ENTEROBACTERIACEAE

Most of the organism studied here belong to the family Enterobacteriaceae with some
exceptions. The diagnosis of infections with enteric pathogens (including carrier state) is generally
made by isolating the infective agent from blood, feces or urine. Special media have been
developed in order to facilitate the isolation of the pathogen. Some of these are enriched for the
desired organism. This is accomplished either by using ingredients that stimulate the growth of the
pathogen more than that of the non-pathogen or by using substances more toxic to the non-
pathogen to the pathogen. The enrichment media normally employed are: GN broth, selenite broth
and tetrathionate broth.
In addition some special media contain ingredients that indicate other chemical capabilities
of the bacteria (generally by a color change) thus providing more insight as the possible nature of
the bacterial growth.

ENTERIC ORGANISMS AND OTHERS

PURPOSE: To furnish the student with a working guide in the isolation of enteric organisms, the
following series of experiments will be performed during the next lab periods. The overall plan is
as follows: Each group will receive known culture of enteric organisms. Media for their isolation
and identification will be used.

PROCEDURE:
FIRST LABORATORY PERIOD
1) Each group will get their specimens from the counter.
2) Inoculate the text organism to primary media: EMB, SSA, BSA, BGA using the streak
plate method.
3) Incubate at 37 ºC for 24 hours.
4) After 24 hours examine the plates and record the results.
SECOND LABORATORY PERIOD
1) Examine each plate carefully and look for the plate showing the most typical growth and
use this plate as the source of the inoculum.
2) Make a suspension of the organism in sterile NSS and use this suspension in the
biochemical tests to be done.
3) Inoculate the biochemical tests and incubate at 37 ºC for 24 – 48 hours.
THIRD LABORATORY PERIOD
1) Read the results of the biochemical tests. Record.
2) Compare the results with the results with theoretical results of known organisms.
3) From the comparative study the identity of the organism is known.
 Laboratory Activity #13
BIOCHEMICAL TEST FOR THE IDENTIFICATION OF ENTERICS

Bacterial metabolism is a fundamental part of the science of bacteriology, which attempts

to understand the function of bacterial processes. This described in terms of the biochemical
activities of the organisms which are almost unlimited being the most obvious manifestations of
the existence.
So varied are those characteristics that the catalysis of particular chemical reactions by the
presence of specifying enzymes has provided the major means of classifying and identifying
microorganisms.

I. FERMENTATION OF CARBOHYDRATES
Microorganisms ferment carbohydrates in different ways depending upon the enzymes they
possess. Pyruvic acid maybe considered as the key compound formed during the process of
fermentation since it is at this point that the pathways of carbohydrate breakdown diverge with
different species of bacteria. Pyruvic acid maybe converted into a variety of end products.
1) These tests differentiate the fermentative capacity of different microorganisms.
2) Gas maybe detected and measured in Durham’s fermentation tube as bubbles in the
inverted submerged tube.
3) A change in the color of the medium to a pink color is indicative of acid production.

PROCEDURES:
a) LACTOSE BROTH IN DURHAM’S FERMENTATION TUBE
1) Inoculate 1 loopful of the test tube organism into lactose broth in Durham’s
Fermentation tube.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period observe the results.

OBSERVATION: The medium contains Andrade’s indicator. In the presence of acid the indicator
turns pink. Gas production can be detected by the presence of bubbles in the Durham’s
fermentation tube.

b) SOLID SUGAR MEDIA

1) Inoculate the test organism into the medium by stabbing halfway to the bottom of the
medium.
2) Incubate at 37 ºC for 48 hours.
 3) Next lab period observe the results.

OBSERVATION: Acid production is detected if the medium turns pink as the indicator present is
Andrade’s indicator. The presence of cracks in the medium denotes gas production.

II. INDOLE PRODUCTION

Indole is produced as a putrefactive product from tryptophan and other amino acids containing
indol ring and only under anaerobic conditions and the absence of sugars from the medium.
The ability of an organism to give a positive indole test depends whether or not the organism
possess the enzyme tryptophanase.

PROCEDURE:
KOVAC’S TEST
1) Inoculate 1 loopful of the test organism into a tube of tryptone broth.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period add 1 cc Kovac’s reagent so that it forms a layer on the culture medium.

OBSERVATION: If a purple color forms at the junction of the medium and the reagent the test is
positive. A yellow color is negative.

III. METHYL RED TEST

PURPOSE: Methyl red test is used to determine the amount of acidity produced as a result of
bacterial fermentation.

PROCEDURE:
1) Inoculate 1 loopful of the test organism into MRVP medium.
2) Incubate at 37 ºC for 45 hours.
3) Next lab period add 5 drops of methyl red indicator to the medium.
4) Mix thoroughly and observe the color reaction.

OBSERVATION: A distinct red color is positive. A yellow color is negative.

IV. VOGES-PROSKAUER TEST
PURPOSE: This reaction depends upon the ability of certain bacteria to produce
acetylmethylcarbinol from glucose. Under alkaline conditions acetylmethylcarbinol oxidized
to diacetyl. This will produced a red color complex. Alpha naphthol greatly intensifies the
color.

PROCEDURE:
1) Inoculate MRVP medium with 1 loopful of the test organism.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period add 1 cc of KOH followed by 1 cc of alphanaphthol. Shake the test
tube slightly and let it stand for about 15 – 30 minutes. Observe for any color change.

V. CITRATE UTILIZATION TEST

PURPOSE: This test depends upon the ability of the organism to utilize citrate as the sole
source of carbon. Hence organisms utilizing citrate grow on this medium. The indicator used
in the medium is bromthymol blue.

PROCEDURE:
1) Inoculate the surface of Simmon’s citrate agar by the streak method by the use of the
wire loop with the test organism.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period observe for color change.

OBSERVATION: If the medium turns blue the test is positive.

VI. PRODUCTION OF HYDROGEN SULFIDE

PURPOSE: Cysteine is attacked by some organisms which the liberation of hydrogen sulfide.
Ferrous sulfate in incorporated into the culture medium to detect this bacterial property. The
hydrogen sulfide reacts with ferrous sulfate to form ferrous sulfide which is black in color.

PROCEDURE:
TRIPLE SUGAR IRON AGAR (TSI)
1) Inoculate the slant portion of the medium by the use of the wire needle with the test
organism. The butt portion is inoculated without getting a new inoculum by stabbing
halfway through the butt portion without reaching the bottom with the wire needle.
2) Incubate at 37 ºC for 24 hours.
3) Observe for color changes in the medium.
 OBSERVATION: TSI contains 3 sugars (glucose 0.1%, lactose 1%, sucrose 1%). Phenol red is the
indicator used. The presence of a pink color in either slant or butt indicates an alkaline reaction. A
yellow color in either slant or butt indicates an acidic reaction. Cracks in the medium indicate the
presence of gas production. Presence of a black color indicates hydrogen sulfide production.

VII. RAPID UREASE TEST

PURPOSE: Some bacteria possess the ability to split urea into ammonia and carbon dioxide.
This is accomplished by means of enzyme urease. This test is done for the rapid identification
of proteus organisms.

PROCEDURE:
1) Inoculate the urea broth with 5 loopfuls of the test organism.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period observe for any color change.

OBSERVATION: If the medium turns to a dark pink or purple color the test is positive. The dark
pink or purple color is due to the presence of ammonia.

VIII. MOTILITY TEST

PURPOSE: To demonstrate the presence or absence of motility.

PROCEDURE:
1) Inoculate the test organism by the stab method into SIM medium.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period observe the results.

OBSERVATION: Motility is noted if there is haziness of the medium. Absence of motility can be
determined in as much as the medium will remain clear and growth is confined only to the line of
stab. SIM medium also contains ferrous sulfate so this medium can also determine hydrogen
sulfide production by the formation of ferrous sulfite. The presence of a black color in the medium
denotes hydrogen sulfide production.
IX. REDUCTION OF NITRATES TO NITRITES
PROCEDURE:
1) Inoculate to nitrate agar 1 loopful of the test organism.
2) Incubate at 37 ºC for 48 hours.
3) Next lab period add 2 drops of sulfanilic acid followed by 2 drops of
alphanaphthylamine solution.
4) Observe for any color reaction.

OBSERVATION: In the presence of nitrites a red azo compound is formed. This color disappears
in a few seconds.