sample processing

SAMPLE PROCESSINGDr. Yuting Deng

Pearl River Fisheries Research Institute (PRFRI), Chinese Academy of Fishery Sciences (CAFS), China

[email protected] 2021.07.29

Processing of sample from aquatic environment 5.1.2

OUTLINE

Processing of tissue sample from aquatic animals5.1.1

Processing of tissue sample from aquatic animals

5.1.1

In AMR survellance, we should obtain representative farm isolates

to determine the prevalence of resistance in particular

bacteria–antibiotic combinations.

Once the samples have been brought to the laboratory ,

they are processed to isolate and identify target bacteria.

0102

In the cases of diseased fish or shrimps, direct inoculation of infected

organ sample is viable for isolation of pathogenic bacteria.

Typical symptoms of motile Aeromonas septicemia (MAS) caussed by A. hydrophila.

---Zhang D et al., Aquaculture Reports, 2016https://www.grobest.com/my/news/detail/1159

PROCEDURE

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1. Euthanization

1.1 Purpose of euthanization

Euthanasia is a common requirement in aquaculture and fisheries

research for the lethal collection of tissues and blood.

If a fish has to be killed, then death must occur with the least

possible anxiety, pain and distress.

1.2 Three methods:

Ø Chemical methods

e.g., tricaine methane sulfonate (MS-222)

Ø Physical mehthods

e.g., decapitation, pithing, thermal shock

Ø A combination of both

https://share.usask.ca/go/ethics/SOPs/Guideline/SOP%20E201(AquaticsEuthanasia).pdf

1.2.1 Chemical methods

The most humane method is by administering an euthanasia agent,

e.g. MS-222.

, is the only anesthetic approved for use on fish that could be

used as human food.

https://www.fau.edu/research-admin/comparative-medicine/files/guidelines-for-the-preparation-and-use-of-ms222-final.pdf

Tank immersion method:

Place the fish in a buffered solution

of MS-222,

at a concentration of 250 mg/L,

for at least 10 minutes.

Ø In any case, death needs to be assured before

discarding the animal best by following with a

secondary method such as pithing.

-- photo by Deng YT

-- photo by Deng YT

1.2.2 Physical methods

Decapitation

Use a sharp scissors or knife to

separate the head from the body

rapidly and completely.

-- photo by Deng YT

Pithing

Distroy the proxima spinal cord

by a sharp scissors.

-- photo by Deng YT

Thermal shock（Rapid chilling)

Ø the process of chilling is commonly

considered to be effective, as

crustaceans subjected to chilling do

not show the behavioural signs of

stress that occur when some other

killing methods are used.

https://kb.rspca.org.au/wp-content/uploads/2019/01/Humane-killing-of-crustaceans-for-human-consumption-%E2%80%93-RSPCA-Information-Paper-May-2018.pdf

-- photo by Deng YT

2. Disinfection / Sterilization

Disinfection and sterilization are necessary in order to

prevent microorganisms surviving in cleanrooms (and for

maintaining a level of hygiene in labs).

2.1 Purpose of disinfection / sterilization

Three different processes (cleaning, disinfection, and sterilization)

are commonly referred to as “disinfection”.

There are different levels of decontamination.

Ø Cleaning simply removes a proportion of organisms present.

Ø Disinfection removes most pathogenic organisms.

Ø Sterilization is the killing or removal of all organisms.

2.2 Definition of disinfection and sterilization

When we perform an experiment, we need to disinfect when

there is existing or possible contamination from pathogenic or

harmful microorganisms.

2.3 Methods of disinfection and sterilization

Ethyl alcohol (70~75%) is a

powerful broad-spectrum

germicide, which is often used to

disinfect small surfaces and

occasionally external surfaces of

equipment .

Ø Disinfectant spray

-- photo by Deng YT

-- photo by Deng YT

Simply place instruments in gently boiling water for 30 minutes.

But it may not eliminate some bacterial “spores” and could

cause issues with rusting over time, especially on sharp

instruments like scissors or knives.

Ø Boiling

Soaking in disinfectant solution for 15-30 minutes will disinfect

instruments but no longer or rusting will occur.

Instruments must be rinsed in sterilized water afterward.

Ø Soaking

The best way to guarantee sterility in an austere setting is autoclave,

which uses steam to clean instruments, surgical towels, bandages, and

other items.

The minimum exposure period for

sterilization requires 15-30 minutes at 121℃ 。

Ø Steam sterilization

Ø Filtration

Filtration is used for media particularly heat labile in nature (e.g.

sera, antimicrobial solution).

üIf the study warrants bacteria free filtrates it can be obtained

through 0.45 micron sized filter membranes.

üIf the study requires viral particle free solution, then 0.22

micron sized filter membranes are use.

Dissect the aquatic animals to collect tissues that can be used

to isolate the bacteria. Tissues will vary between and within

species (e.g. size).

Determine the number of animals to be collected, and how

much tissue is required from each animal for the analyses to

be conducted.

3. Dissection

3.1.1 Anatomy of fish

https://www.visitflorida.com/en-us/things-to-do/florida-fishing/internal-and-external-anatomy-of-a-fish.html

3.1 Dissection of fish

https://www.visitflorida.com/en-us/things-to-do/florida-fishing/internal-and-external-anatomy-of-a-fish.html

3.1.2 Dissecting procedure

（1）Lightly pat the fish dry on a paper towel and place it on a dissecting pan.

-- photo by Deng YT

（2）Lay fish flat on one side with the dorsal fin facing away from you, and

disinfect the surface of fish .

-- photo by Deng YT

（3）Make a small cut just in front of the anus by a sharp sterile scissors.

anus

-- photo by Deng YT

-- photo by Deng YT

（4）Make a vertical cut from the anal region up to the lateral line.

Remember not to cut deeply,

otherwise, you will damage the

internal organs.

-- photo by Deng YT

（5）Cut across below the lateral line, to the gills.

-- photo by Deng YT

（6）Behind the operculum, cut the body wall from dorsal to ventral.

-- photo by Deng YT

（7）Carefully expose the internal organs.

liversp leen

in testine

kidney

-- photo by Deng YT

3.2 Dissection of shrimp

Shrimp external anatomy

comprises a rostrum, carapace

(cephalothorax), abdomen

segments, telson, pleopods,

pereiopods (legs), maxilliped,

and antennule. https://www.researchgate.net/figure/Anatomy-of-the-shrimp-The-midline-of-the-third-abdominal-segment-is-where-the-body_fig3_329029213

3.2.1 Anatomy of shrimp

The cephalothorax contains the

internal organs including the

hepatopancreas, the heart, and

the gonads.--Duarte-Restrepo E et al., PLOS ONE, 2020

3.2.2 Dissecting procedure（1）Rip off the shell of the head by a sterile forcep.

-- photo by Deng YT-- photo by Deng YT

（2）Then expose the internal organs.

hepatopanceas

-- photo by Deng YT

4. Inoculation

Ø In the cases of diseased aquatic animals, direct inoculation of

infected organ sample is viable for isolation of pathogenic bacteria.

Ø Make use of streak plating technique for the isolation of pathogenic

bacteria. It is a useful method to separate bacteria from a mixed

population.

P rocedure o f inocu lation（1）Flame the inoculating loop until it is red hot.

-- photo by Deng YT

（2）Insert the loop into the infected organ.

Twist the loop to collect some sample.

-- photo by Deng YT -- photo by Deng YT

（3）Immediately streak the loop very gently over a

quarter of the plate using a back and forth motion.


（4）Flame the loop again and

allow it to cool.

Going back to the edge of area

that you just streaked.

Extend the streaks into the

second quarter of the plate.

Repeat the procedure, streak the

remaining area.-- photo by Deng YT

The purpose of this procedure is to

obtain well isolated colonies from

a specimen by creating areas of

increasing dilution on a single

plate.

Streak plating technique

https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/

（5）Label the bottom of the plate with the sample name and the inoculated date.


（6）Placed the plates in inverted position, and Incubate at

28℃ for 18 to 24 hours.

The most appropriate incubation temperatures and

time differ by organism.

-- photo by Deng YT

Carefully dispose of the dead animals after sample poccessing.

Burial might be a probable method.

You'll need to check your local regulations for the requirements for burial.

They will tell you how deep you need to bury the dead animal (usually at

least 3 or 4 feet) and other requirements.

5. Disposal

In the cases of healthy fish and shrimps, some opportunistic pathogen

involving Aeromonas spp. and Vibrio spp. are mainly distributed in the

intestines of fish or the hepatopancreas of the shrimps.

Therefore, homogenate is another sample pocessing method for

bacterial isolation.

PROCEDURE

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Sample processing depends on the size of sampled animals.

• Individuals > 10 g in weight, only collect the intestines samples

• Individuals < 10 g in weight, collect trunks samples

Ø FOR FISH

Ø FOR SHRIMP

• collect hepatopanceas samples

Homogenate method for sample processing

(1) Individuals > 10 g in weight of fish

1. Sample collection

Taking care to avoid cross contamination.

Separate the intestines of several fish from other internal organs.


Collect the intestine sample for no less than 5 g.

-- photo by Deng YT

(2) Individuals < 10 g in weight of fish

Cut off the head and tail of several fish.


Collect the fish bodies (trunk) for no less than 5 g.

-- photo by Deng YT

(3) Shirmp sample collection

Separate the hepatopancreas of several shrimps from other

internal organs.

Collect the hepatopancreas sample for no less than 5 g.

-- photo by Deng YT

2. Sample homogenate

(1) Slice the sample and transfer it to a clean blending bag.


The Stomacher® Bag has been

designed to fullfil all applications

from sample blending, straining

and storage.

https://etconanalytical.com/stomacher-bags/

(2) Label the sample name and the weight.

-- photo by Deng YT

(3) Dilute the sample in 1 in 10 (w/v) in sterile saline.


(4) Seal the bag using a sealer and check for any leakage of fluid.


(5) Homogenize the sample with the paddle blender homogenizer.

-- photo by Deng YT -- photo by Deng YT -- photo by Deng YT

The paddle blender is convinient for

separating bacteria from tissues

with gentle blending action, and also

ensures the minimal damage to cell

and tissues.https://www.thomassci.com/Equipment/Blenders/_/MiniMix-100mL-Lab-Blenders?q=Stomacher%20Lab%20Blender

(6) Transfer 10mL of the filterate to a sterile centrifuge tube.

The sealed bag can use a syringe Open the bag and use a pipettehttp://www.labplas.com/en/products/filtra-bag

-- photo by Deng YT

3. Homogenized sample dilution

ü Serial dilution is the simplest technique for obtaining manageable

concentrations of a desired organism and it is complemented by

petri dish streaking and spreading.

ü The benefit of this approach is that the experimenter can harvest

pure strains of a single species or separate strains from a mixed

population.

(1) Prepare a series of at least 3 sterile centrifuge tubes containing

9 mL of sterile saline.

-- photo by Deng YT

(2) Using a sterile pipette, add 1 mL of filterate in the first tube of the set.

Label it as 10-1. Mix the contents well by swirling the tube upside down a

few times.

the filter fluid (100) the first diluted fluid (10-1)

-- photo by Deng YT

-- photo by Deng YT

(3) From the first diluted tube, take 1 mL of the mixture and transfer it to

the second tube. Label it as 10-2.

the second diluted fluid (10-2)

-- photo by Deng YT

(4) Repeat the procedure with the thrid tube and label it 10-3.

the third diluted fluid (10-3)

-- photo by Deng YT

9 mL sterile saline in each tube

10-1 10-2 10-3 10-4 10-5

Serial Dilution Ø To prepare decimal dilutions,

add 1 mL of the initial

suspension to tube 1 which

contains 9 mL of sterile saline,

and mix well.

Ø Repeat by aliquoting 1 mL of

the newly created solution1

and adding it to tube 2.

Ø Aliquoting and resuspension

continues until the final tube

is reached.

--Alvex GM and Cruvinel PE. Sensors & Transducers, 2016

（1）Label the sample name, diluted factors and the incubated date.

4. Spread plating

-- photo by Deng YT

(2) Pipette out 0.1 mL from the appropriate desired dilution series

onto the center of the surface of an agar plate.

-- photo by Deng YT

(2) Spread the sample evenly over the surface of agar using

a L-shaped spreader.

Carefully rotate the plate

underneath at an angle of 45

degree at the same time.

-- photo by Deng YT

Spread plating method

Pipette out 100 microlitre of the diluted sample, and spread it evenly. --Image by Macedo, 2016


(4) Allow the liquid soak in, and place the plates in inverted position.

Incubate at 28℃ for 18 to 24 hours.

The most appropriate incubation temperatures and time differ by

organism.

-- photo by Deng YT

Sample processing of aquatic environment 5.1.2

Aquatic environment constitutes important

antibiotic resistance reservoirs where

anthropogenic pressures may promote the

dissemination of antibiotic resistance genes and

bacteria. -- photo by Deng YT

Water and seiment samples should be collected

from at least 3 sampling sites in the same

aquatic environment.

Spread plate technique is a viable method

employed to plate a liquid sample for bacterial

isolation.

No more than 3 strains of the same

morphological type should be isolated from

each sample.-- photo by Deng YT

PROCEDURE

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1-1 Water Sample Pooling

(1) Transfer the same volume of each water sample to a 500- mL

volumetric flask.


(2) Mix the contents well by swirling the flask a few times.

-- photo by Deng YT

(3) Trasfer 10- mL of the mixture to a sterile centrifuge tube for

preparation of further dilution.

-- photo by Deng YT

(1) Dilute the sediment sample in 1 in 3 (v/v) in sterile water. Mix them well.

1-2 Sendiment Sample Pooling


(2) Leave the mixture for 30 min to 1 hour.


(3) Transfer the same volume of supernatant from each

sediment sample to a sterile centrifuge tube.

-- photo by Deng YT

(4) Mix the contents well by swirling the tube upside down a few times.

-- photo by Deng YT

2. Dilution

9 mL sterile water in each tube

10-1 10-2 10-3 10-4 10-5

Serial Dilution

1 mL mixture

Prepare tenfold dilutions of the sample with sterile saline.--Alvex GM and Cruvinel PE. Sensors & Transducers, 2016

Spread plate method

3. Inoculation

Pipette out 100 microlitre of the diluted sample, and spread it evenly.


--Image by Macedo, 2016

4. Incubation

Allow the liquid soak in. Incubate the plates at 28℃.

The most appropriate incubaiton temperatures and time differ by

organism.

-- photo by Deng YT

A quantity of water (e.g. 100 mL) is passed

through a specialized membrane filter

(pore size of 0.45 µm) , facilitating the

capture of bacteria.

Following filtration, the membrane is

carefully applied to a agar plate, and

incubated in an appropriate condition.

TIPS: Membrane filtration for water sample

Ø Membrane filtration method is not applicable to test the turbid water.

https://microbenotes.com/water-quality-analysis-by-membrane-filter-mf-technique/

--Waqalevu V, Batissa violacea, 2015

REFERENCE

Spread plate technique: https://microbeonline.com/spread-plate-technique/

Dissection of organs from fish:https://www.jove.com/cn/t/1717/dissection-of-organs-from-the-adult-zebrafish

Euthanasia in fish: Saint-Erne N, Tranquilisation, anaesthesia and euthanasia in pet fish.Companion Animal, 2014 ,19(12):658-662.

Streak plate method: https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/

Sample techiniaque of finfish: Buller, N.B. Bacteria from fish and other aquatic animals: a practical identification manual (Chapter 2.1), 2004

Sample techiniaque of crustaceans: Shields et al . Collection techniques for the analyses of pathogens in crustaceans Journal of Crustacean Biology 37 (6), 753-763,

Pearl River Fisheries Research Institute (PRFRI),

Chinese Academy of Fishery Sciences (CAFS), China

sample processing

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