ISOLATION OF MICROBES

Sidarningsih,drg.,M.Kes
 Inoculation

 is an attempt to grow microbes from one source (Sample) into

sterile growth media.

 Is introducing microorganisms into a medium where they can

grow and reproduce

 Culture. Microbes that grow together with the medium.

 Pure culture : if the microbe that grows is 1 type

 Mix culture : if microbes > 1 type

 Isolat is a pure culture of microbes that come from one type

 isolation is an attempt to get isolate.
 Isolation source → Inoculation → Mix Culture
→ Isolation → Isolate (pure culture)

 Mix culture Pure culture

CULTURING/ INOCULATION

On artificial media

The purpose of growing, breeding, storing microbes

Requirements for microbial growth :

suitable medium
pH
temperature
humidity
Oksigen + or no
METHODE OF BACTERIA INOCULATION ON
ARTIFICIAL MEDIUM

1. Do aseptically. Avoid the contamination

all materials and tools used must be sterile

medium must steril : otoklaf, filter,tyndalisasi

work is done near the fire

Ideally : in a Laminar flow
2. Materials and tools prepared for the inoculating process
Material:
Spiritus brander, loop (oese), micropippete (a) . Sterile
medium : solid (b), liquid(c), semi solid (d), culture / sample
a b c d
 STEP OF INOCULATING
 1. Materials taken as samples
 A. Specimens
› B. Culture

 2. Inoculating material on the media

 3. Incubation
 1. A. Specimen
as material for microbial examination

saliva Serum/blood Skin Urin

Crevikular plaque jar karies pus periodontal tissue

Dan sebagainya
 Swab
.

Methode of
sampling
speciment

Swab

Scraped
Aspiration
off
1. B. From culture :
a. Liquid culture
b. Solid culture
2. Inoculating material on the media .

From cultur to liquid media

 From solid culture – liquid medium
liquid culture – solid medium

Incubator

Streaked Plate Technique

Poured plate Technique
Spreader Technique
Incubation
Microorganisms should be cultured under conditions that best
support their growth including atmosphere, temperature, and
duration of incubation

 An incubator is a device used to grow and

maintain microbiological culturesThe incubator maintains optimal
temperature, humidity and other conditions such as the CO 2 and
oxygen content of the atmosphere inside

 Temperatur is setting in 37o C, 24,48 hour etc

 For anaerobic microba : put in anaerobic jar

anaerbic jar
 Microbial Isolation

The purpose is
to dilute out and separate microbe present to get isolated coloni
from a large number of a different microbes

Separated individual cells from large numbers of microbes and to

allow these cells to grow into pure cultures

Technique of Microbial Isolation :

A. Streaked Plate Technique

B. Poured plate Technique
C. Spread plate Technique
 Steps of Microbial Isolation
1. Take a sampel

Mucosa of the tongue 2.

2. Put
Put these
these
sampel in 3.Inoculated
(swab) liquid media in agar plate
medium
medium

Streak /
 Skin : swab Pour plate/
Spreading

Water

Air INCUBATION
Open the agar plate
Separate individual cell from largr number of microbe
ISOLATE ONE TYPE OF MICROBES

PURE CULTURE
 Inoculated in agar plate medium

1. STREAK METHODE
Streak the loop in a zig zag patern on the agar plate

take the inoculum

using loop(oese)

There are 4 types of methode used to streak a plate:

1. T Streak 2. Radiant Streak

Streak the inoculum on

3. Quadrant Streak 4. Continuous Streak
the agar media
With certain pattern
 Inoculated in agar plate medium

The way of Sreaking

T streak Continuous streak

 Radiant Streak Quadrant Streak

 Inoculated in agar plate medium

 Appearance of coloni after incubation

 T - Streak

Continuous streak Quadrant Streak

 Inoculated in agar plate medium

2. POUR PLATE METHODE

The bacterial culture and liquid agar medium are mixed together.

After mixing , the medium containing the culture poured into sterilized
petridishes , allowed solidifying and then incubated.

After incubation colonies appear on the surface.

 PROCEDURE OF POUR PLATE

Melt agar in waterbath, get a melted agar,

Transfer a singel loop of the inoculum to the melted agar

2
Appearance of pour plate coloni after incubation
 Inoculated in agar plate medium

3. SPREAD PLATE METHODE

In this technique, 0,1 ml of sample taken from diluted mixture of culture ,
which is placed on the surface of the agar plate and spread evenly over the surface by
using L spread glass rod called spreader.

Incubate the plates.

After incubation, colonies are observed on the agar place

 Appearance of spread plate coloni after incubation

Appearance of the colonies of air sample after incubation

Separate individual cell from large number of microbe
From the result of mix culture, select of
one desired colony , separate it from the mix

Inoculate it on the medium

PURE CULTURE
(ISOLAT)