MICROBIAL ASSESSMENT OF SOME MARKETED FISH IN DAMANHOUR CITY

A. SALEH, OMAIMA; EL-KEWAIEY, I.A.

doi:10.21608/avmj.2008.175913

MICROBIAL ASSESSMENT OF SOME MARKETED FISH IN DAMANHOUR CITY

Authors

OMAIMA A. SALEH ¹
I.A. EL-KEWAIEY ²

¹ Animal Health Research Institute, Damanhour Branch.

² Animal Health Research Institute, Damanhour Branch

10.21608/avmj.2008.175913

Abstract

Seventy-five random samples of marketed fish (25 of each fresh Tilapia nilotica, fresh Mugil cephalus and frozen Mackerel) were collected from different fish marketes in Damanhour city for microbiological examination. The results revealed that the mean values of total counts of aerobic mesophilic bacteria, psychrotrophic bacteria, Enterobacteriaceae, Coliforms, Staph. aureus, Pseudomonas, Aeromonas, as well as Moulds and Yeasts of examined fresh samples of Tilapia nilotica were 2 x 10⁵ ± 1 x 10⁴, 2 x 10⁴ ± 9 x 10³, 2 x 10⁴ ± 7 x 10³, 3 x 10² ± 2 x 10², 4 x 10⁴ ± 2 x 10⁴, 1 x 10⁴ ± 4 x 10³, 7 x 10³ ± 2 x 10³, 1 x 10³± 4 x 10² and 1 x 10³± 4 x 10² respectively. While that of examined fresh samples of Mugil cephalus were 1 x 10⁴ ± 1 x 10³, 1 x 10⁴ ± 1 x 10³, 7 x 10³ ± 1 x 10³, 2 x 10² ± 2 x 10², 6 x 10³ ± 2 x 10³, 6 x 10² ± 2 x 10², 2 x 10⁴± 2 x 10⁴, 2 x 10² ± 5 x 10 and 6 x 10² ± 3 x 10² respectively, and that of examined frozen Mackerel fish samples were 4 x 10³ ± 1 x 10³, 4 x 10³ ± 2 x 10³, 3 x 10² ± 9 x 10, 3 x 10 ± 1 x 10, 4 x 10³± 9 x 10², 7 x 10³± 5 x 10³, 4 x 10³ ± 4 x 10³, 1 x 10³ ± 1 x 10³ and 5 x 10³ ± 2 x 10³ respectively. The results of most of examined fish samples were in compliance with the Egyptian and the international permissible standard limits. The recommended measures for improving the microbial quality of the marketed fish were discussed to become more safety for the consumers.

Keywords

Full Text

Animal Health Research Institute, Damanhour Branch.

MICROBIAL ASSESSMENT OF SOME MARKETED FISH IN DAMANHOURCITY

(With 4 Tables)

By

OMAIMA A. SALEH and I.A. EL-KEWAIEY

(Received at 18/5/2008)

التقييم الميکروبي لبعض الأسماک المعروضة للبيع بمدينة دمنهور

أميمه عبد الفتاح صالح ، إبراهيم على القويعى

أجريت هذه الدراسة على عدد 75 عينة عشوائية من أسماک البلطي الطازج والبوري الطازج وأسماک الماکريل المجمد (25 عينة لکل نوع) تم جمعها من أسواق الأسماک المختلفة بمدينة دمنهور وذلک لتقييمها ميکروبياً وأوضحت الفحوص الميکروبية أن نسب تواجد البکتريا الهوائية عند 37º م/48 ساعة والتي تنمو عند 7º م/10 أيام والبکتريا المعوية والعصيات القولونية والميکروب العنقودي الذهبي وميکروبات السودوموناس والأروموناس والفطريات والخمائر في أسماک البلطي کانت کالآتي : 88% ، 72% ، 64% ، 56% ، 84% ، 36% ، 32% ، 76% و60% بمتوسط عد کلى 2 × 10º ± 1 × 10⁴، 2 × ⁴10 ± 9 × 10 ³ ، 2 × 10 ⁴ ± 7× 10 ³ ،3 × 10 ² ± 2 × 10 ²، 4× ⁴10 ± 2× 10 ⁴ ، 1 × 10 ⁴ ± 4 × 10 ³ ، 7× 10 ³ ± 2 × 10 ³ ، 1× ³10 ± 4×10 ² و 1 × 10 ³ ± 4 × 10 ²على التوالي. أما في أسماک البوري فکانت النسب کالآتي: 100% ، 96% ، 88% ، 48% ، 88% ، 16% ، 40% ، 60% و 40% بمتوسط عد کلى 1× ⁴10 ± 1 × 10 ³ ، 1 × ⁴10 ± 1 × 10 ³ ، 7× ³10 ± 1× ³10 ، 2× 10 ² ± 2 × 10 ² ، 6× 10 ³ ± 2× 10 ³، 6 × 10 ² ± 2× 10 ²، 2× 10 ⁴ ± 2× 10 ⁴ ، 2× 10 ² ± 5× 10 و 6 × 10 ² ± 3× 10 ² على التوالي. بينما في حالة أسمـاک الماکريل المجمد کانت نسب الميکروبات بترتيب ذکرها کالآتي : 92% ، 60% ، 12% ، 24% ، 88% ، 20% ، 16% ، 60% و44% بمتوسط عد کلى کالآتي: 4× 10 ³ ± 1 × 10 ³ ، 4× 10 ³ ± 2 × 10 ³ ، 3× 10 ² ± 9 × 10 ، 3× 10± 1× 10 ، 4× 10 ³ ± 9× 10 ²، 7 × 10 ³ ± 5 × 10 ³، 4 × 10 ³ ± 4 × 10 ³ ، 1 × 10 ³ ± 1× 10 ³و 5× 10 ³ ± 2 × 10 ³ على التوالي. هذا وقد تم مناقشة وتقييم النتائج وملاحظة عدم تجاوز غالبية العينات سواء الأسماک الطازجة أو المجمدة للمواصفات القياسية المصرية والعالمية. هذا وقد تم وضع التوصيات اللازمة لجعل هذه الأسماک أکثر أماناً لصحة المستهلک.

SUMMARY

Key words: Fish, Tilapia nilotica, Mugil cephalus, Mackerel.

INTRODUCTION

Fish and shellfish are second to meat and poultry as stable animal protein foods in most of the world. Fish have protein of high biological values as they contain essential amino acids and good source of minerals such as calcium, phosphorus, iron and trace elements like iodine as well as vitamins in addition to the high content of polyunsaturated fatty acids (Sedik et al., 1989).

Fish is subjected to many risks of contamination from different sources either during their aquatic environment, sewage pollution of harvesting areas and/or after being harvested by workers, utensils and equipments during transportation, distribution and food preparation (National Academy of Science, 1985 and El-Leboudi, 2002).

The counts of Enterobacteriaceae have the potential indicator for not only the health hazard but also as an indicator of spoilage (Gorczyca et al.,1985). Members of family Enterobacteriaceae are of potential public health importance as it causes diseases for humans during lowering of their resistance. Also this group contains most members of food poisoning microorganisms (Edwards and Ewing, 1972 and Collins, 1984).

The increasing demand of consumers for fish and fish products makes it necessary to assess the microbial contamination of the fresh water fish, Tilapia nilotica “Bolti” and Mugil cephalus “Bouri” and frozen Mackerel fish marketed in Damanhour city to establish the best utilization at a high quality level and to safe-guard the human health.

MATERIALS and METHODS

A total of 75 random samples of fish (25 each of Tilapia nilotica and Mugil cephalus) and 25 of imported frozen mackerel fish “Scomber scombrus” were collected from different fish markets in Damanhour city. Each sample was wrapped separately in sterile polyethylene bag and transferred directly to the laboratory without delay in an ice box. In the laboratory, each sample was put on a sterile plate and the whole skin surface of each fish sample was sterilized by ethyl alcohol and flaming, the skin surface was removed aseptically and the following examinations were performed.

1. Preparation of samples according to ICMSF (1978):

Ten grams of prepared fish sample were transferred to a sterilized homogenizer flask containing 90 ml of 0.1% sterile peptone water. The contents were homogenized at 14000 r.p.m. for 2.5 minutes to provide a dilution of 10‾¹. The homogenate was allowed to stand for 5 minutes at room temperature, then 1 ml of homogenate was transferred with a sterile pipette into a sterile test tube containing 9 ml of 0.1% sterile peptone water to obtain a dilution of 10‾². Then further decimal ten fold serial dilutions up to 10‾⁶ were prepared.

2. Total aerobic bacterial count according to APHA (1992):

One ml from each dilution was transferred into duplicate sterile Petri dishes and mixed with about 10 ml of sterile plate count agar medium “melted and kept at 45ºC”. After solidification, cultivated plates as well as control one were incubated at 37ºC for 48 hours in an inverted position. Average count was calculated as a total aerobic count per gram of sample.

3. Total Psychrotrophic bacterial count according to APHA (1992):

The same steps, as in total aerobic bacterial count, were carried out but the incubation was done at 7ºC for 10 days.

4. Total Enterobacteriaceae count according to ICMSF (1978):

0.1 ml from each prepared dilution was transferred and distributed over the surface of dried violet red bile glucose (VRBG) agar poured plates, then overlaid by a thin layer of VRBG agar. Inoculated plates as well as control one was incubated at 37ºC for 48 hours. The colonies which showed a purple colour surrounded by a purple zone were counted.

5. Total coliform count (MPN/g):

The multiple tube method recommended by ICMSF (1978) was applied. Most probable numbers (MPN) of coliforms per gram of the examined samples were calculated by using MPN table. A loopful from each positive MacConkey broth tube was picked up and spread over the surface of MacConkey agar plate. Plates were incubated at 37ºC for 48 hours. Suspected colonies were picked up and inoculated into sterile semisolid nutrient agar tubes for further biochemical identification.

6. Total Staphylococcus aureus count (ICMSF, 1978):

0.1 ml from each dilution was spread over the surfaces of duplicate dried Baired Parker agar plates. The inoculated plates were incubated at 37ºC for 48 hours in an inverted position. The black shiny colonies with narrow white margines and surrounded by a clear zone were counted. Suspected colonies were stabbed in semi-solid agar for further morphological and biochemical identification (catalase, mannitol, coagulase, thermostable nuclease production and oxidation-fermentation of glucose)

7. Pseudomonas and Aeromonas count: Pseudomonas-Aeromonas selective agar base (GSP) recommended by Kielwein (1969):

Suspected colonies were picked from GSP medium then identified according to method recommended by Macfaddin (1980) morphologically (films stained with Gram’s stain and motility test) and biochemically by the following tests (indole, methyle red, Voges-Proskauer, TSI, citrate utilization, oxidase, O/F of carbohydrates (glucose, mannitol, maltose and sucrose), nitrate reduction and esculin hydrolysis.

8. Total mould and yeast count (Konemen et al., 1994):

One ml from each original dilution was streaked onto Sabouraud dextrose agar an incubated at 25ºC, and examined daily for 5 days.

Results

Table 1: Statistical analytical results of icrobiological counts (cfu/g) of examined fresh fish “T. nilotica” and M.cephalus Samples. (N = 25 of each)

Microbial counts	T. nilotica					M.cephalus
	Positive Samples		Minimum	Maximum	Mean ± SEM	Positive Samples		Minimum	Maximum	Mean ± SEM
	No	%	Minimum	Maximum	Mean ± SEM	No	%			Mean ± SEM
- Aerobic bacterial count :
Mesophilic	22	88	1 x 10²	2 x 10⁶	2 x 10⁵ ± 1 X 10⁴	25	100	7 x 10²	2 x 10⁴	1 x 10⁴ ± 1 x 10³
Psychrotrophic	18	72	8 x 10²	2 x 10⁵	2 x 10⁴ ± 9 x 10³	24	96	1 x 10	2 x 10⁴	1 x 10⁴ ± 1 x 10³
- Enterobacteriaceae	16	64	1 x 10²	1 x 10⁵	2 x 10⁴ ± 7 x 10³	22	88	5 x 10	2 x 10⁴	7 x 10³± 1 x 10³
- Coliforms ( MPN/g )	14	56	3	2 x 10³	3 x 10² ± 2 x 10²	12	48	4	2 x 10³	2 x 10² ± 2 x 10²
- Staphylococcus aureus	21	84	1 x 10²	3 x 10⁵	4 x 10⁴± 2 x 10⁴	22	88	2 x 10²	3 x 10⁴	6 x 10³ ± 2 x 10³
- Total Pseudomonas count	9	36	3 x 10²	3 x 10⁴	1 x 10⁴ ± 4 x 10³	4	16	3 x 10²	1 x 10³	6 x 10² ± 2 x 10²
- Total Aeromonas count	8	32	2 x 10³	2 x 10⁴	7 x 10³ ± 2 x 10³	10	40	1 x 10²	2 x 10⁵	2 x 10⁴ ± 2 x 10⁴
- Total mould count	19	76	1 x 10	8 x 10³	1 x 10³± 4 x 10²	15	60	1 x 10	8 x 10²	2 x 10² ± 5 x 10
- Total yeast count	15	60	4 x 10	5 x 10³	1 x 10³± 4 x 10²	10	40	1 x 10	3 x 10³	6 x 10²± 3 x 10²

N = number of examined samples. SEM = Standard error of mean.

Table 2: Statistical analytical results of microbiological counts (cfu/g) of examined forzen Mackerel fish samples. (N = 25)

Microbial counts	Frozen Mackerel
	Positive Samples		Minimum	Maximum	Mean ± SEM
	No	%	Minimum	Maximum	Mean ± SEM
Total aerobic bacterial count:
- Mesophilic	23	92	2 x 10²	2 x 10⁴	4 x 10³ ± 1 x 10³
- Psychrotrophic	15	60	1 x 10²	2 x 10⁴	4 x 10³ ± 2 x 10³
- Total Enterobacteriaceae	3	12	2 x 10²	5 x 10²	3 x 10²± 9 x 10
- Total Coliforms ( MPN/g)	6	24	3 x 10	9 x 10	3 x 10 ± 1 x 10
- Total Staphylococcus aureus	22	88	6 x 10	2 x 10⁴	4 x 10³ ± 9 x 10²
- Total Pseudomonas count	5	20	2 x 10²	2 x 10⁴	7 x 10³ ± 5 x 10³
- Total Aeromonas count	4	16	1 x 10²	2 x 10⁴	4 x 10³ ± 4 x 10³
- Total mould count	15	60	3 x 10	2 x 10⁴	1 x 10³ ± 1 x 10³
- Total yeast count	11	44	3 x 10	2 x 10⁴	5 x 10³ ± 2 x 10³

N = number of examined samples SEM = Standard error of mean.

Table 3: Incidence of coagulase positive Staphylococcus aureus isolated from examined fish samples. (N = 25)

Examined Fish	Samples positive for Staphylococcus aureus		Coagulase positive Staphylococcus aureus
	No.	%	No.	%
	No.	%	No.	%
- Tilapia nilotica	21	84	9	43
- Mugil cephalus	22	88	7	32
- Frozen mackerel	22	88	11	50

N = number of examined samples.

Table 4: Incidence of isolated microorganisms from examined fish samples.

Microbial species	T.nilotica		M. cephalus		Frozen Mackerel
Microbial species	No.	%	No.	%	No.	%
Coliform group:
E.coli	2	8	1	4	1	4
E.coli, inactive	2	8	1	4	0	0
Escherichia fergusoni	1	4	1	4	0	0
Citrobacter freundii	1	4	1	4	2	8
Citrobacter diversus	2	8	1	4	2	8
Klebsiella pneumoni subsp
Pneumoni	3	12	5	20	1	4
Klebsiella oxytoca	3	12	1	4	1	4
Enterobacter aeragenes	2	8	0	0	2	8
Enterobacter intermedium	0	0	2	8	0	0
*Pseudomonadaceae:*
P.alcaligenes	2	8	1	4	1	4
P.pseudoalcaligenes	4	16	2	8	2	8
P. aeruginosa	0	0	1	4	1	4
P. fluorescens	1	4	0	0	0	0
P. stutzeri	2	8	0	0	0	0
P. putida	0	0	0	0	1	4
*Aeromonadaceae:*
A.hydrophila	2	8	2	8	0	0
A.caviae	2	8	3	12	2	8
A.schubertii	3	12	2	8	1	4
A.sobria	1	4	3	12	0	0
A.veronii	0	0	0	0	1	4

DISCUSSION

Fresh fish:

Table (1) revealed that the mean values of total aerobic, mesophilic and psychrotrophic bacterial counts of the examined T.nilotica (Bolti) samples were 2 x 10⁵ ± 1 x 10⁴ and 2 x 10⁴ ± 9 x 10³ while for the examined M. cephalus (Bouri) samples were 1 x 10⁴ ± 1 x 10³ and 1 x 10⁴ ± 1 x 10³ respectively.

These findings are in agreement with the results obtained by Goda et al. (1980), Farouk (1989) and Roushdy et al. (1996) while, lower results were reported by Thabet (1972) and Abdel-Hafiez (1991).

Amin (1973) suggested that the spoilage of fish could be detected when APC was reached 10⁹/g.

The ice in which the fish are to be preserved, is usually contaminated (10²/ml of ice melt water) and the holds of the fishing vessels normally have an indigenous flora (FAO, 1996).

The results given in Table (1) showed that the mean values of total Enterobacteriaceae counts of the examined T.nilotica and M.cephalus were 2 x 10⁴ ± 7 x 10³ and 7 x 10³ ± 1 x 10³ with percentages of 64% and 88%, while that of total Coliform counts (MPN/g) were 3 x 10² ± 2 x 10²and 2 x 10² ± 2 x 10² with percentages of 56% and 48% respectively.

Nearly similar results were reported by Ahmed et al. (1986), Farouk(1989), Roushdy et al. (1996), Yehia (1996) and Mousa and Mahmoud (1997), but higher counts were reported by Abdel-Galil et al. (1988) and Mahmoud (1990).

Coliforms are intestinal and non intestinal inhabitants, so their presence in food give an index of poor sanitation as well as possible presence of enteric pathogens (Matthes, 1984).

As shown in Table (4) the incidence of isolates of Coliforms group from the examined fresh fish, T.nilotica and M.cephalus were as follow:
E. coli (8% & 4%), E.coli, inactive (8% & 4%), Escherichia fergusoni (4% & 4%), Citrobacter freundii (4% & 4%), Citrobacter diversus (8% & 4%), Klebsiella pneumoni subsp. pneumoni (12% & 20%), Klebsiella oxytoca (12% & 4%), Enterobacter aerogenes (8% & 0%) and Enterobacter intermedium (0% & 8%) respectively.

In polluted waters, high numbers of Enterobacteriaceae may be found but in clean temperate waters, these microorganioms disappear rapidly and it has been shown that Escherichia coli and Salmonellae can survive for very long periods in tropical waters and once introduced may almost become indigenous to the environment (Fujioka et al., 1988).

Also, the resulls shown in Table (1) cleared that the mean values of Total Staph. aureus counts of the examined samples of T. nilotica and M. cephalus fish were 4 x 10⁴ ± 2 x 10⁴ and 6 x 10³ ± 2 x 10³ cfu/g with percentages of 84% and 88% respectively.

Nearly similar results were reported by Ahmed et al. (1986) and Roushdy et al. (1996) but lower counts were reported by Hafez (1989) and Yehia (1996).

It is evident from Table (3) that the incidences of the isolated coagulase positive Staph. aureus from the examined T.nilotica and M. cephalus fish samples were 43% and 32% respectively.

Studies indicated that large numbers (usually greater than 1 million cfu/g) of coagulase positive Staph. aureus must contaminate the food for producing sufficient enterotoxin to cause food poisoning (Liston et al. 1971 and Gilbert et al. 1972).

The results recorded in Table (1) showed that the mean value of Pseudomonas and Aeromonas counts of the examined T.nilotica samples were 1 x 10⁴ ± 4 x 10³ and 7 x 10³ ± 2 x 10³ with percentages 36% and 32%. While of the examined M.cephalus samples were 6 x 10² ± 2 x 10² and 2 x 10⁴ ± 2 x 10⁴ with percentages of 16% and 40% respectively. Similar results were reported by El-Kelish (1995) but slightly lower counts were reported by El-Atabany (1995), Hassan (1998) and El-Mossalami et al. (2004).

Table (4) showed that the following species could be isolated from the examined T.nilotica and M.cephalus samples at percentages as: Pseudomonas alcaligenes (8% & 4%), Pseudomonas pseudoalcaligenes (16% & 8%), Pseudomonas aeruginosa (0% & 4%) Pseudomonas fluorescens (4% & 0%), Pseudomonas stutzeri (8% & 0%), while Aeromonas hydrophila (8% & 8%), Aeromonas caviae (8% & 12%), Aeromoras Schubertii (12% & 8%) and Aeromonas sobria (4% & 12%) respectively. Nearly similar results were reported by Soliman (1988) and Bastawrows and Mohammed (1999).

Aeromonas hydrophila is an apportunistic pathogen in persons with impaired immune function, but it has been detected frequently in stools of patients having diarrhea (Brayan, 1992). Also, it was isolated as causative agent in a case of sever gastroenteritis, including sever abdominal pain, fever and bloody stools, involving a 10 years old girl (Rosner, 1964).

Table (1) showed that the mean values of total mould and yeast counts of examined T.nilotica were 1 x 10³ ± 4 x 10² and 1 x 10³ ± 4 x 10²_, while for the examined M.cephalus were 2 x 10² ± 5 x 10 and 6 x 10² ± 3 x 10²cfu/g respectively. Simlar results were recorded by Hassan and Abdel-Dayem (2004) from the examined fresh local M. cephalus muscles samples.

The mode of handling of fish in the market also contributes to the incidence of fungi on the fish. The possibility of the incidence of toxic fungi or fungal metabolites leading to food poisoning can not ruled out unless proper care is taken (Vishwanath et al., 1998).

Frozen mackerel fish:

Table (2) showed that the mean values of aerobic bacterial counts “mesophilic and psychrotrophic” of frozen mackerel fish, were 4 x 10³ ± 1 x 10³ and 4 x 10³ ± 2 x 10³ cfu/g respectively. Nearly similar results were obtained by Alian et al. (1969) and Hassan (1998) but higher results were reported by Lee et al. (1967) and Morshidy and Hafez (1986). The comparatively lower aerobic mesophilic counts of frozen fish in comparison to fresh fish could be attributed to that these microorganisms could not adapt to the cold environment. In the meantime the psychrotropic count had been established and began to multiply. In this respect, Frazier and Westhoff (1978) reported that freezing kills some but not all microorganisms present in fish where psychrotrophs can survive freezing and are ready to grow on thawing. Egyptian standard (1991) stated that, the permissible limit for the total bacterial count for frozen fish was no more than 10⁶ colonies/g fish muscles.

The microbial activity is one of the main causes of quality deterioration of fish, so the spoilage pattern of fish depends upon the initial bacterial count, in addition to those acquired during handling and storage (Cobb and Vanderzant, 1971).

As regard to total Enterobacteriaceae and Coliform counts, Table (2) showed that all the examined frozen mackerel fish samples were negative for total Enterobacteriacea count except 3 samples (12%) were exhibited the mean count 3 x 10² ± 9 x 10 cfu/g. Also for the Coliforms count (MPN/g), only 6 samples (24%) were having a mean value of 3 x 10 ± 1 x 10 cfu/g. Nearly similar results were obtained by lee (1967), Abdel -Hafeez (1991), Awad et al. (1993) and Hassan (1998)but higher countswere reported byGorczyca et al. (1985), Morshidy and Hafez (1986), (Hasan 1991) and Mahmoud (1994).

The obtained results were within the permissible limit (100 colonies/g) recommended by the Egyptian Standard (1991) for frozen fish. Using the Coliform count as an index of pollution in frozen food has been criticized because of the susceptibility of this group of microorganisms to freezing injury resulting in gradual disappearance in their numbers in frozen food during continued storage (Licciardello and Hill, 1978).

Table (4) showed that 9 isolates of Coliform group as follow: E. coli (1), Citrobacter freundii (2), Citrobacter diversus (2), Klebsiella pneumoni subsp. Pneumoni (1), Klebsiella oxytoca (1) and Enterobacter aerogenes (2) with incidences 4%, 8%, 8%, 4%, 4%, and 8% respectively. Kosev et al. (1990) found that Coliforms and aerobic psychrotrophic bacteria were not detected in muscle tissue at any time of frozen storage at -20ºC for 190 days for samples of carp, trout and fresh water fish, while these bacteria decreased only in count on fish surfaces by 32-57% during storage.

Table (2) show that the total Staph. aureus counts of the examined frozen mackerel fish samples were ranged from 6 x 10 to 2 x 10⁴, with a mean value of 4 x 10³ ± 9 x 10² cfu/g and with a percentage of 88%. Similar findings had been reported by Awad et al., (1993) but lower counts were reported by Nickelson et al. (1980), Morshidy and Hafez (1986) and Hassan (1998). It was cleared from table (3) that Staph. aureus can grow best in foods in which the competing microorganisms are present in low numbers.

Table (3) declared that the incidence of coagulase positive Staph. aureus isolated from the examined frozen mackerel fish samples marketed at Damanhour city fish markets was 11 (50%).

Enterotoxigenic strains of Staph aureus can give rise to food borne intoxication. Hence, such contaminated frozen fish may at times constitute a public health hazard and this agree with the statement reported by Thatcher and Clark (1978).

Results present in Table (2) reveal that the mean values of total Pseudomonas and Aeromonas in examined frozen mackerel were 7 x 10³ ± 5 x 10³ and 4 x 10³ ± 4 x 10³ cfu/g, the percentages of positive samples were 20% and 16% respectively.

Hassan (1998) found that the mean values of Pseudomonas and Aeromonas of the examined frozen mackerel were 3 x 10³ ± 1 x 10³ and 3 x 10² ± 24 cfu/g in winter and 2 x 10⁴ ± 2 x 10³and 5 x 10² ± 23 cfu/g in summer, respectively.

Pseudomonas alcaligenes (4%), Pseudomonas pseudoalcaligenes (8%), Pseudomonas aeruginosa (4%) and Pseudomonas putida (4%) could be isolated (Table 4). Similar findings were obtained by Edward and Kraszewski (1991) and Mahamoud (1994). Pseudomonas aeruginosa produces a variety of extracellular enzymes, including proteases and lipases that are responsible for most of the histopathological effects of the infection (Wilson and Miles, 1975 and Blackweed et al., 1983).

On the other hand, Aeromenas veronii (4%), Aeromonas schubertii (4%) and Aeromonas cavia (8%) could be isolated (Table 4).

Results recorded in Table (2) showed that the mean values of total mould and total yeast counts of examined frozen mackerel were 1 x 10³ ± 1 x 10³ and 5 x 10³± 2 x 10³ respectively.

In moist foods, yeasts and moulds tend to grow slower than other microorganisms (Hobbs, 1983). Low mould count may also be due to that the moulds are strictly aerobes, while the yeasts can grow in either aerobic or anaerobic environments.

The following bacterial reference values for raw fish for total mesophilic count vary from 10⁴ to 10⁷ cfu/g (as a rejection limit for spoilage). Reference values for the control of hygienic measures range from 10²-10⁴ cfu/g for Enterobacteriaceae, from 0-400 cfu/g for total faecol Coliforms and E. coli, and from 100-2000 cfu Staph.aureus/g and absence of Salmonellae and Vibrio parahaemolyticus in 25g in all raw fish (Friedhoff, 1994).

Therefore, good water quality is the key to improve the production and hygiene of fish as a food. An abundant water supply will avoid many problems associated with aquaculture by dilution, in addition to the prevention of pollution of the fish growing farms through the treatment of sewage water and careful determination of the discharge points seems to be a successful strategy to improve fish quality and to protect the consumer from fish borne enteric diseases. With regard to the catched fish, the delay between catching and marketing should be as short as possible and there must be some form of refrigeration to hold the product at 0ºC for fresh fish such as boxing and icing and then the boxes held in an insulated chilled room at between 1 and 2ºC but the temperatare of the cold stores of the frozen fish should ideally be at -18ºC.

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