The document provides information on identifying an unknown organism through a series of biochemical tests. A number of tests were performed on the unknown organism including oxidase, lactose fermentation, triple sugar iron, indole, methyl red, Voges-Proskauer, citrate, motility, and hydrogen sulfide tests. Based on the results, the unknown organism was identified as Bacillus cereus. The tests help determine an organism's metabolic capabilities and pathways, which can be used to differentiate between organisms.

1. UNKNOWNORGANISM
The UNKNOWNorganismis Bacillus cereus
TEST THAT ARE IMPORTANTINIDENTIFICATIONOF UNKNOWN
Oxidase
Starch hydrolysis
Sugar fermentation
Lactose fermentation
Triple sugar iron agar
Hydrogen sulphide(H2S)
Indole
Methyl red Voges-Prokauer(MRVP)
Citrate
motility
IMPORTANTFACTS
IMViCtest are used to study the physiologicalcharacteristics of bacteria.
The biochemical characteristics of bacteria can be used to demonstrate their
metabolic diversity. Bacteria exhibit a very large range of metabolic capabilities,
partially explaining why these organisms can be found in virtually every
environmental habitat. Not only are bacteria capable of obtaining energy by
variety of pathways, someof which are unique to bacteria, they are capable of
utilizing a very large number of different metabolites.
The reactions determine if certain carbohydrates areused (glucose,
lactose, sucrose)
and whether or not the sugars can be fermented with the production of acids and
gas, or
only acid. pH indicators are used in the media to determine whether acids are
produced

2. Bacteria that producethe enzyme typtophanasecan convert the amino
acid to by products that include indole.when indole is combined with kovac’s
reagent (which contains hydrochloric and dimethylaminobenzaldehydein amyl
alcohol) the solution turns fromyellow to chery red. Because amyl alcohol is not
water soluble, the red y will m in an oily layer at the top of the broth.
The MRVP tests are run together in the samebroth and then split into
two tubes when ready to be tested for the end products. The methyl red test
determines the useof glucose, with the subsequentproduction of acid, tested for
by the pH indicator methyl red. The voges-proskauer testalso determines glucose
use, but for different end product- not acid but a neutral productcalled acetoin
(or acetylmethlcarbinol). The VP is really important for identification of many but
it is a picky test, and mustbe done exactly right.
The citrate test identifies the use of citrate as a sole carbon source, since
there are no other nutrients in this medium. Yhe basic end products (carbonates,
bicarbonates, and ammonium hydroxide) will cause the brom thymolblue
indicator in the medium to turn fromforestgreen to royalblue.
INTRODUCTION
Normal enteric flora
An enteric flora refers to a group of organisms thatare usually found in
interstine.. The enteric organisms area large heterogeneous group of organisms
whosenatural habitat is the intestinal tract of humans and animal.
Escherichia coli are part of the normal flora. Other enteric bacteria (proteus,enterobacter,
klebsiella, morganella, providencia, citrobacter, and
serratia species)are also found as members of the normal
intestinal flora. Soil also contain a lot of enteric organisms.
Enteric Pathogen
Pathogen as is rightly defined means any diseasecausing-microorganism. This
may include viruses and many bacteria, fungi, and protozoans. Fromthis
definition and explanations so far, enteric pathogens can be said to be enteric
diseasecausing microorganism. They rangefrombacteria, viruses to other

3. microbes. Enteric pathogens range fromEscherichia coli, Enterobacter aerogenes,
Salmonella, Serratia marcescens, Enterobacteraerogenes etc. they are enteric
diseasecausing organisms.
Enteric disease
An enteric disease is an illness of the gastrointestinaltract, most often caused by
bacteria or viruses thatenter the body primarily through eating of or drinking of
contaminated food or water, or by contact with vomit or faeces. People with an
enteric illness most often experience symptoms such as nausea and vomiting,
diarrhea, fever and abdominal pain.
Serious complications of enteric illness, such as the development of chronic
conditions or death, are more likely to occur in young children, the elderly and
people with suppressed immunesystem, though they can develop in healthy
individual.
Some enteric diseases follow a seasonalpattern:
* Campylobacter enteritis cases typically peak in July, Augustand September
* Cryptosporidiosistypically peaks in August
* Giardiasis is historically reported most frequently in September
* verotoxin producing E. coli typically peaks in August
The sources of exposure are not always identified, even when a thorough
investigation is completed. Food, water and contact with animals are frequent
sources of exposure. Below is a table that shows someknown sources of exposure
of someenteric diseases.
KNOWNSOURCES OF EXPOSUREOF SOMEENTERICDISEASES
DISEASE SOURCE OF EXPOSURE
Salmonellosis Food, especially chicken and eggs.
Animals, including reptiles
Cryptosporidiosis Recreation water, contact with animals

4. Yersiniosis Meat, typically pork products
Hepatitis A Food, contact with infected person
Purposeof identification of enteric organisms
The importance in identifying enteric organisms cannotbe overemphasized. This
will enable one to know the different approach to take when one is to handle
issues concerning enteric diseases. Proper diagnosis can be carried out when
enteric organisms havebeen identified.
MATERIALS USED INIDENTIFICATIONOF UNKNOWN
Before incubation
* 1 Numbered unknown slantculture
*1 lactose tryptone brot
* DCA plate
*Peptone water
* 1Sterile test tube
* 1Ureasebroth
* 1MRVP broth
*1 Simmon citrate agar slant
*1 Triple Sugar-Iron agar slant
*MacConkey’s agar plate
*Hydrogen sulphide
*Glass slide
After incubation
*Kovac’s reagent
*Barritts reagent A (alpha-naphthol) and B (KOH)

5. *methyl red reagent.
PROCEDURES/METHOD
INDOLETEST
* Obtain unknown culture.
* Obtain a sterile tube of tryptone broth.
* Using an inoculating loop and aseptic technique, transfer a loopful of unknown
organism into the tryptone broth.
N.B Be sure to shake the loop in the broth and touch it to the side of the tube to
remove excess broth.
*Incubate the tube at 35 degree Celsius for 24-48 hours
*Obtain the inoculated tryptone broth tube from the incubator
* Add 10 drops of Kovac’s reagent to the tube inoculated with unknown organism.
* Allow the tube to stand for 5 minutes.
N.B A red ring will form at the top of the broth if it is positive for indole production.
N.B a yellow or green ring will form at the top of the broth if it is negative for indole
Production.
HYDROGEN SULPHIDE TEST
*Obtain unknown culture
*Drop 3% Hydrogen sulphide on a glass slide
*Inoculate organism in the slide
*Presence of gas burbles shows positive test while absent show negative test
MOTILITY TEST
*Obtain unknown organism
*Using a straight wire loop, take a stab of unknown organism and stab straightly in a
solid media provided.

6. *Incubate at 35˚C for 24-48 hours
N.B Motile organisms will show some growth while non motile organism will not.
STARCH HYDROLYSIS TEST
*In a given petri dish media given, streak unknown organism in it.
*Incubate at 35˚C for 24-48 hours
*Take the dish and pour iodine solution
N.B Blue black coloration shows that organism did not utilize the starch hence negative
result while a non blue black coloration shows that starch has been utilize.
SUGAR FERMENTATION TEST
Obtain unknown organism.
Streak organism in a test tube containing dunhams tube provided
Incubate at 35˚C for 24-48 hours
N.B A change from purple coloration to colorless coloration and presence of gas
burbles in dunham’s tube shows a positive test for glucose and lactose while no
change indicate negative.
LACTOSE FERMENTATION TEST
Obtain unknown organism.
Streak unknown organism on a DCA plate provided
Incubate at 35˚C for 24-48 hours
N.B If the organism turns the DCA plate from brown coloration to colorless color the
organism is positive to lactose fermentation while otherwise shows negative test.
OXIDASE TEST
Take a loopful of unknown organism from broth

7. Spread on a filter paper
Observe colour changes if there is any
METHYL RED AND VOGES-PROSKAUER TEST
* Inoculate unknown organism into the MRVP broth.
* Incubate at 35˚C for 24-48 hours..
* AFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: run
the MR test in the tube with 2/3, and the VP test in the open tube with 1/3.

for methyl red: Add 6-8 drops of methyl red reagent.

for Voges-Proskauer: Add 12 drops of Barritt's A, 4 drops of Barritt's B, mix
gently for 1 minute to aerate the solution (the reaction requires O2).

Let sit, undisturbed, for at least 20 minutes, maybe more.
N.B A light pink colouration at the top of tube indicate a positive test for voges-
proskauer test.after the addition of methyl red reagent in the methyl red test, a red pink
colour of acid from glucose use indicate positive test. A yellow colour indicate a
negative test.
CITRATE TEST
* Streak up the slant with the unknown inoculum.
* Incubate at 35˚C for 24-48 hours
N.B The presence of growth and a blue colour represent a positive test for citrate
utilization. The absence of growth and a green colour represent a negative test for
citrate utilization.
TRIPLE SUGAR IRON AGAR TEST
Gram stain unknown organism and observe gram morphology

8. Inoculate with loop full of unknown organism
Stab butt and streak surface of TSI agar slant.
Incubate at 35˚C for 24-48 hours
N.B If organism utilize lactose and produce acid and colour changes from red to yellow,
it is a positive test but when there is no change the organism is negative to TSI agar.
RESULT OF TEST
A TABLE SHOWING TEST RESULT OF VARIOUS TEST IN IDENTIFICATION OF AN
UNKNOWN ORGANISM`
TEST OBSERVATION ± RESULT INTERPRETATION
Oxidase There was no colour
change
- Organism could not
act on the oxidase
reagent to produce
blue colour
Glucose Purple colouration
with no gas burbles
- Organism cannot
utilize glucose
Lactose A colour change
from brown to
colourless colour
and presence of gas
burbles observed
+ Presence of gas
burbles confirmed a
positive test hence
organism can utilize
lactose
TSI slant No colour change
observed
- B This shows that the
solution is alkaline
and organism could
not act
Agar Butt A colour change
from red to yellow
was observed
+A This shows that the
solution became
acidic due to the
presence of the
unknown organism
H2S There was no gas
bubbles
- Unknown organism
could not utilize the
hydrogen sulphide
Indole A red ring
colouration at the
top of the broth
+ Organism was able
to react with kovac’s
reagent to produce
the red colour at the
top. This indicate
the presence of

9. indole.
MR A yellow colouration
observed
- Organism could not
use glucose hence
there was no acid
production which
the yellow colour
indicates.
VP A yellow colouration
observed
- Organism could not
produce acid to
bring about a red
colour hence was
negative
Citrate There was presence
of growth and a blue
colouration
observed
+ The organism was
able to utilize citrate
hence the presence
of growth and blue
colouration
Motility The straight stab
line was intact,
there was no growth
of bacteria
- The original stab
line could not
diffuse out into the
medium as the
bacteria could not
spread out
throughout hence
there was no
growth.
MR=Methyl red; VP= voges-proskaves; A= acid; B= alkaline
UNKNOWN ORGANISM: Bacillus cereus
DISCUSSION
How I identified unknown organism
A positive rod bacteria was given to me, having carried out all the tests so far, I
compared my result with a table provided at page 94 of the manual on Laboratory
exercises for introduction and general Microbiology by university of Port Harcourt.
A careful analysis and contrast was carried out and compared with the table.
REASONS FOR DIFFERENCIES IN ACTUAL AND THEORETICAL RESULT

10. LABORATORY REPORT
ON MCB 200.1
PRACTICAL.
TITLE: UNKNOWN
THIS LABORATORYREPORT WORK TALKSABOUT THE IDENTIFICATION OF
BACTERIA.THE TEST USE IS THE IMVIC TEST <INDOLE, METHYL RED, VOGES-
PROSKAUERANDCITRATE> WHICH ALSOCOMPRISESOF THE TSIA AND CITRATE
TEST.
09 05
2013
user
BY AGGREH ERHOVWON PETER U2011/5555010
MICROBIOLOGY
5/9/2013