Two methods, the 2-(4-Iodophenyl) 3-(4-nitrophenyl) 5-phenyltetrazolium chloride (INT) method and the direct viable count (DVC), were tested and compared for their efficiency for the determination of the viability of bacterial populations. Use of the INT method results in the formation of a dark spot within each respiring cell. The DVC method results in elongation or swelling of growing cells that are rendered incapable of cell division. Although both methods are subjective and can result in false positive results, the DVC method is best suited to analysis of waters in which the number of different types of organisms present in the same sample is assumed to be small, such as processed waters. The advantages and disadvantages of each method are discussed

Huff, T. L.

Rodgers, E. B.

English

NASA Technical Reports Server

NASA
Technical
Memorandum
NASA TM - 103592
COMPARISON OF EPIFLUORESCENT VIABLE BACTERIAL
COUNT METHODS
By E.B. Rodgers and T.L. Huff
Materials and Processes Laboratory
Science and Engineering Directorate
July 1992
(NA3A-TM-I03592) COMPARISON OF
EPIFLUORESCENT VIABLE BACTERIAL
COUNT METHODS (NASA) 17 p
N92-30305
Unclas
N/ A
Nat,onal AeronautLcs and
Space Administration
George C. Marshall Space Flight Center
G3/51 0110797
MSFC- Form 3190 (Rev. May 1983)
https://ntrs.nasa.gov/search.jsp?R=19920021062 2020-03-17T11:24:59+00:00Z
Ii
|
Form Approved
REPORT DOCUMENTATION PAGE OM8 No. ozo4-o18e
Public reDOrting burden for this collection of information is estimated to average I hour per response, including the time for reviewing Instructions, searching existing data sources.
gathering and maintaining the data needed and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this
collection of information, including suggestions for reducing this burden, to Washington Hezldquarters Services. Directorate tot Information Operations and RepOrts, 1215 Jefferson
Davis Highway. Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget. Paperwork Reduction Project (0704-0188), Washington, DC 20503.
1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE | 3. REPORT TYPE AND DATES COVERED
July 1992 11 I Technical Memorandum
4. TITLE AND SUBTITLE S. FUNDING NUMBERS
Comparison of Epifluorescent Viable Bacterial Count Methods
6. AUTHOR(S)
E.B. Rodgers and T.L. Huff*
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
George C. Marshall Space Flight Center
Marshall Space Flight Center, Alabama 35812
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES)
National Aeronautics and Space Administration
Washington, DC 20546
8. PERFORMING ORGANIZATION
REPORT NUMBER
10. SPONSORING/MONITORING
AGENCY REPORT NUMBER
NASA TM - 103592
11. SUPPLEMENTARY NOTES
Prepared by Materials and Processes Laboratory, Science and Engineering Directorate.
*Sverdrup Technology, Inc.
,12a. DISTRIBUTION/AVAILABILITY STATEMENT
Unclas sifiedmUnlimited
_13. ABSTRACT (Maximum 200 words)
12b. DISTRIBUTION CODE
Two methods, the 2-(4-Iodophenyl)-3-(4-nitrophenyI)-5-phenyhetrazolium chloride (INT)
method and the direct viable count method (DVC), were tested and compared for their efficacy for the
determination of the viability of bacterial populations. Use of the INT method results in the formation
of a dark spot within each respiring cell. The DVC method results in elongation or swelling of
growing cells that are rendered incapable of cell division. Although both methods are subjective and
can result in false positive results, the DVC method is best suited to analysis of waters in which the
number of different types of organisms present in the sample is assumed to be small, such as processed
waters. The advantages and disadvantages of each method are discussed.
14. SUBJECTERMS
Bacteria, Epifluorescent Microscopy, Acridine Orange, Direct Viable
Count, INT
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION
OF REPORT OF THIS PAGE OF ABSTRACT
Unclassified Unclassified Unclassified
NSN 7540-01-280-5500
15. NUMBER OF PAGES
17
16. PRICE CODE
NTIS
20. LIMITATIONOFABSTRACT
Unlimited
Standard Form 298 (Rev. 2-89)

TABLE OF CONTENTS
INTRODUCTION .........................................................................................................................
MATERIALS AND METHODS ..................................................................................................
Water Sources/Cultures .....................................................................................................
INT Method .......................................................................................................................
DVC Method .....................................................................................................................
RESULTS ......................................................................................................................................
Wastewater Samples ..........................................................................................................
Processed Water Samples ..................................................................................................
Laboratory Isolates .......................................................................................... . ..................
DISCUSSION ...............................................................................................................................
CONCLUSIONS ...........................................................................................................................
Page
1
1
1
1
2
2
2
2
3
3
4
_I_...IN_NIIONmY A_Nt
o°o
IU PRECEDING PAGE BLANK _IOT FILMED
LIST OF ILLUSTRATIONS
Figure
1.
2.
3.
4.
5.
6.
7.
Title Page
INT positive cell (wastewater) ..................................................................................... 6
DVC assay ................................................................................................................... 7
Acridine orange stain (wastewater) ............................................................................. 8
INT positive cell (processed water) ............................................................................. 9
DVC positive cell (processed water) ........... . .............................................................. 10
DVC assay (gram-positive laboratory isolates) ........................................................... 11
DVC assay (gram-negative laboratory isolate) ............................................................ 12
LIST OF TABLES
Table
1.
2.
Title Page
Ratio of reagents used in direct viable count methods ................................................ 5
Analysis of water recovery test samples using the INT and DVC viable
count methods .............................................................................................................. 5
iv
TECHNICAL MEMORANDUM
COMPARISON OF EPIFLUORESCENT VIABLE BACTERIAL COUNT METHODS
INTRODUCTION
Epifluorescent microscopy is a useful tool in the rapid determination of total bacterial popula-
tions in aqueous environments. An acridine orange (At) dye that binds Deoxyribonucleic Acid (DNA)
and Ribonucleic Acid (RNA) enables rapid bacterial enumeration under a microscope adapted for
fluorescent illumination. Among the dye's limitations, however, is the inability to distinguish viable
from nonviable cells. The Marshall Space Flight Center (MSFC) is currently conducting a water
recovery test to determine the suitability of processing wastewater for crew reuse. The epifluorescent
technique is one method being used to assess the microbial content of the water. The ability to further
define the viable population present would be advantageous.
The purpose of this study was to test and compare two methods for the determination of viable
populations using epifluorescent microscopy. These are the 2-(4--Iodophenyl)-3-(4-nitrophenyl)-5-
phenyltetrazolium chloride lINT) and the direct viable count (DVC) methods. In the INT method,
respiring cells reduce a tetrazolium dye, forming a dark spot within each cell. The DVC method consists
of a reaction mixture containing two major components, a nutrient and naladixic acid. Cells that respond
to the nutrient grow, but are unable to divide due to the naladixic acid. This results in elongation of the
cell or, in the case of gram-positive cocci, sweIling.
MATERIALS AND METHODS
Water Sources/Cultures
Two wastewater (condensate and hygiene) and five processed water samples collected from the
MSFC water recovery test (WRT) were examined. Samples were taken directly from the collected water.
Two bacteria isolated from earlier stages of the WRT, Enterobacter agglomerans, a gram-negative rod,
and an unidentified gram-positive coccus were also chosen for this study. These bacteria were plated on
brain heart infusion (BHI) agar and incubated for 24 h at 35 °C. Isolated colonies were placed in 99 mL
of sterile phosphate buffer, which then served as the sample source for evaluation of the INT and DVC
methods.
INT Method
A l-mL water sample was placed in a sterile test tube, and 0.1 mL of INT reagent (2 percent)
was added to the tube. The tube was incubated in the dark at room temperature for 30 min. The bacteria
were then inactivated by the addition of formaldehyde (4-percent final concentration), and the sample
was filtered across a 0.2-1am black polycarbonate filter. The INT-treated/preserved sample was stained
for 2 rain with AO (0.01 percent). The filter was then rinsed with sterile buffer and examined under the
microscope for the presence of dark spots (indicative of respiring bacteria) within the green to orange
fluorescing cells.
DVC Method
Several DVC methods are available in the literature, differing primarily in the proportion of each
reagent used in the reaction mixture. Table 1 shows the reagent ratios for the three DVC methods tested
for this study. Method 2 was found most effective in a preliminary analysis of the types of samples
studied here and was used for all subsequent analyses. A 3-mL volume of the reaction mixture was
added to 1 mL of sample in a sterile tube which was incubated at 35 °C for 6 h. The bacteria were then
inactivated by the addition of formaldehyde (4-percent final concentration), and the sample was filtered
across a 0.2-I.tm black polycarbonate filter and was stained for 2 min with AO (0.01 percent). The filter
was then rinsed with sterile buffer and examined under the microscope for green to orange fluorescing
bacteria that were elongated to at least t_wice their original length or, in the case of cocci, swollen. =
RESULTS
Samples from the WRT were scored as positive or negative based on the presence of any bacteria
fitting the criteria described above for the two viable count methods (table 2).
Wastewater Samples
Some bacteria from the condensate wastewater initially appeared positive for the INT method as
indicated by the presence of one or more dark spots within the cells (fig. 1). Hygiene wastewater gave
similar results. These samples were also positive by the DVC method (fig. 2). In no instance was a
sample positive for only one method. During analysis of DVC samples, it was observed that both
responsive (elongated) and nonresponsive cells appeared very similar to cells that would have been
scored as positive by the INT method due to the presence of dark spots (fig. 2). The spots were more
readily apparent in the DVC responsive cells for which the increased size allowed better observation of
cell stracture. These samples were then tested using AO alone and again were found to contain dark
spots (fig. 3).
Processed Water Samples
Of the five processed waters examined, two were positive for the INT method (fig. 4). These two
samples were also positive using the DVC method, although the response was much less pronounced
than that seen in wastewater samples (fig. 5). As was true of the wastewater sources, no sample was
found to be positive for only one method alone. In addition, black spots were again seen in DVC-treated
samples. Four of the samples tested were found to contain populations of ultramicrobes (microbes that
will pass through a 0.2-pm filter), which is not surprising considering the low-nutrient environment of
the processed waters. Because these cells were reduced in size, they had insufficient detail to reveal the
presence of any INT-positive bacteria.
2
Laboratory Isolates
The unidentified gram-positive isolate was negative for INT, but some bacteria appeared to swell
using the DVC method (fig. 6). There was no evidence of the false INT response that had been seen in
WRT samples analyzed by the DVC method. The gram-negative bacterium (Enterobacter agglomerans)
appeared positive for both the INT and DVC methods. A few of the cells mimicked the INT response in
the DVC procedure (fig. 7). After being exposed to starvation conditions for 5 days, numerous E.
agglomerans cells mimicked the INT response using the DVC method. Elongation of these cells was
much less pronounced than in 24-h cultures.
DISCUSSION
Both methods evaluated for determining viable populations of bacteria using epifluorescent
microscopy appear largely unusable. Determination of positive and negative responses was found to be
quite subjective. Because both methods are based upon the ability of the organism to respond to certain
conditions such as nutrient type, temperature, and/or pH, there exists the possibility that some organisms
are viable but nonresponsive to the conditions employed.
The DVC method appeared to work for both gram-negative and gram-positive laboratory iso-
lates, although it was much easier to distinguish the gram-negative bacterium. A major disadvantage to
the DVC method is the problem that may be encountered in a mixed population of bacteria. Because this
method is based on the ability of a viable cell to elongate two to three times the size of a nonviable cell,
one would have to assume that all cells in the mixed population were roughly the same size prior to
analysis, which is often not the case. For instance, a nonviable Bacillus sp. may actually be as long as a
viable E. coli. Because species cannot be distinguished using this method, both cells would be scored
incorrectly as positive. Another disadvantage of the DVC method is that an incubation period of at least
6 h is required. Previous studies have indicated that, in general, gram-negative bacteria require a shorter
incubation time (3 to 4 h) in the presence of the reaction mixture than gram-positive bacteria. Also, the
DVC reaction mixture itself is not standardized. One combination of the reaction mixture components
used provided much better results for the samples tested. However, samples containing other types of
organisms might not respond as favorably. Singh et aI, 1 using pure cultures of organisms, found that
changes in the concentration of nalidixic acid in the reaction mixture improves the response of certain
species of bacteria over others. It is possible that the black spots originally seen in wastewater samples
analyzed by the DVC method represent cell damage due to the effects of nalidixic acid, which at higher
concentrations results in cell destruction and death.
The INT method requires much less time to perform (30 to 40 min total) than the DVC method.
The major problem with this method is the inability to distinguish the black spot, indicative of respira-
tion, from unrelated nonuniformities observed within the cell. This was especially apparent with stressed
organisms. The resolution of the microscope (x 1,000) was not sufficient to detect black spots, if present,
in stressed organisms that had reverted to an ultramicrobial state. Nonstressed organisms analyzed by the
use of AO alone were also found to occasionally contain these cell nonuniformities. In addition, this
method was unable to reveal viable gram-positive cocci.
o Singh, A., Yu, F.-P., and McFeters, G.A.: "Rapid Detection of Chlorine-Induced Bacterial Injury by
the Direct Viable Count Method Using Image Analysis." Applied and Environmental Microbiology,
vol. 56, No. 2, 1990, pp. 389-394.
3
CONCLUSIONS
The two viable count methods examined appear unsuitable for use in the WRT. Both are quite
subjective and could readily result in false positive results. Reading of results is tedious, especially for
the INT method, and requires training of laboratory technicians. Each method uses epifluorescent
microscopy for enumeration of bacteria, and is, therefore, subject to the same constraints of that tech-
nique, including insufficient sensitivity to meet current WRT specifications for number of bacteria
allowed in reclaimed water.
If a viable method is necessary for the WRT, the DVC method would appear to be the best suited
to analysis of waters in which the number of different types of organisms present in the sample is
assumed to be small, such as processed waters. Even so, this study suggested that elongation of starved
gram-negative laboratory isolates is much less dramatic following starvation conditions, which would
presumably be those of processed water samples.
The reduced size of bacteria in processed WRT samples and starved laboratory isolates made
scoring of these samples using the INT method difficult. Wastewater samples from the WRT were better
suited to this method, because of nutrient conditions that promote cell uniformity (i.e., less cell degrada-
tion). However, nonuniformities were occasionally found in both these samples and the 24-h laboratory
isolates without the addition of INT. Thus, analysis of processed and wastewater sources using this
method is subject to inaccuracies.
4
Table 1. Ratio of reagents used in direct viable count methods.
Method a
Reagent 1 2 b 3 e
Yeast extract (mg) 0.25 -- --
Yeast extract (gm) -- 0.5 0.3
Naladixic acid (mg) 0.02 0.128 0.025
HEPES (0.1 m) (mL) m 3.0
BHI (mL) -- 50.0 m
BHI (gm) m -- 3.0
Phosphate buffer (mL) m __ 10.0
a. All methods use 1-mL sample volume.
b. Mix yeast extract in BHI and add 1 mL to sample.
c. Mix all reagents and add 1 mL to sample.
Table 2. Analysis of water recovery test samples using the INT and DVC viable count methods.
WRT-4*5-22-2-METM-020-M-TNK
WRT-4*5-23-1-METM-021-M-TNK
WRT-4*5-10-1-METM-008-M-PU 1
WRT-4*5-39-1-MEEH-006-M-PT 1
WRT-4*5-23-1-METM-006-M-PT2
WRT-4*5-39-1-MEEH-002-M-HT3
WRT-4*5-22-1-MEEH-002-M-H'I2
1.
Source
Hygiene wastewater
Condensate wastewater
Processed potable water
Processed potable water
Processed potable water
Processed hygiene water
Processed hygiene water
INT1 DYe
+ +
+ +
+ +
+ +
m w
Samples were judged positive or negative based on criteria described in Materials and Methods
section. Refer to Discussion section for interpretation of results.
5
L)
eL)
:=.
O
_)
,1,-wLT..,
6
ORIGINAL PAGE
BLACK AND WHITE PHOTOGRAPH
Ur,j "-'
.,-,, _
I r_
_ ,--4
O_ 0 "_
,_ _ <.,-.,
;>
fT.,
ORIGINAL PAGE
BLACK AND WHITE PHOTOGRAPH
?
ol,-q
"l:::
<
LF.,
ORIGINAL F,,-X,3E
BLACK AND WHITE PHOTOGRAPH
0ORIGIN'S[ "_ ": '-"
BL_CR AND WHITE PH...,TOL_RAPH
9
L.
",,=1
°_,=d
O
rj
;:;,.
,,,-;
o1-.i
10
OR_GINAL P_GE
BLACK AND WHITE PHOTOGRAPH
r,_
O
0 _...q
L"
:=.
>.
,,6
C,,_:GIN,_.L P,:_CE
BLACK AND WHITE PHOTOGRAPH
11
OO
¢)
IZl
1:1
12
BLACK AND WHITE:PHOTOGRAPH
APPROVAL
COMPARISON OF EPIFLUORESCENT VIABLE BACTERIAL COUNT METHODS
By. E.B. Rodgers and T.L. Huff
The information in this report has been reviewed for technical content. Review of any informa-
tion concerning Department of Defense or nuclear energy activities or programs has been made by the
MSFC Security Classification Officer. This report, in its entirety, has been determined to be unclassified.
P.H. SCrtLrERER
Director, Materials and Processes Laboratory
"_ U. S. GOVERNMENT PRINTING OFFICE 1992 -- 631-- 060 / 60156
13
i= =
7 2


Comparison of epifluorescent viable bacterial count methods

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server