APPLICATION OP FLUORESCENT ANTIBODY METHODS

FOR THE ENUMERATION AND IDENTIFICATION OF

BACILLUS CEREUS

APPROVED:

Major Professor

Minor Professor

i i

Director of the Department; of Biology

Dean of the Gradua'te School

APPLICATION OF FLUORESCENT ANTIBODY METHODS

FOR THE ENUMERATION AND IDENTIFICATION OF

BACILLUS CEREUS

THESIS

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

By

Robert Newton Ferebee, B. S

Denton, Texas

August, 1969

TABLE OP CONTENTS

Page

LIST OP TABLES iv

INTRODUCTION 1

MATERIALS AND METHODS ' 5

RESULTS AND DISCUSSION 12

CONCLUSION 21

LITERATURE CITED 22

i i i

Lisa? OF TABLES

Table Page

1 Rabbit Anti-Bacillus Sera Titers . . . . . . . . 12

2 P. A. Reactivity with B. cereus Antisera-Slide Technique . . . . . . . . . . . . . . 13

3 Absorbed - B. cereus ATCC # 10876 and

10987 Antisera . . . . . . 15

% MFFA - with B. cereus ATCC # 10876 Antisera . . . 18

5 MFFA - with B. cereus ATCC # 10987 Antisera . . . 19

6 MFFA - Bacillus cereus 10987 Added to Raw Pond Water . 20

iv

INTRODUCTION

There is substantial evidence that many strains of

Bacillus cereus are effective in removing Actinomycete-

formed taste and odor problems in fresh water supplies.

Taste and odor in water are primarily aesthetic problems,

but in an attempt to furnish users with better quality

water, considerable attention and expense ,has been expended.

The deletion of these undesirable qualities, attributed to

one group of microorganisms, and removable by some function

of another, seems a natural and desirable process. If,

however, the addition of bacterial organisms is to be used

for water treatment, whatever the reason, there should be

an efficient method for tracing their progress.

Since its development by Coons and Kaplin (10), the

fluorescent-antibody technique has become well established

as a sensitive, rapid, and potentially specific method

for the identification of microorganisms. Carter and

Leise (8) adapted the fluorescent technique for the identi-

fication of bacterial organisms on Millipore membrane filters.

Recent work has been accomplished by Guthrie and Reeder

(13) for the enumeration, as well as identification, of

water pollution indicator organisms. Their studies explain

the fluorescent-labeled colony count technique. Rapid

identification, enumeration, and versatility of the

fluorescent antibody method make it a logical choice for

the tracking and study of B. cereus in water.

Other members of the family Bacillaceae, to which

B. cereus belongs, have been successfully labeled and

specifically identified with fluorescein conjugates.

Beigelisen, Cherry, Skally, and Moody (2) demonstrated

B. anthracis by the fluorescent-antibody technique.

This same organism was studied by Dowdle and Hansen (12),

employing a phage-fluorescent antiphage system. Carter

et al. (8) identified B. anthracis by labeling colonies,

grown on black Millipore membrane filters, with specific

fluorescein-tagged antiserum. Kreig (15) traced B. thur-

inglensis in microbial preparations by immunfluoreacent

publication, suggest that B, cereus is a non-virulent

strains of B. anthracis.

Much of the taxonomy of the genus Bacillus is based

on the physical and biochemical differences ([}., 16) be-

tween strains. The genus has been divided by cell size into

the "large-celled species" and the "small-celled species"

(i|, 16).

There has been difficulty in establishment of the

antigenic differences in the genus Bacillus. Lamanna,

191+0 (16), working on the taxonomy of these organisms,

shows difference in spore and vegetative cell antigens

of the "small-celled species", The "large-celled species"

fail to show these antigenic differences, but are described

as having "demonstrated a disquieting heterogeneity".

This same worker reports B. cereus delimited by spore-

formed antibodies fr*om the other "large-celled species",

but vegetative cell antigenic differences are not reported.

Sievers and Zetterberg (20) suggest, in their investigations

of the antigenic characters of B. meseriterieus and B. sub-

tilis, that B. subtilis, B. mesenterieus, B. vulgatus,

B. mycoides, and 3. cereus can be characterized by anti-

genic structure differences, but also list common antigens

for B. subtilis and B. mesenterieus. The literature gives

little else as to the antigenic separation of the genus

Bacillus.

A variety of other methods have been proposed to iden-

tify B. cereus. Burdon, Stokes, and Kimbrough (6) showed

separation of B. cereus and B. megaterium from B. subtilis

and B. mesenterieus by staining fat reserves in B. cereus

and B. megaterium with Sudan Black B-Safranin, A more

recent paper by Mossel, Koopman, and Jongerius (18) gives

MY-agar, with polymyxin added, as a differential medium for

the identification on B. cereus from foodstuffs. Bonventre

and Eckert (3) used toxin production as a criterion for

k

differentiating B. cereus from B. anthracis. These methods,

as well as the biochemical reactions, involve a considerable

amount of time.

This particular work is proposed as a test of the

expedience of using the fluorescent-antibody technique as

a method for enumeration and identification of certain

strains of B. cereus that have been found to be effective

in preventing taste and odor in water supplies resulting

from certain Actinomycete blooms. In practice, the ex-

perimental design of this study was to determine the com-

petence of two strains of B. cereus, as antigens as well

as their susceptibility to fluorescent labeling; to make a

limited evaluation of the antiserum specificity; and to

make filter enumerations of B. cereus, by use of techniques

reported by Guthrie, et al., 1969 (13)•

MATERIALS AND METHODS

Cultures for this work were obtained from the North

Texas State University stock culture laboratory. These

cultures are designated, by numbers of the McBryde Collection

(MC), Dr. J. B. McBryde, Denton, Texas, the American Type

Culture Collection (ATCC), Washington, D. C., or the North

Texas State University Collection (NTSU), Denton, Texas.

The following nine organisms were utilized in this work:

Bacillus cereus. ATCC IO876, B. cereus, ATCC 10987# B.

megaterium, ATCC 9885, B. subtilis, NTSTJ 7, B. mycoides,

MC, B_. anthracis, MC, Clostridium sporogenes, MC, Escherichia

coli 1 ATCC 10586, and Streptococcus faecalis, ATCC 105̂ -1.

The ATCC IO876 and 10987 strains of B. cereus were

chosen as antigens, as they were found to be ninety per cent

effective in deleting Actinomycete-caused taste and odor

in water (lij.).

The antisera were produced in rabbits of the two-to

three-pound weight range, locally obtained, and without

regard to sex. Essentially following procedures by Camp-

bell, Garney, Cremer, and Sussdorf (7)> twenty-six-gauge

disposable needles were used to inject dilutions of the

antigen, equivalent to the number three McFarland nephelo-

meter tube, into a marginal ear vein. The animals were

injected a total of five times over an eighteen-day

period, and were exsanguinated and sacrificed on the

twenty-third day following the first injection.

Antigen preparation is also described by Campbell,

et al. (7)« The strains of B. cereus were cultured in

tryptic soy broth (Difco Laboratories, Detroit, Michigan)

for eighteen hours at 30 C, harvested by centrifugation

at 10,000 rpm for ten minutes, then suspended and washed

three times in 0.85 pe*1 cent sterile saline. The cells

were then resuspended in 0.85 per cent saline, heated in

a boiling water bath for thirty minutes, after which time

phenol crystals were added to give a 0.5 per cent solution,

and once again heated for thirty minutes. The finished

antigens were tested for sterility by inoculation on

tryptic soy agar plates and in thioglyc-olate broth (Difco

Laboratories, Detroit, Michigan) on three consecutive days.

Blood for antiserum was taken via heart puncture from

rabbits that were ether-anesthetized. The bleeding was

accomplished with an eighteen-gauge needle on either end

of a four-inch lengbh of plastic tubing. One needle was

used for the heart puncture; the other was inserted into

the rubber stopper of an air-evacuated 1.5-ml glass test

tube. All materials except the test tubes were siliconized,

and all equipment was sterile. Approximately 80 to 100 ml

of whole blood can be obtained in this manner. The blood

obtained, was allowed to clot under refrigeration at 5 C

and the serum separated by centi'ifuging for ten minutes

at 10,000 rpm.

Proteins were determined by use of the biuret reagent and

procedure (?)• A standard curve was prepared with

crystallized egg albumin, and after a thirty-minute reaction

period, readings were made on the Spectronic 20, set at

540 mu.

Serum fractionation and gamma globulin separation

were accomplished by saturated ammonium sulfate salting.

Sulfates were subsequently removed by dialysis against

0.85 pe? cent sterile saline until tests with a two per

cent barium chloride solution were negative (9).

The gamma globulin fractions of the antisera were

conjugated at C, for thirty minutes, according to

Spendlove (21), by using fluorescein isothiocyanate (FITC)

obtained from the Nutritional Biochemical Corporation,

Cleveland, Ohio. A twenty mg/ml concentration of the

powdered FITC was added for each gram protein in the total

volume of antiglobulin. Final purification of the conju-

gated antiserum was by passage through a Pharmacia. 18-

inch Sephadex DEAE anion exchange column, using G-50 fine

resin equilibrated with phosphate-buffered saline (pH 7-2)

(21). The final conjugate was diluted to a ten mg/ml

protein concentration, as is recommended for most bacterial

labeling (19). Pooled whole serum from two rabbits, which

' 8

exhibited no titer against B. cereus, was conjugated for

control serum. Goat-anti-rabbit sera (Colorado Serum

Company, Denver, Colorado) was FITC-conjugated for indirect

labeling tests.

Titers on all sera, as shown in Table 1, were by standard

tube agglutination methods (7).

Specificity testing necessitated the use of absorption

procedures (7)« All absorptions were reacted overnight

at 5> C, using a cell suspension equivalent to the number

three McParland nephelometer tube> and an equal volume of

antisera. These heterologous absorptions were used in an

attempt to remove those antibodies which caused non-specific

fluorescence.

Both the direct slide technique, essentially described

by Moody, Goldman, and Thomason (17), and the indirect slide

technique of Weller and Coons (22), were used in testing

reactivity and specificity of the B. cereus antisera.

Slides were made of the desired organism, allowed to air

dry, then fixed for ten minutes in acetone. At this stage,

the slides could be used at once or stored for later use.

Smears for the direct technique were overlayered with

normal rabbit serum for five minutes, to accomplish pre-

inhibition, as proposed by Bergman, Forsgren, and Swahn

(1). This step decreases non-specific labeling. They were

then washed with phosphate-buffered saline (PBS; pH 7«0), and

9

finally overlayerad with the specific-labeled and pooled

antisera for thirty minutes to one hour. The smears were

then washed for two minutes each in PBS, carbonate-bicar-

bonate buffer, and distilled water. Finally, the cells

were covered by a glass slip using a carbonate-bicarbonate

buffered glycerol mounting fluid, as recommended by Pital

and Janowitz (19). These prepared slides were observed

for fluorescence with a Nikon SKE microscope (Nikon Inc.,

Garden City, New Jersey) equipped with a darkfield con-

denser (N.A. 1.^0). A 200-w mercury arc lamp (Bausch and

Lomb, Inc., Rochester, New York) was the light source.

Green filters were used for visible lights, and Corning

5-5>8 filters were used for ultraviolet illumination.

Nikon Y-8 yellow barrier filters were used for light

filtration.

The indirect method essentially follows the procedure

described for the direct technique. The primary difference

is reaction of B. cereus cells or colonies with B. cereus

antisera that were not FITC-conjugated. This antigen-anti-

body complex was then reacted with a separate antiglobulin

system, which was FITC-conjugated. The separate system, goat-

anti-rabbit sera, labeled the cells for fluorescence.

The membrane filter-fluorescent-antibody technique

(MFFA) (11, 13) was used as the method to provide enumera-

tion and identification of the B. cereus strains. The

10

results of the studies are given in Tables ij. and This

technique incorporated the use of black-gridded filters,-

HABG OI4.7 (Millipore Corporation, Bedford, Massachusetts),

in glass filter bases and 2$0 ml glass funnels. Steri-

lized distilled water, as well as sterilized, but other-

wise untreated, pond water was used to dilute known con-

centrations of the organisms utilized in this study. These

dilutions were filtered through the membrane filters; the

filters were then removed and placed ontryptic soy agar

plates. Incubation was carried out at 35 C for twelve hours.

At the end of the incubation period the membrane filters

were placed back on the filter base, a negative pressure was

applied, and the colonies were wetted with phosphate

buffered saline (pH 7»0). The pressure was then allowed

to equalize and the colonies were overlayered with normal

rabbit serum for five minutes. This serum was removed by

negative pressure and the colonies were again overlayered,

this time with the specific-labeled antiglobulin for

15 to 20 minutes. The antiglobulin was removed by negative

pressure, and the colonies were washed free of excess antiglo-

bulin with carbonate-bicarbonate buffer (pH 9.^). Glycerol

mounting fluid, buffered (pH 9.0), was used to cover the

colonies to prevent drying. Counts were made on a Nikon

dissecting microscope (10 X magnification), with indirect

illumination. Visible-light counts gave the total colony

11

count, and ultraviolet was used for the fluorescent number.

All bacterial colony counts were controlled by deliver-

ing 0.1 ml of a viable cell suspension, of the appropriate

dilution, on the center of a tryptic soy agar-filled

petri plate. The aliquot was then spread evenly over the

agar surface by use of a sterile glass, hockey-stick-

shaped spatula. Controls of vegetative cell and colony

fluorescence were made by the addition of FITC-conjugated

normal rabbit sera.

RESULTS AND DISCUSSION

Antisera produced by the ATCC IO876 and ATCC 10987

strains of Bacillus cereus, gave only moderate titers,

as shown in Table 1. A titer as low as 1:128 shown in

rabbit number five, was found to produce fluorescent

reactivity. The antisera titers, even after conjugation,

as evidenced by the fluorescent reactivity, Table 2,

gave sufficiently good labeling results.

TABLE 1: Rabbit anti-bacillus sera titers

Rabbit # Antigen B. cereus # Preinjection 18th day 23rd day

1 10876 1:8 1:2^6 1:512

2 10876 1:2 1:2£6 1 .*512

3 10876 0 1:2^6 1:10214.

k 10876 0 1:614- 1:128

5 10987 0 1:256 1:2014.8

6 10987 l:k 1:512 1:2014.8

*•7 10987 1:3 2 1:2014.8 __

**8 None 1:2 __ —

*-*-9 None 0 __

^-Injection completed but antiserum not used, due to elevated prexnjection titer.

•K-«-Animals bled for "normal" control serum.

13

TABLE 2. F. A. reactivity with B. cereus antisera-slide technique

P. A. Reaction-Direct-;:- P. A Reaction Indirect-s*-B. cereus Antisera B. cereus Antisera Organism

ATCC # 10876 - 1098?

ATCC # 10876 - 10987 Control

B. cereus ATCC 10876 2+ 3+ 3+ 3+ 0

B. cereus ATCC 10987 3+ 3+ 3+ k* 0

E. coli 0 0 0 (±) 0

Strep, faecalis 0 - 0 0 (±) 0

B. subtilis ' 3+ 3+ 3+ 1+

B. megaterium 3+ 3+ 3+ 3+ (±)

B. mycoides 2+ 2+ !(.+ 3+ 0

c l o s* sporogenes 0 0 (±) 0 0

B. anthracis 0 (± ) (i) (±> 0

^Direct reactions are averages of five duplicate slides. -̂"-Indirect reactions are averages of three duplicate slides

Table 2 provides evidence that B. cereus vegetative

cells reacted with the two antisera to give fluorescence.

Lack of reactivity with Escherichia coli and Streptococcus

faecalis showed a trend toward family specificity. These

particular organisms were chosen because of their use as

water pollution indicators. Possibility of genus specificity

34

was provided by the negative reactions of Clostridium sporo-

genes. In these results only a slight increase in intensity

of the fluorescence was noticed with the indirect method.

The direct labeling technique appears slightly more specific

then the indirect. Specificity, as expected from the studies

previously mentioned and as results with B. subtilis, B.

megaterium, and B. mycoides showed, was a problem.

Table 3 data show the failure of heterologous absorption

to resolve the lack of species specificity. Multiple ab-

sorption with the three species that showed cross-reactivity

was not successful. Absorption with the three organisms

individually gave the same results. Homologous absorption

with a mixed suspension of the two B. cereus, cells, and

antisera,rendered antisera that gave no fluorescence with

the same B. cereus strains or with the B. mycoides strain.

This sarae absorbed antiserum shotted positive results with

B. subtilis and B. megaterium. It seems possible that the

animals used had antibodies for the omnipresent B. subtilis,

and perhaps for the B. megaterium, but pre-immunization

titers were not studied.

Smears for the fluorescent slide method were made from

eighteen-hour-old cultures, as older cultures showed con-

siderable fragmentation and atypical cells. Time studies

for the MFFA cultures gave best results at ten to twelve

15

TABLE 3. Absorbed - B. cereus ATCC # 108'7o"" and 10957 "ant i s e r a

1. -"-Absorbed with B. subtilis, B. mycoides, B. negate riurn

Organism Dilution F. A. Reaction to Antisera

1 0 8 7 6 1 0 9 8 7 Controls-"--:

B. cereus 1 0 8 7 6 1 : 1 0

+ + 0

n 1 : 1 0 0 0 0 0

B. cereus 1 0 9 5 7 1 : 1 0 + + 0

ft 1 : 1 0 0 +

+ 0

f! 1 : 1 0 3 0 0 0

B. subtilis 1 : 1 0 +

Hh 0

tr 1 : 1 0 0 0 0 0

B. mycoides 1 : 1 0 + + 0

1 : 1 0 0 +

0 0

1 : 1 0 3 0 0 0

B. megaterium 1 : 1 0 +

+ 0

tt 1 : 1 0 0 0 0 0

£-All absorptions were reacted in a 1:1 cell-to-antisera ratio,

•KHC-Dilutions do not apply to controls

0 = No Fluorescence - = Trace (detectible) + = Good Fluorescence

16

TABLE 3- Continued.

2. -̂ -Absorbed with B. subtilis

P. A. reactions to Antisera Organism Dilution 10876 10987 Controls

cereus o . , 10876 1:103 - t 0

1:10^ 0 0

£• cereus _

10987 1:10-* - . + 0

" 1:10^ 0 0 0

£• subtilis 1:10^ + + 0

" 1:103 t t 0

" 1:10^ 0 0 0

3. ^-Absorbed with B. mycoides

P. A. Reaction to Antisera Organism Dilution 10876 10987 Control'

B. cereus

10876 1:102 ± t 0

" l:lo3 0 ± 0

1:10^ 0 4" 0

B. cereus 0

109B7 1:10<~ + ± 0

" 1:103 0 0 0

B. mycoides 1:10^ + i 0

" 1:103 ± + 0

" 1:10^ 0 0 0

17

TABLE 3« Continued

lj.. *Absorbed with B. megaterium

P. A. Reaction to Antisera Dilution 10876 10987 Controls** Organism

B. cereus 10876 1:102 + +

0

!! 1:10^ + + 0

B. cereus 10987 1:102 + +

0

1! 3 1:10 0 0 0

B. megaterium 1:102 + t 0

it 1:103 0 0 0

hours incuhation. Incubation for longer periods yielded the

typical colony-spreading tendency of the genus Bacillus.

Tables if. and 5 give the MFFA results for the B_. cereus

strains under investigation. These results indicate the

efficiency of both enumeration and genus identification for

B. cereus. Members of the genus Bacillus tested presented

some difficulty with the MFFA method^ as they form dry, crinkled

colonies that tend to become dislodged from the mem-

brane and float on the surface of both preinhibition

and labeling sera. This difficulty was almost entirely

18

alleviated by wetting the colonies with PBS prior to the

addition of the more viscid antisera. Wetting of the colon-

ies also allowed the addition of antisera without the use

of negative pressure, thus requiring a lesser volume for

the contact time. Necessity of immediate colony counting,

because of rapid fading of fluorescence, was, as reported

(13), a problem. Allowing a thin film of the buffered glyc-

erol to remain over the entire surface of the membrane was

found to preserve the fluorescent intensity for at least

ten minutes.

TABLE I4.. MFFA - with B. cereus ATCC W 10875" antisera

Organism (s)

Calculated #

j

Cells Added -

{ l

TJ 0

1—! 0

c5 #

0 § U O PH O F

luorescent

Count-;:-

Fluorescent

Reaction

I 1

0 -P

iH O Pn O

O *

<J -p a * 0

fo 0

B. cereus 10876 30 43 kl 2+ 66 0

B. cereus 10987 30 39 kl 2+ 87 0

B. cereus 10876 + E. coli 30+15 68 19 1+ 125 0

B. cereus 10876 4. + Strep, faecalis 30+15 120 Ik 1+ 206 (-)

B. cereus 10876 JL

+ B. subtilis 30+15 22 29 3+ 51 (-)

•sc-Colony counts are averages from five duplicate counts.

19

TABLE 5. MFFA - with B. cereus ATCC iTToW/ antisera

ra

<0

GO U O

*d © -p T$ aJ <J 3 a O rH H H OS <D O O

<D rH <D Cd # ^ 4i <D P o O

-P d <D a w <D # fc p o d 3 3 H O o

a o •H • -p

<1 O cd • <D

*4* 0 «P -P fl aS p r~ j o PU o

rH O * u • o

o

B. cereus 10987 30- Sk 14-0 2+ 89 (-)

B. cereus 10876 30 21 29 2+ 63 0

B. cereus 10987 + E. coli 30+15 92 6 1+ 160 ( - • )

B. cereus 10987 + Strep, faecalis 30+15 78 10 3+ 71 1+

B. cereus 10987 +B. megaterium 30+15 60 55 3+ 9i| (±)

*-Colony counts are averages from three duplicate counts

B. cereus ATCC 10987 was added to jars of raw water

obtained from a local surface pond. Each of five jars

containing 50 ml of the water received dilutions of B.

cereus amounting to 300 cells per ml of raw water. Only

one duplicated count was made. This study was conducted

to ascertain if the B. cereus could be demonstrated by the

MFFA method from an untreated water. Results, Table 6,

indicate that the normal flora of a raw water do not

interfere with the fluorescent-antibody labeling of the

20

B. cereus. After seventy-two hours, in a static condition,

the total per cent of B. cereus increases, while the normal

flora decrease, but insufficient tests were performed for

valid assumptions.

TABLE 6. MFFA - Bacillus cereus 10987 added to raw pond water

'

Incubation

Time (Hours

1

Dilution

Raw H0H-::-

Pre-Label

Count

Fluorescent

Count

F. A.

Reaction

Plate

Count

F. A.

Control

106 2k 6 1 + 35 t - )

1 10 6 66 39 2+ 49 ( i )

18 10^ 26 8 2+ 30 0

2k 10 3 16 1+ 86 (±)

k.Q 103 17 Ik 2+ 36 ( i )

72 10 3 57 52 1+ 75 <i)

#Zero incubation time has no added B. cereus.

•K-«-0.1 ml of each dilution was plated.

CONCLUSION

A review of this work indicates that the strains of

Bacillus cereus employed as antigens provided antisera of

sufficient titer for the fluorescent procedures used. The

members of the genus Bacillus are readily labeled and both

vegetative cells and colonies give good fluorescence.

Neither the absorption procedure nor the change in

conjugation concentration provided a solution for the lack

of antisera species specificity, and more work will be

necessary in this area.

Results indicate that the MFFA method is quite effic-

ient for the rapid enumeration, and provides a degree of generic

identification for B. cereus in water.

It is plausible to say that the MFFA methods can be

used in following the activity of B. cereus that has been

added to water for treatment of taste and odor. Since the

majority of Bacillus species are soil-inhabitants, the use

of this MFFA procedure could be considered in order to

detect those organisms which may be added in the control of

tastes and odors. Normal flora Bacillus species counts

in waters would be expected to remain somewhat low and re-

latively stable.

LITERATURE CITED

2.

3.

k-

5.

Bergman, S., A. Forsgren and B. Swahn. 1966. Effect of normally occurring rabbit antibodies on fluo-rescent-antibody reactions. Journal of Bacter-iology. 91:166I[.-1665 •

Biegeleisen, J. Z., W. B. Cherry, P. Skally, and M. D. Moody. 1962. The demonstration of Bacillus anthracis in environmental specimens by conven-tional and fluorescent antibody techniques. American Journal of Hygiene. 75^230-239-

Bonventre, P. P. and N. J. Eckert. 1963* Toxin pro-duction as a criterion for differentiating Bacillus cereus. and Bacillus anthracis. Journal of Bac-teriology. 8iT~T2) :i}.90.

Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957-Bergey's manual of determinative bacteriology. 7th edition. The Williams and Williams Company, Baltimore, Maryland.

Brown, E. R., M. D. Moody, E. L. Treece, and C. W. Smith. 1957- Differential diagnosis of Bacillus cereus, Bacillus anthracis, and Bacillus cereus var. mycoio.es W - 5 0 9 7 ~

"Journal" of Bacteriology. 75̂ "

6. Burdon, K. L. 19lj.8. The Potential pathogenicity of Bacillus cereus and its relationship to Bacillus anthracis. Journal of Bacteriology. 5V"TlT^57

7. Campbell, D. H., J. S. Garvey, N. E. Cremer, and D. H. Sussdorf. 1963* Methods in Immunology, W. A. Benjamin, Inc. New York.

8. Carter, C. H. and J. M. Leise. 1958. Specific stain-ing of various bacteria with a single fluorescent antiglobulin. Journal of Bacteriology. 76:152-I5tf..

9. Clark, H. P. and C.C. Shepard. 1963* A dialysis technique for preparing fluorescent antibody. Virology. 20:61\.2»

22

23

10. Coons, A. and M. Kaplin. 1950- Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluo-• rescent antibody. Journal of Experimental Med-icine. 91:1-13»

11. Danielson, D. and G. Laurell. 1965- A membrane filter . method for the demonstration of bacteria by fluo-rescent antibody technique. II. The application of the method for detection of small numbers of bacteria in water. Acta. Pathol. Microbiol. Scand. 63:60i|-608.

12. Dowdle, W. R. and P. A." Hansen. 1961. A phage-fluo-rescent antiphage staining system for Bacillus anthracis. Journal of Infectious Disease. 108: T25^I3FT

13* Guthrie, R. K. and D. J. Reeder. 1969. Membrane filter-fluorescent- antibody method for detection and enumeration of bacteria in water. Applied Micro-biology. 17 (3):399-J^01.

llf. Hoehn, R, C. 1963* "Some relations between certain aquatic Actinomycetes and Bacillus cereus," unpublished master's thesis, Department of Biology, North Texas State University, Denton, Texas.

1f>. Krieg, Aloysius. 1965* Identification of Bacillus thuringiensis in microbial preparations by com-bination of immunofluorescence and phase contrast technique. Zentral Bakteriol. Parasitenk Infektions Krankheiten Hyg. ABT I Org. 197 (k): 527-532.

16. Lamanna, C. I9I4.O. The taxonomy of the genus Bacillus, Journal of Infectious Disease. 67:193-205.

17. Moody, M. D., M. Goldman, and B. M. Thomason, 1956. Staining bacterial smears with fluorescent antibody. I. General methods for Malleomyces pseudomallei. Journal of Bacteriology. 72l3Fl-3^1. *

18. Mossel, D. A., M. J. Koopman, and E. Jongenius. 1967-Enumerations of Bacillus cereus in foods. Applied Microbiology. 15 (3 ) : 6^0-65jfT~

2i|'

19-. Pital, A. and S. L. Janowitz. 1963• Enhancement of staining intensity in the fluorescent-antibody reaction. Journal of Bacteriology. 86:888-889.

20. Sievers, Olof, and B. 0. Zetterberg. 19i)-0. A pre-investigation into the antigenic characters of spore-forming aerobic bacteria. Journal of Bac-teriology. l\.0 :ij.5-56.

21. Spendlove, R. S. 1966. Optimal labeling of antibody with fluorescein isothiocyanate. Proceedings for the Society of Experimental Biology and Medicine. 122:580-^83.

22. Weller, T. H. and A. H. Coons. 1954-- Fluorescent antibody studies with agents of varicella and Herpes Zpster propagated in vitro. Proceedings for the Society of Experimental Biology and Med-icine. 86:689—69i4- •