General information of microbiology.ppt.pdf

Microbiology methods and types

1. Chapter 3
Tools of the
Laboratory:
The Methods for
Studying
Microorganisms

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display.

2. *
Subculture
INOCULATION
INCUBATION
ISOLATIONINSPECTION
INFORMATION
GATHERING
The sample is placed into a container of medium
that will support its growth. The medium may be in
solid or liquid form, and held in tubes, plates,
flasks, and even eggs. The delivery tool is usually a
loop, needle, swap or syringe.
Inoculated media are placed in a
controlled environment (incubator)
to promote growth. During the hours or
days
of this process, a culture develops as the
visible growth of the microbes in the
container of medium.
Some inoculation techniques can separate
microbes and spread them apart to create isolated
colonies that each contain a single type of microbe
This is invaluable for identifying the exact species
of microbes in the sample, and it paves the way for
making pure cultures.
Cultures are observed for the macroscopic
appearance of growth characteristics. Cultures
are examined under the microscope for basic
details such as cell type and shape. This may
be enhanced through staining and use of
special microscopes.
Additional tests for microbial function and
characteristics are usually required. This may
include inoculations into specialized media that
determine biochemical traits, immunological
testing, and genetic typing. Such tests will
provide specific information unique to a certain
microbe.
SPECIMEN
COLLECTION
One goal of these procedures is to
attach a name or identity to the
microbe,
usually to the level of species. Any
information gathered from inspection
and investigation can be useful.
Identification is accomplished through
the use of keys, charts, and computer
programs that analyze the data and
arrive at a final conclusion.
IDENTIFICATION
Staining
Streak plate
Keys
Blood bottle
Incubator
Bird
embryo
Biochemical
tests
Drug
sensitivity
Immunologic tests
DNA
analysis
Pure culture
of bacteria
Microbiologists begin by sampling the
object of their interest. It could be nearly
any thing or place on earth (or even Mars).
Very common sources are body fluids,
foods, water, soil, plants, and animals,
but even places like icebergs, volcanoes,
and rocks can be sampled.
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3. *
The Microscope
Key characteristics of a reliable microscope are:
•Magnification – ability to enlarge objects
•Resolving power – ability to show detail

4. *
Magnification in most microscopes results from an
interaction between visible light waves and the
curvature of a lens.



–The extent of
enlargement is the
magnification.

Magnification

5. *
Parts of the Microscope
Ocular
(eyepiece)
Interpupillary adjustment
Body
Arm
Coarse
adjustment knob
Fine focus
adjustment
knob
Stage adjustment knobs
Nosepiece
Objective lens (4)
Mechanical stage
Substage condenser
Aperture diaphragm control
Base with light source
Field diaphragm lever
Light intensity control
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© Leica Microsystems Inc.

6. *

–The objective lens
forms the magnified
real image

–The real image is
projected to the
ocular where it is
magnified again to
form the virtual
image
Magnification in Two Phases
Retina
Brain
Objective lens
Specimen
Ocular lens
Eye
Condenser lens
Light source
Virtual image
Formed
by ocular lens
Real image
formed
by objective
lens
Light
rays
strike
specimen
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7. *

•Total magnification
of the final image is
a product of the
separate magnifying
powers of the two
lenses

objective
power
ocular
power
total
magnification
x =
Total Magnification
Retina
Brain
Objective lens
Specimen
Ocular lens
Eye
Condenser lens
Light source
Virtual image
Formed
by ocular lens
Real image
formed
by objective
lens
Light
rays
strike
specimen
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8. *
The capacity to distinguish or separate two
adjacent objects and depends on
–The wavelength of light that forms the image
along with characteristics of the objectives


Resolution
(a) (b)
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9. *
Quantifying Resolution

•Visible light wavelength is 400 nm–750 nm
•Numerical aperture of lens ranges from 0.1 to 1.25
•Shorter wavelength and larger numerical aperture
will provide better resolution
•Oil immersion objectives resolution is 0.2 μm
•Magnification between 40X and 2000X

Resolving Power
(RP)
Wavelength of
light in nm
2 X Numerical aperture
of objective lens
=

10. *
The Purpose of Oil

Air Oil
Objective lens
Slide
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11. Magnification & Resolution
0.2 µm
1.0 µm
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12. Concept Check:
In order to get the best resolution possible with a
light microscope, you would want:

A. Long wavelengths of light
B. Short wavelengths of light
C. The wavelength doesn’t affect resolution

13. *
Variations on the Optical Microscope
•Bright-field – most widely used; specimen is
darker than surrounding field; used for live and
preserved stained specimens

14. *
Variations on the Optical Microscope

•Dark-field – brightly illuminated specimens
surrounded by dark field; used for live and unstained
specimens

15. *
Variations on the Optical Microscope

•Phase-contrast – transforms subtle changes in light
waves passing through the specimen into
differences in light intensity, best for observing
intracellular structures

16. *
Fluorescence Microscope
•Modified microscope
with an ultraviolet
radiation source and
filter.

•Uses dyes that emit
visible light when
bombarded with
shorter UV rays -
fluorescence

•Useful in diagnosing
infections
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© Invitrogen Corporation

17. *
Scanning Confocal Microscope
•Uses a laser beam of
light to scan the
specimen.

•Integrates images to
allow focus on
multiple depths or
planes.

18. *
Electron Microscopy
•Forms an image with a beam of electrons that can
be made to travel in wavelike patterns when
accelerated to high speeds

•Electron waves are 100,000 times shorter than the
waves of visible light

•Electrons have tremendous power to resolve
minute structures because resolving power is a
function of wavelength

•Magnification between 5,000X and 1,000,000X

19. Comparing Microscopes:
*
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Light Microscope
Lamp Electron gun
Electron
beam
Ocular lens
Viewing screen
Eye
Image
Transmission Electron
Microscope
(a) (b)
Condenser lens
Light rays
Specimen
Objective lens

20. cilia
MA
C
macro-
nucleus
pre cv
chambe
r
cv
fv13
pa
k
mic
pa
k
myoneme
pi
Courtesy Dr. Richard Allen
*
2 Types of Electron Microscopes
•Transmission electron
microscopes (TEM) –
transmit electrons
through the specimen.
Darker areas represent
thicker, denser parts
and lighter areas
indicate more
transparent, less dense
parts.

Viruses
(a)
Hazelton, PR and Gelderblom, HR. 2003. Electron microscopy for
Rapid diagnosis of Emerging Infectious Agents. Emerging Infectious
Diseases 9:294-303.
0.1
mm
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(b)

21. *
2 Types of Electron Microscopes

•Scanning electron
microscopes (SEM) –
provide detailed
three-dimensional
view. SEM bombards
surface of a whole,
metal-coated specimen
with electrons while
scanning back and
forth over it.

22. Concept Check:
What kind of microscope was used
to get this image of Rotavirus?

A. Brightfield
B. Phase Contrast
C. Scanning Electron
D. Transmission Electron

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Colony with zone
Of beta hemolysis
© Kathy Park Talaro

23. *
Specimen Preparation for
Optical Microscopes
•Wet mounts and hanging drop mounts –
allow examination of characteristics of live
cells: size, motility, shape, and arrangement

•Fixed mounts are made by drying and
heating a film of specimen. This smear is
stained using dyes to permit visualization of
cells or cell parts.

24. *
Staining

•Dyes are used to create
contrast by imparting
color

•Basic dyes – cationic,
positively charged
chromophore

•Positive staining –
surfaces of microbes are
negatively charged and
attract basic dyes

(a) Basic Dye
Cell envelope
Positive-Type Staining(a and b)
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25. *
Staining

•Acidic dyes – anionic,
negatively charged
chromophore

•Negative staining –
microbe repels dye, the
dye stains the
background
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Cell
envelope
Cell
envelope
Acidic
Dye
(b)
(c) Acidic
Dye
Negative Staining(c)

26. *
Staining
•Simple stains – one dye is used; reveals
shape, size, and arrangement

•Differential stains – use a primary stain and a
counterstain to distinguish cell types or parts
(examples: Gram stain, acid-fast stain, and
endospore stain)

•Structural stains – reveal certain cell parts not
revealed by conventional methods: capsule and
flagellar stains

27. Staining Examples
*
Methylene blue
stain of Corynebacterium
(1,0003)
(a) Simple Stains (b) Differential Stains
Gram stain
Purple cells are gram-positive.
Red cells are gram-negative
(1,0003).
Acid-fast stain
Red cells are acid-fast.
Blue cells are non-acid-fast
(7503).
Spore stain, showing spores (green)
and vegetative cells (red)
(1,0003)
(c) Structural Stains
India ink capsule stain of
Cryptococcus neoformans
(5003)
Flagellar stain of Proteus vulgaris.
Note the fine fringe of flagella
(1,5003).
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© Harold J. Benson
CD
C
© Jack Bostrack/Visuals Unlimited
© Steven R. Spilatro, Department of Biology,
Marietta College, Marietta, OH
© A.M. Siegelman/Visuals Unlimited
© Dr. Rita Colwell & Dr. Munirul Alam

28. *
The 6 I’s of Culturing Microbes
Inoculation – introduction of a sample into a
container of media to produce a culture of
observable growth
Isolation – separating one species from another
Incubation – under conditions that allow growth
Inspection
Information gathering
Identification

29. *
Isolation
•If an individual bacterial cell is separated from other
cells and has space on a nutrient surface, it will grow
into a mound of cells— a colony. A colony consists of
one species.

Mixture of cells in
sample
Microscopic
view
Cellular level
Macroscopic
view
Colony level
Incubatio
n
Parent
cells
Separation of
cells by
spreading
or dilution on
agar
medium
Microbes become
visible as isolated
colonies
containing
millions of cells.
Growth increases the
number of cells.
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30. *


–Streak plate
technique


–Pour plate
technique


–Spread plate
technique
Isolation Techniques
1
Steps in a Streak Plate; this one is a four-part or quadrant streak.
2 3 4 5
(a)
(c) Steps in Loop Dilution; also called a pour plate or serial dilution
21
"Hockey stick"
(e) Steps in a Spread Plate
21 3
21 3
Note: This method only works if the spreading
tool (usually an inoculating loop) is resterilized
(flamed) after each of steps 1–4.

Loop containing sample
Loop containing sample
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© Kathy Park Talaro
© Kathy Park Talaro
© Kathy Park Talaro
(b)
(d)
(f)

31. *
Inspection
•If a single species is growing in the container, you have
a pure culture but if there are multiple species than
you have a mixed culture.
•Check for contaminants (unknown or unwanted
microbes) in the culture.

32. *
Ways to Identify a Microbe:
•Cell and colony
morphology or
staining
characteristics
•DNA sequence
•Biochemical tests to
determine an
organism’s chemical
and metabolic
characteristics
•Immunological tests

Oxidase –
Acinetobacter
spp.*
Moraxella spp.
Oxidase +
Ferments maltose
Grows on
nutrient
agar
Reduces nitrite Does not reduce
nitrite
Does not grow
on
nutrient agar
Does not ferment
sucrose or lactose
Ferments sucrose;
does not ferment
lactose
Ferments lactose;
does not ferment
sucrose
Gram-negative
cocci and
coccobacilli
Neisseria
meningitidis
Neisseria
lactamica**
Neisseria sicca N. gonorrhoeae
Branhamella
catarrhalis
Does not ferment
maltose
Scheme for Differentating Gram-Negative Cocci and Coccobacilli
(a)
(b)

33. *
Media: Providing Nutrients in the
Laboratory
Media can be classified according to three properties:

1.Physical state – liquid, semisolid, and solid

2.Chemical composition – synthetic (chemically
defined) and complex

3.Functional type – general purpose, enriched,
selective, differential, anaerobic, transport, assay,
enumeration

34. *
Physical States of Media
Liquid – broth; does not
solidify

Semisolid – contains
solidifying agent

Solid – firm surface for
colony formation
–Contains solidifying
agent
–Liquefiable and
nonliquefiable

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© Kathy Park Talaro

35. *

–The most commonly used
solidifying agent

–Solid at room temperature,
liquefies at boiling (100
o
C),
does not re-solidify until it
cools to 42
o
C

–Provides framework to hold
moisture and nutrients

–Not digestible for most
microbes

Agar

36. *

–Nutrient broth – liquid medium containing
beef extract and peptone

–Nutrient agar – solid media containing
beef extract, peptone, and agar

Most Commonly Used Media

37. *
•Synthetic – contains pure organic and inorganic
compounds in an exact chemical formula

•Complex or nonsynthetic – contains at least
one ingredient that is not chemically definable

•General purpose media – grows a broad range
of microbes, usually nonsynthetic

•Enriched media – contains complex organic
substances such as blood, serum, hemoglobin, or
special growth factors required by fastidious
microbes
Chemical Content of Media

38. Examples of Enriched Media
*
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Colony with zone
Of beta hemolysis
© Kathy Park Talaro © Kathy Park Talaro

39. *
Selective
media: contains
one or more
agents that
inhibit growth of
some microbes
and encourage
growth of the
desired
microbes


Differential
media: allows
growth of several
types of microbes
and displays
visible differences
among those
microbes
Selective & Differential Media
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(a)
General-purpose
nonselective medium
(All species grow.)
Selective medium
(One species grows.)
Mixed sample
(b)
General-purpose
nondifferential medium
(All species have a similar
appearance.)

Differential medium
(All three species grow but may
show different reactions.)
Mixed sample

40. Concept Check:
CHROMagar contains several dyes and is used to
diagnose Urinary Tract Infections. The patient’s sample is
inoculated and based on the color of the colonies you can
identify the pathogen. CHROMagar is best described as:

A. Enriched
B. Selective
C. Differential
D. Complex
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© Kathy Park Talaro

41. *
Some media can be both
Selective & Differential

42. *
Miscellaneous Media
•Reducing medium –
contains a substance
that absorbs oxygen
or slows penetration
of oxygen into
medium; used for
growing anaerobic
bacteria

43. *
Miscellaneous Media

•Carbohydrate
fermentation medium
– contains sugars that
can be fermented,
converted to acids,
and a pH indicator to
show this reaction
Gas
bubble
Outline of
Durham
tube
Cloudines
s
indicating
growth
© Harold J. Benson
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