DISCUSSION QUESTIONS

1. Why should the motility test be done at the log phase of bacterial growth?
Motility test should be done at the log phase of bacterial growth because it is the
optimal phase to test for motility. If did on the lag phase, the motility may not be seen
as bacterial still trying to survive in the new media.
2. Explain the principle of TSI test
Triple sugar iron agar (TSI) is a differential medium that contains lactose, sucrose, a
glucose ,ferrous sulfate, and the pH indicator phenol red. It is used to differentiate
enterics based on the ability to reduce sulfur 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 and Shigella.
3. Explain the principle of Urea test
Urease broth is a differential medium that tests the ability of an organism to produce
an exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide.
The broth contains two pH buffers, urea, a very small amount of nutrients for the
bacteria, and the pH indicator phenol red. Phenol red turns yellow in an acidic
environment and fuchsia in an alkaline environment. If the urea in the broth is
degraded and ammonia is produced, an alkaline environment is created, and the
media turns pink.
4. Explain the principle of Litmus milk.
Litmus milk is done to differentiate bacteria based on various reactions that occur in
skim milk suplemented with a litmus pH indicator. Milk is a complex nutritional source
that contains proteins (mainly casein) in an aqueous solution of lactose and
minerals. Bacterial enzymes alter the media and may bring about various changes.
Litmus is added to the medium to detect pH changes that may occur as a result of
these enzymatic reactions. Above a pH of 8.3 litmus is blue, while below a pH of 4.5
litmus is red.
CONCLUSION (Tak habus)
Based on the experiment, we managed to find the characteristics of gram negative
rod shaped bacteria. Gram negative rod shaped bacteria have a thin layer of
peptidoglycan held between two membranes. They also have outer membrane
containing lipopolysaccharides. Additionally, gram negative rod shaped bacteria
have cytoplasmic membrane. The gram-negative bacteria have a thinner, less
complex cell wall compared to their gram-positive counterparts. Most or not all
spoiled food have bad odour due to chemicals that are released from the food as the
microbes decompose it, or chemicals produced directly by the microbes themselves.

REFERENCES
1. http://infectionnet.org/notes/gram-negative-bacilli/
2. https://microbeonline.com/tag/biochemical-tests-for-gram-negative-bacteria/
3. http://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211436.pdf
4. http://microbesinfo.com/2013/05/triple-sugar-iron-agar-tsi-test/
5. http://www.austincc.edu/microbugz/urease_test.php
6. https://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Litmus
%20Milk%20Results.html
DISCUSSION
The results of the study confirm the hypotheses that some gram negative rod
shaped bacteria are involved in the spoilage of food. Different types of bacteria will
produce different-looking colonies, that’s why some of the experiment colonies form
are different for example E.coli and Serratia marcescens have circular form but
Salmonella sp and Klebsiella aerogenes have irregular form. This also applies
elevation, margin, and texture. We also performed motility test. Motility testing is
performed by preparing a wet mount and is then observed under the microscope. It’s
done to find out whether the sample is motile or non motile. Based on the test, we
find out that E.coli, Serratia marcescens and Salmonella sp are motile meanwhile
Klebsiella aerogenes non motile. Klebsiella aerogenes are non-flagellated means
they don’t have flagella to propel themselves thus making them non-motile.
We also perform Biochemistry test on the samples. First we did Urea Broth test,
Urea broth is a differential medium that tests the ability of an organism to produce an
exoenzyme, called urease, that hydrolyzes urea to ammonia and carbon dioxide.
Then we did litmus milk test. Litmus milk is done to differentiate bacteria based on
various reactions that occur in skim milk suplemented with a litmus pH indicator. All
of the sample shows positive results. For Klebsiella aerogenes it shows pink to red
color which is Acid pH. For Salmonella sp it shows Rennet curd which shows soft
curd followed by peptonization. For Serratia marcescens shows Acid curd which
shows hard curd with clear supernatant. For E. coli shows Rennet curd which soft
curd followed by peptonization.
We also did Triple sugar iron agar (TSI) is a differential medium that contains
lactose, sucrose, a glucose ,ferrous sulfate, and the pH indicator phenol red. It is
used to differentiate enterics based on the ability to reduce sulfur and ferment
carbohydrates. All of them shows positive acid in TSI agar butt but E. coli and
Klebsiella aerogenes shows acid and Salmonella sp and Serratia marcescens
shows alkaline in slant agar. This is accurate according to the theory. For lactose, E.
coli and Serratia marcescens shows negative results. This shows that Serratia
marcescens can’t ferment lactose but E. coli suppose to ferment lactose. This maybe
because our E.coli sample is contaminated when we did our experiment due to our
careless. This can be easily avoided by perfoming aseptic technique properly. For
sucrose test we get all positive results except for Klebsiella aerogenes. Klebsiella
aerogenes suppose to ferment sucrose . We failed in achieving this may due using
an old sample that’s denatured. This can be fix by using newer sample. Next we did
glucose test which all results shows positive. After that we did Maltose test, which
shows all but Klebsiella aerogenes positive results. Klebsiella aerogenes suppose to
ferment maltose . We failed in achieving this may due using an old sample that’s
denatured.
We examined the spoiled food . We managed to find that all the sample produced
bad odour. Most spoiled food have bad odour due to chemicals that are released
from the food as the microbes decompose it, or chemicals produced directly by the
microbes themselves. Most of them produce sticky slimy texture due to the growth of
the bacteria. All of the food sample changed color also for the same reason .
 LABORATORY REPORT

FOOD MICROBIOLOGY
MIC254

EXPERIMENT 4: FOOD BACTERIA: GRAM NEGATIVE ROD

SHAPED BACTERIA

Muhammed Hakim Bin Abd Razak (2016378735)

Mohd Zahid Irfan Bin Jakaria (2016935847)
Nurul Nabihah Binti Mohamad Tahir (2016329013)

GROUP CLASS : AS1144A1

DATE OF EXPERIMENT : 10/10/2018
DATE OF SUBMISSION : 17/10/2018
LECTURER : MADAM ILYANIE HJ YAACOB