Chapter 8 Introduction To Laboratory Technique

DNS 0303
Microbiology & Parasitology

Chapter 8
Introduction to Laboratory Technique
What is microscope?
Different types of microscope
Parts and uses of microscopes
Procedures of using microscope
Handling of microscope

11/10/2014 DNS 0303 Microbiology & Parasitology 2
Contents
What is inoculation?
Different types of Culture medium / Growth
medium
Aseptic techniques
Inoculation of culture media
Principle of staining
What is simple staining, Gram staining and
acid fast staining?

Contents (Cont.)
Upon completion of this chapter, the
students will be able to:
(a) Describe the differences between light
and electron microscope.
(b) Discuss the parts and uses of
microscope.
(c) Briefly explain how to observe
specimen by using a light microscope.
(d) Describe how to handle the
microscope.
Learning Outcomes
(e) Discuss the inoculation of culture
media.
(f) Define, describe and give examples of
the different kinds of media.
(g) Explain the uses of various types of
media.
(h) Explain the importance of aseptic
techniques.
(i) Describe the principle of staining and
differences between simple staining,
Gram staining and acid fast staining.
Learning Outcomes (Cont.)
Micro = small Scope = to view

A scientific instrument with one or more lenses
that allow you to observe small specimens which
is not visible to the naked eye.
It magnifies the image of the object to be
visualized through it.

Normally the laboratory microscopes provide a
magnification of 40X (scanner), 100X (low
power), 400X (high power) & 1000X (oil
immersion)
What is microscope?

What is microscope?

Different Types of Microscope
Types of microscope
Light / Optical
microscope
Bright-field microscope well
suited for viewing stained
specimens, eg. Stained blood
smears
Phase-contrast microscope
Epi-fluorescence microscope
Electron microscope
Transmission electron
microscope (TEM)
Scanning electron
microscope (SEM)
Optical microscope
(a) function through the optical theory of
lenses in order to magnify the image
generated by the passage of a wave
through the sample
(b) most simplest & most widely used
type of microscope
(c) typical magnification of a light
microscope is up to 1500x
Different Types of Microscope (Cont.)
Electron microscope
(a) uses electrons to illuminate a
specimen & create an enlarged image
(b) have much greater resolving power
than light microscopes
(c) can obtain much higher
magnifications (~ 2 millions X)

Variants of Electron microscope
(a) Scanning Electron Microscope (SEM)
- looks at the surface of bulk objects by
scanning the surface with a fine electron
beam & measuring reflection
(b) Transmission Electron Microscope
(TEM)
- passes electrons completely through
the sample, analogous to basic optical
microscopy


Different Types of Microscope
(Cont.)

Parts of Microscope (Cont.)
Ocular / eyepiece
- monocular or binocular
- the ocular / eyepiece are located at the top
of the microscope, are attached to a barrel or
tube connected to the microscope arm
- each ocular, through which the object is
viewed, contains a magnifying lens.
- Usual magnification = 10X

Objective lens
- underside of the microscope arm
contains a revolving nosepiece to
which the objectives are attached
- at least 3 objectives
(a) low power objectives:
magnifies 10X
(b) high power objectives:
magnifies 40X
(c) oil immersion objective:
magnifies 100X

To determine the degree of
magnification, the magnification listed on
the ocular (10X) usually is multiplied by
the magnification listed on the objective
being used.

Eg. Object viewed on 10X ocular & high
power 40X = would be magnified 400X

Total magnification = magnification of objective x magnification of eyepiece

Light source, condensor & diaphragm
- The microscope arm connects the
objectives & eyepiece to the microscope
base, which supports the microscope.
- The base also contains the light, which
illuminate the object reviewed. Located
above is the moveable condenser & iris
diaphragm.
- Condenser: focuses or directs the
available light into the objective as it is
raised or lowered & enhances specimen
contrast.

- Iris diaphragm: located in the condenser
unit, regulates the amount of light that
strikes the object being viewed. Iris
diaphragm can be adjusted by a movable
lever.

Coarse adjustment
- focus with low-power objective only

Fine adjustment
- give a sharper image after the object is
brought into view with the coarse adjustment.

The higher the magnification of the
objective, the shorter the working distance
will be.

Stage
- is supported by the arm & is located
between the nosepiece & the light
source

- serves as the support for the object
being viewed & has stage clip to keep
slides stationary

Light microscope
(a) Low power:
- examine small living animal & plant
cells
(b) High power:
- examine bacteria
- view drug particles / shape of the
crystals
Uses of Microscope
Electron microscope
(a) image, characterize & manipulate
material structures at exceedingly small
scales including features of atomic
proportions.
Uses of Microscope (Cont.)
1. Turn the revolving nosepiece to engage the
10X objective. Make sure that the revolving
nosepiece stops with an audible click.
2. Lower the stage using the coarse adjustment
& gently place a prepared slide on the stage in
the specimen holder clips. The specimen holder
clips are spring loaded & if forcibly
released may break the slide.

Procedures of using Microscope
3. Turn the mechanical stage controls to adjust
the position of the slides. Do not move the
stage manually without adjustment knobs.

Procedures of using Microscope (Cont.)
4. Switch the main switch to ON and adjust the
brightness with the light intensity knob.
5. Look through the eyepiece, turn the coarse
adjustment knob to bring the specimen into
focus. When you have optimized the focus
with the coarse adjustment controls,
use the fine adjustment knob
to improve the focus.

6. Start with the most light & gradually lessen it
until the specimen image has clear, sharp
contrast. Then, adjust the diaphragm to get
the best lighting.

7. Scan the slide (right to left & top to bottom) at
low power to get an overview of the specimen.
Then centre the part of the specimen you want
to view at higher power.

8. Engage the objective to be used for
observation by turning the revolving
nosepiece. Refocus by rotating the coarse
adjustment knob to reach the pre-focusing
position. This is limited by the engaged pre-
focusing lever. Make fine adjustments with the
fine adjustment knob. Always focus up,
moving the objective away from the slide.

9. Rotate the nosepiece to the 10X objective for
100X magnification. Refocus & view your
specimen carefully. Adjust the lighting again
until the image is most clear (you will need
more light for higher power). Repeat with the
40X objective for 400X magnification, which
will enable you to see all of the specimen detail
that is necessary for high school biology lab
work.


Handling of microscope
First clear your desk to receive the microscope, then grasp
its arm firmly, lift & support under the base with other
hand, set on a cleared desk.
Remove & store its dust over in cabinet under desk.
Unwrap power cord, loop once around gas outlet at rear of
desk, plug into electrical outlet in front of desk.
To carry a microscope
Use ONLY lens paper.
Polish the objectives & oculars.
Clean the lenses
Always begin slide set-up with the stage lowered & the lowest
power objective (4x) in place.

Handling of microscope (Cont.)
Focus initially only by LOWERING the stage to the
focal point using the coarse focus.
Make only minor changes in focus when
necessary with the fine focus knob.
If you totally lose focus, return to a lower
power objective to find the focal point.
Do not use the 100X objective unless you have
received specific instructions on its use.
Use only the fine focus with higher power
objectives.

Handling of microscope (Cont.)
Clean the lens
with lens paper
The act of introducing microorganism /
suspension of microorganisms (eg.
Bacteria) into a culture medium
The introduction of microorganisms into a
growth medium, to cause the growth &
multiplication of the microorganisms
What is inoculation?
Inoculum:
- microbes which are introduced into a
culture medium to initiate growth
- also known as inoculant

Culture:
- Microbes that grow & multiply on a
culture medium

Definition
= A nutrient material prepared for the
growth of microorganisms in a
laboratory

Depending the special needs of particular
bacteria, a large variety & types of
culture media have been developed with
different purposes & uses.

Culture medium
Culture media are employed in the
isolation & maintenance of pure
cultures of bacteria & are also used for
identification of bacteria according to
their biochemical & physiological
properties

Culture medium (Cont.)

Type of
media
Agar Broth

Solid medium
A complex polysaccharide derived from a
marine alga
Usually contained in test tubes or Petri
dishes
Liquid media are often mixed with agar &
poured into petri dishes to solidify.
Agar

Types of agar media
Types of agar media
Chemically defined media
Complex media
Selective media
Reducing media
Differential media
Enriched media

Types of agar media (Cont.)
Exact chemical composition is known
Chemoheterotroph & autotrophic (directly
use sources of energy - light to produce
organic substrates from inorganic CO
2
)
Contain organic growth factors that serve
as a source of carbon and energy

Eg. Glucose, Ammonium phosphate,
sodium chloride, magnesium
sulphate, potassium phosphate and
water is included in the medium for
growing Escherichia coli
Other: Neisseria, Lactobacillus

Complex media
Heterotrophic bacteria & fungi
Made up of nutrient including extracts from
yeasts, meat or plants / digests of proteins
Energy, carbon, nitrogen & sulphur
requirements of the growing
microorganisms are primarily provided by
protein

Complex - constituents
Peptone (partially digested protein)
Beef extract
Sodium chloride
Agar
Water

Complex media
If a complex medium is in liquid form, it is
called nutrient broth.
When agar is added, it is called nutrient agar.
Also known as undefined medium
Why? amino acid source contains a variety of
compounds with the exact composition being
unknown

Complex media
Nutrient agar

Selective media
Is used to suppress the growth of unwanted
bacteria & encourage the growth of the
desired microbes
Eg. Bismuth sulphite agar
- isolate the typhoid bacterium, gram-negative
Salmonella typhi from faeces
- inhibits gram-positive bacteria and most gram-
negative intestinal bacteria

Selective media
Eg. Sabourauds dextrose agar (SDA)
- pH 5.6
- isolate fungi that outgrow most
bacteria at this pH

Selective media
Sabourauds
dextrose agar

Reducing media
Anaerobic bacteria which might be killed
by exposure to oxygen
= Anaerobic Growth Media
Ingredients: sodium thioglycolate, which
chemically combine with dissolved oxygen
& deplete the oxygen in the culture
medium

Differential media
Distinguish colonies of the desired organism
from other colonies growing on the same plate
Eg. Blood Agar (contains red blood cells)
- identify bacterial species that destroy red
blood cells
- Streptococcus pyogenes : causes sore throat,
show a clear ring around their colonies - lysed

Differential
media
Staphylococcus aureus colonies on BA
- The bacteria have lysed the red blood cells,
causing the clear areas around the colonies

Differential
media
Staphylococcus epidermidis colonies on BA

Differential media
Eg. Eosin Methylene Blue (EMB) media
- differential for lactose and sucrose
fermentation
- inhibits the growth of Gram-positive bacteria
and provides a colour indicator distinguishing
between those organisms that ferment lactose
versus those that do not. Organisms which
ferment lactose display "nucleated colonies" --
colonies with dark centres

Differential
media
Escherichia coli on EMB
medium

Selective & Differential media
Sometimes, selective & differential characteristics are
combined in a single medium
Eg. MacConkey agar (MAC)
- selective and differential media used to differentiate
between Gram negative bacteria while inhibiting the growth
of Gram positive bacteria. The addition of bile salts and
crystal violet to the agar inhibits the growth of most Gram
positive bacteria, making MacConkey agar selective.
Lactose and neutral red are added to differentiate the
lactose fermenters, which form pink colonies, from lactose
nonfermenters that form clear colonies.

Mac Conkey (MAC)
agar

Mac Conkey agar
E. coli and Proteus on
MAC

Enriched
Bacteria present in small numbers can be missed,
especially if other bacteria are present in much larger
numbers
Liquid
Provides nutrients & environment conditions that
favour the growth of a particular microbe but not
others
Also a selective medium, but it is designed to
increase very small numbers of the desired type of
organism to detectable levels

Enriched
Isolate from a soil sample a microbe that
can grow on phenol & is present in much
smaller numbers than other species
If the soil sample is placed in a liquid
enrichment medium (phenol is the only
source of carbon & energy), microbes
unable to metabolize phenol will not grow

Type Purpose
Chemically defined Growth of chemoautotrophs and
photoautotrophs; microbiological assays
Complex Growth of most chemoheterotropic
organisms
Reducing Growth of obligate anaerobes
Selective Suppression of unwanted microbes;
encouraging desired microbes
Differential Differentiation of colonies of desired
microbes from others
Enrichment Similar to selective media but designed to
increase numbers of desired microbes to
detectable levels


Broth
Types of broth
Peptone water
Nutrient broth
Tryptic soy broth
Selenite-F broth

Types of broth media
Peptone water
Non-selective enrichment medium
Coagulase test
Can be used for fermentation
studies with various
carbohydrates

Types of broth media (Cont.)
Nutrient broth
Complex medium in liquid form
Contains nutrients, vitamins,
minerals and other growth factors
Suspension of microorganisms

Tryptic soy broth
Basic medium used for culturing many
kinds of microorganisms
Tryptic soy broth is used mostly to
generate a large supply of bacteria for
certain biochemical tests.
It can also be used in the determination of
bacterial numbers.

Tryptic soy broth

Selenite-F broth (SF)
For the isolation and cultivation of
Salmonella species from faeces
and other specimens.

Aseptic Techniques
Before inoculation with the desired
microorganisms, microbiological media &
all materials coming into contact with it
must be sterile.

During any subsequent handling of the
bacterial cultures, unwanted /
contaminant organisms must be
excluded employing aseptic techniques.
Aseptic Techniques (Cont.)
When handling specimens / cultures,
aseptic technique is important to avoid
their contamination & to protect the
worker from infection.

In-house training to demonstrate the skills
of aseptic technique should be given to
staff who will process specimens /
cultures.
Sterilization implies the complete
destruction of all microorganisms
including spores.
This is accomplished by the use of
(a) heat
(b) chemicals
(c) radiation
(d) filtration
The streak plate method works well
when the organism to be isolated is
present in large numbers relative to the
total population.

However, when the microbe to be isolated
is present only in very small numbers, its
numbers must be greatly increased by
selective enrichment before it can be
isolated with the streak plate method.

Inoculation of Culture Media
The object of any streaking pattern is the
continuous dilution of the inoculum to give
many well isolated colonies.

For multi-phase streaking it is crucial to
flame the loop before starting the next
phase. Note the slight overlap into the
previous phase to pick up a small
inoculum.


1. Flame the loop and wire &
streak a loopful of broth as at
A in the diagram.
2. Reflame the loop & cool it.
3. Streak as at B to spread
the original inoculum over
more of the agar.
4. Reflame the loop & cool it.

5. Streak as at C.
6. Reflame the loop
and cool it.
7. Streak as at D.
8. Label the plate and
incubate it inverted.


Most bacteria grow well at the
temperatures favoured by humans.
Mesophiles, with an optimum growth
temperature of 25 to 40C, are the most
common type of microbe.
The optimum temperature for many
pathogenic bacteria is about 37C, and
incubators for clinical cultures are usually
set at about this temperature.

After inoculation, the specimen should be
retained for at least 48 hours after the
laboratory has issued the final report.

Most positive cultures plates can be
discarded within 24 48 hours of issuing
a final authorized report.

In preparation of staining, a small sample
of microorganisms is placed on a slide &
permitted to air dry.
The smear is heat fixed by quickly
passing it over a flame.
Heat fixing kills the organisms, makes
them adhere to the slide & permits them
to accept the stain.
Smear

Bacteria have nearly the same refractive
index as water.
Therefore, when they are observed under a
microscope they are transparent or nearly
invisible to the naked eye.
Different types of staining methods are used
to make the cells & their internal structures
more visible under the light microscope.
The cells are then visible against a light
background

Principle of Staining
Stains
Basic stains
Cationic (positively
charged), react with
material that is
negatively charged.
Bacterial cell walls
have a slight
negative charge
attract & bind with
basic dyes
Eg.
Crystal
violet,
safranin,
basic
fuchsin,
methylen
e blue
Acidic stains
Negatively
charged
chromophores
& are repelled
by the leave
the microbe
transparent
Eg.
Nigrosin &
congo red
Stains
Simple stains use one dye that stains the
cell wall.

Simple Staining
Differential Staining
Differential
Stain
Gram stain Acid fast stain
Differential stains use two or more stains
& categorize cells into groups
4 different reagents are used & the results
are based on differences in the bacterial cell
wall.
Gram positive bacteria have a relatively
thick cell wall composed of a special
carbohydrate called peptidoglycan
Gram negative bacteria have a much
thinner cell wall composed of the same
carbohydrate, peptidoglycan, but with certain
chemical differences, eg. the presence of
lipopolysaccharides (LPS)

Gram Staining

Gram Staining (Cont.)
Procedures
1. A heat-fixed smear is covered with a basic
purple dye (crystal violet). Because the
purple stain imparts its colour to all cells, it is
referred to as a primary stain.

2. After 1 minute, the purple dye is washed off,
& the smear is covered with iodine, a
mordant (1 minute).
When the iodine is washed off, both Gram
positive & Gram negative bacteria appear dark
violet/purple.

3. Next, the slide is washed (3 10 sec) with
alcohol / alcohol-acetone solution
(decolourizing agent), which removes the
purple from the cells of some species but
not from others.

4. The alcohol is rinsed off, & the slide is
then stained with safranin (basic dye) for
1 minute.
The smear is washed again, blotted dry &
examined microscopically.




Gram staining results
- Indicate type of stain used, the reaction, &
the morphology of the cells observed
- Eg.
(a) Round (spherical), purple (or dark blue)
cells are reported as Gram positive
cocci (GPC)
(b) Rod-shaped, purple (or dark purple) cells
are reported as Gram positive bacilli
(GPB)

Gram positive cocci (GPC)
Gram positive bacilli (GPB)
Gram negative cocci (GNC)
Gram negative bacilli (GNB)
The standard abbreviations for the 4 types
of Gram stain & morphology are
= Ziehl-Neelsen staining
Mycobacteria have waxy coats on their cell
walls that prevent them taking in the dye
from the Gram staining procedure
Acid fast staining detergents are applied
which remove this waxy coat
Bacteria are stained hot Carbol-Fuchsin
(red dye which contains detergents) slide is
gently heated for several minutes All
bacteria are then stained red
Heating enhances penetration & retention
of the dye

Acid Fast Staining
The bacteria are washed with acid alcohol, a
de-colourizer, which removes the red stain
from bacteria that are not acid-fast.
The acid-fast microorganisms retain red
colour because the carbol-fuchsin is more
soluble in the cell wall lipids than in the acid-
alcohol.
Then stained with methylene blue (blue
dye). Those bacteria that retain the red dye
from the original stain are known as acid-fast
bacteria, all others go blue.

Acid Fast Staining (Cont.)



Acid fast bacilli
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