NEW DIRECTIONS IN THE IDENTIFICATION
OF MICROORGANISMS

A Report for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
RODRIGO SAMAYOA R.

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NEW DIRECTIONS IN THE IDENTIFICATION

OF MICROORGANISMS

BY

Rodrigo Samayoa R.

A REPORT

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Microbiology and Public Health

1975

Dedicated to my father who made it possible
for me to come to this country, my mother
for her advice, and my wife for all these

years away from home.‘

ii

ACKN OWLE DGEMENTS

I wish to take this opportunity to express my
sincere appreciation to Dr. Gordon E. Carter, my academic
adviser, for his guidance and assistance in the preparation

of this report.

iii

TABLE OF CONTENTS

MULTI-BIOCHEMICAL TEST SYSTEMS FOR DISTINGUISHING
ENTERIC AND OTHER GRAN-NEGATIVE BACILLI. . . . .

Enterotube . . . . . . . . . . . . . . . . . .
R/B Enteric Differential System. . . . . . . .
API Microtube System . . . . . . . . . . . . .
Auxotab. . . . . . . . . . . . . . . . . . . .
Reagent-Impregnated Test Paper Strips. . . . .

DETECTION AND IDENTIFICATION OF BACTERIA BY GAS
CHROMATOGRAPHY O O O O O O O C O C O O O O O O .

AUTOMATION OF COLONY IDENTIFICATION AND MUTANT
SELECTION O O O O O O O O O O O O C C O O C C O .

CULTIVATION OF MICROORGANISMS ON A.SUBSTRATE TYPE

MONITORING OF BACTERIAL ACTIVITY BY IMPEDANCE
MEASUREMENTS O O O O O O O O O O O O O O O .4 O O

BACTERIAL IDENTIFICATION BY MICROCALORIMETRY . l .
AUTOBAC I, AUTOMATED ANTIMICROBIAL SUSCEPTIBILITY

SYSTEM 0 O C O O O I I O O O O O O O O O O O O O
LITEMTURE CITED . O . O O O O O O O O O C O 0— ‘. 0
APPENDIX: MECHANISM AND PROCEDURES OF BIOCHEMICAL

TESTS WIDELY USED FOR THE IDENTIFICATION OF
BACTERIA. O O O C O O . O O O C O O O O O O O 0

iv

Page

14
15

19

25

32

35

39

42.

46

51

58

Table of Contents (cont'd.)

Ammonia Production . . . . .

Arginine Dihydrolase . . . .

Carbohydrate Fermentation Tests.

OF Test. . . . . . . . . . .
Catalase Test. . . . . . . .
Citrate Test . . . . . . . .
Gelatin Test . . . . . . . .
Hydrogen Sulfide Production.
Indole Test. . . . . . . . .
KCN Test . . . . . . . . . .
Lysine Decarboxylation . . .
Malonate Utilization . . . .
Methyl Red (M.R:) Test . . .

Nitrate Reduction. . . . . .

Ortho-Nitrophenyl-B-D—GalactOpyranoside (ONPG)

Test . . . . . . . . . . .
Ornithine Decarboxylation. .
Oxidase Test . . . . . . . .
PhenylaIanine Deamination. .
Triple sugar Iron Agar (TSI)

Urease Test. . . . . . . . .

Page

64

65

65

67

68

69

69

71

72

73

74

75

76

77

80

81

82

84

86

86

A

Table of Contents (cont'd.)

Page
Voges-Proskauer (V.P.) Test. . . . . . . . . . . . 87

Appendix Bibliography. . . . . . . . . . . . . . . 88

vi

LIST OF TABLES

Table I Page

1. ACCURACY OF IDENTIFICATION OF UNKNOWN ENTERIC
CULTURES BY THE ORIGINAL AND REDESIGNED
INTEROTUBE . O C O C O O O . O C C . C C O O O 6

2. ACCURACY OF IDENTIFICATION OF UNKNOWN ENTERIC
CULTURES BY THE R/B SYSTEM . ,,, . . . . . . . 13

3. AGREEMENT BETWEEN API SYSTEM AND CONVENTIONAL
IDENTIFICATION O. O O. O O C O O O O O O O O O 0 l6

4. ACCURACY OF IDENTIFICATION WITH THE AUXOTAB
SYSTEM 0 O O O O O O O O O O C O O O O O O O O 20

5. ACCURACY OF IDENTIFICATION OF UNKNOWN CULTURES
BY PATHOTEC SYSTEM . . .r. . . . . . .,. . . . 24

Appendix

6. KEY INGREDIENTS AND APPLICATION OF COMMON
BIOCHEMICAL TESTS. . . . . . . . . . . . . . . 59

vii

LIST OF FIGURES

Figure Page
1. Enterotube . . . . . . . . . . . . . . . . . . . 8
2 O BaSic R/B system 0 O O O O 0 O O O O O O O 0 O O 12

3. API (sustrate card). . . . . . . . . . . . . . . 17

4. The dumbwaiter (large scale colony processor). . 34
5. The automatic plating machine. . . . ... . . . . 38
6. Detail of the plating mechanism. . .‘. . . . . . 38
7. Bacterial heat profiles by microcalorimetry. . . 45

8. Autobac I. . . . . . . . . . . . . . . . . . . . 50

viii

MULTI-BIOCHEMICAL TEST SYSTEMS FOR
DISTINGUISHING ENTERIC AND OTHER

GRAM-NEGATIVE BACILLI

Enterotube

The first type of "enterotube" (Roche Diagnostic)
which appeared on the market was a "ready-to-use" test
system for the routine identification of the enterobacteria.

The enterotube is a plastic tube with one round and
a contiguous side flat. The flat side consists of a thin
plastic cover. The enterotube has 8 sections, each of
which contains a different biochemical test medium. A
single inoculating needle extends lengthwise through the
center of each compartment and protrudes at both ends of
the tubes. One end of the wire and tube is covered by a
blue screw cap, and the other is covered by a white cap.
Aerobic conditions during inoculation are insured by 3
small air holes on the side of the tube. These are covered
by a blue strip which is removed at the time of inoculation.
The enterotube is inoculated by removing the white plastic

1

cap, touching the center of an isolated colony with the
straight wire, and then drawing the wire through the tube.
The wire inoculates each of the test media in the compart—
ments. The question of sufficient inoculum arises when
inoculating the tube from a single bacterial colony (17).
With rare exceptions, each of the 8 compartments were
inoculated satisfactorily after drawing the inoculating
needle through the enterotube (17). This elegant, quick,
and safe inoculation method avoids contamination of the
media, as well as errors inherent in multiple-tube tech—
niques. After incubation overnight, 9 biochemical tests

can be recorded and used for identification. The tests are:

 

 

Substrate (in medium) Reaction Detected
Glucose Acid production
Citrate Citrate utilization
TryptOphan Indole and H28 formation
Lysine Lysine decarboxylase
Dulcitol Acid production
Lactose Acid production
Urea Urea cleavage

Phenylalanine Phenylalanine deaminase

To detect indole, Kovacs reagent is injected into
the SHz-indole compartment, after incubation at 37°C for
18 to 24 hours. Likewise, the phenylalanine deaminase
reaction is read after injecting 10% ferric chloride solu-
tion through the cellophane covering.

Discrepancies were noted in the lysine decarboxylase
results between the enterotube and the conventional methods
(29). The agreement in the lysine decarboxylase reaction
was only 87.6%, whereas all other tests showed a reliability

between 95.0% and 98.8% when compared with conventional

methods (9).

Improved Enterotube

The arrangement of the media has been changed
markedly in the improved enterotube. Now 8 media permit
the detection of 11 biochemical reactions of the entero-
bacteria, viz., acid and gas from glucose, lactose, dul-
citol, citrate utilization, and the production of hydrogen
sulfide, indole, phenylalanine deaminase, urease, ornithine
decarboxylase, and lysine decarboxylase. An ornithine

decarboxylase compartment has been added. This was made

possible by combining in one compartment the phenylalanine
deaminase (PA) test with the dulcitol reaction, since all
Proteus species, which always are PA positive, are all coin—
cidentally dulcitol negative. Also the incorporation of
ferric ammonium citrate into the PA/dulcitol compartment
medium eliminates the need to add the 10% ferric chloride
solution after incubation. Also advantageous is the possi-
bility of detecting the gas reaction in the glucose chamber.
The wax overlay in the lysine and ornithine compartments
ensures anaerobic conditions for glucose fermentation. The
latter results in gas formation and lifting of the wax
overlay from the surface of the agar. The wax overlay in
the lysine and ornithine compartments prevents false-
positive reactions. The handling of the improved entero-
tube is unchanged with the following exception: after
drawing the needle through all 8 compartments, the user
must re-insert the needle through the glucose, lysine, and
ornithine compartments, so that the tip of the needle can
be seen in the HZS-indole compartment.

Morton and Monaco (41) compared the enterotube and
routine media for the identification of 147 clinical cul-

tures belonging to Escherichia, Enterobacter, Citrobacter,

 

 

 

Klebsiella, Proteus, Providencia, Pseudomonas, Salmonella,

 

 

 

and Shigella. They reported an agreement of 82.3% with the
two methods. Other studies have confirmed the value of the
revised enterotube for bacteriological laboratories. The
overall agreement with conventional methods observed by
several workers as 92% (15), 64.4% (25), and 95.1% (57).
Elston et a1. (9) reported that the lysine decarboxylase
was not an acceptable alternative to the conventional
methods but found no difference in the indole test and only
small discrepancies were noted in the citrate test.

Martin et a1. (29) reported excellent agreement be-
tween the 2 test systems with the following: hydrogen sul-
fide and indole production, citrate utilization, and acid
and gas from glucose lactose. Agreement was not as good
with urea, phenylalanine deaminase, and dulcitol reactions
(85%). With the exception of the lysine decarboxylase and
urease tests, in general the results obtained in this eval—
uation (29) indicated good agreement between the two systems.

The redesigned enterotube has been reported to have
better agreement with the conventional test procedures than

the original enterotube system (57), as shown in Table l.

TABLE 1

ACCURACY OF IDENTIFICATION OF UNKNOWN ENTERIC
CULTURES BY THE ORIGINAL AND
REDESIGNED ENTEROTUBE (57)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Egiigiizi: Redesigned Enterotube
Organisms No.
% Correct/ %
Correct No. Correct
Tested

Arizona 82.8 28/28 100

Citrobacter 69.7 ‘23/23 100

Klebsiella 79.7 30/30 100

Proteus mirabilis 90.0 27/27 100

P. vulgaris 63.6 11/11 100

Serratia 97.2 27/27 100

Shigella 73.2 19/19 100
Providencia 84.8 30/31 96.8
Enterobacter hafniae 80.0 29/30 96.7
E. cloacae 20.0 28/29 96.6
E. aerogenes 28.6 27/28 96.4
Escherichia coli 95.4 27/28 96.4
P. morganii 83.3 15/16 93.8
E. liquefaciens 12.5 20/22 90.9
P. rettgeri 75.0 19/21 90.5
Salmonella 87.2 25/28 89.3
Edwardsiella 90.0 14/16 87.5
Total 399/414 Avg. 96.4

 

aResults obtained in a previous study with a total of

624 tests.

From the literature, one can conclude that the
enterotube will accurately identify between 80% to 100% of
the enteric bacteria isolated in the clinical laboratory.
It permits the rapid identification of 5 groups and 17
species of the family Enterobacteriaceae with one tube.
Compared with the conventional methods, the advantages of
this useful device for the routine bacteriological labora-
tory include the following: provision of the routinely
used media in a single tube, simplicity in use and reading,
and a marked saving in time in carrying out procedures and
media preparation, elimination of glass washing, and a

saving of reagents, media, and storage space.

R/B Enteric Differential System

 

This is a two-tube system which incorporates in one
tube the test for hydrogen sulfide production, phenylalanine
deaminase, lysine decarboxylase, lactose utilization, and
gas production from glucose, and in the other tube, the
tests for indole production, ornithine decarboxylase, and
motility. Sellers (50) concluded that this system provides

essentially the same answers as the conventional system and

 

 

 

Fig. l.--Enterotube

within 24 hours usually provides a genus identification and
often a species identification. O'Donnell et al. (42) con-
cluded that the R/B system provides an acceptable alterna-
tive to conventional techniques, even though they found the
motility reaction unreliable, and the indole reaction less
sensitive than that of standard procedures. Martin et a1.
(28) obtained very poor results with the R/B system: 44%
of all cultures tested could not be accurately identified
because half gave atypical or produced conflicting reactions
in the R/B system and half did not ferment glucose. They
concluded that the disadvantages of the R/B system out-
weighed its advantages and did not recommend its use.

Smith and co-workers (53) at the Center for Disease
Control, tested the 8 components of the R/B tube exactly as
prescribed supplementing the system when indicated with 10
additional tests suggested by the manufacturer. In the
comparison, 18 tests conventionally used were employed.
Good agreement was obtained except with gas from glucose,
motility, and lysine decarboxylase. An average of 89.6% of
these cultures were correctly identified by the R/B system,
whereas 98.2% were accurately identified by their conven-

tional procedures. They concluded that the R/B system is

10

not an alternative to our conventional system but that it
should perform reasonably well with typical enteric bac-
teria, provided the manufacturer's instructions are fol-
lowed precisely and the user does not attempt to make the
system perform beyond its capabilities. The R/B system
must not be considered as a substitute for classical
methods, but rather an initial step in identification which
may save the careful user both time and money. In most
cases, the R/B system was quite accurate in placing the

test cultures in proper general groups.

Modified R/B System

The product was repackaged in the "Beckford Tube,"
a tube constricted near the base, to improve the performance
of the system. The constriction on the primer tube sep—
arates the lysine decarboxylase test from the phenylalanine,
lactose, glucose, H25 tests in that tube. In the second
tube, the constriction confines the indole-ornithine—
motility medium which contains less agar than used pre-

viously. Another difference in the modified system is that

R/B tubes are inoculated with an extremely small, flattened

11

100p, not over 1 mm. wide. The revised R/B instructions

include differentiation of Enterobacter species, Serratia,

 

and non-SHz-producing Salmonella species using additional

 

tests for deoxyribonuclease production and Sorbitol,
rhamnose, and raffinose fermentation.

Smith et al. (52) usingthe modified R/B system,
reported the correct identification of 95.5% of the 200
cultures tested, as shown in Table 2, and a correlation of
the modified R/B system and conventional tests of 96.3%.

Isenberg and Painter (22) thought that the modified
R/B system performed well in the primary identification of
various species of enterobacteria. The modified system
tested by McIlroy (31) gave reactions comparable to con-
ventional tests. These reactions were hydrogen sulfide
production, lysine decarboxylase, ornithine decarboxylase,
and acid and gas from glucose. The findings obtained in
different studies indicate that the R/B system in its re-
cently modified form represents a useful alternative ap-

proach for the identification of the enterobacteria.

12

Positive
: Pos. or Neg.
——:: Negafive

0+
n

O

 

Phenylalanine
Lactose

Glucose
_-,, Ornithine
Lysine , . Motility

i}.

Fig. 2.--Basic R/B system

ACCURACY OF IDENTIFICATION OF UNKNOWN
ENTERIC CULTURES BY THE R/B SYSTEM (52)

13

TABLE 2

 

 

 

 

 

 

 

 

 

 

 

Organism No. correct/ Per cent
No. tested correct
Arizona 16/16 100
Klebsiella 29/29 100
Proteus (all) 24/24 100
Providencia 12/12 100
Serratia 10/10 100
Shigella 12/12 100
Escherichia coli 10/11 91
Citrobacter 16/17 94
Salmonella 15/17 88
Enterobacter (all) 39/42 93
Enterobacter hafniae 8/9 89
Edwardsiella 8/10 80
191/200 95.5
(Total) (Avg)

 

l4

API Microtube System

 

The Analytab Products Inc. miniaturized system (API)
utilizes a strip containing several small plastic chambers.
Each test is performed within one of these small sterile,
plastic compartments. Each chamber contains the appropriate
substrate and reagents and is affixed to an impermeable
plastic backing. The 20 tests are for: ONPG, arginine
dehydrolase, lysine decarboxylase, ornithine decarboxylase,
tryptophan deaminase, citrate utilization, hydrogen sulfide,
urease, indole, acetoin, gelatinase production and fermen-
tation of glucose, mannitol, inositol, sorbitol, rhamnose,
sucrose, melibiose, amigdaline, and arabinose. The system'
requires incubation at 37°C for 24 hours.

Results of the comparison of the API method with
the standard System showed an agreement of the order of 95
'to 100% (51); citrate was the only test that had an agree-
ment under 95%. Both Washington et a1. (59) and Smith
et a1. (51) found that the conventional system detected
more urease positive cultures than the API system.- Gardner
et al. (12) were able to identify only 25% of the entero-
bacteria recovered in a clinical laboratory with the API

system. They concluded that the system was not a reliable

15

substitute for the standard biochemical methods of identi-
fication employing tube media. Brooks et al. (6) reported
that the API system as a whole is not without certain dif-
ficulties. The task of filling the incubation chamber with
water, preparing the bacterial suspension and inoculating
each capsule was cumbersome and required an average of 3
minutes per organism if a series of 10 to 15 organisms were
prepared and inoculated at the same time. Smith et a1. (51)
reports an agreement of 96.5% between the API system and

conventional identification as shown in Table 3.

Auxotab

The system consists of a card with 10 capillary
units, each containing a specific biochemical test. One
card provides the following tests: viability control
(resazurin reduction), malonate utilization, and the pro-
duction of phenylalanine deaminase, hydrogen sulfide, acid
and gas from sucrose, O-nitrophenyl—beta-D-galactopyranoside,
lysine decarboxylase, ornithine, decarboxylase, urease, and

tryptophan (indole).

16

TABLE 3

AGREEMENT BETWEEN API SYSTEM AND
CONVENTIONAL IDENTIFICATION (51)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Organism C/Ta % Correct
Enterobacter cloacae 25/25 100
Enterobacter hafniae 19/19 100
Edwardsiella 18/18 100
Klebsiella 21/21 100
Proteus mirabilis 16/16 100
Proteus morganii 20/20 100
Proteus vulgaris 11/11 100
Providencia 28/28 100:.
Salmonella 28/28 100 I
Shigella 12/12 100
Enterobacter aerogenes 21/22 95.5
Proteus rettgeri 18/19 94.7
Arizona 27/29 93.1
Escherichia coli 26/28 92.9
Citrobacter 21/23 91.3
Enterobacter liquefaciens 19/21 90.5
Serratia 23/26 88.5

Avg: 96.4

 

aC/T, No. correct per no. tested.

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17

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l. YE. HEF («announcement mm" mo» ANALYYAO Pnoouctsmc

 
   

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WM). .0 "U‘IIQE’ISBSIQJ

 

Fig. 3.--API (sustrate card)

18

A colony from an agar plate is transferred to a
5 ml. volume of brain-heart infusion and incubated at 37°C
for 3.5 hours. The broth culture is centrifuged (350 X g,
5 min.), after which the sedimented cells are suspended in
1.8 ml. volumes of distilled water at pH 6.7. The cell
suspension is then inoculated into the upper opening of
each capillary tube on the card and incubated at 37°C for
18 hours. The viability control tube containing resazurin
is examined, and if there is positive reduction, the card
is incubated for an additional 6 hours at 37°C.

Washington et a1. (60) found the use of this
product to be laborious and potentially hazardous to those
working with it. The percentage of correct identifications
was 83.8% at the species level and 90% at the generic level.
The obvious need for modification of this product was
pointed out by Rhoden et al. (47). Their study indicated
that the "auxotab" system (Colab Laboratories, Inc.), al-
though offering the user the advantages of "same day"
identification and moderate to good identification accuracy,
had several disadvantages which made it a poor alternative
to the conventional system or to other similar products on

the market. The disadvantages were: the potential danger

19

of infection of laboratory personnel when using the card,
long preparation time, variability of test sensitivity, and
subjective interpretation of results. The accuracy of
identification with the auxotab system was 87.1% (47), as

shown in Table 4.

Reagent-Impregnated Test Paper Strips

 

A strip of specially purified filter paper having
certain arbitrary dimensions, and used as an inert support,
can be impregnated with specific quantities of specific
reagents, forming a simple test system. Reagents can be
applied to the paper with high levels of precision ensuring
producibility. Following reagent applications, strips are
dried to a determined moisture level and maintained at that
level by a dessicant. Some reagents are unstable in solu-
tion, but are stable when dry and maintained under proper
conditions of reduction.

The strip provides intimate contact between bac-
teria and substrate for a period of time before a reaction
is measurable. Specific substrates and a pH indicator are

applied to the strip and a color identification band is

ACCURACY OF IDENTIFICATION WITH THE

20

TABLE 4

AUXOTAB SYSTEM (47)

 

 

No. correct/

 

 

 

 

 

 

 

 

Organism No. tested % Correct
Citrobacter 23/23 100
Klebsiella 29/29 100
Providencia 28/28 100
3. vulgaris 11/11 100
Salmonella 28/28 100
Shigella 19/19 100
E. coli 27/28 96.4
B. morganii 19/20 95.0
E. hafniae 26/29 89.7
g. mirabilis 23/26 88.5
E. Earda l4/l7 82.3
g. rettgeri 18/22 81.8
Arizona 23/29 79.3
E. cloacae 21/29 72.4
Serratia, E. liquifaciens,

E. aerogenes 54/79 68.4
Total 363/417 Avg. 87.1%

 

21

added. Since time is one of the real differences between
strip technology and conventional media, the amount of
material applied to paper is important in addition to the
nature of the substrate. Since it takes less time for an
organism to metabolize a small amount of a substrate than

a larger amount of substrate, micro amounts of substrate
plus the indicator are applied to the strip. There are
several biochemical reactions in which the reagents re-
quired to detect an end product of metabolism are toxic to,
or interfere with, the production of the end product. Such
reactions include those involving nitrate reductase,
phenylalanine deaminase, and indole, and the Voger-Proskauer
Test. In these circumstances, the detection reagents must
be separated from substrate. Since the design flexibility
of an inert paper support allows impregnation of reagents
at any point, such reagents as acidified P-dimethyl-amino
benzaldehyde naphthylamine and sulfanilic acid, and or a
ferric salt, are applied to the paper in an area separated
from the substrate by a water-proof barrier. A second
waterproof barrier is then applied to the strip to localize

color formation.

22

One technique using paper strips is the Pathotec
reagent system (General Diagnostics). It consists of dry
paper strips impregnated with reagents that detect the
presence of specific enzymes or metabolic end products.

The following tests can be carried out: nitrate reduction,
malonate utilization, esculin hydrolysis, oxidase, pheny-
lalanine deaminase, urease, indole, lysine decarboxylase
and acetoin production;

The gram-negative enterobacteria were tested by
Matsen and Sherris (33) using 7 paper strips and their con-
ventional counterparts. Excellent correlation was obtained
with the oxidase, phenylalanine deaminase, and Voger-
Proskauer tests; good correlation with oxidase, acetoin
and urease, and disagreement with the lysine decarboxylase
test. Grunberg et a1. (17) also reported a rather good
correlation with the test for indole, phenylalanine deami-
nase, and urease. However, the best correlation between
the paper strips and the standard media was reported by
Rosner (48), who found a 99.3% correlation between results.

The strip tests which provided the most accurate
and consistent results were those in which the color

changes resulted from specific interaction between metabolic

23

products or enzymes of the organisms and the substances in-
corporated in the strip. The results with test strips which
depended on pH change due to action of the organisms on a
substrate contained in strips were generally less satisfac-
tory (33).

The Pathotec system offers an identification system
giving results in good agreement with the conventional pro-
cedures. Equipment has been developed that will accurately
apply specific volumes of reagents per unit area of paper
and thus paper strip systems are highly reproducible as well
as precise. Since growth is not a factor in measuring a
biochemical event, data can be obtained in 4 hours or less.

Test strip systems are stable for at least 2 years
at 4°C. The pathotec requirement for a heavy loopful of
bacterial suSpension is considered a disadvantage compared

with other systems like the enterotube that require only a

simple multiple inoculation.

24

TABLE 5

ACCURACY OF IDENTIFICATION OF UNKNOWN
CULTURES BY THE PATHOTEC SYSTEM
(10 TESTS) (54)

 

 

No. correct/ %

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

organism No. tested Correct

Arizona 28/28 100.0
Citrobacter diversus 10/10 100.0
Proteus vulgaris ll/ll 100.0
Providencia 31/31 100.0
Shigella l9/l9 100.0
Klebsiella pneumoniae 31/32 96.9
Enterobacter hafniae 27/28 96.4
Proteus mirabilis 26/27 96.3
Citrobacter freundii 24/25 96.0
Serratia liquefaciens/

Serratia marcescens 47/49 95.9
Proteus rettgeri/P. morganii 34/36 94.4
Salmonella 30/32 93.8
Escherichia coli 28/30 93.3
Enterobacter cloacae 27/29 93.1
Xgrsinia 9/10 90.0
Edwardsiella 14/16 87.5
Enterobacter aerogenes 24/28 85.7
Enterobacter agglomerans 8/10 80.0
Klebsiella ozaenae 8/10 80.0

 

Klebsiella rhinoscleromatis

 

8/10

80.0

 

DETECTION AND IDENTIFICATION OF BACTERIA

BY GAS CHROMATOGRAPHY

The isolation and identification of an individual
bacterial species in a sample from a natural environment or
in a clinical specimen is commonly a time-consuming process.
The conventional methods usually identify the organism in
question correctly; however, the identification is not
available until a day or 2 after receipt of the sample by
the laboratory. To speed up identification of microorgan-
isms, efforts have been directed toward developing rapid,
automated identification procedures, included among the pro-
cedures are: immunologic techniques (13), bioluminiscent
reactions (26), radioisotopes (30), electrophoresis (19),
and pyrolysis gas-liquid chromatography (46).

Gas chromatography has become the preferred method
for rapidly and accurately analyzing any volatile substance.
In essence, the volatile material is injected into a column
containing a liquid absorbant supported on an inert solid.

The basis for the separation of the components of the

25

26

volatile material is the difference in the partition coeffi-
cients of the components as they are carried through the
column by an inert gas such as helium. The carrier gas
transports the injected volatile material into a column,
where the components partition into the liquid absorbent
and separate; eventually a fraction passes through a suit-
able detecting device, which sends signals to a recorder,
which in turn converts the signals into a useful sequence
of peaks.

Gas chromatographic techniques have been employed
to some extent for detection or identification of micro-
organisms for some years. Oyama (43) proposed a technique
involving a chromatographic characterization of the products
of pyrolysis of bacteria or substances of biological origin.
To determine the possible existence of life in extraterres-
trial environments, Reiner (46) used pyrolysis coupled with
gas chromatography for the purposes of differentiating be-
tween bacterial strains. Abel et al. (1) and Yamakawa and
Veta (61), Henis et al. (18) observed that distinct patterns
of volatile metabolic products characterized the various

bacteria examined by means of a gas chromatograph fitted

27

with an electron detector* (ECD) and a flame ionization
detector** (FID). An approach to the rapid detection
or differentiation of microorganisms not previously in-
vestigated, involved the use of the gas chromatograph
with highly sensitive detectors for the examination of
bacterial products which are either volatile or can be
converted to volatile derivatives.

Many volatile compounds are synthesized by mi-
croorganisms during their growth (24, 55). Mitruka et
al. (35, 37) reported that gas chromatographic methods
were sensitive and precise enough to detect certain
chemical changes in animal body fluids or tissues or

in in vitro cultures, which were related to characteristic

 

*Electron Detector. This is a detecting device that
responds to sample components which have a strong
affinity for electrons. This detector is extremely
sensitive to halogen-containing compounds and a lim-
ited group of other compounds. The dynamic range is
quite limited and its usefulness is not as universal
as the thermal conductivity and hydrogen flame ioni-
zation detectors.

**Flame Ionization Detector. In theory, when organic
material is burned in a hydrogen flame, electrons and
ions are produced. The negative ions and electrons move
in a high voltage field to an anode and produce a very
small current, which is changed to a measurable current
by appropriate circuitry. The electrical current is
directly proportional to the amount of material burned.

28

metabolic activities of microorganisms. Their approach
to the problem of rapid detection and identification of
microorganisms was based on the assumption that each
type of microbial species forms characteristic or pos-
sibly unique compounds in its growth environments or
may yield typical GC elution patterns on analysis of

the "infected" sample. Gas chromatographic (GC) tech-
niques were employed for identification of microorgan-
isms in clinical specimens in the following ways: 1) de—
tection of one or more characteristic products of micro-
organisms in vitro and in vivo; 2) analysis of the
products of microbial isolates after a brief incubation
in an enriched broth culture medium; and 3) comparison
of metabolic profiles (fingerprints) of microorganisms
in “infected" and “uninfected" specimens.

Mitruka's (34) results showed that highly sen-
sitive GC methods can be employed for the identification
of microorganisms by analysis of specific components
generated either by microbial activity in vivo or by
the specific host responses to a particular infectious
agent. Identification can be facilitated if the early

microbial product detected is a unique metabolite (34).

29

Previous studies on viruses (36, 39), and bacteria (37,
23) seemed to provide evidence to support this.

Comparison of gas chromatographic patterns of
serum or blood from infected and uninfected human pa-
tients was used as a means of rapid identification of
microorganisms or an infectious disease (34). Compar-
ison of GC profiles of human clinical specimens in
conjunction with mass spectrometry* and computer analysis
have been applied in the diagnosis of infectious disease
(23), metabolic disorders (38), and other pathologic
processes (21, 61).

A study was made to test the usefulness of the
method just referred to for the rapid identification of
microorganisms (34). Strains of bacteria were analyzed
for their production of acids from the Kreb cycle and
related compounds. A characteristic pattern was ob-

tained for the organic acids of each strain studied.

 

*Mass Spectrometry. Mass spectra obtained by field
ionization mass spectrometry are generally character-
ized by the presence of prominent molecular ion in-
tensities and minimum fragmentation. In principle,
FI-MS is a suitable technique for the analysis of
multicomponents mixtures of organic compounds that
can be volitilized by evaporation.

30

The results indicated that the identification of unknown
bacteria (or other microbial species) in clinical spe-
cimens can be achieved in less than 6 hours after pri-
mary isolation by examining for the presence or absence
of a few typical products in pure cultures. The appli-
cation of GC methods as a routine diagnostic procedure
would be possible if a large volume of pertinent data
were stored in a computer. Then the data from an un-
known could be compared with the stored data making
possible rapid identification.

The studies suggested that gas chromatography
is a potential useful tool for the rapid, automated,
identification of microorganisms in clinical specimens.
It may be possible in the near future to apply auto-
mated GC methods in diagnostic microbiology labora-
tories for identification of organisms either by direct
analysis of clinical specimens or by analysis of prod-
ucts from pure cultures.

A number of clinical laboratories now use simple
gas chromatography on a routine basis to aid in the
rapid and accurate identification of anaerobic bacteria.

With the information from chromatography and gram's

31

stain, genus identification of isolates is usually com-
pleted in 24 to 36 hours after the specimen is received.
Following chromatography, fewer biochemical tests are

required to speciate each isolate (56).

AUTOMATION OF COLONY IDENTIFICATION

AND MUTANT SELECTION

In this system a large scale colony processor
called "The Dumbwaiter" is used. This machine is able to
pour agar into special small glass trays or compartments
which are mounted on frames which are stacked in heat-
sterilizable magazines, thus, eliminating the need for
large numbers of agar-filled petri dishes. Each tray or
compartment is inoculated with microorganisms in a regular
pattern. As the agar trays move through the dumbwaiter,
at incubation temperature, they can be photographed at in-
tervals. The time-lapse series of photographs are analyzed

by a flying spot scanner* and computer combination.

 

*Flying Spot Scanner. This apparatus is used to scan images
by measuring levels of gray on the photographic image.
After the scanner has found a colony and determined its
boundaries, a measurement of the optical density of the
image is made at several 100 points across each of a few
typical diameters of the colony; thus an optical density
profile is generated for each colony photographs, using
each of 3 different colors.

32

33

The system is able to identify many bactereria by
colony morphology and find specific classes of mutants
based on colony morphology, growth rates under controlled
environmental conditions, response to drugs and nutrients,
and other optically detectable traits. In addition, extra
nutrients or drugs may be sprayed on the agar and a por-
tion of the growing colonies can be replicated onto fresh
agar by mechanically picking and reinoculating clones of
interest.

It is possible to establish desired gaseous envi-
ronments, growth temperatures, and light levels. A design
prototype of the dumbwaiter is now being used for mapping
mutants and for making exhaustive genetic maps of E. coli.

Some other possible applications are the following:
monitoring the type, extent, and routes of contamination
in food, water, air, and other materials, carrying out
clinical microbiological procedures, and providing data
for the understanding of the epidemiology of infectious

disease (14).

34

 

Fig. 4.--The dumbwaiter (large scale colony processor)

CULTIVATION OF MICROORGANISMS

ON A SUBSTRATE TAPE

Agar media can be coated as a layer approximately
2 mm. thick onto a flexible substrate tape over a length
of several meters. All types of conventional semisolid
agar used in petri dishes can be applied in the same way.
Once the agar has attached to the carrier tape the latter
may be rolled up and handled without the danger of separat-
ing the cultivation medium from the carrier. Rolls of
coated cultivation tape can be stored easily preventing
evaporation.

Burger and Quast (7), using dilution'plating of
bacterial suspensions, tested the experimental prototype
of the "plating automate." They reported on the use of
three loops working synchronously in the apparatus follow-
ing the usual microbiological routine inoculation. As the
tape moves through the surface of the plating automate,
the first loop takes up a drop of the liquid specimen from

a test tube and plates it in a meandering path on the

35

36

surface of the agar layer, while the second loop passes
through the lines made by the first and distributes the
bacteria fetched en route in a similar way. The third 100p
repeats the steps of the second loop; thus, depending on
the bacterial density of the sample, single colonies were
obtained after incubation along the lines of the second
loop, or with very high bacterial densities along the lines
drawn by the third 100p. Plating areas of 9 x 60 mm. were
sufficient to obtain single colonies in each dilution plat-
ing test.

The tape can be used to perform bacteriological
counts and sensitivity tests. A broad spatula was used to
inoculate aspecific volume of specimen to the type surface
after prior incubation. The colonies are counted, visually
or optoelectronically. In sensitivity tests, sensitivity
disks are placed on top of the agar tape prior to inocula-
tion.

A carrier tape coating device for applying the
nutrient medium to the carrier tape, as well as a plating
automate for processing the agar-coated cultivation tapes
are presently under construction. Burger and Quast (7)

plan to use the plating automate for the purpose of

37

screening specimens for enteropathogenic bacteria like
Shigella and Salmonella. They found that before plating

the specimen on the cultivation tape it was necessary to

 

enrich Salmonella and Shigella using a conventional enrich-
ment medium. The dilution plating on the cultivation tape
produced single colonies after incubation; these colonies
were screened to determine if they were potential pathogens.
Such colonies are readily available from the cultivation
tape for subculture and further biochemical and immunolog-
ical identification.

Advantages of the use of the nutrient tape in the
immediate future are: the reduction of approximately 85%
in incubator space needed; a saVLng in nutrient medium of
about 60% compared with the use of petri dishes; and a
marked reduction of manpower needed in the microbiological
laboratory.

Because the specimen inoculated is liquid there is
a greater tendency for swarming to take place, e.g., with
Proteus species. The swarming can be suppressed by incor-
porating P-nitrophenyl glycerol-in the semisolid media

layer.

 

 

 

 

4!

 

 

Fig. 6.--Detail of the plating mechanism

MONITORING OF BACTERIAL ACTIVITY

BY IMPEDANCE MEASUREMENTS

Automated methods in microbiology have been slow to
develop. One of the reasons for this is the relatively
long biochemical reaction time of growing cultures. speed-
ing up of microbiological tests would therefore depend on
the development of more sensitive techniques for detection
of growth or activities depending upon growth. The growth
of bacterial cultures can be detected by the changes in
electrical impedance.3

Ur and Brown (58) pointed out the possibility of
using inpedance measurements to monitor bacterial activity
because of the increased electrical resistivity of a medium
containing active microorganisms. A method employing this
principle would have the immediate advantages of high sen-
sitivity offered by impedance measurements and simplicity
of apparatus, as compared with such other methods of moni-
toring bacterial growth as photometric systems or the

coulter counter. The impedance of an inoculated medium

39

40

changes by about 4% from the time of inoculation to the
point at which the stationary phase is reached. Such
changes could be measured easily by using bridge circuits.
To facilitate such fine measurements it is necessary to
eliminate or mitigate irrelevant activities of the micro—
organisms like noise and drift due to evaporation of water,
temperature variation, electrochemical reactions and other
processes that can alter or confuse the signal from active
bacterial cultures.

Ur and Brown (58) detected bacterial activity by
monitoring the changes in electrical impedance of broth
cultures versus the impedance of sterile broth contained
in an identical conductivity measuring cell. The impedance
changes showed that the microorganisms metabolize sub-
strates of low conductivity into products of higher conduc-
tivity (8).

Sensitive measurements of this change can be made,
and the growth of microorganisms and other cells can be
rapidly detected even with a population of only several
hundred cells, long before there is optical evidence of

growth.

41

The indications were that curves obtained with
different organisms in different media may make possible
the rapid identification of bacterial species. Bacteria
in mixed cultures can be identified by specific growth
inhibitors, such as specific antisera and antibiotics.

It was concluded (8) that automated simultaneous
electrical impedance measurements on an organism in many
selective media will provide the characteristic profiles

necessary for identification.

BACTERIAL IDENTIFICATION BY

MICROCALORIMETRY

Interesting data regarding bacterial metabolism can
be obtained by the microcalorimetry of growing cultures.
The continuous thermal monitoring of biological systems
provides a means for detection of subtle changes in metab-
olism.

Microcalorimetric analysis has been specially
useful in ecological studies (40). In 1911 microcalori—
metry was used by Hill to investigate the action of yeast
cells on cane sugar (20). The relation of heat production
to phases of bacterial growth was reported in 1929(4).
Microcalorimetry also has been useful in comparative
physiology, where alterations in thermogenesis may be re-
lated to senescence, metamorphic transformation, and en—
vironmental variations. Prat (45) demonstrated that cer-
tain bacteria generated a fluctuating but specific and
reproductible heat profile when the rate of heat production

was plotted against time. Forrest (11) obtained

42

43

characteristic profiles for different members of the family
Enterobacteriaceae by measuring with the microcalorimeter
(Instrumentation Laboratory Inc.) heat produced during
their growth in a liquid medium. Because a broad selection
of different organisms were studied, a complex culture
medium was used. Boling et al. (5) also described charac-
teristic heat profiles for different members of the family
Enterobacteriaceae.

Russell (49) used a multi-channel microcalorimeter
(Instrumentation Laboratory, Inc.) to obtain profiles of
clinically significant microorganisms. Virtually all were
identifiable by microcalorimetry using a single medium.
Some profiles were identifiable within 3 hours of the onset
of heat production, whereas others required as long as 14
hours. Strain differences within species were detectable
by this method.

Investigation of facets of microcalorimetry are
planned for the future (49). Foremost will be the exami-
nation of many more strains of organisms to increase the
reliability of identification. Another area needing
further study is the development of culture media to

optimize differential heat profiles, with special empahsis

44

on organisms that do not grow well in broth because of
special growth requirements. Heat profiles may be useful
in probing taxonomic problems through characterizing unique

features of given populations of microorganisms.

45

 

 

 

 

 

 

/ a H _- d
/\ 1 I
I" v
/ I, {3,
It _‘ _:fi_—” :17. " ”'7’” If— '—/‘.: — - 7::
I b i '4 e
/ , :
/ I; ,I
// I I .._..»
I / “\w /‘ '3
' c f I
II :3 1'
1 ;I '
//.\/~J i /\’-\

 

 

Fig. 7.--Bacterial heat profiles by microcalorimetry.

Six named heat profiles are shown. For each,
the abscissa represents time, with 2-h intervals indi-
cated by scale marks, and the ordinate represents heat
production; with scale marks at 0, 20, and 40 ucals‘
ml,'1. a, Enterobacter aerogenes; b, Klebsiella; c,
Proteus vulgaris; d, Enterobacter cloacae; e, Escherichia
coli; f, Proteus rettgeri(5).

AUTOBAC I, AUTOMATED ANTIMICROBIAL

SUSCEPTIBILITY SYSTEM

The Autobac I (Pfizer Diagnostic Division) suscepti-
bility testing system can determine within 3 to 5 hours the
susceptibility of a clinical isolate to at least 12 antibi-
otics. Each antibiotic is evaluated against the organism on
a numeric scale of 0 to l, and, simultaneously, the numerical
evaluation is interpreted by the system as resistant, inter-
mediate, or sensitive. Both the numerical result and the
interpretation are printed on a convenient report form.

The system consists of 4 components: 1) an incuba-
tor/shaker, which incubates the cuvette and agitates it over
the 3-hours incubation period; 2) a disk dispenser, which
loads the cuvette with a panel of elution disks; 3) a trans-
parent plastic cuvette, which automatically distributes the
inoculum prepared from a clinical isolate to 12 test
chambers and 1 control chamber that contains the antibi-
otics, distributed in the form of antibiotic-impregnated

"elution disks"; 4) an automated light-scattering photometer,

46

47

which standardizes the original inoculum, reads the amount
of growth in each chamber of the cuvette, and prints out
both the numerical ratio to a resistant, intermediate, or
sensitivity rating correSponding to the usual Kirby—Bauer
interpretive results (3).

The Autobac I susceptibility test method evolved
from light scattering studies of broth suspended bacteria
in the presence and absence of a wide variety of antimicro-
bial agent. Such studies permitted the optimization of
light scattering conditions for detection of changes in the
concentration of bacteria. In addition, a number of micro-
biological parameters were varied in order to maximize the
agreement between the interpretation afforded by this 3-5
hours light scattering method and the 18 hour manual methods
such as the disk diffusion procedure of Bauer (3), the agar
overlay version of the Bauer-Kirby method (2), and the
WHO/ICS agar dilution method (10).

The principle upon which the Autobac I suscepti-
bility testing method is based is the in vitro effect of
antimicrobial agents on the growth of microorganisms in
nutrient media at 30°C. The effects of antimicrobials on

growth are detected by means of light scattering and

48

compared with the control growth after a minimum of three

generations have occurred. The individual antimicrobial

disk masses have been adjusted to insure a high degree of

interpretive agreement between the Autobac I light scat-

tering index (LSI) and the results of the STANDAR Kirby-

Bauer method (3). The photometer automatically reads the

light-scattering value in each chamber and calculates a

light-scattering index (LSI) for each antibiotic. Two

measured values and one constant based on the initial

inoculum

concentration are used in the calculation.

the light scattered in the uninhibited or control
chamber.

the light scattered in each of the twelve test
chambers.

the light scattered by the experiment at time zero.
This value is equal to the initial inoculum concen—
tration set up by the same photometer when the
saline tube was standardized. It is therefore
known, retained in the machine memory, and entered
as a constant in the calculation.

The following parameters are then calculated (44).

light scattered in control chamber.
light scattered in chambers with antibiotic.

light scattered by the initial inoculum.

49

Growth index,

th , tr 1 h mb
control chamber: log G /Gk = log (grow ’ con 0 c a er\
c

initial organism conc.

 

Growth index, rowth control chamb
I r
inhibition chamber: log G /G = log (9 . , . Ce )
c x growth Wlth antibiotic

 

1 <3 G 1 c; - 1 G
09 C/ X 09 C 09 X

Light-scattering

Index (LSI) log G /G log G - log G
c k c k

Complete resistance: log G log G ; LSI = O
c x

Complete susceptibility: log G = log Gk; LSI = l
x

McKie et al. (32) found a high correlation between
the results obtained with currently accepted standard sus-
ceptibility testing and those of the Autobac I. They also
reported that the latter offered an economically desirable
reduction in labor time, increased reproducibility, and
sensitivity, and convenience as compared with currently

used manual procedures.

 

50

 

 

 

 

Fig. 8.--Autobac I

LITERATURE CITED

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

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PRAGLIN, J., C. CURTISS, K. LONGHENRY, and J. E.

MCKIE JR. 1975. Autobac I--a 3 hour, automated anti-
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immunology, John Wiley and Sons, New York.

PRAT, H. 1963. In recent progress in microcalori-
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REINER, E. 1965. Identification of bacterial strains
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RHODEN, D. L., K. M. TOMFOHRDE, P. B. SMITH, and A.
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*Original not seen.

(49)

(50)

(51)

(52)

(53)

(54)

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(56)

(57)

56

RUSSELL, W. J., J. F. ZETTLER, G. C. BLANCHARD, E. A.
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(58)

(59)

(60)

(61)

(62)

57

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APPENDIX

MECHANISMS AND PROCEDURES OF BIOCHEMICAL

TESTS WIDELY USED FOR THE

IDENTIFICATION OF BACTERIA

APPENDIX
MECHANISMS AND PROCEDURES OF BIOCHEMICAL
TESTS WIDELY USED FOR THE

IDENTIFICATION OF BACTERIA

The performance of the common biochemical tests is
described in many texts and manuals but the actual biochem—
ical mechanisms involved are seldom referred to. An
attempt has been made below to describe briefly the bio-
chemical reactions involved.

The tests that will be discussed are listed in
Table 6 under the headings: test, key ingredients, and
principal use. Many of the tests described are ones that
have been adapted to the rapid and/or miniaturized systems

described previously.

58

59

Any aaov manoflumnomm Eoum A+ adamsmsv
maamflmnwax cam uwuomnouwucm oumaucwummmac oa

 

 

 

 

 

 

 

Any EdwcAMUmoau Eoum A+V msaaflomm mumummmm 09

 

AIV mmmomaflomnouomn
Eoum A+v ommomooooouoflz mumummmm 09

man Hosanusoum
mHMHUAU Edflvom

wmmoflxoumm
cmmonomm mm

 

umma mumnufio

 

 

umme mmmamnmu

Honuflwc Ho :Hmucmahmm= u0\©sm
:Mmufloflxo= cm mfl Esfinouomn m ma mcflshoumo 09

Umummu on

on mumnomnonumo
was no we man
IGHMpcoo Hmmd mo

ummB m0

mwumuo>£onumo
mo coflumuflawus powwow on cam .mnwucoEHmMIcoc
Eoum muoucmsumw mumuoxnonumo: nmflsmcflumflc OB

ism“ Heavens
HoumoflUcH
mumuwhnonumu

 

=umma coflumucmfiumm
mumnwwnonumu=

 

 

. mwamomcoeoosmmm mo
cowumoHMHucwvfl may CH Uflm Op cam .AIV mmcwmoumm

.m Eoum A+V mmomoHo Monomnonmucm mumummom 09

 

mDHQ Hemmuofioum
OCHGHOH<

 

umma mcficflmud

t

 

.mumsuo 0cm .momc080©smmm .Edflumuomnomsm
.mofloouasfi maamusummm mo coaumoHMAucmofl CH can 09

 

usmommu m.Honmwz
Spoun ocoummm

 

coauosooum mecoaa¢

 

coflumoflammd

mucmwkumGH hmM

ummB

 

dHMMBUdm ho ZOHBfiUHmHBZMQH mmB mom QmmD VHZOZEOUmEmfifluAfiUHZHmUOHm

o mqmda

60

 

.AIV Haoo .m Eoum
A+ mHHmsmsv Hmuomnoumucm mumaucmummmwp OBIIN

 

 

 

AIV maamcoaamm was mcouflnm
Sony A+v Hmuomnouuflo sunflucmumMMAU calla

 

 

moflommm
msououm uwnuo Scum AIV mHHHQMHHE mdmuoumlum

 

AIV maamflmnmam paw
HoHUMQOHmucm Sosa A+V maam5msv flHoo nmlla
"wumwucwuwmmao OB

 

 

 

.mHHmosHm ..m.m
.mwuwuomn nonuo mcH>MHucmofi ca was OBIIm

 

AIV mmflowmm msmuonm Hmnuo Eouw A+v mflaflnmnfle

 

msmuoum odomflummag msmuoum gunfiucmumwmwc oanum

 

 

A+V mHHmcoEHmm pmoE cam mmcofiou m

 

mHHmHmUHMva .HOOUMQOHUAU .MCONflum uomumc OBIIH

 

TV mwofloflmmgm Rm 596
A+V mpflowcofiamm mmcoaoumd mumaucmnmmmflv OBIIN

 

 

A+V aflumuumm
tam A+ >HUHQMH waamsmsv Mmocflmosuom
mmcoaovsmmm mo coaumoflmaucmofl ca me OBIIH

 

 

50.3 zoz

ucmmmmu
mxm>ox Ho Soaaunm
cosmoummua

mummasm
Edflcosfim
muonuomulm

mvflom

ocflam Hmcwmu.
ucoo HDMHSm

mumumom ommqlla

cflumamu

HmmB ZUM

 

umme maoccH

 

GOHUOSUOHm

mUHmasw
cmmouvwm

 

umoe cflumamw

 

coaumoflammd

mucmfivmumcH mow

ummB

 

A.U.HGOUV 0 GHQMH

61

.mauwuomn o>wummmc IocmuwmouomHmmlo
nomouomH Eoum commawoummouoma oumfipcmumMMHo oe smlamcwgmouuflz|o umma wmzo

 

 

 

any Hmuomnoumsfioa
Eoum A+v wamxmuoz gunfiucmeMMHo OBIIN

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

cowuodcoum
A+V mmoomanmuomnoumucm mo GOHOMOHMHuchw tam OBIIH mOZM mumuuflz
AIV wmomoao am cam suoun omoosHm
mwcwmoumm am soum A+V Haoo am mumaucmeMMHo OB .mcoummm Umuowwsm umwa com dwnumz
AI waamsmsv
mHHmCOEHmm Eoum A+ maamsmsv mcoufludllm
Alv maumuumm 0cm
.Haoo am .mHHmHQOHM Scum A+V HOMOMQOHOOCMIIH wumcoamz COAMMNHHHHD
.mcflumflpcmumMMHc cfl eflm oe ssflcom mumconz
A: maamsmsv
uwuombouuflo Eoum A+ haamsmsv acouflud
tam Aflsmwmmumm am ammoxmv maamcoEHmmllm
A-V madmmflgm
Eon m mums mmma Inmcoomm m .I
m + AA V.M .v a xadw m wamusm
.wmomoHo am Eoum A+v monomoumm HmuUMQOMODCMIIH HomeoEOHQ :oflumammonumomo
umGHDMHucmeMMHU GA can 08 mcflmmq ocflqu
coaumoflammd mucmHUmHmGH mom umma

 

A.©.UGOUV m OHQMB

 

 

 

own Hoconm
.oummasmOHnu 55Ho0m
.oUMMHSm sdflsoasm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

62

 

 

 

 

 

 

 

 

 

 

 

 

msouumm .mmoosam Hmmd couH
.Haaflomn w>flummmclemnm mo GOHHMOHMHquUH Uflm OB .omOHUSm .mmouomq Homqm mamwue
AIV wmmomfluwuomnoumucm Hmnuo Eoum A+v coflumcflammo
“meocotfl>oum cam msmuoumv mmwuoum mumaucmHmMMHU OB msHCMHmHmcmnm ocflcmamamcmnm
.AIV mwmomflnmuomnouwucm
tam .A+ adamsmsv mMQOEOUSomm .A+v mmcoEooum¢ mcflamavmcmamcmzm
tam mwuwmmflwz mo cOADMUAMAucoUH ca tam OBIIH (muawnumsmuume ummB mmmvflxo
Ans mmHHmmHnm “mane scum A+v mHHmmflgm a msouou-m
A+v maamcosamm Hmnuo Eonm
TV 232333 .m 69w 2&3 38858-4
Alv mHHmmHS> am
Eoum A+ >Ham5mov mfiaflnmnfls msmuoumllm
AIV Monomnouuwu Scum A+ waamsmsv maawcoaammulm
i-c mHHmflmnmax
Eoum A+ >Hamsmsv promnoumucmlla coaumwwxonumomo
"mumfiucmummmaw on damp oe mcflnpflcuo mcflruncuo
coflumoflammé mucwflcwuucfi mom umme

 

A.G.UGOOV m OHQMB

63

 

 

Aoonm um : oomm um +v mmficmmn umuomnoumucmuum

 

Auonm um I 00mm Om +V moauwaooonwucm macflmuowlla
"no coflpmofimaucwnfl as 638 09

«IV umuomnoumcfiom Eonw A+meHwamuozllm

 

 

 

.AIV flaoo 2m Eoum
A+ maamsmsv mHHmeQwHM cam HouomnouquMIIH
"hwflusmpfl Ho mumau:muomma© OB

 

 

mmoous

 

Ummfi
nmsmxmoum

mwmo>

.mflumuomn Hmzuo mSOHHm>IIv

.A+V wmmemU H0\wsm
xmo3 on woe 30Hc3 mo Add .mHHmflmanM
.maumuumm .HmpomnouuHU .kuomnoumuCMIlm

 

 

 

a+ Hang
flfimmuoflsmfla mDHHflomQOQwuow .mowumwmfisoconn

 

 

 

maamumouom .MOHvflHooouwuco mafimumm .maamosumllm

 

A+ mapflmmuv msmuoumlla
Humaucmpfl mam: OB

UOH Hocwnm

o
AmmZI Uummzv mmHD

 

umme mmmeD

 

coaumoflammd

mucmfltwumcfl mom

pmmB

 

A.v.ucoov m magma

64

Ammonia Production

 

Purpose and Mechanism: To determine if a bacterium can
produce ammonia from peptone.

Peptone, a digest of certain proteins, serves as the source
of amino acids for NH3 production.

Deamination of peptone is the enzymatic splitting off of
the amino group from an amino acid to yield NH

3.

R - CH - COOH Deaminase

 

I
“r R - CI- COOH + NH

+ .
NHZ H2O a keto acid

Procedure: The organism to be tested is inoculated into
peptone broth and incubated at 37°C overnight. One ml. of
the incubated broth is transferred to another tube and a
few drops of Nessler's reagent are added. A positive test
is indicated by brown, orange, or deep yellow; negative
by faint yellow. Nessler's reagent components:

1--Potassium Iodide (KI)

2--Mercuric Cloride (HgClz)

3--Potassium Hydroxide (K OH)

4--Ammonia—free distilated water.

65

Arginine Dihygrolase

Purpose and Mechanism: To determine if a bacterium is able

 

to produce the enzyme arginine dihydrolase.
Arginine undergoes hydrolytic deamination and decarboxila-

and 2NH .

tion, giving ornithine, C02, 3

Procedure: The arginine is incorporated in M¢ller broth,

 

the medium is inoculated tw'stabbing and each tube is
covered with a 4 to 5 mm layerof sterile oil. It is then
incubated at 37°C overnight, together with a tube of M¢ller
broth without arginine (control). The ammonia (NHB) which
is released is dissolved in water. The alkaline reaction
changes the color of the indicator, bromcresol blue, from

yellow (pH<5.2) to purple or red-purple (pH>6.8).

Carbohydrate Fermentation Tests

 

Purpose and Mechanism: To determine if a bacterium is able

 

to ferment a specific carbohydrate.
Acid production resulting from the breakdown of carbohy-

drates during fermentation (or alkali released by

66

utilization), causes pH change, that is detected
by an indicator.

Carbohydrate broths contain 0.5 or 1.0% of the specific
carbohydrate. Sufficient amounts of beef extract
and peptone are also included to satisfy the ni-
trogen, vitamin, and mineral needs of the organisms.

If peptone is used as the nitrogen source, it may be at-
tacked to give enough ammonia to neutralize the
acid from fermentation and may give a false nega-
tive for acid production.

-With some organisms, e.g., Bacillus species, it is better

to use ammonium salts as the nitrogen source, as

only enough is released for use in cell synthesis.

Procedure: Tubes of carbohydrates broth are inoculated

 

and incubated for usually not more than a week.

Inverted Durham tubes are often used in carbohy-
drate broth cultures to trap any gas producted during
fermentation. Among the products responsible for the
acid reaction are: lactic, acetic, succinic, and formic

acids. The gases produced are CO2 and H2.

67

The widely used indicators are phenol red and
Andrade's (acid Fuchsin + NaOH). The former in the presence
of acid turns from red to yellow; the latter turns from

colorless (near neutrality) to pale yellow.

OF Test

Purpose and Mechanism: To determine if a bacterium is able

 

to split a carbohydrate by fermentation or oxidation, or by
both. Acid may not or may be produced as an end product
of the fermentation or oxidation of a carbohydrate. The
organisms are grown anaerobically to determine the former

and aerobically to detect the latter.

Procedure: Two tubes of semi-solid agar containing 1% of

 

the carbohydrate to be tested-—usua11y glucose--are used.

Both tubes are inoculated by stabbing almost to the bottom.
One of the tubes is covered with sterile mineral oil to pro-
vide anaerobiosis. The tubes are incubated 3 to 4 days be-

fore being reported as negative.

68

 

 

 

Interpretation:

Positive in covered tube only: "Fermenter" e.g. Clostridium

Positive in open tube only: "Oxidizer e.g. Pseudomonas

Positive in both tubes: "Fermenter and oxidizer" e.g.
@2241

Negative in both tubes: "Neither fermenter nor oxidizer"

e.g., Alkaligenes faecalis.

A test is considered positive when the indicator (Phenol red)

changes from red to yellow (pH<6.9).

Catalase Test

 

Purpose and Mechanism: To detect the bacterial production

 

of the enzyme catalase. Some bacteria break down peroxides

to oxygen and water by means of the enzyme catalase.

ataas
2H0 Clek2Ho+oz+

 

N

N
4
N

Procedure: The test is carried out in various ways but in

 

all of them one looks for the presence of 02 usually as

indicated by the presence of bubbles of the gas.

69

Citrate Test

 

Purpose and Mechanism: To determine if citrate can be

 

utilized as a sole source of carbon.

The medium contains sodium citrate and the indicator
bromthymol blue (Simmons). The utilization of
citrate leaves behind sodium residue making the

medium alkaline.

Na + OH ———-'* NaOH (Alkaline)

Procedure: The agar slant is inoculated and incubated

 

overnight; if the organism is able to grow on it (i.e.
utilize citrate), the indicator bromthymol blue changes

from a green to a deep Prussian blue color.

Gelatin Test

 

Purpose and Mechanisms: To determine if a bacterium is able
to produce the enzyme gelatinase.
Bacteria producing extracelular proteolytic enzymes (gela-

tinases) hydrolyse gelatin to less complex

70

compounds and thus alter the latter's original

properties. Gelatin is broken down as follows:

gelatin ———-+ roteoses -——-+ polypeptides -———+ amino acids
P

Gelatin, a native protein, liquifies at 28-30°C. Further-
more, the solution is liquid at 37°C (incubator
temperature) and solid at 4°C (refrigerator temp-
erature)

In the laboratory gelatin liquefaction may be determined

using "tube" or "plate" media.

Procedure: Gelatin medium in tubes is inoculated.‘with the

 

organism in question and, after suitable incubation refrig-
erated. If hydrolysis has occurred, the medium remain
liquid; otherwise, it "gels."

Two types of "plate" media are used and they differ
in that one contains ammonium sulfate and the other does
not. After incubation, plates of the former are examined
for evidence of "clearing" around the lines of growth; the
latter (no ammonium sulfate) must be flooded with ammonium
sulfate solution before "clearing" gelatinase activity)is

evident.

71

Hydrogen Sulfide Production

 

Purpose and Mechanism: To determine if a bacterium is able

 

to produce hydrogen sulfide.

Bacteria may produce H S by two different methods. H S may

2 2

be produced by the deamination of sulfur-
containing amino acids such as methionine, cys-

tine and cysteine.

The production of H S is the initial step in the deamination

2

of cysteine as indicated in the following reactions:

CHZSH CH2 CH3 CH3
I -st II I n20 I
CHNH —* C - NH —‘+ C = NH """"+ C = O + NH
| 2 I 2 I I 3
COOH COOH COOH COOH

Also H28 may be produced by the reduction of inorganic sul-

fur compounds, e.g. thiosulfates, sulfates, or sulfites.

Procedure: H25 is detected using salts of heavy metals

 

(e.g. ferrous ammonium sulfate, lead acetate) which in its
presence produces black metal sulfides. Usually salts are
incorporated in the medium like the T81 used for identifica-
tion of enterobacteria and if H28 is produced the medium

is blackened.

72

In addition, a more sensitive method is used in
identification of some bacteria, e.g. Brucella, Pasturella.
Filter paper strips saturated with the salts may be placed
in the mouth of the inoculated. tube. In this case, H28
evolves from the medium and the filter paper strip turns

black. The paper strip method is 10 times more sensitive

than the test using "agar" medium.

Indole Test

Purpose and Mechanisms: To determine if a bacterium is able
to break down enzymaticallytryptophan with the re-
lease of indole.

The amino acid tryptophan can be hydrolyzed by some microor-
ganizms into several products, one of which is in-
dole. These microorganisms produce the enzyme
tryptophanase which in combination with the
coenzyme,pyridoxal phosphate, converts tryptophan
into indole, pyruvic acid, and NH3 in ratios almost

exactly 1:1:1.

73

CH - CH - COOH

 

/2 I CH
NH 3
‘i 2 H 20 l
_ 3- + c =<)+ NH
N triptophanase \\ri I 3
I COOH
H
H
TryptOphan Indole Pyruvic acid

Procedure: The production of indole from a peptone broth con-

 

taining tryptophan is detected by adding Ehrlich's or Kovac's
reagent to the culture. If indole is present a red layer

forms on top of the culture.

indole + P-dimethyl-amino benzaldehyde'——-“* Quinoidal red-violet
compound

KCN Test

 

Purposg and Mechanism: To determine if a bacterium can grow-
in very dilute potassium cyanide broth (1:13,300).

Organisms that do not grow are poisoned by KCN.

Procedure: A tube containing KCN broth is inoculated with

 

bacteria to be tested and incubated over night.

74

The test is read as positive when visible turbid-
ity indicates growth,or negative when no growth is observed.
The tube should be incubated 48 hours before re-

porting the test to be negative.

Lysine Decarboxilation

 

Puppose and Mechanism: To test the ability of bacteriatx>decar-

 

boxylate the amino acid lysine.
Lysine is decarboxylated by some bacteria to the amine
cadaverine liberating C02.
Since the reaction results in the accumulation of
an amine which is basic in nature, it is possible to detect

decarboxylation by measuring the rise in pH, as indicated by

bromcresol purple.

 

1-- NH NH
2 2
(CL ) lysine decarboxylase I
2 4 + CH + CO 1‘
I (I 2)5 2
H N-C-H NH
2 I 2
COOH
L-lysine Cadaverine
2-- bomcresol purple cadaverine bromcresol purple
(yellow) ~+ (lavender-purple)

 

75

Procedure: The medium is inoculated Mdth.the straight

 

wire by spreading the organism on the slant and then stab-
bing once to the base of the butt. Glucose-fermenters
such as the Enterobacteriaceae produce enough acid in the
butt after incubation for 24 hours at 37°C to change the
bromcresol purple to a yellow color. If in addition the
organism decarboxylates lysine the butt reverts to an
alkaline purple color after 48 to 72 hours.

Organisms that decarboxylate lysine but do not
ferment glucose produce a purple color throughout the
medium from the start. The indicator bromcresol purple

is yellow at the pH<5.4 and purple at pH>7.0.

Malonate Utilization

 

Purppse and Mechanism: To determine if a bacterium has

 

the capacity to utilize sodium malonate.
Utilization of sodium malonate releases Na. The alkalin-
ization of the medium is indicated by the color

change in bromthymol blue from yellow to Prussian blue.

76

l..-
CodSNéa CodCNiC)
CH2 + ATP + COASH m)“ CH2 + ADP + NaOH
I Coenzyme A I
- d d) -
Coo Ni) (re uce , CO SCoA
Disodium Sodium Malonyl
Malonate -SCoA
2—- bromthymol blue bromthymol blue
NaOH
(yellow) '“““""”“—+ (blue)
pH<6.0 pH>7.6

Procedure: A tube containing 0.3% sodium malonate in

 

phosphate broth with bromthymol blue is inoculated and

incubated 48 hours before being reported as negative.

Methyl Red (MR) Test

 

Purpose and Mechanism: To differentiate a mixed acid fer-

 

menting bacterium from a 2—3 butanediol ferment-
ing bacterium.

During glycolysis, pyruvate is usually produced via the
Embden-Meyerhof pathway. From pyruvate different

types of fermentation can be carried out by

77

different microorganisms. One of these is the
mixed acid fermentation in which three acids are
formed in significant amounts, acetic, lactic

and succinic. These acids produce a marked acid-

ity which is detected in the MR test.

Procedure: If the culture produces enough acid to overcome
the neutralyzing effect of the buffer, the culture medium
will be acid (pH<4). If on the other hand only a small
amount of acid is produced, it will be neutralized by the
buffer. If there is mixed acid fermentation, the methyl
red which is added will result in a red color; if the test

is negative the methyl red will be unchanged in color.

Nitrate Reduction

Purpose and Mechanism: To determine the ability of a bac—
terium to reduce nitrate (N03) to nitrite (N02).

Nitrates can act as hydrogen acceptors for many hetero-
trophic and autotrOphic bacteria under reduced

oxygen tension.

78

Procedure: To determine if a bacterium is able to reduce

 

nitrate to nitrite the organism is grown in a nitrite-free
medium containing nitrate (KNO3). After incubation
measured amounts of alfa-naphthylamine and sulfanilic acid
are added to the medium. If nitrites have been formed, a
pink or dark red color deVelbps at once.

nitrate

l-- = inorganic N03- 4+ inorganic N02—
reductase

 

NH2 ‘ GQEN

 

. . - acid
2--inorganic NO2 + ‘
SO H 50 H
3 3
. . . . . . . acid . . .
inorganic nitrite sulfanilic aCid p-diazoniumbenzenesulfonic
acid
3-- G)
NEN
I + I
S NH
03H 2 HO S NEN NH
3 2
p-diazoniumbenzene- -———+- p-benzenesulfonic acid—azo-p-a—
solfonic acid naphthylamine

(bright red color)
a-naphtylamine

79

A negative nitrite test does not necessarily mean
nitrates have not been attacked. Some bacteria are able
to reduce nitrate to nitrite and finally to ammonia. As a
result, all nitrite-negative tubes should be checked for
the presence of nitrates. This can be accomplished'by
adding a small amount of zinc to the tube. If nitrates
are present, they will be reduced to nitrites by the zinc
and a positive test will be obtained for nitrite indicating
that the original negative test was correct, i.e., that the
nitrate was not reduced.

If the zinc yields a negative test it indicates

that the original negative test was false.

 

 

_ - NH
N03 > NO “4 3
bacterial nitratases I reductases
(positive test production) (reduction but test negative)
NO - ZlnC NO -
3 ‘+ 2

 

(True negative test, yields positive test with reagents.)

80

Ortho-nitrophenyl-B-D-Galactopyranoside (ONPG) Test

Purpose and Mechanism: To differentiate delayed lactose-

 

fermenters from non-lactose fermenting bacteria.
An organism may be "lactose-negative" not because it lacks
"lactase," but because it lacks enough permeases (enzymes
permitting passage of material through the cell membrane).
The enzyme Beta-galactosidase splits the Beta-galactoside

bond in both lactose and ONPG.

 

 

l-- CH OH
2
O
NO
2 OH N
H CK -galact051dase ’// 2
H +
H OH fi‘r
O-nitrophenyl O-nitrOphenol B-D-galactose

B—D-galactOpyranoside

(ONPG)

OH
NO

lkal' H
a ine p yellow color

 

O-nitrophenol

81

Procedure: A tube of 0.2% O-nitrophenyl B-D-galactopy-

 

ranoside in peptone water is inoculated with the bacteria
to be tested and incubated overnight. In a positive test
Beta-galactosidase changes colorless O-nitrophenyl-Beta-

D-galactopyranoside to yellow 0—nitrophenol.

Ornithine Decarboxylation

 

Purpose and Mechanism: To test the ability of bacteria

 

to decarboxylate the amino acid ornithine to the

amine utrescine, liberating CO Since the reac-

2.
tion results in the accumulation of an amine which

is basic in nature, it is possible to detect

decarboxylation by measuring the rise in pH.

 

l-- NH
2 NH
I - I 2
(CH ) ornithine decarboxylase (CH ) + C0 +
23 y 24 2
HN-C-H ' I
2 NH
I 2
COOH
L—ornithine putrescine carbon dioxide
2-- bromcresol purple bromcresol purple
Putr '
(yellow) esc1ne . (lavender-purple

 

pH<5.2 pH>5.2

82

Procedure: A tube of 1% L-ornithine in M¢ller broth with

 

a layer of sterile paraffin or mineral oil is inoculated
and incubated overnight. A color change in the medium from

yellow to lavender-purple denotes a positive reaction.

Oxidase Test

 

Purpose and Mechanism: To detect those bacteria that pro-

 

duce indophenol (cytochrome) oxidase.

The flavoproteins and cytochromes are associated in an
organized particulate structure, the electron-
transport particle. Within this particle, elec-
trons pass from one component to the next and
ultimately in a reaction catalyzed by the enzyme

cytochrome oxidase to oxygen.

CYT a CYT a
(reduced) (oxidized)

Cytochrome oxidase

/ N

02 H20

 

83

Procedure: The oxidase test is carried out by adding

 

several drops of aqueous di-cn:tetra-methyl-P-phenylene-
diamine to bacterial colonies. Within a minute or so,
oxidase-positive colonies turn anywhere from a lavender
to dark purple approaching black. Oxidase negative

colonies show no color change.

 

N(CH3)2
N(CH )
32
cytochrome
+ +02 ‘ N +2H20
\\\ _oxidase
OH
O
N.N.-Dimethyl—P a-napthol Indophenol
phenylenediamine blue

"Oxidase disks" available commercially can be
placed on plates in areas of growth. After saturation of
the disk with 2 or 3 drOps of water, the aniline reagent
diffuses from the disk into the surrounding medium, and

oxidase positive bacterial colonies become black.

84

Phenylalanine Deamination

 

Purpose and Mechanism: To test the ability of a bacterium

to diaminate phenylalanine to phenylpyruvic acid.

 

 

H O
| //
CH -C-COOH CH -C

2 2 \
I COOH
NH (phenylalanine + NH +

2 7‘ 3
deaminase)
Procedure: To test for presence of phenylpyruvic acid

 

a tube of 0.25% phenylalanine agar is inoculated
with the bacteria to be tested. After incubation the test
reagent (10% aqueus ferric chloride) is added to the tube.
Phenylpyruvic acid is detected by a reaction with ferric

ions which produces a specific color change.

Triple Sugar Iron Agar (TSI)

Purpose and Mechanism: T81 is used for determining carbo-
hydrate fermentation and hydrogen sulfide produc-
tion as the first step in the identification of

gram-negative bacilli.

85

T81 agar contains the three sugars, glucose, lactose, and
sucrose; phenol red indicator to indicate fermen-
tation; and ferrous ammonium sulfate to demon-

strate hydrogen sulfide production.

Procedure: The media is. inoculated by spreading the cells

 

on the slant with a straight needle and then stabbing once to
the base of the butt.

The glucose concentration is one-tenth of the con-
centration of lactose and sucrose in order that the fermen-
tation of this carbohydrate alone may be detected. The
small amount of acid produced by fermentation of glucose
is oxidized rapidly in the slant, which will remain or re-
vert to alkaline; in contrast, under lower oxygen tension
in the butt, the acid reaction is maintained. To promote
the alkaline condition in the slant, free exchange of air
must be permitted through the use of a loose closure.

The reactions should be read ideally after 18 to 24 hours.

Results are interpreted as follows:

 

 

slant butt sugar fermented
red red none
red yellow Glucose only

yellow yellow Lactose or Sucrose

 

86

Gas production: bubbles present in and below the butt

 

428 production: black precipitate within the butt and

 

slant.

Urease Test

Purpose and Mechanism: To detect the ability of a bacter-
ium to hydrolyse urea (NHz-CO-NHZ).

The enzyme urease hydrolyses urea to carbon dioxide and

 

ammonia.
NH
C/= O + H O urease 2 NH + CO
\\ 2 p 3 2
NH2 ammonia

Procedure: Urea broth is a buffered solution of yeast

 

extract and urea. It also contains phenol red as a pH
indicator. When urease is produced by an organism growing
in this medium, the ammonia that is released raises the pH.
As the pH becomes higher the phenol red changes from

yellow to red.

87

Voges-Proskauer (VP) Test

Purpose and Mechanism: To test for the presence of acetyl-

 

methyl carbinol in the medium so as to determine
whether the bacterium produces large quantities of
2,3 butanediol and ethanol rather than a mixture

of acids from glucose.

 

 

 

 

\ 1--
" CH CH
2 O O H O
- H H Embden-Myerhoff // butylene I //
H . ‘+ 2 H c-c --——+-Ho-c-c + 2 co
0H glycolytic pathway 3 \ 91YC°1 I \\
HO OH COOH pathway CH CH
H OH 3 3
a-D-glucose' ; pyruvic acid___+ acetoin (AMC) carbon dioxide
2—- H o O
HO-ClI-CI KOH H C-C// CH
> 3 ‘\ / 3
l \ c
CH3 CH3 "
O
acetoin diacetyl
3-— 0 NH
H c Cl /cH O \ + HN-(II NH condensation pink-to-red
.. + -
3 3 2 -+ roduct
II R
OH
O
diacetyl a-naphthol guanidino group

(arginine)

88

Procedure: In an alkaline medium, acetylmethyl-carbinol

 

is oxidized to diacetyl, which forms a red complex with
- alfa-naphthol in the medium.

The Voges-Proskauer Test is positive when the
medium appears pink after addition of alpha-naphthol and

potassium hydroxide (Barritt's reagent).

Appendix Biblipgraphy

BLAIR, E. B. Media, Test procedures and chemical reagents.
In H. L. Bodily, E. L. Updyke, and J. 0. Mason (eds.),
Diagnostic procedures for bacterial, mycotic and parasitic
infections, 5th ed. American Public Health Association,
New York. 1970.

BRANSON, B. Method in clinical laboratory. A. Ballows
(ed.). Atlanta, Georgia. -

Difco Manual. Difco Laboratories (ed.). Detroit, Michigan.
1972.

Difco Supplementary Literature. Difco Laboratories (ed.).
Detroit, Michigan. 1968. ‘

McALISTER, H. A. Procedures for the identification of
microorganisms from higher animals. Clinical Microbiology
-Laboratory, Veterinary Clinic, Michigan State University.
1970. i

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