Diffusion coefficients for Pseudomonas aeruginosa and Klebsiella pneumoniae by Yeong-Chul Kim
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Chemical Engineering
Montana State University
© Copyright by Yeong-Chul Kim (1990)
Abstract:
The effects of motility and aggregation on the diffusion coefficient for bacteria were studied in an aqueous system. The effects of capillary tube sizes, exposure times, and dilution rates on the diffusion coefficient were examined.
In general, motile cells can diffuse about 1000 times faster than non-motile cells. Pseudomonas aeruginosa, a motile cell, and Klebsiella pneumoniae, a non-motile cell, were used for this research.
Diffusion coefficients were measured by the capillary tube assay developed by Adler (1969). From this procedure the diffusion coefficient of Pseudomonas aeruginosa was 2.1 x 10^-5 (standard deviation :
1.3 x 10^-5) cmys and that of Klebsiella pneumoniae was 0.9 x 10^-5 (standard deviation : 0.9 x 10^-5) cmVs. The diffusion coefficient of Pseudomonas aeruginosa was about 2.3 times higher than that of
Klebsiella pneumoniae.
The Stokes-Einstein equation cannot be 'used for estimating the diffusion coefficients for Klebsiella pneumoniae and Pseudomonas aeruginosa. The experimental value for the diffusion coefficient of
Klebsiella pneumoniae was about 2000 times higher than that (4.5x10^-9 cm^2 Vs) obtained from the
Stokes-Einstein equation. This discrepancy was due to aggregation of Klebsiella pneumoniae or technical error from this experiment.

DIFFUSION COEFFICIENTS FOR PSEUDOMONAS AERUGINOSA
AND KLEBSIELLA PNEUMONIAE by
Yeong-Chul Kim
A thesis submitted in partial fulfillment of the requirements for the degree
Master of Science in
Chemical Engineering
MONTANA STATE UNIVERSITY
Bozeman, Montana
January 1890
11

X / 3 7 %
© COPYRIGHT
BY
Yeong-Chul Kim
1990
All Rights Reserved

ii
APPROVAL of a thesis submitted by
Yeong-Chul Kim
This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the College of Graduate Studies.
2 . 6
Date
/ 9 ? O
Chairperson, Graduate Committee
/ffa
Date
Approved for the Major Department
He46, Major Department
Approved for the College of Graduate Studies
Date Graduate Dean

STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Montana State University, I agree that the
Library shall make it available to borrowers under rules of the Library.
Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made.
Permission for extensive quotation from or reproduction of this thesis may be granted by my major professor, or in his absence, by the
Dean of Libraries when, in the opinion of either, the proposed use of the material is for scholarly purposes. Any copying or use of the material in this thesis for financial gain shall not be allowed without my written permission.
Signature
/ f f a

iv
ACKNOWLEDGEMENTS
The author would like to thank the following individuals for their help in the completion of this project.
My thesis advisor. Dr. William G. Characklis, whose help and advice in all phase of this work made it possible.
Dr. John T . Sears and Dr. Ronald W. Larsen for their guidance as members of the thesis committee.
Special thanks are also extended to all members in IPA for their help and my family, especially my wife, Eun K. Han, for understanding and support.
-C

V
TABLE OF CONTENTS
Page
1. INTRODUCTION ...................................................... I
Goal and Objectives of Research ............................... 2
2. LITERATURE SURVEY ......... ....................................... 3
Previous Research ..
Model Equation ....
Theoretical Equation
Environmental Effects on Diffusion Coefficients
Chelating Agents ........
P H ....... ...............
Energy Sources and Oxygen
Temperature .............
Viscosity ................
Diffusivities Studied Previously .............................. 10
3. EXPERIMENTAL APPARATUS, METHODS, AND TECHNIQUES ... .............. 13
Nutrient and Dilution Solutions ............................... 13
Apparatus .......................... 14
Experimental Procedure ........... 15
4. RESULTS ................... 17
Diffusion Coefficients for Pi aeruginosa .................... 18
Diffusion Coefficients for jL pneumoniae ...................... 24
Diffusion Coefficients Estimated by
Summary of Results ........................................ 30
5. DISCUSSION ...................... 31
6 . CONCLUSIONS ...................................................... 40
7. R E C O M MENDATIONS... ................ ............................... 42
REFERENCES CITED ..................................................... 43
APPENDICES .................................... 47

vi
TABLE OF CONTENTS - Continued
Page
Appendix A - Cell Numbers for Pi aeruginosa
Counted by Image Analyzer ...................................... 48
Appendix B - Cell Numbers for Jli pneumoniae
Counted by Image A n a l y z e r .................................. 54
Appendix C - Average Cell Numbers for Pi aeruginosa in a Capillary T u b e ....................... 60
Appendix D - Average Cell Numbers for Jli pneumoniae in a Capillary Tube ........................................... 64
Appendix E - Identifications for Pi aeruginosa and
K. pneumoniae ................................................. 68

vii
LIST OF TABLES
Table Page
1. Cell Counts .................................................... 3
2. Velocity of Various Bacteria ................................. 4
3. Diffusivities of Cells ........................................ 10
4. Cell Specificities .......... .......................... ...... 11
5. Nutrient Solution ............................................. 13
6 . Diffusivity for Pi aeruginosa with Different Cell Concentrations .................. ........ 18
7. Diffusivity for Pi aeruginosa with Different Sizes of Capillary Tube .................. 20
8 . Diffusivity for Pi aeruginosa with Different Dilution Rates ............................... 22
9. Diffusivity for Ki pneumoniae with Different Cell Co n c e n t r a t i o n s......... ................ 24
10. Diffusivity for Ki pneumoniae with Different Sizes of Capillary Tube ...................... 26
11. Diffusivity for K i pneumoniae with Different Dilution Rates ................ ............... 28
12. Summary of Results ......... 30
13. Actual Diffusivity for K. pneumoniae from Sample I ........ 34
14. Actual Diffusivity for Ki pneumoniae from Sample 2 ........... 35
15. Diffusion Coefficients for Lost or Gained V o l u m e .......... 39

vii i
LIST OF FIGURES
Figure Page
1. Apparatus .................................................... 14
2. Diffusion Coefficient for Pi Aeruginosa with Different Cell Concentrations ..................... 19
3. Diffusion Coefficient for Pi Aeruginosa with Different Sizes of Capillary Tube ................. 21
4. Diffusion Coefficient for Pi Aeruginosa with Different Dilution Rates ........................... 23
5. Diffusion Coefficient for Ki Pneumoniae with Different Cell Concentrations ..................... 25
!
6 . Diffusion Coefficient for Ki Pneumoniae with Different Sizes of Capillary Tube .................
7. Diffusion Coefficient for Ki Pneumoniae with Different Dilution Rates ........................... 29
8 . Size Distribution of Sample No. 1 for Ki Pneumoniae ......... 32
9. Size Distribution of Sample No. 2 for Ki Pneumoniae ......... 33
10. Penetration Distance for Pi aeruginosa ...................... 37
11. Penetration Distance for Ki pneumoniae ....................... 38
12. Effect on Diffusion Coefficient of Ki Pneumoniae for Lost or Gained Volume .............................. 39
IU i I

ix
ABSTRACT
The effects of motility and aggregation on the diffusion coefficient for bacteria were studied in an aqueous system. The effects of capillary tube sizes, exposure times, and dilution rates on the diffusion coefficient were examined.
In general, motile cells can diffuse about 1000 times faster than non-motile cells. Pseudomonas aeruginosa, a motile cell, and Klebsiella pneumoniae, a non-motile cell, were used for this research. Diffusion coefficients were measured by the capillary tube assay developed by Adler
(1969). From this procedure the diffusion coefficient of Pseudomonas aeruginosa was 2.1 x 10 ’ 5 (standard deviation : 1.3 x 10’5) c m y s and that of Klebsiella pneumoniae was 0.9 x IO "5 (standard deviation : 0.9 x 10'5) cm 2 /s . The diffusion coefficient of Pseudomonas aeruginosa was about 2.3 times higher than that of Klebsiella pneumoniae.
The Stokes-Einstein equation cannot be ^used for estimating the diffusion coefficients for Klebsiella pneumoniae and Pseudomonas aeruginosa. The experimental value for the diffusion coefficient of
Klebsiella pneumoniae was about 2000 times higher than that (4.5x10
" 9 cmV s ) obtained from the Stokes-Einstein equation. This discrepancy was due to aggregation of Klebsiella pneumoniae or technical error from this experiment.

1
CHAPTER I
INTRODUCTION
Systems which are not in equilibrium tend toward equilibrium according to the second law of Thermodynamics. This phenomenon occurs due to a difference in the chemical potential of a component between one region in space and another. There are many factors that can give rise to a difference in chemical potential: concentration, temperature, pressure differences, and differences in potential caused by external f sources (gravity, magnetic, etc.). The only driving force considered in this research is a difference in cell concentrations. Molar flux is proportional to the concentration gradient, and the proportionality factor is the diffusion coefficient.
It is very important to know the diffusion coefficient of cells, if one is to understand the initial events of microbial adhesion in an aquatic system. Microbial cells attach to substrata firmly and cause energy losses (i.e., heat, mass, and momentum transfer resistances), deterioration of water quality, and corrosion of metals. In other cases, attached cells can be used for biological wastewater treatment. Knowing the mechanism of cell attachment to surfaces is useful to the understanding of attached bioprocesses.
First of all we should know actual diffusion coefficients of cells in aquatic systems. Diffusion coefficients of cells available in the

2 literature are rare due to few experimental data, as only Escherichia coll, various Salmonella and a few other bacteria have been studied in recent years.
It is also important to know the difference in diffusion for motile and non-motiIe cells. Here we define the difference between motility and mobility. Colloidal particles, which are mobile, show Brownian motion.
Dead cells or non-motiIe cells exhibit this motion. Living cells are said to be motile when they are able to perform autonomous displacement.
Motile bacteria can generally move much faster than non-motiIe bacteria due to the movement of flagella. Motile bacteria can be attracted by some chemicals, termed "attractants" and repelled by some other chemicals, termed "repellents". These chemicals are detected by "chemoreceptors".
This is chemotaxis. The chemical gradient affects the receptors and changes directions of the flagella. In this research, chemotaxis is not considered.
Goal and Objectives of Research
The goal of this research is to determine the diffusion coefficients for Pseudomonas aeruginosa, which is motile, and Klebsiella pneumoniae, which is non-motiTe.
Two objectives were formulated:
D Establish a technique for determining bacterial diffusivities.
2) Investigate the effect of cell aggregation on measured diffusivities.

3
CHAPTER 2
LITERATURE SURVEY
Previous Research
Observation of chemotaxis of motile cells by Pfeffer in 1884 became the basis of quantitative assessment of bacterial motility to many researchers (Adler, 1972). Ogiuti (1936) measured the time taken for
Escherichia coli to move over a known distance. Shoesmith (1960) developed a simple, rapid, quantitative method to measure bacterial motility, which consisted of counting the number of organisms that pass across a small aperture in a given time. He found that the count was proportional to the suspended cell concentration and also to the average speed of the bacteria. Table I shows his results.
Table I. Cell Counts
Species Cell Cone. C o u n t ^
(#/ml)
Pseudomonas viscosa 2.0x1oj|
Bacillus brevis 0.8x10®
Escherichia coli 0.9x10®
70
32
12
Suspending Solution
(#/min)
Phosphate Buffer (pH:7.4)
Phosphate Buffer (pH:7.2)
Phosphate Buffer (pH:7.2)
Note : (I) Count means the number of cells passing across a small aperture per minute
Vaituzis and Doetsch (1969) developed a motility track technique which is of value in studying the changes of direction, reversals, and rotations observed in motile behavior. In this technique a drop of

4 culture was placed on a glass slide, where he recorded bacterial movements for several cells in time and space on a single photograph. A mean velocity was calculated from the average of the five longest tracks found in one photograph. It was assumed that the track was made by an organism moving parallel to the cover slip and at a uniform velocity.
Table 2 shows his result for the mean velocities of several organisms.
Also, results of Adler and Dahl (1967) and Ogiuti (1936) are Included in
Table 2.
Table 2. Velocity of Various Bacteria
Organism Mean Velocity
(pm/s)
Pseudomonas aeruginosa
Escherichia coli
Bacillus licheniformis
Sarcina ureae
Chromatium okenii
Thiosoiri I Ium .ienense
Flagella
55.8
16.5
Monotrichous
Peritrichous
25 (Ogiuti,1936)
28.8 (Adler & Dahl,1967)
21.4
Peritrichous
28.1
Peritrichous
45.9
86.5
Lophotrichous
Lophotrichous
Temp ( 0 C)
30
30
35
30
30
30
30
Pseudomonas aeruginosa. Chromatium okenii. and ThiosoiriIlum
■ienense. polar flagellated cells, appear to move more rapidly than cells with peritrichous forms (Escherichia coli. Bacillus licheniformis. and
Sarcina ureae) . although extensive and definitive studies have not been made.
Adler (1967,1973) developed a capillary tube assay to measure chemotaxis, based on Pfeffer1s experiment. Adler and Dahl (1967) measured the motility of Escherichia coli in a capillary tube. They also measured the diffusion of glucose and verified the diffusion of glucose can be correctly described by the well-known diffusion equation.

5
Diffusion of glucose under conditions identical to those used for the study of bacterial motility was 4.722x10'6 - 6 . IIIxIO " 6 c m 2 This shows a good agreement with 6.667x10"6 c m 2/s which Gladden and Dole (1953) have obtained at 2S 6 C. Segel et a the average motility of bacterial populations, and he concluded that the assay could be operated with speed, simplicity, and sufficient accuracy and was a valuable tool in the assessment of motility. Slater and Powell
(1981) used a video camera and recorder to monitor the passage of the cells through the capillary tube.
Nossal and Chen (1972) investigated cell movement by laser light intensity correlation spectroscopy. Nossal and Weiss (1973) used light scattering densitometry assay. Dahlquist, Lovely and Koshland (1972) developed a technique of obtaining detailed quantitative data on the chemotactic response in order to study its precise dynamics and to clarify the relationship to chemoreception in higher organisms. Cell concentration is determined by monitoring the intensity of light scattered by the bacteria, using a photomultiplier tube to measure the intensity of the laser light. Berg and Brown (1972) built a microscope which automatically followed individual cells to get tracking image. Stock
(1978) used photon correlation spectroscopy.
Among the methods for determining diffusion coefficients for cells, methods which need laser light or video camera and recorder are difficult to use, and require special equipment. Therefore, the capillary tube assay was selected for investigating bacterial motilities in this research.

6
Model Equation
A classical diffusion equation based on a material balance for cells in three dimensions can be used to calculate diffusion coefficients for bacteria :
S C
S t
0 AB a 2c s 2c
S X 2 0 Y 2 a 2C
S Z 2
( 1 ) here C : Cell concentration .
t : Time
X : Axis
Y : Axis
Z : Axis
D ab : Diffusion coefficient
For one dimension, suitable for a long capillary tube with a small cross-sectional area, Eq. (I) reduces to the following :
S C a 2C jAB
( 2 )
The boundary and initial conditions of the diffusion equation are as follows :
Boundary condition (I) : C =
Co
Boundary condition ( 2 ) : C = 0
Initial condition : C = 0
@ X = 0 and t > 0
@ X = L and t > 0
6 t = 0 and 0 < X < L
The solution for Eq. (I) with the initial and boundary conditions is as follows (Segel, 1977) :

7
D ab = n N 2 / (4 C 02 A 2 t) where N : Total cell number in the capillary tube
C q : Suspended cell concentration
A : Cross-sectional area of the capillary tube t : exposure time
( 3 )
Theoretical Equation
The Stokes-Einstein equation gives good estimates for the diffusion coefficients of large spherical molecules or particles in liquids (for example, polymer molecules or colloidal particles). Bacterial movement can be treated as diffusion of colloidal particles because colloidal
/particles range from approximately 10 A to 1 pm in diameter. Therefore the
Stokes-Einstein equation can be used for estimating a diffusion coefficient for non-motiIe cells, but cannot be used for motile cells.
The Stokes-Einstein equation is as follows :
B = kB T kB T
7 = ----------f 6 Tl Mg R a where kB : Boltzman constant, 1.38062x10"^ J/K f : Frictional coefficient
T : Temperature, K
(4)
Mg : Viscosity of suspended medium
R a : Radius of cell
Environmental Effects on Diffusion Coefficients for Cells
Variations in cultural conditions (i.e., temperature, pH, osmotic pressure, and. nutritional level) may affect motility both quantitatively

4
8 and qualitatively. Adler and Templeton (1967) examined the effects of environmental conditions on the motility of Escherichia coli. and reported the following needs.
1) Chelating agents are necessary to maintain motility in the presence of traces of heavy metal ions
2) A buffer is necessary to maintain pH at the optimum for motility
3) An energy source is necessary to stimulate the motility
Chelating Agents
Motility is highly sensitive to inhibition by trace amounts of heavy metal ions. Amino acids are good chelating agents for metal ions. A chelating agent such as EDTA (ethylene-diamine tetraacetic acid) added with glucose and buffer allowed excellent motility. Bacteria which were highly motile completely lost their motility within 30 minutes when they were washed free from the medium and then placed into a medium which is lacking a chelating agent. Their motility was fully restored at once by adding EDTA or amino acid (Adler et al^, 1967).
Bid
Flagella are known to disintegrate into subunits at pH 3 to 4
(Adler and Templeton, 1967). Cells which were deliberately suspended in buffers with lower pH values had lower cell counts passing through small aperture (Shoesmith, 1960).
Energy Source and Oxygen
Motility requires oxygen if the particular substrate yields energy

9 only aerobically, but motility does not require oxygen if pathways are present for obtaining energy from a substrate anaerobically. Adler
(1973) showed that Escherichia coli which grew without shaking were poorly motile and grew poorTy, but longer shaking in the medium caused serious loss in viability (Adler and Dahl, 1967).
Temperature
An organism grown at above optimum temperature may be very weakly motile and have very few flagella because the synthesis of flagella may be prevented. But the growth of the organism may be almost normal.
Ogiuti (1936) found that the optimum temperature decreased for the motility of Salmonella as the incubation time increased (Adler and
/
Templeton, 1967). Transport itself is a process that is highly dependent upon temperature.
Viscosity
Shoesmith (1960) reported that a slight increase in viscosity of a suspending medium above that of a buffer solution had the effect of increasing bacterial velocity, whereas a further increase reduced it.
Schneider and Doetsch (1974) showed bacterial motility for all motile flagellated bacteria increased in a more viscous solution, reached a maximum, and decreased with higher viscosities. Peritrichously flagellated bacteria had maximum velocity at higher viscosities than polar flagellated bacteria. Schneider and Doetsch (1974) indicated that it is a general behavioral phenomenon of motile bacteria.

10
Diffusivities Studied Previously
Table 3 shows diffusivities for organisms which several researchers have obtained under different environmental conditions. Table 4 shows the cell specificities to help qualify the results in Table 3.
Table 3. Diffusivities of Cells (cm 2 /s)
Reference Bacterial
Species
Diffusivities
(cnr/sec)
Temp. (
Shoesmith (1960)
Pseudomonas viscosa
Bacillus brevis
Escherichia coli
Adler and Dahl (1967)
Escherichia coli
Nossal and Chen (1972)
Escherichia coli
Berg and Brown (1972)
Escherichia coli
Thonemann and Evans (1976)
Escherichia coli
Stock (1972)
Salmonella tvobimurium
Nossal and Weiss (1973)
Salmonella tvobimurium
Lovely and Dahlquist (1975)
Salmonella tvohimurium
Segel et &L. (1977)
Pseudomonas fluorescens
Slater and Powell (1981)
Bacillus cereus
2.60x10®
3.39x10'“
3.77x10'°
7. OxIO "5
5.0x10'9*
8.7x10'?
9.0x10'®
4.3x10"9*
4.4x10'®
1.3x10'®
5.56x10'®
23
30
30
35
25
32
35
37
25
25
?
3.6x10'®
4.6x10
' 9
37
(Note * : Diffusivities for non-motiIe cell)
0 C)

11
Table 4. Cell Specificities
Bacterial
Genus
Cell Size (pm)
Pseudomonas
Escherichia
BaciIlus
Salmonella
Klebsiella
0.5-1.0 by I.5-4.0
1.1-1.5 by 2.0-6.0
0.3-2.2 by 1.2-7.0
0.3— 1.5 by 0.6-6.0
Motility
Motile by polar flagella
Motile by peritrichous flagella or non-motile
Majority motile
(Flagella typically lateral)
Motile by peritrichous flagella
Non-motile
Shoesmith (1960) used nutrient agar for Pseudomonas viscosa and suspended the growth in phosphate buffer. He used peptone medium for
BaciIlus brevis and Escherichia coli . centrifuged, and suspended the deposits in phosphate buffer with NaCl. Also bacterial suspension were aerated by shaking. Diffusion coefficients for three cells were
I calculated by E q . (3) under assumptions that cells diffused in only one direction such as in the case of capillary tube of 10 pm in diameter.
Adler and Dahl (1967) grew Escherichia coli with shaking, and then centrifuged, washed, and resuspended the pellet in potassium phosphate buffer. They repeated this procedure three times. Potassium phosphate buffer, EDTA, MgSO4, (NH 4 )^SO4, and L-serine were filled in the capillary tube.
Nossal and Chen (1972) added 0.01 M CuCl 2 to a sample to cause cessation of the cell movement, therefore movement could be assumed to be that of large Brownian particles. Escherichia coli moved like non-motile cells, and a very low diffusion coefficient was obtained instead of a diffusion coefficient typical of motile cells.
Berg and Brown (1972) grew Escherichia coli at 35°C with rotary shaking, washed the cells twice with sodium phosphate buffer solution,

12 and suspended the cells in a sodium phosphate buffer (pH 7.0). They used the basic equation to predict the diffusivity, which they obtained from molecular theory. They assumed that cell diameter was 2 pm and viscosity of suspension was 2.7 cp at 32°C.
Thonemann and Evans (1976) developed their model equation using the experimental results of Adler and Dahl (1967) to get the diffusivity of cells. They tested three types of distribution (Maxwellian, Uniform, and
Exponential) speeds, and suggested that exponential distribution speeds was the best.'
Stock (1978) grew cells at 37®C with constant aeration, then centrifuged, washed, and resuspended in phosphate buffer or nutrient broth. He obtained non-motiIe bacteria by subjecting the bacteria to an
!
acid wash at pH 1.5. He got very low diffusivity for Salmonella typhimurium, which is almost the same as that of a non-motiIe cell.
Environmental condition for Nossal and Weiss’s research (1973) was the same as that of Dahlquist et aL. (1972).
Slater and Powell (1981) aerated Bacillus cereus by agitation.
Bacillus cereus are peritrichous, therefore its movement is slower than that of cells which is monotrichous or lophotrichous. The cells were not washed. Also the cells were treated with 0.1 M CuCl 2 for 2 hours to get non-motile cells.

13
CHAPTER 3
EXPERIMENTAL APPARATUS, METHODS, AND TECHNIQUES
Nutrient and Dilution Solutions
Table 5 shows the composition of the nutrient solution used in the
Pseudomonas aeruginosa and Klebsiel la pneumoniae cultures. The composition of the dilution solution is the same as that of the nutrient solution except the dilution solution does not contain glucose.
Table 5. Nutrient Solution
Component M.W.
Amount(mg/l)
Glucose
NH4Cl
MgSO 41 TH2O
MnSO 41 H2O
CuSO41S H p
Na 2 B4O V I O H p
FeSO 41 TH2O
(HOCdCH2)3N
CaCl 2 2HU0
(or CaCl2)
Na 2 HPO 4
KH 2 PO 4
180.16
53.49
246.48
1235.86
28T.54
169.0
249.68
381.3T
2T8.05
191.1
14T.02
110.99
142.0
136.09
100.0
36.0
10.0
0.005
0.5
0.04
0.01
0.005
0.56
2 .0
92.0
69.4
568.0
544.0
Note : The pH of the solutions should be adjusted to 6 .8
using strong acid or base.

14
Apparatus
Figure I shows the experimental system. Nutrient solution is used for growing cells, dilution solution for changing a cell concentration in the measurement chamber. The chemostat is used for obtaining constant effluent cell concentration. The measurement chamber is well mixed to meet a boundary condition (I), i.e., constant concentration at x= 0 .
Growth
Medium Dilution
Mediurn
Measurement
Chamber
Capillary Tube
Chemostat
Figure I. Apparatus
Effluent
Silicone Glue
Silicone Tube
Hardened Agar

15
Experimental Procedure
1) Prepare the nutrient solution and dilution solution (10 liters of each) for Pseudomonas aeruginosa (Table 5). (Use a small spin bar (8 mm x
1.5 mm) to mix the contents in the measurement chamber)
2) Set up the chemostat, connect the chemostat inlet to nutrient solution, and the measurement chamber to chemostat effluent. Connect the dilution solution to the measurement chamber.
3) Make sure all connections are tight enough to be autoclaved.
4) Autoclave all experimental equipment for 1 hour at 121°C and about
20 atmospheres.
5) After the experimental equipment cools down to room temperature, inoculate the pure culture of Pseudomonas aeruginosa to the chemostat.
Before the culture is used, make sure that culture is pure. Verifications by API methods were shown in Appendix E.
6 ) Wait for I day, in order to grow the cells.
7) Turn on the pump for the nutrient solution and adjust the flow rate.
8 ) Wait 4 or 5 residence times to get a constant cell concentration from the chemostat effluent.
9) Turn on the pump and adjust the flow rate.for the dilution solution.
10) Take 3-4 ml of the effluent from the measurement chamber.
11) Filter the effluent using membrane filter paper with pore size of
0.1
pm to filter out grown cells.
12) Inject this filtered effluent into a capillary tube (1.5-1 .8
mm ID X
100 mm, Kimex no. 34502, Owens-Illinois, Toledo, Ohio) via a. sterile syringe. Before being used, the capillary tube should be washed with

16 strong acid (Chromerge : Chromic-sulfuric acid cleaning solution, VWR)
13) Block the one end of capillary tubes with agar by plunging the tube into a Petri dish containing hardened agar.
14) Cover the end of the capillary tube blocked with agar with silicone tube (one-end sealed) to prevent the agar from moving out from the capillary tube by the head in the measurement chamber.
15) Plunge the open end of this capillary tube into the sample port of the measurement chamber (make sure there is no air bubbles in the capillary tube) .
16) Remove the capillary tube from the measurement chamber after the designated time (10, 20, or 30 minutes).
17) Remove the entire contents of the capillary tube by applying air pressure on teh agar plug.
18) Stain the sample with acridine orange (Sigma) (or Hoechst 33258 or
33342 (Aldrich) for at least 1 hour) for 2 minutes.
19) Count the cell numbers by Image Analyzer (Quantimet 10, Cambridge /
Olympus) .
20) Repeat the same procedure from 12) with different sizes of capillary tubes (Size : 0.56 mm ID X 75 mm, Thomas Scientific, no. 2413K40 and Size
: 1.10 mm ID X 75 mm, Thomas Scientific, no. 2413K30) .
21) Repeat the same procedure from 8 ) with different flow rates of nutrient and dilution solution.
22) Repeat the whole procedure with Klebsiella pneumoniae.

17
CHAPTER 4
RESULTS
Raw data from the counting procedure by the Image Analysis system are presented in Appendix A, for Pseudomonas aeruginosa, and Appendix B, for Klebsiella pneumoniae. Duplicate runs were always made for each designated time (10, 20, and 30 minutes). Ten fields per sample were taken to count the cell numbers by the Image Analysis system. Also average cell numbers per ml in a capillary tube were calculated based on a result in Appendices A and B. These calculated results are presented in Appendix C, for Pseudomonas aeruginosa, and Appendix D, for Klebsiella pneumoniae. Diffusion coefficients for Pi aeruginosa and Ki pneumoniae were calculated by Eq. (3) from data in Appendices C and D.
One sample calculation for Pseudomonas aeruginosa is presented here. Data for 10 minutes of exposure time (sample no. 1) in Appendix A and C were used.
n N 2
(4) (1.91x10* #/ml ) 2 (0.0132 c m 2) 2 (10 min) (60 s)
= 1.32x10"* c m 2/s

18
Diffusion Coefficients for Pseudomonas Aeruginosa
Table 6 shows diffusion coefficients calculated by E q . (3) for five different suspended cell concentrations from data (I)-(S) in Appendix C.
Flow rates of dilution solution were adjusted to change the cell concentration in the measurement chamber. The size of capillary tube and dilution rate were fixed during this experiment. Dilution rate was
0.19/h and the size of the capillary tube was 1.5 - 1.8 mm ID x 100 mm.
Table 6 . Diffusivitv with different cell concentrations
Cell cone.
(#/ml)
Sample
No.
Exposure Times
1.91x10®
4.95x10?
2.84x10?
I
2
A v g .
1
2
A v g .
I
2
Avg.
10 min.
1.32
1.98
1.65
0.975
0.730
0.853
0.516
0.616
0.566
20 min.
2.01
1.65
1.83
0.967
1.14
1.05
1.73
1.32
1.53
2 .
00 x 10 ?
I
2
Avg.
-
-
—
1.13
11.6
6.37
1.54x10?
I
2
Avg.
-
-
—
0.672
2.73
1.70
3.383
SD 0.502
Average 1.023
Note A v g . : Average
SD : Standard deviation
: Samples contaminated
2.693
30 min.
2.71
2.18
2.45
1.05
2.03
1.54
0.342
2.97
1.66
4.36
2.55
3.46
2.21
3.07
2.64
1.140
2.274
Avg.
1.975
1.149
1.249
4.91
2.171
2.098

19
A plot of the average diffusion coefficient versus suspended cell concentration can be found in Figure 2. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 6 . Via linear regression, the slope of this plot was found to be -0.044 (unit : 1x10 12 cmVtf's).
DlFFUSlVlTY FOR P. AERUGINOSA
(C apillary Tube Dia. : 1 .5 - 1 .8 m m )
□ 10 min x 20 min v 30 min
# Average
Cell Cone.x 1C T-7 ( # / m l)
Figure 2. Diffusion Coefficient for Pi Aeruginosa with Different Cell
Concentrations

20
Table 7 shows diffusion coefficients calculated by Eq. (3) for three different sizes of the capillary tubes from data (1), (6), and (7) in
Appendix C. Suspended cell concentration (1.91x108 #/ml) in the measurement chamber was the same and the dilution rate (0.19 /h) was fixed throughout this experiment.
Table 7. Diffusivity with different sizes of capillary tube
(unit : IxIO "1 cmys)
Tube size
(mm)
Sample
No.
Exposure Times
10 min.
20 min.
1.5-1 x
.8
I.D.
100
I
2
Avg.
1.32
1.98
1.65
2.01
1.65
1.83
1.1 I.D.
X 75
I
2
Avg.
2.61
0.813
1.71
0.945
1.39
1.17
0.56 I.D.
X 75
1
2
Avg.
-
2.04
2.04
0.943
3.33
2.14
SD
0.623
0.826
Average
1.753
Note Avg. : Average
SD : Standard Deviation
: Samples contaminated
I.D. : Inside Diameter
1.711
30 min.
2.71
2.18
2.45
2.51
1.98
2.25
—
2.71
2.71
0.292
2.418
Avg.
1.975
1.708
2.256
1.945

21
A plot of the average diffusion coefficient versus diameter of tube can be found in Figure 3. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 7. Via linear regression, the slope of this plot was found to be -0.234 (unit : 1x1012 c m 5/#'s).
DIFFUSIVITY FOR P. AERUGINOSA
(Cell Cone. :1.91 x 1CT8 # / m l)
10 min
20 min
30 min
Average
Capillary Tube
D iam eter o f Tube (m m )

22
Table 8 shows the diffusion coefficients calculated by Eq. (3) for four different dilution rates from data (1) and (8)-(10) in Appendix C.
One size of capillary tube (1.5-1 .8
mm I.D. X 100 mm) was used and suspended cell concentration (1.91x108 #/ml) was fixed throughout this experiment.
Table 8 . Diffusivity with different dilution rates
(unit : IxIO 8 cmys)
Dilution rate
( V h )
0.11
0.19
0.27
0.35
Sample
No.
1
2
A v g .
1
2
A v g .
I
2
A v g .
I
2
Avg.
Exposure Times
10 min .
—
4.99
4.99
1.32
1.98
1.65
20 min.
4.02
1.87
2.95
2.01
1.65
1.83
2.08
1.81
1.95
3.30
2.29
2.80
SD
Average
Note
1.146
2.539
A v g . : Average
SD : Standard Deviation
: Samples contaminated
0.641
0.427
0.534
1.51
0.724
1.12
1.071
1.607
30 min.
—
-
-
2.71
2.18
2.45
0.683
1.36
1.02
0.843
1.01
0.927
0.739
1.464
Avg.
3.627
1.975
1.167
1.613
1.877

23
A plot of the average diffusion coefficient versus dilution rate can be found in Figure 4. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 8. Via linear regression, the slope of this plot was found to be -8.56 (unit : 1x1012 c m 5/# 's).
DlFFUSIVITY FOR P. AERUGINOSA
(Cell Cone. :1.91 x 10^8 # / m l)
I 0 min
20 min
30 min
Average
Dilution Rate ( / h )
Figure 4. Diffusion Coefficient for Pi Aeruginosa with Different Dilution
Rates

24
Diffusion Coefficients for Klebsiella Pneumoniae
Table 9 shows diffusion coefficients calculated by Eq. (3) for five different suspended cell concentrations from data (1)-(5) in Appendix D.
Flow rates of the dilution solution were adjusted to change the cell concentration in the measurement chamber. The size of capillary tube and dilution rate were fixed during this experiment. The dilution rate was
0.19 /h and the size of the capillary tube was 1.5 - 1.8 mm ID x 100 mm.
Table 9. Diffusivity witji different cell concentrations
(unit : I x 10‘ 5 cmys)
Cell Cone. Sample
No.
10 min.
Exposure Times
20 min.
30 min.
(#/ml)
2.45x1O
8
I
2
Avg.
0.960
1.52
1.24
1.37
0.62
1.00
3.17
0.628
1.90
Avg.
1.38
6.35x10?
3.65x10?
I
2
Avg.
I
2
Avg.
0.034
0.0191
0.027
0.525
0.552
0.539
0.142
0.0805
0.111
0.252
0.563
0.408
0.0409
0.0834
0.062
0.597
0.636
0.613
0.0667
0.521
2.56x10?
1.97x10?
SD
Average
Note Avg
SD
I
2
Avg.
I
2
Avg.
0.541
0.237
0.389
0.198
0.597
0.398
0.713
7.82
4.27
-
0.958
0.958
0.433
0.518
Average
Standard Deviation
Samples contaminated
2.305
1.392
0.548
1.09
0.819
0.529
0.223
0.376
0.857
0.755
1.825
0.501
0.871

25
A plot of the average diffusion coefficient versus suspended cell concentration can be found in Figure 5. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 9. Via linear regression, the slope of this plot was found to be 0.023 (unit : IxIO 12 cm 5 /#'s).
DlFFUSlVlTY FOR K. PNEUMONIAE
(C apillary Dia.: 1. 5 - 1 . 8 m m )
I 0 min
20 min
30 min
Average
Cell Cone.x I C f- 7 ( # / m l)
Figure 5. Diffusion Coefficient for Ki Pneumoniae with Different Cell
Concentrations

26
Table 10 shows the diffusion coefficients calculated by E q . (3) for three different sizes of capillary tube from data ( 1 ), ( 6 ), and ( 7 ) in
Appendix D. Suspended cell concentration (2.45 x10 8 #/ml) in the measurement chamber and dilution rate (0.19 /h) were fixed throughout this experiment.
Table 10. Diffusivity with different sizes of capillary tube
(unit : 1x1 O ’ 5 cmys)
Tube Size
(mm)
Sample Exposure Times
10 min.
20 min.
30 min.
Avg.
I.5-1.
8 I .D.
x 100
I
2
A v g .
0.96
1.52
1.24
1.37
0.629
1.00
3.17
0.628
1.90
1.380
1.1
I .D.
x 75
I
2
Avg.
-
1.23
1.23
0.729
-
0.729
1.16
0.799
0.980
0.980
0.56 I .D.
X 75
I
2
Avg.
7.16
0.202
3.68
0.235
0.101
0.168
0.9323
0.177
0.555
1.468
SD 2.511
0.445
0.955
Average 2.214
0.613
1.144
1.313
Note A v g . : Average
SD : Standard Deviation
I .D. : Inside Diameter
: Samples contaminated

27
A plot of the average diffusion coefficient versus diameter of tube can be found in Figure 6. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 10. Via linear regression, the slope of this plot was found to be -0.0635 (unit : 1x10^ cm®/# "s).
DlFFUSIVlTY FOR K. PNEUMONIAE
(Cell Cone. :2.45x1C T8 # / m l)
D I 0 min x 20 min v 30 min
# Average
D iam eter of Tube (m m )
Figure 6. Diffusion Coefficient for Ki Pneumoniae with Different Sizes of
Capillary Tube

28
Table 11 shows the diffusion coefficients calculated by Eq. (3) for four different dilution rates from data (I) and (B)-(IO) in Appendix D.
One size of capillary tube (I.5-1.8 mm I.D. X 100 mm) was used for this experiment and suspended cell concentration (2.45 x 108 #/ml) was constant.
Table 11. Diffusivity with.different dilution rates
(unit : 1 x 10 "^ cmys)
Dilution rate
( V h )
Sample
No.
Exposure Times
10 min.
20 min.
30 min.
0.11
I
2
Avg.
2.38
1.17
1.78
1.36
0.717
1.04
0.772
0.840
0.806
0.19
1
2
Avg.
0.96
1.52
1.24
1.37
0.629
1.00
3.17
0.628
1.90
0.27
0.35
1
2
Avg.
1
2
Avg.
0.329
0.378
0.354
0.707
0.0974
0.402
0.517
0.906
0.712
0.476
0.623
0.550
0.604
0.552
0.578
0.462
0.648
0.555
SD 0.700
0.335
0.843
Average 0.943
0.825
0.960
Note Avg. : Average
SD : Standard Deviation
Avg.
1.207
1.380
0.548
0.502
0.909

29
A plot of the average diffusion coefficient versus dilution rate can be found in Figure 7. Plots were made for three designated exposure times (10, 20, and 30 minutes). The average diffusion coefficients used in this plot can be found in Table 11. Via linear regression, the slope of this plot was found to be -3.684 (unit : 1x1012 c m 5/# 's).
DIFFUSIVITY FOR K. PNEUMONIAE
(Cell Cone. :2 .4 5 x 1 0 "8 # / m l)
10 min
20 min
30 min
Average
Dilution Rate ( / h )
Figure 7. Diffusion Coefficient for Kl2. Pneumoniae with Different Dilution
Rates

IL I l Il
30
Diffusion Coefficient Estimated bv the Stokes-Einstein Equation for Klebsiella Pneumoniae
It is assumed that the cell is spherical with a diameter of I pm and the viscosity of the suspension liquid is 1 cp at 28 0 C.
From E q . (4),
Kg T
6 Tl R a pB
(1.38062X10"23 J / K H 3 0 1 K)
(6)(3.14)(0.5 pm)(1.0 cp)
4.5 x 10 " 9 cm2/sec
It was found that the calculated value of the diffusion coefficient for
Klebsiella pneumoniae from the Stokes-Einstein equation is in a reasonable range for non-motiIe cells (see Table 3). Most non-motlie cells have diffusion coefficients of an order of IO"9, when units of cm2/s is used.
This calculated value is about 1/2000 of the experimental value (9.0 x
10"6 c m 2 /s)
Summary of Results
Results for this experiment are summarized in following Table 12.
Table 12. Summary of Results
Species
Pseudomonas aeruginosa
Klebsiella pneumoniae
Diffusion Coeff.
From experiment
(cm 2 /s)
2 .
1 x 10"5
9.0x10
" 6
Calculated
Diffusion Coeff.
(cnr/s) ^
—
4.5x10-9

31
CHAPTER 5
DISCUSSION
The diffusion coefficient for Pseudomonas aeruginosa obtained from this experiment is consistent with those of other motile bacteria in Table
3, but the diffusion coefficient for Klebsiella pneumoniae obtained from this experiment was high, compared to the value calculated from the
Stokes-Einstein equation and compared to other non-motlie cells. It is believed that the difference is due to the aggregation of Klebsiella pneumoniae or technical error in this experiment.
First, lots of aggregations were observed during this experiment, compared to those of Pseudomonas aeruginosa. Figures 8 and 9 show size distributions for Klebsiella pneumoniae under the conditions of this experiment. Two samples were taken from the measurement chamber after 20 minutes of exposure time. Size distributions for these two samples were obtained via Image Analyzer. From the size distributions of Klebsiella pneumoniae, we can get the distribution of the diffusion coefficients.

32
SIZE DISTRIBUTION
(S am ple No. I )
2
22 71
10.99
35.07
Size o f A ggregate (u m 2 )
Figure 8 . Size Distribution of Sample No. 1 for Ki Pneumoniae (The number shown above each bar denotes the numbers of cells which can be included in one aggregate for each size.)

33
SIZE DISTRIBUTION
(Sam ple No. 2)
150 -I-------------------------------------------------------- ------------------ -------------
2 2 co ioo -
<D
O
CD
<L> i _
CD
C D
<
13 22
6.15
10.99
Size of Aggregate (u m 2 )
Figure 9. Size Distribution of Sample No. 2 for IL Pneumoniae (The number shown above each bar denotes the numbers of cells which can be included in one aggregate for each size.)

34
Following Tables 13 and 14 show the calculation for the actual diffusion coefficients for Klebsiella pneumoniae. It is assumed that the cross- sectional area of one cell is 0 .5
jW.
Table 13.
Actual diffusivity for Klebsiella pneumoniae from sample I
Area of
Aggregate
(pm2)
No. of
Aggregate ( % C )
Approx. Cell
No. in
Aggregate
Aggregate
No. Diffused in 20 min
(#/ml)
Cell No.
Diffused in 20 min
(#/ml)
0.19
0.34
0.60
1.08
1.93
3.44
6.15
10.99
35.07
24
48
42
54
43
13
I
I
1
1 .
12 x 10 "®
8.49x10®
6.37x10'®
4.75x10";
3.59x10";
2.67x10";
2 .ooxio";
1.50x10";
8.39x10'"
13
22
71
2
2
4
1
I
7
13175
11471
9936
8580
7459
6433
5568
4822
3606
13175
11471
19872
17160
29836
45031
72384
106804
256026
Total 5.71x10s
Data in the third columns in Tables 13 and 14 were calculated by the
Stokes-Einstein equation, and those in the fifth column was calculated by
Eq. (3).
From Eq. (3),
( 0AlP actual
4 C 0 2
(Tt) (5.71x10s)2
(4) (2.45x10s #/ml ) 2 (0.013 c m 2) 2 (20 min) (60 s)
2 .
10 x 10 5 cm2/s

35
0.11
0.19
0.34
0.60
1.08
1.93
3.44
6.15
10.99
Table 14. Actual diffusivity for Klebsiella pneumoniae from sample 2
Area of
Aggregate
(pm2)
No. of
Aggregate
Approx. Cell
No. in
Aggregate
Aggregate
No. Diffused in 20 min
(#/ml)
Cell No.
Diffused in 20 min
(#/ml)
30
7
64
112
113
64
266
2
2
1.47x10'®
1 .
12 x 10 ]
8.49x10]
6.37x10]
4.75x10]
3.59x10]
2.67x10]
2 .
00 x 10 ]
1.50x10"9
7
13
22
I
2
2
4
I
I
15094
13175
11471
9936
8580
7459
6433
5568
4822
15094
13175
11471
19872
17160
29836
45031
72384
106084
Total 3.30x10s
From E q . (3),
CtWactual = 7.03x10 6 cm2/s
Therefore the average actual diffusion coefficient calculated from the theoretical equation is 1.40x10"S c m 2 /s. This value shows better agreement with the experimental value <9.0x10
"6 cm 2 /s).
A long exposure time should not be used for this experiment because cells may grow in the capillary tube, or the movement of cells may be reduced under almost anaerobic conditions inside the capillary tube, or the cells can approach the opposite end of capillary tube, which means the boundary condition (2) (C = 0 at X = L) we used may not be correct.
Adler (1969) used a capillary tube, slide glass, and cover glass for his experiment, but the suspended cell concentration at the entrance may not have been constant during his experiment. A small mixing chamber with small spin bar was used to reduce this effect. The suspended cell concentration should be constant at the entrance to meet

36 the boundary condition ( 1 ).
From Eq. (4) the diffusion coefficient is inversely proportional to the viscosity of suspension medium. But Shoesmith (1960) and Schneider et aL. (1974) reported that motility increased in a slightly more viscous solution and decreased with higher viscosities.
Second, there exists the possibility of lost or gained volume on one end of the capillary tube during this experiment resulting when the capillary tube is taken out from the measurement chamber. Penetration distances for Pseudomonas aeruginosa and Klebsiella pneumoniae for each of the first runs were calculated by the general solution of E q . (2) and with experimental values of diffusion coefficients. From the results shown on Figures 10 and 11 these two cells penetrated less than 12 % of the entire length of the capillary tube, even if 30 minutes of exposure time was allowed. This means that the small amount of liquid lost or gained at the tip of the capillary tube can produce considerable errors.
For the calculation of the results above it is assumed that the liquid surface at the tip of the capillary tube is flat. Table 15 shows the calculated values of diffusion coefficient for Klebsiella pneumoniae for different length of lost or gained liquid volume ( 0 , 0 .
2 , 0 .
3 , 0 .
5 , and
1 mm). Figure 12 was drawn using data from Table 15.

37
PENETRATION LENGTH
(P seudom onas a e ru g in o sa )
•Z3 .4 c .1 -
10 min
30 min
20 min
Distance (c m )
Figure 10. Penetration Distance for Pi Aeruginosa

38
PENETRATION LENGTH
(Klebsiella pneum oniae) c .1 -
30 min
20 min
10 min
Distance (c m )
Figure 11. Penetration Distance for jL. Pneuminiae

39
Table 15. Diffusion Coefficient for Lost or Gained Volume
(unit : 1 X 10 ' 5 cmys) mm 0 .0
0 .2
0.3
0.5
1.0
Diff. Coeff.
(Gained Vol.)
1.0
0 .6 6 0.53
0.32
0.026
Diff. Coeff.
(Lost Vol.)
1.0
1.1
1.95
1.34
1.69
EFFECT ON DIFFUSIVITY
(L o s t o r gained volum e)
Lost Volume
Gained Volume
Length lost o r gained (m m )
Figure 12. Effect on Diffusion Coefficient of _K^ Pneumoniae for Lost or
Gained Volume

---------------------- 1 --'
40
CHAPTER 6
CONCLUSIONS
I. Actual diffusion coefficients for cells may be obtained from a capillary tube assay, based on a method developed by Adler. From this research, diffusion coefficients for Pseudomonas aeruginosa, a motile cell, and Klebsiella pneumoniae, a non-motiIe cell, are as follows :
Organism
P. aeruginosa
K. pneumoniae
Diffusion Coefficient
(cmys)
2.1 (± 1,3) x IO "6
0.9 (± 0.9) x 10 ' 5
The minus sign in the above standard deviation for Klebsiella pneumoniae has no meaning because the value of the diffusion coefficient for that cell cannot be zero.
2. The Stokes-Einstein equation cannot be used for estimating actual diffusion coefficients for motile cells and non-motiIe cells that can attain aggregations. From this research an actual diffusion coefficient for Klebsiella pneumoniae was 9.0 x IO "6 cm 2 /s, which is almost in the range of diffusion coefficients for motile cells, but a diffusion coefficient calculated from the Stokes-Einstein equation was 5.6 x IO "6 cm /s, which is in a reasonable range of diffusion coefficients for non- motile cells.
' i

41
3. Diffusivities for Pseudomonas aeruginosa and Klebsiella pneumoniae decreases with increasing dilution rates (Figures 4 and 7). (The slope for Pseudomonas aeruginosa was -8.56 (unit : 1x10 12 cm 5 / # 's) on Figure 4 and that for Klebsiella pneumoniae was -3.684 (unit : 1x10 12 cm 5 / # 's) on Figure
7 by linear regression.)

42
CHAPTER 7
RECOMMENDATIONS
1. It is recommended to use any chemicals with known diffusion coefficients to test the capillary tube assay for confirmation of the results.
2. It may be helpful to use killed Pseudomonas aeruginosa to illuminate the difference of diffusion coefficients between motile cells and non-
3. It may be helpful to use killed Klebsiella pneumoniae to illuminate the discrepancy due to aggregation from this experiment.
4. It will be very useful to develop a method to measure a lost volume or gained volume on the tip of the capillary tube when that tube is taken out from the measurement chamber.

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Vaituzis, Z. and R. N. Doetsch,"Motility Tracks: Technique for
Quantitative Study of Bacterial Movement", Applied Microbiology, 17, 584-
588 (1969)

47
APPENDICES

APPENDIX A
CELL NUMBERS FOR PSEUDOMONAS AERUGINOSA COUNTED
BY IMAGE ANALYSIS SYSTEM

49
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x10® #/ml
Capillary tube size
Dilution Rate
: I.5-1 .8
I.D. x 100 mm
: 0.19 /h
Cell #/Field 1
10 min 1) 39
2
21
3
20
4
23
20 min
30 min
5 6 7 8 9 10
7 32 18 20 17 27
2) 32 37 32 19 40 26 28 21 21 29
1) 34 37 38 27 44 35 26 48 37 30
2) 34 38 32 34 28 30 29 44 26 27
I) 49 74 69 58 32 47 44 52 49 62
2) 50 38 55 55 59 56 41 64 45 48
(Field area : 20106 pm^)
10 min I)
2 )
20 min 1 )
2 )
30 min I)
2 )
Tube length/100ul Cross-sectional
(mm)
76
Area (cnr)
0.0132
73
83
83
79
0.0137
0.0120
0.0120
0.0127
74 0.0135
Average cell no. per field
22.4
28.5
35.6
32.2
53.6
51.1
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 4.95x10^ #/ml
Capillary tube size : 1.5-1 .8
I.D.
X
Dilution Rate : 0.19 /h
100 mm
Cell #/Field 1
10 min
2 3 4 5 6 7 8 9
D
3 5 10 5 8 3 6 1 7
2 ) 3 3
6
4 2 2 5 4 3
20 min
30 min
D 6 8 6 2 6 6 10
9 6
2 )
6 4 13 4 10 9 3 10 6
I)
12 10 5 2 9 11 8 9
2 ) 7 14 14 15 10 9
8
8 16 10
10
2
7
6
9
9
10
(Field area : 20106 pm')
Tube length/ 100 ul Cross-sectional
(mm) Area (cnr)
10 min I)
20 min
30 min
2 )
D
2 )
D
2 )
76
84
82
78
82
83
0.0132
0.0119
0.0112
0.0118
0.0112
0.0120
Average cell no.
per field
5.0
3.9
6.5
7.4
8.3
11.3

50
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 2.84x107 #/ml
Capillary tube size
Dilution Rate
: I.5-1 .8
I.D. x 100mm
: 0.19 /h
Cell #/Field
10 min
1
1 ) 2
2 ) 2
20 min
30 min
I) 5
2 ) 6
I)
1
2 )
8
2
0
2
3
1
4
3
7
6
7 3
0 14
11
4
3
0
3
11
4
0
5
2
2
13
5
3
7
6
1
1
4
6
$
7
0
2
4
8
0
2
4
9
7
2
10
2
3
7
1
0
2
3
1
11
5
6
I
0
.14
4
7
1
(Field area : 20106 pnv)
10 min I)
2 )
20 min 1 )
2 )
30 min I)
2 )
Tube length/IOOul
(mm)
83
86
79
81
77
84
Cross-sectional
Area (cnv)
0.0120
0.0116
0.0127
0.0123
0.0130
0.0119
Average cell no.
per field
1.9
2 .0
5.2
4.4
2.9
7.8
CELL TYPE : Pseudomonas aeruginosa
Capillary tube size
Dilution Rate
7 4/ml
: 1 .5-1.8 I.D. x IOOnm
: 0 .19 /h
Cell 4/Field 1 2 3 4
10 min
20 min
30 min
5 6 7 8
9 10
D
-
2 )
-
D 2 2 I 4 4 1 3 0
7 3
2 ) 7 8 11 11 7 10 7 11 8 15
D 6 6 7 4 3 10 10 14 6
2 ) 7 4
6 4 8 3 10 6 2
8
7
(Field area : 20106 pnr)
Tube length/IOOul
10 min 1 )
(mm)
74
2 ) .
84
20 min I) 86
2 ) _ 79
30 min I) 76
2 ) _ 75
Cross-sectional
Area (cm2)
0.0135
0.0119
0.0116
0.0127
0.0132
0.0133
Average cell no.
per field
—
—
2.7
9.5
7.4
5.7

51
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.54x10
7 #/ml
Capillary tube size : I.5-1 .8
I.D. x 100 mm
Dilution Rate : 0.19 /h
Cell #/Field 1
10 min 1 ) -
2 3 4 5 6 7 8 9 10
_________________________________________
2) _z_____________________________________ _
20 min 1) 6
2) 5
30 min I) 2
0
4
6
2
4
0
0
1
3
1
4
4
2
7
2
1
0
2
3
5
6
0
5
4
2
2
7
2) 3 5 5 6 5 3 2 7 3
(Field area : 20106 pnr)
7
Tube lenqth/100ul
(mm)
10 min I)
2 ) .
81
20 min 1 ) 81
2 ) .
82
30 min I) 79
2 ) .
79
Cross-sectional
Area (cm2)
0.0123
0.0123
0.0123
0.0122
0.0127
0.0127
Average cell no. per field
—
1.7
3.4
3.9
4.6
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x10® #/ml
Capillary tube size : 1.1 I.D. x 75 mm
Dilution Rate : 0.19 /h
Cell 4/Field
10 min
20 min
30 min
1
1 ) 25
2
3 4 5 6 7 8
22 16 19 30
9 10
2)
8 12 16 11 14 8 17 14 9 18
D
16 25 22 13 21 14 19 15 24 24
2)
26 32 28 30 29 13 26 25 2 23
D
28 45 48 29 40 31 38 53 35 38
2)
14
37
31
42
26
35 26 37 43 34
18
34
26
25
(Field are : 20106 pnr)
10 min
20 min
30 min
Tube lenuth/IOOul
(mm)
D
2 ) .
D
2 ) .
D
2 )
Cross-sectional
Area (cm2)
0.0095
0.0095
0.0095
0.0095
0.0095
0.0095
Average cell no.
per field
22.7
12.7
19.3
23.4
38.5
34.3

52
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x108 #/ml
Capillary tube size
Dilution Rate
: 0.56 x 75 mm
: o.19 /h
Cell #/Field
10 min
20 min
30 min
I) -
1
2 ) 6
2
7
I) 5 5
2 ) 9 10
D 15 5
2 ) -
3
6
5
9
12
4
6
6
14
3
5 6 7 8
2 4 8 4
5
12
6 5 3
8 13 10
13 17 6 11
9
6
7
4
9
10
3
3
15
13
(Field area : 20106 pm2)
Tube length/ 100 ul Crossi-sectional
(mm) Area (cnr)
10 min D
2 )
20 min I)
2 )
30 min D
2 )
0.00246
0.00246
0.00246
0.00246
0.00246
0.00246
Average cell no.
per field
—
5.2
5.0
9.4
10.4
-
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x108 #/ml
Capillary tube size : 1.5-1 .8
I.D. x 100 mm
Dilution Rate : 0.11 /h
Cell tt/Field 1
10 min
20 min
1
2 3 4 5 6 7 8 9 10
) _ =________________________________________________
2) 82 43 13 43 31 60
I) 62
8 45 49 61
66 53 65 55 53 59 61 53 47
30 min
2) 31 28 41 31 40 25 35 37 29 46
1) ________________________________________________
2
) — ---------------------------------------------------
(Field area : 20106 pnr)
Tube length/IOOul
(mm)
10 min I)
2 )
20 min I)
2 )
30 min I)
81
77
83
80
82
2 ) 77
Cross-sectional
Area (cm2)
0.0123
0.0130
0.0125
0.0120
0.0122
0.0130
Average cell no per field
—
43
52.4
34.3
—
-

53
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x10® #/ml
Capillary tube size
Dilution Rate
: 1.5-1 .8
I.D. x 100 mm
: 0.27 /h
Cell #/F1eld
10 min
D
1
14
2
26
3
22
4
13
5
33
2 ) 30 25 28 22 32
6
22
14
7
25
25
8
40
19
20 min
30 min
9
17
28
I) 24 18 25 29 7 30
2 ) 18 18 23 11 17 16
I) 16 28 25 30 26 14
10 21 26
8
24 21
33
2 ) 29 40 31 38 43 33 37
32
33
21
44
10
23
20
23
18
35
41
(Field area : 20106 pnr)
10 min 1)
2 )
20 min 1 )
2 )
30 min I)
2 )
Tube length/1 O O u I Cross-sectional Average cell no.
(mm) Area (cur) per field
91___________________0.0110______________ 23.5
82
79
0.0112
0.0127
24.3
21.3
79 0.0127
17.4
81
82
0.0123
0.0122
26.0
36.4
CELL TYPE : Pseudomonas aeruginosa
Suspended Cell Concentration : 1.91x10® #/ml
Capillary tube size : 1.5-1 .8
I.D. X 100 mm
Dilution Rate : 0.35 /h
Cell #/Field 1 2 3 4
10 min D 43 33 46 28
5 6 7
36 30 19
8
34
2 ) 46 16 22 30 23 51 21 33
9
31
16
20 min 1 ) 27 45 29 32 35 31 42
2 )
20 16 25 28 22 10 37
2 $
9
30
20
30 min I) 26 36 37 26 29 19 30 45 38
2 ) 45 40 25 27 36 29 27 32 37
10
30
40
31
25
27
44
Tube length/ 100 ul Cross-sectional
(mm)
10 min I) 81
2 ) _ 75
20 min I) 79
2 ) 84
30 min I) 75
2 ) _ 75
Area (cm/)
0.0123
0.0133
0.0127
0.0119
0.0133
0.0133
Average cell no.
per field
33.0
29.8
32.7
21.2
31.3
34.2

APPENDIX B
CELL NUMBERS FOR KLEBSIELLA PNEUMONIAE COUNTED
BY IMAGE ANALYSIS SYSTEM

55
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 2.45x108 #/ml
Capillary tube size
Dilution Rate
: I.5-1.8 I.D x 100 mm
: 0.19 /h
Cell #/Field 1
10 min
20 min
30 min
2 3 4 5 6 7 8 9 10 11 12 13
D
40 28 37 20 29 34 27 38 22 7 14 19 31
2) 36 33 30 33 28 25 31 38 34 37
I) 51 34 27
2) 13 44 27
19
22
30
35
50
18
41
16
38
31
45
36 30
36
25
1) 54 80 62
2) 21 39 43
75
40
74
35
73
23
92
48
71
36
83
37
67
24
(Field area : 20106 pm^)
Tube lenqth/IOOul
10 min I)
(mm)
70
2)
20 min 1)
72
87
2)
30 min 1)
2)
79
80
75
Cross-sectional
Area (cnr)
0.0143
0.0139
0.0115
0.0127
0.0125
0.0133
Average cell no.
per field
26.6
32.5
36.3
27.0
73.1
34.6
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 6.35x107 #/ml
Capillary tube size
Dilution Rate
: 1
: 0 .19 /h
. x 100mm
Cell 4/Field 1
10 min I)
2 3 4 5 6 7 8 9
2
2 0 I 0 2 3 0 I
20 min
2) 0 0 4 0 2 0 1 0 2
D
3 4 1 2 6 9 6 3 5
30 min
2) I 0 7 5 1 2 3 2 2
D
1 4 3 4 3 2 2 1 3
2) 1 2
2
0 5 2 5 6 7
(Field area : 20106 pmO
10
1
0
0
3
2
2
Tube lenqth/100ul
(mm)
10 min I) 76
2)
20 min I)
_ 75
67
2)
30 min 1)
2)
_
_
76
69
77
Cross-sectional
Area (cnr)
0.0132
0.0133
0.0149
0.0132
0.0145
0.0130
Average cell no.
per field
1.2
0.9
3.9
2.6
2.5
3.2

56
CELL TYPE : Klebsiel Ta pneumoniae
Capillary tube size
Dilution Rate
: I .5-1.8 I.D. x 100 mm
: o .19 /h
Cell #/F1eld 1 2 3 4
10 min
20 min
5 6 7 8 9
D
6 I 3 2 3 3 0 2 4
2)
2
2 4 2 3 1 2 $ 5
D
4 I
2
6 3 0 5 I 4
30 min
2) 9 3 2 I 5 7 3 2 4
D
5 5 4 4 6 1 3 9 5
2) 8 3 2 8 5 7 3 5 5
10
(Field area : 20106 janr)
1
4
3
7
6
4
Tube lenqth/100ul
(mm)
10 min 1)
2)
20 min I)
82
70
78
2)
30 min I)
2)
79
79
78
Cross-sectional
Area (cm2)
0.0122
0.0143
0.0128
0.0127
0.0127
0.0128
Averaqe cell no.
per field
2.5
3.0
2.9
4.3
4.8
5.0
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 2.56x107 #/ml
Capillary tube size : 1.5-1.8 I.D. X
Dilution Rate : 0.19 /h
100 mm
Cell #/F1eld I
10 min
2
3
4 5 6 7 8 9 10
D .
1 3 I 0 1 5 3 1 0 4
2) 3 I 0 3 I 1 0 0 2 2
20 min I) . 3 6 3 3 4 4 4 3 4 2
1?
30 min
2) 15 8 16 8 8 9 7 8
D .
I 3 2 1 6 3 2 4 6
11
2) 5 5 5 6 1 6 I 9 3
3
(Field area : 20106 pm*)
10 min
20 min
30 min
D
2)
D
2)
D
2)
Tube lenqth/100ul
(mm)
77
74
66
77
82
78
Cross-sectional
Area (cm*)
0.0130
0.0135
0.0152
0.0130
0.0122
0.0128
Averaqe cell no.
per field
1.9
1.3
3.6
10.2
3.1
4.6

57
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration
Capillary tube size
Dilution Rate
I.97x1O7 #/ml
1.5-1.8 I.D. x
0.19 /h
Cell #/Field I
10 min
2 3 4 5 6 7 8 9 10
D ,
2 1 1 3 I 0 0 0 1 0
20 min
2) 2
D
-
0 2 1 I 1 5 I 0 2
30 min
2) . 1
I) . 7
0
2
2
I
4
2
3
2
0
4
4
0
7
2
3
3
3
2
2) . 0 3 2 0 0 2 I 2 0 „ 5
(Field area : 20106 pm*)
10 min
20 min
30 min
D
2)
D
2)
D
2)
Tube lenqth/100ul Cross-sectional
(mm)
75
Area (cm*)
0.0133
79
75
78
77
83
0.0127
0.0133
0.0128
0.0130
0.0120
Average cell no.
per field
0.9
1.5
—
2.7
2.5
1.5
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 2.45x10® #/ml
Capillary tube size : 1.1 I.D. x 75 mm
Dilution Rate : 0.19 /h
Cell it/Field 1
10 min
D
-
20 min
2 3 4 5 6 7 8 9 10
2) 18 25 12 13 24 13 26 20 26 23
D
29 25 22 25 27 16 23 16 23 11
30 min
2)
-
D
42 34 29 23 38 36 29 35 32 38
2) 32 28 22 29 33 39 25 18 26 27
(Field area : 20106 pm*)
10 min
I)
2)
20 min I)
Tube length/100ul
(mm)
30 min
2)
D
2)
Cross-sectional
Area (cm*)
0.0095
0.0095
0.0095
0.0095
0.0095
0.0095
Average cell no.
per field
—
20.0
21.7
33.6
27.9

58
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration :
Capillary tube size
>.45x10® #/ml
: ( ).56 I.D. x 75 mm
Dilution Rate : ( ). 19 /h
Cell #/Field 1 2 3 4
10 min
20 min
30 min
5 6 7 8
I) 15 11 12 8 12 15 14 14 8
2) 2 2 I I 2 0 3 6 2
D
-
2) 2 1 5 I 2 I 2 1 4
D
4 13 10 7 8 6 6 9 6
2) 5 4 I 3 4 I 2 4 4
9 10
16
2
2
9
(Field area : 20106 imr)
6
10 min
20 min
30 min
Tube lenqth/100ul
(mm)
U
2) _
D
2) _
I)
2) _
Cross-sectional
Area (cmr)
0.00246
0.00246
0.00246
0.00246
0.00246
0.00246
Average cell no.
per field
12.5
2.1
2.1
7.8
3.4
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 2.45x1O8 #/ml
Capillary tube size
Dilution Rate
100 mm
Cell 4/Field 1
10 min
2 3 4 5 6 7 8 9 10
D
33 35 34 55 39 35 33 47 38 40
2) 24 34 39 25 27 34 30 19 25 28
20 min
30 min
I) 40 22 39 36 41 42 40 34 36 33
2) 35 26 28 39 35 31
29 32 26 27
D
21 46 35 30 31 44 48 40 40 32
2) 39 19 37 34 46 39 41 47 40 28
(Field area : 20106 pnr)
10 min
D
2)
20 min I)
30 min
2)
D
2)
Tube length/IOOul
(mm)
75
72
86
83
79
81
Cross-sectional
Area (cm*)
0.0133
0.0139
0.0116
0.0120
0.0127
0.0123
Average cell no.
per field
38 .9
28.5
36 .3
30 .8
36 .7
37 .0

59
CELL TYPE : Klebslel la pneumoniae
Suspended Cell Concentration
Capillary tube size
Dilution Rate
; 1.5-1 .8 I. 0. x 100 mm
• 0.27 /h
Cell #/Field 1
10 min I) 20
20 min
30 min
2) 15
1) 24
2) 29
1) 35
2) 29
2
1?
11
25
36
37
30
3
15
19
28
31
18
41
4
11
15
26
38
20
22
5
5
16
23
35
37
33
6 7 8 9 10
19
13
38
37
16
21
30
33
34
10
22
32
19
33
13
17
19
31
40
17
6
26
34
36
38 25 33 28 31
(Field area : 20106 pur)
10 m1n 1)
2 )
20 min
30 min
1)
2 )
1)
2 ) lenqth/100ul
(mm)
79
75
74
79
78
79
Cross-sectional
Area (cm2)
0.0127
0.0133
0.0135
0.0127
0.0128
0.0127
Averaae cell no per field
13.8
15.5
26.0
32.4
32.7
31.0
CELL TYPE : Klebsiella pneumoniae
Suspended Cell Concentration : 2.45x10® #/ml
Capillary tube size
Dilution Rate
:
:
1.5-1.8 I .D. x 100 mm
0.35 /h
Cell #/Field 1
10 min 1) 19
2
22
20 min
2) 19 5
I) 18 19
30 min
2) 30 31
1) 24 26
2) 29 28
3
23
11
24
33
16
39
4
22
11
22
21
23
37
5 6 7 8 9 10
19 22 21 19 17 23
10 5 3 3 9 1
24 32 14 25 27 21
20 29 26 30 23 28
32 39 29 27 33 30
34 57 40 33 33 27
(Field area : 20106 pm2)
Tube lenath/100ul
(mm)
10 min 1) 77
2) _
20 min I)
77
82
2)
30 min 1)
2)
78
80
74
Cross-sectional
Area (cm2)
0.0130
0.0130
0.0122
0.0128
0.0125
0.0135
Averaae cell no per field
20.7
7.7
22.6
27.1
27.9
35.7

APPENDIX C
AVERAGE CELL NUMBERS FOR PSEUDOMONAS AERUGINOSA CALCULATED
IN A CAPILLARY TUBE

61
Pseudomonas aeruginosa
1) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
1.91x10s #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
Avg. Cell #
10 min 20 min 30 min.
I 2 I 2 I 2
2.53X105 3.22X105
4.02x10S 3.64x10S 6.05x10S 5.77x10
2) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
4.95x1O7 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
Avg. Cell #
10 min 20 min 30 min.
1 2 I 2 I 2
5.64x10* 4.40x10* 7.34x10* 8.35x10* 9.37x10* 1.28x10
3) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
2.84x1O7 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
Avg. Cell #
10 min 20 min 30 min.
1 2 I 2 I 2
2.14x10* 2.26x10* 5.87x10* 4.97x10* 3.27x10* 8.81x10
4) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
Exposure time
Sample No.
Avg. Cell
I
10 min
2
2.0x107 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h.
20 min 30 min.
1 2 1 2
3.05x10* 1.07x10* 8.35x10* 6.43x10*

Exposure time
Sample No.
Avg. Cell #
I
—
10 min
—
2
62
5) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
1.54x1O7 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
20 min 30 min.
1 2 I 2
1.92x10* 3.84x10* 4.40x10* 5.19x10
6) Suspended Cell Concentration
Capillary Tube Size
1.91x10® tt/ml
■ : 1.1 mm I.D. X 75 mm
Dilution Solution Flow Rate 0.19 /h
Exposure time
Sample No.
A b g . Cell #
10 min 20 min 30 min.
I 2 I 2 I 2
2.56x10s 1.43x10s
2.18x10S 2.64x10S 4.35x10S
3.87x105
7) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
Exposure time
Sample No.
Avg. Cell #
1.91x10® #/ml
0.56 mm I.D. X 75 mm
0.19 /h
1
10 min 20 min 30 min.
2 I 2 I
5.87x10* 5.64x10* 1.06x10S 1.17x10® -
2
8) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
Exposure time
Sample No.
Avg. Cell #
1.91x10® #/ml
1.5-1.8 mm I.D. X 100 mm
0.11 /h
1
10 min 20 min
2 I 2
4.85X105 5.92x10s 3.87x10S
1
30 min.
2

9) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
63
1.91X108 tt/ml
1.5-1.8 mm I.D. X 100 mm
0.27 /h
Exposure time
Sample no.
A v g . Cell #
10 min 20 min 30 min.
1 2 1 2 I 2
2.65X105 2.74X105
2.40x10s 1.96x10s
2.94x10S
4 . 11x10S
10) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
: 1.91x10s tt/ml
: 1.5-1.8 mm I.D. X 100 mm
: 0.35 /h
Exposure time
Sample No.
A v g . Cell #
10 min 20 min 30 min.
1 2 1 2 I 2
3.73x10s 3.36x10S
3.69x10S 2.39x10S
3.53x10s
3 .86x10S

APPENDIX D
AVERAGE CELL NUMBERS FOR KLEBSIELLA PNEUMONIAE CALCULATED
IN A CAPILLARY TUBE

65
Klebsiella pneumoniae
1) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
2.45x1O8 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
Avg. Cell #
10 min 20 min 30 min.
1 2 1 2 1 2
3.00x10* 3.67x10* 4.10x10* 3.05x10* 8.25x10* 3.91x10 *
2) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
6.35x1O7 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
A v g . Cell #
1
10 min
2 1
20 min
2 1
30 min.
2
1.35x10* 1.02x10* 4.40x10* 2.94x10* 2.82x10* 3.61x10
3) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
3.65x1O7 #/ml
I.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
A v g . Cell #
10 min 20 min 30 min.
1 2 ' I 2 I 2
2.82x10* 3.39x10* 3.27x10* 4.85x10* 5.42x10* 5.64x10
4) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
2.56x1O7 #/ml
1.5-1.8 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
Avg. Cell #
10 min 20 min 30 min.
I 2 1 2 1 2
2.14x10* 1.47x10* 4.06x10* 1.15x10* 3.50x10* 5.19x10*

J V v
IL
66
5) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
: I.97x1O7 #/ml
: I.5-1.8 mm I.D. X 100 mm
: 0.19 /h
Exposure time
Sample No.
A v g „ Cell #
I
10 min
2 I
20 min 30 min.
2 1 2
3.05x10* 2.83x10* 1.69x10*
6) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
Exposure time
Sample No.
A v g . Cell #
2.454x1O8 #/ml
1.1 mm I.D. X 75 mm
0.19 /h
1
10 min 20 min
2 I
2.26x10s
2.45x10-
2
30 min.
I 2
3.79x10S
3.15x10s
7) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
2.45x108 #/ml ]
0.56 mm I.D. X 100 mm
0.19 /h
Exposure time
Sample No.
A v g . Cell #
10 min 20 min 30 min.
I 2 I 2 1 2
1.41x10s 2.37x10* 3.61x10* 2.37x10* 8.81x10* 3.84x10
8) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
2.45x108 tt/ml
I .5-1.8 mm I.D. X 100 mm
0.11 /h
Exposure time
Sample No.
A v g . Cell #
10 min 20 min 30 min.
1 2 1 2 1 2
4.39X105 3.22X105 4. 10x1 0S 3. 48 x1 0 S 4 . 1 4 x 1 0 S 4 . 1 8 x 1 0 5

67
9) Suspended Cell Concentration : 2.45x10** #/ml
Capillary Tube Size 1.5-1.8 mm I.D. X 100 mm
Dilution Solution Flow Rate : 0.27 /h
Exposure time
Sample No.
A v g . Cell #
10 min 20 min 30 min.
I 2 1 2 I 2
1.56x10s 1.75x10s
2.94x10s 3.66X10S
3.69x10S
3.50x10 s
10) Suspended Cell Concentration
Capillary Tube Size
Dilution Solution Flow Rate
: 2.45x10s #/ml
: I.5-1.8 mm I.D. X 100 mm
: 0.35 /h
Exposure time
Sample No.
A v g . Cell #
10 min 20 min 30 min.
I 2 I 2 1 2
2.34x10S 8.69x10* 2.55x10s 3.06x10s
3.15x10S
4.03x10 5
I

APPENDIX E
IDENTIFICATIONS FOR Pi AERUGINOSA AND Ki PNEUMONIAE

Ftef._ Q o p i0Rapid NFT'
Dale.
Dr__
H - f -
P a tie n t.
Source .
S e rv ic e .
4-
+-A lull I IHAUI OXI
-f
24h
48h
.
4-
>.
—
—
Iir-I I
A
J
I"-- -y. j J + j
— —
I
▲ i
Gram
Flagella
Morphology
Motility
Other
* R.<.id E. R w k I STBEP. Rapid CM. STAPH T,ac. Rapid SYS and Rapid NFT a . trad.marks of API S,.t.m. S A
Identification I - C K t f r y ^
/$ju//syy-/Y
_________ c/_/ c>/ sr />-& opi
System
Reference N u m b e r
Source/Site _____
Petient .
. Date .
Physician .
DeptJServica .
O N P G
I
A D H
2
S h
2 4 h
4 8 h
Profile
N u m b e r
+ — +
L D C
4
O D C
I
-
C I T
2
■t-
H 3 S
4
— 4
U R E
I
T D A
2
I N D
4
—
I
—
N O , G A S M O T
I
M A C O F - O O F F
4 I I 2 4
5 h
2 4 h
L _
4 8 h
J
Additional I
Digits = □
□
V P
I
I SkJ/I j
U -
G E L
2
G L U
4
M A N
I
I N O
2
S O R
4
R H A
I
S A C
2
M E L
4
A M Y A R A
I 9
O X I f j r _ f T l
~ t i
4
Additional Information
._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Identification_ _ _ _ _ _ .
4 rTl
4 rTl
|
4 2 - 0 1 2
(12/861
,
§

MONTANA STATE UNIVERSITY LIBRARIES
3 1762 10108751 6