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SCHEFFERLE, H. E. (1966). J . appl. Bact. 28 (3), 403-411.
The Microbiology of Built Up Poultry Litter
HENRIETTA E. SCHEFFERLEI Department of Bacteriology, School of Agriculture, Edinburgh, Scotland
(Received 10 May, 1965)
SUMWY. The numbers of viable bacteria in built up poultry litter were found to be 101o-lO1l/g fresh weight and appeared to be little affected by factors such as age, temperature, moisture content and pH. Counta for unused litter and poultry droppings were lower. In built up litter of high alkalinity coryneform bacteria were predominant; micrococci occurred sporadically and smaIl numbers of nocardias, streptomycetes, aerobic spore formers and streptococci were encountered. A variety of Gram negative bacteria also occurred, the numbers of which appeared to be controlled by alkalinity; they were less abundant in litters where the pH and buffering capacity were high. Strongly alkaline conditions also tended to lower the fungal counts but had no effect on the count of enterococci.
TEE BUILT UP or deep litter system of poultry keeping is one of the intensive methods of management whereby the birds are confined indoors during their productive life. The method consists in starting with about 4-6 in. of peat moss, chopped straw, wood shavings or some other form of litter and adding to this periodically until a depth of 10-12 in. is attained. In this way excess moisture from the droppings of the birds is absorbed and the litter is kept in a moderately dry and friable state. Under these conditions decomposition of the droppings takes place without the production of offensive odours; ammonia is liberated and the material becomes markedly alkaline,
The only recorded investigation into the microbiology of built up poultry litter is that of Halbrook, Winter & Sutton (1951), who examined litter made from ground corn cobs, shavings or bark and found the same trend in their microfloral content. In fresh litter under birds they observed during the first 8 weeks a general increase in the total count at 37" and in the counts of lactobacilli, enterococci, coliform bacteria, moulds and yeasts. In litter in use for over a year, however, the numbers of lactobacilli, coliform bacteria, moulds and yeasts had decreased appreciably, but the enterococci had decreased only slightly. The aerobic pIate count remained virtually unchanged with increasing age of the litter and always at a higher level than could be accounted for by the groups determined; therefore Halbrook et al. (1951) postulated the existence of other bacteria not included in the differential analysis. The general reduction in the counts of lactobacilli, coliform bacteria, moulds and yeasts was thought to be due to the increase in pH of the litter because the effect could be accentuated by the addition of lime. It might also be significant that the total aerobic count was determined at 37", a temperature a t which many organisms originating from soil would fail to grow.
* The present address of the author is: Scottish Home and Health Department, 1 India Buildings, Victoria Street, Edinburgh 1, Scotland.
404 Henrietta E. Schefferle
The work described below is concerned with the numbers and types of micro- organisms found in poultry litter of varying nature; one sample of droppings was also examined. Although lactobacilli are numerous in the intestine of poultry, many grow poorly or not at all under aerobic conditions (Shapiro, Rhodes & Sarles, 1949; Harrison & Hansen, 1950a,b) and would not be expected to multiply under the conditions of aeration and pH existing in built up litter. The occurrence of this group of bacteria has therefore not been considered in the following investigation.
Materials and Methods
Nature of samples examined A description of the samples and details of temperature, moisture content and pH are lieted in Table 1.
T~LBLE 1 Nature of the samples of poultry titter and droppings examined
Mean Month temp. Dry PH
sampled when matter Sample TJrpe Age number of litter
sampled ( %) ("C)
18
lb lo 2 3 4&
4b 40 4d 40 4f 5 6
7
8
Chopped straw and peat moss
1,
,, - -
Chopped straw and peat moss
,* I,
, ,, ,,
Chopped straw Chaff, peat moss and straw Sawdust and oow manure Droppings from birds on litter 4a
Before chicks introduced 3 months 9 months 2 months 4 months 1 week
6 weeks 9 weeks 3 months 44 months 9 months 10 weeks 7 months
9 months
June
Sept. March June May May
June J d Y Aug. Sept. Feb. Oct. June
June
16.0
19.6 18.9 20.0 12.9 8.0 18.6 21.0
26.0
884
79.0 69.6 72.8 76.8 54.2 79.4 87.6
68.2
23.6
4.3
8.4 6.6 8.6 8.9 7.2
7 -0 8.0 7 43 7.6 6.4 7.4 8.1
8.5
Samples having the same number are from the same litter at different ages. - , Data incomplete, but probably chopped straw and peat moss.
Collection of samples The surface layer containing fiesh droppings was first removed. A sample was then taken from the remaining depth of litter, the process being repeated at several points in the poultry house. The samples were thoroughly mixed and a portion taken for bacteriological examination and for pH and moisture determinations.
Microbiology of poultry litter 405 Handling of the samples was done as aseptically as possible. At the time of sampling the temperature was taken at a point midway down in the litter and at random positions throughout the house, the results being averaged.
The sample of droppings was collected from the surface of one litter, care being taken to avoid contamination from the litter material.
Moisture content and p H of samples The moisture content was found by drying 60 g of the material a t 100" overnight. pH was measured electrometrically (Cambridge Instrument Co., Ltd., Grosvenor Place, London, S.W.l) on a portion of the suspension, 1 in 10 in water, prepared for bacteriological examination.
Preparation of samples for bacteriological examination Long lengths of straw were f is t cut into small pieces. A 30 g sample was transferred to a previously sterilized macerator jar, 270 ml of sterile tap water were added and the whole was treated in a top-drive macerator for 1 min. Decimal dilutions were then made in tap water. For most of the work described below only the higher dilutions containing relatively low numbers of bacteria were suitable.
One ml quantities of appropriate dilutions were used for making plate counts. In later work 0.1 ml of the inoculurn was spread over the surface of a dried plate using a bent glass rod. This latter method had the advantage of showing the features of surface colonies. Platings of each dilution were made in triplicate and carried out as rapidly as possible after the dilutions were prepared.
Estimations of numbers of micro-organisms All media were sterilized at 128" (22.5 lb steam pressurelina) momentarily.
Total viable counts and the bacterial types present Total viable counts were found to be best determined on nutrient agar containing ;
peptone (Difco-Bacto), 6 g; peptone (Evans), 6 g; meat extract (Oxoid Lab-Lemco), 10 g; NaCl, 6 g; agar (Davis), 16 g; tap water, 1000 ml; pH 7. Plates were incubated at 22" for 10 days.
From the incubated. plates approximately 30 colonies were picked at random so that the relative abundance of the different bacterial types might be estimated. Each colony was transferred to a nutrient agar slope and incubated at 22"; as soon as growth appeared a Gram stained preparation was made and the young culture was examined for motility.
Gram negative bacteria From the examination of the colonies on the total count plates there was some
indication that the relative abundance of Gram negative bacteria might be influenced by the pH of the sample. To investigate this further, plate counts for Gram negative bacteria were made using nutrient agar containing 1 :600,000 of crystal violet incubated at 22' for 10 days. When the plates were crowded, the dye in this medium became concentrated in many of the colonies and Gram positive bacteria then
406 Henrietta E. Schefferle
developed. To overcome this difficulty plates with (100 colonies were always selected for counting. Streptococci occasionally appeared on these plates but their colonies were tiny and easily recognized.
Enterococci Enterococci are present in considerable numbers in the intestinal contents of
poultry and since they tolerate a high pH they probably withstand the alkaline conditions in built up litter.
The medium used for their detection contained; peptone (Evans), 10 g; meat extract (Oxoid Lab-Lemco), 10 g; glucose, 5 g; yeast autolysate, 50ml; agar (Davis), 15 g; tap water, 1000 ml; pH 6. The autolysed yeast was prepared by holding 1 kg of brewer's yeast in 1 1 of water a t 50" for 24 h ; after centrifuging, the supernatant was sterilized in bottles. Plates were incubated a t 45" for 2 days. The pH of this medium was somewhat critical because a t pH 7 coli-aerogenes bacteria (Escherichia coli type I) tended to outgrow the streptococci.
Moulds and yeasts The colony counts of moulds and yeasts were estimated by surface plating on:
glucose, 10 g; peptone (Evans), 5 g; K,HPO,, 0-5 g; agar (Davis), 15 g; tap water, lo00 ml. The pH was adjusted to 3 4 4 . 0 by adding 1 ml of 0.5 N HaSO, to 100 ml of the melted medium immediately before use. After 5 days a t 22" mould and yeast colonies could be recognized.
Results Total bmtericll counts and the types of bacteria predominantly present
The counts obtained on the various samples are summarized in Table 2. The counts for the built up litter were all of the same order of magnitude, being in the range 10,800~ 1O6-153,0O0x 106/g of fresh material whereas those for unused litter (sample la ) and droppings (sample 8) were somewhat lower.
Examination of the colonies on high dilution plates showed that the majority of organisms could be placed in one of three groups, coryneform bacteria, micrococci or Gram negative types. Other bacteria such as aerobic spore formers, nocardias, streptomycetes and streptococci were found only occasionally. From the results it can be seen that coryneform bacteria became the predominant bacteria in built up litter, particularly if the pH was high; the occurrence of micrococci was more erratic and there seemed to be no relationship between their numbers and the nature of the litter. The percentages of the Gram negative types varied considerably but there seemed to be fewer present in those litters of high pH.
Although the sample of droppings showed a lower total count than did built up litter samples, the proportions of the various types of organisms were very similar, and on more detailed examination the strains isolated appeared to be identical with those normally found in built up litter. This suggests that either the sample of droppings had become contaminated or the organisms ingested from the litter retained their viability when passing through the intestine of the bird. That the
TA
BL
E 2
~ To
tal b
acte
d counts a
nd th
e predominant t
ypes
of b
acteria in
poultry
litte
r and
droppings
Per
cent
age
of
E P
late
cou
nt
-c
Sa
mpl
e ( x
10"g
Cor
ynef
orm
M
icro
- G
ram
A
erob
ic
stre
pto-
St
rept
o-
0 0,
r A
-
w
0
C.
num
ber*
fr
eah
wt)
btMteria
cocc
i ne
gati
ve
spor
e N
ocar
dias
m
ycet
es
cocc
i ty
pes
form
ers
3
35,000
41
3 48
0 0
3 3
5
18
188
16
8 68
0
0
8 0
lb
153,000
83
8
0
3 0
3 3
IC
14,700
66
2
40
2
0 0
0 0
2
49,000
72
3 12
3 5
0
5 r
cd
0
3 21,000
66
31
0
0
0 0
3 pa
86,000
62
0
35
0
0
0
3 4b
4c
30,000
74
0
19
0
0
3 3
4d
36,000
65
0
35
0 0
0 0
48
47,000
76
10
10
3 0
0
0
rlf
10,800
60
0
31
0
6 3
0
6
5 72,000
76
7 14
0
0 3
0
6
35,000
74
17
9
0
0
0
0
7 36,000
80
8 4
0
8 0
0
8 88
72
9 19
0
0
0
0
.c" (D 1
* For
des
crip
tion
of s
ampl
es, s
ea T
able
1.
408 Henrietta E. Schefferle
latter is possible was shown in later work when the caecal contents of a battery kept hen were examined; coryneform bacteria and aerobic Gram negative bacteria were among the isolates from platings on nutrient agar. Unfortunately no similar examination was made on a hen kept under the built up litter system.
Gram negative bacteria Counts of Gram negative bacteria determined on the crystal violet medium were
compared with those estimated from their percentage representation on the total count plates; this showed that the selective medium detected 8-40% of the estimated number. As each calculation was based on the examination of only about 30 colonies it was felt that, although the selective medium was not giving a n absolute count of the Gram negative bacteria, it was giving a roughly constant proportion so that the results could be used for following fluctuations in the number of Gram negative organisms.
No direct relationship appeared to exist between the numbers of Gram negative bacteria (counted either on the selective medium or on the total count plates) and pH of samples but there did seem to be a correlation between numbers and percentages of the total count and the amounts of acid required to adjust the pH of 300 ml of a 1 : l O litter suspension to a value of 5 (Table 3). It would appear, therefore, that the relative incidence of Gram negative bacteria in built up litter is diminished by alkaline conditions and more effectively so if the material is well buffered.
TABLE 3 The incidence of Gram negative bacteria in poultry litter and the
volumes of acid required to adjust samples to p H 5
Sample number*
4f 4d 4b 4a 4c 4e 6 6 7
count ( x loe/@; fresh wt) of Gram negative
bacteria on &ysta l violet medium
% of Gram negative
bacteria on total count plates
PH of sample
1,020 2,900 2,400 8,900 1,000
870 820 560 700
31 36 48 35 19 10 14 9 4
6.4 7.6 7.9 7.2 8.0 7.6 7.4 8.1 8.5
No. of ml of N H,S04 required to adjust 30 g of
litter to pH 6
2.6 8.8 8.9 9.1 9.4
11.8 13.0 13.6 2043
~
For description of samples, see Table 1.
Enterococci Only enterococci grew on high dilution plates prepared from built up litter or
droppings and incubated at 45". They were identified by morphology, ability to grow at 45" and production of ammonia from arginine; all produced acid and reduced the dye in litmus milk, in most cases this was followed by curdling. Species differentiation within the group was not made. Their occurrence in built up litter was consistent (Table 4) and variations in their numbers did not seem to be related to any characteristics of the litter samples.
Microbiology of poultry litter
TABLE 4 The types and numbers of bacteria f r m poatltry litter
and droppings capable of growth at 46"
409
Sample Plate count Types of bacteria number* ( x 10a/g fresh wt) present
Unused litter la 15 Bacillua licltengormis;
Escherichia coli type I; unidentified streptomycetes
Built up litter l b 163,000 2 423,000
4b 13,000 Enterococcoci 4c 14,000 4d 7,000 40 11,600
2,700 60,000
4f 5 7 56,000
Poultry droppings 8 1,100 Enterococci
4a 82,000
* For description of samples, see Table 1.
The count a t 45" on the unused litter (sample la ) was much lower than those for built up litter and did not represent a count of enterococci. They were Bacillus licheniformis (identified by the key proposed by Gibson & Topping, 1938), E . coli type I end unidentified streptomycetes. I n the absence of strongly acid-forming bacteria such as the enterococci, faecal coli-aerogenes bacteria were evidently able to grow on the medium a t pH 6.
TABLE 5 Plate counts of moulds and yeasts in poultry litter and droppings
Plate count Sample p H of ( x 108/g fresh wt) of
Moulds Yeasts number*, sample -
Built up litter 4a 4b 4c 4d 40 4f 5 6 7
8 Poultry droppings
7.2 7.9 8.0 7.6 7.6 6.4 7.4 8.1 8.5
11,000 20,000 5,400 3,100
920 1,300 4,200
70 100
130
88,000 3,000 1,000
200 90
1,000 < 100 180 20
1,050
* For description of samples, see Table 1.
4'0 Henrietta E. Schefferle
Moulds and yeasts Colony counts for moulds and yeasts are given in Table 5. From the figures for
litter sample 4 it may be aeen that there was a slight decrease in the numbers of moulds and y w t s with increasing age of the litter. The reduction in counts observed for this litter may have been due to the continuance of slightly alkaline conditions in the older samples because the counts increased again in sample 4f in which the pH had dropped to 6-4., The other samples of built up litter also show relatively low counts with high pH values. Counts for poultry droppings are included for comparison.
Discussion When the droppings of hens are added to litter rapid multiplication of bacteria appears to take place. The plate counts obtained in the present work from built up litter were somewhat higher than those previously reported by Halbrook et al. (1951) who, however, made counts at a temperature (37") which would inhibit some of the bacteria likely to be present. Generally the total counts found during the present investigation were all of the same degree of magnitude and seemed to be independent of the age of the material. Slightly lower counts were found in two samples collected during the winter; these were both cold and wet. Low temperatures in poultry litter would be expected to restrict multiplication of the bacteria present ; in addition the droppings tend to remain wet and the litter may become sodden, a condition not conducive to the growth of the bacteria, the majority of which are strict aerobes. In normal built up litter bacterial decomposition of uric acid results in a rise in pH. The relatively low pH of the samples collected in the winter possibly resulted from the continual addition of droppings, normally slightly acidic, unaccompanied by active decomposition of the uric acid.
The bacteria found on high dilution plates could be divided into three main groups, coryneform bacteria, micrococci and Gram negative types. Although the total counts showed little variation, the proportions of the different types varied with the alkalinity of the samples. Generally, the greater the alkalinity the higher was the proportion of coryneform bacteria and the lower the proportion of the Gram negative types.
The number of enterococci found in the sample of poultry droppings was com- parable with that reported for the faeces of hens (Johansson, Sarles & Shapiro, 1948) and the colon contents of chickens (Shapiro & Sarles, 1949). The numbers found in built up litter samples tended to be slightly higher and because the droppings become diluted by the litter it seems certain that these organisms multiply after excretion. Their numbers showed no correlation with the nature of the litter. The results obtained in this investigation are in marked contrast to those of Halbrook et al. (1951) whose counts for enterococci in poultry droppings and litter were never >lOO/g. It is difficult to understand why they were so low eince the method of enumeration was the same as that used by Johansson et al. (1948) and Shapiro t Sarles (1949).
Fungi would not be expected to be favoured by alkaline conditions and, in agreement with the results of Halbrook et al. (1951)) this was confirmed in the present work.
Microbiology of poultry litter 41 * I t has been suggested that when using the built up litter system of poultry
management soil or old poultry litter should be used to inoculate fresh material. Possibly some benefit from this practice would be derived in new houses, but in old houses the bacteria responsible for the decomposition processes would be present in adequate numbers and would certainly multiply provided the conditions of temperature, aeration and moisture content were suitable.
The author wishes to express her thanks to Dr. T. Gibson for his guidance during the course of this work and for helpful criticism given during the preparation of the paper,
References GIBSON, T. & TOPPING, L. E. (1938). Further studies of the aerobic spore-forming bacilli. Proc.
HALBROOK, E. R., WINTER, A. R. & SUTTON, T. S. (1951). The microflora of poultry house
HARRISON, A. P. JR. & HANSEN, P. A. (1950a). The bacterial flora of the cecal feces of healthy
HARRISON, A. P. JR. & HANSEN, P. A. (1950b). Lactobacilli from turkeys. J. Bact. 60, 643. JOHANSSON, K. R., SARLES, W. B. & SHAPIRO, S. K. (1948). The intestinal microflora of hens
SHAPIRO, S. K., RHODES, R. A. & SARLES, W. B. (1949). Lactobacilli in the intestinal tract of
SEAPIRO, S. K. & SARLES, W. B. (1949). Micro-organisms in the intestinal tract of normal
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litter and droppings. Poult. Sci. 30, 381.
turkeys. J. Bact. 59, 197.
as influenced by various carbohydrates in a biotin-deficient ration. J. Bact. 56, 619.
the chicken. J. Bact. 58, 689.
chickens. J. Bact. 58, 631.
