Condenser and Iris

Condenser is a large lens mounted below the stage, with

an iris and diaphragm. There may be 2 or more lenses. Its

function is to deliver the light beam to the objective at a

sufficiently wide angle.

FIG. 1.8B: Digital microscope

Microscope Optics

Objective

On objective quality, depends, the quality of the image.

These are usually made up of more than one lens. On each

objective is engraved the magnification power.

Numerical Aperture

Numerical aperture (NA) of the objective is important, for

on this, depends, among other things, the amount of light

which the lens passes and the detail which it can make

visible, on which it is said to resolve.

Oil Immersion Objectives

They are used to avoid bending of light beam (with higher

magnification). The oil used should have the same optical

properties as glass, e.g. cedar wood oil. Liquid paraffin can

also be used.

Objective Aberrations

With increasing magnification certain optical aberrations

creep in:

1. Spherical aberration—edge of the lens gives slightly

higher magnification than its center.

2. Chromatic aberration—blue light is magnified slightly

more than red.

These aberrations can be avoided by using a series

of lenses made of special glass, carefully calculated and

designed.

Objective Qualities

1. Achromatic—are the usual average quality lenses and

are good enough for routine laboratory work.

2. Fluorite (Fi)—are highly corrected and expensive, have

a wider field and are good for searching blood films. FIG. 1.9: Microscope objectives

20 Concise Book of Medical Laboratory Technology: Methods and Interpretations The Mirror

It is placed below the condenser and iris, it can be turned in

any direction. It reflects the light beam from the source to

the iris and condenser. It usually has two mirrors mounted

back to back, one flat and the other concave. Flat mirror

is used in the presence of condenser and the concave

without the condenser.

Light Source

Daylight

Use of direct sunlight is bad for the microscope and the

eye. It is best to use reflected sunlight of a dull white

background. It is not sufficient for oil immersion lens and

it is not available during evening or night.

Electric Light

A 60 watt frosted electric lamp placed 18" away from the

microscope is sufficient for most routine work. Many

microscopes are now provided with built-in sources of

illuminations. In the absence of electricity, a battery lamp

or an oil lamp can be utilized. The light from these artificial

sources is rather yellow but may be used. Best, however,

are halogen lamps.

Special Applications of the Microscope

Phase Contrast Illumination

This is needed to visualize transparent microorganisms

suspended in a fluid. Ray of light travels in a wave form in

a straight line. Two such rays traveling together are said to

be in phase, and they produce a brighter illumination. If,

however, these rays are out of step with each other, they

are said to be out of phase. They interfere and produce less

bright illumination. Phase contrast microscopy makes use of

this property of rays to help or hinder each other and thereby

resulting increased contrast in the microscopic image.

The desired effect is brought about by placing an

annulus in the condenser and a phase plate in the

objective. A circle is engraved in the phase plate which

matches the ring of beam coming through the condenser

and annulus. This circle makes the wave take a longer or

a shorter step, so becoming out of phase with those aves 

 

which pass through the rest of the plate.

Supposing that the specimen is suspension free fluid,

the only light that reaches the eye is that which goes from

the annulus through the phase plate. Whereas presence

of organisms would diffract and scatter the light. The light

passing through the fluid gets out of phase with the light that

has the organisms stand out in contrast to their background.

Equipment Needed

An annulus, a phase plate and a telescope that is needed

for adjusting the rings of both annulus and the phase plate.

Method

1. Focus the specimen with the right objective after

illuminating the microscope.

2. Place the matching annulus at its position.

3. Remove the eyepiece and put the telescope in its place,

adjust it till the two rings, one bright and one dark are

in focus.

4. Adjust condenser screws till the bright annulus ring

fits exactly into the darker ring of the phase plate.

5. Remove the telescope, replace the eyepiece, focus and

examine the specimen.

Importance

This method is made use of for examining live organisms,

for examaple,

a. Cholera vibrios

b. Amebae

c. Trypanosomes

d. Trichomonas, and

e. Other flagellates.

It can also be used for platelet counting and for examining routine urine specimens.

Demerits

a. A halo is seen around each particle, it gives a false

appearance of its structure.

b. In addition, some resolution power is lost but this is

more than compensated for by the increased contrast

that is produced.

Dark Ground Illumination

This method too, is used for visualizing organisms

suspended in fluid, both the structure and the motility

FIG. 1.10: Working of an oil immersion objective

Laboratory 21

of the organisms can be seen. In this method, the light

enters the special condenser which has a central blackedout area so that light cannot pass directly through it to

enter the objective. Instead the light is reflected to pass

through the outer rim of the condenser at a wide angle

which illuminates the microorganisms by a ring of light

surrounding them (Fig. 1.11).

In this method, the light that is seen comes only from

the microorganisms themselves and not from the light

source. Hence, the organisms are brightly illuminated

against a dark background. Though useful, this method is

rather cumbersome.

Equipment Needed

1. An oil immersion dark ground condenser with the

centering screws.

2. A funnel stop for insertion in 100X objective to reduce

its NA and exclude light coming directly from the

source.

3. A brightly illuminated microscope lamp.

4. Scratchless slides not more than 1 mm thick.

Method

1. Fit the dark ground condenser and raise it to stage level.

2. Place the coverslipped specimen on the thin polished

glass slide. Both, the coverslip and the slide should be

absolutely clean.

3. Place a drop of immersion oil between the condenser

and the slide.

4. Adjust light source and the mirror properly.

5. Focus 10X objective and observe.

6. Focus condenser up or low, so that the ring ultimately

becomes just a spot of light. Focus this spot right in the

center.

 

Micro = Small, Scope = to view.

It magnifies the image of the object to be visualized

through it. Normally, the laboratory microscopes provide

18 Concise Book of Medical Laboratory Technology: Methods and Interpretations a magnification of 40x (scanner), 100x (low power),

400x (high power) and 1000x (oil immersion). The total

magnification is obtained by multiplying the magnification

of the objective with that of the eyepiece.

Parts of the Microscope

It has three sets of parts. They are the:

1. Stand,

2. Mechanical adjustments, and

3. Optics or the lenses.

Stand

It consists of:

1. The tube—supports objectives and eyepiece.

2. The body—gives support to the tube.

3. The arm—gives correct height and angulation to the

body and the tube.

4. The stage with a pair of spring clips or a mechanical

stage.

5. The substage holds the condenser lens with its iris

diaphragm and a holder for light filters and stops.

6. The foot on which other parts rest, can be in tripod or

horseshoe shape.

Mechanical Adjustments

Focusing Adjustments

These are coarse and fine adjustments.

Coarse Adjustment

Controlled by a pair of large knobs, one on each side of

the body. On rotating this, the tube moves with its lenses.

Some microscopes have this attached to the stage; so that

instead of the tube, the stage moves up and down. Coarse

adjustment is enough for low power lenses.

Fine Adjustment

Necessary for high power and oil immersion lenses. This is

usually controlled by two smaller knobs on each side of the

body. They may be graduated to indicate the movement in

microns.

Draw Tube

It is used to adjust the distance between the objective lens

and the eyepiece lens.

Inclination

The arm can be tilted upon the foot by a hinge.

Condenser Adjustments

Focusing of condenser is done by rotating a knob present

on one side below the stage.

Aperture Adjustment

It is done by the iris diaphragm (made up of leaves).

Centering of Condenser

It is done to bring the light beam accurately through the

instrument. In some microscopes, it is permanently fixed.

Mechanical Stage

It has knobs for moving the slide across or along the stage.

Monocular, Binocular and Digital Microscopes

Monocular—has only one eyepiece (Fig. 1.7).

Binocular—has 2 eyepieces, the only advantage it offers is

that it causes less strain on the eyes (Fig. 1.8A). Nowadays

digital microscopes are available, here digital image is

projected onto a digital display device (Fig. 1.8B).

FIG. 1.8A: Binocular microscope with substage lamp

FIG. 1.7: Monocular microscope with substage lamp

Laboratory 19

3. Apochromatic (Apo)—are very highly corrected and

costly and are only of value in special work.

Spring-loaded Objectives

The high power objectives (40X and 100X) of most modern

microscopes are spring loaded, i.e. the front mount of the

objective will be pushed in rather than pushed through

a specimen, if such an objective is accidentally pressed

against a specimen when focusing (Fig. 1.9).

Working of Oil Immersion Objectives

A beam of light passing from air into glass is bent; and

while passing from glass to air, it is bent back again. The

bending effect and its limitations can be avoided by

replacing the air between the specimen and lens with an

oil which has optical properties similar to that of glass,

i.e. immersion oil. When an appropriate oil is used, the

light passes in a straight line from glass through the oil

and back to glass as though it were passing through

glass all the way. Whenever possible, the immersion

oil recommended by the manufacturer of a microscope

should be used (Fig. 1.10).

Eyepiece

The most commonly used eyepiece is known as Huygens

eyepiece which has 2 lenses mounted at a correct distance

apart, with a circular diaphragm between, which give a

sharp edge to the image. These are available in different

magnifications. Lesser the magnification, brighter and

sharper is the image. For routine work, a 10X Huygens is

good enough. The 15X eyepieces are also available, as are

wide field ones.

 

¾ Medical practitioners delivering new service without

any consideration in a charitable hospital or medical

camps are exempted from the provisions under CPA

¾ As per clause 2(d) (ii) of the CPA 1986, a consumer

implies any person who hires or avails of any service

for a consideration, which has been paid or promised,

or partly paid and partly promised, or under any

system of deferred payment, and includes any

beneficiary of such services other than the person

who hires or avails of the service for consideration

paid or promised or partly paid and partly promised

or under any system of deferred payment, when such

services are availed of with the approval of the first

mentioned person

¾ The time limit stipulated for filing a complaint is 2 years

from the date of alleged negligence

¾ Patients can be dealt with severely if they file frivolous and

false complaints just to harass the medical practitioner

¾ Free services provided are exempted under CPA

¾ A laboratorian is also a consumer as he buys various

instruments, equipment, diagnostic kits/reagents/

devices. He too can file a complaint under CPA for any

defect or deficiency in service related to that purpose

¾ Ignorance is not held as an excuse as an established

legal principle. Concurrently, law does not expect a

very high degree of knowledge but expects only average

knowledge from a medical practitioner

¾ Medical negligence is a civil wrongdoing classified as

‘tort’, where a medical practitioner fails to take proper

care in respect of examination, diagnosis, investigation,

treatment, etc. resulting in injury or mortality

¾ Laboratorians are expected to keep all reports

confidential (legally and ethically). The reports can

be divulged to the referring clinician or to the patient

or the relatives of the patient (with patient’s onsent).

Reports pertaining to sexually transmitted diseases or

HIV/AIDS should be handed over only to the patient

¾ Legally, only authorized or registered blood banks can

supply units of blood. All mandatory information must

be clearly mentioned on the bottle label legibly

¾ These days doctors have ‘Malpractice Insurance Covers’.

In case a legal notice is received by such a doctor, he

should immediately notify the insurance company. The

insurance company must take all necessary actions in

such a case. The company should appoint a lawyer to

give reply or to take legal steps and inform the doctor

about it. The doctor, by permission of the company, can

appoint a lawyer of his choice

¾ What constitutes a legal notice? Any letter received by

a medical practitioner from a patient or a voluntary

registered organization or an advocate, demanding

explanation about treatment given or demanding some

explanation about treatment for alleged injury or death

constitutes a legal notice

¾ Section 27 of the Civil Procedure Code provides that

when a suit has been duly instituted, a summon may

be issued to the defendant to answer the claim, and

such summon is to be served in the prescribed manner.

When a complaint is lodged before the commission or

the forum, the defendant practitioner is informed by a

registered letter by the office, which is called a summon

in legal parlance. In this summon, time for the reply

and date of hearing is mentioned. Usually, the time

given for filing the reply is 30 days.

LABORATORY INSTRUMENTS

Microscope

 

Much of the contamination in the laboratory occurs as

a result of penetrating injuries caused by sharp objects and

the spilling and splashing of specimen materials.

Components of UWP

1. Handwashing.

2. Barrier precautions (mask, cap, plastic apron and

protection of feet).

3. Careful handling of all kinds of sharps and needles.

4. Effective infection.

5. Sterilization.

6. Correct disposal of different kinds of wastes generated

in a health care facility.

Guidelines of Basic Practices and Procedures

¾ Prevention of puncture wounds, cuts and abrasions and

protection of existing wounds, skin lesions, conjunctiva

and mucosal surfaces

¾ Application of simple protective measures designed

to prevent contamination of the person and his/her

clothing

¾ Good basic hygiene practices, including regular

handwashing

¾ Control of surface contamination by containment and

disinfection procedures

¾ Safe disposal of contaminated waste.

Biosafety Regulations for Laboratory Procedures

¾ Wear gloves when handling infectious materials or

where there is a possibility of exposure to blood and

other body fluids. All laboratories that work with

material that is potentially infected with HIV require a

generous supply of good quality gloves.

¾ Discard gloves whenever they are thought to have

become contaminated or perforated, wash your hands

and put on new gloves. Alternatively, where there are

economic constraints, wash gloved hands whenever

they get contaminated with blood/body fluids before

collecting further samples

¾ Do not touch your eye, nose, or other exposed

membranes or skin with your gloved hands.

Sterilization (for Nondisposable Items)

¾ For sharps, reusable blades, cystoscopy instruments,

endoscopy instruments, use CIDEX (2% glutaraldehyde)

or 5% Korsolex. Disinfection usually occurs in

30 minutes

¾ Use autoclaving for other reusable items (e.g. needle

holders, gowns, etc.)

¾ Wherever, autoclaving is not possible, boiling must be

for 30 minutes at the least.

Waste Disposal

Divide waste into three parts at source.

i. Household type noninfectious waste:

• Not to be decontaminated

• To be disposed off as such.

16 Concise Book of Medical Laboratory Technology: Methods and Interpretations ii. Infected sharp waste disposables (needles/surgical

instruments):

• Place in puncture-proof container containing

disinfectant (1% bleach prepared every morning). Needles should ideally be burnt (machines

are available that operate on electricity)

• Final disposal.

iii. Infected nonsharp waste:

• Is to be decontaminated

• Placed in disinfectant 5 to 10% bleach as the case

may be (left over blood, tissues, etc.).

 

Final Disposal

¾ Purchase of needle destroyer if resources permit

¾ Incineration of all infected waste

¾ Deep burial in controlled land fill sites (protected from

all sides)

¾ Shredding of disposable plasticware waste.

Postexposure Care

¾ Minor bleed with percutaneous inoculation, open skin

wound, breached skin, exposed mucous membranes.

First Aid

¾ Allow to bleed by squeezing

¾ Wash with water

¾ Antiseptic.

Report

¾ Employee identification, date, time with place of

accident

¾ Circumstances around accident

¾ Action taken.

Initial Consultation

¾ Easy access to medical advice with counseling. Consult,

physician for AZT prophylaxis regime if medication

available.

Laboratory Testing

¾ After consent with counseling within 2 weeks, 5 weeks,

12 weeks, or 24 weeks.

Clinical Follow-up

¾ For fever, pharyngitis, rash, malaise, lymphadenopathy,

myalgia and arthralgia within 6 months

¾ Do not leave the workplace or walk around the

laboratory while wearing gloves

¾ Wash hands with soap and water immediately after

any contamination and after work is finished. If gloves

are worn, wash your hands with soap and water

after removing the gloves. This is a vital and simple

precaution that is often overlooked

¾ Wear a laboratory gown or uniform when in the

laboratory. Wrap-around gowns are preferable. Remove

this protective clothing before leaving the laboratory

¾ When work with material that is potentially infected

with HIV is in progress, close the laboratory door and

restrict access to the laboratory. The door should have

a sign BIOHAZARD: NO ADMITTANCE

¾ Keep the laboratory clean, neat and free from

extraneous materials and equipment

¾ Disinfect work surfaces when procedures are

completed at the end of each working day. An effective

all-purpose disinfectant is a hypochlorite solution

with a concentration of at least 0.1% available chlorine

(1 g/L, 1000 ppm)

 

the involved part with pure carbolic acid.

Precautionary Measures

1. A fire extinguisher should always be handy.

2. Keep sand bucket in the laboratory.

3. Take measures to prevent electrical short circuiting.

4. No smoking in the working zone of the laboratory. FIG. 1.6: Mouth-to-mouth respiration

Laboratory 15

5. Breakable items should be kept in proper racks and

never at the edge of the working table.

6. Do not suck anything with the mouth, use rubber teats

and bulbs for sucking.

7. Do not place eatables on the working bench.

8. Keep fingernails short.

9. At the end of the day, clean all working benches with

a disinfectant. See that nothing except the required

electrical appliance is on.

10. Dispose all infected material properly. Can put such

material in hypochlorite solution or in an acidic

solution, e.g. diluted sulfuric acid (25%). Burn off all

dried contaminated articles, e.g. filter papers.

11. The glassware should be disinfected with a suitable

disinfectant and be cleaned thoroughly with running

water.

12. Use rubber gloves and a nose mask while working with

infective samples, e.g. serum of viral hepatitis patient.

UNIVERSAL WORK PRECAUTIONS (UWP) FOR

LABORATORY PERSONNEL (ESPECIALLY IN

RELATION TO HIV TRANSMISSION)

Introduction

Healthcare personnel (HCP) can acquire certain illnesses

beyond those acquired by all others who live and work

in our society, by virtue of their profession. HCPs are

at risk of acquiring any of the whole gamut of infections

from patients/specimens, which may be viral, bacterial,

parasitic or fungal. However, this risk due to occupational

exposure can be minimized if not obliterated altogether, if

we follow universal work precautions (UWP).

Today, with the WHO estimates of above 5 million HIV

positive persons in India, there is an urgent need to review

UWP. Besides HIV, there is the very real danger of acquiring

Hepatitis B and Hepatitis C in exactly the same way as

HIV and could also be fatal. Hepatitis B is 100 times more

infectious than HIV. Besides, Hepatitis B is also far more

prevalent in India in comparison to HIV with estimated

carriers being between 30 and 40 million, a considerable

number being infectious. However, fortunately, effective

vaccination is available for hepatitis B; therefore, it is

strongly recommended for all levels of healthcare workers.

 

2. Heating glassware and liquids

a. Test tubes: Never heat the bottom of a test tube.

The liquid inside might sputter. Heat the middle

of the tube, shaking gently. The mouth of the tube

should be facing away from the worker and any

other person, towards an empty space or a sink.

b. Ordinary glass and Pyrex: Only Pyrex glassware

and porcelain receptacles can be heated over a

Bunsen flame. Ordinary glass will break.

c. Inflammable liquids: Only small quantities of inflammable liquids such as ether, ethanol, acetone,

benzene, toluene and carbon disulfide should be

kept in the laboratory.

 Warning: Ether will ignite at a distance of several

meters from a flame. Never place a bottle of ether

on a workbench where there is an open flame

(Bunsen burner, spirit lamp, etc.). Carbon disulfide is even more dangerous.

d. Butane gas: When lighting a gas burner, always

light the match and hold it to the burner before

turning on the gas tap. Turn off the main valves of

all butane gas cylinders every evening. Replace

the rubber connecting pipes once a year.

3. Do not use broken, cracked or chipped laboratory

glassware.

4. Put clear labels on poisons. Keep them in a locked

cupboard.

5. Do not use nylon clothes while working as these are

easily inflammable. Always use a laboratory apron.

6. Always ensure that electrical wiring and electrical

appliances are in good condition.

Suggested List of First Aid Equipment for

Laboratory

1. 5% aqueous sodium carbonate

2. 2% aqueous sodium bicarbonate in an eye drop bottle

3. 5% acetic acid

4. Saturated solution of boric acid in an eye drop bottle

5. Soap powder solution (5 g per liter of water)

6. Acriflavine ointment

7. Mercurochrome 2%

8. Antiseptic lotion

9. Cotton wool

10. Gauze

11. Roller bandage

12. Adhesive tape

13. Scissors.

Contamination from Infective Material

If contamination has occurred, then:

1. Disinfect the part with the disinfectant available in the

laboratory. Thoroughly clean the affected area with a

stream of running water.

2. Sucking the contaminated material: Spit out all that

has been sucked. Use a disinfectant liquid (e.g. diluted

dettol) for mouth washing. If the infected material has

been swallowed accidentally, forced vomiting to be

done, ascertain the kind of infection and take advise

from a medical person.

3. If skin is infected by highly virulent organisms, touch

 

any flame. Use a water bath or electric hot plate.

Control of Fire Caused by Flammable Chemicals

Best controlled by smothering them. Use sand, thick blanket

or the now available multipurpose fire extinguishers.

Pouring water on such fires will spread them. Every

laboratory should be equipped with the commercially

available fire extinguishers. If these are not available, there

should be sand buckets in accessible places.

Corrosive Chemicals

These include strong acids, e.g. concentrated sulfuric

acid, hydrochloric acid, nitric acid, glacial acetic acid,

trichloroacetic acid, orthophosphoric acid, and strong

alkalies like sodium hydroxide and potassium hydroxide.

Storage

Store these at low levels.

Safe Use

Never attempt mouth pipetting. Accidental swallowing

can be lethal as these chemicals cause destruction of

living tissue. Always pour a corrosive chemical at below

eye level, slowly, and with great care to avoid splashing.

Wear protective eye glasses/eye shields while opening

such containers. Always add the corrosive substance to

water and that too slowly. The addition of small amount of

water to sulfuric acid is enough to produce sufficient heat

to break a glass container.

Laboratory 9

Toxic, Harmful, and Irritating Chemicals

These are chemicals that can cause death or serious illhealth if swallowed or inhaled or if they come in contact

with skin. Examples are potassium cyanide, mercuric

nitrate, sodium azide, sodium nitroprusside, formaldehyde

solution, chloroform, barium chloride and methanol.

Iodine and sulfuric acid also fall in this category. Skin and

mucous membrane irritants are xylene, formaldehyde and

ammonia vapors.

Storage

Store highly toxic chemicals, e.g. potassium cyanide in a

locked cupboard. Stock solutions should also be stored

safely in a cupboard, not on an open shelf.

Safe Use

Always wear protective gloves and after working with

them immediately lock them up. Always wash your

hands after using a toxic or harmful chemical. Keep fume

forming chemicals in a fume cupboard. Never mouth

pipette them.

Oxidizing Chemicals

These include chlorates, perchlorates, strong peroxides,

potassium dichromate, and chromic acid.

Storage

Keep these away from organic materials and reducing

agents. They can produce much heat when in contact with

other chemicals, especially flammable chemicals.

FIG. : General laboratory

SIGNS FOR MEDICAL LABORATORIES

10 Concise Book of Medical Laboratory Technology: Methods and Interpretations Safe Use

Handle them with utmost care. Most of them are dangerous

to skin and eyes and when in contact with reducing agents.

Explosive Chemicals

These chemicals can explode on being heated or on getting

exposed to flame or friction. A good example is picric acid,

which must be stored under water. If picric acid is allowed

to dry, it can explode.

Carcinogens

These chemicals can cause cancer by ingestion, inhalation,

or by skin contact. Such chemicals include benzidine,

O-toluidine, O-dianisidine, a and b naphthylamine, nitrosamines, nitrosophenols, nitronaphthalenes, and selenite.

The carcinogenic risk is directly proportional to the length

and frequency of exposure and the concentration of the

chemical.

Storage

Label their containers “CARCINOGENIC” and handle

with special precautions.

Safe Use

Must wear protective plastic or rubber gloves, a facemask

and eyeshields when handling carcinogenic chemicals.

Do not let them come in contact with skin. After handling

a carcinogen, wash well in cold water all the apparatus,

bench, bottles and protective gloves (before removing

them) and change your overall. Rinse your hands in cold

running water before using soap. Should a carcinogen

come in contact with skin, wash the affected part in cold

running water for 5 minutes.

ACCIDENTS IN THE LABORATORY

They may be caused by:

1. Acids }

• Splashes on the skin

or • Splashes in the eye

2. Alkalis • Swallowing

3. Toxic substances

4. Heat • Open flames

• Hot liquids

• Inflammable liquids

• Explosions

FIG. : Laboratory cautionary

Laboratory 11