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CHAPTER 3

Content
purification of cultures
morphological and pureculture studies biochemical tests

Purification of cultures
The only way to purified culture are by getting a pure

culture. Have a control procedure in collecting specimen, preparing media, microbiological tecniques, staining and reagents and equipment used.

Collecting specimen
Sterile procedure using a correct media for

transportation and stock. Main source for bacteria should be microbial food. Which are?? The transport media used to preserve and ensure the viability of bacteria during the transportation period Besides also provide a particular temperature for the viability of bacteria Crucial for cerebrospinal fluid, blood culture and fecal specimens, etc.

Preparation of media
The media should be packed well to prevent from

leakage and breaks, protected from moisture and sunlight and excessive heat The expiry date should be noted and the instruction of storage should be followed The mix bacterial colonies should be sub cultured until the culture are purified the bacterial colony characteristic should only derive from a single colony

Pure colony

Morphological and pure culture studies

In nutrient agar

In MacConkey agar

In MacConkey agar

In Blood agar

3 types of hemolysis

Biochemical tests
Catalase test
Oxidase test Coagulase test

Sugar fermentation test


MRVP test Indole test Citrate test Motility test H2S test

Catalase test
Produce bubble just

after attaching the bacteria to the reagent To differentiate staphylococci and streptococci

Oxidase test
Have 2 methods:Filter paper/Sterile swab
To help identify Vibrio, Neisseria, Pasteurella and

Pseudomonas sp. Oxidase enzymes oxydize phenylenediamine. Deep purple colour on reagent paper

Oxidase test

Coagulase test
To identify S.aureus The enzyme coagulase

clots plasma Tube : fibrin clot Slide: clumping of bacterial cells

Sugar fermentation test


Glucose test Maltose test

Sucrose test
Lactose test Some will appear with

gas production

Voges-Proskauer test
To differentiate

enterobacteria Organism ferments glucose with acetoin production. Acetoin is oxidised to diacetyl which reacts with creatine Brick red colour develop slowly

Methyl Red test


To differentiate

enterobacteria Org. ferment glucose, producing sufficient acidity in buffered medium to give a colour change of indicator Brick red medium

Indole test
Using Kovac reagent. To differentiate Gram

negative rods, especially E.coli Tryptophan is broken down wt the release of indole which reacts wt dimethylaminobenzalde hyde Reddening of strip or medium

Indole test using other reagent

Citrate test
To differentiate

enterobacteria from other bacteria Org uses citrate as its only source of carbon, producing an alkaline reaction wt colour change indicator Blue and turbid medium

Motility test

Please do revision for your 1st test..next week!!