Group 4

1.Karimatul Aini
2.Sylvia Anggraeni
3.Retno Dwi P.
4.Nurvita Wahyu K.

s
t
i
n
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Metric

Light Properties

Wavelength

Polarity
Light is a wave

Filters can block waves in off axis planes

Waves can be
added
+

Light
Properties

Resolution

Wavelength/Resolution
Interaction

Light Properties

Reflection

Transmission

Light Properties

Absorption

Refraction
bending

Light Microscopy
Types
Compound
Bright Field

10

Oil immersion

Oil with intermediate

refractive index
With oil

without

Some info lost

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Microscopy Dark
Field

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Microscopy Phase
Contrast

Dual Beam

13

Phase contrast

Phase-contrast microscopy was invented in 1936 by Frits

Zernike, a Dutch mathematical physicist. It is based on the
principle that cells differ in refractive index (a factor by which
light is slowed as it passes through a material) from their
surroundings. Light passing through a cell thus differs in phase
from light passing through its surroundings. This subtle
difference is amplified by a device in the objective lens of the
phase-contrast microscope called the phase ring, resulting in a
dark image on a light background (Figure 2.5b). The ring
consists of a phase platethe key discovery of Zernikethat
amplifies the minute variation in phase. Zernikes discovery of
differences in contrast between cells and their background
stimulated other innovations in microscopy, such as
fluorescence and confocal microscopy (discussed below). For
his invention of phase-contrast microscopy, Zernike was
awarded the 1953 Nobel Prize in Physics.
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Microscopy DIC

Differential
Interference
Contrast

15

DIC differential
interference contrast
Similar to phase contrast, but input light is polarized

16

Microscopy
Fluorescence

Ultraviolet light

flourescein

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Advantages of
fluorescence
Can use specialized chemical probes that target specific features
and then tag with fluorescent dyes

Downside: must use expensive filters and excitory

frequencies
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Microscopy
Confocal

Confocal

Allows 3 dimensional viewing

Allows multiple dyes to be overlaid

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Confocal microscopy

Allows 3 dimensions

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Combined confocal and

fluorescence

Antibody labeling
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Microscopy Imaging

Digital

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Fig. 2-15

23

Electron Microscopy

Transmission
(TEM)

Scanning (SEM)

Scanning
Tunneling (STM)

24

TEM

Most popular for

bacteria. Allows imaging
internal features, but
requires heavy metal
staining.

25

Electron Microscopy
Images

26

Microscopy
Techniques

Wet Mounts
Smears
Staining

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Spread culture in thin

film over slide

Dry in air

I. Preparing a smear

Pass slide through

flame to heat fix

Flood slide with stain;

rinse and dry

II. Heat fixing and staining

100
Slide

III. Microscopy

Oil

Place drop of oil on

slide; examine with 100
objective lens

Staining cells
contrast

increases

Simple stain - one dye - shows size, shape,

and arrangement
Methylene blue -

yeast
Cheek cell

29

Common stains
Safranin (*basic, + charge)
red

Crystal violet

30

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Differential stains
Use multiple dyes or dyes that interact with organisms
differently.

Primary stain / counterstain

32

Gram Stain

Gram Stain The single

most important stain in
microbiology. Set the initial
taxonomy of bacteria.

Crystal violet *(basic stain)

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Gram Stain

35

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Acid Fast Stain

Carbol-fuchsin stains acid fast organisms

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Acid Fast

The Ziehl-Neelsen stain, also known as the acid-fast stain,

was first described by two German doctors; Franz Ziehl
(1859 to 1926), a bacteriologist and Friedrich Neelsen (1854
to 1894), a pathologist. It is a special bacteriological stain
used to identify acid-fast organisms, mainly Mycobacteria.
Mycobacterium tuberculosis is the most important of this
group, as it is responsible for the disease called tuberculosis
(TB). It is helpful in diagnosing Mycobacterium tuberculosis
since its lipid rich cell wall makes it resistant to Gram stain. It
can also be used to stain few other bacteria like Nocardia.
The reagents used are Ziehl-Neelsen carbolfuchsin, acid
alcohol and methylene blue.

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Acid Fast of
Mycobacterium
tuberculosis

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Negative Stain

India ink or nigrosin

Sometimes referred to as capsular stain

41

42

Flagellar Stain
Salmonella typhimurium

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Endospore Stain

Used on spore
forming bacteria
such as Bacillus
sp.
Malachite green
stains spores
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WASSALAMUALAIK
UM

THANK YOU

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