THE UNIVERSITY OF ZAMBIA
SCHOOL OF VETERINARY MEDICINE
PARACLINICAL STUDIES DEPARTMENT
VMP 4300 MICROBIOLOGY/IMMUNOLOGY
LAB NUMBER 3:
IDENTIFICATION OF ENTEROBACTERIA BY BIOCHEMICAL TESTS
IDENTIFICATION OF ENTEROBACTRIA BY BIOCHEMICAL TESTS
To identify enterobacteriaceae in culture A and B by biochemical tests
The identification of bacteria is a careful and systemic process that uses many different techniques to
narrow down the types of bacteria that are present in an unknown bacterial culture. It produces
benefits for many aspects of the research of microorganisms and helps the physician to correctly treat
Various steps are involved in the identification of unknown bacteria in the laboratory.
Isolation is the first one. The importance of this step is to isolate pure colonies of bacteria. The streak
plate is a quantitative isolation method. The inoculation of the culture is made on the agar surface by
back and forth streaking with the inoculation loop over the solid agar surface. This will make a dilution
gradient across the agar plate. The characteristic features in the identification of unknown bacteria
being; the shape, size, elevation, surface, edges, color, degree of growth and nature.
The second method being microscopic and staining reactions method, Staining is a simple basic
technique that is used to identify microorganism. For example, Gram staining is a differentialstaining
technique that imparts different colors to different bacteria or bacterial structures. Usually it
differentiates bacteria into two groups: Gram positive and Gram negative bacteria. The primary stain
crystal violet and mordent iodine form strong Crystal violet iodine (CVI) complex. Gram positive bacterial
cells due to their thick peptidoglycan layer will retain the CVI complex even after it is subjected to
decolorization by acetone or alcohol. Hence, the counter stain safranin has no action on the gram
positive cells. But in the case of gram negative bacterial cells, the thin peptidoglycan layer and more lipid
contents in the cell will easily make them susceptible to the action of the decolorizer and hence the CVI
complex is easily washed out and therefore the gram negative bacteria cells will retain the color of the
counter stain safranin. Hence, after gram staining, the gram positive cells appear purple and the gram
negative cells will appear pink. Then it can be said that the study of morphological features and staining
characteristics help in the preliminary identification of the isolate.
Biochemical reactions are also used in the identification of enteric bacteria. Gram negative enteric bacilli
play an important role in the contamination of food. Hence, they are the main causative agents of
intestinal infection. Gram negative family includes Shigella, Salmonella, Proteus, Klebsiella, Esherichia,
and Enterobacter. Four biochemical tests were carried out in this practical, these being: Triple sugar –
iron (TSI) test, Citrate test, SIM (H2S, Indole, Motility) and the Urease test.
TSI test is designed to differentiate among the different groups or genera of the Enterobacteriaceae,
which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to
distinguish them from other gram negative intestinal bacilli. This differentiation is based on the
differences in carbohydrate fermentation patterns and hydrogen sulfide production by the various
groups of intestinal organisms. Carbohydrate fermentation is detected by the presence of a gas and a
visible color change (from red to yellow) of the pH indicator, phenol red. The production of hydrogen
sulfide is indicted by the presence of a precipitate that blackens the medium in the butt of the tube. TSI
Agar contains three fermentative sugars, lactose and sucrose in 1% concentration and glucose in a
concentration of 0.1%.
Citrate Utilization test was also done. This test determines the ability of microorganisms to utilize
citrate. Some bacteria have the capacity to convert the salts of organic acids, for example, Sodium
citrate to alkaline carbonates. Sodium citrate is one of the most important metabolite of Krebs cycle.
Certain bacteria use citrate as the sole carbon source. Citrate utilization requires a specific membrane
transport and citrate lyase activity. Citrate is converted to Oxalo acetic acid by citrate lyase and
oxaloacetate decarboxylase activity will convert oxaloacetate to pyruvate with the release of carbon
dioxide. The carbon dioxide reacts with sodium and water to form sodium carbonate.
Urease test was also one of the biochemical tests looked in this practical. Urea is a nitrogen containing
compound that is produced during the decarboxylation process of the amino acid arginine in the urea
cycle. Certain bacteria produce the enzyme Urease during its metabolism process and that will break
down the urea in the media and carbon dioxide:
Some enteric bacteria produce the enzyme Urease, which splits the urea molecule into carbon dioxide
and ammonia. The Urease test is useful in identifying the genera Proteus, Providentia and Monganella,
which liberate this enzyme.
(H2S) 2CO + 2H2O >>>>>>>>>> CO2 + H2O + 2NH3
SIM test stands for Hydrogen sulphide, Indole, Motility test, is a semi – solid media meant to test for
hydrogen sulphide gas production and the metabolism of the amino acid tryptophan. This test is also
solely known as the Indole test which looks for the presence or absence of tryptophanase enzyme
production of the bacteria. If the enzyme is present, it will degrade the amino acid tryptophan in the
media and will produce Indole, ammonia and pyruvic acid. Indole will react with Kovac’s reagent to
produce a cherry red complex, which indicates a positive Indole test. The absence of red color is
indicative of tryptophan hydrolysis due to the lack of tryptophanase enzyme. SIM agar may also be used
to detect the presence of H2S production. Hydrogen sulphide is liberated as a result of the degradation
of sulphur containing amino acids. A black precipitate forms due to the reaction of hydrogen sulphide
with the iron salts in the medium.
It can be said then that through biochemical tests performed in the laboratory, a more conclusive
identification of the different enterobacteriaceae species can be done.
Cultures A & B
Inoculating wire –straight wire
Test tubes & racks
A. TSI (triple sugar iron) test for CHOs fermentation
The inoculating loop was firstly sterilized by heating on a flame until it was red hot, the mouth part
of the tube containing the agar was also sterilized by being passed over the Bunsen burner fame.
The loop was then cooled and thereafter a single colony was picked using the loop, then slant was
then streak followed by the butt. The lid was then placed over the test tube. This procedure was
repeated for cultures A & B.
B. SIM (H2S, Indole, Motility) tests
In the same way as in A. above the inoculating loop was used. Firstly sterilized using the Bunsen
burner flame and the test tube containing the media, mouth was also sterilized. A single colony
was then picked using a sterilized loop. The butt was streaked. This was done to both cultures A &
C. Citrate test
A straight wire was first sterilized, cooled and then mouth of the tube containing the media was
sterilized as well. A single colony was collected from the culture plate and then the slant of the
citrate test sample was gently inoculated. This was done to both cultures A & B.
4) UREASE TEST
The straight wire was first sterilized by heating till red hot over a Bunsen burner flame. The mouth
part of the test tube containing the urea agar was also sterilized by being passed over the flame.
Thereafter, a single colony was picked using a cooled straight wire from the culture plate then the
butt was stabbed and the wire was removed via the same path of entrance. The lid was then placed
over the test tube. This was done for both culture A and B.
Table1: showing results of laboratory biochemical bacterial identification tests
Colors of media before inoculation were as follows;
TSI agar --- brown
Green= citrate utilized
Pink= Indole present
Blue= citrate utilized
Culture A: E.coli
Culture B: Salmonella
The four biochemical tests were carried out in order to differentiate and identify enterobacteria
and in this lab practical the bacteria identified was E.coli and Salmonella.
In TSI test, culture A had an acidic slant and an acidic butt (A/A), indicating that there was
glucose, sucrose and/or lactose fermentation. In this test tube, hydrogen sulphide was not
produced butt there was the presence of gas. Culture B had an alkaline slant and acidic butt
(K/A), which indicated that glucose was fermented anaerobiaclly and showed that there was the
production of hydrogen sulphide and gas.
In the citrate test, culture A tested negative for citrate utilization and culture B tested positive
shown by the formation of the blue color.
In the Urease test, culture A and B tested negative for urea production showing that these
bacteria had no ability to produce Urease enzyme.
The SIM test, three things were being tested for thus, hydrogen sulphide, Indole and motility.
Culture A tested negative for hydrogen sulphide while culture B tested positive. For Indole test,
culture A tested positive while culture B tested negative. Finally for Motility test both culture A
and B tested positive.Kovac’s reagent is is an isomyl alcohol scientifically known as
Therefore, based on the total of these results, culture A and B were identified as E.coli and
The cultures A and B of enterobacteriaceae were identified as E.coli and Salmonella respectively.
1) Ikram M. and Hill E. (1991), Microbiology for Veterinary Technicians, 1st Edition,American
Veterinary Publications Incorporated, California.
2) Nester W.E et al (2004), Microbiology: A Human Perspective, 4th Edition, McGraw Hill
Companies, New York.
3) http://www.scienceprofonline.com/microbiology/bacteria-colony-morphology-identificationunknown bacteria.html retrieved at 14:05 hrs, 24/11/2013.s