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)

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

The microscope is an instrument most widelyused and most useful in microscopylaboratories. With this tool acquiredmagnification making it possible to see theorganisms and structures appears to the nakedeye. Memungkan microscope magnification in awide-range of a hundred to hundreds ofthousands of times.

Electron microscope uses electrons insteadof bundles of light waves to acquirebayanagn enlarged. Type-type ofmicroscope is used to study, for theprocedures in diagnostic microbiologylaboratories, and for other special purposes.

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.

The focus achieved by using a lens system, berlawannawith moderate Leeuwenhoek microscope, which usesonly a single lens. Its there on the condenser lens,objective and ocular (eye lens). Condenser lenscentering cone of light on the field specimens. Most ofthe files in the light of this light cone directly penetratesthe objective lens to form a light background or lightfield. File a PhD in Straits light objects(mikroosganisme) on the specimen to be bent in focusby the objective lens to form a shadow objects arrive.Bayanagn is on view by lens okuler. so that the systemprovides the initial magnification of the objective lens,then the zoom lens system further by okuler.

• 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

• 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

◦ 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-

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

◦ 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

Fotomograf Treponema pallidum for the dark field, the cause of syphilis note the spiral shape of each organism mostThe shape of syphilis

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.

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.

Electron microscopeElectron 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 (UI)

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

Mikroskop Cahaya Dan Mikroskop Elektron

Many of the techniques developed for mikoorganismeexamination 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)

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

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.

Figure 1.2 The difference in MO and SEM

Figure 1.3 places the sample in the scanning

electron microscope

The first is an organism in a liquid suspension.

The second use or spread a thin layer of dried specimens,

at fixation, and stained

Wet mount or hanging drop preparations allowingexamination of living organisms tersupensi the flow ofsubstances.

Wet preparations obtained by placing a drop of the flowof substances containing organisms on the glass objectand close it with a very thin glass lid Glass said.

To reduce the rate of evaporation and negate thedroplets in the air stream with a circled "Petroleumjelly" or similar material so that the object glass andglass lid pressed glass rapat.Tersedia special objectwith concave regions into preparations for the hangingdrops.

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

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)

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

Defferential colouring

• Colouring technique which show the differential between

microbe cell or part of the cell is called by differential

colouring.

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

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.

TABLE. GRAM COLORING

SOLUTION USES AND

SEQUENCELooks 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

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.

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.

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.

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.

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.

METHODS OF PURE CULTURE

Scratch Grail Scatter GrailPour the

cup

Enrichment culture

Isolation of single cells

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.

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.

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.

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 .

Characteristic method of microorganism