The document discusses various microscopy techniques used in microbiology laboratories, including: - Bright field microscopy, which produces up to 1000x magnification - Dark field microscopy, used to view organisms like Treponema pallidum that cause syphilis - Fluorescent microscopy, which uses fluorescent dyes to stain specimens - Phase contrast microscopy and electron microscopy, which provide higher magnifications - Methods for staining, culturing, and isolating pure cultures of microorganisms are also described.

1. METHOD IN
MICROBIOLOGY
Group 1
1. UTAMI INDRIASTUTI ( A1D011006)
2. WIDIA GUSTINA ( A1D011029)
3. PANI ASWIN ( A1D011024)
4. FEBRANDI EKANANDA ( A1D011030)
5. SITI KURNIAWATI ( A1D011026)
6. TIARA NINDIA TRISNA ( A1D011027)

2. INTRODUCTION
 Microbiology is the science that is determined by
the techniques than the learned subject. Many
techniques amount, and used all kinds of laboratory
equipment to implement.
 Much progress has been made in equipment for
laboratories for microbiology since the early 1900s.
Instruments such as the present can identify a very
detailed chemical composition of a microbial cell,
and also compounds, chemical compounds
produced by a cell

3. The microscope is an instrument most widely
used and most useful in microscopy
laboratories. With this tool acquired
magnification making it possible to see the
organisms and structures appears to the naked
eye. Memungkan microscope magnification in a
wide-range of a hundred to hundreds of
thousands of times.

5. Electron microscope uses electrons instead
of bundles of light waves to acquire
bayanagn enlarged. Type-type of
microscope is used to study, for the
procedures in diagnostic microbiology
laboratories, and for other special purposes.

6. 1. The bright field microscopy
In bright field microscopy dangan illuminated
lit up the objects that are in the study appear
to be darker than the background. In general,
this kind of microscope produces a maximum
useful magnification of about 1,000 diameters.

8. The focus achieved by using a lens system, berlawanna
with moderate Leeuwenhoek microscope, which uses
only a single lens. Its there on the condenser lens,
objective and ocular (eye lens). Condenser lens
centering cone of light on the field specimens. Most of
the files in the light of this light cone directly penetrates
the objective lens to form a light background or light
field. File a PhD in Straits light objects
(mikroosganisme) on the specimen to be bent in focus
by the objective lens to form a shadow objects arrive.
Bayanagn is on view by lens okuler. so that the system
provides the initial magnification of the objective lens,
then the zoom lens system further by okuler.

9. • The microscope is commonly used in
microbiology usually equip with 3 objectives in
each providing different degrees of
magnification.
objective
nomination
magnification
of the
objective
magnification
okuler
total
magnification
low power 10 10 100
high power 40 10 400
oil immersion 100 10 1000

10. • Separation of power is the ability of an
objective to separate the two points are very
close together in the structure of the object.
This separation of power is determined by the
wavelength of light and the numerical aperture
(numerical aperture, NA) lens.
Split power formula:
Power split = wave length of light
NA kondensor+ NA objective

11. ◦ Dark field microscope used to look like
spirochaete Treponema (syphilis
causes) Leptospira (leptospirosis). This microscope
has a condenser which prevents the light
transmitted through the material, but instead
causes the material to reflect light at certain angles,
so that objects look bigger shines with a dark
background.
◦ Dark field microscopy obtained from the same kind
of microscope that is used for bright field
microscopy except that the device was equipped
with a dark field kodensor and a low NA objective
air-

12. Figure 2.5 With withstand partial beams
of light that enters the condenser, just
skip the dark field ring light beam on the
object slid into the specimen so that the
object (microorganisms) be lit in the
microscopic field that should be dark

13. ◦ Fluorescent microscopy (Fluorescence Microscopy):
a fluorescent microscope and the procedure has
become widely used in clinical laboratories.
◦ Some biological substance is basically fluorescent,
but other materials can also be colored with
fluorescent dyes and observed with a microscope
using ultraviolet light source
◦ This microscope is widely used in microbiology and
immunology

14. Fotomograf Treponema pallidum for the
dark field, the cause of syphilis note the
spiral shape of each
organism most
The shape of syphilis

16.  Laboratorisnya ways work can be carried out
quickly. For example, if a patient has a wound
and in fear that the injury caused by the
syphilis bacterium substance can flow from
the wound examined microscopically with a
technique called fluorescent antibody.

17.  Phase contrast microscopy is a type of light
microscopy that allows a greater contrast
between substances with different thickness or
the refractive index range. Phase contrast
microscopy (Phase-contras Microscopy): This
microscope is rarely used in diagnostic
laboratories.
 Phase contrast microscope is a type of
microscope that allows light occurs greater
contrast in the refractive index and density of the
fluid between the cells of living organisms which
are, resulting in a better picture contrast than
that seen in the bright field microscope.

18. Electron microscope
Electron microscopy provide useful magnification
is much larger than that may be obtained with
light microscopy. This is made possible by the
greater separation power obtained for beams of
electrons are used for magnification possessed a
very short wavelength compared with the light.
Electron microscopy contained
in the University of Indonesia
Electron beam used in electron
microscopy has wavelengths
ranging from 0.005 to 0.0003
nm, very short when compared
to the wavelength of visible light
used in light microscopy

19. Mikroskop Cahaya Dan Mikroskop Elektron

20. Many of the techniques developed for mikoorganisme
examination by electron microscopy. Among these are
the methods of the new staining, the method of slicing
the microbial cells, into thin slices for microscopic
examination and radioactive techniques. All of these
procedures applied to transmission electron microscopic
(MET)

21. observation with Scanning Electron Microscope
OM-4x magnification 1000x
SEM 10x-3000000x
application:
• Observing the structure and shape of the surface of the finer scale
• Equipped With EDS (Electron Dispersive X-ray Spectroscopy)
• Can detect unsur2 in the material.
• The surface must be conductive electrons observed

22. Advantages of SEM to OM
Depth of Field Resolution magnification OM-
1000x15.5mm 4x-10x 0.19mm ~ 0.2mm SEM
3000000x4mm-0.4mm1-10nm depthoffield SEM has a
large, which can focus more number of samples at a time
and produce a good image of the sample three
dimensions.
SEM also produces high-resolution image, which
means approaching shadow that can be tested
with high magnification
Main Application SEM
•Topography •Morphology
•Composition •Crystallographic Information
When SEM is used, the electron-optical column and
sample chamber must always be under vacuum.

23. Figure 1.2 The difference in MO and SEM

24. Figure 1.3 places the sample in the scanning
electron microscope

26. The first is an organism in a liquid suspension.
The second use or spread a thin layer of dried specimens,
at fixation, and stained

27.  Wet mount or hanging drop preparations allowing
examination of living organisms tersupensi the flow of
substances.
 Wet preparations obtained by placing a drop of the flow
of substances containing organisms on the glass object
and close it with a very thin glass lid Glass said.
 To reduce the rate of evaporation and negate the
droplets in the air stream with a circled "Petroleum
jelly" or similar material so that the object glass and
glass lid pressed glass rapat.Tersedia special object
with concave regions into preparations for the hanging
drops.

29. Have developed procedures for staining procedures:
 Observing with good looks rough morphology of
microorganisms
 Identify the parts of the cell structural parts
microorganisms
 Help identify and / or distinguish similar organisms

30. The main steps in preparing specimens for the
microbes in the paint for microscopic examination
are:
1. Placement smear, or a thin layer of the specimen on
a slide
2. Fixation smear it on glass objects, usually by
heating, causing the microorganisms attached to the
object glass
3. Application of single staining (simple staining) or
serankaian dye or reagent solution (differential
staining)

31.  The color on the bodies of other microorganisms
bacteria using a single solution of a dye on a thin
single layer or smear that has been called the fixation
on simple staining. Flood the last layer in the dye
solution for a certain period, then the solution was
washed with water and dry with a glass object in
blotter. Usually it tewarnai cells evenly.
 Dyes used for coloring alkolin generally simple. With
a simple staining can determine the shape and range
of bacterial cells. Alkaline dyes commonly used for
simple staining is methylene blue, crystal violet, and
carbolic fuehsin

32. Defferential colouring
• Colouring technique which show the differential between
microbe cell or part of the cell is called by differential
colouring.

33. GRAM COLORING
 In this process the fixed smear of bacteria subjected to the
following solutions in the order listed: crystal violet, iodine
solution, alcohol (bleaching lye), and safranin dye or some
other appropriate counter.
 Gram method is divided into two groups:
1. gram-positive bacteria
2. gram-negative bacteria

34. CONTINUED ...
 Gram-positive bacteria, retain crystal violet dye and
therefore looks dark purple.
 Gram-negative bacteria, purple crystals lost when
washed with alcohol, and when given a match with
red dye safranin, was colored red.
 Why gram staining procedure bacteria purple
coloring and a few others into the red? This seems
to be due to differences in the chemical structure of
the surface.

35. TABLE. GRAM COLORING
SOLUTION USES AND
SEQUENCE
Looks of REACTION AND BACTERIA
Gram Positive Gram Negative
1. Purple Crystals (UK) Purple cells Purple cells
2. Iodine Solution (Y) UK-Y complex is formed in the
cells; cell remains purple
UK-Y complex is formed in the
cells; cell remains purple
3. Alcohol Cell wall of dehydrated, shrink
pores; Power permeable cell
wall and membrane decreases,
UK-Y not get out of the cell, the
cell remains purple
Extracting lipids of the cell wall, the
pore expands, UK-Y complexes out
of the cell; cells become colorless
4. Safranin Cells unaffected, still purple Cells absorb the dye, red

36. PURE CULTURE TECHNIQUES
 Microbial populations in the natural world around us
large and complex.
 Decent research on microorganisms in different
habitats requires a technique for separating
complex mixtures of this population, or a mixture of
cultures, species become different as a pure
culture.
 Pure culture consists of a population of cells that
are all derived from a single stem cell.

37. CULTURING AND ISOLATION OF PURE
CULTURES
 Microorganisms are cultured in the laboratory on
nutrient material called a medium.
 Materials are inoculated on the medium
called inoculum.
 With inoculating nutrient agar medium with scratch
plate method or pour plate method, the cells will
separate on their own. After incubation, individual
microbial cells will multiply so quickly form colonies.
 More direct method to isolate a single
microorganism is by using manipulatormikro tool
called kuarmikro (microscopic probes) to move one
cell from the cell suspension flow substances.

38. SOME TECHNIQUES FOR ISOLATING PURE
CULTURES
 The cup is scratch: inoculum streaked on nutrient
agar surface, into a petri dish using the needle
move (inoculation loop).
 Grail stocking: A drop of inoculum placed in the
middle of the nutrient agar medium in a petri dish,
using a sterile glass rod bent inoculum spread on
the surface of the medium.

39. CONTINUED ...
 Pour the cup: the inoculation loop (suspension
origin) move into a tube, rolled between the hands
to mix. Perform removal of tube A to B, from B to C.
The contents of each tube was poured into petri
dishes separately. After incubation colonies were
isolated check.
 Enrichment cultures: Scratch, scatter, or cast by
inoculum through transfer to a medium with a
composition that promotes the growth of
undesirable microorganisms.

40. CONTINUED ...
 Isolation of single cells: With the help of
manipulatormikro, can be used kuarmikro
(microprobe) to take a microorganism of the
suspension flow of substances containing cells with
a microscope, examining preparations. Then the
single cell was transferred to a sterile medium.

41. METHODS OF PURE CULTURE
Scratch Grail Scatter Grail
Pour the
cup
Enrichment
culture
Isolation of
single cells

42. CONSERVANCY AND PIKLING BREEDING
NATURE
 After a microorganisem can be isolated inside
breeding nature, so we require to coservancy
breeding it condition on life for long time. Really
more then microbiology laboratorium be
canservancy collection breeding nature, it often
called collection breeding suply.

43. The American Typr Culture
Collection, at Wanshington D.C.
The American Typr
Culture Collection
in Washington
D.C. collect
thusands species
of
microorganisme,
including virus.

44.  Many procedure used for pickling and conservancy
breeding organism
 The conservancy activity at short time, pickle can
save on refrigretor with temperature 0 until 10
celcius. And then, for saving with long time, must
used nitrogen liquid on temperature -196 C. Or we
can also used didehidration metode insed tube,
freezering and to be closed by cover. This proses is
name liofilisation.

45. Characteristic method of
microorganism
 If already get breeding a small organism, so we
must do check everything need of it. Each
laboratorium test can given notive of
microorganism .

46. Characteristic method of microorganism