UNIVERSITI SAINS MALAYSIA

JIB 221 MICROBIOLOGY

LAB PRACTICAL 4: BACTERIA ISOLATION AND BIOCHEMICAL IDENTIFICATION

TECHNIQUES (DEMONSTRATION)

Name: .............................. Matric number: .............................. Group:

OBJECTIVE:

• Observe the demonstration and describe the applications of identification techniques

using morphologial and biochemical tests.

INTRODUCTION:

Isolation of microorganisms: The isolation of bacteria and fungi from natural materials
depends on their use of appropriate medium and incubation method. The usual procedure is
to streak materials on nutrients and collect the isolated colonies for further observation.
When there is more than one organism, different colonies are produced making it easier
to identify using a medium supplemented with a indicator material ie the medium that
contains material that can change as a result of the growth of microorganisms. If there is one
organism whose number exceeds another, then, the differential medium or the enrichment
medium should be used to promote the growth of the species.
Bacteria Identification Techniques: Once the pure culture of a bacterium has been isolated,
several tests are performed for its identification. In this context, several identification systems
have been introduced.
Primary exam: Among the tests performed were colony examination for features
such as size, shape, color, etc., as well as bacterial morphological features such as shape
and cell arrangement, Gram staining, acid fastness, the presence of spores, motility and
etc. Other biochemical tests such as oxidase, catalase, sugar fermentation tests, indole test,
citrate tests and other tests.

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Secondary test: Secondary tests are performed after the primary test. By performing some
specific tests certain organisms can be classified into genus level. For identification to the
species level, more tests are required.
Additional Testing: The complete identification of some species is the work of an expert.
Additional information of characteristics such as physiological, antibiotic resistance patterns,
phage typing and others are required.

4a. BACTERIA ISOLATION TECHNIQUES

The effect of environmental conditions on bacterial
growth

Growth of microorganisms on different growth mediums.

E.coli on brilliant green agar
E.coli and Ps. aeruginosa on MacConkey agar

Temperature effect
Inoculate 2 nutrient agar medium with E.coli and Candida albicans. Test one plate at each of
the following temperatures: 37oC, 55oC and 40oC. Observe the plate after 3 days.

4b. BIOCHEMICAL IDENTIFICATION TECHNIQUES

Oxidase test
This test is performed on aerobic bacteria: it separates the bacteria that have
cytochrome c (positive oxidase) to those that do not have cytochrome c (negative oxidase).
Some bacterial assemblages in particular enteric organisms (eg E.coli) are oxidase negative
while other groups (such as Pseudomonas spp) are oxidase positive.
Wet a piece of filter paper with oxidase reagent (l% tetramethyl-phenylenediamine)
in water with 0.l % ascorbic acid to slow down the auto-oxidation). Using a
platinum wire / glass rod, spread a bit of bacteria off the plate (E.coli / Pseudomonas
aeruginosa) onto reagent-added filter paper. Positive response shown
with the production of purple colour in 10 seconds.

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Catalase test
Most aerobic bacteria produce hydrogen peroxide as a result of their metabolism. It is toxic
to cells and is degraded by the enzyme catalase as soon as it is formed. A small number of
aerobic bacteria do not produce catalase but is relatively insensitive to hydrogen peroxide.
Obligate anaerobes do not produce catalase but hydrogen peroxide is not produced in the
anaerobic metabolism. Emulsify one full loop of Pseudomonas spp with one loopful of
hydrogen peroxide onto the slide. Bubbles released are caused by the production of
free oxygen as a gas bubble indicates the presence of catalase in the tested culture.

Indole Test
This test depends on the indole production from the amino acids tryptophan. So this test
is best done in a medium rich in tryptone. This test is important for distinguish between
specific coliform bacteria. Inoculate a tryptone tube with E.coli (from young culture) and
incubate it in optimal growth temperature for 2-7 days. Add 0.5 ml of Kovac indole reagent,
shake the tube slowly and put it aside. Dark red color is produced by the presence of indole
that separates in the alcohol layer.

Citrate Test
The ability to use citrate as a major carbon source is used to distinguish the family
Enterobacteriaceae. Simmons citrate agar is usually used. It is a medium that uses a
bromothymol blue as markers / indicators. Positive growth (use of citrate) results in an
alkaline reaction and changes the medium color from green to light blue. Inoculate the
Simmons citrate agar with E.coli. Incubate the tube at 37oC for 2-5 days and notice the
colour change in the medium.

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