MICROBIOLOGY AND

PARASITOLOGY

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
knowledge about the mode of spread of infection. Some
III PPE & SAFETY IN LAB
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
o 3 – typical size of an animal cell would be from
body fluids.
o Centrifuging without covering with biological 10 – 100 µm
hood.
o Not wearing a proper face shield when working MICROSCOPE
● Uses light
with specimens.
o Exposure to sharps, such as needles and ● Few types
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 to
● The adaption of microorganisms to certain treatments or 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 illumination to enhance
setting. Microbiology helps a healthcare professional to contrast in specimens that
have better understanding of these microorganisms so are not imaged well under
that a patient whose immune system lowers down and is normal illumination
always at risk of being affected by any microbe, can conditions.
remain safe and healthy when he is in the hands of a Phase-contrast ● an optical microscopy
nursing professional. Microscopy technique that converts
● Microbiology helps a nursing professional to understand phase shifts in light passing
the basic concepts of reproduction, morphology, through a transparent
biochemical characteristics and genetics. Microbiology specimen to brightness
makes aware about new diseases and modern changes in the image.
molecular

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

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
COMPOUND LIGHT MICROSCOPE
microscope and can
● The image is magnified again by ocular lens have a magnification of
o Total magnification = objective lens x ocular 4x, 5x, 10x, 20x, 40x,
lens 50x, and 100x.
● Resolution – ability of lenses to distinguish 2 points Stage ● The platform below the
o E.g RP of 04 nm can distinguish between 2 objective lens on which
points > 0.4nm the object to be viewed
● Shorter light wavelength provides greater solution is placed. A hole in the
● Refractive Index – Light bending ability of a medium stage allows light
● Light may bend in air that it misses the small high- beam to pass and
magnification lens illuminate the
● Immersion oil is used to keep the air from bending. specimen.
Types of Microscopes Stage Clips ● These are two stage
Light Microscope ● Found in most schools, clips on each side of
uses compound the stage. Once the
lenses to magnify slide containing the
objects. The lenses specimen is placed on
bend or refract the the stage, the stage

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clips are used to hold
the slide in place PREPARING SMEARS FOR STAINING
Diaphragm ● It is located on the ● Staining – coloring microbe with a dye to emphasize
lower surface of the certain structure.
stage. It is used to ● Smear – A thin film of a microbe solution on a slide, a
control the amount of smear is usually fixed to attach microbes to the slide and
light that reaches the kill microbes.
specimen through the
hole in the stage.
o Also known
as the iris
diaphragm.
Illuminator ● Simple compound
STAINING
microscopes have a ● Stain usually consists of +ve and -ve ion
mirror that can be ● Basic dye – chromophore is a cation
moved to adjust the ● Acidic dye – chromophore is an anion.
amount of light that ● Staining the background instead of the cell is called
can be found on the negative staining.
specimen. However,
some advanced types
of compound
microscopes have their
own light source.
The Adjustments ● 2 adjustment knobs:
fine adjustment &
coarse adjustment
knob; refine the focus
of the lenses. The
coarse adjustment
knob helps in
improving the focus of ● Stains are classified broadly as basic, acidic, or neutral
the low powers stains. The chemical nature of the cells under
whereas the fine examination determines which stain is selected for use.
adjustment knob helps Cell staining is important in the diagnosis of
in adjusting the focus microorganisms because bacteria can be identified by
of the lenses with the color differentiation of stains (dyes).
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.

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GRAM STAINING
● Gram staining 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.

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.

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● Each person will make a smear and Acid-fast stain o Membrane filter set
mixed broth containing:
Mycobacterium smegmatis (Gram +) & DESCRIPTION
Staphlococcus epidermis

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

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PPE & SAFETY IN LAB

● Personal Protective Equipment (PPE)
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
o Non-sterile Gloves
o Heavy-duty Gloves
STERILE GLOVES
● Disposable, individually wrapped.
● They are mostly used during surgery and during
performing invasive 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).

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MASKS
● Places over nose mouth and chin.
o Standard mask
▪ Used when there is risk of exposure to droplets
HEAVY-DUTY GLOVES containing infectious agents.
● Reusable after decontamination
● Used for:
o Handling medical waste
o Performing environmental cleaning

o N95 masks
▪ Used when there is risk of exposure to airborne
infectious agents such as M. tuberculosis

APRONS AND GOWNS

● Used during procedures that generate splashes of
blood or body fluids.

CLOSED FOOT WEAR

● In operating theatre

● Over-shoes
o NOT RECOMMENDED
o Ideal way of transferring microorganisms from
floor and shoes to the hand.

EYE GOGGLES
● Used for protection against splashes of blood or body
fluids.
o (eg. During certain surgical procedures)

OVERHEAD
● Used to contain hair during surgical procedures

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● 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.

● Important prevention of:

o Infection
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 ● Laboratory personnel should not wear sandals
leaving the laboratory at the end of the session.

PERSONAL PROTECTIVE EQUIPMENT (PPE)

USED
● Gloves are essential.
● Lab coats are required.
● Safety glasses (goggles) may be required to avoid
splashes.

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● Report all accidents, no matter how minor, to your

supervisor.
● Be careful & be safe.
● Smoking in the lab is essentially prohibited.

● Eating and drinking in the laboratory are absolutely

prohibited.

● 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.
o Discard in a red bag.

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REFERENCES
Notes from the discussion by VMUF powerpoint
presentation

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