For bacterial sampling of raw unprocessed fish and frozen fishery products, spread plate method is preferable to pour plate method; incubation of plates at 30°C gives a higher count than incubation at 37°C. Analysis of variance of the data shows that sample variation between different types of fishes is highly significant whereas the variations between triplicate plates are not significant at 5 % level

Krishna Iyer, H.

Mahadeva Iyer, K.

Thampuran, N.

Aquatic Commons

Fish. Technol. (1981) 18, 95-100 	 95 
T InflucAce of Plating Technique and Incubation 
Temperalt:re on Bacterial Cc 'ant DIM Fish and 
Fishy Products 
NIRMALA THAMPURAN, H. KRISHNA IYER and K. MAHADEVA IYER 
Central Institute of Fisheries Technology, Cochin-682 029 
For bacterial sampling of raw unprocessed fish and frozen fishery products, spread 
plate method is preferable to pour plate method; incubation of plates at 30°C gives a 
higher count than incubation at 37°C. Analysis of variance of the data shows that 
sample variation between different types of fishes is highly significant whereas 
the variations between triplicate plates is not significant at 5 % level. 
Increasing attention is paid nowadays 
to the microbiological quality of food-
stuffs and this necessitates a thorough study 
of the techniques adopted for assessing their 
microbial content. Total aerobic bacterial 
count (TABC) or total plate count (TPC) 
is the most important means of establishing 
whether a product has been prepared under 
strict sanitary condition. 
Different techniques are in use for deter-
mining the TPC of which the spread plate 
technique and the pour plate technique 
are the most common and widely accepted. 
Pour plate technique involves the use of 
melted agar maintained at 45°C, while 
spread plate technique employs pre-set 
plates of agar equilibrated to the desired 
temperature. 
The ISI method for the bacteriological 
examination of fresh fish as well as frozen 
fish products stipulates that pour plate 
technique be used along with an incubation 
temperature of 37°C (IS: 1977 a, b). The 
same procedure is also suggested for micro-
biological analysis of shell fish and its 
products (AOAC, 1970). 
There are several reports on the role of 
transient warm agar temperature in decreas-
ing the microbial recoveries by pour plate 
technique (Zobell & Conn, 1940; Stapert 
et al. 1962; Van Soestbergen Lee, 1969; 
Klein & Shenyu Wu, 1974). Majority of the 
marine micro-organisms are found to suc-
cumb when exposed to a temperature of 
30-40°C for 10 min. Recently several 
communications have come forth, which  
claim spread plate method to be superior 
to pour plate method especially in the case 
of seafoods (Lee & Harward, 1970; Not-
tingham et al. 1975; Shenyu Wu at Klein, 
1976). On the other hand Mossel & Van 
der Moosdijk (1963) found no difference 
between pour plate (agar 44-46°C) and 
spread drop plate (agar at room temperature) 
of 42 food samples. 
A study was undertaken in this laboratory 
to find out the suitability of these two pro-
cedures for use in bacteriological sampling 
of fish and fishery products and to evaluate 
their performance. 
Materials an Methods 
Raw fish samples were procured either 
from local country crafts or landing centres 
near Cochin. Samples were analysed within 
2h after collection. Raw fish samples ana-
lysed were in very fresh condition as judged 
by their appearance. 39 samples of raw fish 
and 48 samples of frozen materials were 
studied during 1976-79. Frozen samples 
were obtained from a local seafood proces-
sing plant and hence of unknown history. 
The frozen blocks were kept in cold storage 
till required. Muscle homogenates of the 
raw or frozen samples were prepared by 
grinding about 10 g of the material with 
90 ml of normal saline in a sterile mortar. 
Subsequent dilutions were prepared in the 
same diluent by mixing 1 ml of the sample 
dilution with 9 ml of the diluent in a vortex 
mixer. Sea water agar (SWA) for sampling 
raw fish and tryptone glucose agar (TGA) 
for frozen material were used. The agar 
Vol. 18, 1981 
96 	 NIRMALA T A PURAN, H. K. IYER AND K. M. IYER 
media for 	 spread plating had been 
poured 6 h before the experiment and excess 
moisture evaporated by keeping the plates 
at 45°C for one hour. Care was taken to 
prevent syneresis fluid on the agar surface 
to assure that colony spreading would be 
minimised. 0.5 ml of the appropriate dilu-
tions were spread evenly on the agar surface 
with an alcohol flamed bent glass rod. 
Pour plating was done using 1 ml of the 
dilution. The molten agar was maintained 
at 45°C in a thermostatic water bath. All 
samples were plated in triplicate. One set 
of plates from each method was kept at 
room temperature, (29 ± 1°C; RT) and 
another at 37° C. In addition, a set of 
spread plates was also kept at 8°C for psy-
chrotrophic count. RT and 37°C counts 
were taken at the end of 48 h of incubation 
and low temperature count after three 
weeks. 
Results and Discussion 
Average counts from three spread plates 
and three pour plates were used for statis-
tical analysis. For raw minced fish, the 
microbial count varied from 8.4 x 10 4 /g 
of the muscle to 1.3 x 10 8 /g (Table 1.) 
The maximum count was noted in spread 
plates incubated at room temperature. The 
lowest count was obtained by pour plating 
and incubating at 37°C. For frozen pro-
ducts the count varied from 3 x 10 3/g of 
the material to 1.2 x 10 6/g (Table 2). In 
frozen and raw fish, spread plates incubated  
at room temperature gave highest count 
and pour plates at 37°C the lowest. The 
count at 8°C was used to estimate the pre-
valence of psychrotrophic bacteria in the 
sample. Analyses of variance for different 
fishes are presented in Tables 3 to 10. 
From Table 3, it is found that for sardines 
(Sardinella longiceps) there is no significant 
difference in bacterial count between tem-
peratures. But difference existed between 
methods, spread plate count :being greater 
than pour plate count. The analysis of 
variance (Table 4) for mullets (Mugil 
cephalus) sliovved significant difference in 
bacterial count between temperatures, RT 
giving highest count than 37°C. However 
there was no significant d_ ifference with 
methods. In silver jew fish (Johnius dussu-
mieri) and Indian halibut (Psettodes erumei) 
the trend was similar (Tables 5 and 6). The 
variation in bacterial count between tem-
peratures as well as methods were highly 
significant. Room temperature and spread 
plates yielded higher bacterial counts.- 
Regarding frozen sardines, significant 
difference in count was observed between 
the two temperatures as well as the two 
methods (Table 7). RT and spread plates 
consistently gave higher counts. Fillets of 
frozen jew fish (Pseudosciaena sp.).. and 
the mixed fish muscle paste popularly known 
as `Kheerna' (Tables 8 and 10) showed more 
or less similar behaviour. But for fillets 
of cat fish (Tachysurus sp.) differences 
Table 1. 	 Total bacterial count for raw fish (total plate count/g x 104 ) 
(29 ± 1)°C 	 37°C 
S.P. 	 P.P. 	 S.P. 	 P.P. 
8°C 
S.P. 
Sardine 230* 190 200 203 
Sardine 6812 3110 1900 1400 48 
Sardine 1275 594 4300 3400 3005 
Sardine 17 21 20 27 13 
Mullet 170 780 85 91 68 
Mullet 93 781 8 8 
Mullet 540 87 96 160 
Indian halibut 13100 13100 12000 11000 6000 
Silver jew fish 742 306 290 280 102 
* Average of three values S.P—Spread plate method 
P.P—Pour plate method 
FISHERY TECHNOLOGY 
PLATING TECHNIQUE AND TEMPERATURE ON ACTERIAL COUNT 
	 97 
existed only between the two temperatures, 
RT giving higher bacterial count than 37°C 
(Table 9). 
In all, sample variation was highly signi-
ficant, but variation between triplicate plates 
was not significant at 5% level. .A similar 
finding was also reported by Nottingham 
et al. (1975). 
For raw fish as well as frozen samples, 
room temperature gave higher count than 
37°C. The . only exception was raw 
sardines for which the count variation due 
to temperature difference was not very much 
marked. Identical counts obtained at RT 
and 37°C for sardine can be accounted by 
the fact that the samples had been exposed 
to ambient temperature for a longer time. 
Jan Gjerde (1975) had reported that the 
higher count at 37°C for raw fish can be 
considered as an indication that the fish 
has been exposed to higher temperatures, 
since normally psychrotrophic bacteria 
predominate in raw fish. It has also been 
suggested that a comparison of count at 
25 and 37°C gives a measure of relative 
proportion of psychrotrophs and mesophiles 
(Nottingham et al. 1975). 
able 2. Total bacterial count for frozen 
fish (total plate count/g x 10 3 ) 
Sardine 
(29 ± 1)°C 
S.P. 	 P.P. 
21* 	 6.7 
37°C 
	
S.P. 	 P.P. 
	
g 	 g 
	
13 	 7.6 
8°C 
S.P. 
g 
- 
Sardine 180 109 160 108 4.6 
Sardine 181 76 74 68 37 
Sardine 65 46 56 32 3.2 
Sardine 21.0 150 190 65 
Sardine 21 9 16 8 
Jew fish 46 19 36 11 14 
Jew fish 57 81 39 48 21 
Cat fish 1200 638 530 570 30 
Cat fish , 188 170 108 77 
Cat fish 314 206 29 20 8 
Kheerna 449 317 278 242 11 
*Average of three values 
S.P- Spread plate method 
P.P- Pour plate method 
Table 3. 	 Analysis of variance for raw sardine 
Source 	 S.S. 
Total 	 47.9681 
Samples 	 44.1282 
Temperature 	 0.0040 
Methods 	 0.8828 
Triplicate 	 0.0002 
Error 	 2.9529 
c - Significant at 0.1 % level 
df 
71 
5 
1 
1 
2 
63 
MS 
8.8256 
0.0040 
0.8828 
0.0001 
0.0469 
188.18° 
0.085 
18.82. 
0.002 
Table 4. 
	 Analysis of variance for raw mullet 
Source S.S. df MS lr 
Total 20.4909 59 
Samples 9.1154 4 2.27885 15.45° 
Temperature 3.6608 1 3.6608 24.819° 
Methods 0.1937 1 0.1937 1.313 
Triplicate 0.0003 21 0.00015 1 
Error 
c - Significant at 0.1 ° level 
7.5207 51 0.1475 
Vol. 18, 1981 
98 	 NIRMALA THAMPURAN, H. K. IYER AND K. M. IYER 
Table 5. Analysis of variance for raw Indian halibut 
Source 	 S.S. 	 df 	 MS 
Total 0.0119 11 - - 
Temperature 0.0094 1 0.0094 67.14e 
Methods 0.0012 1 0.0012 8.57a 
Error 0.0013 9 0.00014 
a, c - Significant at 5 and 0.1 % respectively 
Table 6. An 
 
lysis of variance for ri w silver jew fish I 
 
Source 	 S.S. 	 df 	 MS 
Total 0.3691 11 
Temperature 0.1483 1 0.1483 13.12b 
Methods 0.1188 1 0.1188 10.51a 
Error 0.1020 9 0.0113 
b - Significant at 5 and 1 % respectively 
Table 7.• nalysis of variance for frozen sardine 
Source 	 S.S. 	 df 	 MS 
Total 11.8636 71 - 
Samples 9.9333 5 1.98666 179.46. 
Temperature 0.2351 1 0.23510 21.24. 
Methods 1.0061 1 1.00610 90.89. 
Triplicate 0.0027 2 0.000135 21 
Error 0.6864 62 0.01107 
c - Significant at 0.1 % level 
Table 8. Analysis of variance for frozen jew fish 
Source 	 S.S. 	 df 	 MS 
Total 1.6288 23 - 
Samples 0.7593 1 0.7593 27.51. 
Temperature 0.2280 1 0.2280 8.26a 
Methods 0.1450 1 0.1450 5.25a 
Triplicate 0.0003 2 0.0002 - 
Error 0.4962 18 0.0276 
a, c - Significant at 5 and 0.1 % respectively 
FISHERY TECHNOLOGY 
PLATING TECHNIQUE AND TEMPERATU E ON BACTERIAL C UNT 
	 99 
TC.:le 9. 
	 Analysis of variance for frozen cat fish 
Source 	 S. S. 
Total 	 9.5148 
df 
35 
MS 
Samples 5.8035 2 2.9028 63.80c 
Temperature 2,2526 1 2.2526 49.51 
Methods 0.1383 1 0.1383 3.04 
Triplicate 0.0002 2 0.0001 1 
Error 
c — Significant at 0.1 % level 
1.3182 29 0.0435 
Table 10. Analysis of variance for frozen kheema 
Source 	 S.S. 	 df 	 MS 
Total 0.1191 11 
Temperature 0.0793 1. 0.0793 113.29c 
Methods 0.0334 0.0334 47.71c 
Error 
c — Significant at 0.1 % level 
0.0064 9 0.0007 
Since the primary objective of the aerobic 
plate count is to evaluate the sanitational 
aspect of food handling, aerobic meso-
philic count at 37°C would be deemed 
sufficient for this purpose. ut such a 
count will not reflect the actual microbial 
load on the food where psychrotrophic 
organisms are also involved as in the case 
of raw fish or frozen products. The micro-
bial flora of raw fish contained species of 
which a good part was classified as psychro-
trophs. ur earlier studies had indicated 
that raw fish (Sardinella longiceps) con-
tained about 44 % of psychrotrophs while 
mesophiles constituted only 56 %. It was 
also found from their growth temperature 
studies that 31 % of the bacteria isolated 
from fresh fish could not grow at 37°C 
(Thampuran & Iyer, 1979). It was not 
surprising therefore that 30°C incubation 
is giving higher counts. 
In frozen samples, the predominating 
flora is Gram positive cocci belonging mainly 
to genus Micrococcus (Thampuran & Iyer 
unpublished data). Selection of cold resis-
tant species due to freezing and frozen 
storage coupled with the elimination of a 
large number of strict mesophilic types 
would have been the reason for getting 
higher counts at 30°C. 
Generally it was found that spread plates 
yielded more colonies than pour plates for 
raw and frozen fish. Clark (1967) had 
suggested that spreading by means of a 
glass rod would allow the clumped cells to 
get disintegrated thereby improving the dis-
persion of cells result Ong in higher counts. 
ut analysis of our data shows that the 
count difference between spread plates and 
pour plates at 30°C is greater than the 
count difference at 37°C. Had the effect 
been due to clumping of the cells alone this 
difference would not have arisen. It 
appears therefore that higher counts obtained 
in spread plates could be due to other 
factors as well. 
ne possibility may be the sensitivity of 
certain marine micro-organisms to the 
action of hot agar used in pour plating. 
Zobell & Conn (1940) pointed out the 
harmful action of agar at 45°C on certain 
marine bacteria. The extreme heat sensi-
tivity of a psychrotrophic bacterium Pseu-
domonas flourescens was recently shown by 
Gray et al. (1973). Nottingham et al. 
(1975) suggested that the lower count in 
pour plates could be attributed to the ina-
bility of psychrotrophs to survive in the hot 
agar media used in pour plating. 
Vol. 18, 1981 
100 	 NIRMALA THAMPURAN, H. K. IYER AND K. M. IYER 
Postgate (1967) 	 stated that 	 bacteria 
subjected to a stress becomes hypersensi- 
tive to a secondary stress. It has been 
shown that microbial starvation which can 
occur in low nutrient environments can 
also lead to increased susceptibility to a 
transient secondary warming stress (Klein & 
Shenyu Wu, 1974). This was shown by 
decreased recoveries of heterotrophic micro- 
organisms from aquatic environments in pour 
plate as compared to spread plate method. 
In frozen foods, stress due to intense cold 
can cause the microbial flora to be more 
sensitive to warm agar temperature of 45°C 
leading to decreased recoveries with pour 
plate method. 
The implication of this study was limited 
by the number of samples analysed and 
methods of enumeration adopted. 	 The 
data nevertheless points out the significance 
of adopting a procedure by which greater 
accuracy is attained in the estimation of 
bacterial count of sea foods. 
The authors are grateful to the 	 late Shri. 
G. K. Kuriyan, former Director, Central Institute 
of Fisheries Technology for his keen interest and 
encouragement. They are also thankful to the 
technicians in the laboratory for specimen colle-
ction and laboratory assistance. 
References 
AOAC (1970) Official Methods of Ana- 
lysis, 11th edn. Associatio of Official 
Analytical Chemists, Washington 
Clark D. S. (1967) Can. J. Microbiol. 
13, 1409 
Gray, R. S. H., 	 'it -ter, L. D. & Ordahl, 
Z. J. (1973) Appl. Microbial. 26, 78 
IS: 8653 (1977a) Indian Standard Specifi-
cation for Sardines, Fresh. 1st edn. 
Indian Standards Institution, New Delhi 
IS: 8652 (1977b) Indin Standard Spec-
cation for Sardine, Frozen. 1st edn. 
Indian Standards Institution, New Delhi 
Jan Gjerde (1975) Fisk Dir Skr. 1, 17 
Klein, D. A. and Shenyu Wu (1974) J. 
Appl. Microbiol 27. 429 
Lee, J. S. & Lee ann Harward (1970) J. 
Milk Ed Technol. 33, 237 
Mossel, D. A. A. & Van der Moosdijk, A. 
(1963) J. appl. Bact. 26, 10 
Nottingham, P. M., Rush brook, A. J. & 
Jury, K. E. (1975) J. Ed Technol. 10, 273 
Postgate, J. R. (1967) Advanced Micro-
bial Phys. 1, 1 
Shenyu, Wu & Klein, i. A. (1976) J. 
Appl. Environ. Microbiol. 31, 216 
Stapert, E. M., Sokolski, W. T. & Northam, 
J. I. (1962) Can. J. Microbiol. 8, 809 
Thampuran Nirmala & Iyer, K. M. (1979) 
Fish. Technol. 16, 15 
Van Soestbergen, A. A. & Lee, C. H. 
(1969) Appl. Microbiol. 18, 1092 
Zobell, C. E. & Conn, J. E. (1940) J. 
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FISHERY TECHNOLOGY 


The influence of plating technique and incubation temperature on bacterial count from fish and fishery products

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The influence of plating technique and incubation temperature on bacterial count from fish and fishery products

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