A day book is ideal as it has the necessary ruled lines. In

your record put the date, reference number, patient’s

name, name of the referring doctor, investigations asked

for, reports given and payment status (if privately owned

laboratory).

Laboratory Reporter

An ideal laboratory computer program helps in reporting

and recording diagnostic center, pathology lab and other

diagnostic imaging fields. The program should also keep

history records of the patients. It must have facilities of

making and reporting profiles, e.g. lipid, renal, cardiac,

hepatic and diabetic profile. A program can be called ideal if:

FIG. 1.29: Serological water bath

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 1.30A: Incubator

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 1.30B: Hot air oven

(Courtesy: Yorco Sales Pvt. Ltd)

It Reduces Overload

¾ Avoids manual operations by printing booking slips,

receipts, bills, envelopes, etc.

¾ Prints daily register of patients

¾ Prints rate lists

¾ Reports only the tests required and not the whole group

of tests

¾ A comprehensive reporting option for day end

operations including daily collection report and doctor

wise daily collection.

28 Concise Book of Medical Laboratory Technology: Methods and Interpretations Graphs

¾ Prints graphs, e.g. GTT for to the point reporting

¾ Can make/design your own graphs

¾ Can see the graph on screen as well as print.

Accounts

¾ Maintains your bank, cash accounts

¾ Provides all ledgers

¾ Makes trial balance for final accounts.

Address Manager

¾ A mail list program, keeps address details.

¾ Provides easy working on the basis of name

¾ Keeps addresses for Labmate program of referencing

doctors, patients and reporting doctors

¾ Prints address directory with telephone numbers, etc.

¾ Prints labels for sticking on your mail

¾ Can group your addresses as per nature of address such

as friend, relative, doctor, patient, etc.

The features given above are complete. All records

can be retrieved date wise or name wise. Any program

that provides the above-mentioned capabilities can be

considered as an ideal laboratory reporter.

Caution: All medical electronic diagnostic devices need a

stable constant voltage, therefore, proper protective cover

must be provided. CVT (constant voltage transformer),

servo stabilizers, and UPS (uninterrupted power supply)

should be installed in the mainline or with specific

instruments.

¾ It helps referencing doctor

¾ Provides clear reports with normal values

¾ Abnormal values are underlined or highlighted

automatically

¾ Prints history reports of the patients.

It Makes Working Easy

¾ Reports as per your own method of grouping of tests,

profiles, etc.

¾ Automatic calculation of charges

¾ Keeps list of referencing doctors

¾ Maintains daily collection on referencing doctor/

institution

¾ Provides workload report.

The Computerized System is Easy to Operate

¾ Simple menu-based operations and does not require

any detailed knowledge of computers

¾ Help facility at every stage of working for beginners

¾ Can find a patient detail based on of reference number,

name, date and referencing doctor.

Features and Provisions

¾ Keeps the results for as long as you want

¾ Keeps normal values for male/female and adult/child

for all tests

¾ Can change any normal value as per your equipment,

techniques and methods

¾ Provision for reporting by different doctors

¾ Reports can be printed on simple paper or on preprinted

letter heads (computer stationery).

2

 

Sterilization

C H A P T E R

The terms sterilization and disinfection are used to indicate

the treatment of material so as to destroy or otherwise

eliminate any living organisms present. However, the

term sterilization is used where physical methods are

used and disinfection is used where chemical agents are

made use of.

METHODS COMMONLY USED FOR STERILIZATION

The methods used commonly in practice are:

1. Killing organisms by heat: Heat may be dry or moist

2. Destroying organisms by employing chemical antiseptics,

e.g. lysol, phenol, perchloride of mercury, etc.

3. Removing organisms mechanically by filtration, e.g.

Seitz, unglazed porcelain.

Sterilization by Heat

Adequate heat is the most certain and rapid method for

sterilization. The time needed for sterilization is inversely

related to the temperature of exposure—the higher the

temperature, the shorter the time needed. High temperature

kills bacteria by coagulating their proteins. Different

types of bacteria show considerable differences in heat

susceptibility. In general, vegetative forms are destroyed

at lower temperatures, whereas high temperatures are

needed for sporing organisms.

Dry Heat

This is the preferred method for sterilizing glassware, e.g.

of glass syringes and of materials such as oils, jellies and

powders which are impervious to steam. Dry heat requires

a much higher temperature or a much longer time at the

same temperature than does moist heat. Dry heat can be

used in the following ways:

Flaming

The articles are passed through the Bunsen flame, without

letting them become red hot. It is used for scalpels, needles,

mouths of culture tubes, glass slides, coverslips and points

of forceps. Only the surfaces actually touched by the flame

are sterilized.

Red Heat

Platinum loops, inoculating wires and needles are heated

in the Bunsen flame until red hot.

Hot Air Oven

These are electrically heated and thermostatically

controlled. The oven itself is a double-walled steel chamber

with a stout door. The top or side contains a ventilator

which is left open during sterilization to disperse any

moisture or volatile matter. Air circulates within the oven

by convection currents. Suitable sterilizing times in the

hot air oven are 3 hours at 140°C, 1 hour at 160°C and

30 minutes at 180°C. All dry glassware, such as test tubes,

petri-dishes, flasks, pipettes and throat swabs, etc. are

made sterile by using hot air oven.

This method is not suitable for sterilizing culture

media, liquids, rubber connections, glass to metal fitting

and fabrics, e.g. masks, towels or gowns.

Moist Heat

Temperature

A temperature of 60 to 65°C kills most vegetative bacteria

(made use of in pasteurization of milk and preparation of

vaccines).

Boiling

Boiling is frequently used for sterilizing syringes, etc. but is

not adequate as many spores withstand this temperature.

30 Concise Book of Medical Laboratory Technology: Methods and Interpretations

Steam

Steam is the most effective technique of moist heat

sterilization. Steam may be employed in three ways.

Steam at 100°C

The apparatus used commonly is called Koch’s steamer. It

has a vertical metal cylinder with a conical lid. It is fitted

with a thermometer and has a small opening for escape

of steam.

Sterilization by free steam can be done in two ways.

Prolonged exposure: For 1½ hours, used for broth or

nutrient agar.

Intermittent heat or tyndallization: It involves exposure for

20 minutes on three successive days and is used to sterilize

sugars and gelatin which decompose on higher temperatures.

Principle: Spores would germinate after first steaming and

destroyed on the next, three steamings would eliminate all

spores and their vegetative forms.

Low Temperature Steam

This method is employed for sterilizing materials (blankets, polyethene tubing, etc.), which would be damaged at

higher temperatures.

Steam at Temperatures above 100°C (Autoclaving)

Autoclaves are made of strong metal jackets; strong enough

to withstand high pressures required (Figs 2.1A to C). The

autoclave door is hermetically sealed. It has a safety valve

set to blow off at a predetermined pressure. The principle

is that water boils when its vapor pressure is equal to the

 

a. Specimen bottles—with top screws, e.g. the

universal type containers.

b. Reagent bottles—have ground glass or plastic

stoppers, available in different sizes and may be

made of amber colored glass (Figs 1.21A and B).

c. Drop bottles—fitted with special tops through

which drops can be delivered (Fig. 1.22).

4. Funnels—used to hold filter papers when filtering fluids

or for pouring liquids into narrow neck containers

(Figs 1.23A and B).

5. Cylinders—used for measuring liquids, they have a

pouring spot (Fig. 1.24).

6. Tubes—are of various sizes; of the test tube or

centrifuge (conical) type, with or without a top rim

(Figs 1.25 and 1.26).

7. Pipettes—are used to measure and deliver a given

volume of fluid.

FIG. 1.17: Conical flasks

FIG. 1.18: Volumetric flasks

FIGS 1.19A AND B: (A) Round bottomed flask and

(B) Flat bottomed flask

FIG. 1.20: Beakers

A B

Laboratory 25

FIG. 1.21A AND B: (A) Specimen bottles and (B) Reagent bottles

A B

FIG. 1.22: Drop bottles

a. Volumetric pipettes—have a bulb shape in the

stem. Each pipette is marked to show the given

volume of fluid, it contains or delivers (Figs 1.27A

and B).

b. Graduated pipettes—are of various sizes. They

may be of the non-blow out or the blow out

type.

c. Blood pipettes—have a white back and include

the 0.02 mL pipette used for hemoglobin, red cell

and platelet counts, and also the 0.05 mL pipette

for white cell counts (Fig. 1.28A).

 FIGS 1.23A AND B: (A) Separating funnel and (B) Funnel

A B

FIG. 1.24: Measuring cylinder

d. Pasteur pipettes—have multiple uses. They are

not graduated or marked. These can be bought

or made in the laboratory (Fig. 1.28B).

Other Necessary Equipments

Serological Water Bath

It is electrically heated and has a thermostatic temperature

regulator. It can provide temperature ranging from room

temperature to 100°C. Various sizes to suit various

workloads are available (Fig. 1.29).

26 Concise Book of Medical Laboratory Technology: Methods and Interpretations FIG. 1.25: Test tubes

FIG. 1.26: Centrifuge tubes

FIGS 1.27A AND B: (A) Volumetric pipette and (B) Measuring pipette

A B

FIGS 1.28 A AND B: (A) Blood pipettes and (B) Pasteur pipttes

A B

Incubator

Works on electricity and regulates temperature thermostatically. Necessary for various investigations where body

temperature 37°C (or otherwise) incubation is required

(Fig. 1.30A).

Hot Air Oven

This is used for drying and sterilizing glassware. This too

is thermostatically controlled and electrically heated. It

looks like an incubator (Fig. 1.30B).

Reporting Laboratory Tests and Keeping Records

Standardization

Standardization in the reporting of laboratory tests

contributes to the efficiency of the laboratory service

and is of great value when patients are referred from one

place to another. Whenever possible, request forms and

other laboratory printed stationery should be prepared

and issued by a central stationery office.

Laboratory 27

Use of Rubber Stamps

When stationery is not supplied from a central source,

standardization in presenting and reporting results can be

achieved by the use of rubber stamps. Adequate ink must

be used and the stamp must be positioned carefully.

Format

The top part of the report card must prominently give the

name, address and telephone numbers of the laboratory. It

should then have place for printing the patient’s name, age,

sex, name of the referring doctor, the laboratory reference

number and date. Next, the title of the report should be

mentioned, e.g. urinalysis, stool examination, hematology,

biochemistry, etc. After this, print the investigation name,

leave space for patient’s values, print normal values

followed by the units. The report must end with the

signatures of the person in-charge of the laboratory.

Keeping Records in the Laboratory

A record of all test results must be kept by the laboratory

as carbon copies, work sheets, or in simple exercise books.

 

Importance

1. For identifying mycobacteria.

2. It is used extensively in fluorescent antibody techniques

used in parasitology and bacteriology. FIG. 1.11: The principle of dark ground illumination

22 Concise Book of Medical Laboratory Technology: Methods and Interpretations 3. It is also used widely in histopathology of kidney, skin,

etc. where immune/autoimmune basis of disease is

expected. In fact, anything can be confirmed with

high degree of sensitivity and specificity, if antibodies

against it (later tagged with a fluorescent dye) can be

produced.

4. Used widely in cytogenetics.

Electron Microscope

Basic Principle

The resolution of the light microscope has been shown

to be limited by the NA and the wavelength of light

employed. As the degree of correction in glass lenses is

very high, the main limitation is imposed by the light

(e.g. half wavelength of light), giving a normal resolution

of approximately 250 nm; and when UV light is used,

a resolution of about 100 nm. By the substitution of an

electron beam for light rays, a much greater degree of

resolution can be obtained; since at an acceleration of

50,000 volts, electrons have a wavelength of only 0.001 nm;

therefore, a theoretical resolving power of 0.0005 nm could

be attained, which would enable molecules to be seen.

Unfortunately, the degree of correction that is currently

feasible with transmission electron microscope (TEM)

lenses will permit a resolution of only 0.25 nm, but this is

still a thousand times greater than that possible with the

light microscope. A further difficulty with the TEM is that,

since electrons have poor penetrating power, the sections

to be examined must be very thin, less than 50 nm thick.

This necessitates the use of special hard embedding media

(plastics) and special ultra-microtomes to cut such thin

sections. Steel knives cannot be used to cut these sections;

either glass or diamond knives are used.

Weighing Scales or Analytical Balance

Weighing scales: For weighing large quantities.

Analytical balance: For accurate weighing of smaller

quantities.

Use and Care

1. The weighing equipment must be placed on a firm

bench, away from vibration, draughts, direct sunlight

and dust.

2. It should be kept perfectly horizontal by altering the

screws on which the equipment stands.

3. Chemicals, etc. should never be placed directly on the

pans. Weigh them in a container.

4. Never touch the weights with hands, handle them with

forceps.

5. The balance should be at rest before adding or

removing the weights or chemicals.

6. Before taking the reading, the glass window of the

instrument should be closed.

Electronic analytical balances are also available. Made

by various companies, these are very accurate.

Centrifuge

Centrifuge is used to sediment or deposit rapidly particles

such as cells which may be suspended in a fluid. The speed

is expressed as rpm, i.e. revolutions per minute.

Relative Centrifugal Force (RCF)

More important than rpm is relative centrifugal force

(RCF). RCF is expressed as the acceleration due to gravity

or G (dynes per cm). The formula is:

G = 0.00001118 × (r) × (n)2

where r = radius in centimeters

and n = revolutions per minute.

The time of centrifugation is equally important. The tubes

should be spun for a definite period to obtain the desired

effect.

Types of Centrifuge

Hand Centrifuge

Fixed to the bench, the handle is rotated manually. It gives

low speeds only.

FIG. 1.12: Components of fluorescence system

Laboratory 23

Motor-driven Centrifuge

Operated through mains electricity supply. The tubes

may be kept in a fixed angle head or in a swing out head

(Figs 1.13 and 1.14).

Microhematocrit Centrifuge

Also motor driven for finding out packed cell volume

(PCV) of red blood cells (RBCs). In this, blood-filled

capillary tubes are spun and later the percentage of

RBC-filled column is estimated (Figs 1.15 and 1.16).

Use and Care

1. Use centrifuge tubes made of strong glass and they

should not be too long.

2. The opposite tubes should be balanced properly.

3. The centrifuge speed should be increased gradually.

4. The instrument should be kept clean. If something

spills over inside, it should be cleaned and the

instrument disinfected, if necessary.

FIG. 1.13: Swing out head centrifuge

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 1.14: Motor driven centrifuge with rpm. indicator and auto

(timed) shut off

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 1.15: Dual centrifuge routine centrifuge with microhematocrit

attachment

(Courtesy: Yorco Sales Pvt. Ltd)

FIG. 1.16: Microhematocrit centrifuge and its parts

(Courtesy: Yorco Sales Pvt. Ltd)

24 Concise Book of Medical Laboratory Technology: Methods and Interpretations Glassware (Many Items are now Made of Plastic)

1. Flasks—are of different sizes and shapes.

a. Erlenmeyer or conical flasks—for heating and

boiling liquids (Fig. 1.17).

b. Volumetric flasks—are graduated for getting

exact volume of liquids (Fig. 1.18).

c. Round and flat-bottomed flasks for preparing

solutions (Figs 1.19A and B).

2. Beakers—available in different sizes (Fig. 1.20).

3. Bottles

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.