-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
1/100
DNS 0303
Microbiology & Parasitology
Chapter 8
Introduction to Laboratory Technique
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
2/100
What is microscope?
Different types of microscope
Parts and uses of microscopes
Procedures of using microscope
Handling of microscope
11/10/2014DNS 0303 Microbiology & Parasitology2
Contents
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
3/100
What is inoculation?
Different types of Culture medium / Growth
medium
Aseptic techniques
Inoculation of culture media
Principle of stainingWhat is simple staining, Gram staining and
acid fast staining?
11/10/2014DNS 0303 Microbiology & Parasitology3
Contents (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
4/100
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. 11/10/2014DNS 0303 Microbiology & Parasitology4
Learning Outcomes
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
5/100
(e) Discuss the inoculation of culturemedia.
(f) Define, describe and give examples of
the different kinds of media.(g) Explain the uses of various types ofmedia.
(h) Explain the importance of aseptictechniques.
(i) Describe the principle of staining anddifferences between simple staining,
Gram staining and acid fast staining. 11/10/2014DNS 0303 Microbiology & Parasitology5
Learning Outcomes (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
6/100
Micro = small Scope= to view
A scientific instrument with one or more lensesthat allow you to observe small specimens which
is not visible to the naked eye.
It magnifies the image of the object to bevisualized through it.
Normally the laboratory microscopes provide amagnification of 40X (scanner), 100X (lowpower), 400X (high power) & 1000X (oilimmersion)
11/10/2014DNS 0303 Microbiology & Parasitology6
What is microscope?
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
7/100
11/10/2014DNS 0303 Microbiology & Parasitology7
What is microscope?
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
8/100
11/10/2014DNS 0303 Microbiology & Parasitology8
Different Types of Microscope
Types of microscope
Light / Opticalmicroscope
Bright-field microscopewellsuited for viewing stainedspecimens, eg. Stained blood
smears Phase-contrast microscope
Epi-fluorescence microscope
Electron microscope Transmission electron
microscope (TEM)
Scanning electronmicroscope (SEM)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
9/100
Optical microscope
(a) function through the optical theory oflenses in order to magnify the image
generated by the passage of a wavethroughthe sample
(b) most simplest & most widely usedtype of microscope
(c) typical magnificationof a lightmicroscope is up to 1500x
11/10/2014DNS 0303 Microbiology & Parasitology9
Different Types of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
10/100
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)
11/10/2014DNS 0303 Microbiology & Parasitology10
Different Types of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
11/100
Variants of Electron microscope
(a) Scanning Electron Microscope (SEM)
- looks at the surface of bulk objects by
scanning the surface with a fine electronbeam & measuring reflection
(b) Transmission Electron Microscope(TEM)
- passes electrons completely throughthe sample, analogous to basic optical
microscopy
11/10/2014DNS 0303 Microbiology & Parasitology11
Different Types of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
12/100
11/10/2014DNS 0303 Microbiology & Parasitology12
Different Types of Microscope
(Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
13/100
11/10/2014DNS 0303 Microbiology & Parasitology13
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
14/100
Ocular / eyepiece- monocularor binocular
- the ocular / eyepiece are located at the topof the microscope, are attached to a barrel ortube connected to the microscope arm- each ocular, through which the object isviewed, contains a magnifying lens.
- Usual magnification = 10X
11/10/2014DNS 0303 Microbiology & Parasitology14
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
15/100
Objective lens- underside of the microscope armcontains a revolving nosepiece towhich the objectives are attached
- at least 3 objectives(a) lowpower objectives:
magnifies 10X
(b) highpower objectives:
magnifies 40X(c) oil immersion objective:
magnifies 100X
11/10/2014DNS 0303 Microbiology & Parasitology15
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
16/100
To determine the degree of
magnification, the magnification listed on
the ocular (10X) usually is multipliedby
the magnificationlisted on the objectivebeing used.
Eg. Object viewed on 10X ocular & high
power 40X = would be magnified 400X11/10/2014DNS 0303 Microbiology & Parasitology16
Parts of Microscope (Cont.)
Total magnification = magnification of objective x magnification of eyepiece
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
17/100
Light source, condensor & diaphragm
- The microscope arm connects theobjectives& eyepieceto the microscope
base, which supports the microscope.- The base also contains the light, whichilluminatethe object reviewed. Locatedabove is the moveable condenser & iris
diaphragm.- Condenser: focuses or directstheavailable light into the objectiveas it israised or lowered & enhances specimen
contrast. 11/10/2014DNS 0303 Microbiology & Parasitology17
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
18/100
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
19/100
Coarse adjustment
- focus with low-power objective only
Fine adjustment- give a sharper image after the object isbrought into view with the coarse adjustment.
The higher the magnification of theobjective, the shorterthe working distancewill be.
11/10/2014DNS 0303 Microbiology & Parasitology19
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
20/100
Stage
- is supported by the arm & is located
between the nosepiece & the light
source
- serves as the supportfor the object
being viewed & has stage clip to keep
slides stationary
11/10/2014DNS 0303 Microbiology & Parasitology20
Parts of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
21/100
Light microscope
(a) Low power:
- examine small living animal & plant
cells
(b) High power:
- examine bacteria- view drug particles / shape of the
crystals
11/10/2014DNS 0303 Microbiology & Parasitology21
Uses of Microscope
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
22/100
Electron microscope
(a)image, characterize & manipulate
material structures at exceedingly small
scales including features of atomicproportions.
11/10/2014DNS 0303 Microbiology & Parasitology22
Uses of Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
23/100
1. Turn the revolving nosepiece to engage the10X objective. Make sure that the revolving
nosepiece stopswith an audible click.
2. Lowerthe stageusing 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.
11/10/2014DNS 0303 Microbiology & Parasitology23
Procedures of using Microscope
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
24/100
3. Turnthe mechanical stage controls to adjustthe positionof the slides. Do not move the
stage manually without adjustment knobs.
11/10/2014DNS 0303 Microbiology & Parasitology24
Procedures of using Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
25/100
4. Switchthe main switch to ONand adjustthebrightnesswith the light intensity knob.
5. Lookthrough the eyepiece, turnthe coarse
adjustment knob to bring the specimen intofocus. When you have optimized the focus
with the coarse adjustment controls,
use the fine adjustment knob
to improvethe focus.
11/10/2014DNS 0303 Microbiology & Parasitology25
Procedures of using Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
26/100
6. Start with the most light & gradually lessen ituntil the specimen imagehas clear, sharp
contrast. Then, adjustthe diaphragmto get
the best lighting.
7. Scanthe 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 wantto view at higher power.
11/10/2014DNS 0303 Microbiology & Parasitology26
Procedures of using Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
27/100
8. Engagethe objectiveto be used forobservation by turningthe revolving
nosepiece. Refocusby 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.
11/10/2014DNS 0303 Microbiology & Parasitology27
Procedures of using Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
28/100
9. Rotatethe nosepieceto the 10X objective for100X magnification. Refocus & view your
specimen carefully. Adjustthe lightingagain
until the image is most clear (you will need
more light for higher power). Repeatwith the
40X objective for 400X magnification, which
will enable you to see all of the specimen detail
that is necessary for high school biology labwork.
11/10/2014DNS 0303 Microbiology & Parasitology28
Procedures of using Microscope (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
29/100
11/10/2014DNS 0303 Microbiology & Parasitology29
Handling of microscope
First clear your desk to receive the microscope, then graspits arm firmly, lift & support under the base with otherhand, set on a cleared desk.
Remove & store its dust over in cabinet under desk.
Unwrap power cord, loop once around gas outlet at rear ofdesk, plug into electrical outlet in front of desk.
To carry a microscope
Use ONLY lens paper.
Polishthe objectives & oculars.
Clean the lenses
Always begin slide set-up with the stage lowered & the lowestpower objective (4x) in place.
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
30/100
11/10/2014DNS 0303 Microbiology & Parasitology30
Handling of microscope (Cont.)
Focus initially only by LOWERINGthe stage to thefocal point using the coarse focus.
Make only minor changesin focus whennecessary with the fine focus knob.
If you totally lose focus, returnto 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 powerobjectives.
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
31/100
11/10/2014DNS 0303 Microbiology & Parasitology31
Handling of microscope (Cont.)
Clean the lens
with lens paper
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
32/100
The act of introducing microorganism /suspensionof microorganisms (eg.Bacteria) into a culture medium
Theintroductionof microorganisms into a
growth medium, to cause the growth&multiplication of the microorganisms
11/10/2014DNS 0303 Microbiology & Parasitology32
What is inoculation?
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
33/100
Inoculum:
- microbeswhich are introducedinto a
culture medium to initiate growth
- also known as inoculant
Culture:
- Microbesthat grow& multiply on a
culture medium
11/10/2014DNS 0303 Microbiology & Parasitology33
Definition
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
34/100
= A nutrient material prepared for thegrowthof microorganisms in a
laboratory
Depending the special needs of particular
bacteria, a large variety& types of
culture mediahave been developed withdifferent purposes& uses.
11/10/2014DNS 0303 Microbiology & Parasitology34
Culture medium
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
35/100
Culture media are employed in theisolation&maintenanceof pure
culturesof bacteria & are also used for
identification of bacteria according totheir biochemical& physiological
properties
11/10/2014DNS 0303 Microbiology & Parasitology35
Culture medium (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
36/100
11/10/2014DNS 0303 Microbiology & Parasitology36
Culture medium (Cont.)
Type ofmedia
Agar Broth
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
37/100
11/10/2014DNS 0303 Microbiology & Parasitology37
Culture medium (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
38/100
Solidmedium
A complex polysaccharidederived from a
marine alga
Usually contained in test tubes or Petri
d ishes
Liquid media are often mixed with agar &
poured into petri dishes to solidify.
11/10/2014DNS 0303 Microbiology & Parasitology38
Agar
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
39/100
11/10/2014DNS 0303 Microbiology & Parasitology39
Types of agar media
Types of agar media
Chemically defined media
Complex media
Selective media
Reducing media
Differential media
Enriched media
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
40/100
11/10/2014DNS 0303 Microbiology & Parasitology40
Types of agar media (Cont.)
Chemically defined media
Exact chemical composition is known
Chemoheterotroph& autotrophic (directlyuse sources of energy - light to produceorganic substrates from inorganic CO2)
Contain organic growth factors that serve
as a source of carbonand energy
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
41/100
11/10/2014DNS 0303 Microbiology & Parasitology41
Types of agar media (Cont.)
Chemically defined media
Eg. Glucose, Ammonium phosphate,
sodium chloride, magnesiumsulphate, potassium phosphate andwater is included in the medium for
growing Escher ich ia co l i Other: Neisseria,Lactobaci l lus
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
42/100
11/10/2014DNS 0303 Microbiology & Parasitology42
Types of agar media (Cont.)
Complex media
Heterotrophicbacteria & fungi
Made up of nutrient including extracts fromyeasts, meat or plants / digests of proteins
Energy, carbon, nitrogen & sulphurrequirements of the growing
microorganisms are primarily provided byprotein
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
43/100
11/10/2014DNS 0303 Microbiology & Parasitology43
Types of agar media (Cont.)
Complex - constituents
Peptone (partially digested protein)
Beef extract
Sodium chloride
Agar
Water
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
44/100
11/10/2014DNS 0303 Microbiology & Parasitology44
Types of agar media (Cont.)
Complex media
If a complex medium is in liquid form, it iscalled 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 beingunknown
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
45/100
11/10/2014DNS 0303 Microbiology & Parasitology45
Types of agar media (Cont.)
Complex media Nutrient agar
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
46/100
11/10/2014DNS 0303 Microbiology & Parasitology46
Types of agar media (Cont.)
Selective media
Is used to suppress the growth of unwantedbacteria& encouragethe growth of the
desired microbes Eg. Bismuth sulphite agar
- isolate the typhoid bacterium, gram-negativeSalmonella typhi from faeces
- inhibits gram-positive bacteria and most gram-negative intestinal bacteria
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
47/100
11/10/2014DNS 0303 Microbiology & Parasitology47
Types of agar media (Cont.)
Selective media
Eg. Sabouraudsdextrose agar (SDA)
- pH 5.6
- isolate fungi that outgrow most
bacteria at this pH
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
48/100
11/10/2014DNS 0303 Microbiology & Parasitology48
Types of agar media (Cont.)
Selective media
Sabouraudsdextrose agar
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
49/100
11/10/2014DNS 0303 Microbiology & Parasitology49
Types of agar media (Cont.)
Reducing media
Anaerobic bacteria which might be killed
by exposureto oxygen = Anaerobic Growth Media
Ingredients: sodium thioglycolate, whichchemically combine with dissolved oxygen
& deplete the oxygen in the culturemedium
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
50/100
11/10/2014DNS 0303 Microbiology & Parasitology50
Types of agar media (Cont.)
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 redblood cells
- Streptococcus pyogenes : causes sore throat,show a clear ring around their colonies - lysed
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
51/100
11/10/2014DNS 0303 Microbiology & Parasitology51
Types of agar media (Cont.)
Differential
media
Staphyloco ccus aureus colonies on BA - The bacteria have lysed the red blood cells,
causing the clear areas around the colonies
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
52/100
11/10/2014DNS 0303 Microbiology & Parasitology52
Types of agar media (Cont.)
Differentialmedia
Staphylococcus epidermidis colonies on BA
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
53/100
11/10/2014DNS 0303 Microbiology & Parasitology53
Types of agar media (Cont.)
Differential media
Eg. Eosin Methylene Blue (EMB) media
- differential for lactoseand sucrosefermentation
- inhibits the growth of Gram-positive bacteriaand provides a colour indicator distinguishingbetween those organisms that ferment lactose
versus those that do not. Organisms whichferment lactose display "nucleated colonies" --colonies with dark centres
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
54/100
11/10/2014DNS 0303 Microbiology & Parasitology54
Types of agar media (Cont.)
Differential
media
Escherichia coli on EMBmedium
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
55/100
11/10/2014DNS 0303 Microbiology & Parasitology55
Types of agar media (Cont.)
Selective & Differential media
Sometimes, selective & differential characteristics arecombined in a single medium
Eg. MacConkey agar (MAC) - selective and differential media used to differentiate
between Gram negative bacteria while inhibiting the growthof Gram positivebacteria. The addition of bile salts andcrystal violet to the agar inhibits the growth of most Gram
positive bacteria, making MacConkey agar selective.Lactose and neutral red are added to differentiate thelactose fermenters, which form pink colonies, from lactosenonfermenters that form clear colonies.
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
56/100
11/10/2014DNS 0303 Microbiology & Parasitology56
Types of agar media (Cont.)
Selective & Differential media
Mac Conkey (MAC)agar
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
57/100
11/10/2014DNS 0303 Microbiology & Parasitology57
Types of agar media (Cont.)
Selective & Differential media
Mac Conkey agar
E. coli and Proteus on
MAC
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
58/100
11/10/2014DNS 0303 Microbiology & Parasitology58
Types of agar media (Cont.)
Enriched
Bacteria present in small numbers can be missed,especially if other bacteria are present in much larger
numbers Liquid
Provides nutrients& environment conditions thatfavour thegrowthof a particular microbe but notothers
Also a selective medium, but it is designed toincrease very small numbers of the desired type oforganism to detectable levels
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
59/100
11/10/2014DNS 0303 Microbiology & Parasitology59
Types of agar media (Cont.)
Enriched
Isolate from a soil sample a microbe that
can grow on phenol & is present in muchsmaller numbers than other species
If the soil sample is placed in a liquidenrichment medium(phenol is the only
source of carbon & energy), microbesunable to metabolize phenol will not grow
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
60/100
11/10/2014DNS 0303 Microbiology & Parasitology60
Types of agar media (Cont.)
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
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
61/100
11/10/2014DNS 0303 Microbiology & Parasitology61
Types of agar media (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
62/100
11/10/2014DNS 0303 Microbiology & Parasitology62
Broth
Types of broth
Peptone water
Nutrient broth
Tryptic soy broth
Selenite-F broth
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
63/100
11/10/2014DNS 0303 Microbiology & Parasitology63
Types of broth media
Peptone water
Non-selective enrichment medium
Coagulase test
Can be used for fermentation
studies with variouscarbohydrates
f (C )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
64/100
11/10/2014DNS 0303 Microbiology & Parasitology64
Types of broth media (Cont.)
Nutrient broth
Complex medium in liquid form Contains nutrients, vitamins,
minerals and other growth factors
Suspension of microorganisms
T f b h di (C )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
65/100
11/10/2014DNS 0303 Microbiology & Parasitology65
Types of broth media (Cont.)
Tryptic soy broth
Basic medium used for culturing many
kinds of microorganisms Tryptic soy broth is used mostly togenerate a large supply of bacteria forcertain biochemical tests.
It can also be used in the determination ofbacterial numbers.
T f b th di (C t )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
66/100
Tryptic soy broth
11/10/2014DNS 0303 Microbiology & Parasitology66
Types of broth media (Cont.)
T f b th di (C t )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
67/100
11/10/2014DNS 0303 Microbiology & Parasitology67
Types of broth media (Cont.)
Selenite-F broth (SF)
For the isolation and cultivation ofSalmonellaspecies from faecesand other specimens.
A ti T h i
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
68/100
11/10/2014DNS 0303 Microbiology & Parasitology68
Aseptic Techniques
A ti T h i (C t )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
69/100
Before inoculation with the desiredmicroorganisms, microbiological media &all materials coming into contact with itmust be sterile.
During any subsequent handling of thebacterial cultures, unwanted /
contaminant organisms must beexcluded employing aseptic techniques.
11/10/2014DNS 0303 Microbiology & Parasitology69
Aseptic Techniques (Cont.)
A ti T h i (C t )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
70/100
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 tostaff who will process specimens /
cultures.
11/10/2014DNS 0303 Microbiology & Parasitology70
Aseptic Techniques (Cont.)
A ti T h i (C t )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
71/100
Sterilizationimplies the completedestruction of all microorganisms
including spores.
This is accomplished by the use of(a) heat
(b) chemicals
(c) radiation
(d) filtration
11/10/2014DNS 0303 Microbiology & Parasitology71
Aseptic Techniques (Cont.)
I l ti f C lt M di
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
72/100
The streak plate method works wellwhen the organism to be isolated ispresent in large numbers relative to thetotal population.
However, when the microbe to be isolatedis present only in very small numbers, its
numbers must be greatly increasedbyselective enrichment before it can beisolated with the streak plate method.
11/10/2014DNS 0303 Microbiology & Parasitology72
Inoculation of Culture Media
I l ti f C lt M di
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
73/100
The object of any streaking pattern is thecontinuous dilution of the inoculum to give
many well isolated colonies.
For multi-phase streaking it is crucial to
flamethe loopbefore starting the next
phase. Note the slight overlap into theprevious phase to pick up a small
inoculum.
11/10/2014DNS 0303 Microbiology & Parasitology73
Inoculation of Culture Media
I l ti f C lt M di
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
74/100
11/10/2014DNS 0303 Microbiology & Parasitology74
Inoculation of Culture Media
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 spreadthe original inoculum over
more of the agar.
4. Reflame the loop & cool it.
I l ti f C lt M di
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
75/100
11/10/2014DNS 0303 Microbiology & Parasitology75
Inoculation of Culture Media
5. Streak as at C.
6. Reflame the loopand cool it.
7. Streak as at D.
8. Label the plate andincubate it inverted.
I l ti f C lt M di
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
76/100
11/10/2014DNS 0303 Microbiology & Parasitology76
Inoculation of Culture Media
Inoc lation of C lt re Media
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
77/100
11/10/2014DNS 0303 Microbiology & Parasitology77
Inoculation of Culture Media
Inoculation of Culture Media
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
78/100
Most bacteria grow well at thetemperatures favoured by humans.
Mesophiles, with an optimum growth
temperature of 25 to 40C, are the mostcommon type of microbe.
The optimumtemperature for manypathogenic bacteria is about 37C, and
incubators for clinical cultures are usuallyset at about this temperature.
11/10/2014DNS 0303 Microbiology & Parasitology78
Inoculation of Culture Media
Inoculation of Culture Media
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
79/100
After inoculation, the specimen should beretained for at least 48 hours after the
laboratory has issued the final report.
Most positive cultures plates can be
discarded within 2448 hours of issuing
a final authorized report.
11/10/2014DNS 0303 Microbiology & Parasitology79
Inoculation of Culture Media
Smear
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
80/100
In preparation of staining, a small sampleof microorganisms is placed on aslide &
permitted to air dry.
The smear is heat fixed by quicklypassing it over a flame.
Heat fixing killsthe organisms,makes
them adhereto the slide& permitsthemto accept the stain.
11/10/2014DNS 0303 Microbiology & Parasitology80
Smear
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
81/100
11/10/2014DNS 0303 Microbiology & Parasitology81
Principle of Staining
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
82/100
Bacteria have nearly the same refractiveindex as water.
Therefore, when they are observed under amicroscope they are transparent or nearly
invisible to the naked eye.
Different types of staining methods are usedto make the cells & their internal structures
more visibleunder the light microscope. The cells are then visible against a light
background
11/10/2014DNS 0303 Microbiology & Parasitology82
Principle of Staining
Stains
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
83/100
Stains
Basic stains
Cationic(positivelycharged), react with
material that isnegatively charged.Bacterial cell walls
have a slightnegative chargeattract & bind with
basic dyes
Eg.Crystalviolet,
safranin,basic
fuchsin,methylene blue
Acidic stains
Negativelycharged
chromophores& are repelledby the leavethe microbetransparent
Eg.Nigrosin &
congo red
11/10/2014DNS 0303 Microbiology & Parasitology83
Stains
Simple Staining
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
84/100
Simple stains use one dye that stains thecell wall.
11/10/2014DNS 0303 Microbiology & Parasitology84
Simple Staining
Differential Staining
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
85/100
11/10/2014DNS 0303 Microbiology & Parasitology85
Differential Staining
Differential
Stain
Gram stain Acid fast stain
Differential stains use two or more stains& categorize cells into groups
Gram Staining
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
86/100
4 different reagents are used & the resultsare based on differences in the bacterial cellwall.
Gram positive bacteria have a relatively
thick cell wall composed of a specialcarbohydrate called peptidoglycan
Gram negative bacteria have a much
thinner cell wall composed of the samecarbohydrate, peptidoglycan, but with certainchemical differences, eg. the presence oflipopolysaccharides (LPS)
11/10/2014DNS 0303 Microbiology & Parasitology86
Gram Staining
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
87/100
11/10/2014DNS 0303 Microbiology & Parasitology87
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
88/100
Procedures1. A heat-fixed smear is covered with a basic
purple dye (crystal violet). Because thepurple 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 darkviolet/purple.
11/10/2014DNS 0303 Microbiology & Parasitology88
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
89/100
3. Next, the slide is washed (3 10 sec) withalcohol / alcohol-acetone solution(decolourizing agent), which removes thepurple from the cells of some species but
not from others.
4. The alcohol is rinsed off, & the slide isthen stained with safranin(basic dye) for
1 minute.The smear is washed again, blotted dry &examined microscopically.
11/10/2014DNS 0303 Microbiology & Parasitology89
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
90/100
11/10/2014DNS 0303 Microbiology & Parasitology90
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
91/100
11/10/2014DNS 0303 Microbiology & Parasitology91
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
92/100
11/10/2014DNS 0303 Microbiology & Parasitology92
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
93/100
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)11/10/2014DNS 0303 Microbiology & Parasitology93
Gram Staining (Cont.)
Gram Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
94/100
Gram positive cocci (GPC)
Gram positive bacilli (GPB)
Gram negative cocci (GNC)
Gram negative bacilli (GNB)
The standard abbreviations for the 4 typesof Gram stain & morphologyare
11/10/2014DNS 0303 Microbiology & Parasitology94
Gram Staining (Cont.)
Acid Fast Staining
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
95/100
= Ziehl-Neelsen staining Mycobacteria have waxy coats on their cell
walls that preventthem taking in the dyefrom the Gram staining procedure
Acid fast stainingdetergents are appliedwhich remove this waxy coat
Bacteria are stained hot Carbol-Fuchsin(red dye which contains detergents)slide is
gently heated for several minutesAllbacteria are then stained red
Heatingenhances penetration & retentionof the dye
11/10/2014DNS 0303 Microbiology & Parasitology95
Acid Fast Staining
Acid Fast Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
96/100
The bacteria are washed with acid alcohol, ade-colourizer, which removesthe red stainfrom bacteria that are not acid-fast.
The acid-fast microorganisms retain red
colour because the carbol-fuchsin is moresoluble in the cell wall lipids than in the acid-alcohol.
Then stained with methylene blue (bluedye). Those bacteria that retain the red dyefrom the original stain are known as acid-fastbacteria, all others go blue.
11/10/2014DNS 0303 Microbiology & Parasitology96
Acid Fast Staining (Cont.)
Acid Fast Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
97/100
11/10/2014DNS 0303 Microbiology & Parasitology97
Acid Fast Staining (Cont.)
Acid Fast Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
98/100
11/10/2014DNS 0303 Microbiology & Parasitology98
Acid Fast Staining (Cont.)
Acid Fast Staining (Cont )
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
99/100
Acid fast bacilli
11/10/2014DNS 0303 Microbiology & Parasitology99
Acid Fast Staining (Cont.)
-
8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx
100/100
Next lesson:
Revision