Lab pro learning objectives: Lab 1-4

Describe the seven qualities of laboratory design

1. Large enough to accommodate equipment and personnel

2. Room-temperature and draft-free

3. Sink

4. Storage

5. Electrical supply

6. Internet access

7. Safety

List safety equipment for a lab in a veterinary facility

Eye wash, fire extinguisher, spill cleanup kits, biohazard disposal containers, protective gloves, PPE

Describe the purpose and benefits of, and the Veterinary Technologist’s role in a Veterinary Lab

• collect specimens from the patient

• able to identify them as cells, organisms, bacteria, or parasites

• help the DVM diagnose health issues with patients and develop a treatment plan.

Be able to list equipment, instruments, supplies and requirements of a Veterinary Lab

Microhematocrit centrifuge, clinic centrifuge, refractometer, compound light microscope

Be able to define Quality Assurance/Control and describe steps to achieve

Quality assurance: the procedures established to ensure clinical testing performed is in compliance with accepted standards

Control: must be assayed regularly to help verify accuracy of test results

List the parts of a compound microscope

Coarse and fine adjustment knob, eyepiece / ocular lens, objective lens, stage, mechanical stage controls, iris diaphragm, substage condenser

Explain the importance of lab safety during fecal analysis

PPE must be worn (lab coat, gloves, face masks) because there is zoonotic potential for some parasites and parasitic eggs

List the characteristics to note on gross examination of feces

color, consistency, mucus, odor, adult parasite, presence of blood, foreign bodies

pros and cons of sugar solution

PROS: inexpensive, can be made anywhere, doesnt effect the eggs

CONS: sticky to work with

pros and cons of Sodium nitrate

PROS: Most efficient solution, commercially available

CONS: forms crystals, distorted eggs, costly

pros and cons of Zinc sulfate

PROS: floats protozoal organisms with least distortion

CONS: must be used with an additional solution to identify species

pros and cons of Sodium chloride

PROS: none

CONS: corrodes lab equipment, forms crystals, severely distorts eggs, poor flotation medium; max specific gravity = 1.200g/mL

pros and cons for magnesium sulfate

PROS: inexpensive, readily available

CONS: forms crystals on slide

Explain the significance of specific gravity in fecal flotation solutions

• allows the eggs to be recovered by floating on top of the solution

• flotation solutions have a specific gravity between 1.200-1.250g/mL, and most parasitic eggs have SG  between 1.1-1.200g/mL

• specific gravity of the solution must be higher than the egg and water.

steps of Simple fecal flotation (fecalyzer)

1. 3-5g of fecal sample with fecalyzer and fill up halfway with fecal flotation solution

2. Twist green part to mix the feces and solution to create an emulsion

3. Fill up fecalyzer with more os the same flotation solution and create a positive meniscus

4. Place coverslip on top of meniscus and wait for 10-20min

5. Place cover slip on slide; view on 10x - 40x objective lens

steps of centrifugal flotation

1. 3-5g of fecal sample in a cup and mix with 10-20mL of fecal flotation solution to create an emulsion

2. Strain emulsion by placing cheesecloth over the centrifuge tube, and pour emulsion over the cheesecloth into the tube

3. Place tube in centrifuge, make sure its balanced. 

4. Spin in centrifuge on the urine setting for 5 min

5. Let sample sit for 10-20min

6. Pipette sample from the top layer and place 1-2 drops onto a slide

7. Place cover slip on top and view under 10x- 40x objective lens

steps for fecal sedimentation

1. 3-5g of fecal sample in a cup and mix with 10-20mL of warm water to create an emulsion

2. Strain emulsion by placing cheesecloth over the centrifuge tube, and pour emulsion over the cheesecloth into the tube

3. Let sit in tube undisturbed for 20-30min

4. Pour off the supernatant,

5. Repeat steps C and D 4-5 times

6. Take a pipetted sample from bottom of sample, and place 1-2 drops onto a slide

7. Place a cover slip on top and view under 10x-40x objective lens

How to approximate the size of parasitic eggs observed

• Take your field of view measurement in mm or um,

• apprx. how many time the egg can fit in your field of view along x-axis and y-axis.

• Divide the FOV measurement with number times the egg fits across. Will equal size of the ova.

common intestinal parasites of dogs and cats

Toxocara sp., Ancylostoma sp., Trichuris sp.,  Dirofilaria immitis.

Describe the set up and use of the Baermann apparatus

• funnel, cheesecloth, funnel holder, beaker, and warm water

• used when larva are suspected of lungworm (Stongyloides sp.)

• can use a fecal, soil, or tissue samples with this apparatus

Describe the different techniques and stains used to detect protozoal trophozoites and coccidi

Direct smear: used to identify Giardia and trophozoites; using Lugol’s iodine or new methylene blue

Acid-fast: used to identify coccidial parasites such as Cryptosporidium spp

Describe the importance of using the correct objective lens when examining sample

Differentiating size of the ova and certain characteristics

Buffy coat

a concentration technique for protozoal and rickettsiae in WBC’s

Direct drop

• simplest blood evaluation

• Least accurate

• Drop of anticoagulated whole blood examined microscopically

• Detects movement of extracellular parasite: heartworm microfilariae

Filter test

• method to concentrate microfilaria in blood

• Quicker and easier than modified Knotts

Modified Knott’s test

• used to concentrate microfilariae

• Uses larger sample

• Hemolyzes RBC’s so microfilariae can be observed

• Can help differentiate Dirofilaria  and Acanthocheilonema

• Cannot detect heartworm antigens

ELISA principle

• highly accurate and precise

• Can detect occult infections

• Fast and easy to perform

Describe lifecycles of specific endoparasites

• developmental stages: egg, 4 larval stages, sexually mature adult.

• Infective stages: egg with a larva, a free-living larva, or a larva within an intermediate host.

• direct lifestyle (no intermediate host), or indirect one (requires intermediate host).

• transmission occurs through ingestion, skin penetration, ingestion of intermediate host, or deposit on the skin by intermediate host

Estimate the size of parasitic eggs from Toxocara spp., Trichuris spp., Dipylidium caninum and Ancylostoma spp

Toxocara canis- 75-90um in diameter

Toxocara cati - 65-75um diameter

Toxocara leonina - 75x85um