Identification of The Unknown Bacterium

Aim
The objective of this project was to identify unknown
bacteria from the set of nine different bacteria. The
purpose was to conduct various biochemical tests in order
to obtain biological properties underpinning bacterial
identification.

Introduction
Based on bacteria cell wall structures, they can be
classified into two main groups; Gram positive and Gram
negative.
Gram positive bacteria have a thick multi-layered
peptidoglycan component of the cell wall on the other hand;
gram negative bacteria have a single layer of peptidoglycan
component surrounded by an outer membrane containing
lipopolysaccharide. Bacteria exhibits various shapes and
sizes
such
as
;coccus(spherical),different
cocci
arrangements (cluster ),bacillus(rod shape),a refractive
endospore within bacterium, peritrichous flagella.

Methodology
To identify the unknown bacteria a combination of
selective and differential media, gram staining and
selected biochemical tests were conducted (LSC-10036
Microbiology labs 2014-2015).
Oxidase Test (OX)
Bacteria can be classified into aerobic and anaerobic. A
deep violet compound results from the oxidation of
tetramethyl-p-phenylenediamine
dichydrochlorine
by
aerobic bacteria. This indicates a positive result.
Catalase Test (CAT)
Some bacteria are able to produce catalase. Addition of
3%v/v hydrogen peroxide to a bacterial slope culture,
results an immediate bubbles production and indicates
catalase positive bacteria.
LV Test
Gram positive bacteria can be differentiated based on their
ability to break down lecithovitellin. Around a halo like
opalescence produced by the positive bacteria colonies; egg
yolk due medium turbidity disappears.

Bacterial Motility Test (MOT)

Some bacteria can exhibits surface flagellar motors.
Bacterial surface flagella enable chemotaxis, the
movement towards nutrients and away from toxins. Motile
bacteria show a positive result.
Carbohydrate Fermentation Test
Bacterial sugar fermentation results in acids and /or gas
production.
Glucose (GLU)- Change of colour from purple to yellow
indicates an acid production and therefore a positive
result.
Lactose (LAC) - The presence of brownish/orange colour
indicates very little acid production and therefore a positive
result.
Sucrose (SUC) - The presence of deep purple colour
indicates a negative result.
Mannitol (MAN) Bubble production inside the Durham
tube indicates a gas formation and therefore a positive
result.
Gram Staining
Bacteria can be classified into; Gram positive and Gram
negative. Gram positive remains purple colour whereas
Gram negative bacteria change colour to pink. Staining
bacteria allows to microscopically determining bacterial
shape and size.

Results

Conclusion
The results obtained from various biochemical tests were
compared with different bacterial properties given in
appendix Table 11 (LSC-10036 Microbiology labs 20142015), part of which is presented below:

Eight out of nine properties were compatible with the

qualities of Bacillus cereus. Only catalase test was
unmatched and indicated that the unknown bacteria is
unable to produce catalase. On the contrary, in the appendix
table 11, it was stated that B. cereus has an ability to
produce enzyme in form of immediate enzyme-substrate
bubbles. According to Public Health England, the Oxidise
test should be conducted on colonies that are cultivated from
18 to 24hr. Organisms lose their catalase activity with age
and therefore a false negative result could be obtained
(Public Health England and National Health Service, 2014).

References
1. Greenwood, D., Barer, M., Slack, R., (2012). Medical
microbiology. . 18th ed. China: Churchill Livingstone
Elsevier.
2. 2. Public Health England and National Health Service
(2014). UK Standards for Microbiology Investigations.
Catalase Test. London : National Health Service.
3. Singleton, P., Saintsbury, D. (1978). Dictionary of
Microbiology. Bath: John Wiley & Sons.