MIC 500: FOOD MICROBIOLOGY

LABORATORY REPORT

EXPERIMENT 4: FOOD BACTERIA – GRAM NEGATIVE ROD SHAPED

BACTERIA

NAME : ERNIE NAJWA NAJIHAH BINTI FAIDI

MATRIC NUMBER : 2017283562

GROUP : AS2463C

GROUP MEMBERS :

1. NOOR ALIAH BINTI BAHARI (2017405672)

2. NURUL FATINI BINTI MOHAMAD HALIMIN
(2017405588)
3. NUR NABILAH BINTI JOHARI (2017405596)

LECTURER’S NAME : DR. KHALILAH BINTI ABDUL KHALIL

DATE OF EXPERIMENT : 19TH APRIL 2018

DATE OF SUBMISSION : 26TH APRIL 2018

INTRODUCTION

The gram – negative bacterial cell is bacteria covered with a three layered coat consisting of
an inner membrane adjacent to the cell cytoplasm, a peptidoglycan or murein layer
encompassing the inner membrane, and an outer membrane layer enclosing the cell. Weidel
and Pelzer (152) coined the word murein in 1964 to describe the wall – like properties of the
bacterial peptidoglycan. It is analogous to protein and nuclein and does not have any
biochemical connotations, but rather refers to the functional aspects of the cell wall. The
surface of a rod-shaped bacterial cell is described as a cylindrical side wall capped by two
hemispherical poles. During the early part of the division cycle, when cells are not
invaginate, the cells elongate and grow only in the cylindrical wall area. In the later part of
the division cycle, cells invaginate and produce two new poles in the middle of the parental
cell.

OBJECTIVES

1. To identify the characteristics of gram negative rod shaped bacteria.

2. To expose students to food spoilage characteristics causing by gram negative rod
shaped bacteria.

MATERIALS AND APPARATUS

1. Cultures provided:
a) E. coli
b) Pseudomonas aeruginosa
c) Serratia marcescens
d) Proteus vulgaris
e) Klebsiella aerogenes
f) Shigella spp

2. Media : Triple Sugar Iron Agar (TSI), Urea Broth, Litmus Milk, Sugar Broths :
lactose, sucrose, maltose, glucose
3. Gram stain reagents
4. Cavity slide
5. Sample of spoiled foods (inoculated with gram negative rod bacteria)
METHODS

1. Macroscopic examination (Table 4.1)

On Nutrient Agar (NA) and MacConkey Agar Plate, the cultural characteristics of the
given cultures were studied.

2. Examine the characteristics of gram negative rod shaped bacteria

a) Motility test (Table 4.2)
A very small drops of log phase broth culture of bacteria was placed at the centre
of a cover glass using sterile inoculating loop of 2mm diameter. Then, a small
drops or water was placed on the corners of the cover glass that was next being
inverted over the cavity of the slide, so that the drop of culture is hanging at the
centre of the cavity slide. The hanging drop of the bacterial culture was then
observed under the microscope for the motility of the bacteria. Darting or zig –
zag motility indicates that the bacteria may have poor flagellation while slow
motility or vibratory motility indicates the peritrichous flagellation.

b) Biochemistry test (table 4.3)

Each organism was inoculated into Urea Broth, TSI Agar of stab butt and streak
slant, into the various sugar of lactose, sucrose, maltose and glucose as well as
into litmus milk.

3. The spoiled food sample was examined. The spoiled sample was compared under
natural conditions and those which were inoculated with each of the organism. The
change on odour, texture and colour was determined.
DISCUSSION

Bacterial growth is an increase in all the cell components, which ends in multiplication
of cell leading to an increase in population. It involves an increase in the size of the cell and
the increase in the number of individual cells. Bacteria divide by binary fission. Most bacteria
divide in a short amount of time and produce a large amount of bacteria. It is easier to represent
these large numbers by logarithmic scales. When a bacterium is added to a suitable liquid
medium and incubated, its growth follows a definite course. If bacteria counts are made at
intervals after inoculation and plotted in relation to time, a growth curve is obtained. During
log or also known as exponential phase, the cells start dividing and their number increases
exponentially. The doubling time is measured during this period. The bacteria are most
susceptible to antibiotics during this time and stop growing due to decrease of nutrients and O2
supply as well as accumulation of toxic metabolites.

Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a
small amount of glucose (dextrose), ferrous sulphate, and the pH indicator phenol red. It is
used to differentiate enterics based on the ability to reduce sulphur and ferment carbohydrates.
As with the phenol red fermentation broths, if an organism can ferment any of the three sugars
present in the medium, the medium will turn yellow. If an organism can only ferment dextrose,
the small amount of dextrose in the medium is used by the organism within the first ten hours
of incubation. After that time, the reaction that produced acid reverts in the aerobic areas of the
slant, and the medium in those areas turns red, indicating alkaline conditions. The anaerobic
areas of the slant, such as the butt, will not revert to an alkaline state, and they will remain
yellow. This happens with Salmonella sp and Shigella sp.

Urea is a nitrogen containing compound that is produced during decarboxylation of the

amino acid arginine in the urea cycle. It is highly soluble in water and is therefore an efficient
way for the human body to discharge excess nitrogen. This excess urea is then taken out of the
body through the kidneys as a component of urine. Some bacteria have the ability to produce
an enzyme urease as part of its metabolism to break down urea to ammonia and carbon dioxide.
While many enteric bacteria have the ability to hydrolyse urea as part of their metabolism,
Proteus vulgaris are considered rapid urease producers due their efficiency in carrying out this
process.

Litmus Milk is differential media containing milk, litmus (a pH and oxidation-reduction

indicator), lactose, and casein (a milk protein) all of which can be metabolized by some
bacteria. Litmus milk also contains sulphur and many nutrients that are found in milk making
it is an undefined media that is used to help differentiate and identify Enterobacteria,
Clostridium, and lactic acid bacteria. A number of results may be obtained in the Litmus Milk
test, making it a rather complex test. There are four main reactions: lactose fermentation, litmus
reduction, casein coagulation, and protein hydrolysis. Some or all of these reactions may occur
at the same time, and many of them can have more than one reaction. For instance, casein
coagulation can be from a solid clot, as in an acid clot, or from the casein being converted to a
solid curd in the bottom below a liquid whey as in curd formation.

CONCLUSION
This experiment is conducted to allow the students to identify the characteristics of
gram negative rod shaped bacteria as well as to expose students to food spoilage characteristics
causing by gram negative bacteria. The objectives of this experiment is successfully achieved.

REFERENCES

1. http://www.columbia.edu/itc/hs/medical/pathophys/id/2009/introNotes.pdf
2. https://www.fsis.usda.gov/wps/wcm/connect/dcbfde80-3d65-4b21-9685-
9d6676367aac/PHVt-Food_Microbiology.pdf?MOD=AJPERES
3. http://cytalabs.com/wp-content/uploads/2015/01/Synthesis-of-the-Cell-Surface-
during-the-Division-Cycle-of.pdf