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 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 H 2 S 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 1 st test..next week!!