Microbiological Quality Evaluation of Seafood

Since fish is harvested from natural water bodies including farms, it harbors a number of micro-organisms found in the environment from where it is caught. These native micro-organisms may include fish spoilage bacteria as well as certain pathogens of aquatic origin. In addition to these inherent microorganisms, the fish can get contaminated with other microorganisms during handling, transportation and processing, right from the point of catch to the end product. These microorganisms include both pathogenic and non-pathogenic bacteria. The pathogenic microorganism can be hazardous to the health of the consumer. The most important pathogens which gain entry into the fish during handling, transportation and processing are Salmonella, Vibrio cholerae, Staphylococcus aureus and Listeria monocytogen. In addition, enteropathogenic Escherichia coli, Clostridium perfringens and Bacillus cereus may also gain entry to the fish.

            In order to determine the acceptability of fish as well as selfish, the assessment of their microbial quality is necessary. The microbial parameters that are generally assessed to determine their consumer acceptability and safety are the following,

Total plate count (TPC)

Total Entrobacteriaceae count (includes all Coliforms, Salmonella & Shigella)

Escherichia coil (E.coli)

Staphylococcus aureus

Faecal streptococci

The level of the total bacteria (TPC) will indicate the freshness of the products, as well as their potential shelf life. The type of bacteria in the product can give information regarding how the product has been handled or processed. A high bacterial count indicates the level of contamination of the product, unsuitable conditions of storage, the extent of spoilage, etc.  Hence TPC gives overall picture of quality of product. A TPC of 10⁶/g or above is considered as a proof of quality of the product.

            The TPC can be determined by microscopic or cultural method is preferred for determination of TPC because it gives an estimate of viable (live) cells. A suitable   culture media, like Tryptone Glucose Ager (TGA) or plate count agar is generally used for determination of TPC. Live bacterial cells are capable of forming colonies on a suitable solid medium. This property of the bacteria is made use of the determination of TPC.

            Total Entrobacteriaceae count and total fecal streptococci count gives an indication to whether there was any fecal contamination in the fish at any stage; E.coli is a direct fecal indicator organism. Staphylococcus aureus can arise mainly from the human handlers; Salmonella and V. cholerae are pathogenic organisms, implicated in food-borne infections.

Table 1 gives the various microbial parameters to be studied for fish products and the media used for their sampling.

 Table 1. Microbial parameter and the media for sampling

Parameter                                                      medium

Total plat count (TPC)                                     Tryptone glucose agar (TGA)

Total Entrobacteriaceae count

 (Include all Coliforms, salmonella                 violate red bile glucose agar (VRBGA)

& Shigella)

Escherichia coli (E-coli)                                  Tergitoll 7 agar (T 7)

Staphylococcus aureus                                    Baired parker medium (BP)

Fecal Staphylococci                                         Kenner fecal Staphylococci agar (KF)

Salmonella                                                      A set of media

Vibrio cholera                                                             A set of media

Total plate count (TPC)

In the procedure for sampling for TPC, a known quantity of the sample is macerated well with a know volume of a suitable diluents and one ml of the appropriate dilutions are cultured in the plating medium. For this 10g of the sample is mixed with 90 ml diluents or 25 g  sample with 225 ml diluents i.e.1 part sample with nine parts diluents (i.e. 1+9=1:10 ratio). This gives 10 times dilution of the sample, i.e. 10^-1 dilution. For further dilution, 1ml from 10^-1 dilution is mixed with 9 ml of the diluents (10^-2 dilution) and so on to get the appropriate dilutions for plating. This type of dilution is called serial decimal dilution. Usually, 3 dilutions are plated in duplicate or triplicate. The selection of the dilutions sampling depend on the bacterial load in the sample i.e. highly contaminated or spoiled samples require higher dilutions than a very fresh one.

^Method 

            10g of the sample is aseptically cut into a sample dish and macerated with 90ml normal saline (NS) in a sterile glass mortar. (Alternatively, 25g sample is blended with 225ml NS in a stomacher blender)

Sampling scheme 

10g sample +90ml NS: 10^-1 dilution

1ml+ 9ml NS: 10^-2 dilution (0.5 ml each to BP and T 7; 1ml each to KF and VRBGA)

1ml +9ml Ns:10^-3 dilution (0.5ml each to BP, T 7;1 ml each to KF, VRBGA and TGA)

1ml +9ml NS; 10^-4 dilution (1ml to TGA)

1ml+9ml NS; 10^-5 dilution (1ml to TGA)

For compositions of media refer any standard manual on Microbiological methods for food analysis.

TPC:  Pour plating on TGA, 1ml of 10^-3, 10^-4 and 10^-5 dilutions in duplicate is recommended. For KF and VRBGA, pour plating technique is followed. One ml each of 10^-2 and 10^-3 dilutions is plated. For T7 and BP, pre- set plates have to be prepared 100ml Tergitol-7 agar (T 7) is melted in water bath, cooled to about 50⁰ c and aseptically added 0.25ml of 1% sterile TTC solution. Poured into sterile Petri dishes (15-20ml each,) allowed to set and dried at 56⁰C for 45 min. Cooled to room Temperature (RT).

100ml Baird –Parker medium (BP) is melted & cooled to about 50⁰C; aseptically added 1ml of sterile 1% potassium telluride solution, followed by 5ml of 50% egg yolk  emulsion. Mixed well, poured into sterile petridishes, and allowed to set; dried at 56⁰C for 45 min. cooled to RT.

Arrange 6 petridishes for TGA & 4 each of KF and VRBGA. Also, arrange 4 plates each of preset T- 7 and BP agar. Label appropriately, viz: sample name, dilution, medium, and date.

For TGA, KF and VRBGA, 1ml each of the appropriate dilutions are pipetted and pour plated with the corresponding medium.  Plates are allowed to set, inverted and incubated at 37®C.

For T 7 and BP plates, 0.5ml each of the appropriate dilutions are surface plated using sterile bent glass rod.  Plates are inverted and incubated 37®C for 18-24 hrs. The plates are examined after the incubation.  Observe VRBGA and T 7 plates after 18-24hrs.

Observations and results

VRBGA plates:-

Red, small (2-4 mm dia) colonies are counted as Entrobacteriaceae colonies. Take average count of duplicate plates.

VRBGA is a medium considered specific for Entrobacteriaceae, which includes the Coliform group, Salmonella, Klebsilla and Citrobacter. All these bacteria ferment glucose with the production of acid.  The medium contains neutral red which turns red in presence of acid.  So, all glucose fermenting colonies appear red.

Total Entrobacteriaceae count / g = Average count x dilution factor.

T 7 plates

E. coli colonies are lime yellow, occasionally with rust brown center and a yellow zone around.  (Note: Yellow slimy, raised or convex colonies are not be considered as E. coli colonies.) Take average of duplicate plates.

E. coli/g =Average count x2 x dilution factor.  Average count is multiplied by two because only 0.5ml of the sample dilution was added to the plates.

Tergitol-7 agar containing TTC is a selective and differential medium for Escherichia coli and Enterobacter aerogenes, which allow their detection in 18-24 hrs.  So, this medium has been recommended for routine analysis of water and food.  The Tergitol-7, in the medium inhibits the growth of Gram positive bacteria.  E. coli ferment lactose to produce acid, which change the color of bromothymol blue from green to yellow.  E. coli does not reduce TTC, but other Coliform and Enterobacter aerogenes reduce TTC to red dye formazan.  Hence, E. coli colonies will appear yellow, with depended yellow center and with a yellow halo around.  Colonies with a red tinged center should not be confused for E. coli.

Confirmation of E.coli.

The lime yellow colonies, occasionally with rust brown center and a yellow zone around, on T 7 plates are counted as E. coli; however, to confirm them as E. coli, the following procedure has to be adopted.

1.      Streak  on Eosin -Methylene Blue (EMB) agar

EMB agar is melted, cooled to 50®C, poured into Petri dishes and allowed to set. The set plates are dried at 56®C for 45 min and cooled to room temperature.  Typical yellow colonies from T 7 plates are picked with a sterile platinum loop and streaked on to EMB plates, by the streak-dilution method, incubated at 37®C for 18-24 hrs.  Well isolated colonies, 2-3mm dia with  a greenish metallic sheen by reflected light and dark purple center by transmitted light is picked and subcultured on TGA slants and incubated at 37®C for 18-24 hrs.  The green metallic sheen on EMB agar is caused by the precipitation of methylene blue in acid pH due to lactose fermentation by E.coli

2.      IMViC tests

From the TGA slants above, inoculate to the following media.

a)      Tryptone broth (Indole medium) Inoculate a little of the culture to Tryptone broth and incubate at 37®C for 48 hrs.

b)      MRVP medium: Inoculate each culture into 2 tubes of MRVP medium and incubate at 37®C for 48 hrs.

c)      Simmons citrate Agar:- streak a little of culture to Simmons citrate agar slants and incubate at 37⁰C for 48 hrs.

Results

Observe results after 48 hrs.  of incubation.

1.      Tryptone broth

Test for indole production using Kovac’s indole reagent (add 0.5 ml and shake, allow to stand).  A red or pink colour at top indicates positive test.  Indole forms red dye with p-dimethyl amino benzaldehyde of the Kovac’s reagent.

2.      MRVP MEDDIUM

MR Test

       Into one tube, add Methyl Red indicator. A red color indicates positive MR test. E.coli Ferments glucose to produce acid, which brings down pH of the medium to less than 4.4 indicated by the red color of methyl red indicator. If the color is orange, pH is 5.0-5.8 and if yellow, pH is more than 6.0.

VP Test

       Using 1ml of the culture from the second tube, do the VP test (To 1ml culture, add 0.6ml 5% α -napthol and 0.2ml 40% KOH, add a pinch of creatine and mix, allow to stand up to 4hrs.). Eosin pink color indicates positive VP test. As an end product of glucose fermentation, some bacterial groups produce acetyl methyl carbinol (acteoin). Addition of potassium hydroxide oxidizes this compound to diacetyl which gives an eosin red colour with guanidine nucleus present in peptone in presence of α- napthol. The reaction is accelerated by the addition of creatine.

3.      Simmons citrate agar

Growth indicated by a change in colour of the medium from green to blue indicates a positive test for citrate utilization by bacterial culture. A positive growth indicate that citrate is utilized as the sole source of carbon, and the colour of the medium turns blue due the alkaline pH produce by the growth of bacteria. E-coli do not utilize citrate and hence give a negative reaction.

 Culture giving the following results is confirmed as E-coli.  

Indole                     positive

Methyl red             positive

VP                           negative

Citrate                    negative

  i.e., IMViC:           + + -  -

Eijkman’s test:

This is used as an optional confirmatory test for E-coli. E-coli culture will grow and produce gas in EC both at 44.5 + 0.5⁰ C in 24-48 hrs.

BP agar plates

Observe after 36-48 hrs.  Staphylococcus aureus colonies are black with thin white margin and a zone of clearance around.

Staphylococcus aureus count per gram = Average count x 2 x dilution factor

Average count is multiplied by two since the quantity of sample dilution added is only 0.5 ml.

On BP agar, Glycine, lithium chloride and tellurite suppress of the growth of bacteria other than Staphylococcus spp. S. aureus reduces tellurite to form grey black or black shiny colonies. A clearance zone around the colonies is produced by the action of lecithinase present in S. aureus on the egg yolk. By the action of lipases /phospholipases on lipids or phospholipids S. aureus produces a white precipitate as a margin around the blank colonies. Typical S. aureus gives all the above three characteristics; but the white margin may not be produced by all strains of S. aureus.

Confirmation of Staphylococcus aureus-

S. aureus is confirmed by Coagulase test, Coagulase is a thrombin like substance which coagulates blood plasma. Most of the pathogenic S. aureus produce Coagulase. Difco Bacto-coagulase –EDTA is used for the test. To 0.5 ml. of Coagulase reagent in a small sterile test tube, add 2 drops of 24 hrs. old bacterial culture (grown in Brain Hearth Infusion broth, BHI; incubate in a serological water bath at 37⁰C . Observe every 30 min up to 4 hrs. Coagulation (jell formation) of the contents of the tube indicates a positive reaction for Coagulase.

KF Agar plates-

Observe after 36-48 hrs. Count all surface and sub-surface red to pink colonies (some will be with a thin white margin) as faecal streptococci. On KF agar, the sodium azide accts as a selective agent and streptococci reduces TTC to give red colored formazan dye.

Confirmation of faecal streptococci-

Faecal streptococci (enterococci) group include Streptococci faecalis and Streptococcus faecium.

They are confirmed by catalase test. Pick 5-6 typical colonies from KF agar to BHI broth and incubate at 36 + 1⁰C for 24-48 hrs. Mix 3ml of the culture with 0.5 ml of dilute Hydrogen peroxide (H₂O₂). (Usually  H₂O₂  is available as 30 vol/vol  solution, i.e. 30% . Dilute 2 ml to 5 ml with distilled water for the test). Note the evaluation of gas bubble (oxygen). No evaluation of gas bubble indicates negative reaction. Faecal streptococci are catalase negative. A negative result in catalase test confirms faecal streptococci.

MPN method for Coliform   -

Coliform, including E. coli are determined by Most Probable Number (MPN) method, when present in very low numbers.

The plating methods have limitations to detect low numbers of bacteria in water or food, because the inoculums size is only one ml./ Petri dish. Pathogenic or indicator bacteria may not be present in sufficient large numbers in water or food to be detected by planting methods. For example, in the case of potable water permitted level of Coliform is less than one per 100 ml. In such cases, MPN methods are used, where larger volumes of sample can be used for inoculation. MPN is only a statistical approximation of the test bacteria in the given sample and not the actual number. MPN is defined as “that bacterial density which, if it had been actually present in the sample would, more frequently than any other, have given the observed analytical results” (Hoskns & Butterfield). Usually an MPN method is used to detect Coliform bacteria in water or food provided the expected number of these bacteria in the food product is less than 10³ per gram. Otherwise the result may not be meaningful. MPN method for Coliform is a three step process and all the media used are liquid media.

Step I: (for presumptive total Coliform)

Requirements

1.      Double strength Macconkey broth (DSMC broth)  -50 ml -1 flask

2.      Double strength Macconkey broth (DSMC broth)  -10ml   -5 Tubes

3.      single strength Macconkey broth (SSMC broth)  -10 ml -10 Tubes

4.      10 ml and 1 ml pipettes

5.      Serological water bath at 37⁰C and 44.5⁰C.

6.      Incubator at 37⁰C.

Arrange the DS and SS Macconkey broths in a test tube rack. 50 ml of the given water sample is inoculated into 50 ml. DSMC broth, 10ml each of the water sample is inoculate into 5 tubes DSMC broth, 1 ml each into 5 tubes of SS MC broth. Make a decimal dilution of the water sample by adding 1 ml of the water to 9 ml of normal saline and add 1 ml each of the first dilution (equivalent 0.1 ml of the original sample) to the next five tubes of SS MC broth. Labels appropriately, incubate in the serological water bath at 37⁰ C for 24 hrs.

After 24 hrs, observe the tubes for growth and gas production. If the colour of the MC broth has turned towards yellow with gas production in Durham’s tubes, the reaction is noted as positive. Note the result as numbers of positive in each set of 50ml, 10ml,1 ml and 0.1ml tubes. Compare the result with standard MPN table (for 5 tubes MPN) and note the MPN values for presumptive total Coliform.

Step- II (for confirmed total Coliform)

Inoculate one loopful of culture from the positive tubes of step-I, to BGLB 2% broth and mark with the corresponding label. Incubate at 37⁰ C in a serological water bath for 24 hrs. note growth and gas production. Results are noted as positives if there are growth and gas production. Compare with 5 tube MPN table and obtain the result as MPN conformed total Coliforms.

Step- III (for faecal Coliform and E-coli)

From the positive tubes of step-II, inoculate one loopful each to EC broth and Tryptone broth (indole medium). Label appropriately. Incubate at 44.5^{0 +}  0.5⁰ C for 24 hrs.

EC broth:   Note growth and gas production. Note the number of positives in each set. Compare with 5 tube MPN tables. The value obtained gives MPN faecal Coliform.

Tryptone broth: Test for indole production by adding 4 drops of Kovac’s indole reagent. A pink or red colour at the top layer indicates a positive test for Indole. Note the number of positives in each set. Compare with 5 tubes MPN table. Coliform bacteria which produce gas in EC broth and indole in Tryptone broth at 44.5 + 0.5⁰  C are considered as E.coli.

Detection and confirmation of pathogens in seafood

Salmonella

The genus Salmonella belongs to the family Entrobacteriaceae.  The bacterium is Gram negative.  It is motile due to the presence of Peritrichous flagella (with a few exceptions).  At present more than 2000 serotypes of Salmonella are known to exist.

From the epidemiological point of view, Salmonella can be classified into three main groups.  The first group is the Typhoid group comprising of Salmonella typhi and S. paratyphi A and C.  They infect only man and are spread either directly or indirectly (via food and water) from person to person.  The second group is mainly a veterinary group which includes serovars that are host adapted for particular species of vertebrates, e.g., S. gallinarum in poultry, S. dublin in cattle, S. choleraesuis in swine. Some of these are also pathogenic to man (especially S. dublin, S. choleraesuis). The third group is the food poisoning group (non-typhoid group) which contains the majority of other salmonella serovars with known particular host preference. They infect both man and animal. This third group includes the principal agents of salmonellosis i.e. gastroenteritis due to salmonella. Fish and fish products are only occasionally associated with salmonellosis although fish meal for animal feed often contain salmonella as a result of contamination from rodent and birds. Filter feeding shellfish harvested from polluted waters and frozen pre-cooked prawns have been identified as higher risk products.

Detection and identification of salmonella

Pre- enrichment

Macerate 25g of the fish/prawn sample with 225ml of pre-enrichment medium (lactose broth/buffered peptone water /nutrient broth). Incubate at 36 + 1⁰ C for 18-24 hrs.

Selective enrichment  

Pipette 1ml of culture from the Pre-enrichment medium (above) to 10ml of selective enrichment medium (Selenite Cysteine broth and tetrathionate broth). Incubate at 36 +1⁰C for 18-24 hrs.   

Selective Plating

Streak one loop full from the selective enrichment medium (above) on to pre-dried selective plating medium, Viz. (I) Brilliant green agar (BGA), (II) Bismuth Sulphite agar (BSA), (III) Hekton’s enteric Agar ( HEA), (IV) Xylose lysine Deoxycholate Agar (XLD). Incubate at 36 + 1⁰C for 24 hrs.

Examine the plats for typical salmonella colonies

Salmonella colonies will appear as follows on each of the selective agar

BGA:  (smooth, low, convex, moist pink colonies, surrounding medium bright red)

All salmonella spp. except S. typhi are recovered on BGA. The medium contain lactose and sucrose as sugar and phenol red as acid base indicator brilliant green suppresses gram + ve bacteria. Salmonella (and other bacteria) which do not ferment lactose and sucrose form red pink white, opaque colonies surrounded by brilliant red zones lactose / sucrose fermenting organisms like E-coli / Klebsilla / Enterobacter group may form yellow per greenish  yellow colonies with intense yellow green zones (usually, they are inhabited  by brilliant green).

BSA:-

Brown, gray to black colonies with metallic sheen, surrounding medium brown to black. This medium contains glucose as the fermenting sugar. Brilliant green and Bismuth sulphite suppress the growth of Gram positive organisms and Coliform, while permitting growth of salmonella, including S. typhi.  The metallic ions (Bi⁺⁺ and Fe⁺⁺) present in the medium stains the salmonella colonies and surrounding medium black or brown in presence of H₂S. Salmonella forms brown /black colonies with metallic sheen and surrounding medium turns brown/black. Coliform and other members of Entrobacteriaceae are inhibited on BSA, but occasionally, dull green or brown colonies without metallic sheen may be formed.     

 HEA:-

Colonies are blue-green with black centre. This medium contains the sugars, lactose, sucrose and salicin. Acid fuchsine and bromothymol blue act as acid /base indicators and ferric salt as H₂S indicators. Salmonella do not ferment these three sugars. Hence salmonella colonies on HEA are blue-green with or without black centre (H₂S production). Shigella appears as green moist raised colonies. Coliform and other lactose /sucrose /salicin fermenters produce salmon –pink or orange colonies.

Caution:-Proteus may produce Salmonella-like colonies.

XLD:-

Colonies Red (pink) with or without black centers. Xylose, lactose, sucrose and lysine are the critical ingredients of XLD. Phenol red is the acid-base indicator and ferric ammonium citrate, the H₂S indicator. Salmonella do not ferment sucrose and lactose, but rapidly utilize Xylose, changing the pH of medium to acidic range, but the active decarboxylation of lysine by salmonella produces the base cadavenine, which neutralizes the acidic pH and changes the reaction to alkaline.

Hence Salmonella colonies will appear red (pink) with or without black centre (due to H₂S production by certain salmonella spp.) Other members of the Entrobacteriaceae (like E.coi) ferment sugars and produce yellow opaque colonies. 

Caution: - Shigella form red colonies on XLD.

Inoculation to Triple sugar Iron (TSI) agar, Lysine Iron Agar (LIA) and Urea agar:-

Select 2 or 3 typical (or suspected) colonies form each selective agar. Lightly touch the center of the colony to be picked with sterile needle. Inoculate TSI agar by streaking the slant and stabbing the butt. Without flaming the needle, inoculate LIA by stabbing the butt twice and then streaking the slant. Incubate at 36 + 1⁰C, TSI for 24 hrs and LIA for 48 hrs. Inoculate urea agar slants by streaking and incubate at 36 +1⁰C, for 24 hrs. Observe the reaction. Salmonella gives the following reactions.

TSI – Alkaline (red) slant acid (yellow) butt with or without blackening (due to H­₂S), sometimes breaking of the agar (gas production).

The TSI agar contains 3 sugars- Glucose (0.1%) sucrose and lactose (1% each), with phenol red (acid base indicator) and ferric citrate (H₂S indicator). Two types of sugar reaction take place- aerobic in the slant (slope) and anaerobic in the butt. Salmonella readily ferments glucose, but lactose and sucrose are not fermented. In the slant, salmonella gives either alkaline reaction (red) or no change, even though it is glucose fermentative. This is because, in the thin area of slope, the amount of glucose is very low and the acid produced in the beginning gets neutralized by the alkaline products of amino acid metabolism. But in the butt, there is a higher volume of medium and hence higher quantity of glucose. Hence the acid produced does not get neutralized soon and the butt is acidic (yellow).  All the salmonella give alkaline slant and acid butt. Most of the salmonella strain (except S. typhi) produces gas indicated by break in medium. H₂S produced by most of the Salmonella (except S. typhi and S. paratyphi) indicated by black. Some time black color masks the acid production in the butt.

LIA – Alkaline (purple) butt and slant.

A distinct yellow (acid) in butt indicate negative reaction. Other reaction should be considered suspected positives. A black precipitate indicates H₂S production fermentation of glucose, decarboxylation of lysine and H₂S production are detected in LIA. Bromocresol purple (BCP) is the acid base indicator and ferric citrate is included for testing H₂S production. Possible reactions in the media are acid production indicated by yellow colour, alkaline reaction indicated by purple colour, no reaction indicated by red colour and H₂S production indicated by black colour. Salmonella ferments glucose and rapidly decarboxylates lysine. So, gives alkaline reaction (purple colour) in both slant and butt (acid from glucose in totally neutralized by bases from lysine decarboxylation).

Urea agar:- No colour change

Positive growth with no change of colour indicates a urea’s negative reaction. Growth with pink colour indicates a urease positive reaction. The phenol red indicator in the media turns red in alkaline pH due to the production of ammonia by hydrolysis of urea by bacteria possessing urease enzyme. Salmonella do not possess urease and hence gives a negative reaction.         

Retain all culture giving typical reactions in TSI, LIA and Urea agar.

For salmonella by biochemical test

Purification

a.      Before biochemical test, purify the culture by streaking on Macconkey agar, by streak dilution method. Typical salmonella colonies will be transparent and colorless. (Occasionally with dark center). Pick the typical colonies to NA slants and incubate at 36 + 1⁰ C.

Biochemical tests

Inoculate the following media for biochemical tests.

1.      Lysine decarboxylase broth

2.      Malonate broth

3.      Indole medium (Tryptone broth)

4.      Glucose broth

5.      Lactose broth

6.      Sucrose broth

7.      Dulcitol broth

8.      Salicin broth

9.      MRVP medium (2 tubes; one each for MR and VP tests)

10.  Simmons’s citrate agar.

Also test for Grams reaction and motility.

Table2. Reaction of salmonella

1.      Gram’s stain                       Gram negative; short rods

2.      Motility                               Motile

3.      TSI                                       Alkaline slant, Acid Butt, H₂S positive, Gas Positive

4.      LIA                                       Alkaline slant, alkaline Butt, H₂S positive

5.      Urease                                Negative

6.      Indole                                  Negative

7.      Glucose                               Acid and Gas

8.      Lactose                               Negative

9.      Sucrose                               Negative

10.  Dulcitol                               Acid and Gas

11.  Salicin                                 Negative

12.  MR Test                              positive

13.  VP test                                Negative

14.  Lysine decarboxylase         Positive

15.  Malonate utilization           Negative

16.  Citrate                                Positive

Serological confirmation

All culture giving typical biochemical reactions are confirmed by agglutination test with salmonella polyvalent somatic (0) antiserum.

Mark two section about 1x2 cm each on a glass slide. Emulsify a loopful of the isolated culture with 2ml of 0.85% saline in a test tube. Add one drop of culture suspension to both the marked section on the slide. Add one drop of saline solution to one section only. Add one drop of the salmonella polyvalent O antiserum to the other section only. Using a loop mix culture suspension with saline solution in one section and with the antiserum on the other section. Tilt mixture in back and forth motion for 1 min and observe against dark background in good illumination. Consider any degree of agglutination i.e., clumping together of the bacterial cell as a positive reaction.

Positive: Agglutination in test mixture; no agglutination in saline control

Negative: No agglutination in test mixture; no agglutination in saline control.

Nonspecific: Agglutination in test and in control mixture.

Vibrio cholera

V. cholera comes under the genus Vibrio belonging to the family Vibrionaceae. V. cholera is divided into 2 biotypes classical and El Tor based on certain biochemical properties. Each biotype of V. cholera is further classified into 2 sub-group based on the somatic (0) antigenic profile as 01 and non 01 based on agglutination with specific 0 antisera. V.cholera “O” group has three known serotype each possessing distinct antigenic factor. They are Ogava, Inaba and Hikojima with O antigenic factors AB, AC and ABC respectively.

Detection and identification

1.      Enrichment

25 gm of sample is blended with 225 ml alkaline peptone water (APW); transfer aseptically to a sterile 500 ml conical flask and incubate at 36± 1⁰ C.

2.      Streak on Thiosulphate Citrate Bile salt Sucrose agar (TCBS agar)

After 6-8 hours and 16-24 hours of incubation (do not shake the flask) streak loopful from the surface growth (Pellicle) on to pre-set TCBS agar. Incubate the TCBS plates at 36 + 1⁰ C for 18-24 hrs. Examine the plates for typical V. cholera colonies.

Typical V. cholera colonies are large (2-3 mm dia), smooth, yellow slightly flattened with opaque centers and translucent peripheries (Vibrio spp. Do not form tiny, creamy yellow colonies on TCBS).

3.      Pick typical colonies (2-3) to Nutrient Agar Slants (NA) and incubate 36 + 1⁰ C for 24 hrs.

4.      Triple Sugar Iron (TSI) and Kligler Iron Agar (KIA)

Inoculate into TSI and KIA by stabbing butt and streaking slant. Incubate at 36 + 1⁰ C for 18-24 hrs.

Observe for typical reaction of V. cholera.

TSI - Acidic Slant (yellow) and Acidic butt (yellow); No blackening

KIA – Alkaline Slant (red) and acidic butt (yellow); No blackening

On TSI, V. cholerae ferments both glucose and sucrose, but not lactose and it do not produce gas and H₂S. Hence both slant and butt will turn yellow due to acid production from glucose and sucrose.

KIA contains glucose (0.1%0 and lactose (1%) with phenol red indicator. The level of glucose in the thin slant area is so low that the acid produced by V. cholerae by fermentation will be neutralized by the bases produced from peptones during bacterial growth. So, slant will be red (alkaline). But in the butt, the acid produced will be in sufficiently large quantity and will not be completely neutralized by bases at the end of 24hrs. So, the butt will be yellow (acidic).

Cultures giving typical reactions are confirmed as V. cholera by biochemical tests.

Biochemical tests

Salt tolerance

            Inoculate into T₁N₀ and T₁N₃ broths and incubate at 36 +1⁰ C for 18-24 hrs. V. cholerae will grow in T₁ N₀ and T₁N₃ broths. (T₁N₀ contains zero salt and T₁N₃ contains 3%salt)

(Note:  Some V. cholera Non 01 will grow only at T₁N₃)

H and L glucose O/F test

            Incubate by stabbing with a long needle, into H&L glucose O/F medium and incubate at 36 +⁰ C for 18-24 hrs. A yellow colour throughout, indicates a fermentative reaction typical of Vibrio spp. Vibrios do not produce gas.

Cytochrome oxidase test

            Vibrio cholera gives a positive test for cytochrome oxidase.

  Gram reaction and motility:- V. cholera is gram negative short or curved rods and actively motile.

Fermentation of carbohydrates

Inoculate the following sugar media and incubate at 36+1⁰C for 48 hrs.

(i) Glucose, (ii) Sucrose (iii) Arabinose (iv) Mannose (v) Mannitol (vi) Inositol

Examine for the production of acid and gas from these medium.

Decarboxylase tests

Inoculate the following three amino acid media, add sterile liquid paraffin (approximately 1cm height) and incubate at 36± 1⁰ C for 4 days. Examine for colour change.

A change of colour to purple (alkaline) indicates a positive test for decarboxylation. A yellow colour (acid) indicates negative test.

(i)                 Lysine decarboxylase medium

(ii)               Arginine dihydrolase medium

(iii)             Ornithine decarboxylase medium

The medium contains 0.1% glucose in addition to the test amino acid. In the initial stages glucose is fermented to produce acid (yellow). On further incubations, bases produced by amino acid decarboxylation changes the pH to alkaline, producing purple colour. So if yellow colour is obtained, continue incubation for 4 days for a conclusion.

Sensitivity to vibriostatic agent O/129.

Vibrios are sensitive to the vibriostatic agent 0/129 (2,4-Diamino-6,7- diisopropyl  pteridine  phosphate). Sensitivity of Vibrio cholerae to two levels of O/129, viz. 10 microgram and 150 microgram, are tested. Vibrio cholerae is sensitive to both the levels of O/129.

            A heavy inoculums of the test culture is smeared uniformly on the surface of pre-set Nutrient Agar (NA) plate. One disk each containing 10 Mg and 150 Mg. O/129 compound is placed in each half of the seeded plate and incubated at 36± 1⁰C for 24hrs. A zone of clearance around the disk indicates sensitivity to that level of O/129.

Serological confirmation

            The culture indentified as V.cholerae by cultural and biochemical characteristics as above are confirmed as V.Cholerae by agglutination test using polyvalent V.cholerae, O.antiserum.