University of Nigeria Research Publications

NZEAKO, Basil Chukwuma

Aut

hor

PG/M.Sc.\98\310

Title

The Microbiology of Smoked Fish

Facu

lty

Biological

Dep

artm

ent

Department of Microbiology

Dat

e

July, 1981

Sign

atur

e

THE MICROBIOLOGY OF SMOKED FISH

NZEnKO , B* i S I L CHUK'JJUMii REG. NO- PG/M,S~,/78/310

DEPARTMENT O F MICROBIOLOGY

UNIVZRSITY OF NIGERIA

NSUKKA

J U L Y 1981,

CERTIFICATE

MR. BASIL CHUKWUMA NZEAKO, a P o s t g r a d u a t e S t u d e n t

i n t h e Depar tment of Mic rob io logy , m a j o r i n g i n f o o d

m i c r o b i o l o g y h a s s a t i s f a c t o r i l y comple ted t h e r e q u i r e -

m e n t s of c o u r s e work and P r o j e c t R e p o r t for t h e d e g r e e

of Master of S c i e n c e (M.SC.) i n Mic rob io logy , The

work embodied i n h i s Project Report i s o r i g i n a l and

h a s n o t been s u b m i t t e d i n p a r t o r f u l l f o r any o t h e r

d ip loma o r d e g r e e of this or any o t h e r U n i v e r s i t y ,

J u l y 1981 P r o f , Nduka Okafo r P r o f e s s o r o f M i c r o b i o l o g y U n i v e r s i t y of N i g e r i a , Nsukka

CONTENTS

DEDICATION o o

ACKNOWLEDGEMENT

ABSTRACT o 0 0

LIST OF TABLES m o o

LIST OF- FIGURES

LIST OF PLATES m o m

CHAPTER ONE

100 INTRODUCTION o 0 0 o o o

CHAPTER TWO

200 STUDIES ON THE MICROBIOLOGY OF SMOKED FISH PURCHASED AT NSUKKA MARKET 00.

I n t r o d u c t i o n m o o .me g O m Materials and methods 00- 00. F i s h s a n p l e s u s e d Om. O O m Media u s e d f o r i s o l a t i o n of o r a a n i s m s C l o s t r i d i u m i s o l a t i o n B a c i l l u s . i s o l a t i o n S t a p h y l o c o c c u s i s o l a t i o n L a c t o b a c i l l u s i s o l a t i o n

2,2,8 T r y p t i c a s e s o y a g a r OO. 2,2,9 I s o l a t i o n o f m i c r o o r g a n i s m s 202010 D e t e r m i n a t i o n of w a t e r ccntsnt;

f i s h s a m p l e s 0 0 0 0.0 2,2,11 I d e n t i f i c a t i o n o f o r g a n i s m s 2,2,1101 Gbam's S t a i n o 0 o o o 2m2011.2 O x i d a s e test O Q ~ O O o 2,2.1lm3 M o t i l i t y tes t o 0 Q 20201104 O x i d a t i o n F e r m e n t a t i o n test 20201105 C a t a l a s e test 0 0 0 20201106 C o a g u l a s e test o O o 20201107 DNase t e s t 0 0 0 o m 0 20201108 L i t m u s m i l k test 00. 20201109 Nagler r e a c t i o n m e 0 20201%10 S p o r e s t a i n 9 0 0 g e e

Paqe

io

iio

iii,

v.

vim

viio

CHAPTER THREE

300

Pigment production ,,, o O O 17 I n d o l e test @ m a .em 17 C i t r a t e utilisakion , .. e 17 Carbohydrate u t i l i s a t i o n 6 rn * 18 Urease activity * m e 0 . 19 Decarboxylases test ... *.a 29 M e R e V o P . test O O ~ 0.. 39 Phenylalanine deamination ... 20 Starch h y d r o l y s i s ., .em 20 Hydrogen sulphi.de p r o d u c t i o n 2 1 Gelatin liquefaction m e * 21 Animal i n o c u l a t i o n for toxin 21

Organisms isolated om. 22

I d e n t i t y of o r g a n i s m s i sola ted 22 Coun t s o f v a r i o u s bacteria isolated 23 Frequency of i so la t ion (%I ,,, : 4 Water c o n t e n t of f i s h .Om 25 Summazy me. ~ O O 27

STUDIES ON THE MICROBIOLOGY OF FISH SMOKED IN THE LABORATORY ~ e e 29

I n t r o d u c t i o n 9 9 D e e 28 M a t e r i a l s and methods 00- 28 F i s h s a m p l e s u s e d m o a rn o a 28 The smoker 0 0 0 . o * 31 Smoking o f f i s h s a m p l e s i n t h e l a b o r a t o r y om. .om 31 D e t e r m i n a t i o n o f m i c r o b i a l f l o ra o f f i s h .00 o O e 32 W a t n r c o n t e n t of smoked f i s h 33 Suaaary o O I 000 33 Microbial f l o r a of f i s h smoked in t h e 1 a b o r a t o r y 00. 0.. 33 F i s h smoked at 3 8 O ~ - 3 3 O ~ 33 F i s h smoked at 65 - 78 C . . o 3 3 Water content of smoked f i s h 34 M i c r o b i a l f l o r a of fresh f i s h 37

CHAPTER FOUR

4.0 GENZRAL DISCUSSION 0 . - o 0 40

APPENDIX

REFERENCES

DEDICATION

T o m y l a t e F a t h e r , Mre J o s e p h Okoye Nzeako,

ii,

ACKNOWLEDGEMENT

T h i s p r o j e c t was made p o s s i b l e by the u n t i r i n g effort

of my s u p e r v i s o r , P r o f . Nduka Okafo r who guided m e g r e a t l y

t h r o u g h o u t t h e p e r i o d of t h e p r o j e c t , I have to thank

him for a l l t h e p a i n s h e took t o p r o o f read the m a n u s c r i p t .

I a l s o have t o t h a n k Prof, Gugnani , t h e former Head

of Microbiology for g i v i n g m e t h e much needed r e a g e n t s f o r t h e

assignment.

I thafik my d a r l i n g wife for h e r moral encouragemen t

and patience d u r i n g t h e p e r i o d o

B.C. NZEAKO DEPARTMENT OF MICROBIOLOGY U N L V E R S I T Y OF N I G E R I i i NSUKKA

iii.

ABSTRACT

The m i c r o b i a l f l o r a o f smoked C l a r i a s lazera f i s h bough t -.c-

a t Nsukka market was examined b a c t e r i o l o g i c a l l y a l o n g w i t h

f r e s h l y c a u g h t C l a r i a s f i s h from Ogurugu P i v e r , F r e s h f i s h

was examined b e f o r e and a f t e r smoking a t 3 8 O ~ - 4 3 O ~ and 65 - 7 8 O ~ r e s p e c t i v e l y .

The m i c r o b i a l t o t a l p l a t e c o u n t f o r f i s h bough t a t Nsukka

market was 8.5 x 10'' organisms/g. The m i c r o b i a l p l a t e c o u n t

5 f o r f r e s h l y c a u g h t f i s h was 4 x 10 /g w h i l e f i s h smoked a t 38 - 43'~ and 65 - 7 8 ' ~ had c o u n t s o f 1 x 104/cj and 1 X I.Q~/CJ

From f r e s h f i s h , t h e organisms i s o l a t e d were M i c r o c o c c u ~ ,

L a c t o b a c i l l u s , K l e b s i e l l a , Aeromonas, E n t e r o b a c t e r , Pscudornona~,

Flavobacterium, Moraxel l a , A c l n e t o b a c t e r and (Srynofom spp.

Fish bought: from Nsukka market c o n t a i n e d M i c r a C C-LCU~ Bncil lus_, J -

P r o t e u s , L a c t o b a c i l l u s , Ac ine tobac tc r , and E s c h e r i c h i ~ spp-

F i s h smoked i n t h e L a b o r a t o r y a t 38 - 4 3 O ~ c o n t a i n e d Micrococcus, L a c t o b a c i l l u s and Acinetobacter s p p while o n l y Micrococcus was

i s o l a t e d from f i s h smoked a t 65 - 78% The e l i m i n a t i o n of Pseudomonas, Aeromonas, Moraxe l l a , aorynefcrm , Flavobac te r ium ,. and K l e b s i e l l a spp from smoked f i s h i n d i c a t e s t h a t smoke and

o r t e m p e r a t u r e has a v i t a l e f f e c t i n e l i m i n a t i o n o f m i c r o b i a l

flora of f i s h o However i t is n o t known why o n l y Micrococcus was

i s o l a t e d i n f i s h smoked a t 65 - 7g0c,

iv.

The w a t e r c o n t e n t o f f i s h b o u g h t a t Nsukka m a r k e t v a r i e d

be tween 37% and 60% w h i l e t h a t smoked a t 38 - 4 3 ' ~ and 65 - 78'~ was be tween 40% - 50% and 24% - 36% r e s p e c t i v e l y . The h i g h i n c r e a s e i n t h e amount o f water left a f t e r smoking

of f i s h may be a v i t a l f a c t o r c o n t r i b u t i n g t o r a p i d l y

d e t e r i o r a t i o n of smoked f i s h when i t i s l e f t a t room t e m p e r a t u r e

a f t e r smoking,

A l though B. cereus was found i n t h e fish bought from -- Nsukka m a r k e t , t h e c o m p l e t e absence of othcr food p o e s o n i n g

o r g a n i s m s s u c h a s S a l m o n e l l a , Shiqclla a n d C l o s t r i d i u m spp

i n d i c a t e s t h a t l o c a l l y smoked fish i s f i t f o r human consumpt ione

L I S T OF T A B L E S

TABLES

2 0 1 CHARACTERS O F ORGANISMS ISOLATED FROM SMOKED F I S H 0 0 1 0 1 . 0 0 1 am.

COUNTS OF V A R I O U S B A C T E R I A FROM 25 SMOKED FISH SAMPLES BOUGHT AT NSUKKA MARKET ,., FREQUENCY OF I S O L A T I O N (%I OF BACTERTX FROM 25 SAMPLES O F SMOKED F I S H 0 . . a m

WATER CONTENT O F 25 SMOKED FISH ( % ) 0 - 0

ORGANISMS FROM F I S H SMOKED AT LOWER AND HIGHER TEMPERATURES ' 0 0 1 o o o 0 0 0

WATER CONTENT O F SMOKED FISH o o o o o o

CHiiRACTERS O F ORGI.INISP~S I S O L A T E D FROM FRESH F I S H SAMPLES 0 . 0 e e e 0 0 .

FREQUENCY O F I S O L i i T E S AMONG THE SAMPLES OF F R E S H F I S H e e o m o o 0 0 0 o a e

LIST O F F I G U R E S

F I G U R E Paqe

3 e 1 THE FISH K I L N 0 0 0 m o o 0 0 , 30

401 THE M I C R O B I A L FLORib O F FRESH AND SMOKE@ FISH m o o 0 . 0 0 . . 39

LIST OF PL,\TES

PLATE

1. FISH BOUGHT FROM NSUKK, \ M;\RKET 0 0 0

20 L I V E FISH BOUGHT FROM OGURUGU e o o

Page, - 10

CHAPTER ONE

INTRODUCTION

I n 1976 , l o c a l p r o d u c t i o n of f i s h i n N i g e r i a was

700,000 t o n n e s b u t c u r r e n t demand f o r f i s h amounts t o a b o u t

1 , 2 m i l l i o n t o n n e s p e r annum f o r an e s t i m a t e d p o p u l a t i o n o f

a b o u t 80 m i l l i o n , The s h o r t f a l l i n p r o d u c t i o n was made up by

i m p o r t s of 280,000 t o n n e s v a l u e d a t d98 m i l l i o n (Rehman 1980).

icccording t o Rehman (I?&! 24% t o 31% o f t h e t o t a l

d o m e s t i c f i s h p r o d u c t i o n came from i n l a n d waters, lakes a n d

ponds i n N i g e r i a ,

Sagua (1976) d i s c u s s e d a t l e n g t h t h e various prob lems

f a c i n g increased p r o d u c t i o n o f f i s h i n Nigeria, One o f t h e s e

p rob lems i s g r o s s absence of c o - o r d i n a t e d r e s e a r c h on

f isheries . I n a d d i t i o n , l i t t l e i s known about t h e t y p e of

m i c r o o r g a n i s m s a t t a c k i n g t h e f i s h s e e d l i n g s used f o r t h e e s t a b -

l i s h m e n t of va r ious aqua c u l t u r e s i n Niger ia ,

It i s knolr? t h a t f i s h may carry human pathogens such as

S a l m o n e l l a s p , S h i q e l l a s p , V, cholera, V . p a r a h a e m o l y t i c u s

C l o s t r i d i u m b o t u l i n u m , C l o s t r i d i u r n p e r f r i n q e n s , Paraqonimus and - Aeromonas (F;iO/WHO 1974) . The mode of a c q u i s i t i o n of t h e s e

o r g a n i s m s by f i s h i s from c o n t a m i n a t e d waters o r from s e c o n d a r y

c o n t a m i n a t i o n of fish d u r i n g l a n d i n g , p r o c e s s i n g , s t o r a g e ,

d i s t r l b u t i o n or p r e p a r a t i o n for consumpt ion (Fit0 1 9 7 3 ) ,

2, W i l l i a m and Hollis (1974) d i d show that various human

p a t h o g e n s c a n be i s o l a t e d from p r e s e r v e d fish and f i s h products,

H e examined m a r k e t c a t f i s h and found t h e bacteria count to exceed

t o l e r a n c e l i m i t s set by local h e a l t h o f f i c i a l s for s t a p h y l o c o c c i ,

H c found no S a l m o n e l l a - o u t of t c n f i s h examined. The totah 6 a e r o b i c c o u n t was 2 x 10 organisms p e r gram. Out of t h i s c o u n t ,

s t aphy lococc ica~bs r sd100 o r g a n i s m s per gram and Coliforrns 500/grarn.

E a r l i e r w o r k e r s s u c h a s R a j and Liston (1963) found t h a t 60%

of f i s h s t i c k s a m p l e s were c o n t a m i n a t e d b y S o a u r e u s * 'The s a m p l e s

examined shoxed no c a s e o f Sa lmone l l a . It was l a t t e r d i s c o v e r e d

t h a t t h e s o u r c e o f S t a p h y l o c o c c u s aureus was from t h e p r o c e s s i n g

p l a n t and n o t f rom t h e f i s h , Thatcher and C l a r k (1968) s t a t e d

6 t h a t when f i s h h a s a C O U ~ I L of more t h a n 10 o r g a n i s m s p e r gram

t h a t i t i s c o n s i d e r e d t o have a very short potential s h e l f l i f e

o r may be an e a r l y s i g n of i n c i p i e n t s p o i l a g e ,

i iccording t o Fi-\O/WWO 1974 reports , b a c t e r i a l flora of

f r e s h l y c a u g h t f ' s h contains different g e n e r a of m i c r o o r g a n i s m s

namely Pseudomonas spp, Moraxella spp, A c i n e t o b a c t e r b

( ; rchrornobacter) , Vibrio spp, 2terorncpZ Spp, F l a v o b a c t r i u m , and 0

Cytophaqa , When f i s h i s c a u g h t closa t o l and , m i c r o b i a l flora

may a l s o contain Micrococcus, L a c t o b a c i l l u s , S t r e p t o c o c c u s and

B a c i l l u ~ , Eve lyn and Mcdermott (1961) c o l l e c t e d 350 f r e s h

3,

w a t e r f i s h and found specLes of P s ~ u d o m o n a s , heromonas,

Mic rococcus -- and L a c t o b a c i l l u s to be p r e s e n t mos t o f t e n . They

also n o t e d t h a t t h e w a t e r from where the f i s h were c a u g h t b ~ d

h i g h i n c i d e n c e of ~ e u d o r n ~ n a s ~ I n Zambia, Watanabe (1971)

examined f i s h f rom L a k e s K ~ r i b a and Tanganyika and found an 5 a v e r a g e a e r o b i c p l a t e c o u n t of 1 x l o 3 - 1 x 10 o r g a n i s m s

per gram of f i s h . Out of this count 40% - 80% of t h e o r g a n i s t i s were G r a m p o s i t i v e s namely micrococci, c o r y n e f o r m s , b a c i l l i ,

a c t i n o m y c e t e s and s t r e p t o c o c c i , 30% to 61% were G r a m n e g a t i v e rods.

I n v e s t i g a t i o n i n t o f l s h spoilage h a s shown i t t o be m a i n l y

d u e t o m i c r o b i a l a c t i o n (Lobben and Lee 1968). Env i ronmen ta l

c o n d i t i o n s a s s o c i a t e d w i t h fish spoilaqc a r e pH, m o i s t u r e

content, osmotic p r e s s u r e and redox p o t e n t i a l w i t h i n t h c f i s h

(Eskin, Hsndersen, Townxand 19711. Fish s p o i l a g e s t a r t s when

t r i m e t h y l a m i n c o x i d e p s e s ~ n t i n fish t i s s u e s i s b r o k e n down t o

t r i m e t h y l a m i n e i n the presence of l a c t i c a c i d der ived from b r e a k

down of fish g l y m g e n by micr~rganlsms. When n o more g l y c o g e n

i s left i n t h e f i s h , st-ict a n a e r o b e s as w e l l as aerobic o r g a n i s m s

d e a m i n a t e t h e amino acids derlved from fish p r o t z i n s b y and

r e a c t i o n i n t o ammonia, hydrogen sulphide, organic a c i d s

carban dioxi.de,, The presence of these p r o d u c t s of f i age

p r e s e n t t h e c h a r a c t e r i s t i c smell of s p o i l t f i s h (~eatty a n d

C o l l i n s 1939 , C o l l i n s 1941, Rh.'r 1980) . T h e s e r e a c t i o n s are

, S t i c k l ,

, and

.sh spoil

represented as follows:-

T r i m e t h y l a m i n e ox ide T r i m e t h y l m i n e

CH3 - CH - COOH - --3 CH3-CO-CWH + NADH + H+ A .A

OH Lactic acid Pyruvic aCid

CH3-CO-COOH OXIDATIVE --- --.-- . ._-____- 5 -COOH + C02 -> Pyruvic acid DECiiRBOXYLlrTION AcetW Acid.

THE OVERALL REACTION

TMiiO Lactic acid.

.- - . 2CH-3 COOH + Cop

acetic a c i d

The above reactions determine the fate of tr imethylmine

o x i d e and glycogen+ I n t h e case of amino acids derived from f i s h

p r o t e i n s , the reaction is as follows:-

L - . \ l a n i n e G l y c i n e

acetate + ammonia + C02.

5,

To p r e v c n t spoilage o f f i s h and ensure i t s p r 3 s e r v a t i o n

v a r i o u s methods are used, These methods a c c o r d i n g to FJ;O/~JHO

1 R e f r i g e r a t i o n a t 5 ' ~ - ~ O ~ C . ( 2 ) F r e e z i n g below O'C

( 3 ) S a l t i n g

( 4 ) Drying

(5) Smoking

( 6 ) Canning

R e f r i g e r a t i o n of f i s h i s n o t u n i v e r s a l l y d o n e i n N i g e r i a

b e c a u s e n o t e v e r y town i s provided w i t h e l e c t r i c i t y and n o t

e v e r y o n e c a n a f f o r d a r e f r i g e r a t o r , But i n c o u n t r i e s where t h e s e

f a c i l i t i e s a r e a v a i l a b l e , i t i s found that s p o i l a g e o r g a n i s m s of

r e f r i g e r a t e d f i s h are Pseudomonss spp, Moraxclla spp and A l k a l i q e n e s

s p p (Shewzn and Georgala 1957). Shewan and G e o r g a l a (1957) storcd

f r e s h f i s h a t 5 ' ~ f o r 1 0 - 1 2 d a y s am found t h e c o u n t of Pscudomonas to i n c r e a s e at t h e expense of o t h e r g a n e r a , -

T h e n o n - a v a i l a b i l i t y of cold staraQe and the predilection

o f many N i g e r i a n s especially the Ibos f o r t h e f lavour and t a s t e

of smoked fish h a v e r e s u l t e d i n an e x t e n s i v e smoking of f i s h

i n N i g e r i a especially i n t h e Ibo s p c a k i n g a r e a s of N i g e r i a ,

Smokcd fish m e a l s are s e r v e d i n naming c e r e m o n i e s of babies,

i n i n i t i a t i o n o f peop le i n t o v a r i o u s t r a d f t i o n a l rites and

a t b u r i a l ce remon ies ,

6.

The e s s e n t i a l i n c r e d i e n t s n e c e s s a r y to k i l l microorganisms

i n c l u d i n g p a t h o g e n s i n smoked fish i s e i t h e r smoke a n d / o r

h e a t h t t a i n e d d u r i n g smoking ( C l i f f o r d e t a1 1980). Earl ier workers

l i k e P e t t e t and Lane (1940) had a n a l y s e d the c o n t e n t of smoke.

P e t t e t and Lane (1940) found that smoke c o n t a i n s more t h a n 170

s u b s t a n c e s . F u n c t i o n a l group classes i n c l u d e a l d e h y d e s , ketones,

a c i d s , l a c t o n e s , p h e n o l s and other aromatic compoundso T h e s e

compounds are c o l l e c t i v e l y known as p y r o l i g n i n o u s a c i d s , The

amount, t y p e and n a t u r e of each compound is d e p e n d e n t on t h e t y p e

of wood u s e d (Eyo 1979). Hardwood pravidas l c s s smoke, b u t gives

more i n t e n s e h e a t t h a n s o f t wood depending on the d e g r e e of

a e r a t i o n of t h e f i r e .

Smoke also behaves as an a n t i o x i d a t i v e a g e n t on micro-

organisms (Lea 1933), Lea discovered t h a t unsmokcd bacon showed

pronounced surface r a n c i d i t y af ter 98 days a t - 10'~. Whereas smoked bacon showed no s i g n o f h a v i n g surface r a n c i d i t y u n d e r

the same c o n d i t ~ m s . H e a t k r i b u t e d this effect to t h e p r e s e n c e

of p h e n o l i c compounds i n smoke, Emmanuel (1955) and Dann (1969)

f o u n d t h a t p h e n o l i c compounds act as electron a c c e p k which

terminate t h e r a n c i d i t y chain reactions, Barytko e t a1 (1976)

found m i c r o c o c c i s e n s i t i v e t o smoke w h i l e K s r s t e n (1974) found

l a c t o b a c i l l l and some s p e c i e s of staphylococci r e s i s t a n t ,

Smoke a t l e v e l s of 45 mg/kg of f i s h has a n inhibitory effect on 0

S , aureus equivalent to a reduction i n t e m p e r a t u r e from 20 C - .

t a

t o loOc ( O l s e n 1976) . T h e i n h i b i t i o n of growth o c c u r s a t t h e

l a g p h a s e r a t h e r k h a n on t h e e x p o n e n t i a l g rowth phase. Eyo

(1979) w h i l e e v a l u a t i n g the effect of smoke o n f i s h f o u n d t h a t

t o t a l p h e n o l l e v e l s on t h e s u r f a c e l a y e r s of h a t smoked f i s h

may r e a c h 40 - 50 mg per kg weight of f i s h , Keshvani (1964) at Aba d r i e d fish a t 90°c i n a n a i rwood

oven f o r 24 hour s . A t 9 0 O ~ d r y i n g , h e c o u l d r e d u c e t h e w a t e r

c o n t e n t f rom 80% t o 16% when d r y i n g w a s d o n e f o r 2 4 hour s .

K e s h v a n i ' s dried f i s h grewdould on s t o r a g e a n d was a t t a c k e d

by beetles. Keshvani prevented i n f e s t a t i o n o f smoked f i s h by

beetles by s t o r i n g them i n s ter i le p o l y t h e n e b a g s , Kleickman and

S c h e Z l a a q C 1979) e v a l u a t e d t h e m i c r o b i a l numbers of smoked f i s h 6 and d i s c o v e r e d that t h e a v e r a g e p l a t e c o u n t amounted to 10 /gram

5 o f fish. Out of t h i s number a count of 10 / g of f i s h was d u e t o

e n t e r o b a c t e r i a . On t h e b a s i s of his f i n d i n g s , h e recommended

t h a t smoked f i s h s h o u l d b e g r a d e d as medium or very perishable

food ,

I n Zambia, Watanabe ( 1 9 7 1 ) smoked f i s h a t two t e m p e r a t u r e s : -

( a ) 90°c t o 1 1 0 ~ ~ f o r 3 - 5 h o u r s (b) 45% to 60°c f o r 4 - 7 h o u r s o The m i c r o b i a l f lo ra of t h e smoked f i s h w a s n o t de t e rmined .

On storage, they grewmou3.de and w e r e a t t a c k e d by beetles,

(Dermestes spp) . Al though Watanabe ' s work g a v e some c l u e s as t o

what happens t o f r e s h f i s h from t r o p i c a l waters a f t e r smoking h e

d i d n o t d e s c r i b e t h e m i c r o b i a l s t a t u s o f t h e f i s h before

smoking, S i m i l a r l y K e s h v a n i t s work a t Aba p r o v i d e s no a n s w e r s

t o t h e above problem since h e d i d n o t examine h i s f i s ' h

b a c t c r i o l o g i c a l ~ y a f t e r d r y i n g .

The p r e s e n t p r o j e c t i s aimed therefore a t d e t e r m i n i n g

what constitutes t h e m i c r o b i a l f l o r a of f r e s h f i s h from i n l a n d

waters before and a f t e r the f i s h i s smoked, An a t t e m p t was

made t o e x p l a i n t h e n a t u r e o f t h e f l o r a e n c o u n t e r e d , The

i n f o r m a t i o n o b t a i n e d t h r o u g h t h i s p r o j e c t may be v e r y v i t a l t o

l o c a l f i s h i n d u s t r i e s in Nigeria and t o v a r i o u s governments

i n t c r c s t e d i n a q u a c u l t u r e ,

F u r t h e r m o r e , b e c a u s e smoked f i s h i s e a t e n i n p a r t s of t h e

E a s t e r n States of N i g e r i a e s p e c i a l l y i n Ibo s p e a k i n g areas

w i t h o u t f u r t h e r cooking, t h e presence o f human p a t h o g e n s i n

smoked fish was a l s o s t u d i e d .

T h e f i s h used in this project is C l a r i a s l a z e r a . T h i s was c h o s e n

lazera (1) C l a r i a s F-are e x t e n s i v e l y u s e d a s f i s h s e e d l i n g s

i n N i g e r i a f o r v a r i o u s a q u a c u l t u r e (Rehman 1980)

(2) T h i s t y p e of f i s h i s more r e a d i l y a v a i l a b l e a t Nsukka

and i t s e n v i r o n s than a n y o t h e r t y p e of f i s h

( 3 ) They are c o m p a r a t i v e l y c h e a p e r t h a n o t h e r t y p e s of f i s h

( 4 ) M a r k c t women who sel l smoked f i s h c o m p l a i n t h a t

C l a r i a s spp s p o i l f a s t e r t h a n a n y o t h e r t y p e of

smoked f i s h o

CHAPTER TWO 9 .

STUDIES ON THE MTCKOBIOLOGY OF FISH PURCHirSED AT N S U K K A MARKET

2.1. INTRODUCTION

As p r e v i o u s l y mcn t loned i n c h a p t e r one , smoked f i s h

is e a t e n o c c a s i o n a l l y w i t h o u t f u r t h e r cook ing . I t i s n i a t ~ n

a l o c a l meal known a s "abachsIt o r " j i g b o w made f

s h r e d s . I t i s p o s s i b l e t h a t o n e c a n a c q u i r e food-po i son ing

o r g a n i s m s s u c h as C1, p e r f r i n q e n s , C1, b o t u l i n u m , B e c e r e u s

or S. a u r e u s a f t e r s u c h m e a l s s i n c e i t i s known t h r i t f - -l h a r b o u r s u c h o r g a n i s m s (WHO/FAO R e p o r t lore

:ram c a s s a v a

d e c i d e d t o s t u d y t h e c a r r i a g e of food-p

o t h e r o r g a n i s m s i n smoked f i s h bough t from the l o c c . 1 Nsukka

marke t .

2.2, MATERLLS AND METHODS

20201 FISH SAMPLES

25 s e l e c t e d samples o f smoked C l a r i a s l a z e r a were b o u g h t

from Nsukka and c 4 u d i e d o v e r a p e r i o d of one y e a r from November

1979 t o November 1980. They were s e l e c t e d so t h a t t h e y were

a b o u t t h e same size measuring between 28 c m and 32 c m i n length

and 2*4 crn and 4 c m i n diameter. Zdencck (1977) h a s s t a t e d i n t erme

g e n e r a u t h a t f i s h of the same s i z e a r e a l so of t h a same a g e o

2.2.2, MEDI,r. U S E D FOR 1SOLIJTLON OF' ORG~INISMS

S i x d i f f e r m t media were u s e d for t h e i s o l a t i o n o f o r g a n i s m s

i n th: f i s h s amples , The c o n s t i t u e n t s o f @ash medium w i l l be f o u n d

10,

SMOKED C L ~ ~ R I I ~ S LIiZER>i FISH USED I N THIS

EXPERIMENT

x 0,25 Magnification.

in the Appendix. I s o l a t i o n s on t h e v a r i o u s media are

d e s c r i b e d below,

2,2,3 CLOSTRIDIUM ISOL~LTION

T r i p l i c a t e p l a t e s of b l o o d agar c o n t a i n i n g 1 0 0 ug

ncomycin s u l p h a t e were u s e d fo r t h e i s o l a t i o n of C l o s t r i d i u m

s p p ( C r u i c k s h a n k e t a1 19691, N e a r l y a l l c l o s t r i d i a p a t h o g e n i c

to man p r o d u c e h a e m o l y s i s on human blood.

2 ,, 2 4 - BiiCLLLUS ISOLATION T r i p l i c a t e p l a t e s of ammonium salt medium c o n t a i n i n g 10%

egg y o l k were u s e d for t h e isolation of B a c i l l u s spp

;immonium s a l t medium with egg yolk i s o l a t e d l e c i t h i n a s e p r o d u c e r s

among + c i l l u s spp ( G i l b e r t and Tailor 19761, Organisms p r o d u c i n g

l c c i t h i n a s e cause hydrolysis of t h e l e c i t h i n of egg y o l k

(Cruickshank et a1 1969). T h e h y d r o l y s i s r e s u l t s i n p r e c i p i t a t i o n

of c r e a m - l i k e h a l o s around t h e c o l o n i e s .

2,2,5 STAPHYLOCOCCUS ISOLATION

Triplicate I ,ates of B a l r d - P a r k e r s medium ( 0 x 0 1 ~ ) c o n t a i n i n g

10% egg y o l k were used, Thls medium i s e x c e l l e n t f o r the i s o l a t i o n

of micrococci as w e l l ( S i n e l l 1978, L a c h i c a 19801, The i n c o r p o -

r a t i o n of egg-yolk e n a b l e d l i p s s e producing So a u r m s t o be

i s o l 3 t e d . Lipase p r o d u c i n g S. a u r e u s p roduced z o n s o f c l e a r i n g

a round t h e i r colonies,

2.2 06 ., LACTOBACILLUS ISOLrrTION T r i p l i c a t e p l a t e s of MRS (Man, Rogosa and S h a r p e 1960)

agar (OXOID) were prepared a c c o r d i n g t o m a n u f a c t u r e r s recommen-

d a t i o n and used for t h e i s o l a t i o n o f L a c t o b a c i l l u s .

2,2.7 irEROBIC GR,-rM N E G t T f VE ROD ISOL, ,TION

T r ip l i ca t e p l a t e s of MacConkey agar (0x01~) were used

f o r t h e i s o l a t i o n of a l l aerobic a t a m n e g a t i v e o r q m i s m s t o be

found i n t h e fish samples.

2,2,8 TRYPTICitSE SOY ; G r R

T r i p l i c a t e p l a t e s of t h i s medium were u s e d f o r t h e

d e t e r m i n a t i o n of b a c t e r i a l total c o u n t , T h i s medium a l l o w e d

a n e x c e l l e n t g rowth of a l l major aerobic o r g a n i s m s common i n

f i s h ,

2,2.9 ISOLIiTION OF MICROORGANISMS FROM MiiHKGT SPIOKED FISH

The 25 fish s a m p l e s were each t r e a t e d as fo l lows : -

10 grems from v a r i o u s p a r t s of each f i s h namely, t h e m i d d l e p a r t ,

t h e neck r e g i o n Qnd t h e a r e a about 3 c m from t h e tail were

s e p a r a t e l y cut o u t from each fish, T h i s a r r a n g e m e n t w a s

necessary i n o r d e r t o e f f e c t i v e l y cover t h e d i s t r i b u t i o n of

m i c r o o r g a n i s m s on t h e f i s h , The c u t o u t p i x e s w e r e t h e n

amaPgnmated and 10 g weighed auk and m a c e r a t e d

a t l o w s p c c d (1000 r.p.m.l i n a s t i r i le w a r i n g b lendor c o n t a i n i n c

3 90 c m of 0,1% p e p t o n e saline ( O b l i n g e r , Kennedy 1976),

T e n f o l d d i l u t i o n s were made f r m o a c h macerated fish from lo-'

t o 10'~ d i l u t i o n s . 0.1 ~1 o f each d i l u t i o n was t r a n s f e r r e d

onto t r i p l i c a t e p l a t e s of t h e s i x media d e s c r i b e d above,

Each inoculurn was spread w i t h a sterile s p r e a d e r s t a r t i n g

from t h e h i g h e s t d i l u t i o n . The blood a g a r p l a t e s f o r C l o s t r i d i u m

and Rogosa medium f o r L a c t o b a c Z l l u s were i n c u b a t e d a n a e r o b i c a l l y j

a t 3 0 ' ~ f o r 72 hours in Mclntosh and F i l d e s L w h i l e o t h e r

p l a t e s were incubated a e r o b i c a l l y f o r t h e same p e r i o d ( R o b e r t o

1980). The t e m p e r a t u r e of 30% was u s e d i n t h e s e e x p e r i m e n t s

f o r t h e i n c u b a t i o n o f isolates from fish i n p r e f e r e n c e t o

37'~ because 30'~ i s c l o s e r t o the ambient t e m p e r a t u r e .

A f t e r incubation, counts of a l l o r g a n i s m s g rowing o n t h e

p l a t e s were taken, Representatives of t h e v a r i o u s m o r p h o l o g i c a l

t y p e s were s e l e c t e d and p u r i f i e d on t r y p t i c a s e s o y a g a r , They

werc s t u d i e d and i d e n t i f i e d as d ~ s c r i b e d i n 2.2.11 below.

2.2.20 DETERMIN&TION OF W.bTER CONTENT OF FISH SAMPLES - One af the f .ctors l e a d i n g to fish s p o i l a g e i s i n c r e a s e d

water a c t i v i t y on f i s h tissues. To d e t e r m i n e t h e w a t e r c o n t e n t

o f any f i s h , 20 grams of each f i s h were d r i e d t o c o n s t a n t w e i g h t

after t h e i n i t i a l w e i g h t was ob ta ined , Drying was d o n e a t

70% oven for 5 days,

2.2.21 IDENTIFICATION OF ORG.*NISMS

Except where men t ioned p u r i f i e d i s o l a t e s werc s t u d i e d

following the method of Cowan and Steel (1970) and identified

according to Bergeyws Manual (1974). Only brief description

o f the methods will be g iven below.

Gram's stain according to Preston and Morrellfs (1962)

modificztion was done,

2.2.11.2 OXIDfrSE TEST

;i loopful of oxidase reagent (tetramethyl-phenclene

diarnine dihydrochloride) was dropped on a pure discrete colony

of growths of isolates on trypticase soy agar plates.

Appearance of deep blue calour indicated a positive case. No

colour development indicated a negative case (Kovacs 1956).

2.2e11.3 MOTILITY TEST

This was done on four hour growth culture of Gram negative

and Gram positive rods grown in peptone water.

2 *2.1ie4 O-F (OXIDATION-FERMENT~TION TEST)

This test was done to determine the mode of utilization

of glucose by the isolates. id1 the isolates were separately

inoculated into a pair of Hugh and Leifson's (0-F) medium.

One tube of each pair was filled with sterile paraffir? to 3 cm

thick. The tubes were incubated at 30'~ for 72 hours. (Hugh

and Leifson 195314

2.2 . 12.5 CiiT, iLiiSE TEST J-L l a o p f u l of 3% hydrogen p e r o x i d e w a s dropped on a c o l o n y

o f each organism grown on t r y p t l c a s e soy a g a r , P r e s e n c e o r

absence o f effervescence in a l l t h e o rgan i sms was noted .

Ze2.11,6 COAGULnSE TEST

T h i s test was done on a l l G r a m p o s i t i v e cocci t h a t are

catalase p o s i t i v e and which utilise g l u c o s e f e r r n e n t a t i v e l y

0.5 ml of undi luted rabbit plasma was mixed w i t h an e q u a l volume

o f an 18 - 24 hour growth cul ture of e a c h i s o l a t e i n a test tube . The c u l t u r e s were i n c u b a t e d a t 37'~ fo r 4 hours .

Examination o f t u b e s far f o r m a t i o n of v i s i b l e c l o t s was noted

and f u r t h e r i n c u b a t i o n a t room t e m p e r a t u r e w3s done for f u r t h e r

20 hours .

2e2.11.7 D N i G E TEST

L o c h i c a (1ye0) recommended t h e u s e of 0.1% t o l u i d i n e

blue i n c o r p o r a t e d i n deoxyribonucleic a c i d n u t r i e n t aga r .

P r a d u c t i o n of d ~ d x y r i b o n u c l e a s e by the organ i sm w i l l r e s u l t i n

pink halo p r o d u c t i o n , O n l y S. aureus can produce t h i s c o l o u t

formation. The deoxyribdnuclease produced by 3. a u r e u z

i s a thermonuclease enzyme which c a n w i t h s t a n d t h e t e m p e r a t u r e

of 60°c f o r % hour. M o d i f i c a t i o n o f t h e test which d e t e c t e d

b o t h l a b i l e (LT) and thermostable (ST) was done. The

i so la tes were i n o c u l a t e d onto DNase a g a r p l a t e s and incubated

a t 3 0 O ~ for 72 hour s . .,it t h e e n d of t h i s p e r i o d , 5 N Hcl

was us2d to f l o o d a l l t h e plates. Excess H c l was d i s c a r d e d

i n t o a bowl of lysol. P r e s e n c e or a b s e n c e of c l e a r i n g a r o u n d

t h e zones of i n o c u l a t i o n was no ted ,

2,2,11.8 LITMUS MILK TEST

A 1 1 the Gram p o s i t i v e , non-mot i le r o d s were i n o c u l a t e d

into l i t m u s m i l k o The i n o c u l a t i o n was d o n e i n p a i r s * One set

of l i t m u s m i l k c o n t a i n e d h o t i r o n n a i l s which were e s s e n t i a l

f o r creating a c o n d i t i o n o f a n a e r o b i o s i s . The o t h e r set

c o n t a i n e d no i r o n n a i l s . The sets were i n c u b a t e d 8 t 30'~ for

72 hours. The f o l l o w i n g r e a c t i o n s were n o t e d ,

(a) acid p r o d u c t i o n i n d i c a t e d by p i n k c o l o u r

(bl alkali p r o d u c t i o n ind ica t ed b y b l u e c o l o u r

( c ) r e d u c t i o n af t h e indicator shown by c o l o u r l e s s (white) medium

( d l r e n n e t :lot shown b y a s o f t c l o t which r e t r a c t e d and e x p r e s s e d a c l e a r g r e y i s h f l u i d ( w h e y ) ,

2.2.22m9 NAGLER REACTION

A 1 1 t h e Gram p o s i t i v e rods and oxidase p o s i t i v e Gram

n e g a t i v e r o d s were i n o c u l a t e d s e p a r a t e l y o n t o n u t r i e n t a g a r

p l a t e c o n t a i n i n g 10% egg yolk. The Gram n e g a t i v e r o d p l a t e s were

17,

incubated aerobically at 30'~ f o r 72 hours w h i l e the Gram

p o s i t i v c rod p l a t e s were i n c u b a t e d a n a e r o b i c a l l y a h 30°c for

t h e same period. Ability of t h e i s o l a t e s t o produce t u r b i d i t y

o n t h e p l a t e s was observed ,

Gram positive rod i s o l a t e s were i n o c u l a t e d onto two

sets o f n u t r i e n t agar p l a t e s , One se t o f p l a t e s was i n c u b a t e d

a e r o b i c a l l y w h i l e t h e other was i n c u b a t e d a n a e r o b i c a l l y a t

30% for 72 hours . S p o r e s t a i n was done on colonies growing

on both p l a t e s of n u t r i e n t agar.

2.2021.11 PXGMENT PRODUCTION

A b i l i t y o f some i s o l a t e s to produce pigments were observed

on g rowths of i s o l a t e s on trypticase soy agar.

2.2.11.12 INDOLE TEST

T h i s t es t was done to d i s t i n q u i s h t h o s e i s o l a t e s which

l i b e r a t e d i n d o l e from pep tone from t h o s e t h a t d i d n o t , A 1 1 t h e

G r a m n e g a t i v e rc -? i s o l a t e s were i n o c u l a t e d i n t o p e p t o n e water

and i n c u b a t e d a t 3 0 O ~ f o r 72 hours, 0.5 m l o f Kavncs r e a g e n t

(P- dime4hgl arninobenzaldehydd i n amyl a l c o h o l was added, Pink

devclopmcnt was indiuative of a p o s i t i v e case,

2 -2 . l l , l 3 CITRATE UTILISiiTION

Some organisms e s p e c i a l l y Gram n e g a t i v e rods u t i l i s e

18 a

c i t r a . t e a s a s o u r c e of carbon far growth w h i l e o t h e r s d o n o t ,

A l l the G r m n e g a t i v e rods w e r e inoc.&ated i n t o Simrnangs ci trate

agar, Development of deep b l u e c o l o u r a l o n g l i n e of growth

is i n d i c a t i v e of growth.

2.2 a 11,14 CikRBOHYDRdiTE UTILLSIITION

The following s u g a r s were p r e p a r e d i n 10% c o n c e n t r a t i o n ,

filtered and added aseptically i n t o s te r i le ;indradels p e p t o n e

w a t e r , I

The overal l c o n c e n t r a t i o n of sugar was 1% after a d d i t i o n

i n t o s teri le kndradc's p e p t o n e water,

G l u c o s e

Lactose

Dulcitol

Manni to1

Inositol

Sorbi t o 1

S a l i c i n

Sucrose

Arabinose

Xylose

Galactose

Sorbose

Maltose

Treha losc

Raffinose.

A l l the Gram negative rod isolates a s w e l l as Gram

p o s i t i v e non-spor ing rods were inoculated i n t o these sets of

s u g a r s and i n c u b a t e d a t 30°c f o r 72 hours. Fe r rnun tq t ion of

v a r i o u s carbohydrates was read a f t e r t h e i n c u b a t i o n periodo

2,2 O l l , t S UREkSE XTIVITY

A b i l i t y of the isolates to h y d r o l y s e Urea w a s tested

i n Christensen's Urea agar ( C h r i s t e n s e n ' s 1946).

2,2,11,16 DECARB0XYL;iSES TEST

T h i s test was done according t o Falkow CZ958). The

Falkow's b a s a l medium was prepared and d i v i d e d i n t o f o u r a l i q u o t

p a r t s *

(i.1 0.5% L - a r g i n i n e h y d r o c h l o r i d e ( 3 . i ) 0,5% L - l y s i n e Hydroch lo r ide

(iii) 0,5% L - o r n i t h i n e h y d r o c h l o r i d e (ivl No aminoaci

T h c tubes c o n t a i n i n g t h e four media were i n o c u l a t e d w i t h

o x i d a s c n e g a t i v e , G r a m n e g a t i v e r o d s and i n c u b a t e d a t 30°c for

72 hours. Decarboxy la t ion was i n d i c a t e d by a p u r p l e c o l o u r

change w h i l e n e g a t i v e tubes appeared yel low,

2.2.11.17 M.R. (METHYL RED) V m P m (VOGES PROSKAUER) REACTIONS

T h i s test d i s t i n g u i s h e s t b o s e organisms which l iberate

acetyl methyl ca rb ina l from g l u c o s e from t h Q s e t h a t do n o t

do so. iiftcr inoculation and i n c u b n t i o n of c u l t u r e s , 2 d r o p s (

methyl red s o l u t i o n were added t o e a c h tube , T h e t u b e s were

shaken and examined, Appearnce of red c o l o u r was i n d i c a t i v e b

of a c i d p r o d u c t i o n . A f t e r t a k i n g t h e r e a d i n g of met'yl red test, 11

0.6 m l o f a l p h a n a p h t h o i was added t o e a c h t u b e f o l l o w e d b y

0.2 m l s of 40% K o H , aqueous s o l u t i o n , The t u b e s w e r e s h a k e n

a n d a l l o w e d t o s t a n d f o r 15 m i n u t e s , A p o s i t i v e r e a c t i o n was

i n d i c a t e d b y a s t r o n g r e d c o l o u r o

2 2 s 11.18 PHENYLI~LI+NINE DEc'd4INzrTION

T h i s t es t was done t o c o n f i r m t h o s e i s o l a t e s s u s p e c t e d

t o be P r o t e u s spp as o n l y P r o t e u s s p p d e a m i n a t e p h e n y l a l a n i n e

t o p h e n y l p y r u v i c a c i d , The medium was p r e p a r e d a c c o r d i n g to

m a n u f a c t u r e r s recommendat ion , i n o c u l a t e d and i n c u b a t e d a t ~ G ' C

for 72 h o u r s , 0.2 mls o f a c i d i f i e d 10% F e r r i c c h l o r i d e were

a d d e d t o each t u b e and e a c h t u b e s h a k e n i n t u r n , Appea rance

o f g r e e n c o l o u r which f a d e d w i t h i n t w o m i n u t e s was i n d i c a t i v e

o f a p o s i t i v e c a s e ,

2.2,11.19 STd-bRCH H Y D R O L Y S I S (OXOID) - S t a r c h a g a r w a s p r e p a r e d a c c o r d i n g t o m a n u f a c t u r e r s

recommendat ion , O x i d a s e p o s i t i v e , Gram n e g a t i v e r o d s were

s e p a r a t e l y s t r e a k e d on e a c h p l a t e and i n c u b a t e d a t 3oUc f o r 72

h o u r s , The p l a t e s w e r e e a c h f l o o d e d w i t h L u g o l t s i o d i n e s o l u t i o n .

The medium t u r n e d b l u e where s t a r c h had n o t S e e n h y d r o l y s e d

w h i l e h y d r o l y s i s w a s i n d i c a t e d b y clmr c o l o u r l e s s z o n e s o

210

2 0 2 0 1 1 0 2 0 HYDHOGEK SULPHIDE FRODUCTION ( 0 ~ 0 3 ~ )

The medium uscd f o r t h i s test was t r i p l e s u g a r iron a g a r

which was p r e p a r e d a c c o r d i n g to m a n u f a c t u r e r s recommendat ion.

They w c r e s e p a r a t e l y s t a b b e d a t t h e b u t t w h i l e the s l o p e was

s t r e a k e d w i t h t h e Gram n e g a t i v e rod i s o l a t e s , The s lopes were

i n c u b a t e d and o b s e r v e d f o r 48 hour s . P o s i t i v e cases showed

b l a c k e n i n g of t h e medium d u e t o hydrogen s u l p h i d e p r o d u c t i o n ,

2 2.11,21 GELiiTIN LIQUEF"\CTION

A b i l i t y of some o f t h e isolates to l i q u e f y g e l a t i n was

o b s e r v e d i n n u t r i e n t g e l a t i n a g a r , ~ ' ~ f t e r i n c u b a t i o n of t h e

i s o l a t e s i n n u t r i e n t g e l a t i n a g a r , t h e medium was f l o o d d d

w i t h 10% a c i d i f i e d F e r r i c C h l o r i d e . L i q u e f i e d area was i n d i c a t e d

by m n c s of c l e a r i n g a r o u n d t h e c o l o n i e s of t h e o r g a n i s m s o

2,2.11.22 ANIM/LL ::TNOCUL.\TION FOR TOXIN IISS;~Y

The motile G r a m positive rods i s o l a t e d a n a e r o b i c a l l y were

i n o c u l a t c d into R.:bertson s cooked meat medium and i n c u b a t e d

for 7 days at 37'~. This was to allow p r o d u c t i o n of t o x i n i f

t h c i s o l a t e s were t o x i n producers. On t h e 8 t h day, 0.2 c m 3

o f t h e b r o t h cu1 tu r e w e r e i n o z u l a t ed h t r a p o r i t o n a a l y into 3

mice after t h e coo'ced meat had been s h a k e n ayrh msat p a r t i c l e s

a l l o w e d to settle down, The mice were f e d and o b s e r v e d fo r

o n e wcek,

2,3,1 IDENTITY OF 3RGINISMS ISOLlrTED

The c u l t u r a l and biochemical c h a r a c t e r i s t i c s o f these

i s o l a t e s a r e r e p r e s e n t e d i n Tab l e 2,1, It w i l l be seen from

t h e t a b l e t h a t the isolated organisms were

B. cereus, !roteus r n i r a b i l i s , Proteus rettqeri., Acinetobacter - iwof-f i , - E s c h e r i c h i ~ ; coli type I ,Micrococcus spp and Lactobacillus SPP 0

2.3.2. COUNTS OF ViiRIOUS BACTERIAL GROUP ISOLATED -- FROM D R I E D FISH The average total count per gram of t h e 25 samples of

d r i e d f i s h was 8.5 ~10'~. The average coun t pe r gram f o r

L a c t o b ~ d l l u s was 1.2 x lo5, ,Staphylococcus was 2.7 x 10 7 -- per gram. The highest counts were found among & h r n e and

B a c i l l u s spp w h i c h had c o u n t s o f 3.5 x 10' and 3.2 x 10 9

r e s p e c t i v e l y ,

T h i s i s g ive? i n Table 2.2.

SP

EC

IME

N

NO

-

GR

OW

TH

IN

AIR

GRAM

RE

AC

T1

ON

CA

TA

LA

SE

OX

1 DASE

GL

UC

OSE

(G

AS

)

LA

CT

OS

E

SUCROSE

MA

NN

ITO

L

INO

SIT

OL

I

SA

LIC

IN

i D

UL

CIT

OL

,

CIT

RR

TE

22

IND

OL

E

rtrt

Y.

Y.

VO

PO

SO

RB

ITO

L

UR

EA

i

GE

LA

TIN

AR

GI N

INE

LY

SIN

i5

OR

NIT

HIN

E

PHE

NY

LA

-+L

AN

INE

CV

I\~

UL

IIS

C;

DN

I'ISE

LE

CIT

HIN

AS

E

ST

.iH

CH

AR

~~

BI

N

OS

E

XY

LO

SE

LIT

MU

S M

ILK

Table 2-2, COUNTS OF VARIOUS B ' C T E R I i FROM 25 SMOKED FISH SAMPLGS BOUGHT ,-rT N S U K K l * M~IRKET

ORGANISMS ISOLATED - ( i ) S taphylcrcoccus spp o 1 (ii) Micrococcus spp, 1

(Baird-F'arkerl s medium) ) 1

(ilil Bacilluq spp. thrnonium Sal t medium)

TOTILL COUNT PER GRkM,

(iv) Coliforrns (MacConkey agar) 3.5 x 10 9

(v) Lactobacillus spp, (Man, Roqosa and Sharpe . (MRS) medium)

(vi) Trypticase soy agar ( a l l organisms named above)

2-3-3, FREQUENCY OF ISOLj tT ION (%)

T h e frequency of isolat ion of various groups of bacteria

was calculated as a percen tage of the number of times each

bacter ia l t y p e was found from the 25 samples of

frequencies a re g iven on Table 2.3-

TABLE 2*3, FREQUENCY OF 1SOL;tTION (%) OF B;\CTKi?I r FROM 25 Sr,MPLZS O F SMOKZD FISH

i.

ii,

ifi"

iv,

V o

v i e v i i ,

v i i i .

i x e

X o

x i *

B,, cereus

S t c 7 p h y l o c o c c u ~ (non- - Coi- ;gulase P o s i t i v e spp. 1 Micrococcus spp,

P r o t e u s m i r a b i l i s -- P r o t e u s rettgeri _ . - 4 - i c i n e t o b a c t e : ~ i w o f f 1 - - L a c t o b a c i l l u s - spp.

E s c h e r i c h i a c o l i .- C1, w e l c h i i

C1, bmtul inulq -- Se a u r e u s -

No. OF POSITIVE SI'\MPLES

12

20

17

15

22

0

18

5

nit,

n i l

n i l

FREQUENCY OF ISOLsTION

(%I

80

68

60

88

32

72

20

n i l n i l

n i l

2e3,4* ,WAtTEkl CONTENT 3F SMOKED FISH+ -. The water c o n t e n t of t h e 25 samples o f f i s h is g i v e n i n

T a b l e 2.4, It was f o u n d to be between 37% and 60% o f the d r y

w e i g h t of f i s h .

TABLE 2,4, -- WATER CONTENT O F 25 SMOKED FISH (96) --

- Dates -- 7m11a79

9,11079

14o11e79

20.11,79

13e12e79

15.12.79

17e12e79

2102,80

1,3080

4m3e80

12,4&l

14e4a80

19-4e80

24,4080

3,5,80

5m5e80

6.6.80

34o6m80

20.8m80

23.8-80

25o9m80

11elOo80

18,11,80

-

-- F i s h Sample -II-

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

F13

F14

F15

FIE

F17

F18

F19

F20

F21

F22

F2 3

- - % Water -.

45

43

60

46

44

37

45

43

41

50

48

43

47

48

44

40

49

60

50

47

42

46

39

-

Date Fish Sample

F24

F25

-

% Water

2.3.5. Summary of F.F;?s,u~ 5

h wide v a r i e t y of o rgan i sms w a s found on d r i e d f i s h

bought from t h e market. O f the u s u a l food-poisoning o rgan i sms

namely C1. botu l inum, C1. w e l c h i i , and c o a g u l a s e p o s i t i v e

S taphy lococcus and ,B , cereus o n l y the l a s t named was p r e s e n t ,

On t h e other hand, E r o t e u s rnirabilxs, P r o t e u s r e t t q e r r i ,

Micrococcus spp.' and Lactobacillus spp; were isolated most oftenb

Since t h e h i s t o r y of t h e market f i s h was n o t known it was

d e c i d e d to s t u d y t h e f i s h of known h i s t o r y by s t u d y i n g f i s h

m i c r o f l o r a b e f o r e and a f t e r smoking, These s t u d i e s a r e r e c o r d e d

i n t h e n e x t Chapter.

CHAPTER THREE

3mO STUDIES ON THE MICROBIOLOGY OF FISH SMOKED IN THE LABORATORY

3,1. INTRODUCTION

The examina t ion o f d r i e d f i s h bought a t Nsukka m a r k e t

r e v e a l e d t h a t t h e major food-poj Lsoning o rgan i sms

were absent. I n t h e exper iment t o be d e s c r i b e d , Lne ~ n ~ ~ r u r ~ u r - a

of f r e s h l y c a u g h t f i s h was de te rmined b e f o r e and a f t e r smoking,

ML7\TERI-'iLS I'rND METHODS

3.2.1. FISH SAMPLES USED - l azera

L i v e f i s h o f -- Clarias-were Ogurugu r i v e r a s soon a s t h e f i s h 1 2

t h e l a b o r a t o r y i n a p l a s t i c b u c k e t h

wate r . I n t h e l a b o r a t o r y , t h e y were Kllled by stabblnu t h e medul la

0b langa t : t a a r e a w i t h a s h a r p poir

method of Zdenek ! l977) . The eni

- / 2ted k n i f e i n accordance w i t h thc

t r a i l s were removed and t h e f i s h

washed i n runn ing t a p water a o b e r t o 1980). As i n t h e p r e v i o u s

e x p e r i m e n t all t h e f i s h were of t h e same s ize measuring between

28 c m and 32 c m i n l e n g t h and 2,4 c m and 4 c m i n t h e w i d e s t r e g i o n ,

Two f i s h a t a time were s t u d i e d f o r t h e f l o r a of f r e s h f i s h

w h i l e two e a c h were s t u d i e d a t t h e lower and h i q h e r t e m p e r a t u r e s

of smoking r e s p e c t i v e l y . The exper iment was performed o v e r a

p e r i o d o f s i x months from November 1980 t o May 1981,

PLATE 11,

LIVE CLARI iG LA\ZERI:\

FISH BOUGHT FROM

OGURUGU

x 0.25 magnification

I

FIG. 3 - I

3.202. THE SMOKER

ri smoker d e s i g n e d to SUIUAC~LC AuLaI A Y ~ I I I U A A I 19 IIICLI IVUD

was made. I t c o n s i s t e d of a n a l l o y of t i n and z i n c and measured

45 cm i n l e n g t h , 30 cm deep and 45 cm h i g h , Ii

made of a g r i d s y s t e m which would p e r m i t t h e f k c c u p w a r u r i w v e w r t I t

of smoke. T h e s e s h e l v e s were 1 5 c m a p a r t w h i l e t h e d i s t a n c e

be tween o n e g r i d and t h e o t h e r was 1 c m . The d i s t a n c e be tween

the f i r e p l a c e ( h e a r t h ) a n d t h e l o w e r s h e l f was 1 5 c m . The d o o r

of t h e smoker was h i n g e d so t h a t a e r a t i o n of f i r<

c o n t r o l l e d . The t o p o f t h e smoker was p r o v i d e d c e

r o u t e s (Chimney) for t h e smoker w h i l e a t t h e real

s l i t s measu r ing 1 c m by 1 c m were p r o v i d e d . T h i s was ro e n s u r e

minimum a e r a t i o n when t h e d o o r was c l o s e d and f i r i n g was c o n t i n u e d ,

Fig* 3.1 shows t h e d e t a i l o f t h e smoker,

3.2.3. SMOKING OF FISH SiiMPLES I N THE L-\BOR.rTORY

600 grams of wood s h a v i n g s were p u t i n t o bottom p a r t o f t h e

smoker ( h e a r t h ) , ; idmixturr o f 70% a l c o h o l and k e r o s e n e were

C h l o r o p h o r e x c e l s a s p r i n k l e d on t h e s h a v i n g s , C h l o r o ~ h o r a Oxcelsa

which were'rromIL(rrok= w e r e t h o r o u g h l y mixeu. lllr uvvr

o f t h e smoker was l e f t s l i g h t l y a j a r f o r a e r a t i o n of f i r e to t a k e

place. The s h a v i n g s were s u b s e q u e n t l y l i g h t z d . Th? f i s h t o be

smoked were f e d i n t o t h e smoker. Two f i s h were f e d o n t o t h e

u p p e r chamber and a n o t h e r t w o o n t o t h e lower chamber r z s p e c t i v e l y .

,lfter a l l o w i n g t h e s h a v i n g s t o burn for t w o minu tes ,

t h e door of t h e smoker was closed. The o n l y s o u r c e f o r oxygen

s u p p l y to t h e smoker was th rough t h e s l i ts a t t h e s i d e s and back

of t h e smoker. Smoking u s u a l l y l a s t e d f o r s i x hours . l i f t e r

smokinq for abou t 20 minutes , t h e t e m p e r a t u r e o f t h e smoker was

checked w i t h a thermometer th rough t h e s l i t open ings a t t h e

s i d e s . T h e r e a f t e r , t e m p e r a t u r e s were checked on a n h o u r l y b a s i s

and an average t e m p e r a t u r e f o r each f i s h smoked found a t t h e end

of smoking. Should t h e , p m l d e r i n g of wood s h a v i n g s s t o p , i t

w a s u s u a l l y r e v i v e d by s p r i n k l i n g i n few mi l l i l i t r e s of k e r o s e n e

on t h e s h a v i n g s and r e l i g h t i n g t h e shavings .

Through p r e l i m i n a r y experiments, i t w a s found t h a t w i t h

600 gr71ms of wood, t h e lower t e m p e r a t u r e 38'~ - 4 3 O ~ was rna in t f i ined i n t h e l o w r s h e l f w h i l e 65 - 78Oc was m a i n t a i n e d i n t h e upper s h e l f , F i s h w a s t h e r e f o r e smoked a t two t empera tu res : -

302.4, DETERMINarTION OF FLORit OF FISH

F r e s h f i s h and f i s h smoked a t t h e lower and h i g h e r

t e m p e r a t u r e s were macera ted 3s p r e v i o u s l y d e s c r i b e d i n C h a p t e r 2

and p l a t e d o u t on t h e v a r i o u s media a l r e a d y d e s c r i b e d ,

The e x p x i m e n t w a s r u n txve t i m e s and a t o t a l of 30 f i s h

s a m p l e s were used*

3,2,5, WtITGR CONTENT OF SMOKED FISH

The water c o n t e n t of t h e smoked f i s h was d e t e r m i n e d a s

p r e v i o u s l y d e s c r i b e d i n C h a p t e r 2.

3.3.1,, FLORA OF FISH SMOKED IN THE LABORATORY

Based on the c u l t u r a l and b i o c h e m i c a l c h a r a c t e r i s t i c s

of t h e o r g m i s m s isolated, t h e bacteria e n c o u n t e r e d were

i d e n t i f i e d a s Acinatobacter i w o f f i , Mic rococcus spp, and - - L a c t o b a c i l l u s spp.

3.3.1.1. FISH SMOKED AT 3 8 * ~ - 4 3 ' ~ From t h i s sample o f f i s h , t h e o r g a n i s m s r e c o v e r e d were

m a i n l y -- A c i n e t o b a c t e r j - w o f f i, M i c r o c o c c u s s p p , and Lactobacil l u s spp. o n l y , These i s o l . a t i o n s and t h e i r t o t a l c o u n t s are r e p r e s e n t e d

i n Table 3,1.

TABLE 3 m 1 , ORG;+NISMS FROM F I S H SMOKED iiT LOWER - AND HIGHER TEMPERliTURES

FISH SMOKED ,IT 3 8 O ~ - 4 3 O ~ ORGIN1 SMS

ISOLirTED

i. Micrococcus spp ( B a i r d - P a r k e r s medium )

ii, ircinetobacter iwof f i m n k e y agar)

iii, L a c t o b a c i l l u s SPPm ( Rogosa medium)

+iv. T r y p t i c a s e soy agar

AVERAGE

3T4-J, COUNT

FISH SMOKED iiT 65 - 7 8 O ~ ORGlrNISMS

ISOLitTED TOTiiL COUNT

M i c r o c o c c u s spp ( B a i r d P a r k e r s I

N i l

N i l

1 x l o 2

N i l

Nil

1 :

'Note: - Used i n this e x p e r i m e n t f o r d e t e r m i n a t i o n of total c o u n t only,,

3,3.1..3, WATER CONTENT OF SMOKED FISH

The water content o f t h e f i s h smoked a t lower temperature

was f o u n d t o be b e t w e e n 40% and 50% w h i l e t h e f i s h smoked a t a

high t e m p e r a t u r e was between 24% and 36%- T h e s e are r e p r e s e n t e d

i n T a b l e 3,2,

TABLE 3 , 2 . ----- WAT3R CONTENT OF SMOKED FISH - -u--C-. - FISH SMOKED AT 38 - 4 3 O ~ Y - FISH -7

% SAMPLES I WATER FDLl

FDL2

FDL3

FDL4

FDL5

FDL6

FDL7

FDL8

FDL9

FDLlO

FDLl1

FDL12

FDL13

FDL14

FDL15

FDL16

FDL17

FDL18

FDL19

FDL20

FFDLl

FFDL2

FFDL3

FFDL4

FFDL5

FFDLG

FFDL7

FFDL8

F F D L ~

FFDLIO

FFDL11

FFLL12

FFDL13

FFDL14

FFDL15

FFDL16

FFDL17

FFDL18

FFDL19

FFDL20

3 6 . I 1

CHARACTERS O F ORGANISMS ISOLATED FROM FRESH FISH TABLE 3 , 3 ,

IDENTITY

K 1 . edwards i i - Ea c l o a c a e .- R c i n e t o b a c t e r i w o f f i

Aeromonas hyd roph i l a

tie, s h i q a l l o i d e s

A e spp. - Pseudomonas

Flavobacter ium

Moraxel la

M i c r o c o c c , ~ ~ ~ spp

Lactobacillus-

Ea c o l i

Coryneform

rd

E

,.a 0 z z .$ Z

+ + v t

- t

I

-

t

u l t , Lre -R 9 means Gram n e g a t i v e rod ,

W V1 cx

--

c;l 0

E v)

--I

+ . - .

- .

- - + . + - - .

- a

- .

- A n e q a t i v e r e s u l k , V - Stands f o r a ' v a r i a b l e r e s u l t , + R - ~ r & p o s i t i v e r o d , - Cb means Gram n e g a t i v e c o c c o c o b a c i l l a r y ? Shows growth on blood a g a r o n l y 0

'

V-CH, AGG - V, 0.1olera a g g l u t i n a t i o n Y

W 2

~ H W

:ans

I Z Z E - I - r ' H H k H i u r n z u I f Y > ( r Z W . Z O d

- + + -

+ - + -

Z

- +

H 5:

- + - + -

+

I o x i d a t :

303e204. FLORA OF FRESH FISH

The c u l t u r a l and b iochemica l c h a r a c t e r i s t i c s of t h e

i s o l a t e s from f r e s h f i s h a r e g i v e n i n T a b l e 3,3. They were

i d e n t i f i e d a s -- Micrococcus spp , K l e b s i e l l a e d w a r d s i i , A c i n c t o b a c t e r iwoff i, keromonas, &

pigment ing s p p ) , S t e r o b a c t e r c l o a ~ ~ ~ , L I U ~ ~ ~ , A-ILJL Q A C L ~ ~ , L a c t o b a c i l l u s

f r e q u e n c y of d

from f r e s h f i

TABLE 3.40

y d r o p h i l a , Pseudomonas (non

. P l -...fik-r-L-.":..." M---...-I

i. Micrococcu:i s p p 3-1 x 10 2

(Ba i rd -Parkers Medium)

ii. Gram n e g a t i v e r o d s 2.2 x 10 5

(MacConkey a g a r )

iii, ' T r y p t i c a s e s o y a g a r ( A l l o rganisms

iv. Lac tobac i1 : lus - spp (Rogosa medium)

Note: *The Moraxe l l a - and Coryneform b a c t e r i a were i s o l a t e d on t r y p t i c a s e soy a g a r b u t t h e i r i n d i v i d u a l t a t a l c o u n t

c o u l d n o t be done because of p r e s e n c e of o t h e r o rgan i sms

on t h e p l a t e ,

TABLE 3.5* FREQUENCY OF ISOLiiTES i\MOWG - a T H E .-.- - SI'tMPLES OF FRCSK FISH

ORG. iNISMS I SOLiiTED L ~ ~ o OF POSITIVE FREQUENCY OF 1 S;rMPLES I SOL.'iTI ON ( % ) i. Micrococcus spp i 11 i 7 3 - 3

ii, Lactobacillus- spp

iii. Klebs ie l l a eclwardsii 5

i v l i d n e t o h a c t e r iwof fi

v o Xeromonas hydrophila

v i , Pseudomonas spp

vii. E n t e r o b a c t e ~ cloacae

. --

v i i i , Flavobacterium

i x , Moraxella

x o Coryneforms

40. CHAPTER FOUR

GENERI'LL DISCUSSION

Figo 4,1. r e p r e s e n t s t h e m i c r o b i a l f l o r a of b o t h f r e s h

a n d a l l t h e smoked f i s h examined i n this p r o j e c t between

November 1979 and May 1981.

The m i c r o b i a l l o a d of f r e s h l y caugh t f i s h from I

r i v e r were M i c r o c o c x , L a c t o b a c i l l u s , K l e b s i e l l a , &rur~~ur:aa

Pseudomonas, F lavobac te r ium, 5a.

E a r l i e r workers (Shewan and w=vl y u A a A , J l , Y1lGWaII aIIu IIWLJLIO 1967,

1971, Lobben and Lee 1968, S h o t t s and Bul lock 1975, Rober to 1979)

had found t h e same organisms on f i s h from sea w a t e r w i t h v a r y i n g

f r e q u e n c i e s , Some organisms, n o t a b l y K l e b s i e l l a e d w a r d s i i ,

Aerornonas h y d r o p h i l a , Pseudomonas, Moraxe l l a , F lavobac te r ium - and Cmvne~~ctrns found i n the f r e s h , e a t i n t h e f i s h

i

bought from Nsukka market , However P r o t e u s r e t t q e r f , P r o t e u s

- -

f i s h were abs -. .

p i r a b i l i s , S t a ~ h y l o c o c c u s (non c o a g u l a s e p o s i t i v e ) and El, c e r e u s

n o t found i n f r - s h l - y c a u g h t f i s h were p r e s e n t i n l o c a l l y smoked

fish.

The f i s h smoked a t 38 - 4 3 O ~ c o n t a i n e d a l l m i c r o b i a l f l o r a found i n l o c a l l y smoked f i s h e x c e p t P r o t e u s , Bacillus, Staphy lo -

coccus and E s c h e r i c h i a c o l i which were p r e s e n t i n l o c a l l y smoked

f i s h , From t h e f i s h smoked a t 65 - 78%, o n l y Micrococcus spp. were i s o l a t e d .

The el i r n i n a t i m of Klebsiell a spp,

monas Flzrvobacterium, Moraxel la and Corvne; LII,U AVU.IU - 9

41 e f r e s h l y c a u g h t f i s h b u t a b s e n t i n smoked f i s h i s a t t r i b u t a b l e

to t h e effect of smoke o r t e m p e r a t u r e accompanying smoking

since smoke is known t o have an a n t i b a c t e r i a l effect on

m i c r o o r g a n i s m s when t h e l eve l of smoke d e p o s i t e d on f i s h

s u r f a c e s is u p t o 45 mg/kg. of f i s h (Lea 1933, O l s e n 1976,

EYO 1979)m

The p r e s e n c e o f Proteus spp., S t a ~ h y l o c ~ c c u s (non c o a g u l a s e

posit ive) B a c i l l u s and E s c h u r i c h i a c o l i i n t h e l o c a l l v smoked

f i s h i s a t t r i b u t a b l e t o p o o r h a n d l i n g a n d s t o r a g e si

o r g a n i s m s a r e n e i t h e r found i n f r e s h f i s h o r f i s h SK

the L a b o r a t o r y * The h i s t o r y of l o c a l l y smoked f i s h ---- -..

these e x p e r i m e n t s i s unknown. N e v e r t h e l e s s it is c e r t a i n t h a t

t h e t i m e l a g be tween f i s h smoking and f i s h s a l e may a l l o w

c o n t a m i n a t i o n o f t h e smoked f i s h f rom o u t s i d e . T h i s

c o n t a m i n a t i o n c a n be b r o u g h t a b o u t by m i c r o b i a l l a d d e n sand

or flies, Haywood and R o l l i n g s (1962) r e p o r t e d cases of con tamina - '

t i o n of d r i e d f.*Lsh i n N i g e r i a by f l i e s and sand.

I n t h e f i s h smoked a t 65 - 7 8 * ~ o n l y Mic rococcus was i s o l a t e d w h i l e - L a c t o b a c i l l u s and A c i n e t o b a c t e r which a p p e a r e d i n t h e fish smoked a t 3 8 O ~ - 4 3 O ~ were e l i m i n a t e d . It is t h e r e f o r e p o s s i b l e t h a t besides t h e l e t h a l e f f e c t o f smoke, t h e

t e m p e r a t u r e of smoking is also a n i m p o r t a n t v i t a l e lement .

However, w o r k e r s i n this f i e l d do n o t y e t seem to have a t t e m p t e d

t o s e p a r a t e t h e e f f e c t of smoke from t h a t o f t e m p e r a t u r e ,

The average t a k a 1 c o u n t of o r q a n i s m s i n f r e s h f i s h was

5 4 x 10 p e r gram. In the l o c a l l y smoked f i s h t h e a v e r a g e to ta l

c o u n t wqs 8.5 x 10'' per gram w h i l e i n t h e f i s h smoked a t

38 - 4 3 O ~ and 65 - 7 8 ' ~ t h e c o u n t s w e r e 1 x l o 4 and 1 x 10 3 r e s p e c t i v e l y + Though smoked fish is o f t e n e a t e n i n this mrt of t h e country w i t h o u t f u r t h e r c o o k i n g , l o c a l l y smoked f i s h

bough t from Nsukka m a r k e t contained h i g h t o t a l c o u n t qbovc

6 10 /gram. T h a t c h e r and C l a r k (1968) 5tat'Ld t h a t p r o c e s s e d f o o d " 6 and food p r o d u c t s w i t h rnicrmbial c o u n t s above 10 /gram may c o n t a i n

food-po i son ing o r g a n i s m s , However a l l f i s h used i n t h i s p r o j e c t

showed complete a b s e n c e of well-known f o o d - p o i s o n i n g o r g a n i s m s

s u c h as Salmanclb, 2, a u r m s and

B, cereus a n o t h e r food-po i son ing c

smoked fish, The p r e s e n c e sf t h i :

f i s h may b e t h e r e a s o n f o r the rey ic

following a heavy meal of smoked 1

Although a high m i c r o b i a l c o u n t o c c u r r e d i n l o c a l l y smoked

f i s h , s i g n s of i n c i p i e n t s p o i l a g e like o f f - o d o u r s and s o f t

t e x t u r e which Roberto (1979) p o s t u l a t e d accompany f i s h w i t h h i g h

m i c r o b i a l c o u n t were n o t p r e s e n t * W i l l i a m s and Holl ies (1974)

had recommended t h a t p r o c e s s e d f i s h and f i s h e r y p r o d u c t s s h o u l d

contain no Sa lmone l l= , no S h i q e l l a or C l o s t r i d i u r n s p p o and t h a t

C o l i f o r m s , E s c h e r i c h i a -- c o l i and S, a u r e u s if p r e s e n t s h o u l d o c c u r U I -.I

i n t h e ratio of 200/gram for co1

f o r E s c h e r i c h i a coli, less than

t h e a v e r a g e t o t a l c o u n t should

b a s i s of f i n d i n g s i n t h i s proje

r e g a r d e d as excellent f o r humar

The amount of w a t e r l e f t 3

between 37% and 60%, I n t h e f l

65 - 7 8 ' ~ , t h e amounts were 403 The high p e r c e n t a g e of water 1~

i n d i c a t e s t h a t any organism not

smoking may m u l t i p l y i n t h e f i ~

may be r e s p o n s i b l e f o r the r a p j

d u r i n g s t o r a g e a t room temperat

AS p r e v i o u s l y mentioned i t

e x p e r i m e n t s i n t h i s f i e l d will

e x p e r i m e n t a l i s t s t o access the

o f t empera tu re , Th i s w i l l expl

was i s o l a t e d in t h e fish smokec

APPENDIX

MEDItr CONSTITUENTS

B A I R D - PARKER'S MEDIUM. ( O X O I D CM 27510 Tryplone Lab-Lemco Powder Yeast Extract

Sodium Pyruvate G l ycine

L i t h i u m Chlor ide Agar No. 3 D i s t i l l e d water

MACCONKEY AGiiR ( OXOID CMMb )

Peptone Lactose Bile Salt

Neutral red Agar No, :3

D i s t i l l e d water

AMMONIUM SALT BATE . I

Ammonium Hydrogen Phosphate ( NH4) 3HP04 -

Potassium Chloride ( K c l ) - Magnesium Sulphate (hydra-

ted) - (Mg SO4 7H20) - Yeastrel o

Agar o

10 gram 5.0

1.0 " 1000 * 12.0 " 5.0 "

20.0 "

1 litre

20.0 qram

1000 l'

5.0 "

0,075 So

1200 "

1 litre,

L O gram

0.2 "

D i s t i l l e d water - 1 litre.

BLCOD I+G;tR BASE (OXOID C H 3 )

Lab-Lemco Powder

Yeast Extract Peptone Sodium Chloride

Agar No. 3

D i s t i l l e d water

l o o gram 2.0 " 5.0 tt

5.0 " 15.0 "

1 l i tre ,

Trypt icase peptone - 15.0 gram Phytone peptone . 5.0 " Sodium Chloride - 5.0 " Agar - 15.0 D i s t i l l e d water - 1 l i tre .

MRS L~~CTOBHCILLUS - X : r R ( Ml'rN , ROG0S.r Si+ rRPE ) Peptone 10 grams

Beef e x t r a c t 1b (1

Yeast e x t r a c t 5 t t

Glucose 20 (I

Tween 80 1 ml, d i p o t a ~ ,ium hydrogen phosphate ( K ~ H P c ~ ) 2 grams

CH3 COONO 3H20 Sodium a c e t a t e (hydrated) 5 " Triammonium c i t r a t e 2

Magnesium su lphate (hydrated) 0.2 grams Manganese sulphate

(hydrated) 0.05 *' Agar 17 grams Distilled Water 1,000 ml.

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