Identification of Mcroorg TEK BIO 2

IDENTIFICATION of MICROORGANISM

Nurhayati

Email:[email protected]

DEPARTMENT OF AGRICULTURAL PRODUCTS TECHNOLOGY

FACULTY OF AGRICULTURAL TECHNOLOGY

JEMBER UNIVERSITY

2014

2

Microbe Identification

• Identification measures include:

♣ Microscopy (staining)

♣ growth on enrichment, selective, differential or characteristic media

♣ specimen biochemical test (rapid test methods)

♣ immunological techniques

♣ molecular (genotypic) methods.

• After the microbe is identified for clinical samples it is used in susceptibility tests to find which method of control is most effective.

IDENTIFICATION

The identification of micro-organisms requires:

1. microscopic examination

2. cultural tests

3. (may also) serological tests.

Pure cultures are essential

Hifa ditumbuhkan pada gelas obyek yang mengandung

media agar dan ditutup dengan penutup gelas,selanjutnya

dapat diamatii secara mikroskopis untuk mengidentifikasi

morfologinya.

Slide culture for moulds identification

8

General vs Selective Mediafastidious

9

Differential Media

• Differential media grow several types of organisms and display visible differencesamong organisms.

• Differences may show up as colony size, media colour, gas bubble formation and precipitate formation.

1. MICROSCOPIC EXAMINATION

Untuk mengidentifikasi morfologi sel

Sel diamati dengan mikroskop tanpa maupun dengan pewarnaan

a. Methylene blue stain

b. Gram stain

c. Spore staining

etc. (Please find and explain it as task next lecture)

COLONY AND CULTURAL APPEARANCES

2. Cultural Tests

There are three approaches to the identification of bacteria:

a. Traditional

b. Kit

c. Automated

For physiological of microbe cells i.e fermentability test

?

Traditional cultural for anerobic culture

a. Anaerobic jars

b. Anaerobic cabinets

17

Biochemical Tests

• The microbe is cultured in a media with a special substrate and tested for an end product.

• Prominent biochemical tests include carbohydrate fermentation, acid or gas production and the hydrolysis of gelatin or starch.

• Many of these test in rapid miniaturized system that can detect for 23 characteristics in small cups called Rapid test.

• The info from the rapid test are input into a computer to help in identification of the organisms.

18

Carbohydrate Fermentation

. This medium show fermentation (acid production) and gas formation.

The small Durham tube for collecting gas bubbles.

Left- right:

Uninoculated negative control

Centre, positive for acid (yellow) and gas (open space).

Growth but no gas or acid.

19

Methyl Red Test

• This is a qualitative test for acid production.

• The bacteria is grown in MR-VP broth.

• After addition of several drops of methyl red solution a bright red colour is positive and yellow-orange negative.

20

Nitrate Reduction ♫After 24-48 hrs of incubation, nitrate reagents are added.

♫Left to right:♫Gas formation (positive

for nitrate reduction).

♫positive for nitrate reduction to nitrite ( red colour).

♫Negative control

21

Starch Hydrolysis

• After incubation on starch agar, plates are flooded with iodine solution.

• Positive test indicated by colourless areaaround growth.

• Negative test indicated below.

22

Catalase Test

• Place a drop of H2O2

on the culture.

• A positive reaction show gas bubbles.

• Often used to differentiate Streptococcus from Staphylococcus.

Kit for cultural test

a. BBL Crystal for negative/positive Gram bacteria

b. API (analytical profile index) for lactic acid bacteria

Phenotypic Identification of Cellulolitic Bacteria

Isolated from banana spontaneous fermentation

Fluorogenic and chromogenic indicators

API (analytical profile index) kit for lactic acid bacteria

Media for bacteria identification

• Endo agar: contain lactose, Na-sulphite, base-fuchsin.

• Briliant green agar:

• Mac conkey agar

• China-blue lactose agar

• Manitol salt agar

• Agar darah

3. SEROLOGICAL TEST

Menggunakan prinsip antigen-antibodi

- Aglutination test: antigen sebagai sel utuh, untuk membedakan

mikroorganisme yang erat kekerabatannya atau berbagai galur

dalam species. Suspensi mikroba dicampur dengan antiserum

pada gelas obyek dan diamati terbentuknya gumpalan setelah 2-

3 menit

Presipitation test: reaksi antara antigen terlarut dan antibodi

sehingga membentuk presipitat. Antiserum (konsentrasi sama)

dimasukkan ke dalam tabung berisi antigen (berbagai

konsentrasi). Selanjutnya diinkubasi kemudian disentrifuse. Jika

jumlah antigen-antibodi setara/optimal maka jumlah presipitat

lebih banyak.

Klasifikasikan dunia monera (mikroorganisme) .

32

1. Precipitation Reactions

• Precipitation (ppt) is the interaction of a soluble Ag with an soluble Ab to for an insoluble complex.

• The complex formed is an aggregate of Ag and Ab.

• Ppt rxns occurs maximally only when the optimal proportions of Ag and Ag are present.

• Ppt can also be done in agar referred to as immunodiffusion.

• Ppt test uses antibodies to detect for streptococcal group antigens.

33

Precipitation Reactions

34

2. Agglutination Reactions

• Agglutination (Aggn) is the visible clumping of an Ag when mixed with a specific Ab.

• Aggn tests are widely used because they are simple to perform, highly specific, inexpensive and rapid.

• Standardized tests are available for the determination of blood groups and identification of pathogens and their products.

35

Agglutination Reactions

36

Agglutination Reaction

PRESERVATION OF CULTURES

1. Drying cultures: ampole2. Freezing: media+glicerol 1%3. Freeze-drying: biakan manik-manik

OPTIMATION AND SCALE UP OF PRODUCTION in the bioprocess ?