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
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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!!