacillus megaterium.

Step 3. Turn off the Bunsen burner and stop the gas supply if everyone has
finished the previous steps.

Step 4. Stain all three smears with fuchsine adhering to para 3.4.3. Leave a stain on
a smear for 3 min.

Step 5. Examine stained preparations under oil-immersion objective:

a) Set up the microscope; raise the body tube so that the objectives are well
above the stage. Place the test smear on the stage.
b) Place a drop of immersion oil on the smear to be examined.
c) Swing oil immersion lens into position.
d) Lower the optical system (body tube) very carefully, watching from the
side, until the tip end of the objective is immersed in the oil and touches the
slide.
e) Adjust light with diaphragm while looking through the eyepiece.
Raise the optical system (body tube) gently while searchingly looking into the
ocular until the object comes into clear view
f) Then bring the object into sharp focus with the fine adjustment, which
should be turned gently to lower the optical system.

Step 6. When finished, dispose of slides into discard jar.

Step 7. COMPLETE THE DISTANCE TASK you will receive from the
Instructor during 30 min.

Questions for self-control:

1. What is the difference between sterilization and disinfection?

2. What physical agents are used for sterilization?
3. How can we sterilize heat-sensitive products?
4. What part of a Bunsen burner regulates temperature of a flame?
5. What regions are there in a Bunsen flame? Where is the hottest part of the
Bunsen flame?
6. What types of microorganisms are known?
7. What amount of magnification is required to observe different types of
microorganisms?
8. How to calculate total magnification of a microscope?
9. What are the main parts of a bright-field microscope?
10.What is the purpose of using oil immersion?
11.What types of microscopes do you know?
12.How dyes are classified according to the nature of chromogen?
13.What components is every dye composed of?
14.What types of staining do you know?
15.What is the purpose of heat fixation of a slide?

Literature to get prepared for CLASS1:

 Cowan M.K, Bunn J. - Microbiology Fundamentals_ A Clinical Approach
(Part I) – pages 46-53; 233-255.