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NBSIR 73-1 81

Fine Structure in Thermal Growth

Patterns of Bacteria by

Microcalorimetry

B. R. Staples. E. J. Prosen and R. N. Goldberg

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington. D. C. 20234

April 1973

Interim Report

use

i for Institute of General Medical Sciences

Institutes of Health

a. Maryland 20014

73-/0/

If73

NBSIR 73-181

FINE STRUCTURE IN THERMAL GROWTH

PATTERNS OF BACTERIA BY

MICROCALORIMETRY

B. R. Staples, E. J. Prosen and R. N. Goldberg

Thermochemistry Section

Physical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

April 1973

Interim Report

This report presents results of a preliminary study. This report

is to be superseded by a future publication which will receive

general distribution and should be cited as a reference. Please

consult the NBS Office of Technical Information and Publications

to obtain the proper citation.

Prepared for National Institute of General Medical Sciences

National Institutes of Health

Bethesda, Maryland 20014

U. S. DEPARTMENT OF COMMERCE, Frederick B. Dent, Secretary

NATIONAL BUREAU OF STANDARDS, Richard W. Roberts. Director

Foreword

The use of clinical laboratory tests of physiological

fluids for specific components has been rapidly increasing in

the past few years. One aspect of this has been an increase

in the number of tests for well-known particular substances

for which satisfactory tests are available. Another equally

important aspect is the increasing need of the physician and

of the research biochemist for ready methods for determining

other substances present in varying amounts in normal and

pathological states. For this reason, the development of new techniques applicable to problems for which optimum solutions

have not been found are of considerable interest in clinical

and biological chemistry.

The universal occurrence of energy changes in chemical

and physiological processes has caused the calorimeter to be

considered as a potential tool for clinical chemical analysis

and for observing biological processes for some years. The

actual applicability, however, has been limited previously by

lack or insufficiency of one or more necessary factors. Such

factors include: instrument sensitivity and operational noise

levels, instruments adapted to use of small samples, simplicity

of instrumental operation and data reduction, and suitable

mechanisms for sorting out specific processes from a melange

of potential interferences. It was the judgment of the authors

and of the National Institute of General Medical Sciences (NIGMS)

that the understanding of these factors had developed to a point

where a well coordinated attack on the application of microcalorimetry would now lead to successful results.

A program was established in the Thermochemistry Section

(Physical Chemistry Division, Institute of Materials Research)

at the National Bureau of Standards, and after some months was augmented with support from the NIGMS to demonstrate the

feasibility of microcalorimetry in some specific areas useful

in the Clinical Chemistry Laboratory. Dr. Robert S. Melville

is the program officer for NIGMS. Collaborative support has

been provided by the Clinical Chemistry Service (Dr. Donald

Young, Chief), and by the Laboratory for Technical Development

(Dr. Robert L. Bowman, Chief, and Dr. Robert L. Berger)

.

This is a report provided at the end of the first year of

the NIGMS support of this work and summarizes a portion of the

work done. This report is one of four covering different aspects

of the NBS Program. For the information of the reader, the titles

and NBS Report numbers of the four reports are as follows:

NBS Report 73-178 Microcalorimetric Assay for Glucose in

Human Serum and Plasma

73-179 Design and Construction of the NBS Clinical

Mi croca 1 or ime te r

73-180 Testing of the NBS Clinical Microcalorimeter

73-181 Fine Structure in Thermal Growth Patterns

of Bacteria by Microcalorimetry

<}L 2 -j"—-—• —

George T. Armstrong

Principal Investigator

Edward J. Proven

Project Leader

Fine Structure in Thermal Growth Patterns

of Bacteria by Microcalorimetry

by

B. R. Staples, E. J. Prosen, and R. N. Goldberg

Abstract

Thermal growth patterns of Enterobacter cloacae , Proteus

rettgeri, and Klebsiella pneumoniae have been determined with the new NBS Clinical Microcalorimeter. These patterns show more fine structure

than those made by previous calorimeters. This is due to the much

faster time constant (half-time 30 seconds), the use of smaller

volumes (0.4 ml) or thicknesses (2.5 mm) of samples, and the high

sensitivity of the NBS Microcalorimeter.. Furthermore, this fine

structure is shown to be reproducible and thus is valuable for finer

bacterial "fingerprinting" or identification than was previously

available. The simplicity of the apparatus and the ease of insertion

of samples and operation of the calorimeter make this method attractive

for possible routine use in detection and identification of bacteria.

1 . Introduction

There are several possible applications of microca lorimetry to

probe microbial systems. One may study the metabolic pathway of a

microorganism over its lifespan, the thermal patterns of growth can be

used to identify the type of bacteria, the effect of medicinal agents on

the bacteria can be studied, and perhaps the enthalpy of specific growth

processes may be investigated.

Bacterial calorimetry is not new. Recently W. W. Forrest"'" has

done a thorough review on the status of bacterial calorimetry up to 1969.

Bacterial thermogenesis had been studied earlier by a number of bio2 3 chemists: Bayne-Jones and Rhees , Peterson and Wilson , Carlyle and

Norman^, Rahn"', and Steward^. In 1963 Prat^ applied modern calorimetric

techniques to bacterial studies and showed that the behavior of microbial systems gave reproducible thermal patterns of growth.

Quantitative studies were shown to be practical for both growing

8 9 10 and non-growing bacteria, by Battley , Forrest e_t a_l , Belaich , and

„ . . 11

Boivinet

According to Forrest"'", there are various factors which influence the

heat generated by bacteria. Generally, these can be summarized under

two broad headings: (A) Growing cells in pure culture, and (B) Nongrowing cells in pure culture.

A. Growing cells

A standard text which summarizes the extensive literature

12 concerning behavior of bacteria is Lamanna and Mallette

1

According to Forrest, initially there is usually a lag phase, after

which, the rate of heat generation increases exponentially as the cells

begin to divide. This exponential growth is limited by the energy source.

A very good correlation has been obtained^' ^ with the enthalpy change

calculated for the degradation of substrate to the observed products,

after allowing for second-order effects such as heats of dilution,

neutralization, etc. Forrest''", based on works by Wilkie^ and Prigonine''"^,

stated that while a calorimeter, in principle, can detect coupled anabolic

reactions, it is not practical to determine the enthalpy of growth as it

would be expected to be less than the experimental error of the calorimetric measurement. However, the use of newer microcalorimetric techniques

may make enthalpy of growth determinations more practical in the future.

When growth is limited by specific nutrients, heat evolution is

observed at a low level and appears to be limited by a specific

constituent of the medium or an accumulation of toxic products or both.

In addition to heat being evolved from cell division, heat is

produced as the cells metabolize, usually during the period immediately

following the exponential growth. Reserve materials such as ATP (Daves

15 16 and Ribbons , Hungate ) may be accumulated during the latter period and

heat production subsequently may occur from the degradation of these

reserves. This endogenous metabolism tends to last for long periods of

time (Sigal et al^).

2

B. Non-growing cells

In many cases with aerobic bacteria, where respiration occurs,

the metabolism may be followed by a manometer, but in the case of anaerobes

there is no readily measurable change.

The bacterial Streptococcus faecalis is an example of an anaerobic

organism which shows no pH change in the medium and no detectable

respiration, but a substantial enthalpy change was observed calorimet18 rically by Forrest and Walker

Many studies have involved non-growing or resting cells because of

the absence of the complications of growth and cell fission. In this

manner, relationships have been shown for S_. faecalis grown in a medium

with excess energy source which allows an accumulation of reserve

materials such that they could carry on endogenous metabolism in the

resting state. This feature has been contrasted with organisms which

possess no detectable endogenous enthalpy change when grown in a limited

19

energy source (Forrest and Walker ).

This report summarizes the results of some exploratory bacterial

growth experiments conducted at the National Bureau of Standards with

22

the new NBS Clinical Microcalorimeter . We wish to thank Ms. S. Farling

and Dr. W. Russell of the Instrumentation Laboratory, Inc., Lexington,

Massachusetts 02173 for supplying the bacteria and cultures and assisting

in the conduct of the experiments.

3

2. Instrumentation

The microcalorimeter used in this investigation was designed and

constructed at the National Bureau of Standards and will be described in

22

detail in NBS Report

The calorimeter is a heat conduction microcalorimeter utilizing

commercially available heat flow discs. The inner chamber which

surrounds the block is thermos ta ted and the temperature of the block is

thus passively controlled. The reaction cells are able to be placed in

the block and can be removed from the block easily.

The details of the instrumentation and automatic digital data

21 acquisition system have already been discussed in the National

Bureau of Standards Report No. 10 437.

Briefly, the output of the thermopiles contained in the microcalorimeter is followed by a precision nanovolt amplifier as a function of

time. This signal is amplified by a factor of 10"* and digitally

recorded on punched paper tape. This teletype tape is then processed

in a computer to produce a digital record which can be plotted. This

was done to produce the figures illustrated in this Report.

Small plastic vessels of a capacity of about 0.4 ml are used to

contain the material for insertion between the thermopiles of the

microcalorimeter. The inside cell thickness is 2.5 mm. The calorimeter

has a fast response time (half-time of 30 seconds)

.

3 . Experimental

A . Cell disinfection

Clean cells were rinsed 3 or 4 times with "Amphyl" disinfectant,

then rinsed 6 times with sterile distilled water.

B. Preparation of Inoculum

A small amount of growth from the stock culture was placed in

5 ml of brain-heart infusion broth using a platinum loop that had been

sterilized by flaming in a Bunsen burner.

This inoculum was incubated for 4 or 5 hours at room temperature,

at which time the brain-heart infusion had become cloudy.

Using sterile pipets, a dilution of 1:500 of this culture was

made in the brain-heart infusion.

About 4 ml of brain-heart infusion was the inoculated using one

loopful of the above 1:500 diluted growth. Only about 0.4 ml of this

final dilution was used to fill the cell which was inserted into the

microcalorimeter . The inside cell thickness is 2.5 mm.

C. Data Acquisition

The output of the thermopiles of the microcalorimeter was

followed as a function of time. Readings of time and voltage were made

every 30 seconds over a period of 14 to 24 hours for each growth

study. The data were taken and plotted in digital form to produce

the figures discussed in the following section.

5

4. Results and Discussion

Three bacterial growths were run in duplicate. These bacteria were

(1) Enterobacter cloacae , (2) Proteus rettgeri and (3) Klebsiella

pneumoniae . Each growth study was monitored by the automated data

acquisition system.

The Enterobacter cloacae thermograms are shown in Figures 1 and 2.

Figures 3 and 4 illustrate the heat production from the growth of

Proteus rettgeri and Figure 5 shows the thermal growth pattern of

Klebsiella penumoniae . Only one run was obtained on the latter.

As can be seen by comparing the figures for each pair of thermal

growth patterns (Figs. 1 and 2 are superimposed in Fig. 6. Figs. 3 and

4 are compared in Fig. 7) , a reproducible pattern is observed between

duplicate runs. Most of the significant features are duplicated.

Furthermore, the high sensitivity of the microcalorimeter enables one to

discern much finer details in the "fingerprint" of each bacterial growth

20 than had been observed even in the most recent investigation . Due

to the short sampling time used and the rapid response time of the

calorimeter, changes in metabolism could be observed (Fig. 1, 2, 3)

almost instantaneously. Cessation of heat evolution was observed to

occur within a 30 second time lapse in some cases (Fig. 8). Figure 8

is an enlargement of the early portion of Fig. 4. In Fig. 8 the

instantaneous halt in the production of heat was observed which lasted

for a period of 3-5 minutes.

6

These results indicate a possible means of identification for a

large number of microorganisms routinely determined to clinical labora20 tories . The rapidity of measurement appears to have a great

advantage over present methods.

For research laboratories, the kinds of valuable information which

may be gleaned from such microcalorimetric measurements could include

the determination of metabolic levels of activity, comparative studies

of microbial metabolism, determination whether a postulated reaction

occurs, detection of unknown reactions, interpretation of mechanisms of

growth (especially in complex mixtures such as natural systems) and a

possible means of identification of a variety of strains of the same

microorganism.

7

References

[1] Brown, H. D. , ed., "Biochemical Microcalorimetry", Academic Press,

New York, (1969).

[2] Bayne-Jones, S., and Rhees, H. S., J. Bacteriol. 17, 123, (1929).

[3] Peterson, W. H., and Wilson, P. W., Chem. Rev. 8, 427, (1931).

[4] Carlyle, R. E., and Norman, A. G., J. Bacteriol. 41, 669, (1941).

[5] Rahn, 0., "Physiology of Bacteria", McGraw-Hill (Blakiston),

New York, (1932).

[6] Stoward, P. J., Nature 194 , 977, (1962).

[7] Prat, H. , In "Recent Progress in Microcalorimetry" (H. A. Skinner,

ed.), pp. 111-174, Pergamon Press, London, (1963).

[8] Battley, E. H. , Physiol. Plantarum 13, 192, 628, 674, (1960).

[9] Forrest, W. W. , Walker, D. J., and Hopgood, M. F., J. Bacteriol.

82, 685, (1961).

[10] Belaich, J.-P, Compt. Rend. Soc. Biol. 157 , 316, (1963).

[11] Boivinet, P., Ph.D. Thesis, Univ. of Marseilles, (1964).

[12] Lamanna, C., and Mallette, M. F., "Basic Bacteriology", Williams

and Wilkins, Baltimore, (1959).

[13] Wilkie, D. R. , Progr. Biophys. Biophys. Chem. 10, 259, (1960).

[14] Prigogine, I., "Thermodynamics of Irreversible Processes", p. 27

Wiley (Interscience) , New York, (1961).

[15] Dawes, E. A., and Ribbins, D. W. , Bacteriol. Rev. 28, 126, (1964).

[16] Hungate, R. E., J. Bacteriol. 86, 848, (1963).

[17] Sigal, N. , Cattaneo, J., and Segal, I. H. , Arch. Biochem. Biophys.

108, 440, (1964).

8

[18] Forrest, W. W. , and Walker, D. J., Biochem. Biophys. Res. Commun.

13, 217, (1963).

[19] Forrest, W. W., and Walker, D. J., Nature 207 , 46, (1965).

[20] Boling, E. A., Blanchard, G. C, and Russell, W. J., Nature 241,

, (1973).

[21] Goldberg, R. N., Nuttall, R. L., Prosen, E. J., and Brunetti, A. P.,

National Bureau of Standards Report No. 10 437 ; Digital Data

Acquis tion and Computer Computation Applied to Calorimetric

Experiments, (1971).

[22] Prosen, E. J., National Bureau of Standards Report 73-179,

April 1973.

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form NBS-1 14A (i-7i> U.S. dept. of comm. 1. PUBLICATION OR REPORT NO. 2. Gov't Accession

BIBLIOGRAPHIC DATA NBS IR 73-181 N°'

3. Recipient's Accession No.

4. TITLE AND SUBTITLE

Fine Structure in Thermal Growth Patterns of Bacteria

by Microcalorimetry

5. Publication Date April 1973

6. Performing Organization Code

7. AUTHOR(S)

Staples, Bo R» , Prosen, E. J., and Goldberg, R. N.

8. Performing Organization

NBSIR 73-181

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

HFP Ucr Al\ ARTUPMT 1 MclN 1 MPUr rrtMVJPDrP KJMivl 11 1\ l, WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No. 316-0118 and 316-041]

11. Contract/Grant No.

12. Sponsoring Organization Name and Address National Institutes of Health

Bethesda, Maryland 20014

13. Type of Report & Period Covered

Interim

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.) Thermal growth patterns of Enterobacter cloacae , Proteus rettgeri , and

Klebsiella pneumoniae have been determined with the new NBS Clinical Microcalorimeter. These patterns show more fine structure than those made by

previous calorimeters. This is due to the much faster time constant (halftime 30 seconds), the use of smaller volumes (0.4 ml) or thicknesses (2.5 mm)

of samples, and the high sensitivity of the NBS Microcalorimeter. Furthermore,

this fine structure is shown to be reproducible and thus is valuable for finer

bacterial "fingerprinting" or identification than was previously available.

The simplicity of the apparatus and the ease of insertion of samples and operation

of the calorimeter make this method attractive for possible routine use in

detection and identification of bacteria.

17. key words (Alphabetical order, separated by semicolons) Bacterial growth; bacterial detection by

nicrocalorimetry ; bacterial thermal growth patterns of bacteria; fine structure of

pacterial; identification by microcalorimetry.

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