Based on phenotype

Study of diversity of microorganisms and

their relationships

Includes:
clasification (ordering of B. into groups)
nomenclature
identification

Morfological (cels including staining)

Cultural
Physiological
Nutritional
Biochemical
Ecological
Genetic
Antigenic
Phage susceptibility

As many charactes as posible should be

studied

describtion of new species

Number of tests applied should be kept to

a minimum

concetration for simple methods and for

characters with very good distinguishing value

tests must be standardized

1.
2.
3.
4.

Pure culture (make sure to have)

Examine Gram-stained cells
Look for motility or flagela
Examine colonies

5.Test for oxygen requirements

6.Test for dissimilation of glucose
(oxidative/fermentative)
7.Select tests useful for distinguishing
between the genera
8.Select the test apllicable for species
identification

Determination of shape
Gram reaction
Motlity

Oxidase test
Catalase test
Carbohydrate breakdown
O-F of glucose
Acid formation from sugars
Gas formation from glucose
ONPG test (beta-galactosidase activity)

Reduction of nitrate to nitrite

Indole test
VP test (acetoin production)
Arginin dihydrolase test

( Sulphide Indole Motility medium) -combination differential

medium
tests three different parameters-sulphur reduction, indole
production and motility
This media has a very soft consistency that allows motile
bacteria to migrate readily through them causing cloudiness.
In soft agar tubes non-motile bacteria will only grow on the
inoculated region. Motile bacteria will grow along the stab line
and will tend to swim out away from the stabbed area.
Therefore, a negative result is represented by growth in a
distinct zone directly along the stab. A positive result is indicated
by diffuse or cloudy growth mainly at the top and bottom of the
stabbed region.

TheIMViCtests are a group of individual

tests used in microbiology lab testing to
identify an organism in thecoliform group
A coliform is agram-, aerobic or
facultative anaerobic rod which produces
gas fromlactose within 48 hours.
The presence of some coliforms indicate
fecal contamination.

these IMViC tests are useful for differentiating the

familyEnterobacteriaceae, especially when used alongside
theUrease test.
The IMViC results of some important species are shown
below.

Positive

VogesProskauer
Negative

Negative

Negative

Positive

Negative

Negative

Negative

Positive

Negative

Positive

Negative

Negative

Positive

Positive

Positive

Positive

Negative

Negative

Negative

Positive

Negative

Positive

Negative

Negative

Positive

Positive

Species

Indole

Methyl Red

Escherichia c
oli
Shigella
spp.[2]
Salmonellas
pp.
Klebsiellasp
p.
Proteus
vulgaris[3]
Proteus
mirabilis
Enterobacte
r aerogenes

Positive

Citrate

contains general nutrients and 5% sheep blood

It is useful for cultivating fastidious organisms /determining the hemolytic

capabilities of an organism.
Some bacteria produce exoenzymes that lyse red blood cells and degrade
hemoglobin; these are called hemolysins.
Bacteria produce different types of hemolysins. Beta-hemolysin breaks
down the red blood cells and hemoglobin completely.
This leaves a clear zone around the bacterial growth --hemolysis (beta
hemolysis).
Alpha-hemolysin partially breaks down the red blood cells and leaves a
greenish color behind. This is referred to as -hemolysis (alpha
hemolysis)/caused by the presence of biliverdin, which is a by-product of the
breakdown of hemoglobin.
If the organism does not produce hemolysins and does not break down the
blood cells, no clearing will occur. This is called -hemolysis (gamma
hemolysis).
The hemolysins produced by streptococci perform better in an anaerobic
environment.
Because of this, it is standard procedure to streak a blood plate and then
stab the loop into the agar to provide an area of lower oxygen concentration
where the streptolysins can more effectively break down the blood cells.

Examplesofthebloodculture
patternscreatedby(fromleft)
alpha-,beta-andgammahemolyticstreptococci.

Thisstreptococcuspyogenes
exhibits-hemolysis.

Given a mixed broth culture containing

Staphylococcus aureus, Bacillus subtilis,
Salmonella typhi and Shigella
dysenteriae, describe a systematic
approach to separate and confirm the
identity of each of these bacteria.
(15 marks)

Twenty students were hospitalized after

taking breakfast from a school canteen. A
sample of drink from the canteen was sent
to a microbiologist to determine the cause
of the problem. The only information
available is that the sample contains gram
positive cocci bacteria. As a microbiologist
describe a systematic approach to
determine the concentration of bacteria in
the sample and the identity of the bacteria.
(15 marks)

There are three (3) different types of

bacterial colonies growing on a nutrient
agar plate of which one of it is Salmonella
thyphimurium. Discuss other laboratory
tests needed to confirm this bacterial
species.
(12 marks)