__:_:.__:___:. E__:_:_:_=:_::_:_.:_::_§_.

 

.1.

.01

M. c
. a.
C

.0
.1 a

This is to certify that the

thesis entitled

Development of a Negative Stain for a Direct
Microscopic Count for Ruminal Microorganisms.

presented by

Karl Kereluk

has been accepted towards fulfillment
of the requirements for

l‘l,S! degree in BaCteTiOlog‘y

Maj professor

Date June 6, 1952

 

0-169

 

 

 

bar]. Kereluk

WHENT OF A NEBATIVE STAIN FOR A DIRECT MICROSCOPIC
COUNI‘ F011 W MICBDORGLNIS'IS

(in Abstract)

Amberofnethodsforthe countingofruninal
nmormims has been developed (Kohler, 191.03 Van der
ms, 191.1, 191.8; Bortree, 191.8; an, 191.9). Gall has
developed the better method in as much as her total counts
are higher than those by aw of the other methods. The
Irrita- has observed that stains made by her method have
given some difficulty in use. It was the purpose of this
research to find another stain thich would be satisfactory.

A survey of the majority of the negative stains, which
could be applicable to this method, was made. These stains
which were unsatisfactory are: congo red, indulin, netlvl
blue, aniline blue, and rose bengsh

The follofing steinpreparetionnsfoundto be
satisfactory:

Mgrosine, water soluble, certified W the

Commission on Standsrflzstion of Biological

Stains eeeeeeeeeeeeeeeeeeeeeeeeeeee 10.0 m

Distilled Inter eeeeeeeeeeeeeeeeee 100.0 101

Fomslin (W) seeeeeeeeeseeeeeeeee 0.5 In].
One part of the aqueous nigrosine stain is added to three
parts of 95 percent ethyl alcohol and filtered through s
Seits filter tiles;

Otrtl10fDQOQNQdIOIFIF£OrDOIO

IYQQ‘.QF“OOU‘OQ4O

IO$OODIOiOOQPI".'V‘

Karl Kereluk

A control user should be made and mined adore-
scOpioele. The smear should have an ova: distribution
of the gray-black color 1i thout any perforations in the
curtain of stain.

Bell's method has followed with three exceptions:

l. A two .mmmeter loop was used in the place of a three
miJJineter loop.

2. A circle with an area of four square centimeters was
used in place of s. square their, 1951).

3. The above described stain was used in the place of
Gell's.

Comte on feces from cattle were run in dualicate using
the above described stein in Gell's method and the chamber
saluting method. ihe modified stein, a direct. microscopic
count method, showed a higher nunber of organism than did
the chamber counting method.

WHEN! OF A NEGATIVE STAIN FOR A DIRECT MICROSCOPIC

' COURT FOR EWINAL MICIDORGANISMS

Karl Kereluk
\s

AMI!)

admitted to the school of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requiruents
for the degree of

mom 0? SCIENCI

Deparhent of Bacteriology and Public Health

Year 1952

rumor com

Acknowledgments.................................1
I. Introduction...............................2
11. Historical Review..........................3
III. Methods of Procedure.......................8
IV. Experimental Studies......................12
V. Results...................................21
VI. Conclusion................................23
VII. literature Oi.ted.....;....................24

m”.COOOOOOOOOOOOO0.0COOOOOOOOOOOOOOCOO00.0.26

‘9"
(V

Acknowledgments

The author vdshes to express his sincere thanks to
Dr. H. L. MsJJJnann for his helpful suggestions and guidance.

The writer appreciates the financial support given to
him by the United States Department of Agriculture, Agri-
culture Research Administration, Bureau. of Dairy Industry,
Washington, D. 0.

Be is also indebted to Hr. E.‘ S. Churchill, Dr. C. K.
Mth, and Dr. C. 1". Hufman for their assistance in one we:
or another, and to Dr. E. S. Beneke for his help in taking
the photomicrograph. Mr. s. Ellsworth collected the samples.

- Introduction ..

Of all. the symbiotic relationships between manuals and
micro-organisms, the most important to mans' economyis found
in the minants (Hastings, 191.1,); the ruminating mm
such as cattle, sheep and goats construe and digest large
quantities of pasture grass, hay or other cellulose-containing
roughage feeds. It has been shove: that micro-organisms are '
chiefly responsible for the decotaposition of cellulose in
the rum and that investigators have failed to detect the
presence of the necessary cellulose-digesting enzymes in
the digestive secretions of the host animal (Mdnally,
Hillipeon, 19M) .‘

There is a distinct need for a satisfactory method
of determining microscopically the amber of bacteria in
run contents. Methods uploying a podtive staining of
the bacteria have been matisfactory due to difficulty in
distinguishing between debris md microorganisms and
inability to see the minute nicrococci appsrmtly comma
to rush contents.

Although the counting chambernethodisperhapsbetter
than the stained preparation it is difficult to use and the
results may not be too accurate.

A third method consists of a stained preparation using
negative “9131118.

lash of the latter two nethods have boa recon-tended

-2-

for use. Each has its limitations. In this study an attenpt
was made, first , to improve the negative staining technique of
Gall; and second, to check the improved negative staining
techniques derived against the chamber method.

- Historical Review -

There seems little doubt that cellulose-digesting micro-
organisms are extrenelv important in the digestion of plant
materials in ruminants (Tappeiner, 1884, Baker, 1935). However,“
attempts to grow them and to study then under controlled condi-
tions lave been \mifomly unsuccessful. Some of the men proto-
zoa have been shown to digest cellulose (Hungate, 194.2). The re-
moval of these protozoa does not impair the rate of cellulose
digestion and it is concluded that other organisms also exercise
this function (Becker, Schulz, Emerson, 1929).

Henneberg (1922) applied direct microscopy to the de-
tection of the micro-organisms concerned in the digestion
of structural cellulose. He showed the preponderance of
micro-organisms that underwent a blue color reaction with iodine.
He observed the cellulolytic bacteria within the eroded
cavities of plant material. He emphasized the relationship
of the micro-orgaxisms and cellulose on this basis.

Baker and Harris (1947-108) confirmed the work of Henne-
berg. 1316;? observed and distinguished between a free iodo-
phile population consisting of micro-organisms suspended in
the ruminal or caecal liquids and a fixed iodOphile popula-
tion attached to and acting upon structural celluloszbc mate1~

-3-

ial of the ingesta.

The morphological characteristics of the iodophiles
were studied in detail by Baker (191.2) who distinguished
the following forms:

1. W.W a colorless spore-forming
osdflnrian.

2. A mt m, divided by transverse septa into
spherical or ovoidal compartments.

3- has: ”Esta! 233459.210

It. An unidmtified W m (bacterial forming
rosette-shaped oscillations of 5 to 30 units.

'5. m’m of 2 to 8 units.

Elsden (1948) states that true criteria must be
fulfilled before an organism can be said to be a functional
member of the men pepulation. First, the organism must
perform a chemical reaction known to occur in the men;
second, the organism must be present in the rmen in
sufficient numbers to perform this reaction. He also
pointed out that the isolation in pure culture and study
of orgatdsns responsible for the chemical changes in the
runenaremost important, butthat analysis ofruaen
population will. not be achieved by the "haphazard application
of standard bacteriological procedures." ‘

69,11 and Huhtanen (1951) state that not all. bacteria
present in the men are true ruse: organisms. Contamina-
tion of the men is constantly occurring from outside

sou-cos. Since there are about one hundred billion bacteria

-1...

per gram of men content, only the organisms present in one
million per gram, or not outnumbered by more than one to
100,000 by other bacteria, are considered significant. is
the rmen is an anaerobic organ, it would sea that an
organismmustbeableto carrysnanaembicnetabolisnin
order to survive.

The following is their criterion for Judging a true
runen bacterium '1. n1. bacterium must be anaerobic.

2. he bacterium must be duonstrated to be present in

at least one million per gram of runs: contents. 3. the
bacterial must be isolated repeatedly under these conditions.
At least ten isolations of the same pure "type" culture, as
shots! by selected‘screen tests, are considered flmmm.

1.. n1. bacterium: must be isolated in the animals in at
least two geographical localities. 5. The bacterium must
produce lid products thich can be netaholissd by other ma:
bacteria to end products found in the men. All five of
these conditions should be net before a bacteria: is
accepted as a tune runen bacterial."

Since micro-organism play such an important role in
the breakdoun of cellulose in the rumm, a total count of
the bacteria present would help to shed some light on the
complex problu.

Kohler (191.0) was among the first to attack the problm
of finding a method of counting Moro-organisms present in
the men. His technique involved centrifuging a suitable

-5-

dilution of rumen sample at 800 to 1,000 rpm, concentrating
the sample by a bacterial filter, and staining the bacteria
with carbol fuchsin. Then 0.01 ml of the material is spread
on a cover slip over an area 30 x 2700 139:2 and examined
microsc0pically. It was difficult to distinguish cocci from
debris in the sample. Rods and cocci were counted separately.
In the case of chains of organisms, each cell was counted
separately. The direct count for the rods averaged
2,373,900,000 per ml; and for both rods and cocci, the aVerage
count was 12,980,100,000 per ml.

Van der Wath (1941) reported a method of counting the
infusoria from the rumen of sheep. Material for the
infusorial count was always withdrawn at 9 A. M. After
shaking the sample vigorously, one ml was drawn into a wide
mouth pipette and added -to seven or eight ml of corrosive-
sublimate-alcohol fixative. After washing with alcohol-
iodine and 70 percent alcohol, the sample was stained with
borax-carmine. The stained nnterial was suspended in 3 ml
of oil of cloves in which it was preserved. After diluting
to one to ten or one to 100 in oil of cloves, a dr0p of known
volume from a capillary pipette was placed on a glass slide
and covered with a cover slip. The total number of infusoria
per dmp was counted from which the number per m1 of ingesta
was then calculated.

A Petroff-Hauser bacterial counting chamber for making
ruminal counts is described by Van der Wath (191:8). After

-6...

thoroughly shaking the fixed ruminal sample, a final dilution
of one to #00 is made. Three-tenths ml of 1 percent nile blue
sulphate is added to the final dilution as the stain. A Thomas
Zeiss pipette is filled with the final dilution and the counting
chamber filled. The chamber is allowed to stand for three or five
minutes in order to allow the bacteria to settle. Ten blocks
of nine small squares are counted (giving the total number
of bacteria in 90 squares) and the total bacteria computed
by a known formula. Van der Hath states that these counts
are, of course, not necessarily true total counts, since
an unknown percentage of organisms penetrates into or
becomes absorbed by food particles. The total count as
given is, therefore, always considered as less than the true
count.

Bortree 91; _c_a._]_.-. (1911.8) used a chamber counting method
similar to Van der wath‘s, modified by using another stain
and by the number of small squares counted. At present in
this laboratory, the counting method of Bortree 93 9.1.. (1948)
is being used for a ruminal bacteria count. The method will
be described under procedures.

Gall.g§.§;. (19h?) were the first to describe a counting
method employing a negative stain. The principle used in
the counting method is the same as that of the direct milk
count; however, certain modifications were made to adapt
the technique to the Special purpose of counting ruminal

bacteria.

- methods of Procedure -

The counting method of Bortree ,3; a}. (1948) is as
follows:

Preparation of the stain: Ten ml of ethyl alcohol is
esturated with crystal violet (gentian violet). One ml of
this solution is added to 1.9 ml of distilled water. The
stain is'heated to 60 0., filtered and used.

Usual procedure and dilutions: Five ml of men ingests
is added to 10 ml of 10 percent formalin and sluken. Three
ml of this samle is pipetted into 22 m1 of sterile distilled
water and again shaken. One ml is pdpetted into eight ml of
sterile distilled water and one ml of stain is added. This
ismixedwellandheatedoverablmsenburnsrmtilitbmnps
slightly. The counting chamber is filled by using a mona-
Zeiss blood pipette. he counting chamber is of the Petroff-
Hauser type. One~htmdred mill squares on the top and the
bottom of the ruled area are counted and the average of the
umber of bacteria per 100 squares is calculated. h using
the following formula the total number of bacteria in one n1
can be determined: ,

No. of bacteria .. Ave. no. of bacteria per 100' squares I

per m1 of sample ution 20 20 0 000
No. of mall squares counted

 

20x203dseofsma11squares orAOO

50 " depth of material ulth cover slip on the chamber
1,000 " conversion factor to change an to 1:0.

No. of mall squares " 100 (after average has been found)

Recently a slight modification has been made in dilutions

-8...

and in the counting of the organisms in the chamber. A fecal

specimen1 is diluted one to ten and used as the sample.

Ten m1 of the sample is pipetted into a test tube containing

five ml of £50 percent formalin. The dilution is now one to 15.

One ml of the above dilution is pipetted into seven ml of

sterile distilled water. This gives a dilution of one to 120.

One-half ml of the one to 120 dilution is pipetted into a

test tube containing four ml of sterile distilled water and

one-half ml of crystal violet stain. This final dilution is
one to 1,200. A portion of the final dilution is pipetted
into the counting chamber and the organisms are counted. Two
hundred small squares are then counted, and by using a modifi-
cation of the first formula the following calculation is made:

No. of bacteria .. No. of bacteria per 200 squares

per m1 of sample " x dilution (1,200) g 20 million

200 squares
The disadvantages of the Petroff-Hauser chamber counting
method are:

1. Movanent of the bacteria in the chamber. This takes time as
the chamber on the microscope must remain stationary until
the bacterial movanent has stopped. This waiting takes
from two to five minutes.

2. There is a lack of clear differentiation between micro-

organisms and plant debris. The direct staining method

 

1 v
For the work on this problem, fecal material was used in the

place of ruminal material because of the accessibility of the

88111131850
.- 9 -

stains both the bacteria and plant debris making it hard
to distinguish between the two. i . '
3. The filled chambers cannot stand for a long period of time
as evaporation will take place in the chamber.
A. It is very cumbersome to fill the countingfchamber. Time,
practice, and patience are needed to do a satisfactory Job.
Gall g g... (1949) described a counting method for
rmninal bacteria using a counting method similar to a direct
milk count. Certain modifications were made to adapt the
technique to the special purpose of counting ruminal bacteria.
In brief, the method is as follows: "A 0.01 ml portion of
the properly diluted sample is mixed with a 3 mm loopful of
saturated methyl alcohol solution of watemolufle nigrosine
and spread evenly over a 2 x 2 on area of a very clean slide.
This slide is dried quickly on a very hot electric plate and
10 to 20 fields counted.u In following her inadequate descrip-
tion of the counting technique, these difficulties were noted:
1. When using a clean slide (following her cleaning methods),
the sample would be repelled by the loop 3 containing the stain
and run over the 2 x 2 cm square, marked off by a wax pencil,
before contact could be made. This may be due to static
charges. A rapid contact of stain and sample gives a similar
result. 'mis phenomenon occurred frequently enough to

warrant mention.

-10..

2. ThemarkingefaZchmsquareonthe glass slide
rould always leave may particles of pencil 19.x Itithin the
area which has to be used for counting.

3. There is no mention of a control smear made on the stain
alone. As there is no specification made on the filtering,
the filtering was done with ordinary laboratory filter paper.
If bacteria were present in the stain preparation they would,
therefore, be counted on the slide. To rule out doubt that
the organisms seen on the slide are organisms from the sample,
a control smear should be made and the stain preparation
containing bacteria should be discarded.

1.. When the 0.01 ml sample and the 3 mm loopful of stain
are successfully mixed, the slide is rocked side to side to
effect an even color. Slight shrinkage immediately takes
place on the edge of the smear. The slide is placed on the
very hot electric plate to dry. ihe smear dries unevenly or
concentrates toward the center. On microscOpic examination
of the smear, cracking is seen in the concentmted area
(about one-fourth of the total smear). The cracking and

the concentration interfere with the counting of ruminal
organisms.

Gall stated the following about the stain: "A large
supply should preferably be made, so that it. can be used
over a long period, since minor adjustment in the amount
of dye desired to give the proper black background must be
made with each lot, to suit the individual.” As mentioned,

her stain is a saturated methyl alcohol solution of a water
soluble nigrosine. The only adjustment which could be made
would be the dilution. This lightens the background and
reduces contrast. Due to the unpredictable behavior of the
stain, no counts could be made.

Cumley (1935) maintains that the (bra is derived from
varying degrees of sulfonation; and the manufacturers make
little claim as to the possibility of duplication of arm
batch that has proven satisfactory for any special purpose.

This may explain the unpredictable behavior of the stain.
- Experimental Studies -

It was the purpose of this research to find a negative
stain which would overcome the difficulties encountered in
Gail's method.

A direct staining method was ruled out, as a direct
stain only stains the cytOplasmic membrane and protOplasm
while the cell wall and slime layer are not stained. This
decreases the visibility of the organism. A direct staining
method would increase the time necessary to find the smear
with the oil immersion lens, inasmuch as a high dilution
of the sample is needed to secure 10 to 30 organisms per
field and it is difficult to focus on a smear when there are
so few organisms present.

A negative stain will increase the visitility of micro-
organisms. Definition of small micrococci is important.
Baker (1931, 1939) noted the action of small cocci in the

-12..

disintegration of the cell wall substance. ihe cocci were
present in clearly defined zones of erosion. The size of
the cocci was 0.25 to 0.9 E and 0.1 to 0.2 gt}.

Knaysi (l9h5) made a commarative study of the cell width
of My; mug and M coides. heasuranents of
the living cell in the median in which they were growing
agreed with measurements on similar cells stained by a
method shoudng the cell wall. In stained smears in which
the cell wall was not visible, the cell appeared much smaller
and represented the shrunken mass of protoplasm. The cell
wall and slime luer are not stained with a negative stain
but the stain outlines the cell wall and slime layer. "The
organisms measure larger than their true size owing to the
fact that the colloidal We film retracts on drying.

A umber of negative stains was tested for their
suitability in a direct counting method as prescribed by
0.1.1 gt 9;. (191.9). A control smear of the stain and a
wear with a 0.01 ml sample of rumen liquor (diluted one to
100) and the stain were made on the same slide. As no
comparative comts were to be made, the area covered by the
smear was not controlled.

It was important to have a clean slide for the prepara-
tion of the smears. be following method has been used for
cleaning the slides: 11:. slides were stored in a chromic
acid cleaning solution for a few due, then rinsed in distilled
water, dried, and stored in a clean (met-proof bots. Prior to
using the slide, they were flamed and allotted to cool.

-13..

Benian's Congo red method was the first staining tech-
nique to be tried.

stain preparation: Congo red (80 percent dye content) - 2 gm
Matilled water - 100 ml

Staining schedule: 1. Place a drop of the above stain on
the slide.

2. Hi: the stain and the 0.01 ml diluted
rumen sample and spread out into a
rather thick film.

3. After the film has dried, wash with
1 percent hydrochloric acid.

1,. Dry.

5. hairline.
Results: The cells are unstained against a blue background.
be blue background as heavily textured. (A texture similar
to the crinkly finish caused by baking certain types of black
enamel.) m. textured background interfered with the visi-
bility of the organisms. The torturing was found to be due
to the fomalin present in the original. fecal ample. The
fomalin was added as a preservative. Per further studies,
fecal samples without fomslin were used. However, this
staining method was very unsatisfactory because large sheets
of the smear float off when the 1 percent hydrochloric acid
was added. The parts of the smear which ranain on the slide
show a very even distribution of the blue color. The stain
gave a good contrast and the control smears show little or no
interference from other organisms or artifacts. Various
strengths (0.25%, 0.5%, 2%. 5%) 04'- the Wall-0H0 80541
were used to minimise flaking. This was unsuccessful.

-11...

A one-third percent hydrochloric acid solution made with 95
percent alcohol was tried and gave the same results.

Two and one-half, five, and ten percent aqueous solu-
tions of anilin blue, indulin, rose bengal, and methyl blue
were next tried. Each preparation was heated and filtered
before using. Smears were made in the same manner as that
used with the Congo red stain. All the stains showed poor
contrasting backgrounds.

Two staining procedures were used. The first method
was discarded after a series of 21.0 samples were tested be—
cause of a desire to find a method that would eliminate the
objectionable features that were found.

In the first method the stain preparation, designated
as nigrosine stain #1, was as follows: '

Nigrosine, water soluble (American nigrosine certified

by Commission on Standardization of Biological Stains

as orfinarily satisfactory)......................l0.0 gm

Distilled water.................................100.0 ml

Fomalin (AD percent).............................0.5 ml

Ethyl alcohol (95 percent)........................5.0 ml
The nigrosine is added to 100 ml of distilled water and
stirred until the nigrosine has completely dissolved. Next
the formalin and ethyl alcohol are added. The stain is fil-
tered tvdce through a Seitz's filter. The stain should produce
an even dark gray color on the slide. Procedure: The sample

(rumen or fecal) is diluted in distilled water so that there

-15..

are about five to twenty organisms per microscopic field
(a dilution of one to 15,000). Ten ml of a one to ten
dilution of the sample is placed in a test tube containing
five ml of fonnalin, producing a one to 15 dilution. The
sample is shaken vigorously for 30 seconds. One-tenth ml
of the one to 15 dilution is pipetted into a dilution blank
containing 99 ml of distilled water, producing a final di-
lution of one to 15,000. The diluted sample (one to 15,000)
is vigorously shaken for 30 seconds. I

A 0.01 ml sample is pipetted from the dilution blank
and placed on a clean slide. Care should be taken in pipet-
ting the diluted sample onto the slide. The tip of the
pipette is Wt into contact adth the clean slide at the
cutter of the inscribed circle and the 0.01'ml sample is
released. The tip of the pipette is lifted from the slide
and again gently touched to the slide, near the original
sample, todispellth'elastsmalldropwhichvdllcldngto
the pipette.

A 100p made of nichrome wire, with an inside diameter of
2 m is dipped into the prepared stain and is placed in con-
tact trith the 0.01 ml sainple. By means of the nichrome loop
the stain and sample are completely mixed and spread to form
adrclewhichhaeanareaeffourequare cm.

Heir (1951) used the migrosine slide technique des-
cribedbyGallgtg. butmdifiedtheZ‘chnsquaretoa
circle of the same area. He noted that less shrinking diffi-

-16...

culties were apedenced with the circle than with the square
and the resultant slides were more uniform in density. A
circle with a radius of 1.13 on gives an area of four square
n. The slide is placed over a guide and the smear is made.

After the stain and the sample are mixed and spread, the
slide is rocked back and forth and side to side to achieve an
even color. The slide is rapidly dried on a hot plate at
700 P. (A Stmbeam hot plate, model 1h B, was used.) The
drying takes five seconds at the most. The slide is quickly
removed, as a longer time on the hot plate will crack the slide.
It has been found that 3 percent of the glass slides hill
break from the intense heat of the hot plate. The breakage
is usually due to imperfections in the glass slide and not
from the short exposure to the heat. If the break occurs
through the near, a duplicate slide should be made. After
the slide has cooled, it is ready to be examined microscopi-
cal]: and the bacteria counted.

The microscope is calibrated by following the directions

in the 9th edition of W Methods for them p_f_

alumnae.
The bacteria will appear white against the dark gray of .

the negative stain (Fig. 1). Care should be exercised in the
counting of the bacteria. Spherical abberation of the oil
imersion lens will obscure the view of the periphery of the
field. Therefore, by means of the fine adjustment screw the
bacteria, which would be present in the peripheral area, can

-17..

be brought into focus.

Artifacts present in the smear will cause undue crack-
ing, formation of a dark ring of stain around the artifact,
and irregular white forms. Some artifacts will be stained
also. Dirty slides will cause undue confusion as mall
grease spots till appear as bacteria.

"Strip counting" should be anployed. The smear till be
light gray in the periphery which will afford little or no
contrast for the bacteria. The center of the field Idll be
black containing large cracks and the bacteria will be ob-
scured by the heaviness of the stain. The larger area between
the extreme, the light and the dark, is the preferred area
for the counting of the bacteria. Conditions here are cp-
timua for contrast and for ease of vision. The llstrip
counting" should, therefore, be done in this area. Ten or
20 fields are counted.

Williama and Heir (1951) stated: "The relationship
between the numbers of organisms counted in a nigrosine smear
and the numbers actually present in the salple, however, is
not nearly so satisfactory. It is exceedingly difficult to
distinguish with certainty betwem artifacts and bacteria
when their size is less than about 0.5 mg. The counts
present in this stw arbitrarily exclude all bacteria less
than about 0.5 mg, even though the presence of more minute

organisms can be demnstrated in stained preparations. As

-18...

a result, the number of organisms counted are slightly
underestimated."

Baker (1931, 1939) showed the importance of smaLL
micrococci in the rumen and noted their size as 0.25 to
0.90 g, and 0.1 to 0.2 m3. It is therefore indicated that
organisms under 0.5 mg should be counted, othervdse a com-
plete picture of the total free rmninal organisms could not
be made. Thus in the strip counting, all organisms seen are
counted. 7

In the previously described technique there is a valid
objection to the fact that the center area of the stained
preparation is too heavy and that shrinkage occurs around the
edges. This forced strip counting, which may not be a rep-
resentative count of the total smear.

Steps were taken to eliminate these objections by fur-
ther modifications of the stain mixture. .

To keep the mneer in place during the quick drying on an
electric hot plate, several experiments were tried. Varying
concentrations of agar and gelatin (0.5, l and 1.5 percent)
were added to the 99 ml dilution bottles in an attanpt to
increase viscosity. The 0.01 ml sample was pipetted to a
clean glass slide. The usual procedure of mixing and drying
19.8 followed, but neither the agar or gelatin prevented the
smear from concentrating toward the center. The higher con-
centrations of agar five a textured background to the nigro-
sine stain and interfered with the counting of the bacteria.

-19..

A cationic surface active agent was added to the 99 ml dilution
blank; but it, also, was unsuccessful in stepping the concen-
tration.

different dilutions of ethyl alcohol were tested. A stain
was then found which would give a completely countable smear.
The dilution of the aqueous nigrosine stain with ethyl al-
cohol has overcome the difficulty but has sacrificed some con-
trast. The following is the fomula of the stain:2

Nigrosine, water soluble (certified by the Commission

on Standardization of Biological Stains).........l0.0 gn

Distilled water.................................lO0.0 ml

Formalin (40 percent).............................O.5 ml
One part of the aqueous nigrosine stain is added to three parts
of 95 percent alcohol and filtered twice through a Seitz
filter.

The smear is made in the same way as with nigrosine stain
#1 with two exceptions: l. A nichrome loop calibrated so
it will deliver 0.01 ml of the sample is used in the place of
a pipette. 2. The final dilution of the fecal or rumen sample
is one to 3,000.

"Strip counting" is not employed with this stain (nigro-
sine #2) for it does not concentrate forming an uncountable
area in the center of the smear. There is a slight shrinkage

only on the very edge of the smear caused by the initial

 

5
This stain will be referred to as "nigrosine stain #2".

-20..

drying. Ten or twenty fields on the smear are counted.
Fecal samples from cattle were run in duplicate using

the chamber counting method (Bortree, 1948) and the modified

Gail's slide counting method. The nigrosine stain #2 was

used in the place of Gall's stain.
- Results -

Two hundred and forty samples were counted using nigro-
sine stain #1. Sixty-eight percent of these samples were
higher by the direct microsc0pic method than by the chamber
method.

Two hundred and thirty-seven samples were counted using
nigrosine stain #2. The direct micrOSCOpic method gave 89.5
percent higher counts than did the chamber counting method.

Figures 2 to 8 show the fluctuation in counts for each
cow, of the 237 samples tested, over a four months period.

Five two-hour rumen samples were taken from one cow;
the first sample was taken at 8 A. M., the other samples at
10 and 12 A. 14., 2 and it P. M. The counts by the direct
microscopic method showed a higher number of bacteria present

in the samples than did the chamber count (Fig. 9).
- Discussion -

The first rdgrosine stain preparation develOped (nigro-
sine #1) gives a smear with a large uncountable area. When

the smear is dried, this stain shrinks and concentrates toward

-21-

the center of the smear. The center portion cracks and the
stain is so heavy the bacteria are obscured. "Strip counting'
must be uployed.

To overOOIne these problem, the mgrosine stain was

diluted with ethyl alcohol. The increased amount of alcohol
sllnvedthe unearto drynore qtdcklvthus eliminating the
concentrated center area. It was found that one part of aqueous
nigrosine stain to three parts 95 percmt etlvl alcohol gave
the least shunlsage without a great loss of contrast.
In: lesser counts ofettwl alcohol are added to the stain
preparation, cracks will appear in the near. then greater
mounts are added tecthe stain, the contrast is less, making
it difficult to count the ndcroorganisms.

'Ihe direct microscopic counting method using the nigno-
sine stein gave consistently higher counts than did the
chuberootmtingmethod. ImmMgrosine steinflluasused,
onlyBZpercentof 21.0 samplesverehigherbythechamber
counting method. “tn the elimination of the objectionable
features in the nigrosine stain #1, the counts were still
higher. Only 10.5 percent of 237 samples were higher by the
chamber counting method. Not only does the direct micros-
cepic. method give higher counts, but it is essier and faster
to me.

-Inthe aha-her countingnethod, there are three sepa-
rate steps when making the dilutions of the fecal sample.
A Than-Zed." pipette is used to transfer the fecal sample to

{-22-

the counting chamber. The chamber must rest on the stage

of the microscope until movement of the bacteria has stepped.
In the direct microsc0pic counting method, the dilutions take
only two steps, and the use of a nichrome 100p facillitates

a quick transfer of the sample to the slide.

When examirfing the microscopic field of the counting
chamber, it is hard to differentiate between plant debris
and bacteria for this method stains than both. The negative
stain makes possible a rapid differentiation of plant debris
and bacteria. The bacteria are not stained and appear as
white micro-organisms against the black of the stain. The
plant debris is slightly stained and, therefore, becomes

part of the background.
- Conclusions -

l. A direct microscopic counting method using a negative

stain is presented.

2. The formula for the negative stain is presented.

3. The direct microscOpic count using a nigrosine stain

has several advantages over a chamber counting method:
(a) There is no movement of the bacteria.
(b) Smears can be stored for future checking and reference.
(c) The method is faster and simpler.
(d) A rapid differentiation between plant debris and
bacteria is possible.

(e) The counts are usually higher.

-23..

LITERATURE CITED

Baker, F. (1931). Preliminary note on the role of coccoid
microorganism in the disintegration of cell wall substance.
thralbl. m Bak‘beriol. Abbe II, orig. 8153452.

Baker, 3., R. Martin (1939). Studies in the microbiolog
of the caecum of the horse. Zentralbl. Fur Bakteriol. Abt.
11, orig. ‘99sz.

Baker, F. (191.2). Normal rumen microflora and microfauna
of cattle. Nature 11.93220.

Baker, F., S.'1'. Harris (191.748). The role of the micro-
flora of the alimentary tract of herbivore. with special
reference to ruminants. Nutrit. AbBte and Me 17:14

380k”, EORO, JOAO 30111112, HOAO memn (1929). Marina“?!
on the pl'wsiological relationship between the stomach in-
fusoria of ruminants and their hosts, with a bibliography.
Iowa State College J. Sci. M215. '

Bortree, ‘sLe, Cexe anith, Re W, Cope Huffman (l9hB)e
Types and number of microorganisms in the rumen contents of
cattle being fed natural and semi synthetic rations. J.
Animal Sci. 73520.

mean, 3.3. (191.8). The fementations of carbohydrates in
the rumen of the sheep. J. of Exp. Biol. 22: 51.

Gall, L.s., w. Burroughs, P. Gerlaugh, 3.11. Edgington (191.9).
Special methods for ruminal bacteria studies in the field.
Je Animal Sci. 88 51s

Gall, L.S., Cone Huhtanen (1951). 01311561118 for Judging 8
true rumen organism and a description of five rumen bacteria.
J. Dairy Sci. 31.353.

settings, 3.0. (1941.). the significance of the bacteria and
protozoa of the rumen of the bovine. Bact. Rev. 8:235.

Henneberg, W. (1922). Untersuchungen fiber die damflora des
menschen nit Abesonderer berficksichtigtmg der iodophilen
bacterium 1m menschenund tierdam code in konpostdmxgen.
centrabble flit BfiktBl‘iOle Abbe II, orig. 553we

Bungate, 3.3. (191.2). The culture of Didiplodiniun Neglectm,
with emeriments on the digestion of cellulose. Biol. Bull.

8333030

-21.-

Hungate, 3.1:. (1950). The anaerobic mesophillic cellulolytic
bfiOtOI’ile BQCte Me 115310

Kohler, w. (191.0) . Versuche fiber die zaklamassige veraudermg
der natfirlichen bacterienflora in der verdauungs-organen .der
dederka'uer. ArChe MikrObiOIQ 113432.

We Go (1945). (On the microscOpic methods of measuring
the dimensions of the bacterial cell. J. Bact. 1.93375.

Hclnally, Heb, R.T. Phillipson (19M). Ingestion in the
ruminant. Biol. Rev. 1931.1.

Hair, R.J. (1951). The seasonal variation in the ruminal
microorganisms of grazing sheep. Aust. J. Ag. Res. 2:322.

Standard Methods for the Examination of Dairy Products, 9th
ed. American Public Health Association, (191.8).

Tappedner, H. Von (1881.). Cited by Phillipson, 1.13., (191.7%).
III Fermentation in the alimmtary tract and the metabolism
of the derived fatty acids. Nutrit. Abst. and Rev. 17:1.

Van der Hath, J .6. (191.1). Studies on the alimentary tract
of merino sheep in South Africa. VI: The role of inmsoria
in ruminal digestion with some remarks on minimal bacteria.
Ondersterpoort J. of Vet. Sci. and Animal Industry. 17361.

Van der weth, J.G. (191.8). Studies on the alimentary tract of
the merino sheep in South Africa. III: A technique for the
counting of ruminal bacteria. Ondersterpoort J. of Vet. Sci.
and Animal Industry. 23:385. -

Hillisms, V.J., R.J. Mair (1951). Ruminal flora stumes in the
IhOOpe Part III. Aust. Jo 0: Sci. 3690 h33'fle

-25..

 

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BACTERIAL COUNTS OF THE amen: FOR cow #741

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-31...

 

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