CHAPTER 2 | LECTURE & LABORATORY

MICROBIOLOGY & PARASITOLOGY: CHAPTER 2

identification methods. The role of microorganism in

OUTLINE development of certain medications and vaccinations cannot
I BASIC MICROBIOLOGY AND EQUIPMENT AND be ignored either.
PROCEDURES IN THE STUDY OF BACTERIA ● To prevent spread of infection: Nurses should have
II LAB EQUIPMENT STUDY
III PPE & SAFETY IN LAB
knowledge about the mode of spread of infection. Some
of the infections are spread by contact (touch) air
(airborne) droplets (sneezing, coughing) some by eating
contaminated food or drink (food borne) sexual contact
BASIC MICROBIOLOGY AND EQUIPMENT AND
(STDs), by arthropod bite (vector borne) and others by
PROCEDURES IN THE STUDY OF BACTERIA contaminated blood transfusion, etc. This knowledge
● Personal Protective Equipment would help a nurse to look for specific control of infection.
If a nurse knows how the disease producing organism
enters into the body how they are discharged from the
body and how they spread from person to person the
knowledge would help a nurse to use particular measures
to save community and hospital spread of infection e.g.
in tuberculosis case a negative pressure room works in a
positive way for patient health.

● Masks - placed over nose, mouth and chin

o Standard mask – used when there is risk of
exposure to droplets containing infectious HOW DO WE VIEW MICROORGANISMS?
agents ● Units of measurement
o N95 masks – used when there is risk of o When talking about cells and microscopic
exposure to airborne infectious agents such as organisms, you would be measuring using
M. tuberculosis (prevent 95% of particles less) MICROMETRE (abbreviated: µ -- micron) or
stated as: µm (micrometer).
LABORATORY SAFETY ▪ 1 µm = 1 x 10−6 meters / 1 x 10−3 mm
● Correctly dispose of biohazardous waste. ▪ 1 mm = 1 x 103 nanometers / 1 x 103 µm
o Double bag ● To give you the idea of how small a micro metre is,
● Causes of infectious airborne transmission o 1 – a human hair is about 100 µm wide,
o Removing rubber stoppers o 2 – a red blood cell would be around 8 µm wide
o Splashing during transfer of blood or other body o 3 – typical size of an animal cell would be from
fluids. 10 – 100 µm
o Centrifuging without covering with biological
hood. MICROSCOPE
o Not wearing a proper face shield when working ● Uses light
with specimens. ● Few types
o Exposure to sharps, such as needles and Compound Light ● a microscope with more
lancets. Microscopy than one lens and its own
THE IMPORTANCE OF BEING FAMILIAR WITH light source. In this type of
LABORATORY EQUIPMENT’S & THEIR USES microscope, there are
● Microorganisms as the name suggests are microscopic ocular lenses in the
organisms which have an ability to adapt according to binocular eyepieces and
environment for instance, commensals change objective lenses in a
pathogenic in certain changed environments. rotating nosepiece closer
● The adaption of microorganisms to certain treatments or to the specimen.
medicines remains a challenge for a healthcare Darkfield Microscopy ● a technique that takes
professional like a nurse. Microbiology touches life of a advantage of oblique
nurse while he/she works in a medical health care setting. illumination to enhance
Microbiology helps a healthcare professional to have contrast in specimens that
better understanding of these microorganisms so that a are not imaged well under
patient whose immune system lowers down and is always normal illumination
at risk of being affected by any microbe, can remain safe conditions.
and healthy when he is in the hands of a nursing Phase-contrast ● an optical microscopy
professional. Microscopy technique that converts
● Microbiology helps a nursing professional to understand phase shifts in light
the basic concepts of reproduction, morphology, passing through a
biochemical characteristics and genetics. Microbiology transparent specimen to
makes aware about new diseases and modern molecular brightness changes in the

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image. Phase shifts larger specimens.

themselves are invisible, (The spinning
but become visible when microscope at the top
shown as brightness of this page is
variations. stereoscope)
Differential ● a microscopy technique Scanning Electron ● Allow scientists to view
Interference contrast that introduces contrast to Microscope a universe too small to
microscopy images of specimens be seen with a light
which have little or no microscope. SEMs do
contrast when viewed not use light waves;
using brightfield they use electrons
microscopy. The images (negatively charged
produced using DIC have a electrical particles) to
pseudo 3D-effect, making magnify objects up to
the technique ideal for two million times.
electrophysiology Transmission Electron ● Also uses electrons,
experiments. Microscope but instead of scanning
Fluorescence ● highly sensitive, specific, the surface (as with
Microscopy reliable and extensively SEMs) electrons are
used by scientists to passed through very
observe the localization of thin specimens.
molecules within cells, and Specimens may be
of cells within tissues. stained with heavy
Confocal Microscopy ● broadly used to resolve the metal salts.
detailed structure of specific
objects within the cell PARTS OF THE MICROSCOPE

PARTS FUNCTIONS
Eyepiece ● To observe specimen.
Contains two or more
lenses. The most
common magnification
for the eyepiece is 10X.
There are also 2X and
5X. An eyepiece is
removable, can be
interchanged for
different magnification.
o Also known
as ocular.
Objective Lenses ● More than one
objective lenses These
are the primary lenses
of a compound
microscope and can
have a magnification of
4x, 5x, 10x, 20x, 40x,
50x, and 100x.
Stage ● The platform below the
objective lens on which
COMPOUND LIGHT MICROSCOPE the object to be viewed
● The image is magnified again by ocular lens is placed. A hole in the
o Total magnification = objective lens x ocular lens stage allows light beam
● Resolution – ability of lenses to distinguish 2 points to pass and illuminate
o E.g RP of 04 nm can distinguish between 2 the specimen.
points > 0.4nm Stage Clips ● These are two stage
● Shorter light wavelength provides greater solution clips on each side of
● Refractive Index – Light bending ability of a medium the stage. Once the
● Light may bend in air that it misses the small high- slide containing the
magnification lens specimen is placed on
● Immersion oil is used to keep the air from bending. the stage, the stage
Types of Microscopes clips are used to hold
Light Microscope ● Found in most schools, the slide in place
uses compound Diaphragm ● It is located on the
lenses to magnify lower surface of the
objects. The lenses stage. It is used to
bend or refract the control the amount of
light, which makes the light that reaches the
object beneath them specimen through the
appear closer. hole in the stage.
Stereoscope ● This microscope o Also known
allows for binocular as the iris
(two eyes) viewing of diaphragm.

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Illuminator ● Simple compound

microscopes have a
mirror that can be
moved to adjust the
amount of light that can
be found on the
specimen. However,
some advanced types
of compound
microscopes have their
own light source.
The Adjustments ● 2 adjustment knobs:
fine adjustment & ● Stains are classified broadly as basic, acidic, or neutral
coarse adjustment stains. The chemical nature of the cells under
knob; refine the focus examination determines which stain is selected for use.
of the lenses. The Cell staining is important in the diagnosis of
coarse adjustment microorganisms because bacteria can be identified by
knob helps in improving the color differentiation of stains (dyes).
the focus of the low
powers whereas the
fine adjustment knob
helps in adjusting the
focus of the lenses with
higher magnification.

MAGNIFICATION
● Your microscope has 3 magnifications:
o Scanning, Low and High. Each objective will
have written the magnification. In addition to this
the ocular lens (eyepiece) has a magnification.
● The total magnification is the ocular x objective.

STAINING
● Device to examine bacteria smeared on a slide and
allowed to air dry. Specific stains and techniques are
used to observe bacterial morphology.

PREPARING SMEARS FOR STAINING

● Staining – coloring microbe with a dye to emphasize
certain structure.
● Smear – A thin film of a microbe solution on a slide, a
smear is usually fixed to attach microbes to the slide and
kill microbes.

GRAM STAINING
STAINING
● Gram staining is the most important staining procedure in
● Stain usually consists of +ve and -ve ion microbiology. It is used to differentiate between gram-
● Basic dye – chromophore is a cation positive organisms and gram-negative organisms.
● Acidic dye – chromophore is an anion. Hence, it is a differential stain. Gram-negative and gram-
● Staining the background instead of the cell is called positive organisms are distinguished from each other by
negative staining. differences in their cell walls.

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EXAMPLE OF ACID-FAST BACTERIA

4 STEPS IN GRAM STAINING PROCESS

● Flood the fixed and air-dried smear with Crystal Violet (a
purple dye) for 30 seconds.
● Rinse gently with water and cover the smear with Grams
Iodine solution.
● After 30 seconds, wash off iodine with water and
decolorize with Ethanol.
● Counterstain safranin (a bright red dye) for 1 minute,
rinse, dry and examine under oil.

● Each person will make a smear and Acid-fast stain mixed

broth containing:
Mycobacterium smegmatis (Gram +) &
Staphlococcus epidermis

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LAB EQUIPMENT STUDY

● List of equipment/apparatus used in microbiology
laboratory:
o Autoclave
o Incubator
o Hot air oven
o Inoculating loop
o Vortex mixer/shaker
o Water bath
o Heating mantle
o Hot plate with magnetic stirrer
o UV chamber
o Inoculation chamber
o pH meter
o Colony counter
o Microscope
o Refrigerator
o Bunsen burner
o Spirit lamp
o Micrometer (stage and ocular)
o Balance (digital and 4-beam)
o Thermometer
o Membrane filter set

DESCRIPTION

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HEAVY-DUTY GLOVES
● Reusable after decontamination
● Used for:
o Handling medical waste
o Performing environmental cleaning

APRONS AND GOWNS

PPE & SAFETY IN LAB ● Used during procedures that generate splashes of blood
● Personal Protective Equipment (PPE) or body fluids.
o Specialized clothing or equipment worn by an
employee or protection against infectious
materials.
● How can I select PPE?
o PPE selected for use should:
▪ Protect HCP clothing and skin from
contamination by blood and body fluids
▪ Protect patients from acquiring
microorganisms from HCP.
● What are the types of PPE?
o Gloves
o Gowns and aprons
o Eye goggles
o Overhead
o Foot protection
● Types of Gloves
o Sterile Gloves EYE GOGGLES
o Non-sterile Gloves ● Used for protection against splashes of blood or body
o Heavy-duty Gloves fluids.
STERILE GLOVES o (eg. During certain surgical procedures)
● Disposable, individually wrapped.
● They are mostly used during surgery and during
performing invasive procedures.

OVERHEAD
● Used to contain hair during surgical procedures

NON-STERILE GLOVES
● Disposable, single use, usually not singly wrapped
● They are mostly used to protect skin against exposure to
blood and body fluids.
o (e.g. when changing dress & handling
specimens).

MASKS
● Places over nose mouth and chin.
o Standard mask
▪ Used when there is risk of exposure to droplets
containing infectious agents.

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o N95 masks ● Important prevention of:

▪ Used when there is risk of exposure to airborne o Infection
infectious agents such as M. tuberculosis o Injury by harmful chemicals
o Mechanical injury by glassware, open flames,
and sharp objects.
● Personal belongings
o Away from work station
● Wash your hands with soap at the start of the laboratory
session before performing any procedures, before
leaving the laboratory at the end of the session.

CLOSED FOOT WEAR

● In operating theatre

● Over-shoes PERSONAL PROTECTIVE EQUIPMENT (PPE) USED

o NOT RECOMMENDED ● Gloves are essential.
o Ideal way of transferring microorganisms from ● Lab coats are required.
floor and shoes to the hand. ● Safety glasses (goggles) may be required to avoid
splashes.

● Avoid touching objects (e.g., pencils, cellphones, door

handles) while wearing gloves.

SAFETY IN THE LABORATORY

● The laboratory safety procedures must be read prior to
attending the laboratory session.
● Long hair must be tied back or covered to minimize fire
hazard or contamination of experiments.

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● Laboratory personnel should not wear sandals

● All cultures should be handled as potentially pathogenic.
● Liquid cultures must always be kept in a test tube rack.
● Broth cultures must never be pipetted by mouth.

● In case of blood spills

o Cover with paper towel.
o Apply disinfectant.
o Leave for 15 minutes.
o Remove while wearing gloves.
● Smoking in the lab is essentially prohibited. o Discard in a red bag.

● Eating and drinking in the laboratory are absolutely ● Report all accidents, no matter how minor, to your
prohibited. supervisor.
● Be careful & be safe.

REFERENCES
Notes from the discussion by VMUF powerpoint presentation

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