Lecture PowerPoint to accompany

Talaro

Chapter 3
Tool of the Laboratory:
The Methods for
Studying Microbiology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Key characteristics of a reliable microscope are:

 Magnification – ability to enlarge objects

 Resolving power – ability to show detail

2
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

3
Insert figure 3.14
Student microscope

4
 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

5
6
Resolution defines the capacity to distinguish
or separate two adjacent objects – resolving
power
◦ function of wavelength of light that forms the
image along with characteristics of objectives

 Visible light wavelength is 400 nm – 750 nm.

 Numerical aperture 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

7
8
Insert figure 3.16
Wavelength on resolution

9
Insert figure 3.17
Oil immersion lens

10
 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

11
12
 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

13
 (A) E. coli - directly after staining

(B) E. coli - 4 months at 20°C

(C) G. stearothermophilus

Insert figure 3.21 (D) P. mirabilis

Fluorescent staining
(E) R. rubrum

(F) R. rubrum + SEM

(G) M. thermoautotrophicus

(H) Pyrococcus furiosus

Bar, 1 µm

14
Reinhard Wirth et al., 2010
 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

15
 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.

16
Insert figure 3.24b
Transmission micrograph

17
Insert figure 3.25
Scanning micrographs

18
 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.

19
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

20
 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

21
22
 Định danh Cấy/Nuôi cấy

Thu thập Ủ/

thông tin Nuôi ủ

Khảo sát hình thái

Phân lập
vi thể, đại thể 23
24
 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.

25
Insert figure 3.2
Isolation technique

26
 Isolation techniques include:

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

◦ pour plate technique (hộp đổ̉)

◦ spread plate technique (hộp trải)

27
Insert figure 3.3
Isolation methods

28
29
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

30
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

31
 Most commonly used media:

◦ nutrient broth – liquid medium containing

beef extract and peptone

◦ nutrient agar – solid media containing beef

extract, peptone and agar

32
 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

34
 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
35
36
37
 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

38
Insert figure 3.10
Differential media

39
https://www.asm.org/ASM-Agar-Art-Contest

40
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

41
Identification: macroscopic and microscopic
appearance, biochemical tests, genetic
characteristics, immunological testing 42
 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

44