Chapter 3

Tool of the Laboratory: The

Methods for Studying
Microbiology
Key characteristics of a reliable microscope are:

 Magnification – ability to enlarge objects

 Resolving power – ability to show detail

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Magnification in most microscopes results
from interaction between visible light
waves and curvature of the lens.
◦ angle of light passing through convex
surface of glass changes – refraction
◦ Depending on the size and curvature of
the lens, the image appears enlarged.
◦ extent of enlargement - magnification

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Insert figure 3.14
Student microscope

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 Magnification occurs in two phases –
◦ 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.
 Total magnification of the final image is a
product of the separate magnifying powers of
the two lenses.
power of objective X power of ocular = total
magnification

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Resolution defines the capacity to distinguish
or separate two adjacent objects – resolving
power (năng suất phân ly)
◦ function of wavelength of light that forms the
image along with characteristics of objectives

 Visible light wavelength is 400 nm – 750 nm.

 Numerical aperture (khẩu độ số) of lens ranges from
0.1 to 1.25.
 Oil immersion lens requires the use of oil to prevent
refractive loss of light.
 Shorter wavelength and larger numerical aperture will
provide better resolution.
 Oil immersion objectives resolution is 0.2 μm.
 Magnification between 40X and 2000X

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Insert figure 3.16
Wavelength on resolution

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Insert figure 3.17
Oil immersion lens

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 Bright-field – most widely used; specimen is
darker than surrounding field; live and
preserved stained specimens
 Dark-field – brightly illuminated specimens
surrounded by dark field; live and unstained
specimens
 Phase-contrast – transforms subtle changes in
light waves passing through the specimen into
differences in light intensity, best for
observing intracellular structures

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 Modified compound microscope with an
ultraviolet radiation source and a filter that
protects the viewer’s eye

 Uses dyes that emit visible light when

bombarded with shorter UV rays - fluorescence

 Useful in diagnosing infections

 Chùm tia tử ngoại chiếu vào tiêu bản đã nhuộm

màu bằng các chất huỳnh quang. Trong tế bào,
các cấu trúc khác nhau sẽ phát quang với màu
sắc khác nhau. 13
 (A) E. coli - directly after staining

(B) E. coli - 4 months at 20°C

(C) Geobacillus stearothermophilus

Insert figure 3.21 (D) Proteus mirabilis

Fluorescent staining
(E) Rhodospirillum rubrum

(F) R. rubrum + SEM

(G) Methanothermobacter

thermoautotrophicus

(H) Pyrococcus furiosus

Bar, 1 µm
Reinhard Wirth et al., 2010 14
 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

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 Transmission electron microscopes (TEM) –
transmit electrons through the specimen.
Darker areas represent thicker, denser parts
and lighter areas indicate more transparent,
less dense parts.
 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.

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Insert figure 3.24b
Transmission micrograph

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Insert figure 3.25
Scanning micrographs

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 Wet mounts and hanging drop mounts – allow
examination of characteristics of live cells:
motility, shape, and arrangement
(Giọt ép - buồng treo)

 Fixed mounts (Cố́ định) are made by drying and

heating a film of specimen. This smear is stained
using dyes to permit visualization of cells or cell
parts.

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Dyes create contrast by imparting a color to
cells or cell parts.
 Basic dyes - cationic, with positive
charges on the chromophore
 Acidic dyes - anionic, with negative
charges on the chromophore
 Positive staining – surfaces of microbes
are negatively charged and attract basic
dyes
 Negative staining – microbe repels dye,
the dye stains the background

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 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)
 Special stains – reveal certain cell parts not
revealed by conventional methods: capsule and
flagellar stains

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 Định danh Cấy/Nuôi cấy

Thu thập Ủ/

thông tin Nuôi ủ

Khảo sát hình thái

Phân lập
vi thể, đại thể 23
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▪ 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.

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Insert figure 3.2
Isolation technique

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▪ Isolation techniques include:

◦ streak plate technique (cấy ria – vk/nấm men)

◦ pour plate technique (hộp đổ̉)

◦ spread plate technique (hộp trải)

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Insert figure 3.3
Isolation methods

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Liquid – broth; does not solidify

Semisolid – clot-like consistency; contains

solidifying agent (agar 0.3-0.5 % or gelatin)
➔ For motility, hydrogen sulfide production, indole
reaction tests

Solid – firm surface for colony formation

◦ contains solidifying agent agar 1.5-2%
◦ liquefiable and nonliquefiable

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Most commonly used solidifying agent is agar
(a complex polysaccharide isolated from red algae)

◦ solid at room temp, liquefies at boiling (100oC), does not

resolidify until it cools to 42oC

◦ provides framework to hold moisture and nutrients

◦ not digestible for most microbes

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▪ Most commonly used media:

◦ nutrient broth – liquid medium containing

beef extract and peptone

◦ nutrient agar – solid media containing beef

extract, peptone and agar

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❑ Synthetic (MT tổng hợp) – contains pure organic
and inorganic compounds in an exact chemical
formula

❑ Complex/Natural or Non-synthetic (MT tự nhiên)

– contains at least one ingredient that is not
chemically definable

PGA, Cao thịt-pepton,… 33

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❖ 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

❖ 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 desired and undesired microbes
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 Reducing medium – contains a substance
that absorbs oxygen or slows penetration of
oxygen into medium; used for growing
anaerobic bacteria

 Carbohydrate fermentation medium –

contains sugars that can be fermented,
converted to acids, and a pH indicator to
show the reaction; basis for identifying
bacteria and fungi

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 Transport media are used to maintain and preserve
specimens for a period of time before clinical analysis or
to sustain delicate species that die rapidly if not held
under stable conditions. Stuart’s and Amie’s transport
media contain buffers and absorbants to prevent cell
destruction but will not support growth.
 Assay media are used by technologists to test the
effectiveness of antimicrobial drugs, disinfectants,
antiseptics, cosmetics and preservatives on the growth
of microorganisms.
 Enumeration media are used by industrial and
environmental microbiologists to count the numbers of
organisms in milk, water, food, soil, and other samples.
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Insert figure 3.10
Differential media

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https://www.asm.org/ASM-Agar-Art-Contest

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Incubation – temperature-controlled chamber
at appropriate temperature and atmosphere
◦ microbe multiplies and produces macroscopically
observable growth
Inspection – observation; macroscopic and
microscopic
◦ pure culture – grows only single known species of
microorganisms
◦ mixed cultures – hold two or more identified
species or microorganisms
◦ contaminated culture – once pure or mixed
culture that has unwanted microbes growing

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Identification: macroscopic and microscopic
appearance, biochemical tests, genetic
characteristics, immunological testing 43
 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.
 Provide specific information unique to a
certain microbe
Potentially hazardous cultures and
specimens are usually disposed of
in two ways:
◦ steam sterilization

◦ incineration

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