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Procedure for Chemical Analysis of Fish Meal

Preparation of Samples:
Samples for the following analysis should be mixed properly and ground using a
centrifugal mill with a mesh size of 1mm.Ground sample should be mixed thoroughly
and kept in a bottle with lid tightly closed the bottle with date and other necessary details.
Moisture Analysis
Determining the moisture and dry matter content of fish meal.
Apparatus required.
Analytical or Electronic balance.
Aluminum dish
Drying cabinet
1. Weight of the Aluminum dish is taken after drying and dessicating (W1).
2. Take 5 gms (W) of the ground sample in the weighed dish.
3. Place the sample in drying cabinet at 105C for 4hrs or 130C for 90 minutes.
4. Take the sample out from the drying cabinet using tongs after 90 minutes and
place in a dessicator for cooled sample weight (W2).

Moisture in % = M % (W1+W)-W2 X 100
Dry matter in % = 100-M %


Determination of organic nitrogen in the fish meal by Kjeldahl method and multiplied by
6.25 assuming protein contains 16% nitrogen.
Electronic balance
Kjeldahl nitrogen distillation unit.
Pipette 2 ml
Glass beeds
Titration equipment
Acid dispenser
Erlenmeyer flasks 300 ml
Measuring cylinder 50ml
Wash bottle
Conc. Sulfuric acid (Conc.H2SO4)
0.1N Sulfuric acid. ().1H2SO4)
Sodium Hydroxide.30% (NaOH 30%)
Boric acid solution 4% (H3BO3 4%)
Mixed indicator
Hydrogen peroxide or Chloroform 30%
Potassium sulphate (K2SO4)
Copper sulphate (CuSO4.5H2O)
1. 0.5 to 1 gm (W) of the ground sample are weighed in the balance and transferred
to digestion tube containing the glass bead.5 gm of K2SO4 and 0.1 gm of CuSO4
is added to it.2ml of Chloroform or Hydrogen peroxide is added to it using a
piptte.Then add 15 ml concentrated Sulfuric acid from dispenser.
2. Digestion tube is placed in the holder and it is transferred to the pre heated
digester. Heat the solution for one hour till the solution become clear.
3. Put the Digester off and keep the digested sample for cooling for hour. Take 50
50 ml of 4% Boric acid solution in 300 ml Erlenmeyer flask using
Measuring cylinder and add 50 ml of distilled water in it. Add 3 drops of mixed

Indicator into it and mix well by swirling (Receiver Solution).

4. Place the digested sample in the distillation unit and received solution underneath
The distillate outlet. Dilute the digested solution by adding approx.50 ml distilled
water by pressing the button and add 30% NaOH Solution into it till solution
color turns dark brown. Start the distillation unit and continue the distillation till
100 to125 ml distillates id collected in the receiver flask. Stop the distillation and
take out the receiver solution for titration.
5. Titrate the distillate solution with 0.1N H2SO4 till the color of the solution turns
Pink from green. Note the reading of Titrometer (a=ml acid consumed).
Crude Protein % = a x 0.1 x14.01 x100 x 6.25 = a x 0.8756
Extraction of the sample in Petroleum ether and the residue (cr.Fat) is determined
Soxhlet extraction apparatus
Extraction thimbles
Flat bottom flask
Analytical balance
Drying cabinet
Glass Beads
Petroleum Ether-b.p.100-120C
1. Dry the flat bottom flask with glass bead in a drying cabinet and keep in a
Dessicator for for cooling
2. Take the weight (W1) of the cool empty flask with glass bead.
3. Take 5 gm of the ground sample in an extraction thimble. Cover the sample with
fat free cotton.
4. Add petroleum ether in the weighed flask up to two third level (approx:140 ml)

5. Fix the flask and thimble in its respective places on the Soxhlet apparatus. Start
the extraction process and continue for 2 hrs and 18 minutes. After the extraction
is completed, remove the thimbles from the unit. Distillation process to be
continued without the thimble from the unit. Distillation process to be continued
without the thimble till any solvent is left in the flask. Dissemble the extraction
unit and collect the petroleum ether in a bottle for next extraction. Flask is
removed and kept in a drying cabinet (temp.105C) for 1 hr.Take the flask out and
keep in a dessicator for 30 mins for cooling. After cooling, take the weight of the
flask (W2)
Crude fat % = Weight of the fat x100 = W2-W1 x 100
Weight of the sample
Notes on the use of this Test Set.
1. Keep all reagents out of reach of children. Test set should only be used by authorized
users. Do not ingest any reagent, wash hands after use. Always handle chemicals with
2. In general the method is specified on each bottle of test tablets. The one exception is
The measurement is the measurement of cooltreat 700s which is detailed over leaf.
3. It is important that the equipment provided is kept clean and washed out after use.
Ordinary tap water will suffice but the distilled water or demineralized water is to be
preferred. This can be obtained at any chemist or certain garages and car accessory
outlets. If the apparatus cannot be rinsed with demineralized water then it should be
rinsed with the sample prior to analysis to prevent cross contamination.
4. It will be useful to mark the 50,100 and 150ml marks on the test jar with an indelible
felt tip pen or equivalent around the clear part of the jar to assist is accurate
5. A glass stoppered test jar available to special order part no.020 504.
6. Best results will be obtained if tests can be performed in a clean, well lighted area.
this will assist in seeing colour changes.
7. Most of the test are based on the drop count titrations and should be preformed when
you are unlikely to be interrupted.
8. Hold titrant bottles vertically upside down when performing the test, allowing drops.
to form slowly. Do not shake drops off
9. Do not interfere with the aperture of dropper bottles as this will affect the accuracy of
the test.
10. Do not transfer contents of one dropper bottle to another.
11. Rinse the test jar out with sample, discard and refill as per individual test instructions.
12. In the tests requiring 20 or 40ml of sample, fill the test jar so that the meniscus lies on
top of the graduation.

13. Important: During transportation dropper bottles may become statistically charged.
this results in a continuous stream of small uncontrollable drops. Discharge the bottle
by placing a soft cloth or tissue on a bench top or similar. Push the nozzle tip against
the tissue and dispense a small amount of liquid into the tissue. This will discharge the
Bottle and wet the bottle tip.
14. If being used for the first time or after a long period of non use, wet the titrant bottle
Tip as outlined in 7 above.
1. Certain tests require the sample to be filtered either to remove undissolved material
which would dissolve and add the test result (e.g. phosphate and Zinc) or suspended
Matter which would make colour changes to observe.
2. Fir this purpose. Some sets contain a syringe, filter paper holder and filter papers.
The filter papers are made of glass fiber and behave as a depth filter.
3. Fit a filter paper to the holder by unscrewing the two halves of the holder and placing
top of the filter paper and carefully offer up the outlet half to the inlet half
(Female thread).
4. Fill the syringe with sample and fit the filter holder to the outlet male tip. Depress the
syringe plunger slowly to push sample through the filter holder assembly. Initially the
sample may come out cloudy. Continue to pass sample through until the sample comes
out crystal clear before using for test.
Approximate Product Factors For Drop Test Using Cooling P Test Kit.


Cooltreat 103


Cooltreat 104


Cooltreat 113


Cooltreat 114


Cooltreat 123


Cooltreat 124


Cooltreat 313


Cooltreat 314


Cooltreat 323


Cooltreat 405


Cooltreat 415


Cooltreat 514


Cooltreat 518


Cooltreat 524


Cooltreat 528


Cooltreat 572


Cooltreat 603


Cooltreat 613


Cooltreat 614


Polytreat 291


Polytreat 2076


Polytreat 2084


Chemtreat 252


Test Tips

1. In many waters it is convenient to take a 50ml sample in which case;ppm
Chloride = (No. of tablets x 20)-20
2. If a 10 ml sample is to be taken then it is preferable to use the syringe
Concentration Factor.
1. It is important to monitor the concentration factor in cooling systems to
Ensure that the system is operated economically and efficiently.
2. Concentration factor can be measured with chloride tablets.
3. Concentration Factor = ppm chloride in system
ppm chloride in make - up

pH Measurement by Test Strips.

1. Select a test strip in the range.


Immerse strip in sample covering all colour zones.

Leave for 20 minutes.
Remove and compare central test zone with standard zones.
Report pH as the figure on standard zone nearest in colour to central test
Note: If no colour match is obtained, select a strip from a different range
and repeat the test.
Ortho phosphate as PO4 using 30/70 max/min block.



Fill comparator tube to tube to top mark with filtered sample (10 ml)
Add to drops PBI Vanadomolybdate, and 10 drops PB2.
Leave for 2 minutes.
Place tube in comparator and compare colour with the standard provided.
Report as 30, 50 or 70 ppm PO4.
Color reading
Light Yellow
- 20 ppm (low result advice to increase Nalco 72210)
Dark Yellow
- 50 ppm (acceptable)
No Color
- advise to increase the Nalco 72210
Note: For filtration procedure see instructions at the beginning of this


Nitrite as NaNO2 (ppm)

A. 1ml sample in test jar (using syringe)
1. Add 2 drops N1 and mix
2. Orange colour
3. Add drops N2, one at a time, until colour goes blue.
4. Count number of drops N2 used.
5. ppm Nitrite as NANO2 = drops of N2 x 50


With 2 ml sample (using syringe)

ppm Nitrite as NANO2 = drops of N2 x 25
With 5ml sample
ppm Nitrite as NANO2 = drops of N2 x 10
With 10 ml sample
Nitrite ppm as NANO2 =drops of N2 x 5
With 40 ml sample
Nitrite ppm as NANO2 = drops of N2 x 1.25

Determination of Chloride as (ppm)


Fill the test jar to the 20 ml mark

Add 2-3 drops of PA1
Pink colour result, add PA2 until it become colorless
For colorless, add 4-5 drops BC1 or CC1 and mix.
Sample goes yellow

6. Add drops of BC2 or CC2 one at a time, until sample is just orange.
7. Count the drops of BC2 or CC2 added.
8. Chloride ppm as CI
= Drops of BC2 used x20
= Drops of CC2 used x 5
9. Chloride ppm as NaCl = ppm Chloride as CI x 1.6
Note: Different test sets contain different reagents. Use the factor relating
to the reagent present in your test set.


Determination of Total Hardness CaCO3 (ppm)

1. Fill test jar to the 20 ml mark.
2. For Total Hardness, add one TH1 tablet and drop of TH2.Crush the tablet
and mix.
3. For Calcium Hardness (condenser), add one CH1 tablet and drops of CH2.
Crush the tablet and mix.
4. Sample turn blue.
5. Sample turns red/purple.
6. Report as NIL.
7. Add TH3 or TH4 or H, one drop at a time counting the number of drops
required for the sample to go blue.
8. Add CH3 or TH3 or H,one drop at a time counting the number of drops
For the sample to go blue.
9. Hardness = No of drops TH3 used x 1/
Hardness = No of drops TH4 (or CH3 or H) used x10
10. Magnesium Hardness =Total Hardness-Calcium Hardness.

VIII. Determination of Sulphite as Na2 SO4 tablet is dissolved prior to proceeding.

1. Fill test jar to 20ml mark with cooked sample.
2. Add half tablet of S1 and swirl to dissolve.
3. Add drops of S2, one at a time, until blue/grey color is produced.
4. Count number of drops of S2 used.
5. Sulphite ppm =Drops of S2 used x 5
Note: If low result (30-70 ppm)-advise to increase NALCO 780
IX. Determination of Tannin
1. Fill test jar to 20ml mark with filtered sample.

2. Add heaped blade of TN1 and swirl to dissolve.

3. Add drops of TN2, one at a time, until color stays for 30 seconds.
4. Count the drops of TN2 used.
5. Tannin ppm = Drops of TN2 used x15
X. Determination of Phosphonate as product (using combined buffer and Dechlor tablet).
1. Fill test jar 20 ml mark with filtered sample.
2. Add P1-P4 tablet.
3. Crush to dissolve and shake to ensure most of the tablet dissolves.
4. Ensure a clear green color,if not add another tablet (P1-P4).
5. Add P5 reagent one drop at a time until color changes to purple.
6. Count number of drops of P5 added.
7. Repeat procedure on Sample of make up water without product.
8. Product ppm = (drops of P5 sample-drops of P5 make up) x product factor.
Note: appropriate to your site your water treatment representative will
Determine the product factor appropriate to your site.
XI. Determination of Phosphonate as product.
1. Fill test jar 20ml mark with filtered sample.
2. Add 10 drops of P1.
3. Wait 10 seconds,add drops P2 until yellow color is observed.
4. Add drops of P3, one at a time, until yellow color just disappears.
5. Add one tablet P4 and crush to dissolve.
6. Sample turns yellow.
7. Add drops P5, one at a time, until color turns to pink.
8. Count number of drops P5 used.

9. Repeat above procedure on a sample of make up water.

Note: Product ppm = (drops of P5 sample-drops of P5 makeup) x Product
Factor 7.8.
Your water treatment representative will determine the product factor of your
XII. Determination of Organo-Phosphnate.
The principle instructions for this determination are printed on the bottle of
Organo phoshonate No.1 tablets
1. Using the 10 ml syringe, transfer 10 ml of sample to the test jar.
2. Add one organo-Phosphnate No 1 tablet and swirl to dissolve, ensure the
whole Tablet is dissolved prior to proceeding. (In water with an M alkalinity
above 500 mg/l as CaCo3, use 2 organo-phosphonate No.1 tablets).
3. Add organo-phosphonate No-2 solution drop until the color changes from
Green to deep purple. Count the number of drops. Normally 10-15 drops are
Product ppm = No of drops x Product factor
Sample volume
The product factor will vary from site to depend upon the exact characteristics of
Your individual dropper bottle. However an approximate set of factors is
Appended .The factor appropriate to your system will be recommended by
Your water treatment consultant.
NB: For the purpose of this test is important that consistent drop size is
achieved. Test have shown that this is best done by holding the bottle
vertically and slightly inclined to the left to right .The bottle should be squeezed gently to
produce regular drops at a slow rate.
When the reagent bottle is part full and cold, relative to the hand, a stream of drops may
be expelled due to increase in air pressure, this must be avoided by prior warming of the
bottle in the hand and releasing the pressure with the tip uppermost.
The formation of a small air bubble over the tip should also be avoided by wiping with a
clean tissue.
XIII. Determination of Cool treat 700 series.

A sample of treated test cooling water is acidified and a measured excess of

potassium permanganate added. The time taken to discharge the color is a measure of
the concentration of treatment present.

Take 100 ml of test sample at 20-25C.

Add 2 DM No.1 tablets, crush stir to dissolve.
Add 2No.2 tablets, begin timing immediately, and crush to dissolve.
Measure the length of time required for the pink color to be discharged in
CT 725/726 ppm = 600-40t
CT 715 ppm = 300-70t
Where t = time to discharge color in minutes.
Eg:At 20-25 C. and 400ppm cooltreat 725 the pink color is discharged in 4.5-5.0
XIV. Hardness
1. The Hardness LR (BW) tablets can be used to measure:
2. Feed Water Hardness, or Boiler Water Hardness, Or to check the efficiency of a
3. The method is identical in all cases and is specified on the bottle.
4. If a 25 ml sample were used : Hardness mg/L CaCO3 = { (No. of tablets x 8)-4}
XV. Nitrite
1. For most accurate results this test should be carried out promptly after sampling.
XVI. pH Measurement
1. The test strips supplied require a finite reaction time for full color development.
pH 1- 12
- 30 seconds
pH 6.5-10
- 1 10 Minutes
XVII. Sulphite
1. It is important that the sample be cooled to room temperature prior to testing
and that the test be the first carried out .Also that it be carried of sulphite LR
No.2 tablets should be carried out immediately.

SALT ANALYSIS (In Direct Method)

1. Accurately weigh 1.50 g to 2.5g of homogenized sample into 250ml
Erlenmeyer flask. Record the weight.
2. With the use of a pipette, add 10 ml of standard AgNO3 ,then 10ml
Concentrated HNO3.
3. Swirl the solution while boiling until all the samples are dissolved.
4. Add 5 ml distilled water.
5. Add ml ferric alum to the solution.
6. Titrate with standard NH4SCN solution until it shows pale red brown color
After vigorous shaking.
%NaCl = {(NxV) Ag NO3 (NxV)NH4SCN }x 58.45/1000 x 100
Weight of sample
Volume of AgNO3(ml) = 10 ml
Meq weight of NaCl
= MW NaCl/100
Where MW
= Molecular Weight.
Reagent Preparation:
A. AgNO3,0.1 N
Dissolve about 17.5g of AgNO3 in 50ml of distilled water. Filter and add 950 ml
of distilled water in the filtrate to make 1L solution .store in amber bottle.
Standardize as follows: Accurately weigh about 0.0300+/- (0.0005 KCL (moisture
Free) in 250 ml Erlenmeyer. Dissolve in about 40ml of distilled water .Add 0.5 ml
of K2CrO4 as indicator. Titrate with AgNO3 solution until it shows brown red
color after vigorous shaking. Calculate the actual normality using the principle.
[NxV] AgNO3 = g KCL
Analytical Balance
250 ml Erlenmeyer flask
Hot Plate
Base Burette

25 ml Pipette
95% Ethanol
0.1 N KOH (or NAOH) accurately standardized.
Phenolphthalein Indicator
Reagent Preparation
a. Phenolphthalein Indicator
Dissolve 5 gm of Phenolphthalein in 500ml of 95% ethyl alcohol (Ethanol)
Dilute to one liter with distilled water .Add 0.02 N NAOH until faint pink
Color appears.
[1N NaOH-40 gm of NaOH pellets dilute to 1 liter of Dist: water]
0.1 N NaOH 4g of NaOH, dilute to 1 liter distilled water.
0.02 N NaOH take 200 ml from 0.1N NaOH and dilute to 1000 ml distilled water.
a. To about 50 ml conc:Ethyl alcohol in clean dry 250ml Erlenmeyer flask, add 5 drops
Of oil and 2ml of Phenolphthalein Indicator.
b. Place the sample in a water bath of 60-65C temperature until the sample is warm.
c. Add 3 drops of 0.1N NaOH to permanent pink color.
d. Weigh 56.4 g of oil into the neutralized alcohol and shake.
e. Titrate with 0.1N NaOH while shaking mixture until same faint permanent pink color
Appears in supernate alcohol.
f. Calculate % free fatty acid using the following formula:
%FFA = {Vol of 0.1N NaOH used} x {0.05}
Note: 475 ml ethanol
95% ethanol
25 ml distilled water 95 ml ethanol
500 ml ethanol
5ml distilled water
100 ml
(Spectrophotometeric method)
The objective of this procedure is to determine the histamine content of fishery
Products and other food by means of simple Spectrophotometeric method.
Is a simple method based on the formation of an azo-compound of histamine,
Using a p-notroaniline and their later extraction with an ester (ethyl acetate).The

Azo-compound of histamine is transferred to the ester separating the other

Possible azo-compound form like to the histamine.

5% Trichloroacetic Acid
Weigh 5 grams of Trichloroacetic acid with accuracy of 0.01 g and dissolved
in 75ml of distilled water. Transfer it to 100ml appraised flask and evened it
with distilled water up to the mark.


0.2 M Hydrochloric acid

In an upraised flask of one liter it spill 750ml of hydrochloric acid of 37%
Finally it is evened to a liter with distilled water.


Stock solution of 100ppm of Histamine

In a glass weigh 100mg of histamine and let it dissolve with 0.2M
Hydrochloric acid. Transfer the solution in one liter volumetric flask and
add 0.2M hydrochloric acid up to the mark.


2% Sodium Carbonate
In a glass weigh 2 grams of sodium carbonate granules and dissolve with
75 ml distilled water. Transfer the solution to a 100ml volumetric flask and
Add distilled water up to the mark.


5% Sodium Nitrite
In a glass weigh 5 g of sodium nitrite and dissolve with 75ml distilled water.
Transfer the solution to 100 ml volumetric Flask and add distilled water up
to the mark.


0.1N Hydrochloric Acid

Pour 750ml of distilled water in 100ml volumetric flask. Add 8.69ml of
37% hydrochloric acid and add distilled water up to mark.


0.1% P- Nitroaniline
In a glass weigh 100mg of P- Nitroaniline and dissolve with 0.1N
Hydrochloric acid. Transfer the solution in 100ml volumetric flask and add
0.1N Hydrochloric acid up to the mark.



Pipette 1 ml of 5% Sodium Nitrite and add 5ml of 0.1% P-Nitroaniline.

Note: Use immediately do not store.

Ethyl Acetate




Preparation of sample
In a glass weigh 20 grams of previously crushed and blended sample. Add
10 ml of distilled water and 10 ml of 5% trichoroacetic acid.Swril to mix
and filter. Store the extract for histamine determination.
Preparation of pattern.
In 50ppm, 20ppm, 10ppm and 5ppm.pipette 50ml, 25ml, 10ml, 5ml and
2.5ml stock histamine solution into separate 100 ml volumetric flasks and
dilute each to volume with 0.2N Hydrochloric acid.
Histamine Determination.
In a clean and dry test tube, pour 1 ml of distilled water, add 1ml of extract
from the sample and add 1 ml of each of the pattern. Then add 2ml of 2%
diazo-reagent to each tube. Swirl and mix and allow standing for 5 minutes.
After 5 minutes, add 7ml of ethyl acetate. Swirl to mix for 30seconds and
Allow to rest for 5 minutes. Read the absorbance of the layer of the ester
Moisture in a Spectrophotometer using excitation wavelength of 540nm.

With the absorbance values obtained, make a calibration line in which we
Will be able to read the concentration of histamine in ppm of the extract in
Function of their absorbence.The obtained reading multiplies for 20 and
divided by exact weight of the sample in grams.
Mg of Histamine / kg of sample = extract x 20per p.
Where p = Quantity of used sample expressed in grams
(Oven Drying Method)
This method involves the measurement of the weight of the sample after the
Evaporation of water by use of an oven. Although such methods are frequently used
as they give usually give accurate results when considered on a comparative basis, it
should be borne in mind that the figure obtained may not be a true measure of the
water content of the sample.

For example, volatile oil present is also at drying temperature, such as 1000C.
On the other hand, with some foods (e.g. cereals) is lost at the drying
Temperature. The remainder (often referred to as the bound water as it is difficult to
remove) appears to be associated with the proteins present. The proportion of free water
lost increases as the temperature is raised. So it is especially important only to compare
the results obtained using the same condition of drying. Further, if decomposition is
likely to occur, as with food which contains applicable proportion of sugars, it is
advisable to use lower drying temperature in a vacuum dessicator, for a considerable
period of heat causes appreciable losses of carbon dioxide. The loss in weight may vary
according to various other factors, including particle size and weight of sample, type of
a. Dessicator
b. Aluminum pan (3 inches diameter flat)
c. Drying oven- Temperature is adjusted to 100C - 105C
Sample size- 2 5g
a. Weigh accurately approximately 2-5 grams samples into a dried aluminum
b. Place the aluminum pan and its contents in an oven controlled at 100C 105C.
c. Heat for a stipulated time, or until successive show no further loss.
d. Remove the aluminum pan from the oven, and place it in a dessicator, allow
to cool and weigh.
% Moisture = Loss in weight of the sample
Weight of the original sample
A. Applications
1. Empty cans
2. Lids
B. Glasswares and materials

1. 500 ml beaker
2. 500 ml reagent bottle
3. Electronic weighing scale
C. Reagents
1. Copper Sulfate granule
2. Concentrated Hydrochloric Acid
3. Distilled water
D. Preparation of Copper Sulfate solution

Weigh 70 grams of Copper Sulfate granules (CuSO4.5H2O)

Dissolve the weighed copper sulfate in 245 ml distilled water.
Add slowly 30 ml concentrated Hydrochloric Acid
Prepare the copper sulfate solution once a month.

E. Copper Sulfate Test Procedure

1. Place the prepared copper sulfate solution into the empty cans and make
up to flange of the tins. The solution could also be placed on the inner
portion of then lids to detect for any scratches and sulfide staining.
2. Allow the solution to react with the surface for 15 minutes.
3. Check the surface of the tins and lids for black appearance or sulfide
4. Any tins or lids detected to have scratches should be subjected for
250 ml Erlenmeyer Flask
Hot plate
Ice water bath
Screwed test tube
1. Heat sample while stirring to 130C.
B. Fill the oil sample bottle completely full with sample and insert a cork tightly.
C. Adjust to 25C in a water bath.

D. Immerse the bottle containing the sample in the ice water bath so that the entire
bottle is covered with water and ice.
E. Store the container in the refrigerator.
F. After 5 hours, remove the bottle from the bath and examine closely for at
crystal or cloudiness.
G. Do not identify small and finely dispersed air bubbles for fat crystals.
H. To pass the test, the sample must clear and brilliant.

Histamine Testing
A. Histamine Extraction
A1. Weigh 2 grams of homogenized sample and transfer it into the extraction
A2. Vigorously shake the vial for about one minute.
A3. When finished, set the extraction vial down at room temperature for 10
A4. After the waiting period, repeat steps 1-3 above.
A5. At the end of the second 10-minute waiting time, gently pour the liquid
portion into the filter bag.
A6. Set the filter bag down. The sample liquid is now ready to be analyzed.
B. Histaquant Testing: part 1
B1. Prepare the number of wells that you will use.
B2. Mark the side or the tab of the first well. Securely place the test well strip into
the Well Holder.
B3. Collect 50l of sample on the other side of filter bag and transfer it into the
test well. Change the pipette tip after each transfer.
B4. After the last sample liquid has been transferred, carefully add 100l of
Histamine-ALP conjugate into each sample wells. You may use one pipette tip as long
as it does not get in contact with the liquid inside the wells.
B5. When finished, incubate the wells at room temperature for 30 minutes.
C. Histaquant Testing: part 2
C1. After the 30 minute incubation, pour off the contents of the testing wells by
turning the wells upside down. Make sure the testing wells are completely empty.
C2. When finished, fill each testing wells with the Wash Buffer Solution, and then
pour it off the way you did in Step #1.
C3. Repeat this wash step 3 times.
C4. Upon completion of the 3rd wash, remove as much excess liquid from the test
wells by aggressively tapping the wells upside down onto an absorbent material.
Make sure that all the wells are completely empty/no bubbles.
C5. Transfer into each testing well, 100ul of Substrate Solution then incubate for
15 minutes.
C6. When the incubation is finished, transfer 50ul of Stop Solution into each
testing well. Gently tap the well holder to mix the contents, incubate for 2
C7. Read the test wells using a Statfax.
D. Statfax Reading: Part 1
D1. Turn on the Statfax.

D2. As soon as the display says Ready, press the Menu button
D3. Press #2 to select the Histamine Test, and then press Enter.
D4. When display says Plot curve? Press No.
D5. When display says Stored curve? Press Yes.
D6. The display will now says Set carrier. You can now place the testing wells to
the testing well carrier.
D7. When done, press Enter to begin reading the testing wells.
D8. If reading another strip of testing wells, adjust the well carrier so that the strip
being read now aligns with the notch on the entrance to the machine.
D9. After reading the last set of testing wells, press Clear twice to end the session.
D10. Turn the machine off.